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 ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY

ECOTOXICOLOGY IN LATIN AMERICA

CRISTIANO V.M. ARAÚJO

AND
CANDIDA SHINN
EDITORS

New York

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Copyright © 2017 by Nova Science Publishers, Inc.

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 CONTENTS

About the Book ix

Foreword xi
List of Contributors xiii
Acknowledgments xxix
Chapter 1 What Risks Do the Contaminants Really Represent? a Standpoint
on Effects from Organisms to Ecosystems/Landscapes Based on
Non-Forced Aquatic Exposure Scenarios 1
Cristiano V. M. Araújo, Cândida Shinn, Matilde Moreira-Santos
and Rui Ribeiro
Chapter 2 Biomarkers in Native Central American Species 13
Freylan Mena Torres, Margaret Pinnock Branford,
Silvia Echeverría Sáenz, Luisa E. Castillo Martínez
and Rocío Ugalde Salazar
Chapter 3 Aquatic Ecotoxicology: Native Freshwater Gastropods from
Argentina 23
Paula F. Cossi, Lucila T. Herbert, Beverly Boburg
and Gisela Kristoff
Chapter 4 Energy Physiology as Biomarker in
Assessing Environmental Pollution 37
Beatriz Toro Restrepo and Jorge M. Navarro
Chapter 5 Laboratory and Field Based Studies Provide Insights of Copper
Tolerance Mechanisms in Brown Seaweeds and Biotechnology
Tools for Environmental Diagnosis 57
Claudio A. Sáez
Chapter 6 Endocrine Gene Expression in Aquatic Animals as Indicator
of the Presence of Xenobiotic Compounds 73
Maurilio Lara-Flores, Yassir Edén Torres-Rojas
and Jaime Rendón von Osten

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vi Contents

Chapter 7 Sensitivity of Tropical Cladocerans and Chironomids to

Toxicants and Their Potential for Routine Use in Toxicity Tests 87
Arnola C. Rietzler, Odete Rocha, Ana L. Fonseca,
Marcela M. Ribeiro and Mariana F. Matos
Chapter 8 Anthropogenic Impact Modifies the Blood Antioxidant Status
in Pygoscelis adeliae and Pygoscelis papua at Hope Bay,
Antarctic Peninsula 105
Julieta D. Cebuhar, Carla I. Di Fonzo, Laura P. Dopchiz
and Martín Ansaldo
Chapter 9 Metallothioneins as Biomarkers of Contamination by Metals in
Bivalves of Eastern Venezuela 119
Mairin Lemus, Julián Castañeda and Kyung Chung
Chapter 10 Ecotoxicological Studies of Freshwater Ecosystems in Latin
America: Diagnosis, Perspectives, and Proposals 137
Roberto Rico-Martínez, Ignacio Alejandro Pérez-Legaspi,
Gustavo Emilio Santos-Medrano, Jesús Alvarado-Flores
and Mario Alberto Arzate-Cárdenas
Chapter 11 Freshwater Ecotoxicology in Costa Rica 163
Rocío Ugalde, Silvia Echeverría-Saénz, Elba de la Cruz,
Margaret Pinnock-Branford, Freylan Mena and Luisa E. Castillo
Chapter 12 Environmental Risk in a Coastal Zone of Rio de Janeiro State
(Brazil) due to Dredging Activities 183
Ana Paula de Castro Rodrigues, Alexandre Paoliello Lemos,
Christiane Monte, Sarah K. Rodrigues, Ricardo Gonçalves Cesar
and Wilson Machado
Chapter 13 Assessment of the Risk Generated by Natural and Anthropic Agents
on Peruvian Coastal Marine Environments 201
Guadalupe Sánchez and Aldo G. Indacochea
Chapter 14 Assessing the Ecological Effects of Contaminants in Estuaries in
Brazil: Gaps and Future Directions 217
Marcos Krull, Denis M. S. Abessa and Francisco Barros
Chapter 15 Avoidance and Recolonization Responses of the Gastropod Olivella
semistriata Exposed to Coastal Sediments 239
Cristiano V.M. Araújo, David Salvatierra, Victoria Vera-Vera,
Luís A. Cedeño-Macías, Katherine Benavides,
Dayanara Macías-Mayorga, Matilde Moreira-Santos
and Rui Ribeiro
Chapter 16 Pollution in Coastal Areas: An Integrated Perspective
in Environmental Risk Studies 255
Javier García-Alonso and Delfina Castiglioni

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 Contents vii

Chapter 17 Ecotoxicology in the Marine Environment: Bioaccumulation

and Bioconcentration Factor of Polycyclic Aromatic Hydrocarbons 271
Mónica M. Zambrano, Robinson F. Casanova
and Gustavo Arencibia
Chapter 18 Soil Ecotoxicology in Environmental Risk Assessment: A Case
Study in a Metal Contaminated Site in Brazil 289
Júlia Carina Niemeyer, Eduardo Mendes da Silva
and José Paulo Sousa
Chapter 19 Ecotoxicological Assessment of Dredged Sediments from
Guanabara and Sepetiba Bays (Rio De Janeiro, Brazil)
Using Bioassays with Earthworms 309
Ricardo G. Cesar, Ana P.C. Rodrigues, Bianca C.R.C. da Rocha,
Tácio M. de Campos, Christiane N. Monte, Simone Dealtry,
Zuleica C. Castilhos and Wilson T.V. Machado
Chapter 20 Soil Ecotoxicology: Chilean Studies with Soils Polluted
by Copper Mining 325
Alexander Neaman, Rosanna Ginocchio and Carolina Yáñez
Chapter 21 Inter-Laboratory Calibration of the Ostracodtoxkit F Assay
Using the Crustacean Heterocypris Incongruens on
PAH-Contaminated Soils 339
Belgis Chial Z., Guido Persoone, Doris Hirmann
and Andreas P. Loibner
Chapter 22 Mining in Venezuela: Its Effects on the Environment
and Human Health 347
Antonio Machado-Allison
Chapter 23 Environmental Risk of Lead Accumulation in Crops Irrigated
with Water from the Mantaro River, Jauja Sector, Peru 363
Edith Orellana and Raymundo Erazo
Chapter 24 Human Health Risks Due to Air Mercury Exposure Produced
by Artisanal and Small-Scale Gold Mining Activities
in Portovelo, Ecuador 377
María Fernanda Rivera-Velásquez,
Patricio Colón Velásquez-López,
Victor Hugo Gonzalez-Carrasco and Carmine Fallico
Chapter 25 Environmental Impact of Agricultural Activities on Human
and Wildlife Populations from Palizada, Campeche, Mexico 389
Jaime Rendón von Osten, Lucia Guilhermino
and Amadeu M. V. M. Soares

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viii Contents

Chapter 26 Bioaccumulation of Mercury in Yellowfin Tuna (Thunnus

albacares) from Ecuador and Other Worldwide Regions:
A Comparative Study 407
Luis A. Cedeño-Macias, David Villarreal,
Fernando R. Diz and Dayanara Macías-Mayorga
Chapter 27 Lichens and Air Quality in Latin America 417
Jesús Hernández, Raiza Fernandez, Mariana Cárdenas Henao,
Christopher Franco, Henry Gomez, Antonio Roschman-González,
Zully Benzo and Federico Galarraga
Chapter 28 Lichens as Bioindicators of Air Quality in Mining Areas of Latin
America, with Special Reference to Catamarca, Argentina 437
Martha S. Cañas, Raquel C. Jasan and Rita R. Plá
Chapter 29 Dynamics of Priority Pollutants and Adequacy of Wastewater
Treatments in the Lake Maracaibo Basin (Venezuela) 457
Julio César Marín-Leal, Sedolfo José Carrasquero-Ferrer,
María Carolina Pire-Sierra
and Elisabeth Hedwig Behling de Calmón
Chapter 30 Importance of Bacterial Biofilm for Zn Retention in the Sediment-
Water Interface: The Role of Bacterial Zn Resistance 481
Wilson Machado, Elisamara Sabadini-Santos, Katia N. Suzuki,
Edimar C. Machado, Alfredo V. B. Bellido, Ricardo E. Santelli,
Ricardo T. Lopes and Mirian A. C. Crapez
Chapter 31 Overview of the Occurrence, Effects and Risks of Regulated
and Emerging Contaminants to Freshwater Organisms
in Latin American Watersheds 493
Diana Míguez
Chapter 32 Nanomaterials as CECs: From Environmental Interaction
to Regulatory Efforts for a Safe Use in Latin America 509
Pamela Zuniga Fallas, Jaime F. Quesada-Kimzey
and Ricardo A. Morales Vargas
Chapter 33 Pharmaceutical and Personal Care Products (PPCPs)
in the Environment: Latin American Occurrences,
Adverse Effects and Perspectives 537
Luciane Alves Maranho and Camilo Dias Seabra Pereira
Chapter 34 Restoration and Conservation Actions: Chilean Studies
on Phytoremediation of Metal-Polluted, Acidic Soils 551
Alexander Neaman, Rosanna Ginocchio and Carolina Yáñez
Index 563

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 ABOUT THE BOOK

Studies on the interactions between biotic and abiotic components of ecosystems,

structure and functioning of habitats as well as recovery, rehabilitation and recolonization of
the environments cannot neglect the presence of contaminants and their effects at the
individual, population, community and ecosystem levels. Since in Latin America many
countries are in increasing development, and considering that what we called development
assumes, to some extent, contamination and some loss of the environmental quality, it is a
serious error to consider ecotoxicology a luxury for environmental risk assessment, because
the price to pay for this development has been severe environmental disturbances.
Considering that in Latin America there are many vulnerable areas concentrating one of
the most diverse regions in the world, one can question what is the level of disturbance in
these areas? What are the types of studies carried out in those countries? What is going on in
the field of ecotoxicology in Latin America?
Ecotoxicology in Latin America was proposed, therefore, to offer a collection of studies
in ecotoxicology and environmental risk performed in different countries from Latin America.
Researchers from several Latin American countries were invited to submit a chapter focusing
on any of the following topics: (i) Contaminant entrance, transportation, distribution and fate;
(ii) Environmental risk in freshwater ecosystems; (iii) Ecological risk in coastal zones; (iv)
Biomonitoring programs: water, sediment and air; (v) Physiological effects and biomarkers;
(vi) Soil ecotoxicology; (vii) Bioaccumulation and human risk; (viii) Toxicity of emerging
contaminants; and (ix) Frontiers in Ecotoxicology. These issues tried to cover the most
important subjects and applications of ecotoxicology, including classical and novel subjects.
The original idea to develop a book like this arose after our experience with different
groups from Latin America that work in the area of ecotoxicology and environmental risk
assessment, particularly during our researches in Ecuador in 2014. It became evident to us the
notorious and increasing urban and industrial development of Latin American countries and
the impact caused by anthropogenic activities that greatly threaten the biodiversity and
environmental quality of many ecosystems. Although some studies and monitoring programs
have been developed in those countries, the results obtained and decisions taken by different
groups are poorly known because groups fail to publish their studies or do it exclusively at
the local or regional scale, such that they are often difficult to access. In fact, even among
specialized researchers in ecotoxicology working in Latin America, the transfer of knowledge
is incipient and countries treating similar environmental problems do not know how the other
is acting. Moreover, despite the geographic particularity that lead to differences between

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x Cristiano V.M. Araújo and Candida Shinn

countries in landscape, biodiversity, functioning, biotic and abiotic dynamics, and physical
and chemical properties of habitats, that could justify the lack of interest to publish their
studies at a wider scale, problems tend to be very similar and many decisions and
management plans could be applied transversally in different countries. Obviously, the
publication of many studies at the local or regional scale is not incompatible with a
simultaneous international projection. Therefore, we were motivated by the idea that for a
successful monitoring and management for preservation of disturbed ecosystems it is crucial
to optimize efforts, share experiences and transfer knowledge among different groups
working in areas related to ecotoxicology, as well as among them and other internationally
recognized groups. With this book, it is possible to provide a diverse and relevant collection
of the studies performed in important subareas of ecotoxicology and to expose the main work
area of some research groups within which experiences could be shared.
As the main authors of this book are from Latin America, an obvious question that arises
is “why was the book edited in English?” Since this book is envisioned to have an
international projection not only within Latin American countries, it is a great opportunity to
increase the networks not only among Latin American researchers working in similar themes,
but also with teams from other regions. The idea of this book is, therefore, to favor the
connection among groups to quickly improve the development of methods and their
application in ecotoxicological and environmental risk studies in Latin America.
We believe that the book will be useful to important sectors of environmental sciences
(and related areas - agriculture, biology, chemistry, ecology, environmental engineering,
hydrology, limnology, oceanography, soil sciences, public health and others)- and to the
target public such as students (under-graduate students, and Master and PhD degrees),
researchers acting in environmental studies and decision makers (politicians and
environmental organizations), given that it covers themes related to contamination of water
(freshwater and coastal environments), sediment, soil, air and human beings while taking into
account effects produced by different sectors - industries, agriculture, urban discharges and
natural phenomena.
We gathered 34 chapters authored by 111 researchers from 12 Latin American countries
(Argentina, Brazil, Chile, Colombia, Costa Rica, Cuba, Ecuador, Mexico, Panama, Peru,
Uruguay, and Venezuela) and from 6 non-Latin American countries (Austria, Belgium, Italy,
Portugal, Spain, and USA). Although these data may seem to indicate a considerable number
of participants for a book, taking into account the geographical dimension of Latin America,
uncountable research groups were left out and it was not possible to consider many
studies/projects/researches. Regardless of that, we hope that Ecotoxicology in Latin America
helps to considerably increase the consolidation of networks and knowledge transfer to the
target public, and that ecotoxicological and environmental risk assessment studies can be
improved.
As Editors it has been an incredibly enriching experience to collaborate with scientists of
a very diverse research field in an equally diverse region regarding ecosystems, climate,
culture and history. The entire creative process of the book was achieved using virtual tools
for the uncountable online meetings and exchange of hundreds of emails. This is clear proof
that fruitful collaborations are nowadays possible regardless of the geographical distance, so
long as there is respect, strong intention and clear communication.

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 FOREWORD

Ecotoxicology investigates the fate of contaminants in the biosphere and their effects on
constituents of the biosphere, the biota. Environmental pollution is a problem worldwide, not
restricted to any specific region of the earth. Why then is a book devoted to “Ecotoxicology in
Latin America” needed?
Latin America is the region with the highest biodiversity on Earth. It holds highly
productive marine zones and a major fraction of available freshwater resources. Latin
American ecosystems range from tropical, temperate and desert regions to alpine and polar
zones. At the same time, these ecosystems are under increasing pressure of anthropogenic
threats. Progressive urbanization and overall population growth go along with increasing
habitat degradation, waste water production and the concurrent usage of fresh- and
groundwater resources. The strong agricultural expansion which has taken place over recent
decades results in massive deforestation and depends on intensive use of pesticides. In
parallel, industrial activities have strongly developed, including industries with high
environmental impact such as oil and gas drilling or mining activities. These few examples
may be sufficient to highlight that Latin American ecosystems are under stress of man-made
activities and that there is an urgent need to assess the ecological consequences of
environmental pollution in Latin America.
Historically, many pollution-related environmental problems have first become evident
during industrialization of the countries in Northern America and Europe. As a consequence,
ecotoxicological methods and concepts for environmental surveillance and ecological risk
assessment have been designed for the purposes and under the perspectives of these regions.
This raises the question to what extent these methods and concepts can be transferred to Latin
America, and whether their “applicability domain” is universal enough to address the specific
ecotoxicological problems of this continent and its ecosystems. For instance, the difference in
the toxicant sensitivity and vulnerability between tropical and temperate species and
ecosystems has interested scientists for a long time, but currently we have no conclusive
answer to this question. Still, simply because of the relative scarcity of ecotoxicological
data for Latin American species, risk assessments are largely performed with data determined
for North American and European species. To overcome this biogeographic bias,
ecotoxicological research in Latin America has to grow rapidly in order to learn about the
commonalities as well as the differences in the approaches needed to protect and manage the
integrity of Latin American ecoystems.

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xii Helmut Segner, Pedro Carriquiriborde and Gustavo M. Somoza

This book provides an excellent overview of current ecotoxicological research devoted to

the specific problems and needs of Latin America. The book assembles case studies of typical
pollution problems in Latin America, and it informs on current progress in the monitoring,
risk assessment and management of such problems. At the same time, the book serves as a
platform to bring together scientists from the various Latin American countries, thereby
stimulating the collaboration and exchange that is essential to further advance the study field.
As someone from outside Latin America, I found the progress and vitality of ecotoxicological
research in Latin America, as highlighted by the contents of this book, to be most impressive.
I am convinced that it will become a milestone in the development of ecotoxicology in Latin
America.

Helmut Segner (Centre for Fish and Wildlife Health, Universität Bern, Switzerland).

Since the ‘80s, research in Ecotoxicology in Latin America has slowly but steadily
grown, involving professionals from several countries. More recently a burst in the number
and diversity of activities was observed due to periodical meetings of different scientific
societies and the publication of the extended abstracts as grey literature in regional books.
These meetings helped to increase the frequency of the interactions among researchers from
this geographic area. For example, in 2012, a selected number of studies presented in the
Lima (Perú) and Cumana (Venezuela) biennial SETAC-LA (Society of Ecotoxicology and
Environmental Chemistry, Latina America branch) meetings were published for the first time,
as a special section of the journal Environmental Toxicology and Chemistry. This section
aimed to promote the publication of environmental studies conducted in Latin America as
white literature, allowing readers from all over the world to know about current investigations
in Latin America.
In this context, this book will help to show the current status of research regarding
Ecotoxicology in Latin America presenting 34 chapters by authors from 12 Latin American
countries: Argentina, Brazil, Chile, Colombia, Costa Rica, Cuba, Ecuador, Mexico, Panama,
Peru, Uruguay and Venezuela; and from 6 non-Latin American countries: Austria, Belgium,
Italy, Portugal, Spain and USA.
The chapters deal with different ecotoxicological issues: bioaccumulation, biomarkers
and bioindicators, toxicity tests organisms and laboratory intercallibration, and ecological and
human health risks assessments. The environmental stressors included are: heavy metals,
hydrocarbons, pesticides, nanomaterials, endocrine disruptors, pharmaceutical and personal
care products, dredging sediments and sewage effluents. These assessments were conducted
in relation with soil, sediments, fresh and marine waters and air. The following human
activities are also covered: mining, agriculture, wastewater treatment and restoration and
conservation.
We think this book represents a screenshot of research in Ecotoxicology in Latin America
and nevertheless there are still challenges ahead; we hope that it will be a new starting point
for further substantial advances.

Pedro Carriquiriborde (CONICET-Universidad Nacional de La Plata, Argentina).

Gustavo M. Somoza (CONICET-Universidad Nacional de Gral. San Martín, Argentina).

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 LIST OF CONTRIBUTORS

1 Aldo Gonzalo Indacochea Mejía

Universidad Científica del Sur, Facultad de Ciencias Veterinarias y Biológicas,
Carrera de Biología Marina, Lima, Perú
aindacocheam@cientifica.edu.pe
https://www.researchgate.net/profile/Aldo_Indacochea2
ORCID: 0000-0001-8502-8755
ResearcherID: A-9093-2016
2 Alexander Neaman
Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Quillota, Chile
alexander.neaman@pucv.cl
http://agronomia.ucv.cl/?p=514
ORCID: 0000-0002-1819-0872
ResearcherID: D-4007-2016
3 Alexandre Paoliello Lemos
Universidade Federal Fluminense, Chemistry Institute, Environmental Geochemistry
Department, Niterói, Brazil
alexandreplemos@hotmail.com
http://lattes.cnpq.br/7155209988504151
4 Alfredo Victor Bellido Bernedo
Departamento de Físico-química, Instituto de Química, Universidade Federal
Fluminense, Niterói, Brazil
alfredobellido@gmail.com
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787492A7
5 Amadeu MVM Soares
Departamento de Biología and CESAM, Aveiro, Portugal
asoares@ua.pt
http://www.cesam.ua.pt/index.php?tabela=pessoaldetail&menu=198&user=29
ResearcherID: A-8304-2008
6 Ana Lúcia Fonseca
Institute of Natural Resources, Federal University of Itajubá, Itajubá, Minas Gerais,
Brazil
a1fonseca@uol.com.br
http://lattes.cnpq.br/9838747790108988

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xiv Cristiano V.M. Araújo and Candida Shinn

7 Ana Paula de Castro Rodrigues

Federal Fluminense University, Chemistry Institute, Department of Geochemistry,
Niterói, Brazil.
tantufaz17@gmailcom
http://lattes.cnpq.br/3842494688644394
8 Andreas P. Loibner
University BOKU Wien, Department IFA-Tulln, Institute of Environmental
Biotechnology, Tulln, Austria
andreas.loibner@boku.ac.at
http://www.boku.ac.at
9 Antonio José Machado-Allison
Academia de Ciencias Físicas, Matemáticas y Naturales, Instituto de Zoología y
Ecología Tropical, Universidad Central de Venezuela, Caracas, Venezuela
antonio.machado@ciens.ucv.ve
machado.allison@gmail.com
ORCID: 0000-0002-6028-3677
ResearcherID: L-9835-2015
10 Antonio Roschman-González
Centro de Microscopía Electrónica, Facultad de Ciencias, UCV, Caracas, Venezuela
roschman_gonzalez@yahoo.com
https://www.researchgate.net/profile/Antonio_Roschman-Gonzalez
11 Arnola Cecilia Rietzler
Laboratory of Ecotoxicology, General Biology Department, Federal University of
Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
acrietzler@gmail.com
http://lattes.cnpq.br/6139969138546343
12 Beatriz Toro Restrepo
Universidad de Caldas, Facultad de Ciencias Exactas y Naturales, Departamento de
Ciencias Biológicas, Grupo de Investigación BIONAT (Biodiversidad y Recursos
Naturales), Manizales, Colombia
beatriz.toro@ucaldas.edu.co
http://scienti.colciencias.gov.co:8081/cvlac/visualizador/generarCurriculoCv.do?cod_
rh=0000152170
ORCID: 0000-0002-2555-5147
ResearcherID: B-6568-2016
13 Belgis Chial Zambrano
Universidad de Panama, Esc. Biología, Dpto. Biología Marina y Limnología, Panama,
Panama
belgis.chial@gmail.com
http://www.up.ac.pa/PortalUp/index.aspx
ORCID: 0000-0003-4022-5864
ResearcherID: Q-1750-2015

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 List of Contributors xv

14 Beverly Boburg
Laboratorio de Ecotoxicología Acuática: Invertebrados Nativos, Departamento de
Química Biológica, Facultad de Ciencias Exactas y Naturales, UBA, Universidad de
Buenos Aires, Buenos Aires, Argentina
bevboburg@gmail.com
15 Bianca Carolina Resende Carneiro da Rocha
Catholic University of Rio de Janeiro, Engineering Faculty, Department of Civil
Engineering, Geotechnical and Environmental Laboratory, Rio de Janeiro, Brazil
biancacrcr@gmail.com
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K8144314Z7
16 Camilo Dias Seabra Pereira
Universidade Federal de São Paulo, Campus da Baixada Santista, Departamento de
Ciências do Mar, Santos, Brazil
camilo.seabra@unifesp.br
https://www.researchgate.net/profile/Camilo_Pereira
ResearcherID: H-8352-2012
17 Cândida Shinn
(i) Escuela de Ciencias Agrícolas y Ambientales, Pontificia Universidad Católica del
Ecuador Sede Ibarra, Ibarra, Ecuador
(ii) CFE-Centre for Functional Ecology, Department of Life Sciences, University of
Coimbra, Coimbra, Portugal
candida.shinn@gmail.com
https://www.researchgate.net/profile/Candida_Shinn
ORCID: 0000-0001-8184-6451
ResearcherID: K-8699-2015
18 Carla Ianina Di Fonzo
Laboratorio de Ecofisiología y Ecotoxicología, Instituto Antártico Argentino,
Dirección Nacional del Antártico, Buenos Aires, Argentina
carladifonzo@yahoo.com.ar
www.researchgate.net/profile/Carla_Di_Fonzo
ORCID: 0000-0002-0687-8098
ResearcherID: F-1414-2016
19 Carmine Fallico
Universitá della Calabría, Rende, Italia
carmine.fallico@unical.it
http://polaris.unical.it/user/report/visualizzacurriculum.php3?IDutente=911369814
20 Carolina Yáñez
Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
carolina.yanez@pucv.cl
http://biologia.ucv.cl/web/?portfolio=carolina-yanez-prieto
ORCID: 0000-0003-3969-2078
ResearcherID: F-6114-2013

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xvi Cristiano V.M. Araújo and Candida Shinn

21 Christiane do Nascimento Monte

Federal Fluminense University, Chemistry Institute, Department of Geochemistry,
Niterói, Brazil
christiane.monte@yahoo.com.br
http://lattes.cnpq.br/6178162183692365
22 Christopher Franco
Universidad Católica Andrés Bello, Caracas, Venezuela
christopherfranco.1994@gmail.com
23 Claudio A. Sáez
Laboratory of Coastal Environmental Research, Center of Advanced Studies,
University of Playa Ancha, Viña del Mar, Chile
claudio.saez@upla.cl
https://www.researchgate.net/profile/Claudio_Avaria
ORCID: 0000-0002-5037-3484
24 Cristiano V.M. Araújo
(i) DCI-Central Department of Research, Ecuadorian Aquatic Ecotoxicology group
(ECUACTOX), Universidad Laica Eloy Alfaro de Manabí (ULEAM), Manta,
Ecuador
(ii) CFE-Centre for Functional Ecology, Department of Life Sciences, University of
Coimbra, Coimbra, Portugal
(iii) Institute of Marine Sciences of Andalusia (ICMAN-CSIC), Department of
Ecology and Coastal Management, Puerto Real, Spain
cristiano.araujo@icman.csic.es
https://www.researchgate.net/profile/Cristiano_Araujo2;
http://lattes.cnpq.br/8123137381169371
ORCID: 0000-0003-1793-2966
ResearcherID: H-4813-2015
25 David Jesús Villareal de la Torre
Universidad Laica Eloy Alfaro de Manabí, Facultad de Ciencias del Mar, Ecuadorian
Aquatic Ecotoxicology group (ECUACTOX), Manta, Ecuador
davidvillareal@hotmail.cl
david.villareal@uleam.edu.ec
ORCID: 0000-0001-5220-9971
26 David Salvatierra
Facultad de Ciencias Agropecuarias, Universidad Laica Eloy Alfaro de Manabí,
Ecuadorian Aquatic Ecotoxicology group (ECUACTOX), Manta, Ecuador
davidsg93@hotmail.com
https://www.researchgate.net/profile/David_Salvatierra2
ORCID: 0000-0002-9401-8134
ResearcherID: B-6621-2016

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27 Dayanara María Macías Mayorga

Departamento Central de Investigación (DCI), Ecuadorian Aquatic Ecotoxicology
group (ECUACTOX), Universidad Laica Eloy Alfaro de Manabi (ULEAM), Facultad
de Ciencias Agropecuarias, Manta, Ecuador
dayanara.macias@gmail.com
https://www.researchgate.net/profile/Dayanara_Macias-Mayorga
ORCID: 0000-0002-5214-5792
ResearcherID: C-3476-2016
28 Delfina Castiglioni
Departamento de Ecoloygía Teórica y Aplicada, Universidad de la República,
Maldonado, Uruguay
delfina.mca@gmail.com
29 Denis Moledo de Souza Abessa
São Paulo State University, Coastal Campus, Center of Studies on Aquatic Pollution
and Ecotoxicology, São Vicente, SP, Brazil
dmabessa@clp.unesp.br
http://lnepea.wix.com/lab-nepea
https://www.researchgate.net/profile/Denis_Abessa
http://lattes.cnpq.br/2842419319556542
ORCID: 0000-0003-4609-1668
ResearcherID: D-6507-2014
30 Diana Míguez
Water Program, Environmental Projects Department, R&D&I Division,
Technological Laboratory of Uruguay (LATU) and Latitud Research Foundation of
LATU, Montevideo, Uruguay
dmiguez@latu.org.uy
dmiguez@live.com
http://buscadores.anii.org.uy/buscador_sni/exportador/ExportarPdf?hash=445b90dc90
9906ea8db8402cd6764885
https://www.linkedin.com/in/diana-m%C3%ADguez-
a2842021?trk=nav_responsive_tab_profile
ORCID: 0000-0001-5364-5951
ResearcherID: M-4219-2016
31 Doris Hirmann
University BOKU Wien, Department IFA-Tulln, Institute of Environmental
Biotechnology, Tulln, Austria
dhirmann@boku.ac.at
https://www.european-chemicals-agency
32 Edimar Carvalho Machado
Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro, Nilópolis,
Brazil
edimar_machado@yahoo.com.br
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4769317A5

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33 Edith Orellana Mendoza

Universidad Nacional del Centro del Peru, Peru
edithorellana5@yahoo.es
ORCID: 0000-0002-9597-2130
ResearcherID: A-7811-2016
34 Eduardo Mendes da Silva
Federal University of Bahia, Institute of Biology, Salvador, Bahia, Brazil
marenba@gmail.com
http://lattes.cnpq.br/7294945499790680
ORCID: 0000-0003-1031-5957
ResearcherID: B-5382-2012.
35 Elba de la Cruz Malavassi
Universidad Nacional, Central American Institute for Studies on Toxic Substances
(IRET), Environmental Area, Heredia, Costa Rica
elba.delacruz.malavassi@una.cr
http://www.iret.una.ac.cr/
36 Elisabeth Hedwig Behling de Calmón
Departamento de Ingeniería Sanitaria y Ambiental (DISA), Escuela de Ingeniería
Civil, Facultad de Ingeniería, La Universidad del Zulia (LUZ), Maracaibo, Venezuela
elisabeth.behling@gmail.com
37 Elisamara Sabadini-Santos
Departamento de Geoquímica, Instituto de Química, Universidade Federal
Fluminense, Niterói, Brazil
esabadini@geoq.uff.br
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4703855Z4
38 Federico Galarraga
Centro de Geoquímica, Instituto de Ciencias de la Tierra, Facultad de Ciencias, UCV,
Caracas, Venezuela
federico.galarraga@ciens.ucv.ve
39 Fernando Rey Diz
Departamento Central de Investigación (DCI), Ecuadorian Aquatic Ecotoxicology
(ECUACTOX) Group, Universidad Laica Eloy Alfaro de Manabi (ULEAM), Manta,
Ecuador
fernandordiz@gmail.com
https://www.researchgate.net/profile/Fernando_R_Diz
ORCID: 0000-0002-7639-323X
ResearcherID: N-5781-2015
40 Francisco Barros
Laboratório de Ecologia Bentônica, Programa de Pós Graduação em Ecologia e
Biomonitoramento, Universidade Federal da Bahia, Campus Ondina, Salvador, Brazil
franciscobarros.ufba@gmail.com
http://lattes.cnpq.br/1085274416109765
ORCID: 0000-0002-3037-1991
ResearcherID: A-3545-2008

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41 Freylan Mena Torres

Universidad Nacional, Central American Institute for Studies on Toxic Substances
(IRET), Environmental Area, Heredia, Costa Rica
freylan.mena.torres@una.cr
http://www.iret.una.ac.cr/
ORCID: 0000-0002-1215-0378
42 Gisela Kristoff
Laboratorio de Ecotoxicología Acuática: Invertebrados Nativos, Departamento de
Química Biológica, Facultad de Ciencias Exactas y Naturales, UBA, IQUIBICEN,
UBA-CONICET, Buenos Aires, Argentina
giselakristoff@hotmail.com
43 Guadalupe Sánchez Rivas
Universidad Científica del Sur, Facultad de Ciencias Veterinarias y Biológicas,
Carrera de Biología Marina, Lima, Perú
guada.sari@gmail.com
http://dina.concytec.gob.pe/appDirectorioCTI/VerDatosInvestigador.do?id_investigad
or=27106
ORCID: 0000-0002-0728-9869
ResearcherID: B-6650-2016
44 Guido Persoone
Ghent University, Ghent, Belgium
guido.persoone@ugent.be
https://biblio.ugent.be
45 Gustavo Arencibia Carballo
Centro de Investigaciones Pesqueras, La Habana, Cuba
gustavo@cip.alinet.cu
https://www.researchgate.net/profile/Gustavo_Arencibia
46 Gustavo Emilio Santos-Medrano
Universidad Autónoma de Aguascalientes. Centro de Ciencias Básicas, Departamento
de Química, Aguascalientes, AGS, Mexico
gesantos@correo.uaa.mx
https://www.researchgate.net/profile/Gustavo_Emilio_Santos_Medrano
ORCID: 0000-0002-5953-8409
ResearcherID: C-5995-2014
47 Henry Gómez
Centro de Geoquímica, Instituto de Ciencias de la Tierra, Facultad de Ciencias, UCV,
Caracas, Venezuela
geo_henry_david@hotmail.com
48 Ignacio Alejandro Pérez-Legaspi
Instituto Tecnológico de Boca del Río, División de Estudios de Posgrado e
Investigación Boca del Río, Veracruz, Mexico
ialegaspi@yahoo.com
https://www.researchgate.net/profile/Ignacio_Perez-Legaspi
ORCID: 0000-0001-6796-6617
ResearcherID: C-9895-2016

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49 Jaime Francisco Quesada Kimsey

Instituto Tecnológico de Costa Rica, Fundación Technoinnovation, San José, Costa
Rica
jaime.itcr@gmail.com
ORCID: 0000-0003-0413-1473
50 Jaime Rendón von Osten
Instituto EPOMEX, Universidad Autónoma de Campeche, Campeche, México
jarendon1@gmail.com
http://epomex.uacam.mx/?modulo_micrositio=paginas&acciones_micrositio=ver&id_
pagina=eg==
ResearcherID: C-6828-2016
51 Javier García-Alonso
Departamento de Ecologìa Teòrica y Aplicada, Universidad de la Repùblica,
Maldonado, Uruguay
jgalonso@cure.edu.uy
http://buscadores.anii.org.uy/buscador_sni/exportador/ExportarPdf?hash=d436e82215
20297446bec21db1dfc396
52 Jesús Alvarado-Flores
CONACYT – Unidad de Ciencias del Agua, Centro de Investigación Científica de
Yucatán A.C., Cancún, Quintana Roo, Mexico
jesus.alvarado@cicy.mx
https://www.researchgate.net/profile/Jesus_Alvarado-Flores
ORCID: 0000-0002-4376-4671
ResearcherID: C-9904-2016
53 Jesus Ernesto Hernandez Maldonado
Instituto Experimental Jardín Botánico Dr. Tobías Lasser, Universidad Central de
Venezuela, Caracas, Venezuela
jeshernandezm@gmail.com
https://www.researchgate.net/profile/Jesus_Hernandez2
54 Jorge Navarro Azócar
(i) Universidad Austral de Chile, Instituto de Ciencias Marinas y Limnológicas,
Valdivia, Chile
(ii) Centro FONDAP de Investigación en Dinámica de Ecosistemas Marinos de Altas
Latitudes (IDEAL), Punta Arenas, Chile
jnavarro@uach.cl
www.icml.uach.cl
ORCID: 0000-0001-9920-190X
ResearcherID: B-7928-2008
55 José Paulo Sousa
University of Coimbra, Centre of Functional Ecology, Department of Life Sciences,
Coimbra, Portugal
jps@zoo.uc.pt
http://www1.ci.uc.pt/pessoal/jps/
ORCID: 0000-0001-8045-4296

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56 Júlia Carina Niemeyer

Federal University of Santa Catarina, Centre of Curitibanos Curitibanos, Santa
Catarina, Brazil
juliacarina@gmail.com
http://lattes.cnpq.br/8185023532378108
ResearcherID: E-5838-2013.
57 Julián José Castañeda
Universidad de Oriente, Instituto Oceanográfico de Venezuela, Departamento de
Oceanografía, Grupo Estudios Ambientales, Cumana, Venezuela
jjcasta@gmail.com
ORCID: 0000-0002-4705-9910
ResearcherID: M-3636-2016
58 Julieta Denise Cebuhar
Laboratorio de Ecofisiología y Ecotoxicología, Instituto Antártico Argentino,
Dirección Nacional del Antártico, Buenos Aires, Argentina
jucebuhar@gmail.com
ORCID: 0000-0002-9780-7112
ResearcherID: F-1492-2016
59 Julio César Marín-Leal
Departamento de Ingeniería Sanitaria y Ambiental (DISA), Escuela de Ingeniería
Civil, Facultad de Ingeniería, La Universidad del Zulia (LUZ), Maracaibo, Venezuela
jmarin@fing.luz.edu.ve
https://www.researchgate.net/profile/Julio_Cesar_Marin2
60 Katherine Johana Benavides Anchundia
Facultad de Ciencias Agropecuarias, Universidad Laica Eloy Alfaro de Manabí,
Ecuadorian Aquatic Ecotoxicology group (ECUACTOX), Manta, Ecuador
kajobe15@hotmail.com
ORCID: 0000-0001-7361-6664
ResearcherID: B-9277-2016
61 Katia Noriko Suzuki
Programa de Geoquímica, Departamento de Geoquímica, Instituto de Química,
Universidade Federal Fluminense, Niterói, Brazil
ksuzuki@id.uff.br
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4759934Z7
62 Kyung Chung
Departamento de Biología Marina, Instituto Oceanográfico de Venezuela,
Universidad de Oriente, Cumana, Venezuela
kchung@telcel.net.ve

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63 Laura Patricia Dopchiz

(i) Laboratorio de Ecofisiología y Ecotoxicología, Instituto Antártico Argentino,
Dirección Nacional del Antártico, Argentina
(ii) Departamento de Biología, Universidad Argentina J. F. Kennedy, Buenos Aires,
Argentina
lapadop@gmail.com
ORCID: 0000-0003-3668-5999
ResearcherID: F-1435-2016
64 Lucia Guilhermino
ICBAS – Institute of Biomedical Sciences of Abel Salazar, University of Porto,
Department of Population Studies, Laboratory of Ecotoxicology, Porto, Portugal
lguilher@icbas.up.pt
http://www.ciimar.up.pt/member.php?id=30
ResearcherID: E-8361-2013
65 Luciane Alves Maranho
Universidade Federal de São Paulo, Campus da Baixada Santista, Departamento de
Ciências do Mar, Santos, Brazil
lmaranho@gmail.com
https://www.researchgate.net/profile/Luciane_Maranho
ORCID: 0000-0002-0550-9477
ResearcherID: N-8030-2013
66 Lucila Thomsett Herbert
Laboratorio de Ecotoxicología Acuática: Invertebrados Nativos, Departamento de
Química Biológica, Facultad de Ciencias Exactas y Naturales, UBA, IQUIBICEN,
UBA-CONICET, Buenos Aires, Argentina
lucilaherbert@gmail.com
67 Luís Alberto Cedeño Macías
DCI-Central Department of Research, Ecuadorian Aquatic Ecotoxicology group
(ECUACTOX), Universidad Laica Eloy Alfaro de Manabí (ULEAM), Manta,
Ecuador
luis_trilloalbertcema@hotmail.com
ORCID: 0000-0001-8425-1015
ResearcherID: C-3442-2016
68 Luisa E. Castillo Martínez
Universidad Nacional, Central American Institute for Studies on Toxic Substances
(IRET), Environmental Area, Heredia, Costa Rica
lecastillo2010@gmail.com
http://www.iret.una.ac.cr/
69 Ma. del Rocío Ugalde Salazar
Universidad Nacional, Central American Institute for Studies on Toxic Substances
(IRET), Environmental Area, Heredia, Costa Rica
maria.ugalde.salazar@una.cr
http://www.iret.una.ac.cr/
ORCID: 0000-0001-9069-0284

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70 Mairin Josefina Lemus Barrios

Universidad de Oriente, Departamento de Biología, Laboratorio de Biología Celular y
Ecotoxicología, Cumana, Venezuela
mlemus88@gmail.com
ORCID: 0000-0002-1657-0505
ResearcherID: M-3751-2016
71 Marcela Martins Ribeiro
Laboratory of Ecotoxicology, General Biology Department, Federal University of
Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
mrtelita@yahoo.com.br
http://lattes.cnpq.br/3871456850232817
72 Marcos Krull
(i) William and Mary, Virginia Institute of Marine Sciences, Aquatic Health Sciences,
Gloucester Point, VA, EUA
(ii) Universidade Federal da Bahia, Instituto de Biologia, Departamento de Zoologia,
Laboratório de Ecologia Bentônica, Salvador, BA, Brazil
mkrull@vims.edu
http://www.vims.edu/about/directory/students/krull_m.php
https://www.researchgate.net/profile/Marcos_Krull
http://lattes.cnpq.br/9092492295191022
ORCID: 0000-0002-1586-7454
ResearcherID: E-1353-2014
73 Margaret Pinnock Branford
Universidad Nacional, Central American Institute for Studies on Toxic Substances
(IRET), Environmental Area, Heredia, Costa Rica
margaret.pinnock.branford@una.cr
http://www.iret.una.ac.cr/
74 María Carolina Pire-Sierra
Programa de Ingeniería Agroindustrial, Decanato de Agronomía, Universidad
Centroccidental Lisandro Alvarado (UCLA), Barquisimeto, Venezuela
mcpirre@ucla.edu.ve
https://www.researchgate.net/profile/Maria_Carolina_Pire-Sierra
75 María Fernanda Rivera Velasquez
Universidad Nacional de Chimborazo, Riobamba, Ecuador
mafer.rivera@live.com; mariarivera@unach.edu,ec
ORCID: 0000-0001-6453-2493
76 Mariana Cárdenas Henao
Instituto de Zoología y Ecología Tropical, Universidad Central de Venezuela,
Caracas, Venezuela
cardenas.lmariana@gmail.com
https://www.researchgate.net/profile/Mariana_Cardenas2

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77 Mariana de Freitas Matos

Laboratory of Ecotoxicology, General Biology Department, Federal University of
Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
marianafmatos@gmail.com
http://lattes.cnpq.br/5640045466354400
78 Mario Alberto Arzate-Cárdenas
Universidad Autónoma de Aguascalientes. Centro de Ciencias Básicas, Departamento
de Química, Aguascalientes, AGS, Mexico
marzatc@gmail.com
https://www.researchgate.net/profile/Mario_Alberto_Arzate-Cardenas
ORCID: 0000-0001-5224-7687
ResearcherID: D-1015-2016
79 Martha Susana Cañas
Departamento de Formación Básica, Facultad de Tecnología y Ciencias Aplicadas,
Universidad Nacional de Catamarca, San Fernando del Valle de Catamarca, Argentina
marthacanas@tecno.unca.edu.ar
ORCID: 0000-0001-5668-346X
ResearcherID: I-1412-2016
80 Martín Ansaldo
(i) Laboratorio de Ecofisiología y Ecotoxicología, Instituto Antártico Argentino,
Dirección Nacional del Antártico, Argentina
(ii) Departamento de Biología, Universidad Argentina J. F. Kennedy, Buenos Aires,
Argentina
martinansaldo@gmail.com
www.researchgate.net/profile/Martin_Ansaldo
ORCID: 0000-0003-4463-5924
ResearcherID: F-1396-2016
81 Matilde Moreira Santos
CFE-Centre for Functional Ecology, Department of Life Sciences, University of
Coimbra, Coimbra, Portugal
matilde.santos@zoo.uc.pt
http://cfe.uc.pt/index.php?menu=18&language=eng&tabela=pessoaldetail&user=290
ORCID: 0000-0001-7067-5028
ResearcherID: J-2609-2013
82 Maurilio Lara-Flores
Universidad Autonoma de Campeche, Instituto de Ecologia, Pesquerias y
Oceanografia del Golfo de Mexico, San Francisco de Campeche, Campeche, Mexico
maurlara@uacam.mx
epomex.uacam.mx
83 Mirian Araujo Carlos Crapez
Departamento de Biologia Marinha, Instituto de Biologia, Universidade Federal
Fluminense, Niterói, Brazil
miriancrapez@id.uff.br
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783796U6

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84 Mónica María Zambrano Ortiz

Universidad de Antioquia, Corporación Académica Ambiental, Sede Ciencias del
Mar, Grupo de Investigación en Sistemas Marinos y Costeros (GISMAC), Turbo,
Colombia
monica.zambrano@udea.edu.co
http://scienti.colciencias.gov.co:8081/cvlac/visualizador/generarCurriculoCv.do?cod_
rh=0000396982
ORCID: 0000-0002-7498-2997
ResearcherID: C-9130-2016
85 Odete Rocha
Department of Ecology and Evolutionary Biology, Federal University of São Carlos,
São Carlos, São Paulo, Brazil
doro@ufscar.br
http://lattes.cnpq.br/7577441794992601
86 Pamela Zuniga Fallas
Health Technologies Department, Medicine School, Universidad de Costa Rica,
Fundación Technoinnovation, San José, Costa Rica
pzuniga.nanotec@gmail.com
ORCID: 0000-0003-4839-0848
87 Patricio Colón Velásquez López
Universidad Técnica de Machala, Centro de Investigaciones, Machala, Ecuador
cvelasquez@utmachala.edu.ec
88 Paula Fanny Cossi
Laboratorio de Ecotoxicología Acuática: Invertebrados Nativos, Departamento de
Química Biológica, Facultad de Ciencias Exactas y Naturales, UBA, IQUIBICEN,
UBA-CONICET, Buenos Aires, Argentina
paulacossi@gmail.com
https://www.researchgate.net/profile/Paula_Cossi
89 Raiza del Valle Fernández Malavé
Centro de Geoquímica, Instituto de Ciencias de la Tierra, Facultad de Ciencias,
Universidad Central de Venezuela (UCV), Caracas, Venezuela
raiza.fernandez@ciens.ucv.ve
https://www.researchgate.net/profile/Raiza_Fernandez
90 Raquel Clara Jasan
División Técnicas Analíticas Nucleares, Departamento Química Nuclear, Centro
Atómico Ezeiza, Comisión Nacional de Energía Atómica, Buenos Aires, Argentina
jasan@cae.cnea.gov.ar
ORCID: 0000-0002-0953-3538
ResearcherID: I-1385-2016
91 Raymundo Erazo Erazo
Universidad Nacional Mayor de San Marcos, Lima, Peru
ray2erazo@yahoo.es; directorioctei@concytec.gob.pe
ORCID: 0000-0003-1480-7641
ResearcherID: B-3387-2016

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92 Ricardo Alberto Morales Vargas

(i) Health Technologies School, Faculty of Medicine, Universidad de Costa Rica
(ii) Regulatory Unit for Environmental Health Services, Directorate for Human
Environment Protection, Ministry of Health, San José, Costa Rica
ricardomoralescr@yahoo.com
ORCID: 0000-0002-8417-992X
93 Ricardo Erthal Santelli
Departamento de Química Analítica, Instituto de Química, Universidade Federal do
Rio de Janeiro, Rio de Janeiro, Brazil
santelli@iq.ufrj.br
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783252Z4
94 Ricardo Gonçalves Cesar
Federal University of Rio de Janeiro, Geosciences Institute, Department of
Geography, Soil Ecology and Ecotoxicology Laboratory (LECOTOX), Rio de
Janeiro, Brazil
ricardogc.geo@gmail.com
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=W357769
www.geografia.ufrj.br
95 Ricardo Tadeu Lopes
Laboratório de Instrumentação Nuclear, COPPE, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil
ricardo@lin.ufrj.br
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783796U6
96 Rita Rosa Plá
Departamento Química Nuclear, Gerencia Química Nuclear y Ciencias de la Salud,
Comisión Nacional de Energía Atómica, Buenos Aires, Argentina
rpla@cae.cnea.gov.ar
https://www.researchgate.net/profile/Rita_Pla
ORCID: 0000-0002-0826-1972
ResearcherID: I-2690-2016
97 Roberto Rico-Martínez
Universidad Autónoma de Aguascalientes, Centro de Ciencias Básicas, Departamento
de Química, Aguascalientes, AGS, Mexico
rrico@correo.uaa.mx
https://www.researchgate.net/profile/Roberto_Rico-Martinez
ORCID: 0000-0002-7776-9542
ResearcherID: K-7750-2015
98 Robinson Fidel Casanova Rosero
Centro de Investigaciones Oceanográficas e Hidrográficas del Pacífico (CCCP), Área
de Protección del Medio Marino (APROMM), Grupo de Investigación Oceanografía
Operacional, Tumaco, Colombia
rcasanova@dimar.mil.co
http://190.242.114.26:8081/cvlac/visualizador/generarCurriculoCv.do?cod_rh=00003
44796

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99 Rosanna Ginocchio
Facultad de Agronomía e Ingeniería Forestal (FAIF) and Center of Applied Ecology
and Sustainability (CAPES), Pontificia Universidad Católica de Chile, Santiago, Chile
rginocch@uc.cl
https://sites.google.com/site/rosannapuc/
ORCID: 0000-0003-4089-8188
ResearcherID: C-9637-2016
100 Rui Ribeiro
CFE-Centre for Functional Ecology, Department of Life Sciences, University of
Coimbra, Coimbra, Portugal
rui.ribeiro@zoo.uc.pt
http://cfe.uc.pt/index.php?menu=18&language=eng&tabela=pessoaldetail&user=287
ORCID: 0000-0002-0883-1939
ResearcherID: C-7788-2012
101 Sarah Karoline Rodrigues
Universidade Federal do Rio de Janeiro, COPPE, Oceanic Engineering, Rio de
Janeiro, Brazil
sarah.skr@gmail.com
http://lattes.cnpq.br/5498263820542419
102 Sedolfo José Carrasquero-Ferrer
Departamento de Ingeniería Sanitaria y Ambiental (DISA), Escuela de Ingeniería
Civil, Facultad de Ingeniería, La Universidad del Zulia (LUZ), Maracaibo, Venezuela
scarrasquero@fing.luz.edu.ve
https://www.researchgate.net/profile/Sedolfo_Carrasquero
103 Silvia Echeverría Sáenz
Universidad Nacional, Central American Institute for Studies on Toxic Substances
(IRET), Environmental Area, Heredia, Costa Rica
silvia.echeverria.saenz@una.cr
http://www.iret.una.ac.cr/
ORCID: 0000-0001-8214-745X
104 Simone Dealtry Gomes Alcoforado Cavalcanti
Pontificial Catholic University of Rio de Janeiro, Department of Civil Engineering,
Geotechnical and Environmental Laboratory, Rio de Janeiro, Brazil
simonedgs@yahoo.com.br
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4258291U0
105 Tacio Mauro Pereira de Campos
Catholic University of Rio de Janeiro, Engineering Faculty, Department of Civil
Engineering, Geotechnical and Environmental Laboratory, Rio de Janeiro, Brazil.
tacio@puc-rio.br
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787953E8
106 Victor Hugo Gonzalez Carrasco
Universidad Técnica de Machala, Centro de Investigaciones, Machala, Ecuador
vgonzalez@utmachala.edu.ec

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xxviii Cristiano V.M. Araújo and Candida Shinn

107 Victoria Carolina Vera Vera

DCI-Central Department of Research, Ecuadorian Aquatic Ecotoxicology group
(ECUACTOX), Universidad Laica Eloy Alfaro de Manabí (ULEAM), Manta,
Ecuador
karolv-26@hotmail.com
ORCID: 0000-0001-6434-5161
ResearcherID: B-6601-2016
108 Wilson Thadeu Valle Machado
Federal Fluminense University, Chemistry Institute, Department of Geochemistry,
Niterói, Brazil
wmachado@geoq.uff.br
http://lattes.cnpq.br/9250125302396616
109 Yassir Eden Torres-Rojas
Universidad Autonoma de Campeche, Instituto de Ecologia, Pesquerias y
Oceanografia del Golfo de Mexico, San Francisco de Campeche, Campeche, Mexico
yetorres@uacam.mx
epomex.uacam.mx
110 Zuleica Carmen Castilhos
Centre for Mineral Technology, Coordination for Mineral Processes, Laboratory for
Environmental Mercury Speciation (LEMA), Rio de Janeiro, Brazil
zcastilhos@cetem.gov.br
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787737E6
111 Zully Benzo
Laboratorio de Química Analítica, Centro de Química, Instituto Venezolano de
Investigaciones Científicas (IVIC), Caracas, Venezuela
zbenzo@ ivic.ve
https://www.researchgate.net/profile/Zully_Aguilera_De_Benzo

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 ACKNOWLEDGMENTS

We are grateful to many people that in some way or another have contributed to the
development of this project. Firstly we would like to thank NOVA Science Publishers for
accepting the idea: although the main challenge could be for us as Editors, believing in two
young researchers like us is very noteworthy. Secondly, thanks to all the authors that spent
their time writing the chapters that compose this book; logically, they provided the body and
essence of the project. We appreciate the willingness of Pedro Carriquiriborde, Gustavo
Somoza and Helmut Segner to write the preface. Thanks to Fernando R. Diz for the brilliant
art work of the cover page. This was almost a two-year project (since the presentation of the
initial proposal until its final publication), with a lot of work and patience required. We
therefore also give thanks to every single person that directly or indirectly helped and
supported us to keep up this project, mainly our family and friends. C. Shinn would like to
express special thanks to Andreko for understanding the many hours dedicated to the
computer and all the interest, patience and loving support. And last, but not least, we would
like to express a mutual acknowledgment among Editors for the perseverance and
commitment; if we achieve to write the acknowledgment it is because we made it to the end.

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Complimentary Contributor Copy
In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 1

WHAT RISKS DO THE CONTAMINANTS REALLY

REPRESENT? A STANDPOINT ON EFFECTS
FROM ORGANISMS TO ECOSYSTEMS/LANDSCAPES
BASED ON NON-FORCED AQUATIC
EXPOSURE SCENARIOS

Cristiano V. M. Araújo1,2,3,*, Cândida Shinn1,4,

Matilde Moreira-Santos1 and Rui Ribeiro1
1
CFE-Centre for Functional Ecology, Department of Life Sciences, University
of Coimbra, Coimbra, Portugal
2
Central Department of Research (DCI), Ecuadorian Aquatic Ecotoxicology group
(ECUACTOX), Universidad Laica Eloy Alfaro de Manabí (ULEAM), Manta, Ecuador
3
Institute of Marine Sciences of Andalusia (ICMAN-CSIC), Department Ecology
and Coastal Management, Puerto Real, Spain
4
Escuela de Ciencias Agrícolas y Ambientales, Pontificia Universidad Católica
del Ecuador Sede Ibarra, Ecuador

ABSTRACT
The present essay has as main goal to discuss the risks of the contaminants when
their effects are assessed under non-forced aquatic exposure scenarios. Endpoints in
ecotoxicology have initially focused on mortality, but promptly evolved to more sensitive
and relevant sub-lethal responses, although forced exposures to contamination have
continued to be almost exclusive. Recently, a complementary approach using non-forced
exposures, thus allowing organisms to actively avoid contaminants, has been fostered.
This perspective provides a novel paradigm for ecotoxicological studies by shifting the
focus from individuals to higher levels of biological organisation, even under mild
contamination scenarios in which noxious (physiological) effects at the individual level

*
Corresponding Author E-mail: cristiano.araujo@icman.csic.es.

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2 Cristiano V. M. Araújo, Cândida Shinn, Matilde Moreira-Santos et al.

are not reached. Additionally, the concept of habitat selection/preference due to the
simultaneous presence of contaminants with other attractive or repulsive stimuli is
integrated and here discussed in light of a multi-stressor approach. This essay therefore
discusses the above-mentioned concepts and how non-forced exposures can increase
the ecological relevance in environmental risk assessment schemes and resulting
environmental decisions.

Keywords: avoidance, environmental risk assessment, non-forced exposure

ENDPOINTS BEYOND SURVIVAL

For many centuries, contamination has been a theme linked exclusively to human health.
This is the reason why toxicology is a more developed and robust science than
ecotoxicology1. Only in the last century the word “health” has been extended to
environmental questions (Maltby and Calow, 1989; Newman and Unger, 2002). Physical,
chemical and biological conditions became determinant factors to classify an environment as
fit for life or not. Moreover, recent ecological studies have realized that community structure,
ecosystem processes and temporal and spatial patterns become difficult to be understood if
the presence of contaminants is not considered (Cairns, Jr., 1992; Chapman, 2002). Therefore,
concepts, methods and skills from ecology, chemistry, biology, toxicology, and many other
environmental and life sciences have been integrated to compose the field of ecotoxicology.
In general terms, the first steps for a more integrated environmental assessment of aquatic
ecosystems were focused on chemical analysis. Many chemical indicators were (and still are)
used to define the environmental quality by monitoring contaminant concentrations
comparatively to reference or acceptable values and by verifying the presence of xenobiotic
compounds. Besides traditional and simple but very relevant measurements such as pH,
conductivity, redox potential, temperature, organic matter, dissolved oxygen, nutrient levels,
oxygen chemical demand, and oxygen biochemical demand, other more toxicologically-
related parameters (e.g., concentration of metals, hydrocarbons, agrochemicals, nanoparticles,
pharmaceuticals, etc.) have been added to environmental risk assessment schemes. However,
it is widely recognized that the risk of a contaminant is not exclusively related to its presence,
but mainly to its bioavailable concentration. As organisms respond to and recover from
contamination in a species-specific manner, a chemical compound can be risky for one given
species and, at the same concentration, pose no effect to many others. This vision regarding
vulnerability was initially based on survival: how lethal are the contaminants? Thousands of
studies during the 60’s to the beginning of 80’s were mainly centred onthe lethal effects of
contaminants; finding the most sensitive species was the greatest challenge (Maltby and
Calow, 1989). Researchers then realized that a healthy ecosystem could not be defined based
on whether organisms are dead or alive. Being alive is a primary condition for living
beings, and, ideally, no level of mortality should be acceptable (Maltby and Calow, 1989).
Besides, sub-lethal effects occur at lower and more environmentally realistic contaminant
concentrations than lethal effects. From this point onwards, a new paradigm based on sub-
lethal responses pervaded ecotoxicological studies.

1
No difference regarding environmental toxicology has been considered.

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 What Risks Do the Contaminants Really Represent? 3

In this new (sub-lethal effect) context, more sensitive and ecologically relevant
responses started to be considered: biochemical, histological, morphological, physiological,
behavioural, genetic, etc. The vast majority of the studies monitoring such responses,
including those using survival as endpoint, have been almost exclusively performed under
forced exposure scenarios, in which organisms are exposed to contamination in a confined
environment (e.g., aquaria, plates, flasks, tubes). A concentration-response relationship is
then derived and potential effects on organisms can be predicted. Even in in situ studies,
organisms are very often caged, representing also a forced exposure. Such forced approaches
lead experts to consider that adverse effects at the population level due to contamination are a
consequence of a cascade of alterations that go from the biochemical level, to cellular,
physiological, and finally whole population effects (Newman and Unger, 2002; Walker et al.,
2001; Figure 1). However, in natural environments, a forced exposure scenario is not always
the case. Mobile organisms that have a chemical sensorial system may be able to move
towards less polluted areas avoiding contact with contaminants (avoidance2 response; Figure
1). Even organisms that present a limited displacement ability may evade from disturbed
areas by drifting (Beketov and Liess, 2008; Berghahn et al., 2012). Forced exposure could
then represent a limited approach.

CHANGING THE EXPOSURE PARADIGM: RESPONSES

IN NON-FORCED SYSTEMS

One of the first attempts to verify if organisms actively avoid contaminated areas to
escape from toxic effects was performed with the fish Pygosteus pungitius by simulating a
contamination gradient in a tube containing clean water and a contaminant (alcohol,
chloroform, copper sulphate, formalin, mercuric chloride, or zinc sulphate) in the extremities
(Jones, 1947). Since then, different systems have been employed: bi-compartmented (Folmar,
1976; Svecevičius, 1999), steep-gradient, laminar flow chambers (Hartwell et al., 1989),
avoidance/preference chambers (Smith and Bailey, 1990) and fluvarium systems (Richardson
et al., 2001). Many other studies have shown the ability of fish to detect and actively avoid a
large range of contaminants (Sprague, 1968; Gunn and Noakes, 1986; Scherer and McNicol,
1998; Svecevičius, 2001). Based on the assumption that contamination in aquatic habitats
disperses, forming a gradient which is increasingly diluted with increasing distance from the
contamination source (except when an abrupt discharge forms a contamination plume), a
multi-compartmented system was recently proposed (Lopes et al., 2004; Moreira-Santos et
al., 2008). This system simulates a linear contamination gradient through which organisms
can move freely with access to sequential zones containing different contamination levels: the
exposure is facultative and non-forced, provided the organisms are mobile and have the
ability to detect the contaminant. This simulation of a contamination gradient allows for an
estimation of what could occur in the environment when contaminants are discharged.
Therefore, the focus shifts from individual (i.e., noxious physiological effects) to ecosystem
(e.g., pond, stream, estuary, beach) and landscape (e.g., river basin, coastal ocean) levels.

2
Although in some studies spatial avoidance has been indirectly defined as changes in the patterns of specific
movements (swimming velocity, direction, area of use), in the present study spatial avoidance is exclusively
considered as the spatial displacement to escape disturbed areas.

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4 Cristiano V. M. Araújo, Cândida Shinn, Matilde Moreira-Santos et al.

Using the non-forced multi-compartmented exposure system, Lopes et al. (2004) assessed
the avoidance response of the cladoceran Daphnia longispina exposed to a copper gradient. In
that study, a continuous flow was used to maintain the contamination gradient. An
optimization of this system was later on used by Moreira-Santos et al. (2008), who exposed
the fish Danio rerio to a gradient of copper and acid mine drainage effluent, and by Rosa et
al. (2008), who exposed D. magna to a gradient of pulp mill effluents. Rosa et al. (2012)
modified that system to a static condition with no flow, such that a simpler and more practical
avoidance assay system began to be used (Figure 2). Those authors tested the avoidance
magnitude of D. magna when exposed to an atrazine gradient. Recently, several studies have
been performed using this non-forced multi-compartmented static system: copepods exposed
to a polycyclic aromatic hydrocarbon mixture (Araújo et al., 2014a), amphibians exposed to
copper (Araújo et al., 2014b), to the biopesticide abamectin (Vasconcelos et al., 2016) and to
the fungicide pyrimethanil (Araújo et al., 2014c), freshwater fish exposed to pyrimethanil
(Araújo et al., 2014d), and seawater fish and shrimp exposed to copper (Araújo et al., 2016a).
In all these studies, avoidance showed to be a sensitive endpoint with the potential to be used
in ecotoxicological studies and risk assessment schemes.
Non-forced exposures are specially relevant and realistic in the presence of a gradient of
contamination and in the case of non-sessile organisms. For instance, lethal responses are to
be expected when discharges at very high concentrations are suddenly disposed off in the
environment and organisms have no time to avoid them. Hare and Shooner (1995) observed
that the abundance of Chironomus (salinurius gp.) sp. larvae was negatively correlated with
the sediment Cd gradient. This abundance pattern could be related to avoidance behaviour.
However, when larvae of this chironomid species were tested for avoidance to field control
and Cd-spiked sediments, avoidance was not evidenced. The lower abundance of Chironomus
sp. at high Cd concentrations in the field might thus be attributed to the direct toxic effects of
Cd and not to spatial avoidance. Therefore, this new paradigm does not intend to replace the
forced exposure, but to complement it.

Figure 1. Cascade of time-delayed effects expected from the exposure to a contaminant: from
biochemical alterations to sub-lethal responses, to the decline of the population and its disappearance in
the long term. Immediate mortality is observed in parallel to this cascade effect, at higher
concentrations and with the same consequences, but within a shorter time frame. Spatial avoidance can
also occur regardless of the cascade effect, immediately producing similar ecological consequences:
disappearance of the population. Modified from Walker et al. (2001).

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 What Risks Do the Contaminants Really Represent? 5

Figure 2. Schematic representation of a non-forced multi-compartmented static exposure system used in

avoidance experiments (Araújo et al., 2014a).

CHANGING THE EXPOSURE-EFFECTS PARADIGM: EFFECTS

ON ORGANISMS VS. EFFECTS ON ECOSYSTEMS/LANDSCAPES

The idea behind “what risks do the contaminants really represent” is to reflect on how we
perform ecotoxicological studies, what they really indicate and, the most important question:
can non-forced exposure approaches improve our understanding about the effects of
contaminants on the environment? Although initially one could think that the present essay
tries to convey the idea that contamination is not a problem, the idea is completely the
opposite. Given that an extensive number of studies have shown many organisms are able to
escape contaminated habitats, one could suppose that toxic effects become irrelevant since
organisms are not passively exposed to the contaminants. However, a change at higher levels
of biological organisation in biodiversity, functioning and temporal patterns is expected due
to organism’s dispersion caused by the presence of contaminants. This complementary
approach, in which neither the uptake of contaminants nor individual noxious effects are
mandatory, suggests that contaminants can be less dangerous at the organism level than what
is often expected. However, the ability to avoid contamination creates, at community and
ecosystem levels, an effect similar to mortality, as organisms partially or totally disappear (De
Lange et al., 2006; Rosa et al., 2012).
A novel concept called Population Immediate Decline (PID) including the “ecological
death” caused by the population disappearance was proposed by Rosa et al. (2012). The PID
concept indicates that a local population can be immediately reduced not only due to
mortality, but also due to spatial avoidance (ecological death), mainly when the contaminant
concentration is not lethal or does not even cause moribundity. A simple integration of the
immediate mortality and the immediate avoidance can more accurately predict the population
decline exposed to a contaminant. For instance, in a study with D. magna exposed to atrazine,
it was shown that at levels at which lethal effects are minimal (5%, for example), the
predicted local PID was of approximately 50% due to spatial avoidance (Rosa et al., 2012).
The same authors also observed that, at the 48 h-LC50, the predicted PID was more than
90%. In a study with the copepod Boeckella occidentalis intermedia, it was observed that the
concentration of polycyclic aromatic hydrocarbons that triggered an avoidance of 30% caused
25% mortality among non-avoiders; therefore, the PID was of 55% (Araújo et al., 2014a).
Similarly, in a study with tadpoles of the amphibians Leptodactylus latrans, Lithobates
catesbeianus and Pelophylax perezi exposed to copper, Araújo et al. (2014b) showed that, at
copper concentrations lower than 200µg L-1, the PID was exclusively dictated by avoidance;
above this concentration, organisms began first to lose the ability to avoid (moribundity) and

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6 Cristiano V. M. Araújo, Cândida Shinn, Matilde Moreira-Santos et al.

then, due to the continuous exposure, mortality started to occur. In a recent study with marine
organisms, the fish Rachycentron canadum and the shrimp Litopenaeus vannamei avoided
copper contamination at concentrations below those causing lethal effects (Araújo et al.,
2016a).
When considering all the above-mentioned results, it is clear that extrapolations of
immediate toxic effects based on mortality and forced exposure could lead to a severe
underestimation of ecological risks. The proportion of the population expected to disappear at
the short term has to consider both avoiders and dead organisms. Under a long term and sub-
lethal exposure, population decline will also integrate reduction in feeding rate, delayed
growth, impaired reproduction, susceptibility to predation, loss of competitiveness, etc.
Avoidance should therefore be also considered in chronic exposure scenarios. Although the
integration of avoidance in long term experiments with other sub-lethal endpoints has not yet
been performed, avoidance data on L. vannamei exposed to copper obtained by Araújo et al.
(2016a), when compared with other endpoints published in the literature, strongly suggests
that it is more sensitive than predation rate (Wong et al., 1993), immune response (Yeh et al.,
2004), weight gain (Chen and Li, 2001), and osmoregulatory capacity and hyperplasia of gill
tissue (Soegianto et al., 2013) in the same species. It is thus possible to hypothesize that
spatial avoidance of this marine shrimp might be triggered as a mechanism to escape those
damages, since concentrations considered to produce a significant effect are, in general, time
delayed and higher than the concentration triggering avoidance.
When measured in a non-forced exposure system, avoidance represents an important
change in the exposure paradigm. Moreover, it usually is a quick (short term) and sensitive
(low concentrations) response when compared with lethal (observed in the short term and at
high concentrations) and sub-lethal (observed in the long term and at low concentrations)
responses (Figure 3). Although organisms continue to be the key focus in exposure studies,
the final target/receptor of the ecological consequences is the ecosystem, as no toxic effect
at the individual level is (or at least should be) expected. Long term forced and non-
forced exposure studies with observation of sub-lethal responses and avoidance should
be simultaneously carried out to improve our understanding regarding the effects of
contaminants on population decline due to sub-lethal effects and organism evasion.

A MULTI-STRESSOR APPROACH: AVOIDANCE OF CONTAMINANTS

VS. ATTRACTIVE AND OTHER REPULSIVE STIMULI

Undoubtedly, avoidance can, to some extent, give indications about species spatial
distribution and biodiversity in contaminated ecosystems as well as in neighbouring
uncontaminated areas. According to Hansen et al. (1999), the abundance and distribution of
the fish Oncorhynchus mykiis in areas with metal contamination and acidity could be partially
explained by the escape of organisms from those areas. Similarly, the distribution of
amphipods and chironomids has been inversely associated to the sediment toxicity levels that
indicated a contaminant-driven spatial arrangement (Swartz et al., 1982; Hare and Shooner,
1995). However, it is important to consider that the decision to livein a habitat does not
exclusively depend on the presence of the contamination, even if concentrations are
recognizably risky. Factors such as food availability, presence of predators and conspecifics,

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 What Risks Do the Contaminants Really Represent? 7

and habitat complexity may also play an important role. If, on the one hand, the presence of
contaminants represents a repulsive stimulus, on the other hand many other attractive factors
can influence the decision of the organisms to stay or move towards other areas regardless of
the contamination levels. Even in habitats in which contamination occurs at concentrations
expected to produce deleterious effects on organisms, high biodiversity and complex
biological interactions have been observed. Cardoso et al. (2013) observed that density,
biomass and growth productivity of the snail Peringia ulvae along a mercury gradient in a
shallow coastal lagoon (Ria de Aveiro, Portugal) reached higher values in the area with
intermediate contamination. This apparent preference of P. ulvae to Hg-contaminated areas
might be influenced by many factors such as age, resource availability, presence of refuges,
and other biotic or abiotic factors.
Preliminary observations by Araújo and collaborators (Araújo et al., 2016b) have shown
that the habitat selection process by tilapia fry tends to be influenced by contamination levels.
However, in the presence of two conflicting stimulus (contamination and food) organisms
were compelled to intermittently move towards contaminated areas where the food
availability was higher. This decision was taken regardless of the potential toxic effects
caused by contaminants. The tilapia fry were more intensely stimulated by the attractiveness
of the food than by the repulsion of the contamination. A similar behaviour was observed in
rainbow trout (Salmo gairdneri) exposed to copper and food in an attraction-avoidance
experiment using a two-channel system (Pedder and Maly, 1986). This plasticity of the
avoidance response indicates that organisms may make a decision based on the identification
of the most repulsive and attractive stimuli (Harper et al., 2009).
Avoidance experiments, in non-forced exposure systems, should also be conducted
under a multi-stressor approach to improve the understanding of the ecological risk posed
by contaminants. Although the forced exposure approach is important to determine
concentration-response relationships, and fundamental to immobile organisms, the non-forced
exposure including repulsive and attractive stimuli could provide ecologically relevant
data to predict the effects of contaminants on ecosystems and their functional and temporal
dynamics.

FINAL CONSIDERATIONS
The non-forced exposure system does not focus on the health of individuals, but on the
ecological consequences at higher levels of biological organisation due to the effects of
habitat disturbance on the spatial distribution patterns of organisms. If organism’s spatial
distribution is driven by contaminants, a serious restriction of potentially habitable areas can
occur as avoidance modifies the migratory routes and processes of (re)colonization (Gray,
1990; Smith and Bailey, 1990). “Chemical barriers” due to contamination result in habitat
fragmentation and prevent the flux of individuals and, thus, alleles among populations,
making the latter more susceptible to other perturbations, namely inbreeding and mutational
load in small-sized populations (Ribeiro and Lopes, 2013). This approach implies considering
that contaminants can be environmental disturbers, even if no effect is observed at the
individual level. This new paradigm drives us to take a closer look at the role of contaminants
at the wider and more complex ecosystem/landscape level.

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8 Cristiano V. M. Araújo, Cândida Shinn, Matilde Moreira-Santos et al.

Figure 3. Theoretical representation of lethal, sub-lethal and avoidance responses in relation to the
exposure time and sensitivity (concentration). Mortality is expected to occur at the short term and at
high concentrations; sub-lethal noxious effects are expected at the long term (at least longer than
lethality) and at low concentrations; finally, spatial avoidance is expected to be immediate and to occur
at low concentrations.

ACKNOWLEDGMENTS
CVM Araújo and C Shinn are grateful to SENESCYT (Secretaría de Educación
Superior, Ciencia, Tecnología e Innovación, Ecuador) for PROMETEO research
fellowships and, together with M Moreira-Santos, to FCT (Fundação para a Ciência e a
Tecnologia, Portugal) for the postdoctoral fellowship (references SFRH/BPD/74044/2010,
SFRH/BPD/78642/2011 and SFRH/BPD/99800/2014, respectively), funded by Programa
Operacional Potencial Humano of QREN Portugal (2007-2013 and 2014-2020) and by the
Portuguese public/state budget through the Ministry of Education and Science. CVM Araujo
is also grateful to Spanish Ministry of Economy and Competitiveness for the Juan de la
Cierva contract (IJCI-2014-19318).

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 2

BIOMARKERS IN NATIVE CENTRAL

AMERICAN SPECIES

Freylan Mena Torres*, Margaret Pinnock Branford,

Silvia Echeverría Sáenz, Luisa E. Castillo Martínez
and Rocío Ugalde Salazar
Central American Institute for Studies on Toxic Substances (IRET),
Universidad Nacional, Heredia, Costa Rica

ABSTRACT
In an ecotoxicological framework, biomarkers are considered a source of information
that helps to establish exposure-effects relationships. Data at the biochemical or
physiological level can help to elucidate the mechanisms by which a pollutant can cause
negative effect on an organism. Knowing those mechanisms is key in the identification of
signals of effects or exposure to a given compound. In general, ecotoxicological
information is scarce for tropical species compared to standard temperate counterparts.
The application of biomarkers has provided an alternative method for ecotoxicology
research which can be adapted to local or standard species as well. In the tropics, where
higher biodiversity is found, it is very important to develop research in the field of
biomarkers in order to assess the sensitivity of species to the pollutants that diverse
human activities are releasing into the environment. This chapter summarizes the efforts
carried out during the past two decades to characterize the sensitivity and physiological
responses of several native Central American species exposed to pesticides. At the same
time, the biomarker approach has been applied in the field as a means to assess early
warning signals of pollution in ecosystems.

Keywords: Costa Rica, pesticides, ecotoxicology, biomarkers, native species

*
Corresponding Author E-mail: freylan.mena.torres@una.cr.

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14 Freylan Mena Torres, Margaret Pinnock Branford, Silvia Echeverría Sáenz et al.

INTRODUCTION
Biomarkers have become a widely used tool in ecotoxicology for the evaluation of early
effects or early warning signs of pollution on organisms. The use of biomarker techniques has
been increasing since the 1990’s when this concept was adapted to an ecotoxicology context
(Amiard-Triquet et al., 2012).
During almost three decades of intensive application, this low-biological-level endpoint,
related to exposure and/or effects of pollutants to organisms, as defined by Peakall (1994),
has gained weight in ecotoxicology. The inclusion of biomarkers in regulatory frameworks
for protection of aquatic resources is being currently considered as an improvement in
developed countries (Handy et al., 2003; Sanchez and Porcher 2009; Sanchez et al., 2012;
Capela et al., 2016).
In Costa Rica, studies in this field have been carried out during two decades, progressing
with the implementation of analytical methods for the evaluation of different biomarkers
throughout the years. All work developed so far, has been based on the use of local species as
assay organisms for the characterization of their sensibility to relevant pollutants, but also for
the assessment of environmental samples and in situ evaluations (Broman and Hedene, 1996;
Navarro et al., 2014; Echeverría-Sáenzet al., 2016 in press).
A battery of assays has been developed with the implementation of methods for the
evaluation of cholinesterase (ChE), glutathione S-transferase (GST) and catalase (CAT)
activities, and lipid peroxidation (LPO) and vitellogenin (VTG) levels (Mena et al., 2014a;
Lelieveld, 2004). Collaborative work has also allowed the application of molecular
techniques for phase I biotransformation (cytochrome P450) and endocrine disruption
(vitellogenin) biomarkers at a gene expression level (Navarro et al., 2014).
The use of biomarkers contributes to enhance the specificity and complexity of
information produced in ecotoxicology by characterizing and measuring the effects of
pollutants in lower levels of biological organization. For tropical environments where this
kind of information is scarce, the use of biomarkers should be a resource to improve the
protection of such fragile and rich ecosystems. In this chapter we will summarize the
outcomes of biomarker responses (exposure and effects) that have been observed in
organisms exposed in the laboratory to specific toxicants or to environmental samples
collected in polluted sites, and also the responses of organisms that have been exposed in situ
at sites studied for pollution in Costa Rica.

METHODS
Biomarkers have been evaluated in several native freshwater species which include fish
(Astyanax aeneus and Bryconamericus scleroparius, Characidae; Parachromis dovii,
Cichlidae; Atractosteus tropicus, Lepisosteidae; Poecilia gillii, Poecilidae), bivalve molluscs
(Anodontites luteola, Mycetopodidae), freshwater shrimp (Macrobrachium digueti,
Palaemonidae), amphibians (Agalychnis callidryas, Smilisca baudinii and Isthmohyla
pseudopuma, Hylidae) and crocodiles (Crocodilus acutus, Crocodylidae).
The first approach of these investigations is the characterization (in the laboratory) of
biochemical or physiological responses triggered by specific pollutants or environmental

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 Biomarkers in Native Central American Species 15

samples by toxicity tests coupled with measurement of biomarkers in organisms exposed to

sub-lethal concentrations of toxicants.
A second approach has been the evaluation of biomarkers in free-living organisms,
mostly in fish, but also ChE activity in plasma samples from sloths (Bradypus variegatus and
Choloepus hoffmanni), and vitellogenin, as a biomarker for endocrine disruption, has been
evaluated in fish (Oreochromis aureus, Rhamdia guatemalensis, Herotilapia multispinosa,
Dormitator latifrons, Archocentrus nigrofasciatus, A. aeneus, P. dovii and P. gillii) and the
crocodile (C. acutus).
The third approach undertaken is the in situ exposure of caged fish. This method has
allowed the evaluation of both survival and biomarkers, keeping uniformity in the assay
organisms by using laboratory reared animals and measuring the responses under realistic
environmental conditions.
Biochemical tests to measure the activity of the enzymes ChE, GST, CAT and LPO have
been established in the Laboratory of Ecotoxicological Studies (ECOTOX) at the Universidad
Nacional and are applied routinely. An immunological method for VTG determination has
been established in the laboratory and collaborative work with European counterparts has also
allowed the application of molecular biology and proteomic approaches (Tedengren et al.,
2000; Navarro et al., 2014).

Biochemical Methods

ChE activity is measured with the method described by Ellman et al. (1961), based on the
metabolism of the synthetic substrate acetylthiocholine iodide by tissue endogenous enzyme;
the method was adapted to microplate by Guilhermino et al. (1996). GST activity is
determined as described by Habig et al. (1974), measuring the conjugation of reduced
glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). Lipid peroxidation is evaluated
by the thiobarbituric reactive species (TBARS) assay, as described by Oakes and Van Der
Kraak (2003). CAT activity is measured according to Aebi et al. (1974) by the consumption
of H2O2. In all the protocols we use, biomarkers are normalized to protein content in sample
homogenates which is determined by the method of Bradford (1976) using γ-globulin as
standard.

Proteomic, Molecular and Immunological Methods

Differential expression of CYP1A and VTG genes in tissues has been evaluated
with quantitative real-time polymerase chain reaction (qRT-PCR) technique, using β-actin as
a housekeeping gene (Navarro et al., 2014). Differential protein expression in tissues of
animals exposed to pesticides has been measured with two-dimentional polyacrylamide gel
electrophoresis (2D-PAGE) (Tedengren et al., 2000). Presence of VTG in plasma of fish and
crocodiles has been measured with an indirect enzyme-linked immunosorbent assay (ELISA)
(Lelieveld, 2004).

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 Table 1. Biomarker responses observed in native Central American species exposed to pollutants or polluted sites in Costa Rica

Taxonomic Species Measured Main findings Reference

group biomaker(s)
Fish Parachromis ChE Method implementation and application in tissues (brain, muscle and blood) of native species. Broman and
dovii* (L-F) Hedene, 1996
Fish and P. dovii (L) Physiology, Increased respiration in mussels exposed to a mixture of pesticides. Suppression of 15 proteins and Tedengren et al.,
bivalves Anodontites Protein induction of 12 in mussels exposed to a mixture of pesticides. 2000
luteola (L) expression
patterns (2DGE)
Fish P. dovii (L) ChE Comparison of the ChE inhibition caused by terbufos (organophosphate) in three tissues (brain, Abicht and Pfennig,
muscle and blood). Brain showed the major response and lower variability. 2003
Fish Several species VTG Method implementation, optimization and application in native species. High levels of VTG in Lelieveld, 2004
(F) male fish captured near rice and banana plantations.
Fish P. dovii (L) ChE ChE inhibition by ethoprophos in brain and muscle of P. dovii exposed to ethoprophos ChE Sánchez, 2005
Astyanax inhibition in A. aeneus collected at 2 sites downstream from banana plantations
aeneus (F)
Fish A. aeneus (F) ChE Muscle ChE activity in both species correlated inversely with fish standard length. Lower levels of Pfennig, 2006
and Poecilia ChE activity in muscle of P. gillii correlated positively with presence of ectoparasites on fish.
gillii (F)
Fish Atractosteus ChE Dose-response of ChE inhibition after exposure to ethoprophos. EC50 on ChE inhibition. Mena et al., 2012
tropicus (L)
Fish P. gillii(F) and ChE, GST, LPO ChE inhibition in brain and muscle of P. gillii caged at three sites influenced by pineapple Echeverría-Sáenz et
Bryconamericus plantations. ChE inhibition in B. scleroparius muscle caged at one site influenced by pineapple al., 2012
scleroparius (F) plantations. GST induction in P. gillii at four sites near pineapple plantations and LPO increase at
three sites. LPO increase in B. scleroparuis caged at one site influenced by pineapple plantations.
Bivalves A. luteola (L) ChE, GST, LPO, Sediment avoidance, weight loss and increased GST and ChE in mussels exposed to sediment from Arias-Andrés et al.,
Behavior a swine farm. 2014
Fish P. dovii (L) ChE Dose-response of brain and muscle ChE inhibition vs ethoprophos and chlorpyrifos. ChE inhibition Diepens et al., 2014
by exposure to water samples from three sites downstream from banana plantations.
Fish A. aeneus (L-F) ChE Dose-response of brain and muscle ChE inhibition vs ethoprophos. Muscle ChE inhibition in Mena et al., 2014b
muscle of fish collected at a site that receives effluent from banana plantations.
Fish P. dovii (F) and ChE, GST, LPO Increase of GST, CAT and LPO in P. dovii caged at one site associated with rice plantations. ChE Mena et al., 2014a
P. gillii (F) and CAT inhibition in P. gillii caged at a site with presence of organophosphate pesticides. GST, LPO and
CAT variations in P. gillii caged at sites associated with rice and sugarcane plantations.
*
In the original paper, the species was called Cichlasoma dovii, the name of the genus changed afterwards. L=Laboratory study; F=Field study.

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 Biomarkers in Native Central American Species 17

Behavior and Metabolism

Evaluation of avoidance has been carried out in mussels exposed to contaminated

sediments (Arias-Andrés et al., 2014) and respiration has been measured in mussels and fish
exposed to a mixture of pesticides (Tedengren et al., 2000).

RESULTS AND DISCUSSION

Assessment of the toxicity of pollutants and the evaluation of their effects on aquatic
ecosystems of Costa Rica have been approached from different angles: from the detection and
quantification of residues in environmental matrices, to the evaluation of effects at different
biological organization levels (See chapter 11).
Studies with biomarkers in Costa Rica have supplied broad evidence regarding sub-
individual responses (in the lower biological organization level) and the sensitivity of tested
species. Moreover, effects in the field have been evaluated with a diverse group of native
species of different taxonomic groups (Table 1).
Since agriculture is one of the most important economic activities in Costa Rica and
crops are cultivated with high pesticide usages, these compounds are identified as one of the
most relevant groups of environmental pollutants. Presence of pesticide residues in the
environment is constant in agricultural areas and high sensitivity of some native species to
this group of pollutants has already been described (see Table 2 in chapter 11). Regarding
biomarkers, all of the native species tested so far have shown significant responses when
exposed to sub-lethal concentrations of relevant pollutants. This suggests that many of them
are candidates for being used as sentinel species in the region where they are naturally
distributed.
Fish is the group of organisms on which more studies have been done. Three species (A.
aeneus, P. dovii and P. gillii) have shown to be the most suitable to work with. A common
characteristic that makes these species appropriate is their broad distribution in the region
and, in the case of Costa Rica, their presence in both Pacific and Caribbean versants: A.
aeneus ranges from Mexico to Panama, P. dovii from Honduras to Costa Rica and P. gillii
from Guatemala to Colombia (Bussing, 2002). Besides their distribution, A. aeneus and P.
gillii also have the advantage of numerous populations that can be occasionally sampled
without significantly reducing them. When there is the necessity of having fish raised in
controlled conditions, P. dovii and P. gillii can be easily reproduced in the laboratory to
obtain clean and well characterized experimental organisms. Finally, when running in situ
caging experiments or laboratory tests, it is important to maintain minimal stress and
aggression among organisms during exposures. In this regard, behavior of the fish is an
important issue and P. gillii has shown to be the least aggressive among the species tested.
Regarding other taxonomic groups, more work should be done in order to suggest
suitable sentinel species, although bivalves (A. luteola), decapodes (M. diguetti) and
amphibians (S. baudinii) have shown positive results regarding sensitivity to pesticides and
biomarker responses.
A robust battery of biomarkers, integrating different physiological mechanisms related to
effects or responses to pollutants is highly recommended; and the link of sub-individual

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18 Freylan Mena Torres, Margaret Pinnock Branford, Silvia Echeverría Sáenz et al.

responses measured at higher levels of biological organization should be also pursued

(Galloway et al., 2004; Moore et al., 2004). The interpretation of several biomarkers
measured in the field can be difficult and even confusing. In our field studies, significant
responses of biomarkers have been obtained, but not always in direct correlation with the
detected pesticide residues (Fournier et al., 2016 submitted; Echeverría-Sáenz et al., 2016 in
press). In order to overcome this situation, integrative analysis have been developed that help
merge as many responses one can measure in one index (Broeg and Lehtonen, 2006). For our
data, application of the integrated biomarkers response (IBR) (Beliaeff and Burgeot, 2002;
Devin et al., 2014) has helped to elucidate the relationship between biomarkers and gross
pollution of sites. For instance, in a study where no correlation could be established between
individual biomarker responses and pesticide residues in individual samples, IBR responses
were consistently significant at the overall most polluted sites (Echeverría-Sáenz et al., 2016
in press).
There is a continuous innovation and adaptation in the techniques applied for the
evaluation of biomarkers. This evolution enables researchers to improve the specificity and
refinement of the information in this field, but also requires constant updating in order to keep
up with the advances in biochemistry and molecular biology.
So far, data generated with the application of biomarkers shows the suitability of these
techniques to characterize the sensitivity of some native species to pesticides and chemical
pollution.
Many ecotoxicologists have reservations about the value of biomarkers and their
application in the field, mostly because in many cases the responses measured might be
influenced by natural variations and environmental parameters (Forbes et al., 2006;
Guilhermino, 2006). Also, there might have been some frequent misuse or misinterpretation
of this information that is known to represent a low ecological relevance level in an
ecotoxicological framework (Forbes et al., 2006). Nevertheless, in the end, biomarkers should
be used and interpreted as they are intended (early warning signals) and if applied correctly
(Adams et al., 2001; Handy et al., 2003; Guilhermino, 2006), they can be a valuable tool in
the protection of ecosystems.

CONCLUSION
The inclusion of biomarkers in a set of ecotoxicological tests has improved the capacity
of researchers to detect and characterize effects of pollutants (pesticides) in a group of Central
American native species.
From all the studies that have been executed over the years in Costa Rica, the clearer
results have been obtained from the laboratory experiments, where toxicant-effect causality
has been demonstrated and several new potential test species have been recognized. This is
highly valuable information for the tropics. However, more research is needed to elucidate the
different mechanisms and interactions that take place in field studies, where natural and
anthropogenic stressors get mixed.
The use of integrative methods such as IBR helps to process and interpret the results of a
vast battery of biomarkers. This tool has been useful in order to compare responses between

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 Biomarkers in Native Central American Species 19

sites and finding the overall, most intense response to the toxicants that are present in the
field.
This summary of information regarding sensitivity at a physiological level should be used
in order to protect biodiversity, by setting lower limits to the allowed levels of pollutants in
the environment that should not compromise organisms even at a sub-individual level.

ACKNOWLEDGMENT
We thank Prof. David R. S. Lean for his kind revision of this chapter.

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Isthmohyla pseudopuma and Smilisca baudinii tadpoles. Environ. Sci. Pollut. Res. DOI
10.1007/s11356-016-7301-1.
Moore, M.N., Depledge, M.H., Readman, J.W., Leonard, D.R.P., 2004. An integrated
biomarker-based strategy for ecotoxicological evaluation of risk in environmental
management. Mutat. Res. 552, 247-268.
Navarro, A., Mena, F., Casado, M., Pinnock, M., de la Cruz, E., Barata, C., Piña, B., 2014.
Development of quantitative RNA biomarkers for detecting dioxin-like and estrogenic
pollutants in Costa Rican native fish species. J. Environ. Biol. 35, 99-105.
Oakes, F.D.,Van Der Kraak, V.D., 2003. Utility of TBARS assay in detecting oxidative stress
in white sucker (Catotomus commersoni) populations exposed to pulp mill effluent.
Aquat. Toxicol. 63, 447-63.
Peakall, D.B., 1994. The role of biomarkers in environmental assessment (1). Introduction.
Ecotoxicology 3, 157-160.
Pfennig, S., 2006. Cholinesterase activity in tissues from two tropical fish species as possible
indicator for pollution in Costa Rican waters. Final report. Graduate work in biology
course, University of Hannover, Germany.
Pinnock-Branford, M., de la Cruz, E., Solano, K., Ramírez, O., 2014. Pesticide exposure on
sloths (Bradypus variegatus and Choloepus hoffmanni) in an agricultural landscape of
Northeastern Costa Rica. J. Environ. Biol. 35, 29-34.
Sánchez, R., 2005. La actividad de las enzimas colinesterasas como biomarcador de los
plaguicidas utilizados en las plantaciones de banano de Costa Rica [The enzyme
cholinesterase activity as a biomarker of pesticides used in banana plantations in Costa
Rica]. Graduate Thesis, Universidade do Porto [Graduate Thesis, University of Porto],
Portugal.
Sanchez, W., Porcher, J.M., 2009. Fish biomarkers for environmental monitoring within the
Water Framework Directive of the European Union. Trend. Anal. Chem. 28(2), 150-158.
Sanchez, W., Burgeot, T., Perceval, O., 2012. Perspectives from the French workshop on the
development and validation of biomarkers and bioassays for the monitoring of aquatic
environments. Environ. Sci. Pollut. Res. 19, 1345-1347.
Tedengren, M., Castillo, L.E., Ruepert, C., 2000. Protein expression and physiological
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Costa Rica. In: L.E. Castillo. Pesticide impact of intensive banana production on aquatic
ecosystems in Costa Rica. Ph.D. Thesis, Stockholm University, Sweden.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 3

AQUATIC ECOTOXICOLOGY:
NATIVE FRESHWATER GASTROPODS
FROM ARGENTINA

Paula F. Cossi1,2, Lucila T. Herbert1,2, Beverly Boburg1

and Gisela Kristoff1,2,*
1
Laboratorio de Ecotoxicología Acuática: Invertebrados Nativos (EAIN),
Departamento de Química Biológica,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Pabellón II, Ciudad Universitaria, Argentina
2
Universidad de Buenos Aires-Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET), Instituto de Química Biológica de la
Facultad de Ciencias Exactas y Naturales (IQUIBICEN),
Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina

ABSTRACT
The increasing worldwide contamination of freshwater systems with a wide range of
chemical compounds is one of the main environmental problems that humanity faces
nowadays. These contaminants, such as pesticides, disturb natural ecosystems and can
have a great impact on the species that inhabit them. Traditionally, environmental
monitoring programs were based on detecting and determining the concentration of
contaminants. However, they did not necessarily reflect the actual impact on the
environment. Lately, programs have included bioassays and sensitive biomarkers as
effective tools to obtain an integrated overview of aquatic ecotoxicology. Our research
group uses bioassays to study whether environmental concentrations of toxic compounds
used in Argentina can produce effects on native gastropods. Our research lines involve
the native freshwater snails Biomphalaria straminea, widely distributed in northeastern
Argentina, and Chilina gibbosa, endemic to freshwaters of the Argentine Patagonia. The
first studies we carried out were focused on the organophosphate azinphos-methyl, which

*
Corresponding Author E-mail: gkristoff@qb.fcen.uba.ar.

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24 Paula F. Cossi, Lucila T. Herbert, Beverly Boburg et al.

has been detected in freshwater courses at concentrations higher than those recommended
for aquatic life protection. We found different sublethal effects after exposures to
environmental concentrations in both species. B. straminea is resistant to an acute
exposure but shows toxic effects after subchronic exposures, which has driven further
studies on reproduction and offspring. C. gibbosa, however, is highly sensitive to
azinphos-methyl, which has led us to consider it as a sentinel species. Our research
allows us to contribute with a wide range of biomarker information, propose new sentinel
species and provide data to reassess current guidelines for aquatic life protection.

Keywords: pesticides, azinphos-methyl, freshwater gastropods, native species, biomarkers

PESTICIDES
A great number of pesticides are used around the world in gardening, public health and
agriculture to provide protection from pests. Most of them are generally sold as formulations
that consist of an active ingredient, which is responsible for the mechanism by which the
pesticide kills the target pest, plus other compounds that act as solvents, carriers or adjuvants.
Pesticides can easily enter the soil, surface and ground water through direct application,
terrestrial runoff, rainwater, floods and soil leaching, among others. Pesticide, water and soil
properties together with weather conditions determine the destination and persistence of the
compound in the environment. The use of pesticides in agriculture has led to an increasing
concern about the potential implications for human and environmental health and many
strategies have been developed to evaluate them. A traditional strategy is to determine the
presence and/or concentration of an array of compounds of interest in the environment (US
EPA, 2001; Blasco and Picó, 2009; Lam, 2009). However, this approach is limited because it
is impossible to identify every potential toxic compound and it does not reflect how the effect
to organisms is modified by bioavailability, bioaccumulation, continuous and successive
exposures, among other factors. The use of bioassays and sensitive biomarkers arose from the
need to develop new tools to assess the impact of hazardous compounds in the environment
(Wolska et al., 2007; Lam, 2009; Smital and Ahel, 2015; de Castro-Català et al., 2016).
Consequently, the use of both chemical and biological evaluations provides a more integrated
overview of water quality in environmental monitoring programs (Zhou et al., 2008; Blasco
and Picó, 2009; Guasch et al., 2012; Burgess et al., 2013).

Organophosphorus Insecticides: Azinphos-Methyl

Organophosphorus compounds comprise a large and diverse family of organic chemicals.

They are esters, amides or thiol derivatives of phosphoric, phosphonic and phosphinic acids.
Most organophosphorus insecticides are derived from phosphoric acid and present a
phosphorus atom with a P=O or P=S group. Their main mechanism of action involves
the inhibition of the enzyme acetylcholinesterase, responsible for the hydrolysis of the
neurotransmitter acetylcholine. They can also alter the function of other proteins and enzymes
due to their highly reactive nature (Chambers and Levi, 1992; Terry, 2012).

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 Aquatic Ecotoxicology 25

Organophosphorus pesticides have acquired importance for pest control due to their low
to moderate persistence in the environment (Chambers and Levi, 1992) in relation to other
chemicals used; nevertheless, many of them have been banned or restricted in several
countries because of their high acute toxicity to non-target organisms (Chambers and Levi,
1992; Terry, 2012).
Azinphos-methyl is an organophosphate (phosphoric acid ester) insecticide-acaricide that
contains a phosphorothioate group (P=S). Its half-life in water is 26 days at 30 C and pH 7,
and its water solubility is 28 mg L-1 at 20 ºC (US EPA, 2001). It is commonly applied for
agricultural pest control in the Upper Valley of Río Negro and Río Neuquén in North
Patagonia, Argentina. It is applied between October and March mainly to control Cydia
pomonella, an important agricultural pest in fruit production (Soleño et al., 2008).
This pesticide has been reported as the most frequently detected organophosphate
pesticide in surface and ground water of the Upper Valley region (Loewy et al., 2006, 2011;
Tosi et al., 2009). The recommended value of this insecticide for aquatic life protection
according to Argentine legislation is ≤0.02 µg L−1 (Subsecretaría de Recursos Hídricos de la
Nación, 2003). Nevertheless, Loewy et al. (1999) detected a maximum concentration of 79.30
µg L−1 (from October 1995 to September 1997), and Loewy et al., (2011) a maximum
concentration of 22.48 µg L−1 (from September 2008 to October 2009). It has also been
registered in samples from the Luján river, located in east-central Argentina, at concentrations
below 0.634 µg L−1, the detection limit of the analytical method used for its determination
(Castañé et al., 2015) and in Concordia, Entre Ríos, where it is used for pest control in
blueberry crops (Acosta, 2014).

BIOMARKERS
The concept of biological markers or biomarkers has been defined in many ways
(Amiard-Triquet et al., 2013). According to The National Research Council (NRC, 1987), a
biomarker in environmental health research is any biochemical, physiological, histological or
behavioral response in an organism that indicates the presence of xenobiotics. In a more
generalized way, Depledge and Fossi (1994) re-defined the term biomarker as “a biochemical,
cellular, physiological or behavioral change which can be measured in body tissues or fluids
or at the level of the whole organism that reveals the exposure to/or the effects of one or more
chemical pollutants.”
Biomarkers can be classified as biomarkers of exposure, effect or susceptibility (NRC,
1987; Amiard-Triquet et al., 2013). Biomarkers of exposure indicate that the organism has
been exposed to pollutants, through the measurement of the contaminant itself, its
metabolites, or the interaction product with a target molecule (NRC, 1987). A biomarker of
effect is any quantifiable change in an individual after exposure to a contaminant such as
biochemical, physiological, histological or other alterations (NRC, 1987). Biomarkers of
susceptibility, which can be inherited or acquired, indicate that an organism is particularly
sensitive to the effect of a toxic compound (van der Oost et al., 2003; Manini et al., 2007).
In ecotoxicology, bioassays can be defined as biological systems that allow the study of
the impact of chemical compounds, mainly from anthropogenic activity, on ecosystems.
Exposure to pollutants triggers a cascade of biological responses and multiple toxic effects in

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organisms. The use of a wide battery of biomarkers can provide a suitable evaluation of the
toxic responses in organisms and an early warning tool to prevent a significant deterioration
of the biota or of entire ecosystems, due to the presence of contaminants (Cajaraville et al.,
2000; Amiard-Triquet et al., 2013).
An ideal biomarker for environmental monitoring should be easy to measure, specific, of
fast induction and slow recovery to pre-exposure levels (Grandjean et al., 1994; Wu et al.,
2005). Few biomarkers meet all the requirements but they are useful nonetheless because they
provide complementary and relevant information about potential risks of pollutants for
organisms (Amiard-Triquet et al., 2013).

FRESHWATER GASTROPODS
Working with Native Species

Aquatic invertebrate species have been amply recommended and used as bioindicators of
water and sediment toxicity. For example, the oligochaete Lumbriculus variegatus has been
widely adopted as a standard organism mainly for the study of toxicokinetic processes of
organic substances and heavy metals (Phipps et al., 1993; ASTM, 1995; US EPA, 2000;
Liebig et al., 2005). However, the use of native invertebrate species is not always taken into
account in ecotoxicological research as a criterion to select model organisms (Freitas and
Rocha, 2014).
Conducting studies and biomonitoring programs with native species has been suggested
by US EPA (1976) and recommended by several authors (Buikema et al., 1982; Krull and
Barros, 2010; Gagneten et al., 2012; Freitas and Rocha, 2014). The results of such integrated
studies could be considered more ecologically relevant because native species are likely to be
key organisms within the food webs and the ecosystems they are part of (Krull and Barros,
2010, Baird et al., 2007). Thus, working with native invertebrate species could be more
pertinent for extrapolation to natural scenarios.
Also, there are studies that have shown that some native species are less tolerant to
exposure to toxics than exogenous species and have highlighted this as an important factor in
the success of invasive species (Piola and Johnston, 2009; Lenz et al., 2011; Oliveira et al.,
2015; Varó et al., 2015). Consequently, using non-native species in bioassays could lead to
misguided decision-making if, for instance, they are used to determine aquatic guide levels
for different contaminants (Freitas and Rocha, 2014).

Biomphalaria straminea
The freshwater snail B. straminea (Figure 1a,b) (Dunker, 1848) belongs to the
Planorbidae family, a large and diverse family of aquatic pulmonate gastropods distributed
worldwide (Dillon, 2000). In Argentina, specimens of B. straminea were recorded in Salta,
Formosa, Chaco, Misiones, Corrientes, Entre Ríos, Córdoba, Buenos Aires and Río Negro
provinces (Rumi et al., 2008).
B. straminea gastropods have the same type of radula as scraper herbivore organisms but
they are unspecialized feeders (Yipp, 1983; Vera-Ardila and Linares, 2005). This species is
able to survive within a wide variety of habitats, being hard water, warm temperatures and

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eutrophic habitats optimal for its development (Yipp, 1983). Organisms are also able to
withstand periods of drought as their metabolic rates can be reduced to basal levels. Olivier
and Barbosa (1956) found that a population of snails studied in the laboratory was able to
survive for up to 380 days in mud which was left to dry naturally.
They are simultaneous hermaphrodites, reproducing by both self-fertilization and cross-
fertilization, which allows them to easily reproduce and colonize new habitats. Organisms lay
several eggs inside gelatinous capsules (Figure 1b) which they adhere to hard substrates such
as plants, rocks, shells of other snails or other surfaces; they can store these egg masses inside
the shell for some days before oviposition. Embryos develop inside the egg and hatch out as
juveniles without going through larval stages (Yipp, 1983).
B. straminea is a species that can be easily handled and maintained in laboratory cultures.
The snails do not require stringent conditions to survive and reproduce and, under favorable
conditions, they have great reproductive potential, rapid growth and early maturation (Yipp,
1983).

Figure 1. Photographs of Biomphalaria straminea adults (a) and egg mass (b).

B. straminea Bioassays
There are almost no reports in the literature regarding toxicology studies performed with
B. straminea, except for a study conducted in our laboratory by Bianco et al., (2014). These
authors characterized B. straminea cholinesterases (ChEs) and carboxylesterases (CEs), and
evaluated their sensitivity to a 48 h exposure to azinphos-methyl through no observed effect
concentration (NOEC) and 50% inhibition concentration (IC50) estimations. They also
evaluated subchronic effects after exposure to environmental concentrations of this pesticide.
In order to measure CE activity, they used both p-nitrophenyl acetate (p-NPA) and p-
nitrophenyl butyrate (p-NPB) substrates, since CE activity and sensitivity to pesticides
depend on the substrate used (Laguerre et al., 2009; Kristoff et al., 2012). They found that CE
activity of total soft tissue resulted more sensitive to organophosphates when using p-NPA
and p-NPB compared to other substrates (Kristoff et al., 2012; Cacciatore et al., 2013).
After an acute exposure (48 h) to azinphos-methyl at different concentrations (50, 100,
250, 500, 2500, 5000, 6500 and 10000 µg L-1), Bianco et al. (2014) found that CE and ChE
activities, when using p-NPA, were not inhibited even at the highest concentration used
(Table 1). Conversely, CEs measured with p-NPB were inhibited at 500 µg L-1 and above,
being the values of NOEC and IC50 250 µg L-1 and 2200±750 µg L-1, respectively (Table 1).
Lethality or evident neurotoxic signs were not observed in the acute assay.

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A different picture was observed when evaluating the response of the organisms after 21
days of exposure to environmental concentrations of azinphos-methyl (20 and 200 µg L-1). A
decrease in survival, protein content and CE activity (measured with p-NPB and p-NPA) was
registered. In accordance with the acute exposure results, ChEs were not inhibited.
Contrastingly, CE activity using both substrates was inhibited. With p-NPB as substrate it
was reduced by 90% with the highest concentration of the pesticide and, with p-NPA as
substrate, it was inhibited by almost 50% with both concentrations.
Recent subchronic studies (14 days), carried out in our laboratory at environmental
concentrations of azinphos-methyl, showed no significant alterations in several reproductive
parameters when we exposed B. straminea adults and egg masses until hatching (Cossi et al.,
2015a). However, preliminary results showed effects in some oxidative stress parameters in
adults exposed to the pesticide.

Chilina gibbosa
C. gibbosa (Figure 2) (Sowerby 1841) is a freshwater gastropod from the Chilinidae
(Pulmonate) family, endemic to South America and especially abundant in southern Chile and
Argentina (Bosnia et al., 1990; Rumi et al., 2008; Fuentealba et al., 2010; Gutiérrez-Gregoric
et al., 2010).
C. gibbosa can be readily found along the Limay, Neuquén and Negro rivers, as well as
in lakes and reservoirs of the Río Negro and Neuquén provinces (Bosnia et al., 1990;
Valdovinos, 2006; Rumi et al., 2008). The species’ most suitable environment is clear, well
oxygenated waters, associated with macrophytes. It feeds on periphyton and benthic diatoms
from the riverbed or from the macrophytes and is thus benefited by areas with high primary
productivity (Bosnia et al., 1990).
According to Bosnia et al. (1990), C. gibbosa is a semelparous species with an annual
life-cycle, with largest population growth in summer and almost ceasing in winter.
Preliminary histological results indicate that it is hermaphrodite, but the exact reproductive
strategy has not yet been described. Fertilized eggs are laid within capsules (Figure 2b),
which we have observed can host from a few to more than 200 eggs at a time. A single
juvenile hatches from each egg.
These snails have several characteristics that make them potential model organisms for
ecotoxicology bioassays. On the one hand, adults are easy to collect and handle, as they can
usually be found in shallow waters, in an aggregated dispersion pattern (Bosnia et al., 1990).
On the other hand, their limited mobility and reduced ability to excrete pollutants may result
in several negative effects at low environmental concentrations of toxicants (Oehlmann and
Schulte-Oehlmann, 2003), as well as ensuring an effective exposure to any pollutant present
in the environment. Furthermore, they have been described as a relevant part of the benthic
fauna due to their high productivity and turnover rate, which make them an important food
source for different species (Bosnia et al., 1990; Ferriz, 1993).
Nevertheless, we have found that C. gibbosa requires special conditions for surviving and
breeding in the laboratory, such as controlled temperature and day-night cycles and
permanently aerated and clean water, which represent a challenge in order to keep laboratory
cultures. This is made harder by the fact that little has been studied and reported on C.
gibbosa.

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 Aquatic Ecotoxicology 29

Figure 2. Photographs of Chilina gibbosa adult (a) and egg mass (b).

C. gibbosa Bioassays
The first toxicological studies using C. gibbosa as model organisms were carried out in
our group by Bianco et al. (2013). ChE and CE activity were measured on whole organism
soft tissue after a 48 h exposure to a range of azinphos-methyl concentrations (0, 0.001,
0.005, 0.01, 0.1, 5, 100, 500, 1000 and 1500 µg L-1). ChE activity was strongly inhibited,
becoming significant even at low concentrations such as 0.005 µg L-1. NOEC for ChEs was
0.001 µg L-1 and IC50 0.02 ± 0.01 µg L-1 (Table 1). CEs proved to be less sensitive than ChEs,
being significantly inhibited only at concentrations higher than 1000 µg L-1 (Table 1). CE
activity using p-NPB was in turn slightly more sensitive (NOEC 500 µg L-1 and IC50 1300 ±
100 µg L-1) than using p-NPA (NOEC 1000 µg L-1 and IC50 1290±40 µg L-1) (Table 1).
Even though lethality was not significant, a conspicuous neurotoxic sign was observed in
animals exposed to 0.01 µg L-1 or higher concentrations of azinphos-methyl (Figure 3a,b).
The entire head-foot region of these snails protruded outside of the shell (Figure 3b), they
lacked adherence and spontaneous movement and hardly responded to mechanical stimuli.
Bianco et al., (2013) also evaluated antioxidant defenses of animals exposed to two
environmentally relevant concentrations of azinphos-methyl. At 0.02 and 20 µg L-1, azinphos-
methyl causes approximately 50 and 85% of inhibition of ChEs, respectively, but has no
effect on CE activity. An exposure to 0.02 µg L-1 of azinphos-methyl caused an increase in
glutathione (GSH) levels and catalase (CAT) activity. An exposure to the highest
concentration only caused an increase in GSH levels. No effect was found on superoxide
dismutase (SOD) and glutathione S-transferase (GST).
Based on these results, Cossi et al., (2015b) carried out an acute exposure (48 h) of C.
gibbosa to 20 µg L-1 of azinphos-methyl and then monitored their recovery for 21 days after
transferring the organisms to pesticide-free water. They found that while ChE and CE activity
remained constant in control organisms during the bioassay period, in exposed animals ChEs
were inhibited by 85% after 48 h and remained inhibited up to day 21, when a slight recovery
in activity was registered. CE activity in exposed animals also remained constant and
uninhibited during the experiment. Neurotoxicity was observed after 48 h, with most exposed
snails showing weak or lack of adherence to the vessels and almost all of them exhibiting a

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30 Paula F. Cossi, Lucila T. Herbert, Beverly Boburg et al.

protruded head-foot region and an absence of spontaneous movement. Most snails recovered
adherence capacity during the 21 days in water free of pesticide, and by the end of the
bioassay none of the snails showed any sign of head-foot protrusion.

Table 1. No observed effect concentration (NOEC) and 50% inhibition concentration

(IC50) of azinphos-methyl in the total soft tissue of Biomphalaria straminea and Chilina
gibbosa after a 48 h exposure. ChE: cholinesterase; CE: carboxylesterase; AcSCh:
acetylthiocholine iodide; p-NPA: p-nitrophenyl acetate; p-NPB: p-nitrophenyl butyrate

Species Neurotoxic Enzyme (substrate) NOEC+ IC50++ Reference

-1
signs (µg L ) (µg L-1)
B. straminea No ChE (AcSCh) >10000* >10000* Bianco
CE (p-NPA) >10000* >10000* et al., 2014
CE (p-NPB) 250 2200 ± 750
C. gibbosa Yes ChE (AcSCh) 0.001 0.02 ± 0.01 Bianco
CE (p-NPA) 1000 1290 ± 40 et al., 2013
CE (p-NPB) 500 1300 ± 100
+
Experimentally estimated as the maximum concentration causing no significant effect.
++
Calculated using the 4-parameter logistic model using OriginPro 7.5. Model equation: y=A2 + (A1-A2)
/ (1+(x/x0)p); y enzyme activity (expressed as % of control); x inhibitor concentration; A1 and A2
upper and lower bound for function values y; x0 (relative IC50) concentration corresponding to a
response midway between A1 and A2.
* Not reached at the studied concentrations (50-10000 µg L-1).

Figure 3. Organisms of Chilina gibbosa, control (a) and exposed to 20 µg L−1 of azinphos-methyl for 48
h (b).

Boburg et al., (2015) exposed C. gibbosa snails subchronically during 21 days to

20 µg L-1 of azinphos-methyl. By day 21 most of the snails had lost adherence to the walls
and all presented a protruded head-foot region. At 48 h, ChE activity was greatly inhibited
and remained inhibited during the rest of the bioassay. CE activity measured using p-NPB
and p-NPA was only inhibited after 14 days of exposure. Preliminary histological studies
have not shown any differences between control and treated animals.
In current studies we are focusing on questions that arose from the first bioassays. On the
one hand, we intend to find other biomarkers that could be relevant when using an endemic
native species such as C. gibbosa. For instance, we have recently found that an acute 48 h
exposure to 20 µg L-1 of azinphos-methyl affects the immune system of these snails (Castro et

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al., 2015). On the other hand, we are exposing C. gibbosa to other contaminants, such as the
carbamate carbaryl or river water polluted by a natural petroleum spill, in order to determine
whether the conspicuous neurotoxic sign is specific to anticholinesterase insecticides and can
be used as a simple and effective biomarker.

CONCLUSION
Our research group has evaluated the effects of the organophosphate pesticide azinphos-
methyl, a relevant agrochemical used in Argentina and found in its watercourses (Loewy et
al., 1999, 2011; Castañé et al., 2015) at concentrations higher than those recommended for the
protection of aquatic wildlife, on two different species of native freshwater snails. B.
straminea and C. gibbosa, our model organisms, have different natural habitats, life cycles
and lifestyles. Thus, they provide an interesting possibility for comparing toxic effects
between organisms. B. straminea, found throughout South America and widely distributed in
northeastern Argentina, is a species with a great reproductive potential, rapid growth and an
early maturation (Yipp, 1983), that can be easily handled and maintained in laboratory
cultures. On the contrary, C. gibbosa, endemic to freshwater habitats of the Argentine
Patagonia, is a semelparous species with an annual life-cycle (Bosnia et al., 1990) that
requires special conditions for surviving and breeding in the laboratory.
Our results show clear differences in the responses to azinphos-methyl between B.
straminea and C. gibbosa. Whilst B. straminea does not show acute toxic effects at
environmental concentrations, resulting particularly resistant to azinphos-methyl, C. gibbosa
is a highly sensitive species that exhibits a conspicuous physical neurotoxic sign even after an
acute exposure to the pesticide (Bianco et al., 2013, 2014). In B. straminea, however, toxic
effects were observed at environmental concentrations after subchronic exposures, which
further demonstrated the need to evaluate effects at different exposure times (Bianco et al.,
2014). These dissimilar responses have led to individual branching questions. On the one
hand, could C. gibbosa be a suitable sentinel species to indicate the presence of azinphos-
methyl and/or organophosphate pesticides? On the other hand, given that B. straminea
appears to be more tolerant to azinphos-methyl, could there be long-term effects on the
organisms and their offspring?
Our findings also reinforce the notion that different species within a same trophic level
should be taken into account for biomonitoring programs, especially considering that even
species belonging to the same family can respond differently to the same toxicant (Kristoff et
al., 2006; Bianco et al., 2014). Working with bioassays and native species implies that our
results could provide relevant information to be used for re-assessing guideline maximum
levels of water contaminants for aquatic life protection and new tools to evaluate aquatic
toxicity risk.
Further research will allow us to continue contributing with biochemical, histological,
behavioral and physiological information about native gastropods; propose early warning
biomarkers; provide evidence for the potential use of these species as sentinels; and develop a
baseline to perform ecotoxicological assays with water sampled from the field.

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ACKNOWLEDGMENTS
The results of the studies presented here were supported by grants from ORT (561/12),
University of Buenos Aires (20020110100070, 20020100200140 and 20020130200055),
CONICET (112200901000492; 11220120100680CO) and ANPCYT (2007-00214 and 2013-
1415). P. Cossi and L. Herbert are on doctoral fellowships from CONICET, Argentina. We
would like to thank Lucas Defelipe and Pablo Grotto for their valuable input.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 4

ENERGY PHYSIOLOGY AS BIOMARKER IN ASSESSING

ENVIRONMENTAL POLLUTION

Beatriz Toro Restrepo1,* and Jorge M. Navarro2,3

1
Departamento de Ciencias Biológicas, Universidad de Caldas, Manizales, Colombia
2
Instituto de Ciencias Marinas y Limnológicas,
Universidad Austral de Chile, Valdivia, Chile
3
Centro FONDAP de Investigación en Dinámica de Ecosistemas Marinos
de Altas Latitudes (IDEAL), Valdivia, Chile

ABSTRACT
An ecosystem’s health is more and more difficult to assess due to the variety and
amount of xenobiotic substances. Physiological energy measurements are therefore used
together with chemical analysis of pollutants in the body’s tissues to detect and quantify
the effects of pollution. The Scope for Growth (SFG) is the most common measurement
because it integrates basic physiological responses such as feeding, digestion, respiration
and excretion. This is the case of Choromytilus chorus, a native mussel from the Chilean
coast, where it was observed that an increase in the concentration of PAHs (polycyclic
aromatic hydrocarbons) or Ochs (organochlorine pesticides) in tissue produced a negative
effect on their physiological processes. When the concentrations of these pollutants were
low in the tissue, the population responses were better. This chapter describes the SFG in
a gradient of pollution along the coast in spring and summer, finding the high pollution
site with a negative SFG (-4.56 J/h/g in spring and -3.45 J/h/g in summer). The energy
balance in C. chorus in the site with less pollution was positive with values of 6.2 J/h/g in
spring and 25.7 J/h/g in summer. In general, the physiological responses showed a
significant negative correlation with organochlorine pesticides (r=-0.87) and PAHs
(r=-0.89).

Keywords: scope for growth, biomarker, aquatic pollution, bivalve, Choromytilus chorus

*
Corresponding Author E-mail: beatriz.toro@ucaldas.edu.co.

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38 Beatriz Toro Restrepo and Jorge M. Navarro

INTRODUCTION
The assessment of environmental pollution using physiological biomarkers has been
carried out with success for the easiness to collect the data and its ecological relevance
(Bayne, 1985; Barton et al., 2002; Chambers et al., 2002). The physiological energetic
responses not only provide information about fundamental processes of acquisition, spending
and available energy for growth and reproduction, but also reflect some of the main
mechanisms of toxicity (Smaal and Widdows, 1994). For evaluating the energy balance,
physiological processes such as Scope for Growth (SFG), the Oxygen/Nitrogen Ratio and
Condition Index are used. The SFG is the most applied measurement since it integrates basic
physiological responses (feeding, digestion, respiration and excretion), and because available
total body energy may be affected by altering one or more of these processes (Widdows,
1985).
The assessment of SFG in the organism together with chemical analysis in its tissue
allows for the prediction of possible negative impacts at different levels of biological
organization, from the cell to the ecosystem. In addition, this biomarker has high sensitivity to
environmental changes, accuracy, simplicity and the physiological measurements are of short
duration (Bayne et al., 1979; Peck et al., 1987; Widdows and Johnson, 1988; Widdows and
Donkin, 1991; Widdows and Salked, 1992; Quian, 2011).
The SFG has been used to assess contamination with heavy metals such as cadmium
(Poulsen et al., 1982) and lead (Widdows et al., 1990), organochlorines (Toro et al., 2003;
Shuhong et al., 2005), aromatic hydrocarbons (Toro et al., 2003; Halldórsson et al., 2005), tin
compounds (Widdows et al., 1990; Halldórsson et al., 2005) and other highly toxic
compounds such as polychlorinated biphenyls (Widdows et al., 1990). The main marine
organisms used for the evaluation of pollution are bivalves such as Mytilus edulis (Poulsen et
al., 1982; Halldórsson et al., 2005), M. galloprovinciales (Widdows et al., 1997; Albentosa et
al., 2012) and Anadara granosa (Din andAhamad, 1995) as well as the marine gastropods
Thais lima (Stickle et al., 1984) and Haliotis fulgens (Farías et al., 2003). The main goal of
this chapter is to show that Scope for Growth is a useful biomarker for assessing aquatic
pollution and it has great relevance at an ecological level.

METHODS
Study Area

Specimens of the mussel Choromytilus chorus were collected at three locations on the
south-central coast of Chile (Figure 1). Two samplings were carried out at each location: one
in spring 1998 and another in summer 1999. Site A (highly polluted) is in San Vicente Bay
(36°44´S; 73°09´W), a location surrounded by fish processing, steel, and petrochemical
industries. Site B (medium pollution) is in Corral Bay (39°52´S; 73°25´W), which presents a
fishing port and small human populations along its shore. Site C (low pollution) is in Yaldad
Bay (43°08´S; 73°44´W), an isolated location where the main activity was mussel culture
(M.chilensis).

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 Energy Physiology as Biomarker in Assessing Environmental Pollution 39

Sampling

C. chorus specimens were collected from natural banks in San Vicente and Corral Bays,
while individuals from Yaldad Bay were obtained from cultures suspended at 4-8 m depth. At
each station, adult specimens ~120 mm in length were collected for chemical analysis and
individuals ~70 mm long for physiological measurements.

Chemical Analyses

Tissue Preparation
For each sampling site, between 15 and 20 specimens were pooled as a single sample to
minimize natural variance among individuals (n=four analytical replicates). Standard
operation procedures for sample handling and preparation followed guidelines of FAO
(1983). Subsamples of 10 g of pooled material were prepared for analyses of PAHs
(polycyclic aromatic hydrocarbons) and Ochs (organochlorines).

Standards and Solvents

The analytical methodology was tested on Standard Reference Material (SRM 2974)
containing 14 PAHs and seven OChs at known concentrations in mussel tissue (M. edulis),
obtained from the National Institute of Standards and Technology (NIST).

Analysis of Subsamples
The extraction, clean up and analyses of Ochs in the tissue of C. chorus were carried out
as described by Knickmeyer and Steinhart (1988) and FAO (1983). The UNEP (1992)
methodology was followed for extraction, purification, and analyses of PAHs. Detection of
the OChs was performed with a GC-ECD Perkin-Elmer 9000 with a63Ni electron capture
detector. PAHs were identified and quantified by using a HP 6980 (Hewlett Packard) gas
chromatograph equipped with HP 5973A mass selective detector. The Estimated Detection
Limit (EDL) of the analytical method was 5 µg/kg dry tissue for 10 g sample.

Physiological Measurements

Acclimation
Before beginning physiological measurements the mussels were cleaned of epibionts and
maintained for 12-48 h as recommended by Widdows (1993). Test specimens were
maintained at standard laboratory conditions, at a salinity of 30 and temperature of 12 ºC for
spring experiments and 15°C for those performed in the summer.
Since many of the physiological processes are dependent on the body size of the
individual, the size effect was removed by standardization of the physiological rates to per-
gram dry weight values, using the following equation:

𝑊𝑠 𝑏
Y𝑠 = (𝑊𝑒) x Ye

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40 Beatriz Toro Restrepo and Jorge M. Navarro

Where Ys is the physiological rate for a normalized weight, Ws the normalized weight,
We the observed weight, Ye the measured physiological rate and b the weight exponent
(regression potential; Navarro, 1988).

Figure 1. Choromytilus chorus sampling sites: San Vicente Bay (A); Corral Bay (B); Yaldad Bay (C).

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 Energy Physiology as Biomarker in Assessing Environmental Pollution 41

Clearance Rate (CR)

The feeding rate in bivalves is usually measured in terms of clearance rate, and it is
evaluated using a static system, following the methodology described by Bayne et al. (1985).
Fifteen aquariums of 6 L received a diet of 20x106 cell/L of laboratory-cultured Isochrysis
galbana. The clearance rate for each individual was calculated using the following equation:

𝑉(𝑙𝑛𝐶1 − 𝑙𝑛𝐶2 )
𝐶𝑅 =
𝑡

where, CR is the clearance rate, V the volume of water, and C1 and C2 are the I. Galbana
concentrations at the beginning (1) and end (2) of the test interval t.

Absorption Efficiency (AE)

Faecal material produced during the measurements of particle clearance was filtered onto
filters and processed by the same methods as for the diet samples. The ratio of diet ash free
dry weight (AFDW) of the faeces was used to determine the absorption efficiency using the
method of Conover (1966):

(𝐹 − 𝐸)
𝐴𝐸 =
(1 − 𝐸) 𝑥 𝐹

whereF is the ash free dry weight to dry weight ratio for the food and E the ash free dry
weight to dry weight ratio for the faeces.

Absorption Rate (AR)

AR represents the amount of ingested material absorbed per unit of time, and is calculated
as a product of the absorption efficiency and the organic ingestion rate (OIR; Bayne et al.,
1985):

AR (mg/h)=AE x OIR

Oxygen Consumption (OC)

OC was measured by placing each mussel into a sealed 800 mL respirometer chamber
containing oxygen-saturated seawater. Oxygen in the water was measured by the Winkler
method (Strinckland and Parsons, 1972). Oxygen consumption was calculated using the
formula:

[(𝑚𝐿𝑂2 /𝐿𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝑚𝐿𝑂2 /𝐿𝑐ℎ𝑎𝑚𝑏𝑒𝑟) 𝑉𝑐]

𝑂𝐶 (𝑚𝐿𝑂2 /ℎ) =
𝑡

Where mL O2 per L control is the O2 concentration in the chamber without mussels; ml O2

per L chamber is the O2concentration in the chamber containing a mussel; Vc the volume of
the chamber, and t the incubation time (h). Oxygen consumptions were expressed as energetic
values using the oxycaloric conversion value (1 mL O2=19.9 J) given by Elliot and Davinson
(1975).

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42 Beatriz Toro Restrepo and Jorge M. Navarro

Scope for Growth (SFG)

After completion of all the physiological measurements, the soft tissues of each mussel
were removed and dried to constant weight at 60°C for 48 h, and used to convert the
physiological rates to per-gram dry body weight values. To calculate the SFG, all the
physiological values were converted to energetic equivalents (J/h per g) following Bayne et
al. (1985):

SFG=A-R

where SFG is the scope for growth, A the absorbed energy and R the respired energy.

Statistical Analyses

Data on OChs could not be submitted to statistical analyses because many of the
compounds occurred in trace amounts which affected their detectability, producing very high
variations in tissue subsamples. Conversely, PAH compounds were found in relatively high
concentrations in the C. chorus tissues, allowing for statistical comparisons between samples.
Since the data did not fit criteria for applying analysis of variance tests, the non-parametric
Mann-Whitney test was used to compare PAH contents of tissues of mussels from the three
sampling sites for the two sampling seasons. This test was also used to compare physiological
responses of the mussels from the different populations in the two seasons, since the data did
not show homoscedasticity of variances, nor could be normalized by transformation.
Correlation between different physiological responses and SFG of C. chorus and the
concentrations of the organic pollutants (PAHs and OChs) present in their tissues was
determined using a Spearman multiple correlation matrix (Sokal and Rohlf, 1979).

Table 1.Total organochlorines (OChs) and polycyclic aromatic hydrocarbons (PAHs)

accumulated in tissue of Choromytilus chorus from the south/central coast of Chile

OChs (µg/kg dry wt) PAHs (µg/kg dry wt)

Sampling site
Spring Summer Spring Summer
San Vicente 29.5 29.5 3380.0 2993.0
Corral 7.5 6.0 82.0 215.0
Yaldad 17.0 0.5 347.0 49.0

RESULTS
Chemical Analyses

C. chorus individuals from San Vicente, Corral, and Yaldad Bays contained low levels of
OChs in their tissues. The total concentrations of OChs were always higher in tissues of
specimens from San Vicente Bay population both in spring and summer when compared with

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 Energy Physiology as Biomarker in Assessing Environmental Pollution 43

populations from Corral and Yaldad. The values for PAHs were much higher than for OChs,
following the same tendency (Table 1).

Organochlorines

The OChs results obtained from the samples were classified according to the toxicity
groups established by the US Environmental Protection Agency (EXTOXNET, 2000). The
San Vicente mussel population contained the highest levels of the substances in both seasons
measured (Figure 2), and the highest concentrations were of moderate toxicity (Class II), i.e.,
such as chlordane ( and ), heptachlor, heptachlor epoxide, DDT (and metabolites) and
hexachlorocyclohexane isomers. Others chlorinated compounds highly toxic such as aldrin,
dieldrin, endrin, endrin aldehyde, endosulfan and endosulfan sulfate were also recorded at
relatively high concentrations, particularly in individuals from Bay of San Vicente during the
spring and summer, while in the sites of Corral and Yaldad they were below the detection
limit.

25 I
OChs Concentration (µg/kg dry wt)

II
20 III

15

10

0
Yaldad Corral San Vicente
25
OChs Concentration (µg/kg dry wt)

20

15

10

0
Yaldad Corral San Vicente
Population

Figure 2. Concentrations of organochlorine (OChs) compounds in tissues of Choromytilus chorus

grouped according to toxicity level established by the US Environmental Protection Agency
(EXTOXNET, 2000). Upper: spring; lower: summer. I: high toxicity; II: moderate toxicity; III: non-
toxic.

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44 Beatriz Toro Restrepo and Jorge M. Navarro

Polycyclic Aromatic Hydrocarbons

In the scan for 16 PAH, 13 were found and quantified by the GC-MS methods. Total
levels of PAH were always higher than total OCh levels in the mussel tissues, in an order of
magnitude of 10-35 times in individuals Corral Bay, in 20-100 in Yaldad, and 100 -130 in
San Vicente Bay. The population from San Vicente demonstrated the highest total
concentration of PAHs both in spring and summer (Table 1). Mussel population from San
Vicente bay contained higher levels of potential carcinogens (RAIS, 2000) than those of the
other two locations sampled (Figure 3).
The PAHs were grouped according to their aromatic rings. It was found that in the Bay of
San Vicente the highest percentage corresponded to hydrocarbons with 4rings (52%).While
the PAHs with 2-3 rings showed the highest percentage in the bays of Yaldad and Corral (73
and 79%, respectively; Table 2).

2000
I
PAHs Concentration (µg/kg dry wt)

II
1750
III
1500

1250

1000

750

500

250

0
Yaldad Corral San Vicente
2000 Population
PAHs Concentration (µg/kg dry wt)

1750

1500

1250

1000

750

500

250

0
Yaldad Corral San Vicente

Population

Figure 3. Concentrations of polycyclic aromatic hydrocarbons (PAHs) in tissues of Choromytilus

chorus grouped according to carcinogenic potential (RAIS, 2000). Upper: spring; lower: summer. I:
carcinogenic; II: probably carcinogenic; III: non-carcinogenic (n=4).

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 Energy Physiology as Biomarker in Assessing Environmental Pollution 45

Physiological Measurements

During spring, C. chorus from San Vicente Bay had significantly lower CR (0.67 ± 0.32
L/h per g) than those from Yaldad (1.41 ± 0.38 L/h per g; p<0.0005). In summer, individuals
from San Vicente bay only showed significant differences with those of Yaldad bay
(CR 2.25 ± 0.21), obtaining an average value of 0.97 ± 0.42L/h per g, corresponding to 43%
of the CR found for C. chorus from Yaldad (Figure 4).

3,0
3.0
Spring
Clearance rate (L/h/g)

2,5
2.5 Summer B

2,0
1.5 b
b
1,5
1.0 A A
1,0
a
0.5
0,5
0.0
0,0
San Vicente Corral Yaldad

Population

Figure 4. Clearance rate in Choromytilus chorus exposed to organochlorines and polycyclic aromatic
hydrocarbons from Chilean coast. Values represent the mean ± standard deviation (vertical lines;
hereafter SD); letters indicate statistical differences between sites during spring (lowercase) and
summer (uppercase).

120
Spring
Summer
100
Absorption efficiency (%)

80 *
*

60

40

20

0
San Vicente Corral Yaldad
Population

Figure 5. Absorption efficiency in Choromytilus chorus exposed to organochlorines and polycyclic

aromatic hydrocarbons from Chilean coast. Values represent the mean ± one SD; * indicates no
significant differences in relation to other sites.

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46 Beatriz Toro Restrepo and Jorge M. Navarro

Table 2.Concentration of polycyclic aromatic hydrocarbons (PAHs) in tissue of

Choromytilu chorus from the south/central coast of Chile, according to aromaticity.
The number between () represents the percentage of PAH with this number of rings.
DL=detection limit (5 µg/kg)

Concentration (µg/kg dry wt)

Number
San Vicente Corral Yaldad
of rings
Spring Summer Spring Summer Spring Summer
2-3 695 (18) 1339 (46) 55 (67) 147 (73) 245 (79) 25 (60)
4 1325 (35) 1513 (52) 27 (33) 54 (27) 66 (21) 17 (40)
5-6 1787 (47) 54 (2) <DL <DL <DL <DL

2.5
Spring
Summer
2.0
Absorption rate (mg/h/g)

1.5

1.0

*
0.5 *

0.0
San Vicente Corral Yaldad

Population

Figure 6. Absorption rate in Choromytilus chorus exposed to organochlorines and polycyclic aromatic
hydrocarbons from Chilean coast. Values represent the mean ± one SD; * indicates no significant
differences between seasons.
1
Spring

Summer
Oxygen consumption (ml O2/h/g)

0.8

a
0.6

0.4

0.2

0
San Vicente Corral Yaldad
Population

Figure 7. Oxygen consumption in Choromytilus chorus exposed to organochlorines and polycyclic

aromatic hydrocarbons from Chilean coast. Values represent the mean ± one SD; the lowercase a
indicates statistical differences between seasons.

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 Energy Physiology as Biomarker in Assessing Environmental Pollution 47

35
Spring

30 Summer

25
Scope For Growth (J/h/g)
20

15

10

-5
* *
-10
San Vicente Corral Yaldad

Population

Figure 8. Scope for growth in Choromytilus chorus exposed to organochlorines and polycyclic aromatic
hydrocarbons from Chilean coast. Values represent the mean ± one SD; * indicates no significant
differences between seasons.

The absorption efficiencies measured in individuals from Corral and Yaldad Bays were
significantly higher (p<0.005) than those measured for the mussels from San Vicente Bay,
where the values never exceeded 70% during both seasons (Figure 5).
The AR showed the same tendency as the CR, since it represents an integrated response
to the CR, ingestion rate, and absorption efficiency (Figure 6).
The oxygen consumption rates varied between populations and between seasons.
Although there were significant differences (p<0.01) between spring and summer for all three
populations, there were no differences in rates between the populations of San Vicente and
Yaldad in spring, and between Corral and Yaldad in summer (Figure 7).
The SFG of individuals from Corral bay (15.5 and 6.6 J/h per g) and Yaldad bay (6.2 and
25.6 J/h per g) were positive in both spring and summer, in contrast to individuals from San
Vicente bay which showed negative values in both seasons (-4.6 and -3.5 J/h per g).
Significant differences were found (p<0.01) among populations and between seasons except
for individuals from San Vicente which showed similar values in both spring and summer
(Figure 8).
The Spearman multiple correlation matrix showed that the physiological responses
related to energy balance have a significant correlation with the presence of organic pollutants
in the tissues (p<0.001). In the three populations studied, a negative effect on the
physiological parameters was observed with an increase in PAHs and OChs concentrations in
the mussel tissues. The absorption rate showed the greatest correlation to organic pollutants:
r=-0.86 for OChs and r=-0.91 for the PAHs. This was followed by the CR: r=-0.71 for the
OChs and r=-0.82 for the PAHs. The AE showed a lesser correlation (r=-0.66) with PAHs
while the OChs it was -0.75. Oxygen consumption was the only physiological response with a
positive and significant (p<0.001) correlation with the pollutant concentrations: 0.42 for the

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48 Beatriz Toro Restrepo and Jorge M. Navarro

OChs and 0.51 for the PAHs. The index of SFG showed a significant negative correlation to
tissue pollutant levels: r=-0.87 for the OChs and r=-0.89 for the PAHs (Figure 9).

40
SV
rr ==-0.87
-0.87 CO
30 YA
SV
Scope for growth (J/h/g) CO
20 YA

10

-10

-20
-0.5 0.5 1.5 2.5 3.5 4.5 5.5 6.5

Organochlorines (µg/kg)

40
SV
r = -0.89 CO
30 YA
SV
Scope for growth (J/h/g)

CO
20 YA

10

-10

-20
-500 500 1500 2500 3500 4500

Polynuclear
Polycyclic aromatic
Aromatic hydrocarbons
Hydrocarbons (µg/kg)
(µg/kg)

Figure 9. Correlation between the Scope for Growth and concentration of organochlorines and
polycyclic aromatic hydrocarbons in Choromytilus chorus populations from San Vicente (SV), Corral
(CO) and Yaldad (YA) in spring (dark symbols) and summer (clear symbols).

DISCUSSION
Chemical Analyses

Organochlorines
The low concentrations of OChs in tissues of C. chorus may be the result of the banning
of most of these compounds over the last 20 years in Chile. Residues detected in the mussel
may have originated from resuspended sediments as these (lipophilic) compounds may adsorb
to sedimentary organic material, and remain in bottom sediments for many years (EPA, 1982;
Balls and Campbell, 1999).

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 Energy Physiology as Biomarker in Assessing Environmental Pollution 49

None of the compounds detected occurred above action levels established by the Food
and Drug administration (USA-FDA, 1992). Compared with values for OChs burden in M.
edulis obtained from the highly polluted Baltic Sea (Jensen et al., 1969; Lee et al., 1996), the
present values for C. chorus are low, even those of San Vicente which were the highest
among the values from the present study. Present values obtained from Corral and Yaldad are
indeed at insignificant levels, as would be expected of non-industrialized regions.

Polycyclic Aromatic Hydrocarbons

The greater concentrations of PAHs when compared with the OChs is due to the multiple
sources of the former (pyrogenic, diagenetic, petrogenic) which liberate these compounds into
the environment (CEPAL, 1990; EHC, 1996, 1998). Thus the elevated concentrations of
PAHs in the specimens from San Vicente bay during both spring and summer, when
compared with individuals from Corral and Yaldad Bays, are probably related to the
industrialization at San Vicente. This bay is subject to extensive pollution in the form of
petroleum and pyrogenic wastes (Mudge and Seguel, 1999), while the anthropogenic
influence in the other bays is low, particularly at Yaldad, shown by the low degree of these
pollutants in the respective C. chorus tissues.
Values of PAHs for C. chorus populations were within the ranges observed in other
bivalve species such as M. galloprovincialis (Shchekaturina et al., 1995), M.edulis (Baumard
et al., 1999) and Crassostrea virginica (Noreña et al., 1999). The elevated concentrations of
PAH from San Vicente samples in both seasons explain the higher uptake of oil pollution
(Mudge and Seguel, 1999) by mussels when compared with individuals from Corral and
Yaldad. According to the criteria presented by the preceding authors and Axelman et al.
(1999), mussels from the San Vicente population are highly polluted with PAH, while those
from Corral and Yaldad would be considered unpolluted. Populations of C. chorus from
Corral and Yaldad had a qualitative pattern of distribution of hydrocarbons similar to those in
other species such as Arca zebra (Widdows et al., 1990) and Dreissenia polymorpha
(Gossiaux, 1998) where there was a tendency to accumulate PAH of low molecular weight (di
and triaromatics), principally anthracene and phenanthrene. In contrast, mussels from
San Vicente accumulated a higher molecular weight (3-4 ring) hydrocarbon burden,
including indeno(1,2,3-cd)pyrene, dibenzo(a,h)anthracene, and benzo(ghi)perylene. These
hydrocarbons may have been absorbed from resuspended benthic sediments, since the mussel
bed was located in a zone experiencing a high degree of turbulence.

Physiological Responses

The CR measured in C. chorus was significantly (p<0.001) affected by the presence

of OChs and PAHs in their tissues, as is reflected by high correlation coefficients linking
the physiological responses with concentrations of the studied pollutants. Various
ecotoxicological studies have shown that the CR is the component of the energy budget the
most affected by toxic compounds (Howell et al., 1984; Axiak and George, 1987a; Bourdelin,
1996). These pollutants may produce a narcotizing effect on the ctenidial cilia or cause
morphological alterations in branquial filaments and severe disturbances of ciliated epithelial
cells (Bayne et al., 1982; Axiak and George, 1987b; Auffret, 1988). For example,
concentrations of 100 mg/g dry tissue of 1-3 ring aromatic hydrocarbons may produce a 50%

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50 Beatriz Toro Restrepo and Jorge M. Navarro

reduction in CR of M. edulis when the octanol-water partition coefficient (Kow) is less than
five (Donkin et al., 1989; Widdows and Salkeld, 1992). The former suggests the decline in
CR in C. chorus may be due in part to the presence of tri- and tetra-aromatic PAHs which
were detected at high concentrations in the mussels (Table 2).
The significant reduction in the AE, as experimentally observed in mussels from San
Vicente bay when compared with the same species from Corral and Yaldad bays during two
seasons of the year, indicated deficiencies in the functioning of the digestive systems in
mussels from the polluted bay. These results are comparable to results obtained in other
studies on the efficiency of absorption rate in other bivalve species, where this rate declined
in the presence of organic chemical compounds as aromatic hydrocarbons (Widdows et al.,
1982, 1987). In mussels the exposition to aromatic hydrocarbons induces pathologic changes
in digestive and basophilic cells, unstablizing the lysosomal membrane and thus making it
more permeable to hydrolytic enzymes toward the cytoplasm, which accelerates protein
hydrolysis and causes cellular atrophy (Lowe et al., 1981; Moore and Lowe, 1985; Lowe,
1988; Moore, 1988; Lowe and Clarke, 1989).
The high value for oxygen consumption measured in C. chorus, primarily in individuals
from San Vicente Bay, may be related to the greater expenditure of metabolic energy
produced by significant concentrations of pollutants in tissues of this bivalve species.
Increased consumption of oxygen in bivalves has been observed in response to low levels of
petroleum hydrocarbons (Gillfillan et al., 1976). Some authors suggest that the hydrocarbons
may cause an uncoupling of oxidative phosphorylation, increasing the metabolic rate (Bayne
et al., 1982), while others maintain that the increase in the rate of oxygen consumption is due
to enzymatic activity related to the degradation of the petroleum components (Stekoll et al.,
1980).
The negative SFG demonstrated by mussels from San Vicente Bay in spring (-4.6 J/h per
g) and summer (-3.5 J/h per g) indicates severe stress, given that these individuals are
consuming bodily reserves for survival. The reduction of the SFG to negative values is a
result of the low clearance rate observed for individuals from San Vicente bay which were
unable to obtain enough energy to fulfill their metabolic requirements. Exposure of marine
invertebrates to pollutants, principally PAHs and other organic pollutants, often produces a
loss of fitness of the organism, observable as reduction in SFG or reproductive capacity, or
increase in mortality (Bayne et al., 1982; Widdows, 1985). Ultimately this may affect
population dynamics by interfering with material and energy flows (Kooijman, 1998). The
SFG in C. chorus was reduced in proportion to the degree of pollution to which they were
exposed (Figure 9) and agrees with the results of Widdows et al. (1997) who obtained results
for maximum SFG in M. edulis of 20-25 J/h per g in low-pollution environments, 10-16 J/h
per g in coastal regions with urban influence, and -5 J/h per g in industrialized, highly
polluted urbanized environments. The results obtained on the physiological responses of C.
chorus, in addition to the chemical analyses of their tissues, allow to infer that the populations
at Corral and Yaldad are still able to counteract the negative effects of pollutants present in
their environments, since they demonstrate a positive energy budget. It may, therefore, be
supposed that these populations are capable of normal growth and reproduction, and are thus
capable of maintaining the survival of the species within the ecosystem. San Vicente bay is
one of the most polluted sites of Chile, characterized by a strong hydrocarbon pollution
(Mudge and Seguel, 1999). Hence, individuals of this population showing a negative SFG are

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 Energy Physiology as Biomarker in Assessing Environmental Pollution 51

under a constant stress, being more susceptible to diseases, predation and parasites. If these
conditions persist in the bay, the population could disappear in a long term.

CONCLUSION
The mussel C. chorus presented levels of PAHs and OChs that reflected the
concentrations found in their environments. These organisms would therefore be good
bioindicators of local pollution.
The combination of physiological measurements of energy balance and chemical
analyses of pollutants in tissues of C. chorus is a useful tool for the evaluation of
environmental pollution, given the sensitivity and ecological relevance. Since the responses
measured are at the organism level, they have the advantage of being readily interpreted as
beneficial or deleterious. Furthermore, they may be predictive of long-term consequences for
the growth and survival, not only of the individuals but also of populations, with individual
measurements predicting pollution effects at the ecosystem level.

ACKNOWLEDGMENTS
This chapter formed part of the Doctoral Thesis of Beatriz Toro, supported by a
scholarship from the DAAD, as well as the Aquaculture and Marine Biotechnology
Program,1(97) FONDAP, Chile (Invertebrate Subprogram), the Institutes of Zoology, Marine
Biology and Chemistry, and the partial financial support of the Dirección de Investigación y
Desarrollo, Universidad Austral de Chile (Grant 1999203).

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 5

LABORATORY AND FIELD BASED STUDIES PROVIDE

INSIGHTS OF COPPER TOLERANCE MECHANISMS IN
BROWN SEAWEEDS AND BIOTECHNOLOGY TOOLS
FOR ENVIRONMENTAL DIAGNOSIS

Claudio A. Sáez*
Laboratory of Coastal Environmental Research, Centro de Estudios Avanzados,
Universidad de Playa Ancha, Viña del Mar, Chile

ABSTRACT
The model brown alga Ectocarpus siliculosus, a cosmopolitan small seaweed, is
distributed in the subtidal and intertidal ecosystem, and has been identified in different
impacted ecosystems, including copper-polluted locations. In this chapter we describe
different laboratory- and field-based investigation aiming to identify defence mechanisms
behind intra-specific tolerance in E. siliculosus toward copper (Cu) pollution. The E.
siliculosus strain Es524, isolated from a Cu-polluted location in northern Chile, showed
the highest defences compared to other four strains when exposed to up to 2.4 µM copper
for 10 d. The response manifested in less intracellular copper, higher induction and
polymerization of metal-chelating phytochelatins, increased syntheses of antioxidants
glutathione (GSH) and ascorbate (ASC), and higher expression and activities of
antioxidant enzymes such as ascorbate peroxidase (APX), catalase (CAT), and
superoxide dismutase (SOD); all the latter were responsible, at least in part, for the low
signs of oxidative damage and physiological impairment observed in this strain.
Laboratory experiments help identifying specific metabolic responses, but do not
represent what happens in the natural, more complex, environment. Thus, we designed a
novel transplantation technique for field experiments with E. siliculosus. The algae was
enclosed in a dialysis tubing device and deployed in Cu-polluted and pristine locations.
Antioxidant responses of Es524 in the field were similar to those recorded in laboratory
experiments, supporting that the recorded defences were mediated by copper excess.

*
Corresponding Author E-mail: claudio.saez@upla.cl.

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58 Claudio A. Sáez

Moreover, the method was successful for environmental diagnosis and can be applied
globally in temperate coastal ecosystems.

Keywords: copper stress, tolerance mechanisms, inter-population tolerance, brown algae,

biomonitoring

INTRODUCTION
There is growing concern regarding the increasing anthropogenic impacts on marine
ecosystems, among which metal pollution is one of the most important (Brown and Depledge,
1998; Sáez et al., 2012a). In Chile, coastal areas of the north have been the most impacted by
metal contamination, mainly copper (Cu); mining activities of Cu are mostly located in this
area due to the presence of important reservoirs in the Atacama Desert (Ramirez et al., 2005).
Copper pollution in Chile is derived mainly from the disposal of extracting and refinery
residues, as in the bay of Chañaral city, where miners have historically discharged metal
tailings in coastal ecosystems, negatively impacting their biodiversity (Correa et al., 2000).
Records show that copper concentrations in coastal sediments of Chañaral exceed 900 µg g-1
(De Gregori et al., 1996; Andrade et al., 2006); in contrast, in locations near less impacted
cities such as Coquimbo, sediment concentrations of copper have been observed to be around
15 µg g-1 (De Gregori et al., 1996).
Copper is an essential metal, involved in many metabolic functions of living organisms
such as acting as a cofactor of the enzymes superoxide dismutase (SOD) and cytochrome c
oxidase (Leary et al., 2004), and as component of plastocyanin, which takes part in electron
transport in chloroplasts of photosynthetic organisms (Tapken et al., 2012). However, beyond
certain threshold concentrations, copper can become highly toxic and affect the metabolism.
For example, excess copper can induce over-production of reactive oxygen species (ROS)
such as superoxide anions (O2−) from hydrogen peroxide (H2O2) in Fenton reaction, and
through the disruption of electron transport chains in mitochondria and in chloroplasts of
photosynthetic organisms (Gómez et al., 2015; Sáez et al., 2015a). It is important to highlight
that although ROS play an important role in signal transduction for the induction of
gene expression, their over-accumulation can produce oxidative stress and damage to
biomolecules, disrupting the metabolism, and eventually affecting physiological processes
such as growth and reproduction (Roncarati et al., 2015; Sáez et al., 2015a; Gómez et al.,
2016; Laporte et al. 2016; Moenne et al., 2016).
Brown macroalgae are the main primary producers and the base of trophic networks in
coastal rocky shores in temperate environments, providing food, shelter and habitat for a
variety of organisms; thus, their diversity and abundance control the complexity of entire
coastal ecosystems (Sáez et al., 2012a; Ortega et al., 2014). It is because of the latter that
environmental impacts on brown seaweeds will undoubtedly have consequences at higher
levels of biological organization, such as at population and community levels, compromising
the availability of species with high ecological and economic importance (Sáez et al., 2012a;
Ortega et al., 2014). Although brown macroalgae share certain metabolic and ecological
characteristics with other photosynthetic organisms such as green and red macroalgae, and
even plants, they are phylogenetically distantly related (Charrier et al., 2008). In spite of the
economic and ecological importance of brown seaweeds, little is known about their biology at

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 Laboratory and Field Based Studies Provide Insights of Copper Tolerance … 59

low levels of biological organization when compared with other photoautotrophs; therefore,
further research is required to explain the biology of such a relevant and distinctive group of
marine organisms.
The bioavailability of Cu is an important factor mediating development and growth in
photosynthetic organisms and brown algae. For instance, Cu is involved in enzyme activation
and electron transport in photosynthesis (Falkowski and Raven, 2007); however, beyond
certain levels, Cu can represent a risk for the survival of brown seaweeds. For example, it has
been found that photo-inhibition can be caused by the substitution of magnesium (Mg2+) by
copper (Cu+2) in the chlorophyll molecule, incapacitating it to perform photosynthesis
(Kuepper et al., 2002). Besides the latter, the main negative effects of Cu on brown
macroalgae are associated with the induction of an over-production of ROS and oxidative
stress (Sáez et al., 2015a; Moenne et al. 2016). Tolerance to Cu in brown seaweeds
depends on mechanisms that involve both metal-exclusion and intracellular antioxidant and
defence responses. Exclusion mechanisms can be related to different factors, such as the
permeability of the cellular membrane, adsorption to the cell wall and/or epybionts (i.e.,
epiphytic bacteria), and the exudation of organic substances (e.g., polysaccharides, phenolic
compounds) (Gledhill et al., 1999; Sáez et al., 2015a). On the other hand, metal-complexing
compounds such as phytochelatins (PCs), oligomers of glutathione (GSH) synthesized by the
enzyme phytochelatin synthase (PCS), and transcriptionally produced metal-binding proteins
such as metallothioneins (MET) (Roncarati et al., 2015), are also important intracellular
defence mechanisms. Reactive oxygen defences are based on the syntheses of antioxidants
molecules such as glutathione (GSH), ascorbate (ASC), and phenolic compounds, and in the
activation of antioxidant enzymes like ascorbate peroxidase (APX), glutathione peroxidise
(GPX), catalase (CAT), glutathione reductase (GR) and SOD, to prevent (or at least reduce)
oxidative damage (Ritter et al., 2014; Roncarati et al., 2015; Sáez et al., 2015a,b).
While it is widely recognised that metal-pollution induces stress-mediated changes at
population, community, and ecosystem levels, these higher level changes are typically too
complex and far removed from the causative events to be used in developing tools for the
early detection and prediction of the consequences of anthropogenic activity. The impacts of
environmental conditions on organism fitness can be studied by detecting stress signals at the
cellular to molecular level, and linking these to higher level impacts. These stress signals can
act as early warning ‘biomarkers’ of reduced individual health and fitness. Development of
appropriate biomarkers requires an understanding of the cellular and physiological processes
associated with cell injury, molecular damage and impairment of protective systems as well
as how they are manifested as reductions in reproductive output, competitive ability and
survival of individuals (Brown and Depledge, 1998). This understanding could be gained
using a combination of laboratory-based investigations and field studies (biomonitoring)
(Sáez et al., 2015a). Although there is extensive literature demonstrating the effects of
environmental stressors in living organisms, most of the mechanistic and stress metabolism
observations have been conducted in simple models of single exposure under laboratory
conditions. While they can provide valuable information on specific metabolic responses
against stress, it does not necessarily represent the mechanisms by which organisms respond
in natural, more complex, environments. In contrast, the majority of field environmental
assessments display a descriptive approach and lack of mechanistic evidence permitting an
effective identification of the action of a specific stressor (Sáez et al., 2015c).

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Metal biomonitoring using brown seaweeds has been conducted for over 60 years, mostly
because of their sessile nature, relatively high metal-tolerance, and accumulation capacity
(Sáez et al., 2012b). Most studies have been conducted with a ‘passive biomonitoring’
approach, where local populations are sampled, and different parameters such as
accumulation and metabolism changes (biomarkers) are measured (Pawlik-Skowronska et al.,
2007; Sáez et al., 2012b); however, this method could lead to uncertainties and false
environmental diagnosis due to the known intra-specific responses to metal stress observed in
brown seaweed species (Ritter et al., 2010; Roncarati et al., 2015; Sáez et al., 2015a). On the
other hand, ‘active biomonitoring’ has been proposed as a better alternative: species are
cultivated in the laboratory and then transplanted to locations of interest, after which
bioaccumulation and/or biomarkers are measured (Brown et al., 2012; Sáez et al., 2012b).
Advantages of active biomonitoring lie on aspects such as: known populations can be used
and thus avoiding errors raised by intra-specific responses; similar age individuals can be
assessed; transplants can be exposed for the same time-periods in polluted and control sites;
and the assessment can be conducted even if the biomonitoring species is absent in the
locations of interest (Brown et al., 2012; Sáez et al., 2012b). Although scarce, active
biomonitoring using brown seaweeds have been mostly conducted with Fucales or Kelps
(e.g., Serisawa et al., 2002; Hédouin et al., 2008); however, transplantation efforts with these
organisms are time-consuming and logistically demanding due to their slow growth,
morphological complexity, and large size. It is clear now that active biomonitoring efforts
could be improved by using broadly distributed, small, fast growing, and less-complex
species such as Ectocarpus siliculosus.
E. siliculosus is a small filamentous brown alga that belongs to the order Ectocarpales,
closely related to more physiologically complex brown seaweeds such as Laminariales and
Fucales (Charrier et al., 2008). E. siliculosus is distributed worldwide in temperate regions,
especially in marine and low salinity habitats; however, it has also been found in freshwater
(West and Kraft, 1996) and hyper-saline (Geissler, 1983) ecosystems, which makes E.
siliculosus an environmentally versatile species. E. siliculosus was chosen as a model
organism for the study of brown algae, and the genome was recently published (Cock et al.,
2010). There are records of E. siliculosus in Cu-polluted locations, such as in Chañaral in
Chile, and Restronguet Creek in England (Sáez et al., 2015a). Even though it has been found
that E. siliculosus can be highly adaptable to fluctuating environmental conditions, in terms of
Cu-tolerance, an important degree of intra-specific variation has been observed. For instance,
Hall (1980) found that a an E. siliculosus population collected from a Cu-polluted location in
Salcombe, England, had higher growth rates than a population sampled from a pristine site
when exposed to 1.6 µM Cu for 14 d. Similarly, Ritter et al. (2010) observed that the E.
siliculosus strain Es32, from a pristine site in Peru, presented 70% cell death when grown
under 0.8 µM Cu for 10 d; on the other hand, the Es524 strain, isolated from the Cu-polluted
Chañaral, in Chile, presented a similar percentage of cell death only when exposed to 4 µM
Cu.
The aim of this chapter is to present results of a series of laboratory and field studies
conducted with the brown alga E. siliculosus to assess molecular, biochemical and
physiological intra-specific responses to Cu excess, which describe tolerance mechanisms
and, furthermore, provide information suitable for the development of biotechnology tools for
environmental diagnosis.

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 Laboratory and Field Based Studies Provide Insights of Copper Tolerance … 61

Figure 1. Designed transplantation device for active-biomonitoring using Ectocarpus siliculosus. (A)
Model of the transplantation device, and (B) prototype of the transplantation device in the field.
Modified from Sáez et al. (2015c).

METHODS
The investigations were conducted on five different E. siliculosus strains, isolated from
locations with different degrees of Cu contamination. The strains were: LIA (Culture
Collection of Algae and Protozoa (CCAP) accession number 1310/339), sampled at Lon
Liath, Scotland, a pristine location; Es524 (CCAP 1310/333), isolated from a Cu-polluted site
in Chañaral, northern Chile; REP (CCAP 1310/338), sampled at a metal (including Cu)-
polluted location at Restronguet Creek, south-west England; and Es147 (CCAP 1310/340),
isolated from site with a recent history of low Cu-pollution in Caleta Coloso, northern Chile.
To assess for responses to Cu excess in controlled environmental conditions, the E.
siliculosus strains Es524, LIA and REP were exposed in triplicates for 10 d to a range of Cu
concentrations of 0 (control), 0.4, 0.8, 1.6 and 2.4 µM; details of culture conditions in Sáez et
al. (2015a). Different parameters were measured: relative growth rates (RGR), Cu-exclusion
mechanisms (extracellular and intracellular accumulation), expression of enzymes involved in
GSH and PCs syntheses, and concentration and levels of PCs polymerization (see Greco et
al., 2014; Roncarati et al., 2015); oxidative stress and damage responses, levels of
antioxidants GSH, glutathione disulphide (GSSG), ASC, dehydorascorbate (DHA) and
phenolic compounds, and activity and expression of ASC, CAT, SOD and GR (see Greco et
al., 2014; Sáez et al., 2015a,b).

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A B

Figure 2. Concentrations per g dry weight (DT) of intracellular (A), and extracellular (B) Cu in
Ectocarpus siliculosus strains Es524 (black squares), sampled from a Cu-polluted location, and LIA
(empty circles), from a pristine site, after Cu exposure for 10 d. Error bars were calculated from three
independent replicates ± SD. Different letters represent significant differences at 95% confidence
interval. Modified from Roncarati et al. (2015).

To investigate responses to Cu (and other metals)-excess in semi-controlled

environmental conditions, 6 g fresh weight (FW) E. siliculosus strains Es524 and Es147 were
transplanted in triplicates to polluted (Ventanas site) and non-polluted (Quintay site) locations
in central Chile for 10 d, using a novel active-biomonitoring method based on a designed
dialysis tubing device (see Figure 1). Parameters assessed were: oxidative stress and damage,
concentration of antioxidants GSH, ASC and phenolic compounds, and activities of
antioxidant enzymes (see Sáez et al., 2015c).
Significant differences were calculated by one-way analysis of variance (one-way
ANOVA) and post-hoc Tukey Test, previous assessment of the requirements of normality and
homogeneity of variance (Zar, 1999).

RESULTS
Cu exposure laboratory experiments evidenced that physiological parameters are
intra-specific dependent. When exposing the strains Es524 and LIA to increasing Cu
concentrations, although both strains showed a decrease in relative growth with increasing Cu
exposure, above 0.4 µM the growth levels were always higher in Es524 (from a Cu polluted
site) than in LIA (from a pristine site) (Roncarati et al., 2015).
When comparing Cu exclusion mechanisms, the results evidenced that under high Cu
exposure extracellular and intracellular Cu accumulation were higher and lower, respectively,
in Es524 than LIA (Figure 2). Related to intracellular accumulation, we observed higher
levels and degree of polymerization of PCs in Es524 compared to LIA; indeed, Es524
displayed PCs with 2 and 3 GSH oligomers (PC2-PC3), whereas LIA had only PC2 under Cu
excess (Figure 3).
Regarding antioxidant defences, laboratory experiments showed that ASC, DHA, GSH
and GSSG were higher at greater Cu exposure in Es524 in relation to the strains LIA and REP
(Figure 4). Moreover, phenolic compounds increased similarly in Es524 and REP upon Cu

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exposure, but in a greater manner than in LIA (see Sáez et al., 2015a). Continuing the trend,
the activities of the antioxidant enzymes SOD and CAT were higher in Es524 than in LIA and
REP under increasing Cu exposure (Figure 5A and B). In contrast, the pattern of increase of
APX activity was similar between the E. siliculosus strains assessed (Figure 5C).
After transplanting E. siliculosus strains Es524 (from a highly Cu-polluted site) and
Es147 (from a low Cu polluted site) for 10 d to Ventanas (polluted) and Quintay (non-
polluted), metal accumulation was shown to be similarly higher for both stains at the polluted
location in relation to the pristine site. Results on oxidative stress and damage were, as in the
laboratory experiments, different between strains but in agreement with metal pollution
degrees. On the other hand, levels of GSH, GSSG, ASC and DHA were always higher in
Ventanas than in Quintay for both strains, although those levels were usually higher for
Es524 than Es147 (Figure 6). Furthermore, in terms of the activities of antioxidant enzymes,
while the activities of all SOD, APX, CAT and GR increased in Ventanas in relation to
Quintay for the strain Es524, this pattern was only observed for SOD in Es147 (Figure 7).
Indeed, no significant changes were shown in the activities of APX, CAT and GR in Es147
between transplantation sites (Figure 7).

DISCUSSION
Cu exposure laboratory experiments on E. siliculosus have provided valuable information
on tolerance mechanisms that allow this species to thrive in Cu-polluted environments. In
addition, it has been demonstrated that intra-specific tolerance to Cu excess is likely to be
based on inherited features developed in populations after a long history of being subjected to
different degrees of pollution, varying in strength upon the intensity of the exposure. In this
respect, it is clear that Cu-tolerance mechanisms in E. siliculosus and other brown algae are
essentially based on exclusion mechanisms, syntheses of metal-chelators and antioxidant
defences.

A B

Figure 3. Levels per dry weight (DT) of phytochelatins 2 (PC2; A), and 3 (PC3; B) in Ectocarpus
siliculosus strains Es524 (black squares), sampled from a Cu-polluted location, and LIA (empty
circles), from a pristine site, after Cu exposure for 10 d. Error bars were calculated from three
independent replicates ± SD. Different letters represent significant differences at 95% confidence
interval. Modified from Roncarati et al. (2015).

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64 Claudio A. Sáez

A B

C D

Figure 4. Concentrations per fresh weight (FW) of reduced glutathione (GSH) (A), glutathione
disulphide (GSSG) (B), reduced ascorbate (ASC) (C), and dehydroascorbate (DHA) (D) in Ectocarpus
siliculosus strains Es524 (black squares), sampled from a Cu-polluted location, REP (black triangles),
isolated from a metal (including Cu)-polluted site, and LIA (empty circles), from a pristine site, after
Cu exposure for 10 d. Error bars were calculated from three independent replicates ± SD. Different
letters represent significant differences at 95% confidence interval. Modified from Sáez et al. (2015a).

Physiological observations have disclosed that although tolerance to Cu is concentration-

dependent and can vary between strains/populations of E. siliculosus, all organisms were
subject to stress, which was evident upon a decrease in RGRs (Roncarati et al., 2015), but
RGRs were always higher in Es524, the E. siliculosus Cu-tolerant strain. Results on lipid
peroxidation, production of hydrogen peroxide, and content of photosynthetic pigments
disclosed that Es524 is less affected than other strains assessed in terms of ROS production
and subsequent oxidative damage under Cu excess; the latter would explain, at least in part,
why there is less physiological impairment in Es524 when compared with other strains
(Roncarati et al., 2015; Sáez et al., 2015a).
Another important aspect of these investigations was that, for the first time, PCs were
described as important mechanisms to counteract Cu stress in brown algae, presenting inter-
population differences in levels of production and polymerization. It was identified that the E.
siliculosus strain Es524 was capable of synthesizing PC2 and PC3 under Cu excess, whereas
LIA (isolated from a pristine site) was able to produce only PC2 and in lower quantity than
Es524. This is an interesting feature of Es524 since it is well known that the longer the PC the
greater the capacity to chelate metals, as more binding sites are available (see Roncarati et al.,
2015). It was also observed that genes encoding for γ-GCS, GS and PCS, responsible for

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GSH-PCs syntheses, were more up-regulated in Es524 in relation to the strain LIA, which
also suggests that enzymatically-mediated production of GSH dimers and then PCs is a
transcriptionally regulated process during exposure to high Cu levels in this species
(Roncarati et al., 2015).

A B

Figure 5. Activities per fresh weight (FW) of the antioxidant enzymes superoxide dismutase (SOD) (A),
(B) catalase (CAT) (B), and ascorbate peroxidase (APX) (C) in Ectocarpus siliculosus strains Es524
(black squares), sampled from a Cu-polluted location, REP (black triangles), isolated from a metal
(including Cu)-polluted site, and LIA (empty circles), from a pristine site, after Cu exposure for 10 d.
Error bars were calculated from three independent replicates ± SD. Different letters represent
significant differences at 95% confidence interval. Modified from Sáez et al. (2015a).

In relation to the reactive oxygen metabolism of E. siliculosus under Cu excess, intra-

specific differences were also observed. Es524 showed greater production of antioxidants
GSH and ASC, and their respective oxidized forms GSSG and DHA, and higher levels of
phenolic compounds (Sáez et al., 2015a). The fact that reduced and oxidised forms of
glutathione and ascorbate were higher than in other strains suggests that syntheses and
recycling within the Halliwell-Asada cycle are more efficient mechanisms in Es524 (Sáez et
al., 2015a). Indeed, for example, we could observe that the expression of the genes coding for
γ-GCS and GS was higher in Es524 than in LIA under high Cu exposure (Roncarati et al.,
2015). In addition, we observed that the activities of the antioxidant enzymes SOD and CAT
were higher in the Es524 strain than in LIA and REP under Cu excess. Interestingly, APX

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66 Claudio A. Sáez

activity increased similarly for all strains in parallel with Cu exposure, which suggests that
APX activity is a constitutive feature among E. siliculosus populations to withstand Cu
excess (Sáez et al., 2015a). The information is conclusive and indicates that antioxidant
defences are one of the most important mechanisms to control Cu-mediated ROS excess in E.
siliculosus and responsible, at least in part, for intra-specific Cu tolerance (Sáez et al., 2015a).
The latter is in agreement with the fact that the lowest levels of lipid peroxidation and ROS
were found in the Es524 strain, phenomena accomplished by displaying stronger antioxidant
defences and thus higher syntheses of antioxidant compounds and increased activities of
antioxidant enzymes.

A B

C D

Figure 6. Concentrations of reduced glutathione (GSH) (A), glutathione disulphide (GSSG) (B),
reduced ascorbate (ASC) (C), and dehydroascorbate (DHA) (D) in Ectocarpus siliculosus strains
Es524, sampled from a Cu-polluted location, and Es147, from a site with recent history of Cu inputs,
after transplantation experiments for 10 d to the the pristine Quintay (empty bars) and the metal-
polluted Ventanas (black bars), in central Chile. Error bars were calculated from three independent
replicates ± SD. Different letters represent significant differences at 95% confidence interval. Modified
from Sáez et al. (2015c).

Further steps on the study of metabolic responses of E. siliculosus under Cu excess in the
laboratory focus on the transcriptome and investigate the molecular basis of tolerance
mechanisms as a whole. We have recently conducted laboratory experiments on E. siliculosus
strain Es524 exposed to 2.4 µM Cu for 10 d and assessed the transcriptome using Next

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Generation Sequencing (NGS) Technologies (RNA-seq). Preliminary results showed that the
transcriptome of the Es524 strain cultured in control conditions expresses 48 different genes
encoding for heat shock proteins (HSPs) of type HSP33, HSP40, HSP70, and HSP90. In
contrast, when exposed to 2.4 µM Cu we observed the expression of 65 HSPs of the kind
HSP20, HSP40, HSP70, and HSP90 (unpublished data). Our results are in agreement with
findings by Ritter et al. (2014), which observed up-regulation of HSPs of the classes 70, 40,
and 20 in the Es32 strain under 3.9 µM Cu. The authors suggested that the syntheses of HSPs
intended to counteract protein denaturalization due to Cu-mediated oxidative stress.
Regarding the expression of antioxidant enzymes, in Es524 under control conditions, we
identified two genes encoding for APX, two CATs, and seven SODs, while in the strain
exposed to Cu we found two APX, four CATs, and eight SODs (unpublished data).
Observing results on Es524 under control conditions, it appears that HSPs and antioxidant
enzymes are induced, and that these are part of ‘baseline defence-mechanisms’ for
maintaining cellular homeostasis. Despite the latter, the general trend seems to be that more
HSPs and antioxidant enzymes are getting expressed under Cu excess.

A B

C D

Figure 7. Activities of the antioxidant enzymes superoxide dismutase (SOD) (A), catalase (CAT) (B),
ascorbate peroxidase (APX) (C), and glutathione reductase (GR) (D) in Ectocarpus siliculosus strains
Es524, sampled from a Cu-polluted location, and Es147, from a site with recent history of Cu inputs,
after transplantation experiments for 10 d to the pristine Quintay (empty bars) and the metal-polluted
Ventanas (black bars), in central Chile. Error bars were calculated from three independent replicates ±
SD. Different letters represent significant differences at 95% confidence interval. Modified from Sáez
et al. (2015c).

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The novel active-biomonitoring method designed to assess metal pollution in coastal

environments using E. siliculosus transplants was successful for its biomonitoring purpose
and in agreement with the findings on the reactive oxygen metabolism from laboratory Cu-
exposure experiments. Even though both Es524 and Es147 strains showed higher levels of
metal accumulation at Ventanas (impacted area) than at Quintay (non-impacted location),
antioxidant responses were different between them. Es524 displayed higher defences
manifested in greater levels of GSH and ASC than Es147, although phenolic compounds were
higher in Es147. Moreover, the activities of antioxidant enzymes SOD, APX, CAT and GR
were higher in Ventanas than Quintay for Es524, whereas the latter only occurred for SOD in
Es147. The data were also concordant regarding oxidative responses, which showed lower
lipid peroxidation and ROS levels in Es524 in relation to Es147 (Sáez et al., 2015c). In
relation to its biomonitoring purpose, the best biomarker candidates proposed were those that
had similar inter-strain behaviour, in other to avoid false environmental diagnoses. These
were metal accumulation, production of antoxidants GSH, ASC, and phenolic compounds,
and the activity of SOD (Sáez et al., 2015c). As the active-biomonitoring results were similar
to those recorded under controlled Cu-exposure experiments (Sáez et al., 2015a), the
information suggests that the oxidative responses observed in situ were indeed influenced by
metal excess (Sáez et al., 2015c). This novel active biomonitoring method showed to be
effective in representing metal bioavailability in the environment. Moreover, in agreement
with other investigations (Sáez et al., 2012a,b), the transplantation method confirmed that
Ventanas remains as a highly polluted location (Sáez et al., 2015c).

CONCLUSION
In this chapter we have described different laboratory- and field-based investigations on
the model brown algae E. siliculosus, which were conducted in order to identify intra-specific
tolerance mechanisms that allow this species to thrive in Cu polluted environments. Even
though it was observed that defences to Cu excess in E. siliculosus are mainly based on
cellular exclusion mechanisms, syntheses of metal chelators and in the antioxidant
metabolism, these were shown to vary depending on the exposure history of the strains that
were subject to investigation. Indeed, Es524 (isolated from a Cu-polluted location) displayed
enhanced defences that allowed it to withstand Cu excess more efficiently than other strains,
which was manifested as less physiological and metabolic impairment. Moreover, the novel
transplantation method designed for metal assessment in the environment using E. siliculosus
was successful for diagnosis, can be applied globally in temperate ecosystems, is cost-
effective, and logistically straightforward.

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Complimentary Contributor Copy
In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 6

ENDOCRINE GENE EXPRESSION IN AQUATIC

ANIMALS AS INDICATOR OF THE PRESENCE
OF XENOBIOTIC COMPOUNDS

Maurilio Lara-Flores*, Yassir Edén Torres-Rojas

and Jaime Rendón von Osten
Instituto de Ecología,
Pesquerías y Oceanografía del Golfo de México de la Universidad
Autónoma de Campeche, Mexico

ABSTRACT
This chapter provides an overview on how the application of gene profiling can be
used in different aquatic organisms as biomarkers of endocrine disruption in the presence
of xenobiotic compounds. The application of gene expression qualification provides the
means to identify complex pathways and strategies that an organism applies in response
to an environmental stressor. In this study we used four estrogenic genes: Vitellogenin I
and II (Vtg I and Vtg II), estrogen receptor α (Eα) and estrogen receptor ß (Eß). For the
validation of a gene expression as a biomarker of exposure to a xenobiotic compound we
performed various experiments with different animal species: 15 d exposure to17ß-
estradiol using male mosquito fish (Gambussia yucatana); 48 h exposure of the Japanese
pond turtle (Mauremys japonica) to petroleum; and 48 h exposure of males of black
mollies (Poecilia sphenops) to petrogenic and pyrogenic hydrocarbons. A different gene
expression was observed in relation to the compound, species, time and concentration;
therefore, these genes can be used as biomarkers regarding the presence of xenobiotic
compounds in aquatic animals. For the application of these biomarkers to environmental
samples, we performed the analysis with the Atlantic angel shark (Squatina dumeril); the
results indicate that the gene expression can be used as biomarker of the presence of
xenobiotic compounds in the natural environment and of the consequent damage in wild
organisms.

*
Corresponding Author E-mail: maurilio_lara@yahoo.com.mx; maurlara@uacam-mx.

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74 Maurilio Lara-Flores, Yassir Edén Torres-Rojas and Jaime Rendón von Osten

Keywords: endocrine gene expression, estrogenic disruptor, toxicity; biomarkers

INTRODUCTION
An endocrine-disrupting compound (EDC) has been defined by the U.S. Environmental
Protection Agency (EPA) as “an agent that interferes with the synthesis, secretion, transport,
binding, or elimination of natural hormones in the body that are responsible for the
maintenance of homeostasis, reproduction, development and/or behavior” (Kavlock et al.,
1996). This means that endocrine disruptors are chemicals, or chemical mixtures that interfere
with normal hormone function.
The endocrine disruptors are known to mainly alter the hormonal and homeostatic
systems of living system. These systems are of primary importance since they are involved in
the regulation of several significant processes such as metabolism, sexual development,
insulin production and utilization, growth, stress response, gender behavior, reproduction and
even in fetal development (Kabir et al., 2015).
Different studies demostrate that endocrine disruptors are present in the air, water and
even in the soil (Kabir et al., 2015). The evidences indicate that endocrine disruptors are
responsible for different wildlife crisis. However, wildlife is not exposed to individual
contaminants, but to a complex mixture of EDCs. The exposure route of wildlife to EDCs is
also very critical, because many of those compounds do not persist in the environment and
organism. On the other hand, a large number of chemicals persist in the food and habitat of
wildlife. Although it largely depends on the properties and persistent nature of EDCs, wildlife
is normally exposed to them via air, water, food, soil and sediment, and even via skin
absorption (Kidd et al., 2012). The EDCs are degraded in the environment, for example by
sunlight, bacterial activity and chemical processes, while others persist in the environment for
different periods of time. Some EDCs are found to be highly soluble in water and might be
present in water from the level of parts per trillion to parts per billion.
The EDCs such as organochlorines and pesticides, as well as plasticizers, pharmaceutics,
and natural hormones can interact with various receptors, such as the estrogen receptor (ER),
vitellogenin (VTG), androgen receptor, and aryl hydrocarbon receptor (Iguchi et al., 2002).
Clarifying the molecular basis of the EDCs and the endogenous estrogens on developing
organisms is essential, if we are to understand the linkages between exposure levels, timing of
exposure, genes responsive to these chemicals, and adverse effects (Iguchi et al., 2007).
Various modes of action of chemicals and non-traditional targets of the EDCs have been
summarized (Tarrant, 2005). Understanding the effects of the EDCs on various species at the
level of molecular biology is greatly needed to help explain observations at the cellular and
organism levels that typically are obtained with traditional toxicological approaches.
The sub-discipline combining the fields of genomics and toxicology is defined as
“Toxicogenomics” (Inoue and Pennie, 2002), and has become an important field in
ecotoxicology. “Ecotoxicogenomics” is the application of toxicogenomics to organisms that
are representative of ecosystems and is used to study the hazardous effects of chemicals on
ecosystems as well as individuals (Miracle and Ankley, 2005). Although the availability of
genomic information regarding non-model organisms is still limited, the application of

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 Endocrine Gene Expression in Aquatic Animals … 75

toxicogenomics to a variety of organisms could be a powerful tool for evaluating the effects
of chemicals on ecosystems (Watanabe and Iguchi, 2006).
Profiling of transcripts, proteins, and metabolites can help discriminate classes of the
EDCs and toxicants and be helpful in understanding modes of action. Through systematic
efforts to generate mechanistic information, diagnostic and predictive assessments of the risk
of the EDCs and other toxic chemicals will be established in model species for ecological risk
assessment. Toxicogenomics and ecotoxicogenomics also provide important information on
the basic biology of animals (Iguchi et al., 2007).
DNA microarray methodology has been applied to obtain genome-wide analysis of gene
expression stimulated by hormones and/or chemicals (Watanabe and Iguchi, 2003). An
understanding of the patterns of expression of estrogen-responsive genes is essential to
comprehend the mechanisms of action of estrogenic chemicals on non-target organs (Iguchi
et al., 2007).
For the validation of the gene expression as a biomarker of the xenobiotic compounds we
performed various experiments with different animal species and applied these biomarkers to
environmental samples in wildlife.

METHODS
First Experiment: Expression of Estrogenic Response Genes in Male
Mosquito Fish (Gambusia yucatana)

In this study we evaluated the expression of genes involved in vitellogenin production by

analyzing mRNA in adult male mosquito fish (G. yucatana) exposed to different
concentrations of 17ß-estradiol.
The mosquito fish were collected from a small and non-contaminated pond in a Ria
Creek in Campeche, Mexico, and were maintained and cultivated in an artificial pond for
three months. The organisms were fed ad libitum, three times a day with a commercial fish
food (TretaMin, Tetra Holding, USA). The fish were acclimated for 1 week in 20 L glass
aquarium containing 18 L of aerated and dechlorinated water according to Rendón von Osten
et al., (2005). The stocking rate for the in vivo study was 3 fish/aquarium. The fish were not
fed 24 h prior to the experiments and the food was provided during the test before the
medium was renewed.
A total of 15 male fish were exposed to 17ß-estradiol at nominal concentrations of 0.5, 1,
10, and 15 µg L-1 with three replicates per treatment. The test solutions were prepared by
dissolving the appropriate amount of 17ß-estradiol stock solutions in ASTM hard water. Two
controls were included in the experimental design with male fish, one with ASTM hard water
and with ethanol (solvent control) and the other control was performed with female fish
without treatment. The bioassays were carried out for 14 days in a semi-static test design
according to chronic exposure criteria by Orlando et al., (2002). After the experiments, all the
organisms were sampled for analysis of the gene expression by Q-PCR method.
The total RNA was isolated from liver and gonad tissue of male fish according to the kit
instructions (Ultra Clean Tissue and Cells RNA Isolation, Mobio INC.). The RNA was
diluted at 1 mg ml-1 for RT-PCR (Reverse Transcriptase-Polimerase Chain Reaction) and

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76 Maurilio Lara-Flores, Yassir Edén Torres-Rojas and Jaime Rendón von Osten

stored at -80ºC. The RT-PCR was performed with the total RNA extracted from the mosquito
fish livers and gonads. The RT reaction mixture contained 5 µg of total RNA, 1 µl of oligo
(dT) primer and diethyl pyrocarbonate (DEPC)-treated water. The reaction mixture was
heated to 70ºC for 10 min and quickly chilled on ice. After cooling, 4 µl of reaction buffer
containing 25 mM MgCl2, 2 µl of deoxynucleotide triphosphate (dNTPs; 10 mM each), 1 µl
of RNase inhibitor, and 1 µl of ReverTraAce (BIORAD) was added to a total volume of 20
µl, and the reaction mixture was incubated for 60 min at 42 ºC. The reaction mixture was then
heated to 90ºC for 5 min to stop the RT.
The PCR reactions contained 2 µl of the RT reaction mixture as the cDNA template, 5 µl
of 10x PCR buffer, 1 µl of Taq polymerase (5 U µl-1, BIORAD), 3 µl of 25 mM MgCl2, 1 µl
of dNTPs (10 mM each) and 1.5 µl of sense and antisense primers. The primer pair sequences
for the quantification of the genes involves in vitellogenin production are shown in Table 1.
The total volume of the reaction mixture was 50 µl. The PCR conditions were as follows:
initial denaturation at 94ºC for 2 min, 45 cycles of denaturation at 94ºC for 30 s, annealing at
60ºC for 30 s, and extension at 72ºC for 1 min, and final extension at 72ºC for 10 min. The
PCR products were analyzed by electrophoresis on 1.5% agarose gels. The gels were stained
with ethidium bromide (15 µl of a 10 mg ml-1 ethidium bromide solution per 100 ml of
water). The band densities of amplified products were calculated using Quantity One
Software (BIORAD).

Second Experiment: Expression of Estrogenic Response Genes in Japanese

Pond Turtle (Mauremys japonica)

In this study we evaluated the expression of genes involved in the vitellogenin production
by analyzing the mRNA in adult male Japanese pond turtle (Mauremys japonica).
The Japanese pond turtles were acquired in a commercial aquarium and were maintained
and cultivated three weeks in 20 L glass aquarium containing 3 cm of depth of dechlorinated
water. The organisms were fed ad libitum, three times a day with a commercial reptile food
(Reptile sticks, Wardley INC). The stocking rate for the in vivo study was 1 turtle/aquarium.
The turtles were not fed during the experiment.
A total of 6 turtles were exposed to 1 mg L-1 of a sample of a hydrocarbon leaked from
the Usumacinta oil ring as source of petrogenic hydrocarbon; and 3 turtles were kept as
controls. All the turtles were stocked in ASTM hard water. The bioassays were carried out for
36 h in a static test design according to chronic exposure criteria by Orlando et al., (2002).
Every 12 h, two turtles (except those from the control, which were sacrificed at 36 h) were
sacrificed to remove the gonads.
The total RNA was isolated from the gonad tissue of all the tested turtles according to the
kit instructions (GeneJET RNA Purification, Thermo Fisher Scientific). The RNA was diluted
at about 1 mg ml-1 for RT-PCR. The RT-PCR was performed with the total RNA extracted
from the turtle gonads. The RT reaction was performed according to the kit instructions
(TaqMan Reverse Transcription Reagent, Thermo Fisher Scientific).
For the quantification of the gene expression, a Q-PCR was performed in a StepONE RT-
PCR equipment (Applied Biosystem) using a Maxima SYBRGreen/ROX QPCR Master Mix
(Thermo Fisher Scientific). The primer pair sequences are shown in Table 1. The PCR
conditions were as follows: initial denaturation at 94ºC for 2 min, 45 cycles of denaturation at

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 Endocrine Gene Expression in Aquatic Animals … 77

94ºC for 30s, annealing 60ºC for 30 s, and extension at 72ºC for 1 min. Quantification was
performed by using the ß-actin gene as reference and applying the2ΔΔ𝐶𝑇 method (Kenneth et
al., 2001).

Third Experiment: Expression of Estrogenic Response Genes in Black

Mollies (Poecilia sphenops)

In this study we evaluated the expression of the genes involved in the vitellogenin
production by analyzing the mRNA in adult male black mollies (Poecilia sphenops).
The black mollies were acquired in a commercial aquarium and maintained and
cultivated for three weeks in 20 L glass aquarium containing 18 L of aerated and
dechlorinated water according to Rendón von Osten et al., (2005). The organisms were fed ad
libitum, three times a day with a commercial fish food (TretaMin, by Tetra Holding, USA).
The stocking rate for the in vivo study was 3 fish/aquarium. Fish were not fed during the
experiment.
A total of 12 fish were exposed to 1 mg L-1 of burned grass as a source of pyrogenic
hydrocarbon; another group of 12 fish was exposed to 1 mg L-1 of a sample of a hydrocarbon
leaked from the Usumacinta oil ring as a source of petrogenic hydrocarbon; and 12 fish were
kept as controls. All the fish were stocked in ASTM hard water. The bioassays were carried
out for 36 h in a static test design according to chronic exposure criteria by Orlando et al.,
(2002). Every 12 h, four fish from each treatment were sacrificed (except those from the
control, which were sacrificed at 36 h) to remove the gonads.

Table 1. Sequences of primer pairs used in the RT-PCR study

(Ishibashi et al., 2008)

Gene name Primer sequences

ER (AB033491) 5´-GTCAGTCGGGTTACTTGGCC-3´
5´-CATCACCTTGTCCCAACCTG-3´
ERß (AB070901) 5´-GTGGACTCAACTTTCGGC-3´
5´CACGTCGCAGCAGGATCTT-3´
VTG I (AB064320) 5´-TGGAAAGGCTGATGGGGAAG-3´
5´-AACTGCAGGCATGGTGAGCC-3´
VTG II (AB074891) 5´-GTCYYCAGGAGGTCTTCTTC-3´
5´-GGTAGACAATGGTATCCGAC-3´
ß-Actin (S74868) 5´-AGACCACCTACAGCATC-3´
5´-TCTCCTTCTGCATTCTGTCT-3´

The total RNA was isolated from the gonad tissue of tested fish according to the kit
instructions (GeneJET RNA Purification, Thermo Fisher Scientific). The RNA was diluted at
about 1 mg ml-1 for RT-PCR. The RT-PCR was performed with total the RNA extracted from
black mollies gonads. The RT reaction was performed according to the kit instructions
(TaqMan Reverse Transcription Reagent, Thermo Fisher Scientific).
For the quantification of the gene expression a Q-PCR was performed in a StepONE RT-
PCR equipment (Applied Biosystem) using a Maxima SYBRGreen/ROX QPCR Master Mix

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78 Maurilio Lara-Flores, Yassir Edén Torres-Rojas and Jaime Rendón von Osten

(Thermo Fisher Scientific). The primer pair sequences are shown above in Table 1. The PCR
conditions were as follows: initial denaturation at 94ºC for 2 min, 45 cycles of denaturation at
94ºC for 30s, annealing at 60ºC for 30 s, and extension at 72ºC for 1 min. The quantification
was performed by using the ß-actin gene as reference and applying the 2ΔΔ𝐶𝑇 method
(Kenneth et al., 2001).

Fourth Experiment: Presence of Expression of Estrogenic Genes in Wild

Atlantic Angel Shark (Squatina dumeril)

In this study we evaluated the the expression of genes involved in vitellogenin production
by analyzing the mRNA in adult Atlantic angel shark (Squatina dumeril) for the application
of these biomarkers in wildlife.
The samples were taken from the catch of fisherman from San Francisco de Campeche
city, Campeche, México. Ten grams of the liver were sampled from 4 sharks (3 males and 1
female) and the samples were transported on ice to the laboratory and frozen at -80ºC until
further analysis.
The total RNA was isolated from the shark liver tissue according to the kit instructions
(Gene Jet RNA Purification Kit, Thermo Fisher Scientific). The RNA was diluted at about 1
mg ml-1 for RT-PCR. The RT-PCR was performed with the total RNA extracted from shark
gonads. The RT reaction was performed according to the kit instructions (TaqMan Reverse
Transcription Reagent, Thermo Fisher Scientific).
For the quantification of the gene expression a Q-PCR was performed in a Step One RT-
PCR equipment (Applied Byosystem) using a Maxima SYBRGreen/ROX QPCR Master Mix
(Thermi Fisher Scientific). The Primer cycles of denaturation were at 94ºC for 30 s, annealing
60ºC for 30s, and extension at 72ºC for 1 min. Quantification was performed by using the ß-
actin gene as reference and applying the 2ΔΔ𝐶𝑇 method (Kenneth et al., 2001).

RESULTS
First Experiment

The results of the expression of the estrogen receptor transcripts in the liver and gonads
are presented in the Figure 1. The expression of the gene in the liver was greater than in the
gonads for all of the samples, with the exception of the negative control where it was greater
in the gonads. The females exhibited the highest expression in the liver, significantly different
from the other samples. However, in the gonads there were no differences relative to the
negative control and the concentration of 10 µg L-1 17ß-estradiol; this concentration caused
the highest gene expression in both types of tissues when compared to the other tested
concentrations.
The strongest expression of the vitellogenin gene was observed in the treatment with 10
µg L-1 17ß-estradiol, which was significantly different from the other treatments and the
controls (Figure 2). The females had the greatest expression of this gene with the exception of
the treatment with 1 µg L-1 17ß-estradiol (both types of tissue) and the treatment with 0.55 µg
L-1 17ß-estradiol for liver.

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 Endocrine Gene Expression in Aquatic Animals … 79

Second Experiment

The expression of the transcripts showed a linear increment over time, with significantly
higher expression of the VTG I than the females at 24 and 36 h (Figure 3). In the case of the
VTG II the increment is linear but lower than the females (Figure 3). For The estrogen  and
ß the increment was linear and presented higher values than the females only at 36 h (Figure
4).

Figure 1. Mean and standard deviation of the relative intensity of vitellogenin receptor transcript in the
liver and gonads of mosquito fish exposed to 17ß-estradiol. Values with the same superscript are not
statistically different (p > 0.05).

Figure 2. Mean and standard deviation of the relative intensity of estrogen receptor transcript in the
liver and gonads of mosquito fish exposed to 17ß-estradiol. Values with the same superscript are not
statistically different (p > 0.05).

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80 Maurilio Lara-Flores, Yassir Edén Torres-Rojas and Jaime Rendón von Osten

Figure 3. Mean and standard deviation of the relative intensity of Vitellogenin I (Vtg I) and
Vitellogenin II (Vtg II) receptor transcript in the gonads of Japanese pond turtle exposed to petrogenic
hydrocarbon for 24, 48 and 96 h. Values with the same superscript are not statistically different
(p > 0.05).

Figure 4. Mean and standard deviation of the relative intensity of estrogen alpha and estrogen beta
receptor transcript in the gonads of Japanese pond turtle exposed to petrogenic hydrocarbon for 24,
48 and 96 h. Values with the same superscript are not statistically different (p > 0.05).

Third Experiment

The expression of the VTG I gene in black molly in response to petrogenic hydrocarbon
is shown in Figure 5. At 24 h the VTG I expression was maximum, decreasing after 36 h. The

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 Endocrine Gene Expression in Aquatic Animals … 81

maximum expression of the VTG II was observed at 36 h of exposure to the pyrogenic

hydrocarbon; on the other hand, with petrogenic hydrocarbon the maximum expression was at
12 h with a linear decrease over time (Figure 6).

Figure 5. Mean and standard deviation of the relative intensity of vitellogenin I receptor transcript in the
gonads of black mollies fish exposed to Petrogenic (Pet) and Pyrogenic (Pyr) hydrocarbon for 12, 24
and 36 h. Values with the same superscript are not statistically different (p > 0.05).

Figure 6. Mean and standard deviation of the relative intensity of vitellogenin II receptor transcript in
the gonads of black mollies fish exposed to Petrogenic (Pet) and Pyrogenic (Pyr) hydrocarbon.
Values with the same superscript are not statistically different (p > 0.05).

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82 Maurilio Lara-Flores, Yassir Edén Torres-Rojas and Jaime Rendón von Osten

For the estrogen receptor  gene, an increase over time with maximum values at 36 h in
both hydrocarbon sources was observed; the highest expression was observed at 36 h
exposure to hydrocarbon source (Figure 7). Only after 24 h an increase was observed in the
expression of estrogen receptor ß gene in organisms exposed to petrogenic hydrocarbons,
without expression in other samples (Figure 8).

Figure 7. Mean and standard deviation of the relative intensity of estrogen receptor alpha transcript in
the gonads of black mollies fish exposed to petrogenic and pyrogenic hydrocarbon. Values with the
same superscript are not statistically different (p > 0.05).

Figure 8. Mean and standard deviation of the relative intensity of estrogen receptor beta transcript in the
gonads of black mollies fish exposed to Petrogenic (Pet) and Pyrogenic (Pyr) hydrocarbon. Values with
the same superscript are not statistically different (p > 0.05).

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 Endocrine Gene Expression in Aquatic Animals … 83

Fourth Experiment

In the Atlantic angel shark male #2 presented the highest expression in the VTG I and
estrogen receptor . No expression of the other genes (VTG II and estrogen receptor ß) was
observed in the males.

DISCUSSION
The steroid hormones that circulate in the plasma of organisms are essential to the
reproduction and sexual differentiation. The estrogenic hormones increasingly discarded in
the environment are contaminants and detecting them requires efficient, sensitive methods,
such as the use of molecular biomarkers. The species used as indicators in the first study was
the mosquito fish (G. yucatana), a poecilid that has been used to evaluate toxicity biomarkers
such as acetylcholinesterase (AChE) and glutathione S-transferase (GST) (Rendón von Osten
et al., 2005), along with several response parameters such as vitellogenin concentrations,
sperm count and gonodopodial indices. Exposure to 17ß-estradiol stimulated the induction of
vitellogenin and the expression of the respective genes, allowing us to conclude that this
compound causes changes in the endocrine system of the fish similar to those reported by
Lori et al. (2008) for Nile tilapia (Oreochromis niloticus) exposed to 17ß-estradiol
concentrations of 5 µg L1. In the study of Jin et al. (2008) the vitellogenin genes were
expressed at 17ß-estradiol concentrations of 10 to 1000 ng L-1 in the zebra fish (Danio rerio)
liver, with significant differences starting at 50 ng L-1 and the greatest expression at 1000 ng
L-1. In the present study the concentration of 15000 ng L-1 for the male mosquito fish was
comparable to the expression observed for the females. The family of estrogen receptors can
be induced by xenoestrogens and produce different responses: phosphorylation of signals
regulated by kinases, abnormal cell growth and the increased expression of the genes (Albino
et al., 2009). Hawkins et al., (2000) mentioned that the estrogen receptors participate in the
regulation of vitellogenesis and have been isolated in teleost fish; these receptors have been
proposed to be sensitive to variations in estrogen levels. The present study revealed that the
estrogen receptor  was expressed to differing degrees at all of the concentrations of 17ß-
estradiol used, which is in agreement with the results of Yamaguchi et al., (2005); they
observed gene expression at concentrations between 10 and 10 000 ng L-1. In contrast, Choi et
al., (2007) studied the effects of the induction by 17ß-estradiol on estrogen receptors  and ß
in male olive flounder (Paralichthys olivaceus), and observed that gene expression was dose
dependent. A decrease in the intensity of the gene expression was observed when the fish
were exposed to a concentration of 1 000 ng L-1 of 17ß-estradiol. In this regard, Palstra et al.,
(2010) report that when the organisms make an effort and are under stress there is a decrease
in the levels of estrogen gene expression as a consequence of directing energy to their
muscles as a strategy to allow them to flee quickly and/or prevent muscular atrophy.
The induction of the Vtg in oviparous species is used as a standard biomarker of exposure
to estrogenic compounds (Cheek et al., 2001; Irwing et al., 2001; Rey et al., 2006). Many
environmental contaminants are considered to be xenoestrogens since they show an
estrogenic response in different in vivo and/or in vitro assays (Nakada et al., 2004). Little
information exists concerning hormonal profiles in turtles. The synthesis of estrogens in

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84 Maurilio Lara-Flores, Yassir Edén Torres-Rojas and Jaime Rendón von Osten

organs other than ovaries (adrenal and hepatic tissue) has been observed in various reptilian
species, including the turtles (Zaccaroni et al., 2010). Therefore, small amounts of the
estrogens was produced by these organs might trigger VTG synthesis (Pieau and Dorizzi,
2004). Short-term production of the estrogens as a result of pre-activation or preparation of
the gonads rather than ovarian differentiation could also occur. The possible effect of the
endocrine disrupters (organochlorine compounds, heavy metals, tributyltin) on the VTG
synthesis should also be considered. In this study we observed an activation of the VTG gene
over time during exposure to petrogenic hydrocarbon; these results confirm that the turtles
could be used as bioindicators.
The hepatic VTG is a sensitive biomarker for the estrogenic effect of hydrocarbons. The
VTG is synthesized in response to stimulation by estrogenic chemicals that bind to the ERs to
activate the VTG transcription. When the estrogens enter into the liver, they bind to the ERs,
forming ER complexes that then bind to the estrogen response elements of the target genes to
regulate their expression (Watanabe et al., 2009). A recent in vitro study with primary
cultured zebrafish hepatocytes suggested that the hydrocarbons have a common estrogenic
effect but may interact differently with the ER isoforms, perhaps contributing directly to
differential the ER and the VTG gene transcription (Maradonna et al., 2013). Several studies
have reported the induction of the VTG synthesis or transcription in the fish or in cultured
male fish hepatocytes upon exposure to hydrocarbons (Carnevali et al., 2010; Uren-Webster
et al., 2010; Maradonna et al., 2013). Although the induction of the VTG could depend on
species sensitivity, exposure time, and concentrations, these studies demonstrate the
estrogenic activity of hydrocarbons (Wang et al., 2013). In our study the expression of the
vitellogenin-related genes in the black molly fish was observed only when exposure was to
petrogenic hydrocarbon and not to pyrogenic hydrocarbon, probably due to the nature of the
compounds.
There are no studies that demonstrate the effect of pollutants on the expression of the
genes of endocrine disruption in sharks. These results show an important potential use of the
biomarkers in different aquatic species of commercial interest, such as the shark, to determine
the effects of pollutants. The high levels of expression in one of the tested organisms indicate
that it had contact with some kind of pollutant. This can be confirmed by reproductive age,
eating habits and proximity to the coast where the organism was captured. These findings are
being confirmed by the stomach contents and concentrations of pollutants in the organisms.

ACKNOWLEDGMENTS
We gratefully acknowledge all the students that participated in these studies: Magaly
Guillen, Nidia Cruz, Erick Ramírez, Joana Colunga, Sheila Cahuich, Pilar Marquez,
Monserrat Rodríguez, Olga Loria, Braidie Vargas, Alicia Torres, and Karina Sonda.

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 Endocrine Gene Expression in Aquatic Animals … 85

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 7

SENSITIVITY OF TROPICAL CLADOCERANS

AND CHIRONOMIDS TO TOXICANTS AND THEIR
POTENTIAL FOR ROUTINE USE IN TOXICITY TESTS

Arnola C. Rietzler1,, Odete Rocha2, Ana L. Fonseca3,

Marcela M. Ribeiro1 and Mariana F. Matos1
1
Laboratory of Ecotoxicology, General Biology Department, Federal University of Minas
Gerais, Minas Gerais, Brazil
2
Department of Ecology and Evolutionary Biology, Federal University of São Carlos,
São Paulo, Brazil
3
Institute of Natural Resource, Federal University of Itajubá, Minas Gerais, Brazil

ABSTRACT
We conducted toxicity tests with three reference toxicants, following NBR-12713
procedures (2009), with an aim to establish Daphnia laevis and Ceriodaphnia cornuta as
routine toxicity test species by the Brazilian Association of Technical Procedures. To
evaluate the sensitivity of these native cladocerans, standardized species were also tested,
namely D. similis and C. dubia from temperate regions as well as the neotropical C.
silvestrii. EC50 mean values for D. laevis were 0.025 mg L-1 for potassium dichromate,
1.06 g L-1 for sodium chloride and 5.07 mg L-1 for sodium dodecyl sulfate. Mean values
of EC50s for D. similis corresponded to 1.63 g L-1 (sodium chloride) and 5.64 mg L-1
(sodium dodecyl sulfate). Ceriodaphnia spp. EC50 mean values for sodium chloride and
sodium dodecyl sulfate were, respectively: 1.04 g L-1 and 3.27 mg L-1 (C. cornuta); 1.24
g L-1 and 4.42 mg L-1 (C. silvestrii); 1.43 g L-1 and 4.12 mg L-1 (C. dubia). EC50 values
for potassium dichromate and sodium chloride showed higher sensitivity of D. laevis than
D. similis, already used in Brazil. C. cornuta was also more sensitive to sodium chloride
than C. dubia and C. silvestrii. These results, beside high fecundity and easy laboratory
maintenance, support their use in toxicity tests. Regarding sediment toxicity, Chironomus
xanthus 96h-LC50 for instars II, III and IV exposed to potassium chloride were 3.99, 4.50


Corresponding Author E-mail: acrietzler@gmail.com.

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88 Arnola C. Rietzler, Odete Rocha, Ana L. Fonseca et al.

and 4.91, and 4.64 g L-1, respectively. The results obtained for this tropical chironomid
showed a similar sensitivity to their temperate zone counterparts.

Keywords: tropical aquatic invertebrates, sensitivity tests, potential test species

INTRODUCTION
An important aspect in ecotoxicological studies is the choice of test species. In order to
provide precise and reproducible results, a good background knowledge on the biology of the
test species is required as well as sensitivity to a wide range of toxicants; wide geographic
distribution and representativeness of its ecosystem; easy and cost-effective laboratory
culturing; small size; short life cycle; high reproduction rates and genetic stability (Rand,
1995; Santos, 2011).
Most toxicity test procedures used in Latin America are based on protocols developed
by regulatory agencies of temperate climate countries (e.g., USEPA in United States,
Environment Canada in Canada, and OECD in Europe) and utilize species such as Daphnia
magna, D. similis, Ceriodaphnia dubia and Hyalella azteca as test organism (e.g., Sotero-
Barbosa et al., 2007; Rodgher and Espíndola, 2008; Botelho et al., 2013; Maranho et al.,
2014). However, such protocols are inadequate to evaluate the effects of toxicants in tropical
and sub-tropical countries (Daam and Van Den Brink, 2010). They demand physical and
chemical adjustments since their specifications differ from those of tropical environments and
are not necessarily appropriate to their optimum conditions. As an example, D. magna,
cultured in hard water, cannot precisely evaluate impacts on Brazilian environments, mostly
represented by soft waters (Baptista et al., 2000).
Furthermore, the use of exotic species, confined to the laboratory, should be avoided
whenever possible, since their escape to the wild poses a risk of them becoming invasive and
hazardous to native species (Santos et al., 2007; Fracácio et al., 2009; Braidotti, 2014; Freitas
and Rocha, 2014). Nevertheless, the knowledge of the sensitivity of tropical and subtropical
species is relatively small compared to temperate regions. Therefore, studies with tropical
species from different habits and origins remain necessary in order to establish an adequate
database for ecotoxicological studies (Kwok et al., 2007; Daam and Van den Brink, 2010).
On the other hand, ecotoxicological studies conducted in tropical aquatic environments
have shown the potential of native species as test organisms. Trenfield et al. (2012) tested
Hydra viridissima and Moinodaphnia macleayi to evaluate aluminum toxicity in two
Australian aquatic systems, both demonstrating consistent results related to sensitivity. The
same was found for Brazilian water bodies nearby industries, where the native Pseudosida
ramosa showed higher sensitivity to metals Cr and Cd than D. magna, besides similar
sensitivity to D. similis (Freitas and Rocha, 2011a).
Other tropical species, including C. rigaudi, M. macleayi and Diaphanosoma brachyurum
have also shown sensitivity to salinity (Mohammed and Agard, 2007). The potential of C.
rigaudi for ecotoxicological studies was tested in Central America (Trinidad), including life
cycle data, salinity tolerance and sensitivity to toxicants. Results contrasted to those of D.
magna indicated that the use of C. rigaudi is recommended (Mohammed, 2009).

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 Sensitivity of Tropical Cladocerans and Chironomids … 89

Moreover, C. silvestrii, a Brazilian daphnid (ABNT, 2010) has been used for evaluating
the quality of water (Takenaka et al., 2006; Kuhl et al., 2010) and for eutrophic sediment
remediation purposes (Janke et al., 2011). It has also demonstrated to be sensitive to
pesticides (Moreira et al., 2014; Casali-Pereira et al., 2015) and metals (Rodgher and
Espíndola, 2008; Santos et al., 2008).
Although sediment evaluation is still not a basic requirement of Brazilian environmental
agencies, ecotoxicological studies have been carried out, most of which utilized non-native
species and based on international protocols, including bacteria (Vibrio fisheri) and
invertebrates such as amphipods (H. azteca), cladocerans (D. magna; D. similis; C. dubia)
and oligochaetes (Branchiura sowerbi; Tubifex tubifex).
According to Böhrer-Morel et al. (2005), D. laevis, C. silvestrii and Chironomus xanthus
are the autochthonous species whose biology has been studied and which have been
frequently used as test organisms in Brazil. These authors have pointed out that despite this,
routine actions still necessary in many laboratory tests, include sensitivity evaluations and
establishment of control-charts.

Figure 1. Daphnia laevis (a), Ceriodaphnia cornuta (b) and C. silvestrii (c).

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90 Arnola C. Rietzler, Odete Rocha, Ana L. Fonseca et al.

In this context, the present chapter investigated the potential of tropical species as test
organisms, aiming at their routine use in water and sediment toxicity tests using standardized
toxicity-test protocols from the Brazilian Association of Technical Procedures (ABNT). Two
cladoceran species were considered: D. laevis, which is one of the three Daphnia species
found in Brazil (Matsumura-Tundisi, 1984; Rocha and Matsumura-Tundisi, 1990), endemic
to the American continent; and C. cornuta, another daphnid widely distributed in tropical and
subtropical regions. Both species are among the most abundant in tropical freshwaters (Do
Hong et al., 2004; Brandão et al., 2012).

Regarding sediment toxicity studies, the tropical endemic benthic macroinvertebrate C.

xanthus (Diptera, Chironomidae) has high survival rates, short life cycle and easy culturing in
laboratory. These features make it adequate as test organism for sediment toxicity tests
(Almeida, 2002; Fonseca and Rocha, 2004).

METHODS
Zooplankton Culturing and Sensitivity Tests

Tropical cladocerans considered in the present chapter (Figure 1) were cultured in natural
non-reconstituted water with neutral pH, conductivity of 130 to 150 µS cm-1 and hardness
of 40 to 44 mg L-1 CaCO3. The temperate species (D. similis and C. dubia) were included
for comparison. The cultures were fed 106 cells per mL of Raphidocelis subcapitata
and a compound of fermented fish ration (TetraMin/Tetra) and yeast (Saccharomyces
cerevisiae/Fleischmann) every two days.
Sensitivity tests (24h-EC50* and 48h-EC50) were conducted with the following
reference toxicants: potassium dichromate (K2Cr2O7*), sodium chloride (NaCl) and sodium
dodecyl sulfate (C12H25NaO4S). The methodology followed ABNT procedures (2009). In
each treatment 5 to 10 organisms were added to 10-20 mL of tested solution in 3 to 4
replicates. Concentrations of toxicants for Daphnia spp. varied from 0.55 to 2.3 g L-1 (NaCl);
1.5 to 7.0 mg L-1 (C12H25NaO4S) and 0.01 to 0.05 mg L-1 (K2Cr2O7). In the case of
Ceriodaphnia spp., their concentrations ranged from 0.6 to 1.9 g L-1 (NaCl) and 1.0 to 7.0 mg
L-1 (C12H25NaO4S). Controls consisted of laboratory culturing water.

Chironomus xanthus Culturing and Sensitivity Tests

The tropical chironomid considered was cultured in natural non reconstituted water with
neutral pH, conductivity of 130 to 150 µS cm-1 and hardness of 40 to 44 mg L-1 CaCO3.
Culturing was initiated by transferring ca. 100 larvae to plastic trays. The trays contained a 3
cm thick layer of sterilized sand and 4 L of maintenance water. Larvae were fed 105 cells per
mL of R. subcapitata on the first day plus 0.04 mg mL-1 volatile suspended solids/day fish
ration throughout the development of the four larval instars. The system was maintained
under continuous aeration at 24 ± 1C and photoperiod of 12 h. The trays also consisted of a

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 Sensitivity of Tropical Cladocerans and Chironomids … 91

screened wooden support covered by tulle (±100 µm) for adult retention (Fonseca and Rocha,
2004).
C. xanthus larvae sensitivity to potassium chloride (KCl) was tested following USEPA
(2000) procedures. Five KCl concentrations were used with individual addition of 10
organisms (from instars I to IV) to 20 ml of test solution. Controls used laboratory culturing
water. 96h-LC50 values were estimated by Trimmed Spearman-Karber Program based on
Hamilton et al. (1977). Statistical analysis applied to all sensitivity tests included T-test, one-
way ANOVA and Tukey test, using PAST software package (version 2.17c) for data analysis
(Hammer et al., 2001).

RESULTS
Cladoceran Sensitivity Tests

24h-EC50 mean (n=15) value for D. laevis was 0.025 ± 0.009 mg L-1 for potassium
dichromate. 48h-EC50 mean values for D. laevis using sodium chloride and sodium dodecyl
sulfate (SDS) were 1.06 ± 0.18 g L-1 (n=11) and 5.07 ± 0.77 mg L-1 (n=6), respectively (Table
1). These results showed the higher sensitivity of D. laevis to potassium dichromate (t=-
11.043; p=0.000) and sodium chloride (t=-7.192; p=0.000) compared to D. similis, although
similar sensitivity to sodium dodecyl sulfate (t=-1.028; p=0.323). Coefficients of variation
ranged from 15 to 35%, mostly below 30%, expressing adequate results for test validation
(Environment Canada, 1990). Sensitivity control charts for D. laevis were also included
(Figure 2).
For Ceriodaphnia spp., 48h-EC50 mean values using sodium chloride corresponded to
1.04 ± 0.20 g L-1 for C. cornuta (n=13), 1.24 ± 0.27 g L-1 for C. silvestrii (n=23) and 1.43 ±
0.23 g L-1 for C. dubia (n=10). 48h-EC50 mean values using sodium dodecyl sulfate were
3.27 ± 0.61 mg L-1 for C. cornuta (n=10), 4.42 ± 1.46 mg L-1 for C. silvestrii (n=9) and 4.12 ±
0.97 mg L-1 for C. dubia (n=6). Coefficients of variation ranged from 16 to 33%, in general
within ranges expected (0 to 30%; Environment Canada, 1990). Sensitivity control charts for
C. cornuta were also included to complement the results (Figure 3).

Table 1. Mean values of 24 and 48h-EC50 for Daphnia laevis and D. similis using sodium
chloride (g L-1), sodium dodecyl sulfate (mg L-1) and potassium dichromate (mg L-1)
as reference substances

Values Sodium chloride Sodium dodecyl sulfate Potassium dichromate

D. laevis D. similis D. laevis D. similis D. laevis* D. similis**
Average 1.06 1.63 5.07 5.64 0.025 0.138
Standard 0.18 0.23 0.77 1.20 0.009 0.027
deviation
Coefficient of 17% 14% 15% 21% 35% 20%
variation
Sensitivity range 0.70-1.42 1.16-2.09 3.53-6.61 3.25-8.04 0.007-0.043 0.084-0.192
*
24h-EC50; ** Source: Zagatto (1988).

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92 Arnola C. Rietzler, Odete Rocha, Ana L. Fonseca et al.

Figure 2. Mean values of 48h-EC50 ± 2 standard deviations (Sensitivity control charts) for Daphnia
laevis exposed to potassium dichromate, sodium chloride and sodium dodecyl sulfate (LL=Lower
Limit; UL=Upper Limit).

Table 2. Mean values of 48h-EC50 for Ceriodaphnia cornuta, C. silvestrii and C. dubia
using sodium chloride (g L-1) and sodium dodecyl sulfate (mg L-1)
as reference substances

Values Sodium chloride Sodium dodecyl sulfate

C. cornuta C. silvestrii C. dubia C. cornuta C. silvestrii C. dubia
Average 1.04 1.24 1.43 3.27 4.42 4.12
Standard deviation 0.20 0.27 0.23 0.61 1.46 0.97
Coefficient of variation 19% 22% 16% 19% 33% 23%
Sensitivity range 0.64-1.45 0.70-1.79 0.98-1.89 2.05-4.49 1.50-7.33 2.19-6.06

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 Sensitivity of Tropical Cladocerans and Chironomids … 93

Figure 3. Mean values of 48h-EC50 ± 2 standard deviations (sensitivity control charts) for
Ceriodaphnia cornuta exposed to sodium chloride and sodium dodecyl sulfate (LL=Lower Limit;
UL=Upper Limit).

In considering sodium chloride as reference substance, C. cornuta showed higher

sensitivity than C. silvestrii and C. dubia (F=7.214; p=0.002). No differences among these
congeners were found in the case of SDS (F=2.924; p=0.074).

Sediment Sensitivity Tests Using Benthic Organisms

Mean values of 96h-LC50 for instars II (n=10), III (n=13) and IV (n=12) of C. xanthus
(Table 3) corresponded to 3.99 ± 0.63 g L-1, 4.50 ± 0.94 g L-1 and 4.64 ± 0.68 g L-1,
respectively, showing no differences between sensitivity of instars (F=2.08; p=0.14). The
long term results for instar III larvae compared with other tests conducted with C. xanthus
larvae of the same instar aiming at an interlaboratorial calibration, also showed no difference
in their sensitivity (t=1.230; p=0.230). Sensitivity control charts for potassium chloride could
provide more information on the usual instar adopted for toxicity tests with these chironomids
(Figure 4).

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94 Arnola C. Rietzler, Odete Rocha, Ana L. Fonseca et al.

Table 3. Mean values of 96h-LC50 for Chironomus xanthus (instar III) and C. xanthus
(instars II*, III* and IV*), using potassium chloride (g L-1) as reference substance. Data
were obtained in 2015-2016 and 1996-1997 (*)

Values Instar III Instar II* Instar III* Instar IV*

Average 4.91 3.99 4.50 4.64
Standard Deviation 0.80 0.63 0.94 0.68
Coefficient of Variation 16% 16% 21% 15%

Figure 4. Mean values of 48h-EC50 ± 2 standard deviations (Sensitivity control chart) for Chironomus
xanthus, instars III 2014-2015 (a) and 1996-1997 (b) exposed to potassium chloride as reference
substance (LL=Lower Limit; UL=Upper Limit).

DISCUSSION
Tropical Cladocerans as Test Species

There is a general feeling that at higher temperatures and increased runoff from torrential
rainfall, toxicity effects in the tropics can substantially differ from those in temperate regions,

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 Sensitivity of Tropical Cladocerans and Chironomids … 95

in which pollutants toxicity, process rates, compound transformations and fate of residues are
now well understood (Daam and Van den Brink, 2010). Not only are the processes not
directly applicable, but the responses of temperate region selected test organisms might not
show the same sensitivity or be totally appropriate to show the effects of toxic pollutants
whether in laboratory or in complex natural tropical conditions (Baptista et al., 2000; Lopes et
al., 2007; Tables 1 and 2).
An intensification of ecotoxicological studies is required in developing tropical and
subtropical countries in order to cope with the exponential loss of environmental quality
(Soares and Callow, 1993).
Among tropical freshwater zooplankton, a number of species have already been tested
and recommended for ecotoxicological studies, such as M. macleayi (Semaan et al., 2001;
Orchard et al., 2002), P. ramosa (Freitas and Rocha, 2014), D. laevis (Rietzler and Viegas,
2002; Böhrer-Morel et al., 2005; Braidotti, 2014; Sales et al., 2016) and C. cornuta (Lopes et
al., 2011; Ribeiro, 2011). However, the most frequently used species has been C. silvestrii,
for both acute and chronic toxicity tests following standardized procedures recommended by
the Brazilian methodology standardization agency (ABNT, 2009, 2010).
In the present chapter, D. laevis showed higher sensitivity than D. similis for sodium
chloride and sodium dodecyl sulfate (Table 1). When testing similar substances for D.
ambigua (another native species to South America), Harmon et al. (2003) found 48h-EC50
values of 2.00 g L-1 for sodium chloride and of 44.0 mg L-1 for sodium laurel sulfate as
reference substances. A comparison of these values to our data also shows higher sensitivity
of D. laevis. In addition, EC50 mean values for potassium dichromate also showed higher
sensitivity of D. laevis than D. similis (Zagatto, 1988), a temperate species commonly utilized
in ecotoxicological studies in Brazil (Zoratto, 2007; Araújo et al., 2010; Sales et al., 2016),
thus supporting the idea that the use of D. laevis instead of D. similis in ecotoxicological
studies should be adopted.
D. magna, the temperate species most widely used as test organism (Ratte et al., 2003;
Wong et al., 2009), has lower sensitivity than other tropical cladocerans to many pesticides
(Lopes et al., 2007) when assessing its performance as a laboratory surrogate. The authors
found no lethal effects of deltamethrin to this species and sublethal effects were only
observed at the highest concentrations, being less sensitive to the feeding depression endpoint
compared to the tropical and subtropical cladoceran D. brachyurum.
Freitas and Rocha (2011a) have also observed higher sensitivity of P. ramosa than D.
magna when testing NaCl, KCl, Cr, Cd, SDS and atrazine as reference substances. According
to Moreira et al. (2014), Macrothrix flabelligera and C. silvestrii were also more sensitive to
atrazine than D. magna (12.37 ± 2.6 mg L–1, 14.30 ± 1.55 mg L–1 and 50.41 ± 2.64 mg L–1,
respectively).
In considering other daphnids, C. cornuta, a native tropical species, was more sensitive to
sodium chloride when compared with C. silvestrii, another native species, and C. dubia, their
temperate counterpart (Table 2). C. cornuta was also more sensitive to sodium dodecyl
sulfate (3.39 ± 0.50 mg L-1) than P. ramosa (10.30 ± 1.60 mg L-1) (Freitas and Rocha, 2011b).
In previous studies, C. rigaudi contrasted to two other tropical cladocerans, D. brachyurum
and M. macleayi, presented higher sensitivity to Cu, K, Cd, Cr and triton X100 (Mohammed
and Agard, 2006; Mohammed, 2009).
Among invertebrates, cladocerans are an important tool in ecotoxicological studies.
Wogram and Liess (2001), when comparing the sensitivity of different invertebrate groups,

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96 Arnola C. Rietzler, Odete Rocha, Ana L. Fonseca et al.

including arthropods and non-arthropods, concluded that Cladocera was one of the groups
most sensitive to organic compounds and was the most sensitive to metallic compounds.
Their study was based on D. magna which, in fact, is equally or less sensitive than other
daphnids, as shown in the present chapter, or than copepods (Wong et al., 2009).

Tropical Benthic Macroinvertebrates as Test Species

Regarding sediment ecotoxicological studies, most species adopted in monitoring and

risk assessment are temperate species. Little has been done to provide protocols using native
test organisms for tropical freshwaters (Marchese and Brinkhurst, 1996; Ducrot et al., 2010;
Lobo and Alves, 2011).
Among studies carried out in Brazil, Almeida (2007) has evaluated the potential of
Branchiura sowerbyi (Oligochaeta, Tubificidae) as test species and could verify a 96h-LC50
mean value to potassium chloride (0.364 g L-1) in a low sensitivity range (0.177-0.551 g L-1).
High sensitivity of B. sowerbyi was observed by Dhara et al. (2015) when testing Cd.
However, this species showed slower development and reproduction than C. xanthus.
C. xanthus, an endemic chironomid species to the tropics, has been subject of
investigation in many studies carried out in Brazilian aquatic systems (Tonissi et al., 2004;
Santos et al., 2007; Araújo, 2008; Sales, 2009; Janke et al., 2011; Silva, 2013; Alves and
Rietzler, 2015; Sueitt et al., 2015), presenting feasible features in terms of culturing (Fonseca
and Rocha, 2004) and data reproducibility (Almeida, 2002; Dornfeld et al., 2006; Table 3 of
the present chapter). The genus Chironomus is also well represented in the neotropics
(Correia, 2004).
Studies performed to determine sensitivity ranges for C. xanthus found lower 96h-EC50
values than in temperate congeners (C. crassiforceps, C. riparius, C. decorus and C. tentans)
for Cu (0.27 mg L-1, Masutti, 2004; 0.30 mg L-1, Dornfeld et al., 2006). Almeida (2007) also
verified higher sensitivity of C. xanthus to pentachlorophenol (96h-LC50 of 0.111 mg L-1;
sensitivity range of 0.082-0.141 mg L-1) compared with the values from the literature for
Callibaetis okianos, Neuroptera (1.78 mg L-1; 1.27-2.48 mg L-1) and Monhystera disjuncta,
Nematoda (2.10 mg L-1; 0.50-7.0 mg L-1). Considering potassium chloride as toxicant, our
results (Table 3) show similar sensitivity of this tropical species compared with chironomids
C. riparius and C. tentans from temperate regions (USEPA, 1994; Burton Jr et al., 1996).
Thus, C. xanthus is at least as sensitive as the non-native species.
Stoughton et al. (2008) conducted toxicity tests using insecticides (imidacloprid) to
compare the sensitivity of C. tentans and H. azteca, finding much lower EC50 values for the
chironomids (5.75 g L-1 versus 65.43 g L-1). H. meinerti, a tropical amphipod, has not yet
been sufficiently tested to compare it to H. azteca, an exotic species still often used in toxicity
tests in tropical laboratories (Silvério et al., 2005; Favaro et al., 2014).
Sublethal effects have also been important endpoints in ecotoxicological studies related
to the genus Chironomus, including morphological deformities from environmental
exposition (Bonani, 2010) and genotoxic and bioaccumulative effects of cyanobacteria toxins
(Santiago, 2012).

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 Sensitivity of Tropical Cladocerans and Chironomids … 97

CONCLUSION
Our results for D. laevis and C. cornuta, besides high fecundity, easy maintenance in the
laboratory and data reproducibility of both species, support their use as preferential test
species in water and sediment toxicity tests, in order to construct a strong data base for
toxicity evaluation and risk assessments in tropical conditions.
C. xanthus can also be a useful test species, being representative of the benthic
community and appropriate to evaluate sediment toxicity. This species should be more widely
used, together with tropical cladocerans and other tropical macroinvertebrates, for carrying
out much needed ecotoxicological studies in Brazil.

ACKNOWLEDGMENT
We thank Dr. Henri Dumont for kindly reviewing the English of the final version.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 8

ANTHROPOGENIC IMPACT MODIFIES THE BLOOD

ANTIOXIDANT STATUS IN PYGOSCELIS ADELIAE
AND PYGOSCELIS PAPUA AT HOPE BAY,
ANTARCTIC PENINSULA

Julieta D. Cebuhar1, Carla I. Di Fonzo1,

Laura P. Dopchiz1,2 and Martín Ansaldo1,2,*
1
Instituto Antártico Argentino, Buenos Aires, Argentina
2
Departamento de Biología, Universidad Argentina J. F. Kennedy,
Buenos Aires, Argentina

ABSTRACT
Antioxidant blood status was measured in Pygoscelis adeliae (Adélie penguin) and
Pygoscelis papua (Gentoo penguin) populations coming from areas with high and low
anthropogenic impact at Hope Bay, Antarctic Peninsula. Superoxide dismutase (SOD),
catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase (GPx)
enzyme activities, as well as the levels of reduced glutathione (GSH), uric acid (UA),
protein oxidation (PO) and lipid peroxidation (LPO) were measured. The SOD and CAT
activities showed significant differences for areas and species. Adélie and Gentoo
penguins from the high impact area exhibited an increase in GST activity. Only Gentoo
penguins, that inhabit the high impact area, registered an increase in GPx activity. The
GSH levels did not show significant differences, suggesting that these penguins would
not use GSH per se as primary hydrosoluble antioxidant response. However, UA levels
presented differences among the analyzed factors. The oxidative damage levels registered
in erythrocytes (POe and LPOe) showed similar patterns for both species. Adélie penguins
from the high impact area and Gentoo penguins from the low impact area had the highest
levels of oxidative damage. In view of the present results, we conclude that penguin
populations located in the high impact area are affected by human activities. These
disturbances were evident on the upset of the antioxidant defences and/or the oxidative

* Corresponding Author E-mail: martinansaldo@gmail.com.

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damage. Accordingly, the analyzed parameters used in the present study proved to be
reliable biomarkers to reflect anthropogenic impact on penguin populations.

Keywords: Antarctica, Pygoscelis adeliae, Pygoscelis papua, blood antioxidant status

INTRODUCTION
Expeditions to and settlement in Antarctica began in the early 19th century, with seal and
whale hunting in order to obtain their oil (Bargagli, 2008; Tin et al., 2008; Xin, 2009). At the
beginning of the 20th century, the explorers to Antarctica built wooden huts and brought large
quantities of supplies and equipment to support their geographical and scientific studies for
several years. When the expeditions ended and relief ships arrived, a rapid exodus frequently
allowed only essential items to be taken north. The huts and thousands of items were left
behind. Fuel depots with unused containers of petroleum products, asbestos materials, and
diverse chemicals were also left inside the huts (Blanchette et al., 2004). Since then, the
Antarctic continent has been occupied and visited uninterruptedly (Bargagli, 2008), and
nowadays constructions such as tents, shelters, stations and trails can be found everywhere
(Tejedo et al., 2011).
Despite anthropogenic pollutants (such as DDT, among others) being found in Antarctica
(Bargagli, 2008; Aronson et al., 2011), it is possible to focus on local impacts which include:
oil spills and paintings; heavy metals and metalloids coming from waste disposal sites;
materials used in constructions; and chemicals used in effluent treatment plants (Negri et al.,
2006; Corsolini, 2008; Tin et al., 2008; Aronson et al., 2011).
Esperanza station is located at Hope Bay (Antarctic Peninsula) and its activity is on-
going throughout the entire year. Electric supply is provided by a diesel generator that works
continuously during the year. Even though gasoil containers have protection to avoid
occasionally spills, it is possible to find oil slicks in the surroundings of the station. Besides
this type of pollution, the disposal of incinerated waste, which has not been yet fully retracted,
and the historical trash can be found spread around the station. Moreover, light aircraft (Twin-
Otter) and helicopters, used for personal and cargo transportation, fly over the areas causing
another type of perturbation: noise. Such activities are increased during the summer season
supporting the scientific and touristic activities, coinciding with the reproductive season of
different Antarctic species (Acero et al., 1996; Carlini et al., 2007).
Several studies have registered the effect caused by human activities on different bird
species. These may include the decrease in populations, behavioural changes and the
alteration of physiological responses (Harris, 2005; Tin et al., 2008; Ninnes et al., 2010). The
anthropogenic activity could also modify these responses increasing the production of
reactive oxygen species (ROS) which exert an unbalanced state called oxidative stress
(Halliwell and Gutteridge, 2007). It has been seen that the exposure to heavy metals (Koivula
and Eeva, 2010), hydrocarbons (Perez et al., 2010) and even anthropogenic activities such as
nocturnal working and ecotourism (Burger and Godchfel, 2007; Navarra and Nelson, 2007)
produce ROS, increasing oxidative damage (Stohs and Bagchi, 1995; Bagchi et al., 2012). As
a consequence, the antioxidant responses are increased (Berglund et al., 2007). As was noted
by Costantini (2008), the oxidative damage level is closely related to animal fitness, meaning
that high oxidative stress levels may compromise survival and reproduction. Moreover,

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 Anthropogenic Impact Modifies the Blood Antioxidant Status … 107

animal population health may be affected by oxidative stress which has significant effects on
fitness components (Beaulieu et al., 2013).
Many organisms are used to evaluate environmental pollution. They are recognized as
sentinels and include species such as molluscs, crustaceans, fish and birds (Dee Boersma,
2008). Marine birds usually used as sentinels are: gulls, albatroes, skuas, petrels and
penguins. Besides having sentinel characteristics (Costantini, 2008; Dee Boersma, 2008),
penguins are adapted to swimming and diving, so they can also reflect ocean productivity
variation (Furness and Camphuysen, 1997; Burger and Gochfeld, 2004).
The aim of the present study was to assess the physiological state of Pygoscelis adeliae
(Adélie penguin) and Pygoscelis papua (Gentoo penguin) populations that were nesting in
areas with high and low human impact at Hope Bay, using biomarkers of oxidative damage
and antioxidant responses.

METHODS
Samples

P. adeliae and P. papua penguin populations breed sympatrically during the summer
season at Hope Bay, Antarctic Peninsula. The colonies are located near the Esperanza
Argentinean Antarctic station. The nests are placed in areas which have been identified by
Acero et al. (1996) as high and low impact areas. The high impact area is located close to
(sometimes inside) the station, and the low impact area is far from the station and its
associated activities.
Sampling was carried out in the colonies of both penguin species at the different areas,
during the summer 2012/2013. Blood samples of 3 ml were taken from 15 adults with
heparinized syringes and sterile 23G x 1” needles.

Biomarkers

In the laboratory, samples were centrifuged (1500 rpm x 10 min) in order to separate
erythrocytes and plasma. A small portion of erythrocytes was separated for determining
reduced glutathione (GSH) levels. The remaining erythrocyte fraction was diluted with
potassium phosphate buffer (100 mM, pH 7.4; dilution ratio sample/buffer 1:50) for
superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione
S-transferase (GST) activities, and lipid peroxidation (LPOe) and protein oxidation (POe)
levels determination. Diluted plasma (potassium phosphate buffer 100 mM, pH 7.4; dilution
ratio sample/buffer 1:10) was used for measuring oxidative damage (LPOp and POp) and uric
acid (UA).
In the diluted erythrocyte fraction, SOD, CAT, GST and GPx activities were determined
using spectrophotometric methods described by Misra and Fridovich (1972), Aebi (1984),
Habig et al. (1974) and St Clair and Chow (1996), respectively. Specific enzyme activity was
calculated considering the total protein content. One SOD unit is the amount of enzyme
necessary to inhibit 50% of the rate of autocatalytic adrenochrome formation measured at 480

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nm. One CAT unit is the amount of enzyme necessary to degrade 1 µmol of H2O2, measured
at 240 nm. One GST unit represents the amount of enzyme required to conjugate 1 µmol of 1-
chloro-2.4-dinitrobenzene, determined at 340 nm. One GPx unit is expressed as the amount of
enzyme necessary to oxidize 1 pmol of NADPH/mg of protein per minute at 340 nm.
The GSH level was determined by the method of Moron et al. (1979). After
deproteinization with trichloroacetic acid (TCA 50%), free endogenous GSH was determined
using 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) 0.5 mM. The absorbance was read at 412
nm. GSH was used as standard to calculate nmol/ml hemolysate.
The PO level was evaluated according to Reznick and Packer (1994), with minor
modifications (Ansaldo et al., 2007), by detecting the formation of protein hydrazones as a
result of the reaction of dinitrophenyl hydrazine (DNPH) with protein carbonyls. After the
protein hydrazone formation, they were precipitated using TCA 30% (Fagan et al., 1999), and
then washed 3 times with ethanol:ethyl acetate (1:1). After the final wash, the protein was
solubilised in 1 ml of urea (6 M in 20 mM potassium phosphate, pH 2.5). To speed up the
solubilisation process, the samples were incubated in a 37 ºC water bath for 60 min. The final
solution was centrifuged to remove any insoluble material. The carbonyl content was
calculated from the absorbance measurement at 375 nm.
The LPO level was measured according to Buege and Aust (1978), by the formation of
thiobarbituric acid reactive substances (TBARS). Fresh hemolysates were added to the
reaction mixture (trichloroacetic acid 15% (w/v), 2-thiobarbituric acid 0.375% (w/v), and
butylhydroxytoluene 0.147 mM) in a ratio of 1:5 (v/v). The mixture was vigorously shaken,
maintained in boiling water for 60 min, and immediately cooled at 5 C for 5 min (Ohkawa et
al., 1979). It was then centrifuged at 5000 x g for 10 min, and the supernatant was measured
spectrophotometrically at 535 nm.
The UA level was quantified using the uricase enzyme method. Uric acid is converted by
uricase into allantoin, hydrogen peroxide (H2O2) and CO2. The H2O2 initiates the coupling of
4-aminophenazone to 3,5-dichloro-2-hydroxybenzenesulfonic acid to form a chromogen, red
quinoneimine, which is measured at 505 nm and is proportional to the amount of H2O2
generated from the UA (Fossati et al.,1980). Units are expressed as mg/dl hemolysate.
The total protein quantity was measured by the method of Lowry et al. (1951) using
bovine serum albumin as standard.

Statistics

Data were analyzed with a general linear model using as factors the type of area (high
and low impacted) and species (Gentoo and Adélie). The best model was obtained by Akaike
and Schwartz IC comparison (Infostat, 2012). When the interaction was not significant, the
main effects were evaluated and plotted independently on various graphs. On the other hand,
when the interaction is significant, the impact of one factor depends on the level of the other
factor, so they are plotted all together on a graph. Interaction effects were thus evaluated
given that the analysis of the main effect results uncompleted or misleading. When results
were significant, post-hoc comparisons were performed using the Di Rienzo, Guzman, and
Casanoves test (universally referred to as the DGC test; Di Rienzo et al., 2002).

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 Anthropogenic Impact Modifies the Blood Antioxidant Status … 109

RESULTS
The SOD and CAT activities did not show significant interaction between the analyzed
factors (areas and species), therefore the main effects of each factor are plotted (Figure 1).
The SOD activity was higher in Adélie than Gentoo penguins (p<0.0001; Figure 1-I).
Besides, penguins from the low impact area showed higher SOD activity than those located in
the high impact area (p<0.001; Figure 1-II). The Gentoo penguins had increased values of
CAT activity (p<0.0001; Figure 1-III) but, independently of their species, penguins from the
low impact area showed more CAT activity than those from the high impact area (p<0.001;
Figure 1-IV).
The GST and GPx activities were related to the interaction of the studied factors, type of
area and species (p<0.05 and p<0.01, respectively; Figure 2). Both species from the high
impact area presented increased GST activity in comparison with those from the low impact
area. However, Gentoo had higher enzyme activity than Adélie penguins (Figure 2-I).
Moreover, the Gentoo penguins located in the high impact area registered a 25% higher GPx
activity than those from the low impact area and the Adélie penguins from both areas (Figure
2-II).

Figure 1. The SOD (I, II) and CAT (III, IV) activities in penguin erythrocyte fractions. Values are
expressed as means ± standard error, n=15 animals. Different letters mean significant differences (a,b:
p<0.001).

The measured hydrosoluble antioxidants (Figure 3) showed no significant differences for

GSH levels in penguins of both species and from both areas (Figure 3-I), while UA levels
depended on the interaction among the analyzed factors (p<0.05). The Gentoo from both

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110 Julieta D. Cebuhar, Carla I. Di Fonzo, Laura P. Dopchiz et al.

studied areas had the highest UA levels, while Adélie penguins from the high impact area had
50% more UA levels than those from the low impact area (Figure 3-II).
The erythrocyte protein oxidation (POe) and lipid peroxidation (LPOe) levels showed the
interaction of the studied factors (p<0.01 and p<0.05, respectively; Figure 4), indicating that
POe and LPOe levels depend on the nesting area. Adélie from the high impact area had the
highest protein damage levels (16.67±0.89 carbonyl nmol/mg protein; Figure 4-I). The POe
levels for both species belonging to the low impact area were similar (Gentoo: 13.82±1.11
carbonyl nmol/mg protein; Adélie: 12.30±1.57 carbonyl nmol/mg protein). Gentoo penguins
that inhabit the high impact area presented the highest LPOe levels (16.87±0.86 TBARS
mmol/ml hemolysate; Figure 4-II) contrasting with values from the low impact area and with
the Adélie penguins. Considering only the Adélie penguins, those from the high impact area
registered higher LPOe levels (9% more than individuals from the low impact area).

Figure 2. I) GST and II) GPx enzyme activities in penguin erythrocytes from both study areas. Values
are expressed as means ± standard error, n=15 animals. Different letters mean significant differences
(p<0.05).

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 Anthropogenic Impact Modifies the Blood Antioxidant Status … 111

Figure 3. I) GSH and II) uric acid levels in penguin erythrocyte and plasma fractions, respectively.
Values are expressed as means ± standard error, n=15 animals. Different letters mean significant
differences (p<0.05).

Registered plasma oxidative damage levels were about 50% lower than those of the
erythrocytes (Figure 4-III, IV). The Gentoo showed POp levels significantly higher (p<0.01)
than the Adélie penguins, particularly those Gentoo inhabiting the low impact area presented
the highest levels (7.78±0.62 carbonyl nmol/mg protein). Adélie penguins from the high
impact area were more affected (≈54%) than those located in the low impact area (Figure 4-
III). The LPOp levels did not show significant differences between both areas and species
(Figure 4-IV).

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112 Julieta D. Cebuhar, Carla I. Di Fonzo, Laura P. Dopchiz et al.

Figure 4. I) POe, II) LPOe, III) POp and IV) LPOp levels of Gentoo and Adélie penguins. Values are
expressed as means ± standard error, n=15 animals. Different letters mean significant differences
(p<0.05).

DISCUSSION
In the light of the present results, it is possible to assess the physiological status of P.
adeliae (Adélie) and P. papua (Gentoo) populations that are nesting in areas near and distant
to Esperanza Argentinian Station at Hope Bay, Antarctic Peninsula. There is a clear
difference in antioxidant enzyme activities and oxidative damage biomarkers either between
penguin species or the studied areas. It may be argued that penguins from the high impact
area would be more affected by the station activities. These results agree with those reported
by other authors who notice negative effects in many bird species due to human activity (De
Villiers et al., 2006; Burger and Goshfield, 2007; Tin et al., 2008; Isaksson et al., 2009; Perez
et al., 2010).
The SOD and CAT activities varied with the species or with the areas in which birds nest.
Adélie showed increased SOD activity and Gentoo showed increased CAT activity and, in
both cases, enzyme activities were increased in penguins which belonged to the low impact
area. These results are not those to be expected for the disturbed areas: high ROS production
followed by altered enzyme activities. The enzyme activity declines may be correlated with
some inhibitory effect owing to heavy metals such as cadmium (Cd) and lead (Pb) present in
the environment, which were detected by Guerra et al. (2011) in the vicinity of Esperanza
station soil. Furthermore, Stohs and Bagchi (1995) evidenced that some heavy metals such as
Cd, can inhibit SOD and CAT activities. Thus, it would be assumed that the low activity in
SOD and CAT from the high impact area could be due to the inhibitory effect of Cd present
in the environment.

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Herrera-Dueñas et al. (2013) worked with sparrows which came from polluted areas
(industrial zones) and unpolluted areas of Madrid and surroundings. In their work they
compared the SOD and CAT activities, the GSH/GSSG relation, the lipid oxidative
damage, the total antioxidant capacity and the hemoglobin concentrations. Those results only
showed significant alterations for both the total antioxidant capacity and the hemoglobin
concentration, being lowest in urban areas. The rest of the analyzed parameters did not show
any differences between the compared areas. In the present work, differences in SOD and
CAT activities were registered in both analyzed areas. These changes may indicate that
human activities affect penguin oxidant/antioxidant blood parameters.
The GSH is a hydrosoluble antioxidant with low molecular weight, wide tissue
distribution and considered a key molecule in the antioxidant process because of its reduction
capacity (Manduzio et al., 2005; Halliwell and Gutteridge, 2007). No differences were
registered in GSH levels of penguins for either species or area. Thus, GSH possibly would not
be used as an antioxidant per se by these penguin species. In the detoxification process, GSH
works as substrate and cofactor for GST and GPx enzymes, respectively. The GST activity
was higher, as expected, in penguins from the high impact area. The GST and GPx showed
similar response patterns for the Gentoo penguins, while the Adélie penguins only had
differences for the GST enzyme activity.
Plasma UA is a hydrosoluble molecule with antioxidant properties. It is also the main
form of nitrogen excretion in avian species, which is produced from hypoxanthine and
xanthine by xanthine oxidase and xanthine dehydrogenase enzyme catalysis (Halliwell and
Gutteridge, 2007; Costantini, 2008). High correlation between UA levels and total antioxidant
capacity was found in 526 individuals of 92 bird species (Koivula and Eeva, 2010). Uric acid
also displayed a strong and positive correlation with antioxidant capacity in studies with
chickens and greenfinches (Cohen et al., 2007; Hõrak et al., 2007). Hõrak et al. (2007) found
that there is a positive correlation between UA and LPO in greenfinches, which might be due
to the production of UA in response to increased LPO. In the present study, we also found a
correlation between UA and LPOe (Pearson: 0.37, p<0.05) for Adélie penguins but not for
Gentoo penguins. In the same way, the significant differences observed in UA levels between
Adélie penguins suggest that these penguins could be using the UA as its main circulating
antioxidant.
For erythrocyte and plasma fractions, protein oxidation (POe and POp, respectively)
showed higher levels of damage in Gentoo located in the low human impact area when
compared with those located in the high impact area. On the contrary, the Adélie penguins
which inhabit the high impact area showed augmented levels of both POe and POp in
comparison to those from the low impact area. The LPOe was increased in the penguins that
inhabited the area of high impact; being the Gentoo penguins the ones with the highest LPOe
levels. These results differ to those registered by Herrera-Dueñas et al. (2013), because we
found that LPOe levels showed differences between the sampled areas.
The LPOp levels did not show significant differences between the studied species. These
results agree with Perez et al. (2010), whose study was carried on in breeding adults of the
yellow-legged gull (Larus michahellis) stressed by the addition of fuel to diet. They did not
register a significant increase in the LPOp levels from the exposed birds. One possible
explanation given for this fact is that the LPOp damage was partially prevented by
antioxidants such as vitamin E, which protects polyunsaturated fatty acids from the oxidative
attack.

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114 Julieta D. Cebuhar, Carla I. Di Fonzo, Laura P. Dopchiz et al.

In the Antarctic Peninsula, Beaulieu et al. (2013) reported that Adélie penguins had lower
antioxidant capacity (an index for the hydro-soluble antioxidant levels) and increased levels
of oxidative damage compared to Gentoo penguins. In contrast, the present results showed no
differences in GSH levels and oxidative damage was observed for both species at high and
low impact areas. Only GSH and UA were analyzed in the present study while the total
antioxidant capacity, as measured by Beaulieu et al. (2013), involves ascorbic acid, vitamin E
and bilirubin, in addition to the above mentioned antioxidants. The Adélie penguins showed
lower levels of damage than Gentoo in either proteins or lipids for the oxidative damage.
Anthropogenic activity tends to alter the oxidation status in animals (Navarra and Nelson,
2007); in addition, urbanization and its associated pollution are the main activities that affect
the environment (Baydas et al., 2001; Cruz et al., 2003; Beaulieu and Costantini, 2014). The
Esperanza station generates many disturbances that may affect the penguin colonies.
In agreement with the present observations, Barbosa et al. (2013) registered stronger
alterations in Gentoo penguins from the high tourist traffic area than those from the low
traffic area. They observed lower immunoglobulin levels, erythrocyte malformations, and
augmented levels of Pb and Ni in the most transited area. Moreover, Higham and Luck
(2007), in their book Marine Wildlife and Tourism Management, collected many studies on
the responses of different penguin species regarding tourist transit into the colonies. In most
cases, the registered responses lead to heart rate increase and release of corticosterone. These
reports reaffirm that human activities produce negative effects on animals.
In the review done by Carney and Sydeman (1999), a total of 17 studies examined the
effects of scientific activity, tourism, and aircraft operations on physiology, behaviour and
reproductive success of penguins. Due to their apparent indifference to the presence of
humans, penguins were thought to be relatively immune to human disturbance. It is
noticeable that Adélie penguins from protected areas far from disturbances were more
susceptible to minor contacts than those populations where human contact was common. This
means that the more regular the disturbance, the smaller the response. The present study
showed that, although changes in penguin behaviour were not observed, any disturbance
(overflying aircraft and helicopters) might generate oxidative stress by increasing the ROS
generation.
As seen in the present study, Gentoo penguins from the high impact area have higher
GST and GPx antioxidant enzyme activities and lower POe and POp levels than those Gentoo
penguins which inhabit the low impact area. One might then assume that this increment of
enzyme activities would counteract the oxidative damage, which was reflected in its low
levels. On the contrary, Gentoo penguins from the low impact area had low enzyme activity,
such that levels of oxidative damage would not be offset. Thus, for any small disturbance, as
expressed by Carney and Sydeman (1999) in their review, oxidative damage of these animals
will be higher, as reflected in the present results. Both species of penguins from the low
impact area present PO and LPO “baseline” levels. Unlike what might be happening with
Gentoo penguins from the high impact area, the antioxidant defences registered in Adélie
penguins did not reach the balance of the natural (basal) damage, producing an even greater
damage in response to anthropogenic activity.

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 Anthropogenic Impact Modifies the Blood Antioxidant Status … 115

CONCLUSION
Adélie penguins from the high impact area presented higher GST activity and UA levels
than those from the low impact area. Besides, those penguins registered high LPOe, POe and
POp levels. On the other hand, Gentoo penguins from the high impact area presented higher
GST and GPx activities and high LPOe levels.
Penguins are being affected by human activity, which has altered the analyzed
biomarkers such as the blood oxidative damage and the antioxidant responses. These
biomarkers have proved to be useful and effective tools in reflecting antioxidant responses
and the damage to the exposed penguins. Thus, this work gives the first report on the state of
Hope Bay penguin colonies as well as baseline values of oxidative stress related parameters.

ACKNOWLEDGMENTS
The authors acknowledge the Instituto Antártico Argentino staff that collaborated in
sampling. This work was totally funded by the PICTO 0091 (FONCyT/DNA-IAA) project.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 9

METALLOTHIONEINS AS BIOMARKERS OF
CONTAMINATION BY METALS IN BIVALVES OF
EASTERN VENEZUELA

Mairin Lemus1*, Julián Castañeda2 and Kyung Chung3

1
Departamento de Biología, Universidad de Oriente, Cumaná, Venezuela
2
Departamento de Oceanografía, Instituto Oceanográfico
de Venezuela, Universidad de Oriente, Cumaná, Venezuela
3
Departamento de Biología Marina, Instituto Oceanográfico
de Venezuela, Universidad de Oriente, Cumaná, Venezuela

ABSTRACT
Metallothioneins (MTs) are proteins with molecular mass of 6-7 KDa, a high content
of cysteine and lack of aromatic amino acids. They have the ability to bind more metals
than any other protein. For this reason, they have been considered as biomarkers in the
presence of zinc, copper, cadmium and mercury. This chapter presents studies on the
evaluation of MTs in bivalve molluscs under confined conditions and in natural
environments, particularly in eastern Venezuela where the greatest diversity and
abundance of these organisms are found. Although Perna viridis is an introduced species
in Venezuela, it is the most widely used bivalve in ecotoxicological studies in the region.
Other bivalves have also been evaluated, such as Arca zebra, Anadara floridana, Atrina
seminuda, Tivela mactroides, Donax denticulatus and Lima scabra. The laboratory
bioassays have shown that, in general, metals such as cadmium and mercury induce the
expression of MTs. However, studies in natural environments have determined that MTs
have a seasonal variation associated with the reproductive cycles of bivalves. The latter,
in turn, are influenced by the seasonal variation of physical, chemical and biological
parameters in the region. These results highlight the physiological role of MTs in the
biological processes of bivalves. It is necessary to elucidate the mechanisms of
physiological processes and toxicity of metals, particularly MT isoforms that seem to
play a key role in toxicity and life cycles of molluscs.

* Corresponding Author E-mail: mlemus88@gmail.com.

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120 Mairin Lemus, Julián Castañeda and Kyung Chung

Keywords: metallothionein, bivalve, heavy metal, pollution

INTRODUCTION
Metallothioneins (MTs) were isolated for the first time from the cortex of the horse
kidney as a protein that binds Zn (zinc) and Cd (cadmium). Its name is attributed to the high
content of metal and sulfhydryl groups, lack of aromatic amino acids and a molar mass in the
range 6-7 kDa (Margoshes and Vallee, 1957; Kägi and Vallee, 1960).
MTs are present in all life forms, from bacteria to mammals (Capdevilla and Atrian,
2011). Their cysteine content can vary between 25 and 30%; they are heat stable proteins
(Klaassen et al., 2009) and lack stable tertiary structure, determined by the bound metal ions.
Among marine invertebrates, bivalve molluscs are the most studied organisms. A greater
study of MTs in these invertebrates is enabled by the association between the toxic metals and
MTs, once they were isolated and linked to Cd (Kägi and Vallée, 1960). Accordingly, these
proteins have been linked to the detoxification of metals.
Induction mechanisms of MTs have been established from the information available
from higher vertebrates and similar mechanisms seem to be present in all organisms. The
expression of MTs can be induced by a wide variety of physiological agents, environmental
stressors and transition metals (Juárez-Rebollar and Méndez-Armenta, 2014), and is
controlled at the level of transcription. MT gene promoter sites present metal response
elements (MRE) that are present in multiple copies in the promoter regions of all MTs
induced by metals (Culotta and Hammer, 1989).
A protein called MT transcription factor (MTF-1) acts as a mediator to start the MT gene
expression. This factor requires a high concentration of Zn for binding to DNA (Westin and
Schaffner, 1988). Studies in mammals (Heuchel et al., 1994) point out that MTF-1 is a
ubiquitous protein that contains six domains of Zn and some domains of trans activation, rich
in the amino acids proline and serine/threonine, which are required for the inducible
translation by metal. The active form of MTF-1 is initially inhibited by an inhibitor sensitive
to Zn, called metal transcription inhibitor (MTI). In the presence of Zn and Zn-ions, the
complexes MTF-1 and MTI dissociate. This condition allows that MTF-1 interacts with the
MRE in the MT developer site to activate transcription. The binding of the newly synthesized
MT to Zn, promotes the formation of the MTF-1/MTI complex (Palmiter, 1998). At the same
time, the transcription of MTs via MTF-1/MRE is controlled by multiple cascades of
transduction signals that affect the phosphorylation of MTF-1 (Adams et al., 2002).
MTs in bivalve molluscs have been widely described in their structure and function,
particularly as a response to contamination by metals in coastal marine ecosystems, and their
evaluation has been incorporated into pollution biomonitoring programs (Phillips, 1979;
Phillips and Rainbow, 1993) However, it has not always been possible to determine
associations between metal levels and the expression of the protein in natural environments
(Monserrat et al., 2007; Machreki-Ajmi et al., 2008), since previous exposures of organisms
to any type of metal inductor of MTs modulate the expression of the protein in new exposures
(Lemus et al., 2014a). Most studies performed under laboratory conditions show a dose-
response relationship (Baudrimont et al., 1997; Geret and Cosson, 2002). However, it has
been pointed out that induction mechanisms of these molecules are modulated by biotic

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 Metallothioneins as Biomarkers of Contamination by Metals … 121

factors such as developmental stage, sex, reproductive condition, growth rate, and also by
some abiotic factors such as temperature, salinity, dissolved oxygen, light intensity, among
others (Ladhar-Chaabouni et al., 2012; Zapata-Vívenes et al., 2014).
Studies in marine ecotoxicology in Northeastern Venezuela began to increase in the
1980s when research started on the effect of metals to fish (Lemus et al., 1989; Márquez et
al., 2008) and polychaetes (Marcano et al., 1996, 1997), and on levels of metals in bivalves
(Jaffé et al., 1998; Lista et al., 2006). Evaluation of metals in bivalves was initiated in order to
evaluate the human health safety of these organisms due to the large consumption by the
population in general.
Bivalves have been the most widely studied marine invertebrates in the area of
biomonitoring of heavy metals and ecotoxicology. They are ideal organisms in studies of
toxicity and biomonitoring (Phillips, 1979; Phillips and Rainbow, 1993; Rainbow, 2002),
given that they: (a) accumulate high levels of pollutants; (b) are sedentary or able to make
short migrations; (c) can present large abundances; (d) have wide distribution (cosmopolitan);
(e) are long-lived; (f) are easy to sample; (g) are easy to transport, handle and keep under
conditions of confinement; (h) have a good relationship dose-response; (i) are available
throughout the year; (j) have simple feeding habits, usually by filtration; (k) support a wide
range of climatic and environmental conditions; (l) are taxonomically well described; and (m)
are well documented regarding species life history and biology. Other important conditions,
also identified by researchers, are: (a) the patterns of accumulation in the target organism
should also be reflected in some other species in the study area; (b) the studied organism
should preferably be of economic importance; (c) collection or purchase of organisms
must be low cost; and (d) samples should be transportable internationally without legal
impediments (Rainbow, 1995; Zhou et al., 2008; Páez-Osuna and Osuna-Martínez, 2011).
The eastern region of Venezuela is characterized for having the greatest landings of
fishery products of the country, and studies in the field of ecotoxicology, particularly with
marine bivalves, have been performed to evaluate their safety for human consumption and to
assess their physiological responses to metal incorporation. The bivalve species most widely
used for these studies have been Perna viridis (Linneaus 1758), Arca zebra (Swainson, 1833),
Lima scabra (Born, 1778), Anadara floridana (Conrad, 1869), Donax denticulatus (Linnaeus,
1758) and Atrina seminude (Lamark, 1869).
A. zebra is the most commercialized bivalve species in Venezuela, due to its large natural
banks and landings; in second place is the mussel P. perna, which is being displaced by P.
viridis, an invasive species to the Venezuelan coasts with origin in the Indopacific Ocean.
Other species of commercial importance, but with much lower natural density, are A.
floridana, A. seminuda and Tivela mactroides (Born, 1778). On the other hand, L. scabra is a
representative of coralline environments and D. denticulatus of intertidal sandy beaches.
Ecotoxicological studies have been conducted on all of these species under controlled
conditions as well as in their natural environments.
This chapter presents results from studies on MTs in bivalve molluscs of the eastern
region of Venezuela. The use of bivalves in the presence of metals in laboratory bioassays
and in natural environments is evaluated and finally two cases are shown on the seasonal
variation of MTs in P. viridis and A. zebra.

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ASSAYS UNDER LABORATORY CONDITIONS

Toxicological assays under controlled conditions have allowed determining the
relationship between an inductor (heavy metal) and a specific response from the organism.
The responses can range from alterations in the behaviour, to physiological or biochemical
responses. Most bioassays have established a relationship between the biomarker and the
inductor agent (Ma et al., 2008; Li et al., 2015).
Although laboratory bioassays are widely used today, they have little predictive power in
natural environments, due to their low level of environmental realism (Farris and Van Hassel,
2006). In ecotoxicological studies conducted under controlled conditions with organisms
from the Caribbean Sea, specifically from the eastern region of Venezuela, P. viridis has been
the most widely used (Nusetti et al., 2010; Lemus et al., 2012a; Zapata-Vívenes et al., 2012,
2014; Lemus et al., 2014a,b). However, L. scabra (Lemus et al., 2012b), D. denticulats
(Antón et al., 2008), Pinctada imbricata (Nusetti et al., 2004) and T. mactroides (Acosta and
Lodeiros, 2009) have also been considered.
The green mussel, P. viridis, is the mytillid most widely studied as biomonitor, after
Mytilus edulis, attributed to its wide distribution and ease in colonizing environments. P.
viridis is a native species to the Indo-Pacific, which was detected for the first time in Trinidad
in 1990 and has spread to the eastern region of Venezuela, being detected in the Gulf of Paria
and Northern Sucre state, in Margarita Island and the Gulf of Cariaco (Rylander et al., 1996).
Due to its tropical character and its high fertility, this species has easily established along the
coasts of Venezuela and much of the Caribbean Sea.
Studies carried out with P. viridis under confinement conditions at different stages of
development show that the species has a great capacity for acclimatization to long periods of
time. Thus, it is easily manipulated for laboratory tests and shows physiological and
biochemical responses, such as growth and respiration rates, oxidative stress, glutathione,
lipid peroxidation, and MTs, in the presence of heavy metals (Narváez et al., 2005; Lemus et
al., 2012a; Acosta et al., 2013; Lemus et al., 2014a; Zapata-Vívenes et al., 2014).
The modulation of MT expression is determined by a great variety of external factors.
One of them is the pre-exposure of the organism to toxic agents, particularly to heavy metals
(Lemus et al., 2014a). In the case of juveniles of P. viridis exposed to sublethal doses of Cd
and Cu (copper)/Cd under controlled conditions, MT levels increased with exposure time to
Cd, originating a dose-response behavior in the presence of the metal (Figure 1).
The induction of MTs in organisms pre-exposed to sublethal doses of Cu can also be
modulated. In these organisms, the incorporation of Cd shows no relationship with the
expression of MTs, suggesting that the expression of the MTs by pre-exposure to Cu raises
the ability of the organisms to increase the concentration of this protein in the presence of Cd
(Figure 2). This response, under extremely controlled conditions of pH, temperature, salinity,
feeding and photoperiod, does not occur in natural environments. It has thus been proposed
that studies conducted under natural environmental conditions require more complex
analyses, involving factors and variables that may be affecting the biomarker. Many of the
variations shown by MTs in natural environments have been interpreted using multivariate
models (Lemus et al., 2014a).

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 Metallothioneins as Biomarkers of Contamination by Metals … 123

Figure 1. Mean metallothionein (MTs) levels in Perna viridis exposed to Cd and pre-exposed to Cu and
later to Cd (Lemus et al., 2014a). Vertical lines are one standard deviation.

4 MTs = - 0.2584 + 0.3399 Cd A

µg MTs/g protein

0
0 2 4 6 8 10

MTs = 1.1281 + 0.1683  Cd B

5
µg MTs/g protein

0
0 2 4 6 8 10 12
µg Cd/g f.w.

Figure 2. Linear relationship between the content of metallothioneins (MTs) and levels of Cd in Perna
viridis during 21 days of exposure. A. Exposed to Cd (r=0.88; Fs=102.73, p<0.001); B. Preexposed to
Cu and exposed to Cd (r=0.323; Fs=0.323, p=0.0815). Dotted line: confidence interval 95%; dashed
line: prediction interval 95%. After Lemus et al., 2014a.

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124 Mairin Lemus, Julián Castañeda and Kyung Chung

Furthermore, it has been suggested that the levels of MTs could mediate the resistance of
bivalves to anoxic conditions, since it has been shown that the antioxidant system of the
digestive gland of P. viridis is not affected by the acute exposure to Cd in the short term; this
is possibly associated to the high concentration of MTs recorded, which in turn could explain
the resistance to anoxic conditions of the organisms (Zapata-Vívenes et al., 2014).
D. denticulatus is a representative of the benthos from high energy sandy beaches and has
been used with great success under controlled conditions in toxicological assays, showing
good adaptation to confined conditions (maintains similar growth rates to that of wild
individuals). Individuals exposed to Cd show a significant bioaccumulation of this metal,
depending on the length of exposure, and a significant decrease in growth (RNA/DNA ratio).
This is possibly related to the energy expenditure involved in the processes of incorporation,
metabolization and excretion of the metal, compromising the energy for growth (Antón et al.,
2008).
L. scabra, commonly called fire clam, is a bivalve which inhabits coralline environments
in very clear waters. It is widely distributed throughout the Caribbean Sea, from the coast of
North Carolina in the United States to the north-eastern coast of Brazil, in a depth range
between 1 and 40 m (Lodeiros and Himmelman, 1999). The species is a good representative
to assess disruption of this type of environments, although its maintenance under confined
conditions is more limited than other species evaluated. Under confined conditions and
exposed to Cd, MT expression is induced after 21 days of exposure.

METALLOTHIONEINS IN NATURAL ENVIRONMENTS

More recent studies have characterized MTs as a family of multifunctional proteins
related to many routes of intermediary metabolism, given the implications in the homeostasis
of essential metals (Zapata-Vívenes and Nusetti, 2007; Takahashi, 2012). This is achieved by
controlling the levels of free metals that are available for the proteins associated to cellular
processes: they act as a mechanism of cell protection against toxic metals by binding them
through mercapto links and preventing their linking to other molecules, and they protect
against oxidative stress caused by toxic metals (Lemus et al., 2016).
Studies on the function of MTs in bivalves have not advanced at the same speed as those
in vertebrates. By January 2015, more than 9800 nucleotide sequences had been recorded in
the database of nucleotides of the NCBI as MTs, only 155 of which have been identified in 38
species of bivalves (Lemus et al., 2016). Metallothioneins have shown a large number of
isoforms in these invertebrates, with very different functions that can be induced or
modulated by temperature, anoxia, stage of development and essential or non-essential metals
(Lemus et al., 2016). Researchers point out that since the lifecycles of these sessile, or with
limited mobility, organisms, are directly associated with the physical, chemical and biological
variables of the ecosystems where they develop, their metabolism is subject to multiple
variations and adaptive mechanisms.
Of the species of bivalves studied in eastern Venezuela, only for P. viridis are the MT
primary structure and isoforms known. Leung et al. (2014) characterized two types of MT
isoforms in the digestive gland of the green-lipped mussel P. viridis, using Cd and hydrogen
peroxide as inducers of MTs. The two isoforms differed in their deduced protein sequences,

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 Metallothioneins as Biomarkers of Contamination by Metals … 125

with 73 amino acids for MT10-I and 72 for MT10-II (a novel type), but both contained a high
percentage (27.4 to 29.2%) of cysteine. The MT proteins were present in multiple isoform
spots. The MT10-I responded promptly to Cd but had a lagged induction to H2O2 treatment,
while tm10-II was exclusively induced by the Cd treatment.
Studies so far shows that the assessment of concentrations of MTs as biomarkers of
exposure to metals is a field that still needs much development, because the isoforms have
different inductors (Isani et al., 2000; Lemoine et al., 2000; Ivanina et al., 2015) and this
mechanism is possibly fundamental to maintain the homeostatic functions of the organism
and to respond to the presence of metals or other types of stressors (Lemus et al., 2016).
Further knowledge of MTs isoforms is thus essential to elucidate the function of MTs in
bivalves.
The presence of isoforms in bivalves could explain the reason why many studies
evaluating MTs in this group of organisms in their natural environments are so controversial.
Some of them demonstrate associations with their life cycle, while others identify the
presence of metals, or both aspects, as determinants of the expression of MTs.

Perna viridis

Analyses of MTs in P. viridis from eastern Venezuela show seasonal variations, with
higher concentrations between February and March and lower ones between September and
December (Figure 3), coinciding with the periods of high and low phytoplankton productivity
in the area, respectively. On the other hand, immature individuals have higher concentrations
of MTs in comparison to mature and spawned ones (Figure 4), which leads to a negative
relationship between MTs and Condition Index. Results show that MTs of P. viridis are
influenced by physical and chemical factors of the marine environment as well as by the
physiological and reproductive condition of individuals (Lemus et al., 2013).
Studies performed so far show that there is bioaccumulation of metals associated to the
reproductive annual cycles, and that this fact is a response to annual variation of
environmental factors in the coastal marine ecosystems. The latter, in turn, determines
metabolic variations and modulates the reproductive mechanisms of bivalves (Martínez-
Castro and Vázquez, 2012).

Arca zebra

The Turkey wing, A. zebra, is the largest bivalve in northeastern Venezuela. It represents
the second category in terms of volume of fishing landings for the entire artisanal fishery of
Sucre state, after the sardine, being the fundamental support of several fishing communities
that rely almost exclusively on this activity (Jiménez, 1999; Prieto et al., 2001). This species,
which belongs to the Arcidae family, is distributed from the coast of the Gulf of Mexico and
South Florida to the North of Brazil, but is in Venezuela where it forms banks of commercial
importance (Prieto et al., 2001). In Venezuela this bivalve has been successfully used as a
biomonitor of heavy metals (Acagua, 2008; Lanza et al., 2011).

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126 Mairin Lemus, Julián Castañeda and Kyung Chung

3.0 Chacopata

µg MTs/mg protein
2.5

2.0

1.5

1.0

0.5

3.0
Río Caribe
2.5
µg MTs/mg protein

2.0

1.5

1.0

0.5

0
F M A M J J A S O N D

Mo n t h s 2013

Figure 3. Monthly variation of metallothioneins (MTs) in Perna viridis collected in 2013 from two
coastal locations of Sucre state, Venezuela: Chacopata and Rio Caribe (Lemus et al., 2013). Diamonds
denote medians, boxes enclose the 25th and 75th percentiles, and error bars represent the 10th and 90th
percentiles.

3.0

2.5
µg MTs/mg protein

2.0

1.5

1.0

0.5

0
I II III
Maturity stages

Figure 4. Metallothioneins (MTs) at different developmental stages of the gonad of Perna viridis, from
the coastal zone of Sucre state, Venezuela (Lemus et al., 2013). Diamonds denote medians, boxes
enclose the 25th and 75th percentiles, and error bars represent the 10th and 90th percentiles.

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 Metallothioneins as Biomarkers of Contamination by Metals … 127

he concentrations of MTs in A. zebra also showed seasonal variations associated to the

Condition Index of the organisms, registering the highest values during August, a period in
which the Orinoco River increases the discharge into the coastal zone. In this species, there
was not a significant correlation between the concentration of toxic metals (Pb and Cd) and
the level of MTs, but it was significant with the levels of Cu (personal communication).
These results show that the levels of MTs in bivalves of eastern Venezuela are affected
by environmental and physiologic factors that should be taken into account when determining
the sources inducing MTs in the organisms. In this regard, the variations of physical and
chemical parameters as well as the life cycle stages of the bivalves should be evaluated in
order to determine the intervals of annual fluctuation of this protein. That is why knowledge
about the physiological variations of MTs during the life cycle of bivalves is essential to
consider MTs as a biomarker of metal contamination.

Atrina seminuda

The pen shell, A. seminuda (Lamarck, 1819), is an endobenthic bivalve that usually uses
a strong byssus secreted by the animal to adhere to rocks and gravel substrate in areas of high
energy. It can be found along the Atlantic coast of North America, from North Carolina to
Texas and the Caribbean Sea, down to the shores of Argentina (Lodeiros and Himmelman,
1999; Macsotay and Campos, 2001). Within the Venezuelan Caribbean Sea region, it has
been found on the coasts of the states of Vargas, Miranda, Anzoátegui, Sucre and Nueva
Esparta (Ramos and Robaina, 1994). This bivalve shows reproductive activity during most of
the year with several intense reproductive periods (June-July and September-November and
February) as well as a year-round gametogenesis, a behavior of asynchronous type (Freites et
al., 2010).

15 Female Male
Metallothioneins

12
(g/g)

0
G H Mu Ma G H Mu Ma

Figure 5. Levels of metallothioneins (MTs) in somatic tissues of Atrina seminuda. G: gonads; H:

hepatopancreas; Mu: muscle; Ma: mantle.

In A. seminuda (sampled along the north eastern coast of Venezuela), the levels of MTs
tend to be higher in the parenchymatous tissue, particularly in hepatopancreas. However, this
species also presented a high concentration of MTs in the mantle (Figure 5). This is possibly
associated with the highest levels of Cd that were detected in this tissue. It is known that Cd

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128 Mairin Lemus, Julián Castañeda and Kyung Chung

has a greater affinity for mercapto groups of the MTs than Zn, and thus has a great ability to
displace it. At the same time, this free Zn is a strong inducer of the synthesis of MTs and
could thus explain the high levels of MTs in the mantle. On the other hand, it has been shown
that in many bivalves Cd can be removed via synthesis of the shell that takes place in the
mantle.
Nevertheless, when MTs are evaluated in the whole organism a significant relationship is
found between the level of MTs and the gonadosomatic index (Figure 6).

Anadara floridana

Comparing the levels of MTs obtained in A. floridana with those determined in other
bivalve species, hepatopancreas and gills are usually seen to carry higher levels than other
tissues (Amiard et al., 2006; Barrera-Escorcia and Wong-Chang, 2010). However, in A.
floridana, the highest concentrations of MTs were found in muscle, which suggests that this
tissue has a greater synthesis capacity of this protein, and that it is probably involved in
specific processes of this species to regulate bioessential metals or the metabolization and
clearance of toxic metals.
This species of bivalve has the distinction of presenting a very large muscle with great
pigmentation and also with extreme force to close the shells, unlike many bivalves. Even
though all studies performed on toxicity of metals in bivalves have considered the
hepatopancreas as the target organ and have used this tissue to evaluate enzymes, biomarkers
and physiological and biochemical responses, it is possible that there are species-specific
responses which still have not been discussed.

Donax denticulatus

This species is distributed in the West Central Atlantic Ocean from the Caribbean Sea to
Brazil, inhabiting the intertidal zone in the sweep of the swell area of many sandy beaches
with high energy. Populations of this bivalve in the Caribbean Sea vary annually in density,
size and color of the shell. The factors that influence this variation are mainly the size and
arrangement of the sand grains, the slope of the beach, the degree of exposure to the waves
and the organic content of the sand.
Analyzing the levels of MTs in males and females of D. denticulatus during different
times of the year, a significant increase of the concentration of MTs was found during the
month of January (Figure 7), when phytoplankton levels are the highest in the eastern region
of Venezuela (Velásquez-Martínez and Lemus, 2011). This pattern shows once again that the
MTs are modulated by the availability of food.
In the bivalve species studied in eastern Venezuela, it has been determined that there are
seasonal variations in the incorporation of Cd, Cu, Hg, Zn and Pb, which are mainly
associated to the variation of the upwelling periods that characterize the zone. These
variations are regulated by the upwelling promoted by trade winds and by the influence of the
Orinoco River discharge, which is associated to the precipitation regime. Both processes
expand or contract in an alternating way under an annual cycle, dominated by upwelling
during the first months of the year until June, while the river influence prevails approximately

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 Metallothioneins as Biomarkers of Contamination by Metals … 129

from June to December and generally carries a heavy load of suspended matter (Varela et al.,
2003). These two aspects modulate both the incorporation of metals of natural and anthropic
origin. In particular, the Orinoco River discharges considerable amounts of metals to the
coastal zone, especially Hg and Cd, as waste or sub products of the mining and metallurgical
industries established along this river.

2.4

Metallothionein(µg/g)
2.0

1.6

1.2

0.8

0.4

0.4
Glutathione (mmol/g)

0.3

0.2

0.1

0
Lipidic peroxidation (µmol/g)

1.8

1.5

1.2

0.9

0.6

0.3

0
4 6 8 10 12
Gonadosomatic index (%)

Figure 6. Relationship between levels of metallothionein (MT), glutathione or lipidic peroxidation, and
gonadosomatic index, in Atrina seminude. Solid line: regression line; Dotted line: confidence interval
95%; Dashed line: prediction interval 95%.

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130 Mairin Lemus, Julián Castañeda and Kyung Chung

Metallothioneins (µg/g f.w.)

8
Males
6

0
Metallothioneins (µg/g f.w.)
8
Females
6

0
Dec-05 Jan-06 Feb-06 Mar-06

Figure 7. Concentration of metallothioneins (MTs) in Donax denticulatus collected over four months
(from December 2005 to March 2006) at Playa Brava, Araya Peninsula, Sucre state, Venezuela. f.w.,
fresh weight (Velásquez-Martínez and Lemus, 2011).

CONCLUSION
MTs participate in several metabolic routes and there is limited knowledge about them in
marine organisms, particularly in invertebrates. It is known that environmental factors and the
stage of development of the organisms are determinants in the modulation of this protein. The
previous life history of the organism also introduces a modulation to the expression of these
molecules. In the case of tropical bivalves from eastern Venezuela, MTs are modulated by the
reproductive cycles, which in turn are affected by physical, chemical and biological factors.
Studies should be geared towards knowledge of isoforms in the different bivalves and the role
played by each of them. It is essential to establish methodologies of easy reproducibility to
evaluate isoforms routinely.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 10

ECOTOXICOLOGICAL STUDIES OF FRESHWATER

ECOSYSTEMS IN LATIN AMERICA: DIAGNOSIS,
PERSPECTIVES, AND PROPOSALS

Roberto Rico-Martínez1,*, Ignacio Alejandro Pérez-Legaspi2,

Gustavo Emilio Santos-Medrano1, Jesús Alvarado-Flores3
and Mario Alberto Arzate-Cárdenas1
1
Universidad Autónoma de Aguascalientes,
Centro de Ciencias Básicas, Departamento de Química,
Aguascalientes, AGS, Mexico
2
Instituto Tecnológico de Boca del Río,
División de Estudios de Posgrado e Investigación,
Boca del Río, Veracruz, Mexico
3
Consejo Nacional de Ciencia y Tecnología,
Unidad de Ciencias del Agua, Centro de Investigación
Científica de Yucatán, Cancún, Quintana Roo, Mexico

ABSTRACT
Latin America and the Caribbean are the regions with the greatest biological
diversity on earth, and endemism in these regions is very high. Regarding freshwater
ecosystems and water availability, Latin America and the Caribbean hold more than 30%
of earth’s available freshwater resources, but this water is very unequally distributed and
at risk. Most ecotoxicological studies have been performed in countries and ecosystems
in temperate zones, neglecting the tropics. In tropical environments most research has
focused on water quality and aquatic toxicology, with regulations varying from country
to country. Therefore, the present chapter aims at presenting the current status of
ecotoxicological studies regarding the major risks to which Latin American freshwater
resources are subjected, which can be included in six categories: 1) municipal discharges;
2) mining activities; 3) industrial/agricultural contamination; 4) depletion and

* Corresponding Author E-mail: rrico@correo.uaa.mx.

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138 R. Rico-Martínez, I. A. Pérez-Legaspi, G. E. Santos-Medrano et al.

contamination of aquifers/glaciers; 5) petroleum industry contamination; and 6)

cyanobacteria toxins. In addition, information related to these threats is presented as
evidence of the adverse effects caused to freshwater ecosystems: a) loss of biodiversity;
b) eutrophication/bacterial-parasite contamination; c) bioaccumulation, bioconcentration
and biomagnification; d) aquifer contamination and depletion; e) freshwater salinization.
Despite all efforts of the scientific community, information is lacking regarding fate and
effects of contaminants on freshwaters in Latin America. Thus, it is still necessary to
coordinate the participation of several associations (national and/or international) and
authorities at different hierarchical levels to achieve regulatory consensus regarding
pollutants and their effects on Latin American freshwater ecosystems.

Keywords: eutrophication, pesticides, metal toxicity, aquatic toxicology, environmental

toxicology

INTRODUCTION
Latin America and the Caribbean are the regions with the greatest biological diversity
on earth, holding almost 50% of the world’s tropical forests, 33% of mammals, 35%
of reptiles, 41% of birds and 50% of amphibian species, and endemism in these regions
is very high (UNEP, 2010). Regarding freshwater ecosystems and water availability, Latin
America and the Caribbean hold more than 30% of earth’s available freshwater resources
(UNEP, 2010), but this water is very unequally distributed and in risk due to: a) increasing
pollution, b) degradation of watersheds, and c) the depletion and unsustainable use of
aquifers, which are all mainly due to anthropogenic activities. Ecotoxicology has focused
on countries and ecosystems in temperate zones, with little research carried out in tropical
environments. Techniques and procedures developed for temperate environments are often
applied in tropical regions, even though physical and chemical environmental parameters
in the tropics can be very different. The regulatory environment also varies among
countries (Lacher and Goldstein, 1997). With this background, in the present review
we focused on one main question: What are the major risks to which Latin American
freshwater resources are subjected? The answer presented in this chapter is enclosed in six
categories: 1) municipal discharges causing eutrophication; 2) mining activities; 3) industrial/
agricultural contamination; 4) depletion/contamination of aquifers/glaciers; 5) petrochemical
contamination; and 6) cyanobacteria and cyanobacterial toxins.

MUNICIPAL DISCHARGES CAUSING EUTROPHICATION

Still by large the most common threat to freshwater ecosystems in Latin America is
domestic and agricultural discharges that cause eutrophication. In Latin America there are
many illegal and incidental discharges of unknown contaminants. Latin America has 578
million inhabitants (8% of the World population), and provides water supply for 93% of its
population but sanitation for only 79% of its population (120 million lack sanitation) (Noyola,
2013). Wastewater treatment only covers around 20% of total water (Noyola, 2013). Many
countries in Central and South America are highly dependent on transboundary waters
because the origin of most of their water resources is not within their territory (IDB, 1998).

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 Ecotoxicological Studies of Freshwater Ecosystems in Latin America 139

Heads of Environmental Protection Agencies of Latin American countries have expressed

their commitment, through the use of existing transboundary agreements, to the sustainable
use of water resources; there are many potential sources of conflict, but also opportunities for
cooperation (Garcia, 1999). One of these cooperations is the case of the risk assessment of the
Paraguay River Basin (The Nature Conservancy, WWF-Brazil, 2012) which included four
countries (Argentina, Bolivia, Brazil and Paraguay); the risk assessment report concluded that
the central portion of the basin, i.e., the Pantanal and the Dry Chaco, displayed low ecological
risk, but that the floodplain presented a proportionately high risk.

MINING ACTIVITIES
Rising prices and expanding global demand have detonated an explosive growth in gold
mining. Latin American and the Caribbean, mainly in Venezuela, Guyana, Suriname, French
Guiana, Colombia, and Brazil, have experienced some of the highest growth rates in
production since the year 2000 (Hammond et al., 2013). Mining is a legacy of our
Spanish/Portuguese/English/French/Dutch ancestors in their quest for gold, silver and other
precious metals. The largest mining industry globally is Canadian, with the majority of its
investment in Latin America and the Caribbean: this industry increased up to 35% since 1990
to 2014 (Gordon and Webber, 2008). The gold rush in South America was triggered in 1980
with the discovery of a large gold mine in Sierra Pelada, Amazon Region; from there the
fever spread to Venezuela, Guyana, French Guiana and Suriname (Mol et al., 2001). In the
1990s and 2000s, the largest gold exploration in the world was in Latin America, where 12 of
the 25 largest mining investments took place in Peru, Chile and Argentina (Urkidi, 2010).
From the 1980´s, Brazil was the largest producer of gold in South America followed by
Colombia, Venezuela, Peru and Bolivia (Malm, 1998; Nevado et al., 2010).
Mining liquid waste or suspension tailings and deposits of discarded rock and leaks are a
source of metals. When these deposits are in contact with sulfur and oxygen, they result in
Acid Mine Drainage (AMD), with high levels of dissolved metals (for example, pyrite (FeS2)
associated to coal is exposed to air, and is oxidized to H2SO4 by bacterial action producing
AMD). When these leached or filtered particles reach rivers, a wide dispersion of the metals
in solution, and as particles occurs (Salomons, 1995). Most of the new gold mines are open
pits and use cyanide that is released in the melting. Among the major impacts are the high
consumption of water and the acidification of the water drained into the tailings (Urkidi,
2010). However, the ionic form of a metal is usually the most toxic, and when associated with
suspended matter or dissolved ligands its toxicity decreases (Salomons, 1995).

Mercury

In Mexico and South America, mercury has been widely used since colonization for
recovery of gold and silver, where it is directly released to aquatic systems as mining disposal
(Malm et al., 1995a; Malm, 1998). Brazil is the first producer of gold in South America, of
which 90% of mining sites are small-scale or “garimpos” (Nevado et al., 2010). The largest
“garimpo” is located in the basin of the River Tapajós (Brazil) and releases a large amount of

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mercury into the Amazon River (Malm et al., 1995a; Nevado et al., 2010). The second largest
gold mine in South America is the Pascua-Lama project at the borders of Chile and Argentina
and the Andes mountains, which began in 2009 (Urkidi, 2010). Mercury pollution is
recognized as one of the main environmental problems of tropical South America (Mol et al.,
2001).
Mercury is used for the separation of fine particles of gold through the amalgamation in
an open pit at high temperatures (350-600 °C). The resulting waste is then sent to tailing dams
creating concentrated mercury. This process is repeated to improve the extraction (Salomons,
1995). In addition, the use of cyanide for gold recovery results in the formation of highly
soluble mercury cyanide complexes, contributing to their mobilization (Nevado et al., 2010).
Metallic mercury is volatile and when oxidized and methylated it becomes its more toxic
organic form, methyl mercury, which is biomagnified in the food web eventually reaching
human beings (Malm et al., 1995a; Nevado et al., 2010). High mercury concentrations in air
from urban, rural or occupational exposures such as burning amalgam reflect the volume of
discharge to the atmosphere due to gold mining activities. In addition, the Amazonian aquatic
ecosystems environmental conditions favor high rates of mercury methylation (Salomons,
1995; Malm, 1998). Although Malm et al. (1995a) reported that Hg found in sediments in the
Madeira River of Brazil have values below the global average (0.3 µg/g), high levels of
methyl mercury have been found in carnivorous and piscivorous fishes of the Tapajós river
(Akagi et al., 1995; Malm et al., 1995a,b), and in the Madeira river basin, showing levels of
0.7 µg/g which are higher than the 0.5 µg/g maximum established by the Brazilian legislation
(Malm et al., 1995a,b). High levels of mercury have been found even in human hair along the
Tapajós and Madeira rivers, attributed to the consumption of fish (Akagi et al., 1995). Some
authors argue that the Hg is transported over long distances in the inorganic form, associated
with suspended particles or organic matter. They have therefore suggested to avoid the
ingestion of carnivorous fish such as piranha (Serrasalmus spp.), peacock bass (Cichla
ocelaris), piraíba (Brachiplatystoma filamentosum), gilded catfish (B. flavicans), apapá
(Pellona castelnaeana), spotted surubim (Pseudoplatystoma fasciatum), and tiger catfish
(Pseudoplatystoma sp.) (Malm et al., 1995a).
The safety limits for ingestion of mercury are based on the concentration of this metal in
muscle of consumed products (0.5 µg Hg/g, wet weight) (Nevado et al., 2010). In the
Brokopondo reservoir, Suriname, there have been reported exceeding concentrations of
mercury within several fish species, in which the highest concentrations (3.13 and 4.26 µg/g)
were found in the piranha S. rhombeus (Mol et al., 2001). Neurotoxic effects have been
observed in adults with levels of mercury in hair below 50 µg/g, due to chronic exposure of
communities living close to the Amazon River (Passos and Mergler, 2008; Nevado et al.,
2010). In the Tapajós River basin gold mining is the principal economic activity since the
70´s, representing a serious risk to environment and human health (Nevado et al., 2010).
Malm et al. (1995b) suggest forbidding of the use of mercury, followed by a better
understanding of the mining practice and development of alternative clean techniques.

Arsenic

Arsenic is released to aquatic systems, soil and atmosphere by the mining industry and
metallurgy. Its residues can be dispersed by wind or water after its provision, and may be

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 Ecotoxicological Studies of Freshwater Ecosystems in Latin America 141

associated with AMD (Razo et al., 2004). Gold and copper mining in Brazil have contributed
to increasing levels of arsenic for hundreds of years. High levels of arsenic in drinking water
sources in many countries in Latin America such as Argentina, Bolivia, Brazil, Chile,
Colombia, Cuba, Ecuador, El Salvador, Guatemala, Honduras, Mexico, Nicaragua, Peru, and
Uruguay have been detected (McClintock et al., 2012). Bundschuh et al. (2012) mentioned
that the main sources of arsenic in Latin America including for drinking water are geogenic
and anthropogenic, such as mining. In addition, Salomons (1995) mentions that the erosion of
active and inactive mining areas or direct discharge of tailings or waste rock introduce metals
to the aquatic environment; even in pristine rivers more metals in the inert fraction have been
reported than in polluted rivers.
Razo et al. (2004) reported the presence of arsenic (265 μg/L) in rainwater storage dams
105 km from the mining site of Villa de la Paz-Matehuala, S.L.P., Mexico. In Zimapan
(Hidalgo, Mexico), the concentration of arsenic in groundwater is greater than Mexican
drinking water standards, due to the contribution of tailings which can potentially lixiviate
into not so deep aquifers (Ortega-Larrocea et al., 2010). The Pilcomayo basin (Argentina,
Bolivia and Paraguay) presents an average of 50 μg/L of arsenic, while the secondary channel
used for irrigation has a concentration of 200 μg/L. In addition, the arsenic concentration in
fish is related to its concentration in water and is attributed to the mining activities of Sn, Ag,
Zn, and Pb (Bundschuh et al., 2012).

Other Metals and Radionuclides

The Chilean economy depends heavily on the export of Cu, constituting 55.7% of
national exports in 2007. In addition, Chile exported 47.5% of the total amount of Cu sold
worldwide (Urkidi, 2010). The mining of Ni and other mining activities in the Bay of Levisa
(Cuba) has generated high concentrations of metals in sediments such as Ni, Fe, Co and Mn
(Gonzalez and Ramírez, 1995). The mining and milling of ores to produce uranium yields
large amounts of waste, including AMD, waste rock, tailings, and radionuclides, transported
by air, as the 222Rn and its derivatives of short life. Acid mining and drainage waste rock are
the main sources of 226Ra, 238U, Al and Fe (Fernandes et al., 1995).

INDUSTRIAL/AGRICULTURAL CONTAMINATION
The contamination of aquatic ecosystems by industry and agriculture in Latin America is
complex and difficult to eradicate. Many categories of waste are produced: nitrogen,
pesticides, solvents, hydrocarbons, personal care products, analgesics, antibiotics, hormones,
chemicals and agro-industrial waste. Unfortunately all these compounds are difficult to
remove in wastewater treatment plants (Hillstrom and Hillstrom, 2004). Köhler and
Triebskorn (2010) stated that Latin American ecosystems are contaminated with 120 different
pesticides with half a million tons applied without control, and that these pesticides produce
critical adverse effects on aquatic biota.
Latin America generates 40% of the total world production of soybean in a total of
15,000 km2 of land which was burned and cleared for crops. This has caused increasing levels

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of nitrogen in the atmosphere together with the pesticides that were used. It is estimated that
levels of nitrogen in the water will duplicate by 2050 (Austin et al., 2013). The implications
of contamination by nitrogen in aquatic ecosystems are catastrophic, causing: 1) acidification
of rivers and lakes with low or reduced alkalinity; 2) eutrophication of freshwater and marine
ecosystems (with the additional problem of algal toxicity); and 3) direct toxicity of
nitrogenous compounds to aquatic organisms (Camargo and Alonso, 2007). The main
products produced in Latin America and the pesticides that are used or associated are
presented in Table 1.
Countries such as Argentina, Bolivia, Brazil, Paraguay and Uruguay use large amounts of
herbicides like glyphosate because they use genetically modified soybean with resistance to
glyphosate (Fishbein, 2012). In Mexico, Colombia, Ecuador, and Argentina, especially in
flower-producing regions, several pesticides are used, such as: DDT, endosulfan, carbofuran,
methomyl, dimethoate, aldrin, endrin, dieldrin, isodrin, and heptachlor epoxide (Benitez-
Diaz and Miranda-Contreras, 2013). In countries such as Belize, Costa Rica, El Salvador,
Guatemala, Honduras, Nicaragua and Panama, the most used pesticides are: paraquat,
mancozeb, terbufos, methamidophos, methyl, carbofuran, methyl bromide parathion, CCA,
and aldicarb (Wesseling et al., 2003). As a result, pollution from pesticides in Latin America
is evident and the probability increases as the demand for agricultural products increases.
In Mexico it is estimated that 20,000 tons per year of pesticides are used, and in the
2000’s the Mexican states where the most pesticides were used were: Sinaloa, Veracruz,
Jalisco, Nayarit, Sonora, Baja California, Michoacán, Colima, Tabasco, State of Mexico,
Puebla, and Oaxaca (González-Arias et al., 2012).
Finally, several ecotoxicological studies in Latin America indicate the occurrence of high
concentrations of pesticides in heavily populated and industrialized areas (Table 1).

Table 1. Ecotoxicology studies conducted with organic compounds in water (w),

sediments (s) and organisms (o) in different countries of Latin America. The values are
in mg/L for water and in µg/g for sediments and organisms.
Abbreviations: mxv: maximum values, avv: average values,
dw: dry weight, ww: wet weight

Country Site Concentration Reference

Bolivia a) Chanériver w: fluroxypyrmethylhepthyl ester Vargas et al. (2005)
b) Del Palo river a) 9.11, b) 0.025, c) 0.57, d) 0.26, e)
c) Grande river 0.032. mxv
d) Piraí river
e) Well
Brazil Santa Catarina w: Azoxystrobin 0.65 µg/L Stolberg et al. (2015)
Rio Grande do Sul s: Benzo(a)pyrene 1.58. mxv, dw García et al. (2010)
Paranagua Estuarine s: PCB 0.0066. mxv Combi et al. (2013)
System
Costa Grande de Tárcoles o: caudal scutes of American crocodiles, Rainwater et al.
Rica river p,p´-DDE 0.34, p,p´-DDT 254.8, Dieldrin (2007)
0.008, Endrin 0.22, Methoxychlor 0.53.
avv
Mexico Champotón river s: Lindane 70.12. mxv González et al. (2014)

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Additional problems regarding agriculture contamination to aquatic ecosystems are the

intensive use of fertilizers on crops. Fertilizers are applied to remediate the deficiencies of
primary and secondary nutrients and less frequently of micro-nutrients (Fageria et al., 2002).
Other contaminants associated with agriculture and recently created by nanotechnology
materials such as new pesticides, which include several formulations that can consist of
metal nanoparticles, mixtures of metal nanoparticles and pesticides (e.g., imidacloprid or
deltamethrin), nanoemulsions (e.g., permethrin, garlic essential oil, or β-cypermethrin, among
others), nanodispersion (e.g., novaluron and triclosan), among others, could represent an
additional environmental problem to aquatic ecosystems (Kha, 2015).
The industrial problem coming up is the high demand for hydrocarbons and consequently
contamination of aquatic ecosystems. The strong demand for oil will come from transport
since emerging countries are experiencing an expansion of the automobile industry, which
could reach 1,700 million vehicles before 2035 (Gokulakrishnan and Ganeshkumar, 2015).
By then, oil demand will have increased to about 100 million barrels per day (Shell
International, 2008). The intensification of oil extraction increases the environmental risk, for
example: in April 2010 in the Gulf of Mexico 4.9 million barrels of crude oil leaked from the
Macondo well, to which one of the first responses was to apply more than 1 million gallons of
the oil dispersants Corexit 9500® and Corexit 9521A8®; however, when Corexit 9500® and
oil are mixed, the toxicity to Brachionus manjavacas increases up to 52-fold (Rico-Martinez
et al., 2013).
Finally, contamination by endocrine disruptors, pharmaceuticals, and care products is of
concern, and the increase of discharges of these contaminants is complex. For example,
tourism is a sector of great importance and dynamism that generates large profits to many
Latin American countries. Large hotel settlements on coastal zones or waterfronts cause
degradation and pollution of the coastal zone. Additionally, the population pressure and the
development of coastal tourism and infrastructure have significantly affected coastal and
marine ecosystems. Coral reefs have unfortunately been affected: in the Caribbean, 61% of
coral reefs are threatened by the pressure from tourism and pollution (Miguel and Tavares,
2015).
In general, contamination from agriculture and industry in Latin America will be
devastating if it continues with this progress. It is necessary to implement efficient and
continuous monitoring mechanisms to obtain ecological risk of all of these pollutants in Latin
American aquatic ecosystems.

DEPLETION AND CONTAMINATION OF AQUIFERS/GLACIERS

The expansion of mining, industrialization, and other anthropogenic activities has led to
metal contamination in many aquatic ecosystems of the Neotropics (Figure 1). Analysis of
scutes from Morelet’s crocodiles (Crocodylus moreletii) from Belize and American
crocodiles (C. acutus) from Costa Rica revealed the presence of mercury, cadmium, copper,
lead, and zinc (Rainwater et al., 2007) (Table 2).

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1.8

1.6

1.4

1.2

1
mg/L

0.8

0.6 Zn
Pb
Ni
0.4 Mn
Hg
0.2 Fe
Cu Metals
0 Cr VI
Cr III
Cd
As
Al
Sites

Figure 1. Determination of metals in water of different countries of Latin America. Abreviations: LEGR-
Luján, Escobar and Garín Rivers, Argentina; RR-Reconquista River, Argentina; MRR-Matanza-Riachuelo
River, Argentina; STAR-System Tres Arroyos River, Argentina; PLB-Patos Lagoon, Brazil; IAAB-Iguaçú-
Sarapuí System, Brazil; RWB-Refinery wastewater, Brazil; ENDM-El Niagara Dam, Mexico; TRM-Tula
River, Mexico; CAM-Calera aquifer, Mexico; MLN-Managua Lake Nicaragua; ALN-Apoyo Lake,
Nicaragua. Sources: Magdaleno et al. (2001), Silva and Santa (2005), McCrary et al. (2006), Salibián (2006),
Rubio-Franchini et al. (2008), Peluso et al. (2011), Pinhão et al. (2011), Fonseca and Wallner-Kersanach
(2013), Daflon et al. (2015), Rubio-Franchini et al. (2016).

Table 2. Ecotoxicology studies conducted with metals in sediments and organisms in

different countries of Latin America. The values are in µg/g for sediments and
organisms. Abbreviations: mxv: maximum values,
avv: average values, dw: dry weight, ww: wet weight

Country Site Concentration Reference

Sediments Organisms
Argentina Beagle Channel Cu 18.48, Fe 6.0, Amin et al.
Hg 0.41, Pb (1996)
27.65, Zn 62.35.
avv, dw
Bahía Blanca Females of N. granulate, Ni 13.61, Simonetti et al.
Pb 18.23, Zn 35.61. (2012)
Eggs of Neohelice granulate, Ni
6.13, Pb 2.47, Zn 54.07.
mxv, dw
Belize Sapote Lagoon Morelet’s crocodile eggs, Hg 0.23. Rainwater et al.
mxv, ww (2002)
Hondo river Caudal scutes of Morelet’s Buenfil-Rojas et
crocodile. Cd 0.085, Hg 0.0561. al. (2015)
mxv, ww
Gold Button Caudal scutes of Morelet’s Rainwater et al.
Lagoon (GBL) and crocodile. (2007)
the New River GBL, Cu 346.0, Hg 98.7
Watershed (NRW) NRW, Cd 70.7, Cu 451.8, Hg 72.7,
Pb 109.7. avv, ww

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 Ecotoxicological Studies of Freshwater Ecosystems in Latin America 145

Country Site Concentration Reference

Sediments Organisms
Brazil a) Puruzinho lake plankton, Hg Nascimento et
b) Negro and a) 337.0 al. (2007)
Tapajós rivers b) 452.0 and 264.0 Pacheco-Peleja
c) Ribeirão das c) 100.0 (2002)
Lajes dam d) 309.0 Palermo (2002)
d) Hydropower mxv Nascimiento
Plant Reservoir (2006)
Samuel
a) Negro River Hoplias malabaricus, Hg Belger and
b) Santarém and a) 0.35 Forsberg (2006)
Tapajós river b) 0.133 Uryu et al.
c) Madeira river c) 0.38. avv (2001)
Boischio and
Henshel (2000)
Costa Río Grande de Caudal scutes of Morelet’s Rainwater et al.
Rica Tárcoles crocodile, Cd 0.337, Cu 0.125, Hg (2007)
0.093, Pb 0.49, Zn 4.14. avv, ww
Colombia Magdalena river Fish tissues, Hg 0.41 mxv Alvarez et al.
(2012)
Cuba Sagua la Grande Clarias gariepinus, Hg 0.16. mxv, De La Rosa et
River ww al. (2009)
Guacanayabo Gulf Crassostrea rhizophorae, Cu Díaz Rizo et al.
53,000, Fe 278,000, Pb 130, Zn (2010)
219,000 mxv, ww
Guyana a) Konashen river Hg Howard et al.
and creek a) 0.30 (2011)
sediments b) 0.29
b) Iwokrama river mxv, wd
sediments
Mexico SE Gulf of Crassostrea corteziensis, As 11.04 Bergés-Tiznado
California mxv, wd et al. (2013)
a) Chetumal Bay Bone tissue of Antillean manatees, Romero-
b) Coastal region a) Cd 4.9, Cr 10.7, Cu 4.7, Pb 17.7, Calderón et al.
of Campeche Ni 46.5, Zn 129.5, (2015)
b) Cd 4.6, Cr 11.2, Cu 6.3, Pb 16.2,
Ni 11.8, Zn 140.7
mxv, ww
El Niagara dam Pb 20,870. mxv Zooplankton, Pb 1.22 mxv Rubio-Franchini
et al. (2008)
Tula River As 490, Cd 490, Muscle of Oreochromis niloticus, Rubio-Franchini
Pb 8. mxv As 0.0149, Cd 0.06, Pb 0.83. mxv et al. (2016)
Nicaragua a) Managua lake, Fish, Hg McCrary et al.
b) Apoyo lake a) 0.35, b) 0.19. mxv (2006)
Uruguay Montevideo Ag 2.3, Cd 1.6, Muniz et al.
Harbour Cr 253, Cu 135, (2004)
Hg 1.3, Ni 34, Pb
128, Zn 491. mxv
Virgin River Gut Cd 2,400, Cu Ross and
Islands 70,000, Ni 32,000 DeLorenzo
Zn 870, 000. (1997)
mxv, dw

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146 R. Rico-Martínez, I. A. Pérez-Legaspi, G. E. Santos-Medrano et al.

Another way to evaluate water quality is via Toxic Units (TU), frequently used in
ecotoxicology. It represents the ratio between the concentration of a component in a mixture
and its toxicological acute (TUa; e.g., LC50) or chronic (TUc; e.g., long term NOEC)
endpoint. The U.S. Environmental Protection Agency (2004) has recommended a maximal
value of 0.3 TUa and 1.0 TUc. Herkovits et al. (1996) performed a chronic and acute toxicity
test using Bufo arenarum embryos to evaluate the water quality in The Reconquitsa River in
Argentina, obtaining: 10 TUa and 20 TUc. Herkovits et al. (2002) evaluated by means of the
AMPHITOX test acute and chronic exposure in rivers and streams from the Metropolitan area
of Buenos Aires, obtaining a maximal value of 1000 TUc (leach from landfills and solid
industrial wastes) and a minimal value of 1.4 TUa (surface water samples).
In Brazil, De Paiva et al. (2014) sampled effluents of a large steel industry, finding a
maximum value of 3030 TUa. In Aguascalientes (Mexico) Torres-Guzman et al. (2010)
sampled influents and effluents of the most important wastewater treatment plants, obtaining
between 0.1 and 8.3 TUa. Santos-Medrano et al. (2007) determined the toxicity levels in the
San Pedro River in Aguascalientes which were found ranging from 0.36 to 9.91 TUa.
The role of high mountains as ‘cold condensers’ was hypothesized by Calamari et al.
(1991) and confirmed by many authors (Galassi et al., 1997; Blais et al., 1998; McConnell et
al., 1998). High mountains may represent condensation sites for Persistent Organic Pollutants
(POP). Mountain glaciers may be used as ‘natural archives’ for studying historical trends of
pollution. Blais et al. (2001a,b) showed that glaciers are important contributors of POPs to
freshwater systems: melting glaciers supply 50 to 97% of the organochlorine (OC) inputs to
the water system downstream. The prevalence of inputs of OC in the dissolved phase and the
low organic content of glacial-fed waters determines a high bioavailability and toxic risk of
these compounds to aquatic biota. Quiroz et al. (2009) found, in snow samples in the
Aconcagua Mountains (Argentina), polychlorinated biphenyls (PCB) at levels ranging from
0.020 to 0.190 ng/L. These results point out the need of investigating the role of mountains in
the trapping of POPs and the associated risks, including climate change.

PETROLEUM SPILLS/PETROCHEMICAL INDUSTRY

CONTAMINATION IN FRESHWATER
AND COASTAL LAGOONS

With globalization, emerging pollutants are a threat in the region arising from, industry
and mining activities. Surfactants and in particular linear alkylbenzene sulfonates (LAS) are
widely employed in detergent formulations (Penteado et al., 2006).
Although polycyclic aromatic hydrocarbons (PAH) are widespread contaminants, little
information exists relative to most Latin American countries. Available information on PAH
in sediment from Argentina and Uruguay is presented in Figures 2 and 3, respectively. In the
case of Cuba and Guatemala some PAH have been also detected (Figure 4).

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 Ecotoxicological Studies of Freshwater Ecosystems in Latin America 147

Figure 2. Concentrations of PAH in sediments of Bahía Blanca, Argentina. Source: Oliva et al. (2015).

Figure 3. Concentrations of different polycyclic aromatic hydrocarbons in sediment within Montevideo

Harbour, Uruguay. Source: Muniz et al. (2004).

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148 R. Rico-Martínez, I. A. Pérez-Legaspi, G. E. Santos-Medrano et al.

Figure 4. Occurrence of polycyclic aromatic hydrocarbons in Cuba and Guatemala. The left axis
corresponds to Cuba and the right axis to Guatemala. Source: Theodorakis et al. (2012) and Santana et
al. (2015).

Shale Gas

The existence of natural gas in shale has for long been known but technology could not
develop those resources at economically viable costs. Specifically for shale gas, hydraulic
fracturing techniques (fracking) have been developed in which a mixture of water, chemicals,
and sand is pumped into the well to crack open the rock and release the natural gas into the
well (Helms, 2008).
The environmental risks associated with shale gas exploration and production are:

a) Competition for water affects drinking water, wildlife habitat, recreation, agriculture
and industry.
b) Methane leakage into groundwater.
c) Pollution from frackwater disposal on the surface.
d) Induced earthquakes from frackwater injection into disposal wells.
e) Environmental footprint of industrialized landscapes as new wells are constantly
being drilled.

In Latin America, Argentina is attracting the most attention. Preliminary estimates

indicate that only 20% of the prime Vaca Muerta shale basin has liquids (Wilkinson,
2011). Regarding environmental issues, Argentina starts out with an advantage as the shale
gas reserves are largely in the sparsely populated regions of Patagonia and Neuquen.
Nevertheless, there is a growing NGO (non-governmental organization) movement that is
focusing on fracking issues. The growing opposition has linked environmental issues to those

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 Ecotoxicological Studies of Freshwater Ecosystems in Latin America 149

of indigenous rights to create significant obstacles to shale gas development (MercoPress,

2012).
Mexico has the second largest technically recoverable shale gas deposits in Latin
America and the third largest in the world. Mexico’s national oil company (NOC) Pemex
drilled its first well in the Burgos region across the border from Texas, but it cost almost five
times that of those drilled on the Texas side, and came up dry. Pemex produced shale gas for
the first time in March 2011 in Coahuila state (CSUR - Canadian Society for Unconventional
Resources, 2012). The main environmental obstacle for development of shale gas in Mexico
is scarcity of water and the potential contamination of aquifers due to fracking to obtain shale
gas (Mares, 2012).

CYANOBACTERIA AND CYANOBACTERIAL TOXINS

Cyanobacteria blooms are related to high concentrations of nutrients, a process called
eutrophication that is accelerated by human activities such as modern agricultural practices,
fossil fuel extraction and combustion, use of phosphate-containing cleaning agents,
etc. (Serediak et al., 2014). Moreover, due to bloom formation some cyanobacteria genera
can reduce water quality by depleting dissolved oxygen, altering physicochemical and
organoleptic properties, and releasing toxic metabolites (Sivonen and Jones, 1999).
In Latin America, several studies have been conducted to determine cyanobacteria
species distribution and abundance, their ecological role, the effects on the aquatic biota, and
the effects on human population, which in certain occasions have been intoxicated by
cyanobacterial metabolites. Despite the importance of cyanobacteria in freshwater systems,
little information is available regarding bloom formation and persistence in countries from
Central and South America, where limnological studies mainly present algal lists and in
which some bloom-forming cyanobacteria are also included (González, et al. 2004;
Fontúrbel-Rada, 2005). Nonetheless, media and international organizations have documented
the occurrence of blooms in the aforementioned countries (MARN, 2012; Julajuj, 2015; La
Prensa, 2015). Table 3 presents some of the most representative cyanobacterial events in
Latin America.
Some cyanobacteria strains can produce very toxic secondary metabolites, including
toxins such as microcystins, anatoxins, lyngbyatoxin, cylindrospermopsin, microviridins,
among others. The most studied cyanotoxins are microcystins (MC), which have more than
70 chemical variants and the general structure of cyclo(-d-Ala-l-X-erythro-b-methyl-d-
isoAspl-Y-Adda-d-isoGlu-N-ethyldehydro-Ala) (De Figueiredo et al., 2004). Their toxicity
was first described in mice liver. Nowadays there are reports of the potential deleterious
effects on several organs, affecting both phytoplankton and zooplankton, fish, and even
humans due to recreational or accidental exposure, such as that reported in Brazil in which
people died because of the use of MC-contaminated hemodialysis water (Jochimsen et al.,
1998; Carmichael et al., 2001; Azevedo et al., 2002).
Determination of MC has been performed by instrumental or immunological methods
in several countries in Latin America. According to Dörr et al. (2010), there are no official
data or references related to MC occurrence in Colombia, French Guyana, Guyana, Paraguay,
Peru and Venezuela. However, these authors presented a list of MC analogs found in

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150 R. Rico-Martínez, I. A. Pérez-Legaspi, G. E. Santos-Medrano et al.

environmental samples from Argentina, Brazil, Chile, and Uruguay. Additionally, Mexico
counts with few reports regarding MC detection in environmental samples and
ecotoxicological studies assessing the toxic potential of colonial and filamentous
cyanobacteria (Ramírez-García et al., 2004; Arzate-Cárdenas et al., 2010; Sánchez-Chávez et
al., 2011; Pineda-Mendoza et al., 2012; Tomasini-Ortiz et al., 2012).

Table 3. Cyanobacteria found in Latin American freshwater bodies

between 1988 and 2012

Country Dominant cyanobacteria Effects Reference

Argentina Anabaena Bloom formation Dörr et al., 2010
Microcystis Fish mortality
Cyanotoxin production
Brazil Anabaena Bloom formation Azevedo et al., 1994;
Cylindrospermopsis Fish mortality Beyruth, 2000; Bittencourt-
Microcystis Human intoxication Oliveira et al., 2001;
Planktothrix and death Magalhães et al., 2001;
Pseudanabaena Cyanotoxin production Costa et al., 2006; Dos
Anjos et al., 2006
Chile Cylindrospermopsis Cyanotoxin production Campos et al., 1999, 2005;
Microcystis Neumann et al., 2000
Nostoc
Oscillatoria
Planktothrix
Colombia Anabaena Bloom formation Manceraand Vidal, 1994;
Microcystis Cyanotoxin production Rivera-González and
Nostoc Gómez-Gómez, 2010;
Radiocystis Abella and Martínez, 2012
Mexico Arthrospira Bloom formation Oliva-Martínez et al., 2008;
Anabaena Microcystins production Arzate-Cárdenas et al.,
Cylindrospermopsis 2010, Vasconcelos et al.,
Microcystis 2010; Pineda-Mendoza et
Synechocystis al., 2012
Uruguay Anabaena Bloom formation Pérez et al., 1999; Kruk
Microcystis Cyanotoxin production and De Leon, 2002
Nodularia

Cylindrospermosin (CYL) is a very toxic cyanobacterial metabolite that was named after
Cylindrospermopsis raciborskii, although other species can also perform its biosynthesis. It
was first described as a hepatotoxic compound, but it has been proven to be cytotoxic,
dermatotoxic, or genotoxic. CYL is frequently found in tropical water bodies and also in
temperate zones. In Australia it generated the most famous human intoxication due to
consumption of CYL-contaminated drinking water. CYL detection is not a routine analysis
for water quality in Latin America, although it has been found in Brazilian drinking water
supplies. This should be a warning call to improve and increase monitoring and studying of
the causes and effects of cyanotoxins (Bouvy et al., 1999; Bittencourt-Oliveira et al., 2014).
Despite the progress in the description of potential toxic species, limnological studies and
research of the effect of MC on zooplankton, there is not enough information in Latin
America related to other toxins (e.g., anatoxins, lyngbyatoxin, microviridins, nodularins, etc.)

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 Ecotoxicological Studies of Freshwater Ecosystems in Latin America 151

that are also important because of their mode of action. Moreover, cyanotoxins have also been
found in treatment plants where they could not be removed by “conventional” processes.
Instead, tertiary treatments that include advanced oxidation processes have been tested for
their potential use to purify cyanotoxin-contaminated water (Sharma et al., 2012).
Nevertheless, water treatment and research focused on cyanobacterial-blooms effects
should be extended in order to study and implement methods for their prevention. Human
activities have been the main causes that exacerbate the eutrophication process not only in
temperate regions but also in tropical and subtropical areas, where climate easily promotes the
abundance and proliferation of cyanobacterial species. Thus, a synergy between prevention
and remediation should have the desired effect on reducing cyanobacterial related events and
guaranteeing water quality and safety.

CONCLUSION
All the threats that we have presented here have resulted in severe adverse effects on our
freshwater resources. Many of these adverse effects can be categorized as follows:

 Loss of biodiversity in freshwater ecosystems: Almost one quarter of the world’s

inland water fish species are found in Latin America and the Caribbean (UNEP,
2010). However, human use of freshwater resources and water pollution have
adversely affected catches (UNEP, 2010). Many aquatic invertebrate species might
have already been lost due to contamination.
 Eutrophication/Bacterial-Parasite Contamination: Many problems related to human
health issues are related to simple water sewage issues. A part of the population
still has no access to sewage/treatment plants or drinking water systems. Illegal
discharges worsen the problem.
 Bioaccumulation, bioconcentration, biomagnification: These processes affect many
aquatic ecosystems and cause serious human health concerns.
 Aquifer contamination and depletion occurs as a consequence of severe exploitation
of underground water: Arsenic and fluor are found at higher concentrations in water
as result of deeper drilling to obtain water from aquifers. Drinking water and
wastewater treatment costs are rising.
 Freshwater salinization: Construction in coastal areas sometimes causes freshwater
salinization, affecting estuarine and riverine populations. Mangroves are especially
sensitive because they act as detoxification centers in rivers, coastal lagoons, and
estuaries.

In order to face all these adverse effects we need a better integration in the subcontinent.
Despite the existence of several ecotoxicological agencies in Latin America, the lack of
integration is clear. Although there is an entity called SETAC – Latin America, this society is
clearly a South American branch of the Society for Environmental Toxicology and Chemistry
(SETAC); the representation from North America (Mexico) and the Caribbean is very poor.
Regional or national chapters are also present: Asociación Mesoamericana de Ecotoxicología
y Química Ambiental (AMEQA) is a Mexican chapter of SETAC. There is also a SETAC

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Argentina and an ECOTOX Brazil. A real integration of ecotoxicological societies is missing

in Latin America perhaps because they face the same problems that the national chapters
experience: a) lack of governmental support; b) low number of associates; c) economical
penuries; d) lack of interest or conflicts among associates. Many countries have governmental
agencies that deal with environmental issues at the Municipal, State and Federal level. In
some cases, like La Plata River Basin, there is interaction among countries and international
agencies to deal with freshwater resources (Natale, 2005).

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 11

FRESHWATER ECOTOXICOLOGY IN COSTA RICA

Rocío Ugalde*, Silvia Echeverría-Saénz, Elba de la Cruz,

Margaret Pinnock-Branford, Freylan Mena and Luisa E. Castillo
Central American Institute for Studies on Toxic Substances (IRET),
Universidad Nacional, Heredia, Costa Rica

ABSTRACT
Synthetic pesticides were introduced in Costa Rica at the beginning of the last
century, and even though the cultivated area in the country has remained relatively stable
over the last 20 years, pesticide usage has increased substantially. Currently, Costa Rica
is the largest importer of pesticides in Central America (24.8 kg active ingredient
(a.i.)/ha/yr) and one of the biggest importers per capita in the world. Intensive use of
pesticides coupled with the climatic and topographic characteristics of the country have
triggered continuous aquatic pollution, biota mortalities, changes in communities’
structure, and biochemical and physiological effects. Since before 1990 Central American
Institute for Studies on Toxic Substances (IRET) researchers have used ecotoxicological
tools such as bioassays and biomarkers to study pesticide pollution and exposure effects.
Both standard test organisms and native species, found at different levels of biological
organization, have been used. Results of those studies are presented in this chapter,
highlighting the vulnerability of Costa Rica’s freshwaters. Wiser management of
pesticides is necessary to protect the integrity of this tropical ecosystem.

Keywords: Costa Rica, freshwater ecosystems, pesticides, ecotoxicology

INTRODUCTION
Costa Rica, a Central American country, is divided by a middle mountainous chain,
which results in a hydrographic system with two versants, one towards the Pacific and one
towards the Caribbean (Trejos, 1991). The Pacific side has dry (December to April) and rainy

*
Corresponding Author E-mail: maria.ugalde.salazar@una.cr.

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(May to November) seasons, while the Caribbean versant lacks clear seasonality, with rain
(1500–>7000 P/mm) throughout the whole year (Figure 1).
The climatic and topographic characteristics of Costa Rica are suitable for agriculture,
and the country´s economy relies on export of products such as coffee, pineapple, and
bananas. This business requires continuous production, and therefore pesticides were
introduced at the beginning of the 1900s (Hilje et al., 1987).
The import of pesticides has increased greatly and keeps increasing, even though the
cultivated area has remained relatively stable over the last 20 years (de la Cruz et al., 2014a).
Costa Rica is the largest importer of pesticides in Central America at 24.8 kg active ingredient
(a.i.)/ha/yr (Bravo et al., 2015) and one of the largest importers per capita in the world,
resulting in environmental problems in aquatic ecosystems.

Figure 1. The four regions of Costa Rica where ecotoxicological studies have been conducted, with
respective average annual temperature (T/ºC) and precipitation (P/mm).
Geannina Moraga López, IRET-UNA, Costa Rica. 2016. Administrative Division of GADM database of
Global Administrative Areas, 2016. Climatological Atlas, National Meteorological Institute of Costa
Rica, 2010.

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 Freshwater Ecotoxicology in Costa Rica 165

Table 1. Methods used for freshwater ecotoxicological studies at IRET

Type of study Methods

Biomarkers Cholinesterase activity (ChE), lipid peroxidation (LPO), glutathione
S-transferase (GST), catalase activity (CAT), vitellogenin (VTG), and
cytochrome P450 (CYP1A) on organisms exposed to sublethal
concentrations of pesticides (Mena et al., 2014a,b). (See chapter 2,
“Biomarkers in Native Central American Species,” of this book.)
Clam Anodontites luteola Mortality, avoidance, weight loss and biomarker analysis after 96 h
exposure to either oxitetracycline (OTC) or environmental sediment
samples (Arias-Andrés et al., 2014a).
Water flea Daphnia magna Acute toxicity (48 h) (OECD, 2004).
Chronic test (21 d) (EPS, 1990); in situ toxicity for survival and 24 h
feeding rate (de la Cruz et al., 2012).
Green algae Pseudokirchneriella Growth inhibition (72 h) (EPS, 2007a).
subcapitata
Macrophyte Lemna minor Growth inhibition (7 d) (EPS, 2007b).
Onion Allium cepa Root growth test (72 h) (Fiskesjö, 1993).
Hydra attenuata and Hydra Morphological changes (96 h), microplate-based bioassay (Trottier et
viridissima al., 1997).
Nematode Panagrellus redivivus 96 h assay: Survival, growth, maturation, and total fitness values of
nematodes (Samoiloff, 1990).
Lettuce seed Lactuca sativa 120 h assay: Seed germination inhibition, root and stem growth
inhibition (Dutka, 1989).
Muta-ChromoPlate test kit 5 d test: Numbers of positive wells compared to those containing only
(fluctuation kit) sterile water and bacterial test strain (Salmonella typhimurium) (Rao
and Lifshitz, 1995).
Hyalella azteca 10 d standard sediments test (EPS, 2013).
Microalgae indicators Chlorophyll concentration in water (Eaton et al., 1995);
phytoplankton abundance and diversity (Throndsen, 1978);
microphytobenthos abundance and diversity (CHEbro, 2005).
Macroinvertebrate community Abundance, richness, diversity, colonization of artificial substrates
(MC) structure (Castillo et al., 2006) and BMWP-CR (MINAE-S, 2007)
Other biological assessment Riparian vegetation and river habitat evaluated applying indexes
(Acosta et al., 2009) and fish mortalities recorded.
Ecological risk assessment (ERA) Tier I and tier II risk index (RI) based on predicted environmental
concentrations/predicted no effect concentration (Posthuma et al.,
2002; ECJRC, 2003).
BMWP-CR, Biological Monitoring Working Party index modified for Costa Rica.

Before 1995, most studies involving agrochemicals on freshwater ecosystems of Central

America focused only on pesticide presence, with no further analysis on ecotoxicological
responses (Castillo et al., 1997). With creation of the Central American Institute for Studies
on Toxic Substances (IRET) of the Universidad Nacional there have been assessments of the
agricultural import and use of pesticides (Bravo et al., 2011, 2015), levels of human and
environmental exposure (CGR, 2005, 2013; Castillo et al., 2012), and environmental effects
(Castillo et al., 2006; Ugalde, 2007; Castillo et al., 2012; Echeverría-Sáenz et al., 2012a; de la
Cruz et al., 2014b) and risks for human and wildlife populations of tropical ecosystems
(Arias-Andrés, 2011; de la Cruz et al., 2014a,b; Echeverría-Sáenz et al., 2015b). Created in

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1994, the Laboratory for Ecotoxicological Studies (ECOTOX) of IRET was the first
laboratory of its kind in Central America, driving and supporting all the aforementioned
ecotoxicological research.
In this chapter the most relevant results of the investigations conducted by IRET are
presented in five sections, four representing regions of the country—Central, North,
Caribbean, and Pacific (Figure 1)—and a fifth section on studies with native species. Results
address the most important ecotoxicological effects, with relevant conclusions from
ecotoxicological studies in Costa Rica.

METHODS
IRET takes a multidisciplinary approach to its research, combining biology, ecology,
toxicology, chemistry, agriculture, technology, and remote sensing including land use
mapping. Each investigation diagnoses pesticide use in the crops of the study area (Bravo et
al., 2011). Water and sediment sampling follows the standard protocols described by the U.S.
Environmental Protection agency (USEPA, 2013).
Samples were taken randomly and were not associated with pesticide applications, so
pesticide concentration peaks that may occur after applications and rain events were not
always reflected in the results. Pesticide residue analyses in water, sediments, and tissues
were performed at IRET in the Laboratory of Pesticide Residue Analysis (LAREP) according
to the multi-residue method for pesticide analysis in water samples, using solid phase
extraction and gas chromatography, according to SW846 3546 and ASTM 6010 procedures
(Castillo et al., 1998, 2000a; Diepens et al., 2014). All the information was related to land use
so that empirical relationships can be developed to predict levels of pesticide patterns in other
areas. Predictive models can also be developed for management use.
The effects of toxic substances on aquatic biota were assessed using an established
battery of techniques including organisms from different trophic levels at ECOTOX. All tests
were based on available standards for environmental assessments (Table 1).

RESULTS AND DISCUSSION

Central Region

The Central Valley (CV) is the most urbanized, populated (60% of the 4.5 million total
population), and economically active region of Costa Rica. Surface and groundwater of the
CV are receptors of many pollutants from agricultural, domestic, and industrial activities
(Astorga and Coto, 1996; CGR, 2005; Fournier et al., 2010; CGR, 2013; Reynolds, 2013).
Astorga et al. (1997) conducted research of water quality along the basin of the Grande
de Tárcoles River. The study included chemical analysis of industrial and domestic effluents,
river water and sediments, benthic MC composition, and toxkit tests with Thamnocephalus
platyurus, Brachionus calyciflorus, and Selenastrum capricornutum (currently known as
Pseudokirchneriella subcapitata). Taxa richness was higher during the rainy season, and

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decreased during the dry period. According to toxicity tests, water quality decreased from
upstream to the middle and downstream, where there is greater influence of industrial, urban,
and agricultural activities.
Mo (2001) assessed the impact caused by pesticides used in flower and leather leaf fern
production to surface and groundwater, and the effects on Ceriodaphania dubia survival and
reproduction. Acute toxicity was found in one-third of surface water samples, in which the
detected pesticide residues were chlorothalonil, propiconazole, vinclozolin, prochloraz,
diazinon, dimethoate, endosulfan, pirimicarb, malathion, and pirimiphos-methyl.
Fournier et al. (2010) conducted a study to determine agrochemical contamination of
water, soil, and vegetables in an horticulture watershed located in Plantón-Pacayas, in
Cartago Province. Chlorpyrifos was detected in 55% of the water samples ( 0.07 μg/L),
pentachloroaniline in 48% ( 0.03 μg/L), flutolanil in 40% ( 0.66 μg/L), clorotalonil in 36%
( 0.70 μg/L), and quintozene in 24% ( 0.12 μg/L). Soil samples also were positive to
pesticide presence: pentachloroaniline was detected in 90% of the samples ( 0.35 mg/kg),
hexachlorobenzene in 76% ( 0.06 mg/kg), chlorpyrifos in 73% ( 0.15 mg/kg), clorotalonil
in 72% ( 0.24 mg/kg), DDE-pp in 59% ( 0.09 mg/kg), quintozene in 48% ( 0.17 mg/kg),
flutolanil in 41% ( 0.89 mg/kg), prothiofos in 36% ( 0.30 mg/kg), DDT-pp in 35% ( 0.14
mg/kg), and pentachlorobenzene in 24% ( 0.02 mg/kg). Finally, concentrations (mg/kg)
found in vegetables that exceed national and international standards were clorotalonil (0.06),
permethrin (0.15), pentachloroaniline (0.08), hexachlorobenzene (0.04), pentachlorobenzene
(0.04), flutolanil (0.27), and quintozene (0.04).
MC was assessed during the third year at Pacayas (upstream, middle, and downstream)
and at Plantón (upstream and middle). The BMWP-CR index was applied; water quality was
poor, polluted, or heavily polluted at all sampled points. The authors concluded that the water
quality is not suitable for biological communities and ecosystem function.

North Region

The north zone of Costa Rica is characterized by flat lowlands dedicated mainly to
livestock and agriculture (orange, tubers, pineapple, and rice) and a very rainy climate.
Higher irregular lands on the mountain slopes are dedicated to horticulture and milk farming.
During the 1990s the presence and effect were studied of pesticides used in agricultural
areas on the San Juan River (SJR) ecosystems. Chlorothalonil, chlorpyriphos, and cadusaphos
were detected in SJR effluent. However, surface water presented no acute toxicity to D.
magna, T. platyurus, B. calyciflorus, and H. attenuata; nor were effects on MC diversity
observed (Astorga, 1997).
In 2011 an evaluation was started on the Frío River basin (Fournier et al., 2016). The
diagnosis of pesticide use revealed that 58 active ingredients are applied to the main crops in
the area, including herbicides, fungicides, insecticides, nematicides and/or acaricides, and
compounds with antimicrobial activity. Standard tests are used to determine toxicity of a
single compound, but with 58 different chemicals, the synergistic response cannot be
discarded. The Frío, Sabogal, Thiales, Mónico, and Samen Rivers and the Caño Negro
wetland were sampled for presence of pesticides, acute and chronic toxicity to cladocerans,

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biomarker alterations in fish exposed in situ, and effects in the MC. Ametryn, diuron,
bromacil, cypermetryn, diazinon, and ethoprophos were detected in water samples from
the different rivers. No acute effects were observed on the zooplankter Simocephalus
semiserrulatus, and effects on reproduction were obtained in samples from sites where no
pesticides were detected. MC diversity was similar among sites, and the BMWP-CR index
classified most of the sites (60%) as moderately polluted aquatic environments. Biomarkers
measured in fish (fingerlings of Parachromis dovii) demonstrated cholinesterase inhibition at
two of the four evaluated sites (Mónico and Thiales) and GST induction at one of them
(Mónico); these sites had presence of pesticides.
Recently, a study was started in the higher-altitude Zarcero horticulture area (Ramírez et
al., 2015). Evaluation of pesticide use has identified at least 65 a.i. (among fungicides,
insecticides, and herbicides), detecting 21 of them in river and underground water samples.
Acute effects have been observed on primary producers: L. sativa root growth inhibition in
water samples from four sites; inhibition of P. subcapitata growth in a sample taken
downstream from a potato field treated with paraquat; and growth stimulation in samples
taken from sites with high phosphorus content. Acute effects (20% mortality) on D. magna
have been observed in results obtained after exposure to a river water sample with diazinon
residues. The studied sites were classified as moderately polluted, polluted, and very polluted
by the BMWP-CR index.

Caribbean Region

This region has been subjected to agricultural pressure since the late 1800s, when banana
cultivation first started. Today other products, such as rice, ornamental plants, papaya,
cassava, cacao, and especially pineapple are also grown in this region. Pesticide consumption
has reached 73 kg a.i./ha/yr in banana plantations, followed by pineapple (43 kg a.i./ha/yr),
plantain (25 kg a.i./ha/yr), ornamental plants (≈17 kg a.i./ha/yr), and papaya (13 kg a.i./ha/yr)
(Echeverría-Sáenz et al., 2015b).
Pesticide usage combined with high precipitation (3500 P/mm/yr) and very few
soil conservation practices enhance the transport of agrochemicals into surface waters. These
aquatic ecosystems receive pesticides almost every day of the year due to the lack
of a marked dry season, year-round cultivation, and consequent continuous agrochemical
application.
The most used pesticides in the Caribbean region include mancozeb, chlorothalonil,
conazoles (fungicides), ethoprophos, diazinon, carbaryl, terbufos (insecticides/nematicides),
glyphosate, diuron, 2,4-D, bromacil, and ametryn (herbicides). The toxicity of these
substances to aquatic organisms is known to be very high (de la Cruz et al., 2012), and some
of these are regularly detected in water, with the aggravation of being reported in mixtures of
more than five a.i. in most samples (Castillo 2000a, 2006; Echeverría-Sáenz et al., 2012b,
2015a,b, 2016). This scenario poses high risk to aquatic ecosystems in this region and, as
noted above, most laboratory tests involve single chemicals instead of mixtures. Therefore,
acute and chronic ecotoxicology tests have been performed to assess the toxicity of field
water samples in a battery of organisms from primary producers to invertebrates and fish.

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Acute tests have not provided good information on effects in the majority of cases, since
pesticide concentrations in the field are usually below the lowest lethal concentration for 50%
of exposed individuals (LC50). Nevertheless in some cases, acute toxicity was detected.
Echeverría-Sáenz et al. (2012b) found 80% and 60% immobility of D. magna (48 h) when
exposed to water from the Santa Clara and Limbo Rivers, respectively, which contained
(ug/L) diazinon (0.5–1.0), diuron (0.6–2.0), carbaryl (0.7–7.0), ametryn (0.4–9.0), hexazinone
(1.5), bromacil (1.0), and ethoprophos (0.4). The same water samples caused 45% and 26%
stem growth inhibition and 35% and 24% root growth inhibition of L. sativa, respectively. In
another study area, Echeverría-Saénz et al. (2016) found 60% immobility of D. magna (48 h)
when exposed to water from the Caño Azul River (Madre de Dios River watershed). In the
latter site 21 a.i. were detected throughout the study, with highest concentrations (µg/L)
reported for diuron (0.17–3.01), carbaryl (0.98–2.22), azoxystrobin (0.23–2.10), ethoprophos
(0.05–1.56), and diazinon (0.02–0.63).
Chronic tests have helped us understand the effects of field pesticide concentrations
on aquatic biota. Suárez et al. (2008) reported 50% reduction in reproduction of D. ambigua
(15 d bioassay) exposed to water from a banana drainage canal with detected ethoprophos
(0.2–4.0 µg/L), difenoconazole (0.1–3.0 µg/L), and chlorpyrifos (traces to 0.03 µg/L).
Echeverría-Sáenz et al. (2015b), in a 21-day D. magna bioassay, registered 100% mortality
(0% reproduction) when exposing the organisms to three water samples from the Jiménez
and Parismina Rivers. Other samples from the Parismina River watershed showed <50%
reproduction compared to a negative control. The reported diminished reproduction of
D. magna was directly related to pesticide concentrations in the water. A total of 23 pesticide
residues were detected in the study area, but the three samples with the highest
toxicity consistently contained (µg/L) chlorpyriphos (0.05–0.08), diazinon (0.12–0.30),
ethoprophos (0.04–0.36), propiconazole (0.10–0.24), metalaxil (0.1–0.2), ametryn (0.20–
0.55), bromacil (0.8–1.6), and diuron (0.4–1.0). Other pesticides detected in those
water samples included carbaryl, buprofezin, difenoconazole, epoxiconazole, pyrimethanil,
azoxystrobin, and hexazinone in concentrations <0.85 µg/L.
Neurotoxic biomarkers used to indicate the impact of pesticides that affect the nervous
system—such as ChE—have been included in recent studies. Mena et al. (2014a) conducted
96-hour laboratory experiments with the fish Astyanax aeneus with solutions of 0–1 mg/L
ethoprophos, resulting in ChE inhibition and detection of this substance in fish muscle after
exposure. However, in situ experiments (caged A. aeneus, 48 h) did not render consistent
results, as ChE activities did not vary between sites along a pesticide gradient. Echeverría-
Sáenz et al. (2012b) found ChE inhibition in muscle and brain and increased stress enzyme
(GST) in liver of Poecilia gilli exposed in situ along agricultural sites of the Jiménez River in
the same water samples that generated acute toxicity in D. magna. Echeverría-Saénz et al.
(2016) applied a biomarker battery (GST, LPO, CAT, and ChE) (Table 1) and found
intensified responses in highly polluted sites of the Madre de Dios River watershed. More
details are given in “Biomarkers in Native Central American Species,” chapter 2 of this book.
Ugalde (2007) analyzed differences in the abundance and diversity of phytoplankton
between two sites on the Limbo River (influenced by pineapple plantations) and a reference
stream. The results indicated increased abundance of microalgae in the most impacted site,
where pesticide residues (µg/L) of bromacil (0.3–2.3), ametryn (0.1–0.3), diuron (0.06–0.30),
chlorothalonil (0.1), triadimefon (0.1–0.2), diazinon (0.07–0.40), and ethoprophos (0.2) were

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detected, also with higher nitrates. Other results included absence of sensitive species that
were present at the reference sites. Species known to be indicators of pollution were more
abundant in the Limbo River.
Castillo et al. (2006), Suárez et al. (2008), and Echeverría-Sáenz et al. (2012b, 2015a)
collected MC samples in several impacted and reference sites throughout the Caribbean; in all
of them, a clear difference in MC structure between reference and impacted agricultural sites
was noticed. Richness, biodiversity, and percentage of sensitive organisms as well as biotic
indices for organic pollution (BMWP-CR) diminished in agricultural streams. The MC was
also affected by riparian habitat degradation and stream/river channel characteristics. This
community of organisms provided valuable information on effects and sensitivity to
pesticides, in some cases even more than that obtained by laboratory toxicity tests.
Degradation rate (DR) of organic matter was measured by Suárez et al. (2008) as an
ecological functioning endpoint. They compared DR in rivers inside the La Selva biological
station (LSBS) and drainage canals of a banana plantation. Leaf DR was highest in the third-
order streams where higher diversity of macroinvertebrates was also found. Smaller-order
streams inside the LSBS had lower DR than third-order banana drainage waterways.
Increased water velocity and temperature could account for enhanced mechanical and
microbial degradation in that ecosystem.
Overall, more than 30 pesticide residues and two metabolites have been detected in water
samples of the Caribbean region, in mixtures of herbicides, insecticides (the majority of them
being organophosphates), and fungicides. Field concentrations of individual substances rarely
exceed 2 µg/L; however the sum of all the a.i. detected in a single water sample has reached
more than 10 µg/L. Aquatic biota and ecosystems of this region are in high risk.

Pacific Region

Seven of the major crops of the Pacific Versant (PV) cover 71%–98% of the area
cultivated in this region (SEPSA, 2012, 2014). Coffee, palm oil, and some pineapple are
produced in the central and southern PV, sugar cane and melon in the central and northern
regions, and rice and pastures towards the north and the south. Among these crops, melon has
the highest pesticide use (60.5–258.0 kg a.i./ha/yr), followed by pineapple (24.5–73.0 kg
a.i./ha/yr), rice (9.5–18.9 kg a.i./ha/yr), and sugar cane (15.05 kg a.i./ha/yr). All other crops
have pesticide usages below 7 kg a.i./ha/yr (CGR, 2005; MAG, 2007, 2011a,b,c; CNP, 2010;
Bravo et al., 2013; Chaves and Chavarría, 2013; Echeverría-Saénz et al., 2015a; INEC, 2015).
Most commonly used in these crops are herbicides 2,4-D, glyphosate, paraquat, terbutryn,
ametryn, bromacil, and diuron; insecticides permethrin, diazinon, and dimethoate; and
fungicides mancozeb, fosetyl, and epoxiconazole (CGR, 2005; MAG, 2007, 2011a,b,c; CNP,
2010; Bravo et al., 2013; Chaves and Chavarría, 2013). The toxicity of these substances to
aquatic biota is well known (Solomon et al., 2001; Berenzen et al., 2005; Campagna et al.,
2006; Liu et al., 2013; Marin-Morales et al., 2013; de la Cruz et al., 2014a; Lewis et al.,
2016).
Ecotoxicological studies on freshwater systems of the PV have been mostly focused on
exposure and effects and less on the overall risk to the aquatic ecosystem (Castillo et al.,
2012; CGR, 2013; de la Cruz et al., 2014a,b). Before the year 2000, pollution studies were

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primarily on organochlorine (OC) residues and their metabolites. Hidalgo (1986) described
the relationship between OC levels (specifically p,p´-DDE) and egg shell thickness of
waterfowl inhabiting the lower reaches of the Tempisque River basin (TRB). High
concentrations (4.16 mg/kg fw) of OC (including heptachlor epoxide, HCB, p,p´-DDT, p,p´-
DDE, and endrin) and of p,p´-DDE (3.19 mg/kg fw) were found in eggs of wood stork
Mycteria americana. The eggs with higher concentrations of DDE had cracks. Fyfe et al.
(1990) found high levels of DDE in eggs of Nyctiocorax nyctiocorax (4.59 mg/kg fw) and
Hirundo rustica (3.35 mg/kg fw) and low levels (0.03–1.84 mg/kg fw) in other water birds
such as Calidris mauri and Tringa semipalmata, prey of the peregrine falcon. Amphibians
and turtles of the Santa Rosa National Park (where pesticides are not applied) showed low
levels but high frequency of OC pesticide contamination (Klemens et al., 2003). Maximum
concentrations (ng/g fw) of OC reported for amphibians and turtles were p,p´-DDE (55 and
125), delta-BHC (40 and 11), heptachlor (32 and 17), and dieldrin (3.7 and 4.6). Higher OC
levels were also reported in mayflies’ larval tissue (Euthyplocia hecuba) from agriculture-
impacted forests of the upper and middle TRB compared with non-impacted forests (Standley
and Sweeney, 1995). OC pesticide (ng/g ww) found in the mayfly were aHCH (<6–250),
gHCH (<6–250), heptachlor epoxide (38), alfa-endosulfan (51), aldrin (54), dieldrin (100),
beta-endosulfan (150), endosulfan sulfate (2001), DDE (67), DDT (<6–250), aldrin (54),
dieldrin (100), endrin (<12–500), and endrin aldehide (150).
Most of the ecological effects of pesticides in aquatic ecosystems of the PV have
been in the Arenal Tempisque Irrigation District (ATID) of the TRB, which drains into
several protected areas, among them Palo Verde National Park (PVNP), which protects
valuable wetlands. Nearly 28,000 ha of rice, sugar cane, and pastures, among other crops,
are irrigated through a series of channels (de la Cruz et al., 2014a). Twenty-eight a.i. have
been reported in surface waters of this region. Maximum concentration (µg/L) of the most
frequent herbicides are ametryn (3.0), buthaclor (13.3), bentazon (4.39), 2.4-D (1.02), diuron
(8.4), hexazinon (4.6), and terbutryn (6.5); insecticides carbofuran (0.107), cypermethrin
(12.4), dimethoate (8.0), endosulfan (4.0), and triazophos (6.7); and fungicides epoxiconazole
(1.7), propiconazole (2.5), tebuconazole (1.2), organophosphate insecticide-nematicide, and
terbufos (7.2) (CGR, 2005, 2013; Arias et al., 2014c; de la Cruz et al., 2014a).
Martínez (1998), Rizo-Patrón (2003), Fournier et al. (2006), and Echeverría-Sáenz et al.
(2012a) studied the MC of water ecosystems before and after planting of rice and sugar cane
plantations in the ATID. The biodiversity and percentage of pollution-sensitive taxa (e.g.,
Trichoptera and Ephemeroptera) diminished in streams with agricultural influence, as did the
BMWP-CR index. In contrast, the abundance of organisms resistant to poor water quality
organisms such as snails increased. The most common pesticides in those surface waters
were ametryn, cypermethrin, diazinon, dimethoate, diuron, endosulfan, epoxiconazole,
methamidophos, propiconazole, terbutryn, and triazophos.
Laboratory studies reported 16% mortality in D. magna (48 h), but no growth difference
in P. subcapitata (72 h) exposed to water from aquatic ecosystems influenced by rice and
sugar cane production in the northern sector of the PVNP. Water samples contained low
levels of dimethoate (toxic to D. magna) and high nutrient content (Fournier et al., 2006). Up
to 55% mortality was obtained in laboratory tests with D. magna (48 h) exposed to water
from a contaminated Tempisque River site, which drains rice and sugarcane crops, and 3%
mortality from the Barbudal reference site (B). More than six a.i. have been reported in the

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CVT. The same water samples from CVT and B sites show stem growth inhibition of L.
sativa (120 h) of 3% and 8%, respectively, and slight root growth promotion in both sites.
Survival percentage and feeding rate (intake of P. subcapitata cells per hour) of D. magna
from in situ exposure experiments were also higher in B than in the CVT. Risk estimations
(tier 1) of the pesticides in the aquatic ecosystem of the PV were high for almost all the
a.i. detected, and were especially high for cypermethrin, endosulfan, triazophos, diuron,
propiconazole, dimethoate, and terbutryn (de la Cruz et al., 2012).
Pesticide pollution and integrated responses of biochemical markers in transplanted fish
(P. dovii and P. gillii) were used to monitor environmental hazards in the PVNP. High GST
and CAT enzyme activities and elevated levels of LPO in P. dovii were observed at the
Cabuyo (C) site impacted by rice fields. Mena et al. (2014b) associate these effects with HCB
and triazole fungicides in the water. Fish transplanted across pesticide-contaminated sites near
the PVNP presented significant changes regarding hepatic Cyp1A: P. dovii Cyp1A levels
were enhanced in B and C during the rainy season, whereas in P. gillii fish, Cyp1A levels
responded seasonally. VTG induction in field experiments was not observed in either of the
two species (Navarro et al., 2014).
In the ATID, antibiotics are used in pig farms, aquaculture, and rice farming (de la Cruz
et al., 2014b). Water from sediments impacted by pig farming affected burrowing behavior in
A. luteola and caused higher mortality in D. magna (48 h), in comparison to aquaculture and
rice farming. Integrated biomarker responses (ChE, GST, and LPO) in A. luteola tissue
classified the pig and rice farms as the most stressful for exposed organisms. Sulfamethazine
and oxytetracycline (OCT) were detected in effluents of pig farms and OCT in fish farms
(Arias-Andrés et al., 2014b). A laboratory test of several days with tetracycline (TET) and 4-
epitetracycline (ETC) showed functional and structural alteration in sediment microbial
communities from an ATID tilapia farm. When community-level physiological profiles and
phospholipid fatty acid profiles of the exposed microbial communities were contrasted, both
compounds caused opposing metabolite responses. At concentrations >50 mg/kg, TET had a
tendency to inhibit respiration, whereas ETC showed the opposite effect. From fatty acid
analyses it was shown that the sediment analyzed was predominantly colonized by gram-
negative bacteria. The authors concluded that TET and ETC antibiotics impacted the
sediment microbial community in a unique way, with TET effects stronger. Derivative studies
are important in antibiotics ecotoxicology (Granados-Chinchilla et al., 2013). Bacterial
profiles in sediment collected from a protected wetland and from pig, rice, and fish farms
exposed to various concentrations of OTC indicated basal levels of OTC tolerance in the
protected wetland and increased levels in proportion to the intensity of antibiotic exposure:
agriculture followed by aquaculture and swine farming (Arias-Andrés et al., 2014a).
The pesticides most frequently reported in the waters of the Volcan River in the south
Pacific area of Costa Rica were >75% bromacil, >10% diazinon and permethrin, and <1%
chlorpyrifos and fenthion. Laboratory studies reported 50%–70% mortality in D. magna (48
h), and 41%–48% root growth inhibition of L. sativa (120 h) exposed to water samples
collected from the river and streams under the influence of pineapple crops. Pineapple
production does not have a control system to prevent runoff and soil loss, favoring
agrochemical presence in the surrounding water bodies (Castillo and Ruepert, 2001).

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 Freshwater Ecotoxicology in Costa Rica 173

Table 2. Toxicity of relevant pesticides used in Costa Rican agriculture in native species
of different taxonomic groups

Taxonomic Test species Pesticide Native vs. standard species Lethal or Source
group sensitivity* effective
concentration
for 50% of
exposed
individuals
Crustacea Simocephalus Ethoprophos ≈ C. dubia LC50-48 h Not
semiserratus > D. magna (10x) published
Diazinon < C. dubia LC50-48 h
> D. magna
Daphnia Ethoprophos ≈ C. dubia LC50-48 h 1,2
ambigua > D. magna (10x)
Diazinon ≈ C. dubia LC50-48 h 3
> D. magna (10x)
Carbofuran > C. dubia (20x), D. magna (30x) LC50-48 h
Chlorpyrifos ≈ C. dubia LC50-48 h 2,3
> D. magna (2x)
Macrobrachium Ethoprophos < Peneaus (saltwater) LC50-96 h 3
diguetti > decapoda (Uca)
Diazinon ≈ Peneaus LC50-96 h
< Paratya (10x)
Carbofuran > M. kistnensis (6x) LC50-96 h
Fish Parachromis Ethoprophos > Lepomis macrochirus, Cyprinus LC50-96 h 2
dovii carpio, Oncorhynchus mykiss,
Carassius auratus
Carbofuran < Danio rerio (2x) LC50-96 h 3
> Salmo trutta, O. mykiss, L.
macrochirus, Pimephales promelas,
C. carpio
Chlorpyrifos < L. macrochirus (20x), LC50-96 h 2
O. mykiss (13x)
≈ P. promelas, C. carpio
Astyanax Ethoprophos < P. dovii (2x), L. macrochirus LC50-96 h 4
aeneus > C. carpio, A. tropicus, O. mykiss, C.
auratus
Atractosteus < P. dovii, L. macrochirus, A. aeneus, LC50-96 h 4
tropicus C. carpio
> O. mykiss, C. auratus
Amphibians Agalychnis Chlorothalonil >Bufo bufo japonicas (6x), LC50-96 h 5
callidryas Rana limnocharis (10x)
Isthmohyla >Bufo bufo japonicas (6x), LC50-96 h
pseudopuma Rana limnocharis (10x)
Smilisca >B. bufo japonicas (6x), LC50-96 h
baudinii R. limnocharis (10x)
Aquatic Lemna sp. Diuron ≈L. perpusilla, L. minor, EC50-7 d 3
plants L. polyrhiza
1
Arias-Andrés et al., 2014b. 2Diepens et al., 2014. 3Arias-Andrés et al., 2016. 4Mena et al., 2012. 5Méndez et al.,
2016.
*Comparison with species within same taxonomic group, same toxicological endpoint, and similar assay conditions.
For comparison, ECOTOX-EPA database (USEPA, 2015) and PAN pesticide database (PAN, 2015) were
consulted.

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174 Rocío Ugalde, Silvia Echeverría-Saénz, Elba de la Cruz, et al.

Pesticide Toxicity with Native Species

Since the first studies of water quality with standard (temperate) species in Costa Rica
(Castillo et al., 2000b), it became evident that inclusion of relevant native organisms is ideal
for environmental impact assessment in the tropics. Therefore, further investigation on native
species has been conducted at ECOTOX (Table 2) to check their suitability as test species.

CONCLUSIONS
Agriculture is the second most important sector of the Costa Rican economy, and the use
of pesticides has been identified as a major problem for our ecosystems. At IRET, researchers
have been concerned about the high use of toxic substances in agriculture and have been
working on it for several years, trying to determine the impact of these substances on
ecosystems. Considering the current context of global climate change, emergent pollutants
and elucidation of the complexity with which pollution affects ecosystems, ecotoxicologists
should keep adapting and improving the research field in order to characterize and protect
ecosystem functions and services.
It should be a goal in our countries to increase the sociopolitical impact of results
obtained from ecotoxicological studies for creation of new public policies, aimed for better
environmental protection and management of aquatic ecosystems. The current situation of
agrochemical water contamination in Costa Rica makes it imperative to establish national
freshwater quality monitoring to rigorously assess the use and emissions of pesticides,
environmental health, and the effect of the constant agrochemical exposure of biota.
Implementation of good agricultural and soil conservation practices, integrated pest
management, development of alternatives to pesticide usage, and enhancement of clean
technologies in agriculture are key issues of reduction of pesticide loads in freshwaters.
Tourism being a key economic sector in Costa Rica, it is vitally important to ensure that
natural ecosystems are maintained in order to protect this essential industry. In addition, the
health of the workers and the people using the rivers for drinking water and irrigation of their
crops must be protected from the harmful effects of pesticides.

ACKNOWLEDGMENTS
We sincerely thank David Lean for his suggestions and collaboration on the English
review of this chapter.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 12

ENVIRONMENTAL RISK IN A COASTAL ZONE OF

RIO DE JANEIRO STATE (BRAZIL)
DUE TO DREDGING ACTIVITIES

Ana Paula de Castro Rodrigues1,*,

Alexandre Paoliello Lemos1, Christiane Monte1,
Sarah K. Rodrigues2, Ricardo Gonçalves Cesar3
and Wilson Machado1
1
Universidade Federal Fluminense, Geochemistry Department,
Niterói-RJ, Brazil
2
Universidade Federal do Rio de Janeiro-COPPE,
Naval and Oceanic Engineering Department, Rio de Janeiro-RJ, Brazil
3
Universidade Federal do Rio de Janeiro, Geography Department,
Rio de Janeiro-RJ, Brazil

ABSTRACT
This chapter presents an evaluation of the effects caused by sediments resuspension
events on the biogeochemical behaviour of cadmium (Cd), using, as case study, samples
from a highly polluted coastal system (Sepetiba bay, Brazil). These effects were
evaluated on different depths of three sediment cores and on 12 surface sediments
samples collected with increasing distances from the major Cd source. Physical-chemical
characteristics, Fe and Mn concentrations, grain size composition and organic carbon
contents were also evaluated to characterize potential factors affecting Cd mobility.
Cadmium was nearly 100% in the reactive phase (HCl-soluble) and showed extremely
high concentrations (up to 14.1 mg Kg-1), with a clear decreasing gradient from the main
source of contamination towards the bay. The differences in potential bioavailability after
resuspension were low for the sediment cores, but surface sediments presented up to 30%
more Cd in the reactive phase after resuspension. The changes on Cd concentrations
observed after resuspension may be associated to the dynamics of Fe and Mn compounds.

*
Corresponding Author E-mail: tantufaz17@gmail.com.

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184 Ana Paula de Castro Rodrigues, Alexandre Paoliello Lemos, Christiane Monte et al.

It is important to highlight that Cd concentrations are extremely high at this area. So even
a small change in its relative bioavailability can imply an increase on the potential risks
to local biota.

Keywords: bioavailability, trace metals, Sepetiba bay, resuspension

INTRODUCTION
Contamination of coastal aquatic systems is a worldwide problem and the evaluation of
the risks associated to contaminant release for humans and aquatic biota is of utmost
importance. This assessment should include biological, chemical and toxicological indicators
in order to elucidate the complexity of contaminants dynamics within aquatic systems,
considering the water column and sedimentary compartments. Sediment quality assessments
are therefore recognized as critical steps for estimating the risks associated with man-made
pollution in aquatic systems (Castillo et al., 2013), since these environments can be a sink for
most pollutants, including heavy metals. On the other hand, sediments are also considered as
potential sources, in case of resuspension events (Duarte and Caçador, 2012).
The sediment resuspension can occur naturally due to bioturbation, cold front entry and
strong winds, or artificially due to anthropogenic activities such as dredging and watercraft
navigation (Turner and Millward, 2002). Dredging activities are commonly used as a required
operation to navigation channels maintenance and to remove contaminated sediments of
aquatic ecosystems as a remediation action (Cappuyns et al., 2006). However, it causes
sediments resuspension to water column, which implies in metal mobilization, making these
elements potentially bioavailable in the aquatic system. These changes may lead to higher
exposure and it could enhance the risks of biological uptake of contaminants (Morse, 1994;
Machado et al., 2011).
In situ and ex situ studies have been developed to evaluate the mobilization of metals. In
relation to sediment resuspension ex situ evaluations, laboratory tests are used in order to
contribute to the assessment of ecological risks linked to resuspension events, simulating the
effects of dredging in different time intervals of a pre-defined mechanical agitation. For
instance, Machado et al. (2011) observed that the copper (Cu) content in a potentially
bioavailable geochemical phase (extracted in HCl 1 M) increased by more than 10 times after
the resuspension of sediments from a tropical coastal system (Guanabara Bay, SE Brazil).
The dredging effects on contaminants mobility and their availability to biological uptake
are poorly known, especially in tropical ecosystems. Moreover, trace elements, that typically
undergo high incorporation into pyrite, are susceptible to changes in their geochemical
fractionation in case of sediment resuspension in oxidizing water. Pyrite can become a source
of these elements to the water column if oxidized (Morse, 1994; Saulnier and Mucci, 2000).
However, potential changes in the bioavailability of trace metals that generally do not present
elevated pyritization, such as cadmium (Cd) and zinc (Zn) (Huerta-Diaz and Morse, 1992),
can be also a concern in strongly contaminated areas. This is the case of heavily-polluted sites
affected by metallurgical activities, as observed in Sepetiba Bay, SE Brazil (Monte et al.,
2015). It is a tropical coastal system influenced by combined effects of severe Cd and Zn
contamination (mainly due to past activities of a Zn smelting plant), urbanization and
harboring activities (Barcellos et al., 1991; Machado et al., 2008; Ribeiro et al., 2013). Cd and

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 Environmental Risk in a Coastal Zone of Rio de Janeiro State … 185

Zn emissions from this major industrial source were estimated as 24 and 3,660 t yr-1,
respectively, until the metallurgical plant was closed in 1997 (Molisani et al., 2004). These
anthropogenic pollutants were mainly transferred to the bay by a tidal creek (Saco do
Engenho Inlet) that received the drainage from a large refuse pile of the metallurgical plant
(Molisani et al., 2004).
Therefore, the comprehension of the behaviour of contaminants associated to dredged
sediments is essential for an adequate management of dredging areas and monitoring of sites
used for the final disposal of dredged materials (Cappuyns et al., 2006). Given this scenario,
the inclusion of ex situ experiments of sediment resuspension is important to predict risks
associated to resuspension and deposition of contaminated materials in coastal and oceanic
waters. This chapter addresses these issues, presenting a study on Sepetiba Bay sediments
resuspension. Samples from surface sediments and sediment cores from a heavily-polluted
area were used to evaluate possible changes in the biogeochemical behaviour of Cd, and the
potential risks to local biota are discussed.

METHODS
Sampling

The study area is highly impacted by Cd and Zn contamination, located between Saco do
Engenho Creek (SEC) and the Itaguaí Harbor in the northern region of Sepetiba Bay, Rio de
Janeiro Brazil. In July 2012, surface sediment samples (0–10 cm depth) were collected using
a van Veen grab at 12 sampling stations (Figure 1). Four transects (~350m apart) with 3
sampling stations per transect (~350m apart) were delimited to observe possible gradients of
Cd concentration with increasing distances from the main point source of Cd. Station 1 is the
closest sampling station to the old point source of Cd - a Zn smelting plant (Mercantil Ingá
Company). Even after a program of environmental recuperation in response to decades of
contamination due to mining wastes, high Cd concentrations are observed in this area
(Barbosa et al., 2015).
Physical-chemical characteristics were measured in situ using an YSI probe. Sub-
superficial water presented salinity of 32.8, pH of 7.9, dissolved oxygen of 4.6 mg L-1,
dissolved oxygen saturation of 65%, electrical conductivity (EC) of 48.5 mS cm-1 and
temperature of 23.4oC. Water was sampled only at station 12 at approximately 15 cm depth,
using acid-cleaned glass bottles (total of 6 L) and it was used only for the sediment
resuspension experiment, being analysed before (t0) and after resuspension. Station 12 was
chosen due to its higher salinity and dissolved oxygen - conditions commonly observed at
disposal areas of dredged material in Brazil, simulating what would happen if these 12
sediment samples were resuspended under these different physical-chemical conditions.
Three sediment cores (C1, C2 and C3) were obtained using acrylic tubes (10 cm
diameter, 50 cm length). They were collected along a transect starting at SEC and going 1 km
East into Sepetiba bay (Figure 1), with increasing distances from the major Cd source to the
bay. C1 was collected in a tidal flat containing a mangrove ecosystem affected by
anthropogenic sources of cadmium, while C2 and C3 were sampled at subtidal sites on the
margins of the Cação River, responsible for the fresh water input on Saco do Engenho Creek

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186 Ana Paula de Castro Rodrigues, Alexandre Paoliello Lemos, Christiane Monte et al.

and also with lower influence of the Cd point source. All cores were sectioned at 5cm
intervals. C1 had 50 cm depth, C2 had 40 cm and C3 had 45 cm. Samples were stored in
plastic bags and frozen until analysis. Only the sub-samples used for the resuspension
experiment were not frozen, since the resuspension experiment was performed as soon as the
cores arrived at the laboratory, on the same day of the sampling campaign.

Figure 1. Sampling stations in Sepetiba Bay, Rio de Janeiro, SE Brazil. SEC, Saco do Engenho Creek.
Adapted from Monte et al. (2015) and Santos et al. (2007).

Resuspension Experiment

Resuspension experiments were carried out at room temperature (25oC). The experiments
compared short (1h) and long (24h) time periods of resuspension. Wet sediment subsamples
(7g) were transferred to 125-mL Erlenmeyer flasks and were shaken in 100mL of unfiltered
estuarine water in contact with atmosphere. The sediment:water proportion was based on the
study of Morse (1994), adapted by Machado et al. (2011). EC and pH were measured after
each time interval of resuspension. Unfiltered estuarine water was also agitated during each
time interval to be used as analytical blanks. The experiments were made in duplicates.
The choice of these time intervals was based on previous studies, which demonstrated
that in the first hour (t1) most bioavailibility changes occur (Machado et al., 2011). The
second interval (t2: 24h) has been also previously adopted (Morse, 1994), and was used to
evaluate the stability of the contaminant response, simulating longer resuspension events.
Additionally, the initial condition of all sediment samples and of the water used for the test
was evaluated (t0). After the resuspension experiments, the sediment was centrifuged (3,000
rpm/5 min), dried (<40ºC) and homogenized to posterior analysis.

Metal Determination

The reactive Cd phase (adsorbed and/or associated to carbonates, monosulphides and iron
and manganese oxides) was extracted by 16h agitation in 1mol L-1 HCl solution, which is a
usual approach for metal bioavailability evaluation (Huerta-Diaz and Morse, 1992; Morse,

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 Environmental Risk in a Coastal Zone of Rio de Janeiro State … 187

1994; Hatje et al., 2009; Machado et al., 2011; Birch and Hogg, 2011; Monte et al., 2015).
HCl-extracted subsamples were washed with deionized water and centrifuged three times to
remove HCl residues prior to a second-step extraction of strongly bound metal fractions
(Machado et al., 2011). Fe and Mn were also determined, with the purpose of assessing
possible relations of Fe and Mn oxides formation with Cd bioavailability. The differences
observed for analytical replicates (duplicate) for the reactive phase were 6.7% for Cd, 4.4%
for Fe and 6.1% for Mn.
The method adopted for the extraction of strongly-bound metals (applied only for
sediment cores) was a microwave assisted digestion with concentrated HNO3 (USEPA
method 3051A; USEPA, 1994). Cd, Mn and Fe concentrations were determined by
inductively coupled plasma optical emission spectrometry (ICP-OES). The detection limits
were: 0.01 mg Kg-1 for Cd, 3.00 mg Kg-1 for Fe and 0.02 mg Kg-1 for Mn. The differences
observed for analytical replicates for the strongly bound phase were: 11.6% for Cd, 9.9% for
Fe and 8.5% for Mn.

Sediment Characterization

Besides Fe and Mn determination, sediment grain size was characterized using a particle
size analyser CILAS 1064. Total organic carbon (TOC) contents in sediments (after carbonate
removal by acidification) and total carbon concentration in water, before and after the
resuspension experiment, were determined using a Shimadzu TOC analyser. Possible
associations between target variables were evaluated using correlation analysis (Spearman
Test) with a significance level of p < 0.05.

Bioavailability Change Index (BCI)

The relative change in the percentage of trace metals in HCl-extractable fractions

was calculated to evaluate possible differences in bioavailability after sediment profiles
resuspension, hereafter referred as bioavailability change index (BCI). It is expressed by the
formula (Monte et al., 2015): BCI=(%HClAR - %HClBR)/%HClBR x 100, where %HClAR is the
percentage in the HCl-extractable fraction after resuspension and %HClBR is the percentage in
the HCl-extractable fraction before resuspension.
The percentage in the HCl-extractable fraction (%HCl) of Cd used for BCI calculation
was obtained by the following formula: HCl=([CdRP]/([CdRP]+ [CdSBP])) x 100, where [CdRP]
is the Cd concentration in the reactive phase and [CdSBP] is the Cd concentration in the
strongly bound phase (EPA 3051a method).
As we did not evaluate the strongly bound phase of superficial sediments, a simple
calculation of reactive Cd losses and gains as the percentage related to t0 (in natura
samples, before resuspension) was done, as explained in the following equation: (([CdAR] –
[CdBR])/[CdBR])*100, where [CdAR] is the Cd concentration on reactive phase after
resuspension and [CdBR] is the Cd concentration on reactive phase before resuspension. The
same equation was applied for Fe and Mn.

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Contamination Factors (CF) and Environmental Guidelines

Contamination factors (CF) is the ratio between the metal concentration in the present
(superficial sediment) and the background concentration, usually obtained from sediment
cores (Hakanson 1980). Then, an average background value obtained from 210Pb-dated
sediment cores sampled in Sepetiba bay, reported by Gomes et al. (2009) was used (0.27 mg
Kg-1 for Cd) to calculate CF. Moreover, the results were compared to Brazilian legislation
(CONAMA Resolution 454/2012), which establishes guidelines and procedures for the
management of dredged materials of fresh and estuarine waters.
CONAMA 454/2012 establishes two threshold levels for metals. The first threshold
(Level 1) represents a limit for the low probability of any adverse effect occurrence on biota.
The second threshold (Level 2) represents the limit for a high probability of adverse effect
occurrence on biota. For estuarine waters, the Cd concentrations established as Levels 1 and 2
are 1.2 mg Kg-1 and 7.2 mg Kg-1, respectively. According to this resolution, if the sediment
samples show concentrations above level 2, ecotoxicity tests should be performed before
dredging.

RESULTS
Surface Sediments

The surface sediment samples were classified as very coarse silt (stations 1, 8 and 12),
coarse silt (stations 2, 3, 4 and 11), medium silt (stations 5, 6 and 7), very coarse silty fine
sand (station 9) and very fine sandy very coarse silt (station 10). The average for TOC in
sediment from Saco do Engenho Creek was 2.4 ± 1.2% (Table 1). The concentrations in the
reactive phase ranged from 0.9 to 14.1 mg Kg-1 (stations 9 and 1, respectively) for Cd, from
1,870 to 9,871 mg Kg-1 (stations 9 and 12, respectively) for Fe and from 28.5 to 151.9 mg Kg-
1 (stations 9 and 1, respectively) for Mn (Figure 2). Reactive Cd concentrations are correlated

with reactive Fe (Spearman; r=0.68; p < 0.05; n=12) and Mn (Spearman; r=0.82; p < 0.05;
n=12). Besides, Cd concentrations are correlated to TOC (Spearman; r=0.83; p < 0.05; n=12)
and percentage of silt (Spearman; r=0.72; p < 0.05; n=12).
These are very high Cd concentrations, particularly in the reactive phase, reaching an
average concentration one order of magnitude higher than the background concentration
(CF=13.6 ± 11.4; n=12). The CF reached 41.5 at station 1. Even for the most distant station
from the contamination hotspot, the contamination factors were above 5.7, what indicates
high Cd contamination (Figure 3). These concentrations are almost 12 times higher than level
1, established by the Brazilian legislation guideline and 2 times higher than level 2,
considering the most contaminated station (station 1).
For an initial discussion about the effects of resuspension on a bioavailable phase of Cd,
we will discuss some modifications in physical-chemical parameters and TOC contents
(Table 1) and possible losses and gains after 1h and 24h only on Cd reactive phase (Table 2).
The pH decreased almost 3% after 1 h of resuspension and 5.7% after 24 h (Mann-Whitney U
test; p < 0.001). EC presented an important increase after the first hour (42%), as also
observed after 24h (43.8%), and the most significant changes were observed in transect 4

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 Environmental Risk in a Coastal Zone of Rio de Janeiro State … 189

(sampling stations 10, 11 and 12) (Mann-Whitney U; p < 0.001). Also, conductivity was
significantly correlated to Cd concentrations after 1h of agitation (Spearman; r=-0.61; p <
0.05).

18
16 t0 t1 t2
Reactive Cd (mg Kg -1)

14
12
10
8
6
4
2
0
1 2 3 4 5 6 7 8 9 10 11 12
Sampling Stations
18000
16000 t0 t1 t2
Reactive Fe (mg Kg -1)

14000
12000
10000
8000
6000
4000
2000
0
1 2 3 4 5 6 7 8 9 10 11 12
Sampling Stations

250
t0 t1 t2
Reactive Mn (mg Kg -1)

200

150

100

50

0
1 2 3 4 5 6 7 8 9 10 11 12
Sampling Stations

Figure 2. Reactive Cd, Fe and Mn concentrations in 12 surface sediment samples from Sepetiba bay-RJ,
Brazil. t0: concentrations in sediments before resuspension; t1: concentrations in sediments after 1h of
resuspension; t2: concentrations in sediments after 24h of resuspension.

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190 Ana Paula de Castro Rodrigues, Alexandre Paoliello Lemos, Christiane Monte et al.

Table 1. Average (± standard deviation) pH, electrical conductivity (EC) and total
carbon (TC) in water; and, total organic carbon (TOC) in sediments before (t0) and
after 1h (t1) and 24h (t2) of resuspension in laboratory of 12 surface sediment samples
from Sepetiba bay-RJ, Brazil

pH EC TC TOC
Stati-
(water) (water; mS cm-1) (water; mg L-1) (sediment; %)
ons
t0 t1 t2 t0 t1 t2 t0 t1 t2 t0 t1 t2
1 - 7.7 ± 0.0 7.5 ± 0.0 - 68.5 ± 0.9 70.3 ± 0.5 - 10.6 5.0 4.2 ± 0.1 3.9 ± 0.1 4.6 ± 0.1
2 - 7.8 ± 0.0 7.5 ± 0.0 - 68.9 ± 0.3 69.7 ± 0.5 - 9.1 5.8 3.6 ± 0.0 3.7 ± 0.1 4.2 ± 0.0
3 - 7.8 ± 0.0 7.5 ± 0.0 - 69.0 ± 0.3 68.5 ± 0.4 - 10.0 4.6 3.3 ± 0.1 3.5 ± 0.1 3.9 ± 0.0
4 - 7.6 ± 0.0 7.4 ± 0.0 - 68.6 ± 0.6 68.4 ± 0.2 - 9.4 2.4 2.7 ± 0.1 2.7 ± 0.0 2.9
5 - 7.6 ± 0.0 7.4 ± 0.0 - 69.2 ± 0.0 68.8 ± 0.7 - 11.0 5.6 2.5 ± 0.0 2.5 ± 0.0 2.9 ± 0.2
6 - 7.6 ± 0.0 7.4 ± 0.0 - 69.4 ± 0.4 70.2 ± 0.3 - 9.3 3.8 2.5 ± 0.0 2.5 ± 0.0 2.9 ± 0.1
7 - 7.6 ± 0.0 7.4 ± 0.0 - 69.2 ± 0.6 70.5 ± 0.0 - 4.5 4.5 2.4 ± 0.1 2.5 ± 0.1 2.8 ± 0.0
8 - 7.9 ± 0.0 7.5 ± 0.0 - 69.3 ± 0.3 69.3 ± 1.2 - 10.8 10.8 1.4 ± 0.0 1.6 ± 0.1 2.2 ± 0.1
9 - 7.7 ± 0.0 7.7 ± 0.0 - 69.1 ± 0.2 69.6 ± 0.3 - 9.4 6.6 0.2 ± 0.1 0.2 ± 0.0 0.1
10 - 7.7 ± 0.0 7.7 ± 0.0 - 69.0 ± 0.6 71.3 ± 0.6 - 10.1 6.8 0.4 ± 0.0 0.3 ± 0.4 0.8 ± 0.4
11 - 7.5 ± 0.0 7.4 ± 0.0 - 68.6 ± 1.7 70.5 ± 0.6 - 7.3 2.4 3.0 ± 0.0 3.1 ± 0.0 3.1
12 7.9 7.8 ± 0.0 7.5 ± 0.0 48.5 69.6 ± 0.1 70.1 ± 2.1 7.6 7.2 4.0 2.5 ± 0.1 2.6 ± 0.2 2.7 ± 0.1

Regarding TC in water, an increase of almost 20% after the first hour was observed,
followed by a significant reduction (more than 30%) after 24h (Mann-Whitney U test; p <
0.05). An opposite trend was observed for TOC concentrations in sediments, with no
significant loss after the first hour (0.24%) and a relatively accentuated increase (16.3%) after
24h, but not significantly different from t0.
Cd concentrations in the reactive phase increased after the first hour of resuspension
(ranging from 8.68%, at station 1, to 40.66%, at station 10), while Fe and Mn concentrations
also increased but in lower proportions (Table 2). After 24h of agitation, there was an abrupt
increase of Cd concentrations at station 12, not observed at the first hour. Strong correlations
between Cd and both Fe and Mn were observed after agitation (Spearman; r=0.8; p < 0.05).
This may be associated to Fe and Mn changes after 24h of resuspension (they became slightly
more bioavailable after resuspension), suggesting a relation with the Cd dynamic.

Table 2. The percentage of gains (+) and losses (-, in italic) of Cd, Fe and Mn on
reactive geochemical phase after 1 and 24h of resuspension of surface sediments
from Sepetiba bay, RJ, Brazil

Stations Campaign 2011 (Summer) (Monte et al., 2015) Campaign 2012 (Winter)
1h 24h 1h 24h
Cd (%) Fe (%) Mn Cd Fe (%) Mn Cd Fe (%) Mn (%) Cd (%) Fe (%) Mn (%)
(%) (%) (%) (%)
1 6.53 6.17 3.70 -6.34 -3.08 -7.55 8.68 15.04 7.35 11.65 13.73 9.29
2 24.22 13.99 30.17 7.66 3.43 19.70 20.71 11.49 11.27 19.66 17.85 10.5
3 -9.20 0.38 3.97 -11.53 -11.73 -9.45 16.6 11.09 15.67 18.15 15.23 13.34
4 -21.80 -0.82 -0.66 -22.10 -0.86 -3.91 10.3 4.99 -3.09 12.58 13.33 4.12
5 11.65 0.59 5.22 0.58 -8.18 -8.41 21.04 13.48 8.57 23.82 25.94 14.49
6 60.01 44.78 38.46 -3.55 1.12 -0.71 14.8 12.84 8.34 11.73 15.74 5.77
7 15.20 25.27 23.22 -1.38 -4.37 5.48 22.47 10.97 8.91 20.15 15.72 21.73
8 6.95 3.49 6.30 30.49 36.22 41.73 15.71 9.69 9.54 36.22 21.24 12.09
9 16.73 19.51 12.08 12.80 12.76 10.50 23.74 9.72 19.16 -18.69 -18.45 -9.72
10 7.40 29.69 17.01 4.09 20.97 14.63 40.66 27.99 17.61 47.17 35.56 22.85
11 9.01 20.89 10.72 -1.87 5.94 0.57 20.23 13.38 8.46 -66.31 -19.06 -35.52
12 16.31 23.95 6.92 8.38 11.42 0.56 -0.89 0.05 1.23 239.15 41.44 74.48

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 Environmental Risk in a Coastal Zone of Rio de Janeiro State … 191

Cd
45
40 CF Level 1 Level 2
35
30
25
20
15
10
5
0
1 2 3 4 5 6 7 8 9 10 11 12
Sampling Stations

Figure 3. Contamination factors (CF) of Cd and ratios between measured Cd concentrations and
Brazilian guideline values (CONAMA 454/2012 legislation: levels 1 and 2) of 12 surface sediment
samples from Sepetiba bay-RJ, Brazil.

Sediment Cores

The C1 sediment core was mainly composed of fine particles (above 90%), with
occasional sand influences from 15 to 35cm of depth (maximum of 85.3% of sand). TOC
(maximum 9.33% at 0-5 cm and minimum of 0.3% at 30-35 cm) and metals (Cd, Fe and Mn)
showed higher concentrations in the top and in the bottom sections of the sediment profile
(Figure 4). In intermediate layers, where more sand contents generally occurred, metal
concentrations were lower. The bioavailable percentages of Cd were very high - above 90%
in the exchangeable geochemical phase. Positive correlations of Cd in the exchangeable phase
with TOC (Spearman; r=0.73; p < 0.05; n=10), silt (Spearman; r=0.67; p < 0.05; n=10), Fe
(Spearman; r=0.88; p < 0.05; n=10) and Mn (Spearman; r=0.90; p < 0.05; n=10) were found.
In the strongly bound phase, these correlations with Cd were also observed with TOC
(Spearman; r=0.77; p < 0.05; n=10), silt (Spearman; r=0.79; p < 0.05; n=10), Fe (Spearman;
r=0.85; p < 0.05; n=10) and Mn (Spearman; r=0.87; p < 0.05; n=10), which suggests the
influence of grain size on Cd sorption.
The C2 sediment core showed predominance of silt (above 80%), with low variability
along the sediment profile (minimum of silt: 75.8% at 15cm; maximum: 95.5% at the top).
The TOC content ranged from 1.81% (at 35cm) to 5.67% (at 10 cm). Unlike C1, the metals
concentrations in C2 did not follow grain size oscillations (Figure 4). The highest Cd
concentration (81.2 mg Kg-1) occurred at 25cm, decreasing towards the top where the lowest
concentration was observed (14.0 mg Kg-1). The percentages of the exchangeable phases of
Cd and Mn were close to 100%, while Fe was mainly in strongly-bound phases (<50% in the
exchangeable phase). Strongly-bounded Cd was only correlated to Fe concentrations in this
geochemical phase (Spearman; r=0.71; p < 0.05; n=8). Manganese showed significant
correlation with Fe (Spearman; r=0.74; p < 0.05; n=8).
The C3 sediment core also showed a predominance of silt particles (above 70%), with
two peaks of sand contents at 10 cm (73.8%) and 40 cm (83.9%). TOC contents, as observed
for C1 and C2, increased from the base (0.62%) to the top (3.1%) of the profile. C3 showed
higher metal concentrations at the base (Figure 4). The reactive phase of Cd was also close to

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192 Ana Paula de Castro Rodrigues, Alexandre Paoliello Lemos, Christiane Monte et al.

100%. The reactive Cd and Fe were significantly correlated (Spearman; r=0.82; p < 0.05;
n=9) and, unlike for C1, the reactive phase of Cd was positively correlated to sand
percentages (Spearman; r=0.97; p < 0.05; n=9). Cd in the strongly bound phase was only
correlated to the percentage of sand (Spearman; r=0.93; p < 0.05; n=9).

Figure 4. Reactive (losangle in gray) and strongly bound (rectangles in black) metal geochemical
phases for the sediment cores C1, C2 and C3.

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Table 3. Average (Ave), minimum (Min) and maximum (Max) contamination factors
(CF) of Cd and ratios between measured Cd concentrations and Brazilian guideline
values (CONAMA 454/2012 legislation: levels 1 and 2), considering the sediment
cores samples

C1 C2 C3
Cd Ave Min Max Ave Min Max Ave Min Max
CF 142.1 18.1 262.4 130.7 44.2 240.0 25.1 3.6 57.4
CONAMA Level 1 40.3 5.1 74.4 37.0 12.5 68.0 7.1 1.0 16.3
CONAMA Level 2 6.7 0.9 12.4 6.2 2.1 11.3 1.2 0.2 2.7
Background Cd concentration: 0.34 mg Kg-1 (Gomes et al., 2009); Cd guideline values from CONAMA
454/12 legislation: Level 1=1.2 mg Kg-1 and Level 2=7.2 mg Kg-1.

Contamination Factors

The CF (Table 3) calculated using the maximum Cd concentration found in core C1 was
up to 262 times above the background value for this area. Considering the Brazilian
legislation guidelines, Cd concentrations were 74 times above level 1 and 12 times higher
than level 2. For the C2 core, these contamination factors are even higher, corresponding to
240 times above background, corresponding to 68 and 11 times higher than levels 1 and
2, respectively. On the other hand, core C3 contamination factors were lower, with Cd
concentrations up to 57 times above the background, corresponding to 16 and 2 times higher
than levels 1 and 2, respectively. These results clearly show a concentration gradient of Cd at
the study area, considering the distance from the point source.

Resuspension Experiments

A general trend of pH increasing in the first hour and decreasing at 24h of resuspension
was observed (Table 4). Only for the C1 sediment core the pH decreased after the first hour.
Conductivity increased at both time intervals. For TOC in sediment and TC in water, there
was an apparent inverse trend for the C1 and C3 sediment cores, while it was not so clear for
C2. At 0-5 cm depth and 15-20 cm depth of C1 the increase of TC in water was of 42.9% and
11.7%, respectively, after 1h of resuspension. Within 30-35 cm depth, TC decreased 35.1%.
After 24h, all the tested layers showed a decrease of TC in relation to t0 values, in average by
53.7% (maximum: 71%). For C2 and C3, this decrease was also observed, being more
pronounced after the first hour (58.3%) for core C2, and after 24 h (50.9%) for core C3.
Considering the results obtained to metals determination in reactive and strongly bound
phases, it was calculated the percentage in the HCl-extractable fraction (%HCl) for each
metal (Table 4). The percentages of Cd and Fe in the reactive phase did not present noticeable
changes after resuspension (Table 4). In the case of Cd, it was almost 100% present in the
reactive phase even before resuspension, implying no changes in its geochemical partitioning.

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194 Ana Paula de Castro Rodrigues, Alexandre Paoliello Lemos, Christiane Monte et al.

Table 4. Average pH, conductivity, total carbon (TC) in water and total organic
carbon (TOC), cadmium (Cd), iron (Fe) and manganese (Mn) percentages in the
reactive phase in sediments of the three layers of sediment cores from Sepetiba
Bay-RJ (Brazil), before (t0) and after (t1=1 h and t2=24 h) resuspension in local
water under oxidizing conditions. Samples with percentages of reactive phase
more than 10% higher than t0 are in bold and italic

C1 C2 C3
Parameters Time 0-5 15-20 30-35 0-5 cm 15-20 30-35 0-5 cm 15-20 30-35
cm cm cm cm cm cm cm
t0 7.95 7.95 7.95 7.95 7.95 7.95 7.95 7.95 7.95
t1 7.84 ± 7.88 ± 7.92 ± 7.86 ± 8.17 ± 8.21 ± 7.85 ± 8.21 ± 8.27 ±
pH 0.01 0.02 0.00 0.01 0.01 0.01 0.03 0.01 0.05
t2 7.70 ± 7.82 ± 7.96 ± 7.94 ± 7.47 ± 7.64 ± 7.93 ± 7.78 ± 7.82 ±
0.01 0.03 0.00 0.01 0.01 0.02 0.00 0.01 0.02
t0 48.5 48.5 48.5 48.5 48.5 48.5 48.5 48.5 48.5
t1 75.2 ± 74.9 ± 74.3 ± 75.8 ± 74.6 ± 75.2 ± 76.1 ± 75.9 ± 75.1 ±
Conductivity
-1 0.6 0.1 0.8 0.2 0.8 0.1 0.1 0.0 0.6
(mS cm )
t2 78.5 ± 79.1 ± 78.8 ± 79.0 ± 78.6 ± 78.8 ± 78.6 ± 78.1 ± 78.5 ±
0.5 0.5 0.7 0.3 0.1 0.2 0.1 0.9 0.1
t0 7.6 7.6 7.6 7.6 7.6 7.6 7.6 7.6 7.6
TC
-1 t1 10.9 8.5 4.9 1.7 4.5 3.3 4.6 6 3.4
(mg L )
t2 2.2 5.7 2.7 3.5 2.6 4.2 3.9 3 4.2
t0 9.3 ± 2.7 ± 0.2 0.3 ± 0.0 4.5 ± 0.2 5.2 ± 0.2 2.4 ± 0.1 3.1 ± 0.2 2.1 ± 0.1 2.1 ± 0.1
0.2
TOC
t1 9.3 ± 1.9 ± 0.1 0.3 ± 0.0 4.6 ± 0.3 4.9 ± 0.2 2.5 ± 0.3 2.8 ± 0.0 2.0 ± 0.1 2.1 ± 0.1
(sediment;
0.5
%)
t2 9.8 ± 1.9 ± 0.2 0.3 ± 0.1 4.4 ± 0.0 4.1 ± 0.3 3.5 ± 0.4 2.8 ± 0.3 2.3 ± 0.0 2.1 ± 0.1
1.1
Cd t0 95.4 84.6 95.1 93.4 96.7 99.0 91.9 100 87.3
(sediment; t1 98.1 93.8 99.9 97.0 96.4 99.0 98.8 99.2 98.7
%) t2 98.1 95.2 99.9 97.0 98.7 99.0 98.7 98.8 98.9
t0 31.2 30.7 18.6 52.8 27.1 27.6 40.6 32.4 22.5
Fe (sediment;
t1 37.5 32.7 16.7 45.6 29.0 29.6 45.4 26.9 21.6
%)
t2 34.9 33.2 15.7 40.4 23.7 29.5 42.7 29.2 23.9
Mn t0 49.9 36.7 20.2 88.3 77.1 86.1 71.2 37.6 36.5
(sediment; t1 77.9 65.7 37.6 93.6 77.6 83.4 92.1 58.0 58.8
%) t2 73.8 66.5 36.3 92.0 64.5 83.4 91.2 60.3 61.8

Applying the bioavailability change index (BCI), it was possible to observe important
changes on bioavailability of Cd on the basis of C3. Changes on Fe were not observed,
however the bioavailability of Mn increased (by over 20%) for most layers from cores C1
and C3. This trend was not observed in C2, where the results for the index were below 5%
(Table 5).

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Table 5. Bioavailability changes index (BCI, %) for three layers (0-5 cm; 15-20 cm;
30-35 cm) of three distinct sediment cores (C1, C2 and C3) from Sepetiba Bay-RJ
(Brazil), after resuspension experiments (t1=1 h and t2=24 h). Samples with BCI
above 10 or below -10 are in bold and italic

Sediment Cores Time Depth (cm) Cd Fe Mn

0-5 2.6 6.3 28.0
t1 15-20 9.2 2.1 29.0
30-35 4.9 -1.9 17.4
C1
0-5 2.6 3.7 23.9
t2 15-20 10.6 2.5 29.8
30-35 4.9 -2.9 16.1
0-5 3.6 -7.2 5.2
t1 15-20 -0.3 1.9 0.5
30-35 0.0 2.0 -2.7
C2
0-5 3.6 -12.4 3.7
t2 15-20 1.9 -3.4 -12.5
30-35 0.0 1.9 -2.6
0-5 6.9 4.8 20.9
t1 15-20 -0.8 -5.5 20.4
30-35 11.4 -0.9 22.3
C3
0-5 6.9 2.0 20.0
t2 15-20 -1.2 -3.2 22.7
30-35 11.6 1.4 25.3

DISCUSSION
The transects of surface sediments showed a clear trend of Cd distribution, being station
1 the most contaminated, as demonstrated by the contamination factor. The correlations
between Cd concentrations and Fe and Mn, besides the correlation with silt, may indicate the
origin linked to mining residuals or even the process of adsorption to small grain size
particles, being accumulated in sediments and stabilized in oxides and hydroxides of Fe and
Mn. Additionally, the relation found between Cd and total organic carbon in sediments
indicates possible complexation of this element by organic matter. However, the carbon
contents are not so high, as well the sulfides contents, which could maintain Cd linked to
sediments (Rodrigues, 2013).
The observed Cd concentrations are considered extremely high and essentially
correspond to the weakly bound fraction present in these sediment samples. Even studying
only the reactive phase, these concentrations are almost 12 times higher than the Brazilian
legislation guideline, which established its thresholds based on a strongly-bound extraction.
Reminding that this area includes a port activity that demands periodical dredging, this
environmental scenario is critical in relation to Cd pollution, since after resuspension, this
contaminated material suffered considerable changes in Cd bioavailability (Monte et al.,
2015).
Cd concentrations in the reactive phase increased after the first hour of resuspension and,
in comparison with a previous publication discussing the data from sampling carried out in

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196 Ana Paula de Castro Rodrigues, Alexandre Paoliello Lemos, Christiane Monte et al.

the summer of 2011 (Monte et al., 2015) (Table 2), the remobilization was more effective in
the present study (sampling during winter of 2012). Possible explanations for this observation
include: (i) lower pH at this second campaign; (ii) lower carbon contents, in comparison to
campaign of 2011, which may result in less metal-organic matter complexes; (iii) higher
control of the Fe and Mn dynamics on the Cd mobilization, also indicated by the strong
correlations found between these metals, before and after resuspension.
Regarding the sediment cores, the C1 core was collected in a mangrove area, which is
rich in organic carbon. The accumulation of most trace metals is favoured under reducing
conditions of sediments, while Mn has a different behaviour, since it does not form stable
sulphide, showing higher mobility under reducing conditions (Gueiros et al., 2003). All
correlations described for C2 suggest an important function of Fe compounds (such as oxides
and sulphides) in Cd sorption. Analogous to C2, C3 showed higher Cd concentrations in the
core base, possibly due to the similar influence of Cação River (both cores are located in the
margins of this river). Also it was observed an enrichment in Mn concentrations at the top,
which may be expected due to Mn remobilization within pore waters, migration by diffusion
to the surface and reprecipitation under the oxidizing top layers (Gueiros et al., 2003). In
contrast, Cd presents higher mobility under oxidizing conditions and lower mobility in
reducing environments, which is closely linked to its chemical affinity to sulphides
(Levinson, 1980; Gobeil et al., 1997).
Rees et al. (1998) observed higher Cd concentrations in the base of sediment cores taken
near the sampling point of C1 than those found in the present chapter, reflecting the tendency
of recent reduction of anthropogenic sources of cadmium to the Sepetiba bay. Although this
decreasing tendency is observed in more recent layers of sediment profiles, the contamination
scenario is very clear for the northern region of Sepetiba bay, and there are more than 100
publications in the Web of Science related to its contamination by Cd, Zn and Hg (Gomes et
al., 2009). Wasserman (2005), in his report concerning dredging activities in the bay,
indicated a persistency of Cd contamination and its distribution throughout the bay, far from
the original source, especially in northeast area.
Despite all the published works, this contamination remains a challenge to
environmental managers, especially considering discussions regarding metal bioavailability
and ecotoxicology. The high Cd concentration, especially in reactive phases, could be capable
of inducing negative effects on local biota. Higher toxicity responses in surface sediments
from Engenho Creek were found using acute toxicity tests with the amphipod Tiburonella
viscana (Rodrigues, 2013) and with two shrimp species Penaeus schimitti and Penaeus
paulensis (Moraes et al., 2000).
Although the sediment cores of this study were exposed to oxidizing waters during the
resuspension experiment, Cd had low bioavailability changes (BCI < 10% in most samples).
It is important to highlight that almost all Cd was already in reactive phases. Lacerda et al.
(1987) described similarly that most metals transported by the suspended particulate matter at
Sepetiba bay were in the reactive phase, using an HCl 0.1 M extraction to evaluate
bioavailability (10 times weaker than the extraction applied in this chapter). According to the
authors, Cd was completely in the reactive phase (100%) in suspended matter and in surface
sediment, evidencing that its transport and accumulation in bottom sediments was essentially
in an HCl 0.1 M-extractable form along the bay.
These high percentages of Cd in the reactive phase reflect the predominant anthropogenic
contributions (especially the industrial wastes), which appears to occur as oxidized Cd input,

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 Environmental Risk in a Coastal Zone of Rio de Janeiro State … 197

besides the influence of activities that cause sediment oxidation, such as dredging, shipping,
among others, being released in a reactive form. Additionally, when Cd is associated to
oxides and hydroxides forming a stable solid compound or it is co-precipitated with hydrated
iron oxides (even associated to carbonates), there is a lower risk of mobilization, in case of
resuspension in oxidizing waters (Azevedo and Chasin, 2003).
Maddock et al. (2007) performed resuspension experiments with sediments from
Sepetiba and Guanabara bays (SE Brazil) and found significant differences of dissolved
metals only for samples from Guanabara Bay (in agreement with our results), where metals
were initially bound to sulphides. The authors also found no difference of the release of
metals to water after resuspension of Sepetiba bay sediments, which are not bound to
sulphides. Fathollahzadeh et al. (2015) evaluated the effects of dredging at a port area in
Sweden and also observed a low Cd remobilization after resuspension in laboratory.
In some cases, after resuspension, Fe and Mn showed negative BCI values, indicating a
reduction of these elements in the reactive phase (i.e., they were solubilised and/or
incorporated in strongly bound phases). Caetano et al. (2003) also found a decrease in Mn and
Fe concentrations in HCl 6 M after 4 h of resuspension of sediments from the Tejo Estuary,
Portugal.
Other authors evaluated in laboratory the effects of resuspension to metals mobility.
Simpson et al. (1998) observed that anoxic sediments, when resuspended, released Cd and
other metals to water, due to the oxidation of sulphides. According to Calmano et al. (1988),
the sorption of metals to sediments depends not only on ionic exchanges, but also on
complexation reactions, which enable stability at bonding processes on the surface of
sediments. The authors observed, just as the former, during resuspension experiments with
seawater, Cd remobilization to solution and bonding preference of metals such as Cd and Cu
to organic matter (Forstner et al., 1989), where the organic phase could be considered an entry
for these elements into the trophic chain.
Evaluating the role of organic matter on metal mobility, Cantwell et al. (2008) developed
an experiment using artificial sediment, adding different TOC concentrations (0, 2, 1, 3, 5, 8
and 10%). The authors observed that Cd was released to the water column in the samples with
lower TOC concentrations. Thus, the organic matter has particular relevance in the retention
of dissolved metals, making them less available to the solution (Di Toro et al., 2005;
Rickards, 2012; Rodrigues, 2013).
It is important to note that even a small relative change in Cd bioavailability could be of
concern when the absolute concentrations are high. For instance, using a 10% threshold for
the change in Cd bioavailability in the presented study area, this change corresponds to an
absolute concentration of nearly 3 mg Kg-1 being transferred to a reactive phase in the case of
core C1 (0-5 cm depth). This amount is already above the CONAMA Level 1 guideline and
may lead to increased Cd toxicity to local biota. Lopes-Rosa (2011) observed that the
metabolic response of the bacterial community of superficial sediments from the Engenho
Creek was negatively affected after the resuspension, implying higher toxicity.

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CONCLUSION
Resuspension of both surface (0-10 cm) and deeper (0-35 cm) sediment profiles altered
significantly the physical-chemical conditions of water and the TOC contents of sediments,
which influenced on Cd behaviour. The surface sediments had a higher Cd mobility due to
stronger changes in physical-chemical parameters, which increased the Cd bioavailability,
apparently linked to Fe and Mn oxides. The resuspension experiments allowed the evaluation
of Cd behaviour in the case of a dredging activity in Sepetiba Bay (e.g., for remediation
purposes) and demonstrated strong concerns associated to this activity, evidencing that
ecotoxicological studies in association to resuspension experiments are necessary for
dimensioning environmental risks.

ACKNOWLEDGMENTS
The authors would like to thank the Rio de Janeiro State Research Foundation (FAPERJ)
and the Brazilian Research Council (CNPq) for the financial support. A.P.C. Rodrigues
thanks a grant from PNPD/CAPES.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 13

ASSESSMENT OF THE RISK GENERATED BY

NATURAL AND ANTHROPIC AGENTS ON
PERUVIAN COASTAL MARINE ENVIRONMENTS

Guadalupe Sánchez* and Aldo G. Indacochea

Universidad Científica del Sur, Facultad de Ciencias Veterinarias y Biológicas,
Panamericana Sur km 19, Lima, Perú

ABSTRACT
The Peruvian coast is one of the most productive in the world due to the current
system and the rich upwelling of nutrients. Therefore, it sustains the development of large
and diverse populations, especially within the pelagic ecosystem. Different processes and
events caused by natural and anthropic activities occur along that coast, calling for
scientific research and its diffusion to provide a basis for decisions concerning this
national resource. Natural environmental phenomena, such as El Niño, La Niña and
tsunamis (124 over the last six centuries), have caused disasters involving millions of
people and significant economic losses. The displacement of river sediment also threatens
the use of water resources, although drought is possibly the phenomenon that causes most
damage but the extent is difficult to quantify. It is estimated that the economic impact of
this event is greater than the flooding. Among the anthropogenic risks, there are: threats
posed by the exploitation of oil and natural gas from the continental shelf and from the
installations sited in bays; erosion along the coastline that destroys piers and houses; and
discharge of domestic and industrial wastewater. Furthermore, as the regulations for the
management of solid waste are not implemented in many cities, solid waste is also a
serious environmental problem. This problem should be overcome by improving
prevention systems to mitigate the impacts of natural disasters and avoid the risks of
anthropic need, via appropriate planning, monitoring and control of socioeconomic
activities.

Keywords: coastal zone, natural catastrophes, anthropic pressures, environmental risk

*
Corresponding Author E-mail: guada.sari@gmail.com.

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202 Guadalupe Sánchez and Aldo G. Indacochea

INTRODUCTION
A strategic environmental planning based on the analysis of the vulnerability of the
coastal marine area, as well as knowledge of the risks that may be presented by different
factors, allows for proper management of coastal resources for the economic and social
development of any part of the world.
Peruvian coastal waters are known for being one of the most productive fishing areas in
the world, due to the system of currents and upwelling phenomenon, which bring water rich
in nutrients that sustain the development of an abundant and diverse biological community.
The anchovy is the main pelagic resource that represents 90% of the total catch and which is
used mostly for fishmeal and oil production (IMARPE, 2014). The coastal zone presents
specific risks produced by natural agents like El Niño, which due to lack of prevention causes
serious environmental damage and economic losses. During the El Niño of 1982-1983, listed
as the strongest of the century (Jordán, 1985), there were losses of 3,283 million dollars
equivalent to 11.6% of the annual gross domestic product (GDP) of 1983 (Vargas, 2009).
During El Niño 1997 - 1998, there was a lack of an environmental assessment for the main
peak of the global event. It was thus not possible to accurately describe its effects on the
various resources of the Peruvian marine ecosystem, which decreased catches of the main
pelagic species, mainly Peruvian anchovy (Sánchez et al., 2000), causing the collapse of the
fishery and a 3.4 million dollar loss. Moreover, this event had a great impact on the Peruvian
economy as a result of disasters caused by heavy rainfall, droughts and floods; losses are
estimated at 1,000 million dollars, of which 800 correspond to the north of Peru due to floods
and 200 due to drought in the southern highlands (Lavado-Casimiro and Espinoza, 2014).
The city of Trujillo, in the La Libertad Region, also suffered from the lack of water
resources, leading to the setting up of the CRIS USAID project (MINAM, 2014). The 1997-
98 El Niño caused an estimated 3,000 million dollars damage, equivalent to 6.2% of annual
GDP 1998 (Vargas, 2009).
Another major problem is the erosion of the coastline, a process that is occurring
worldwide in 70% of sandy beaches (Bird, 1985), as a result of the intensification of ocean
storms (Zhang et al., 2004), fuelled by the rise of sea levels caused by the melting of the polar
regions. However, coastal erosion is also caused by human activities such as ventures in ports
and which may be manifested even at a great distance from the point of origin.
This chapter provides a review of the main risks to which the marine coastal environment
of Peru is exposed, whether they are naturally occurring or the result of anthropic activities.
For this the most recent publications were consulted regarding recurring natural events
affecting coastal and marine ecosystems and where human activities generate risk situations.

DATA COMPILATION
Documentation and technical regulations of various public and private sectors involved in
the assessment and risk management, as well as publications of international institutions and
non-governmental organizations were reviewed for the preparation of this chapter.
The first step was to determine natural events or human activities that threaten coastal
marine ecosystems by the high degree of vulnerability. As a second step, each of the risks was

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 Assessment of the Risk Generated by Natural and Anthropic Agents … 203

described with examples that occurred recently, reflecting the current situation of the national
contingency when facing these facts.

STUDY AREA
The marine coastal area is an environmentally sensitive and economically valuable area
with a rich biodiversity and benefits such as a reliable source of food and a range of
opportunities for the production of renewable energy.
The Peruvian sea is under pressure from industrial activities, transport, trade, energy
processing, tourism, recreation, whilst also playing an important role in our cultural heritage.
The important role of the services and goods produced by export aquaculture, artisanal
fisheries and the extraction of benthic resources should also be recognized as the
environmental threats they represent.
The coastal zone of Peru, according to the Brack’s (2000) ecosystemic classification, is
shaped by the Tropical Sea, the Pacific Ocean’s Tropical Forest, the Dry Equatorial Forest,
the Pacific Desert and the Cold Sea. The first three ecoregions mentioned are present on the
north Peruvian coast. The Tropical Sea is characterized by its warm waters and the presence
of equatorial and tropical currents. The latter come from the Central American Pacific Ocean,
which is characterized by the existence of mangrove forests along its coast, continuing from
the Colombian Pacific Ocean down to the northern coasts of Peru.
The Dry Equatorial Forest is also present in the northern area, where 7 different types of
habitats, shaped by tidelands (irrigation channels and creeks), have been identified; a great
extension is occupied by prawn farms, which were the most serious threats to the area in the
70s. This ecoregion includes, besides Tumbes, the coast of Piura and Lambayeque, with the
presence of dune landscapes where forests of carob-trees and ceibos predominate. In this
ecoregion are located the most southern mangrove forests of the Southeast Pacific, threatened
by the waters of the river Piura that contain untreated waste water discharges from Piura city
and the populations settled on its banks.
The most extensive and rich ecosystem is the Cold Sea, because the Peruvian Current or
Humboldt Current that runs from the south to Piura is an area of great primary productivity. It
is the basis of a food web of marine biodiversity, which provides a resource of great
socioeconomic importance for the country based mainly on the capture of anchovies.

NATURAL ENVIRONMENTAL RISKS

Hydrometeorological Events El Niño/La Niña

The overall natural climate cycle known as El Niño-Southern Oscillation (ENSO) has
two phases: a warm one known as El Niño, and a cold phase known as La Niña, called this
way because it presents opposite conditions to El Niño (CENEPRED, 2013).
El Niño and La Niña are an obvious example of the natural oscillations occurring in the
region and are an essential part of a much vaster and more complex system of climatic
fluctuations. La Niña is characterized by the presence of long-lasting cold temperatures,

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204 Guadalupe Sánchez and Aldo G. Indacochea

unlike El Niño which is characterized by unusually warm ocean temperatures in the

equatorial Pacific Ocean (CENEPRED, 2013). The warm phase causes local flooding due to
heavy rains and during which warm fronts come together on a general scale for months
creating intense rainfall that saturates the soil on a local scale.
Flooding causes many disasters all over the world, affecting millions of people with
significant economic loss and has been increasing in recent years. In 2010, about 178 million
people were affected by floods (Table 1). The regions most affected are urban areas and
include roads and irrigation infrastructures which suffer due to poor maintenance and
construction type. Associated with this phenomenon are the mudslides and landslides that
occur at the headwaters of rivers and streams because of the pore pressure caused by water
infiltration in a loosely cohesive, friable soil, creating large deforested areas in the northern
and central coastal regions of Peru.
The “El Niño” events of 1982-83 and 1997-98 affected many cities in northern Peru.
Sullana, Piura Region, was one of the cities most affected. Not only infrastructures were
affected, but also agriculture and industries suffered significantly. It is estimated that the
country lost approximately 3,283 million dollars in the 1982-83 event and 3,500 million
dollars in the 1997-98 El Niño (Vargas, 2009).
A project called “Aliados ante inundaciones: Gestión de riesgos y resiliencia ante
inundaciones de Piura y Lima” has been established as a tool for risk management of the
floods caused by such events in Piura and Lima in order to reduce vulnerability to flooding in
the basins of Piura and the Rimac (Figure 1). This works by promoting a greater integration
of the various entities for flood management, a more scientific approach to construction and
other institutional approaches.
Another key point is the operation of the emergency systems and the Early Warning
System, which, in the case of El Niño, is sometimes able to detect the phenomenon in
advance. This measure helps to reduce losses by the implementation of emergency measures
prior to the event, as occurred in 1997-1998. The Civil Defense System issues a “stay on
alert” warning upon the occurrence of external geodynamic phenomena and promotes the
implementation of contingency plans (MINAM, 2014).

Table 1. Positive and negative impacts of El Niño in Peru

Positive impacts Negative impacts

Emergence of other pelagic species. Accelerated glacial retreat.
Increased rainfall and air temperature Loss of farmland.
favoring the development of rice farming Silting of reservoirs.
on the coast. Land salinization.
Heavy rains in a strong El Niño event Destruction of productive infrastructures (irrigation
favor the natural regeneration of dry channels, intakes, dams, etc.).
forests on the north coast. Destruction of roads and collapsing of bridges.
The emergence of temporary pastures on Death or migration of some plant and animal species.
the north coast is important for livestock. High probability of occurrence of forest fires due to high
Excessive rainfall favors restocking temperatures.
groundwater reserves. High temperatures impact livestock (low production of meat
Higher sea temperatures during the fall and milk).
and winter favor a decrease in the The destruction of basic sanitation infrastructures.
intensity of frost in the central and north Displacement of schools of anchovy to deeper zones, which
highlands. cannot be compensated by the presence of a new species.
Source: SENAMHI (2014).

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 Assessment of the Risk Generated by Natural and Anthropic Agents … 205

Figure 1. Heavy rain causing landslides and floods in Piura highlands in January of 1998.
(Photo: A. Indacochea).

While the cold sea conditions during La Niña favors anchovy, it also causes a wider
dispersion of this species away from the coast, which does not benefit the fishermen as they
must thus travel further than usual to capture the resource (Table 1). Furthermore, a decline
in landings of other fish species such as hake, mackerel, horse mackerel and sardine and
other fishery resources such as squid, octopus, scallops, etc. (CENEPRED, 2013) has been
observed.

Tsunamis

Over the past six centuries, 124 tsunamis have been registered in Peru and the most
disastrous were caused by earthquakes. In 1586, an 8.6 Mw magnitude earthquake occurred
off the coast of Lima, causing waves up to 26 meters high which completely destroyed La
Punta (Callao) and entered 10 km inland. The tsunami spread and flooded all the beaches
from Tacna to Piura. In 1604 there was an earthquake with a magnitude of 7.8 Mw causing
16-meter high waves, causing widespread destruction mainly in the ports of Pisco, Camana,
and Arica (Carpio and Talavera, 2002). In 1687 a tsunami resulting from an 8.2 Mw
magnitude earthquake that occurred south of Lima, seriously affected the ports and towns of
Chimbote, Callao, Lima, Cañete, Chincha, Pisco and Camana. Nearly 500 people died, 300 of
them in Callao (Carpio and Talavera, 2002). There was an earthquake west of Lima with an
8.6 Mw magnitude in 1746 that generated waves of up to 24 m in height, killing between
5000-7000 people, mainly the inhabitants of Callao, Chorrillos and La Herradura (Carpio and
Talavera, 2002). In 1868, an earthquake of magnitude 8.8 Ms in the town of Arica in Chile
had several aftershocks that resulted in tsunamis with waves up to 18 m high, destroying
much of the Peruvian and Chilean coast and killing 300 people in Arica and 30 in Arequipa
(Carpio and Talavera, 2002). In 1960, an earthquake that occurred off the coast of Piura with

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206 Guadalupe Sánchez and Aldo G. Indacochea

a magnitude of 6.8 Mw produced 9 m high waves, causing severe damage to the ports of Eten
and Pimentel and Caletas in Santa Rosa and San Jose (Carpio and Talavera, 2002). Finally, in
2001, an earthquake of 8.4 Mw magnitude, with its epicenter in the city of Ocoña in
Arequipa, created a tsunami that also affected the populations of Ocoña, Camana Quilca and
Matarani, causing the destruction of houses in Camana and penetrating more than 1 km inland
(Carpio and Talavera, 2002).

Sediment Transport in Rivers

The mountain basins of the Peruvian coast suffer intense physical-chemical soil
degradation. They are also the main source of water for 65% of the Peruvian population
(Morera, 2014). The main and largest water projects in Peru are located in this region. For
these reasons the use of such water resources (by the population and for hydroelectricity,
irrigation, etc.) is at risk due to high loads of river sediment, which affect aquatic ecosystems,
reduce the lifetime of waterworks and even contribute to social conflicts due to the decreasing
availability of good quality water (Morera, 2014).
A further problem is that, naturally, due to the characteristics of the Andes that have a
polymetallic formation (Soler and Lara, 1983; Tumialan, 2004), many rivers carry clay
sediments with a load of metallic waste.
In this regard, it is important to consider the variability of the contributions of the
sediments brought by the 53 Peruvian coastal rivers, especially in times of flooding as a result
of alternating wet and dry periods throughout the year. The increased use of water due to
population growth and farming must be considered, as well as the fact that sediment
transported from agricultural land or mining areas is also associated with carrying traces of
heavy metals (Sánchez et al., 2010).

Drought

The variation in water resources makes difficult to quantify the millions in losses that
often occur during a period that may last several years. Also, the associated intangible losses
are many and very diverse (ANA, 2010). It is likely that drought is the phenomenon that
causes most damage, but it is difficult to be certain due to such a challenging quantification.
However, it is estimated that the economic impact of drought is greater, in general, to
flooding, even though the latter is of greater intensity and higher losses are registered (ANA,
2010). In 2011, drought events affected areas of the coast such as: Lambayeque, Piura, La
Libertad, Lima, Moquegua and Tacna. The consequences of this drought were the loss of
crops, livestock mortality and the proliferation of pests and diseases. This mainly affects
small farmers and urban populations, and the production of electricity (ANA, 2012).
The Risk Management Plan and adaptation to climate change in agriculture,
PLANGRACC-A (2012) (FAO support) has identified five areas on the coast as being of low
risk of drought, including Lima; 12 regions of medium risk, including, Ancash, La Libertad,
Piura and Tumbes; and 3 regions of high risk: Ica, Lambayeque and Tacna (ANA, 2012).
Regarding the use of water, the trend is growing as sectors requiring water are increasing,
as shown in the growth of the population and GDP of the Piura region (from 1997 to 2010

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 Assessment of the Risk Generated by Natural and Anthropic Agents … 207

economic activity increased by 70%). The latter is thanks to an increase in economic output in
the unregulated basin: intensive agriculture, fisheries, industries associated with the fishing
industry and associated with the ports (ANA, 2012). It should be noted that the valleys of
Zaña, Chancay, La Leche, Motupe and Olmos that supply the major export crops in the
region, have below average discharges and the reservoir that supplies Tinajone’s 15 irrigation
committees is only working at 30% of its storage capacity. In the Piura River, the biggest use
of water is for irrigation, which consumes 80% of the water in the lower reaches of the Piura
basin (ANA, 2012).

Soil Salinization

There are two major sources of soil salinization. One is a consequence of drought that
favors seawater intrusion in coastal areas, causing the salinization of groundwater and wells
as well as degrading soils. This saturations affect agricultural production, mostly in the case
of the lower basins of the Piura and Chira rivers, and generates health problems in the local
population (ANA, 2012).
Another cause of soil salinization on the coast is the use of irrigation in the upper reaches
of the valley. Ramírez et al. (2007) showed that there is a slight soil degradation in the lower
basin of the province of Santa, Ancash Region, namely in the agricultural area of Samanco
Valley, due to the effect of salinity and poor drainage (Figure 2), leaving the land unfit for
agriculture. However, the positive influence of the Special Irrigation Project (CHINECAS)
has led to soils being recovered in the agricultural sector and has changed land use by creating
conditions for permanent crops or grazing.

Figure 2. Photo illustrates agricultural land affected by salinization caused by the lack of drainage of
the irrigation system in Samanco, Nepeña Valley, Ancash Region (Ramirez et al., 2007).

ANTHROPOGENIC ENVIRONMENTAL RISKS

Exploration and Exploitation of Oil and Natural Gas on the
Continental Shelf

Licenses have been authorized along the Peruvian coast for this activity, and although
they employ technologies used quite successfully in other countries, there still remains a risk

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208 Guadalupe Sánchez and Aldo G. Indacochea

of accidents, with varying degrees of threats to marine and coastal ecosystems. There are 18
sedimentary basins with hydrocarbon potential in Peru, seven of which are located in the
coastal marine area (Perupetro, 2015). The hydrocarbon extraction activity takes place mainly
along the coastal strip and on the continental shelf. There is also a significant flow of tankers
carrying petroleum products from refineries in La Pampilla and Conchan to fuel storage tanks
located along the coast, for distribution to cities or places with intense industrial, fishery or
port activity, water sports, etc. The third refinery on the Talara coast is supplied through an
oil pipeline in the Peruvian jungle.
The northwest coast of Peru is the oldest area of oil exploitation in the country with the
province of Talara including the oil centers of La Brea, Pariñas, Lobitos, El Alto, Talara and
Los Organos. This area constitutes a high-risk zone for oil spills. These and other marine
coastal areas where there are refineries and fuel storage tanks are monitored periodically
(Figure 3).
An example of these risks is the burning of the BAP Supe oil tanker on January 30th of
2008 off the coast of Zorritos, Tumbes, creating an oil spill (Sánchez et al., 2010). There was
another spill of oily waste in the sea at Zorritos that occurred on January 20th, 2016 from a
tanker at the La Cruz pier. A total of 550 L was spilled and the Contingency Plan of the Navy
of Puerto Zorritos was immediately activated.

Figure 3. Total petroleum hydrocarbon (TPH) concentrations in the seawater and coastal areas of Peru
(Sánchez et al., 2010).

Gas exploitation takes place primarily in the jungle of Peru. Gaseous products travel
through pipelines to the coast where fractionation plants are located in Pisco and Cañete, with
a processing capacity of 128 thousand barrels per day and a storage unit for natural gas
liquids (NGL). The other liquefied natural gas is the Pampa Melchorita, Cañete, Lima
Region, with an estimated annual production of 4.4 million tons. In each of these plants port
terminals have been built for export of natural gas. This activity can be a risk for the
development of tourism and recreation, mainly in Paracas Bay in Pisco, and in the case of
Pampa Melchorita, Cañete, and the impact would affect artisanal fisheries.

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 Assessment of the Risk Generated by Natural and Anthropic Agents … 209

The Erosion of the Coastline

The wind, waves or tidal currents carry away soil stripped from the cliffs, such that
coastal inclines may suffer a considerable loss of material. This is what is occurring on the
northern coast of Peru, with the inherent risk of part of the beaches or towns located within
the coastal strip disappearing. However, in most cases it is the result of human activity, such
as: construction, building docks, marinas, piers, breakwaters and embankments or even just
with the intention of retaining sediment particles. Such erosion has changed the size of sandy
beaches, sometimes transforming them into narrow pebble beaches and causing the
destruction of coastal embankments.
In the Bay of Paita a port terminal is located, and 10 km north is the beach of Colan,
where houses have been built in the intertidal beach area near the mouth of the Chira River
(Figure 4). The study by Rondón (2011) with data covering 61 years (1946-2007) shows a
clear trend towards sedimentation of a large area in Colan since 1992 to 1999, reflecting the
1997-1998 El Niño event.

Figure 4. Colan Beach, Bay of Paita, Piura Region, showing strong sedimentation caused by natural
events such as El Niño 1997-98 (Photo: A. Indacochea).

The sea erosion on the coast of Trujillo extends from the Port of Salaverry to the beach of
Buenos Aires (Guerrero-Padilla et al., 2013). It has been caused by the construction of a 1 km
long breakwater at the Port of Salaverry, with the objective of preventing the natural
movement of sediment from the south to the north. That breakwater (Figure 5) was not an
effective solution for the port terminal and has destroyed the main resorts of Trujillo. It is
estimated that 60 million m3 of sand have accumulated in the Port of Salaverry as a result.
According to Veneros et al. (2012), the Trujillo beaches have been negatively impacted,
primarily by a combination of factors such as: destruction of the sandy beaches, retreat of the
coastline, destruction of the ecosystem of wetlands and dunes that have triggered similar
erosion processes, and sedimentation.

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210 Guadalupe Sánchez and Aldo G. Indacochea

It is estimated that between 1976 and 1997, the average erosion of the beaches of Las
Delicias and Buenos Aires (Figures 6 and 7) was 3.2 m/year, with a maximum erosion of 7.6
m/year (160 m erosion in 21 years of Las Delicias beach) and a sedimentation rate of 5-10
m/year at the Las Delicias beach during the 1995 (Veneros et al., 2012).

Figure 5. Satellite view of Salaverry Terminal Port, Peru, in January 2015, with about 600 meters of
sand accumulation. Strong erosion is evident from the Las Delicias beach (Image from Google Earth).

Figure 6. Heavy erosion on the beach in Buenos Aires, Trujillo. Photo taken in August 2010.
(Bocanegra, 2014).

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 Assessment of the Risk Generated by Natural and Anthropic Agents … 211

Figure 7. Protection infrastructure with rocks to prevent erosion and destruction of the houses on the
beach in Buenos Aires, Trujillo. Photo taken in September 2015 (Photo: F. Benites).

Discharge of Domestic and Industrial Wastewater

On the Peruvian coast, wastewater resulting from the combination of water residences,
offices, commercial buildings and institutions, together with waste from industries and
agricultural activities. The dumping of domestic sewage along the Peruvian coast is done after
some treatment. In Lima and Callao, 20 m3/s are generated per drain. One of the primary
treatments, performed in the Wastewater Treatment Plant Taboada (PTAR Taboada), started
to operate in 2013, with a treatment capacity of 14m3/s that serves 27 districts, equivalent to
75% of the sewage produced in the northern and central districts of Lima and Callao. This
treatment plant has a submarine emitter, which is 3 m in diameter and 3.8 km offshore from
Oquendo, Callao Bay.
In Lima, 40% of the wastewater of southern zone is treated by 4 PTARs: San Juan de
Miraflores, Villa El Salvador, San Bartolo (the largest but only works for 50% of its capacity)
and La Chira. These four PTARs comprise the San Bartolo project that aims to use the
wastewater treated to irrigate desert lands, cultivated fields, parks and berms (the Pan
American South Highway).
In the city of Huacho, located in Huaura province, 150 km from Lima with a population
of 55,442 inhabitants (INEI, 2007), the municipality’s wastewater is discharged directly into
the sea without any treatment. The Huaura River, polluted by domestic and industrial
wastewater from the upper basin, discharges into Caleta Carquín, which is also part of
Huacho (Sánchez et al., 2010). The Municipal Water and Sewage Company of Huacho
(EMAPA-Huacho) is in charge of two continuous domestic water discharges: one is
downriver from Huaura, with an average discharge flow of 3.2 m3/s, and the other is near
Puerto Huacho with an average discharge flow of 2.4 m3/h. However, the situation is
considerably better at Pisco, which is a coastal city 260 km from Lima and has a population
of 125,879 inhabitants. The wastewater from Pisco and San Andrés is treated at two anaerobic

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212 Guadalupe Sánchez and Aldo G. Indacochea

lagoons, two facultative lagoons and two maturation ponds, using biological treatment
without any chemical agents. The treated water from the Municipal Water and Sewage
Company (EMAPISCO) that is responsible for the collection, transport, storage, treatment
and disposal of wastewater, is discharged into the Pisco River; however, tourism and
recreational beaches located south are not at risk of contamination (Sánchez et al., 2010).

Solid Waste

Waste such as paper bags, candy wrappers, etc. produced by the inhabitants of urban and
rural areas, end up in the sea after being transported by wind, rain water and even rivers,
becoming marine litter. The bulkier group is comprised of domestic waste mixed with organic
waste. This solid waste impacts the coastline by becoming marine debris that is carried far out
to sea by runoffs from the land or in river water.
Between 17,000 to 18,000 tons of waste (Figure 8) are produced in Peru, daily. Lima and
Callao alone produce around 9,000 tons each day and only 25% is treated (MINAM, 2009).
This environmental problem is mainly dealt with by local governments that try to comply
with the General Law of Solid Waste (Law 27314 year 2000) and its regulations (DS 057-
2004/PCM) in order to solve the poor management of solid waste. This Act establishes the
Integrated Environmental Management Plans for Solid Waste (PIGARS) to be implemented
by the municipalities, but by the year 2008 only 25% of local governments had developed
their PIGARS, mainly comprised of coastal municipalities.

Figure 8. Production of solid waste in tons per day in Coastal Regions of Peru during 2008
(MINAM, 2009).

Peru requires approximately 100 landfills to store the waste that people produce.
However, there are only ten such waste storage facilities in the country, which indicates a
considerable deficit. This problem leads to the majority of municipalities to use solid waste
dumps, many of which are located in areas close to population centers. Lima is located near

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 Assessment of the Risk Generated by Natural and Anthropic Agents … 213

five of the ten landfills in the country: Huaycoloro, located in the province of Huarochirí;
Portillo Grande in Lurin; Ancón; Modelo of Callao in Ventanilla and the Zapallal in
Carabayllo (MINAM, 2014).
Meanwhile, in Lima there are 14 districts with selective collection services operated by
solid waste recyclers such as: downtown Lima, Barranco, Chorrillos, San Juan de Miraflores,
Villa Maria del Triunfo, Villa El Salvador, Comas, La Victoria, Pachacamac, Puente Piedra,
Punta Hermosa, San Juan De Lurigancho and San Martin de Porres; 9 cities have this service
in the Piura Region.
The presence of marine debris at sea poses risks to marine life and has serious effects on
ecosystems due to changes in behavior and life cycles, increased amounts of disease,
including the presence of viruses, bacteria, protozoa among other causative agents of death
affecting species of fish, turtles and marine mammals.

CONCLUSION
Peruvian coastal marine ecosystems have been impacted by natural (El Niño, tsunamis,
droughts, loads) and anthropogenic (salinization, erosion, solid waste, domestic an industrial
wastewater) factors that threat their environmental quality, reduce the biodiversity and
represent a risk for human health.
Since the ENSO events are recurring phenomena an economic allocation should be
considered in the annual budget of the nation, within the Program for Disaster Prevention,
which deals with the most sensitive and vulnerable critical points in coastal environments,
mainly focused on infrastructure and capacity building.
The Peruvian coast has often been hit by tsunamis. Despite advances in international
early warning systems, Peru does not have a warning system that efficiently covers the entire
coastline.
Erosion on the Peruvian coast calls for studies to identify the causative agents, using time
series on factors such as ocean currents, waves, climatic variations and human actions which
have altered the geomorphology of a given coastal area.
The problem of salinization of agricultural land can be avoided with the use of technical
irrigation and improved drainage systems, in addition to a proper selection of the type of crop.
Reports from the monitoring conducted by Sánchez et al. (2010) have determined an
increase in pollution by domestic and industrial wastewater in some bays, such as: Végueta,
Carquín, Huacho and Chancay with the inevitable risk of food safety and the populations’
health due to severe contamination. It is important to increase the installation of wastewater
treatment plants in coastal cities, which allow reuse of treated water or discharge to water
bodies by technically designed emitters, avoiding sensitive areas such as those used for
aquaculture and artisanal fisheries.
There is an urgent need to reverse the deficits in infrastructures, human resources and
funding in the short term. It is also necessary to increase the number of landfills, encourage
recycling, promote environmental education, and develop proposals for funding and
strengthening cooperation and technical assistance for local governments in order to facilitate
a better management of solid waste.

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Lavado-Casimiro, W. and Espinoza, J. C., 2014. Impactos de El Niño y La Niña en las lluvias
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MINAM, 2009. Informe anual de residuos sólidos municipales en el Perú, Gestión 2008.
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MINAM, 2014. Ciudades sostenibles y cambio climático: Resumen de experiencias, procesos
e iniciativas desarrolladas en el Perú. Ministerio del Ambiente, Perú, 187 pp.
Morera, S., 2014. Erosión y transporte de sedimentos durante eventos El Niño a lo largo de
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PERUPETRO, 2015. Memoria Anual 2014. Edited by PERUPETRO S.A., pp. 164.
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Plan de Zonificación Ecológica Económica. Informe de Consultoría del Punto Focal del
Plan de Acción. CPPS/PNUMA. Componente II Preparación del Plan de Zonificación
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de la geomorfología litoral: Bahía de Paita, Perú 1946-2007. Graduate Thesis in
Geography, Pontificia Universidad Católica del Perú.
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coastal marine ecosystem off Peru. CalCOFI Rep, 41, 62-86.
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Convenio IMARPE- CPPS. Programa CONPACSE, Lima, Perú, 169 pp.
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morfológicos litorales sobre las comunidades intermareal de tres playas de Trujillo, Perú.
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Clim. Change, 64, 41-58.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 14

ASSESSING THE ECOLOGICAL EFFECTS OF

CONTAMINANTS IN ESTUARIES IN BRAZIL:
GAPS AND FUTURE DIRECTIONS

Marcos Krull1,2*, Denis M. S. Abessa3 and Francisco Barros1

1
Laboratório de Ecologia Bentônica, PPGEcoBio, Instituto de Biologia,
Salvador, BA, Brazil
2
College of William and Mary, Virginia Institute of Marine Science,
Gloucester Point, Virginia, US
3
Núcleo de Estudos sobre Poluição e Ecotoxicologia Aquática, UNESP,
São Vicente, SP, Brazil

ABSTRACT
This chapter reviews published papers from the last 15 years that estimate the effects
of contamination in Brazilian estuaries. The goals were to identify (i) the main classes of
contaminants studied in estuarine systems in Brazil; (ii) the most used lines of evidence
(LOEs) to estimate the effects of contaminants; (iii) the most common experimental
designs and statistical analysis; and (iv) the major gaps in the field and suggest future
directions. A total of 130 papers were reviewed. Metals were the most measured
contaminant (34.6%) followed by organic contaminants (13.1%). Most papers (56.9%)
only used one line of evidence (LOE) and the most common LOE were bioaccumulation
(40.8%) followed by media chemistry (39.2%) and biomarkers analysis (33.8%). Most
papers (59.6%) did not report the salinity range of the studied system, had three or less
spatial replicates (56.4%), and did not include temporal replication (48.1%) or reference
sites (52.6%) in their design. The most used analyses were ANOVA (44.6%) and
correlation (26%). Some suggestions for future studies are: (i) consider the salinity range
of the studied sites when planning the experimental designs; (ii) include appropriate
reference areas; (iii) clearly state the question and the null hypothesis to be tested, (iv)
evaluate the possible effect of covariates when performing bioaccumulation and
biomarker studies; and (v) include more than one LOE in the design if possible.

*
Corresponding Author E-mail: mkrull@vims.edu.

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218 Marcos Krull, Denis M. S. Abessa and Francisco Barros

Enhancing the collaboration among scientists in different states and regions of Brazil may
reduce the gap in our knowledge of the effects of contaminants in many estuaries.

Keywords: ecotoxicology, estuary, pollution, experimental design

INTRODUCTION
Estuaries are among the most degraded coastal ecosystems on the planet (Agardy et al.,
2005). The human impact on these systems has led to the depletion of populations
and extinction of estuarine species in different parts of the world (Lotze, 2006). As a
consequence, many of the goods and services provided by these highly productive
environments, such as viable fisheries and provision of nursery habitats, are threatened
(Worm et al., 2006).
Estuarine systems are subject to a wide variety of human activities as they are sites of
port, industrial, urban, aquaculture, and recreational development. As a consequence, they are
exposed to many different point and non-point sources of contaminants such as oil spills,
untreated sewage discharge, urban and agriculture run-offs, and air pollution deposition
(Kennish, 2002). They often serve as sinks for different types of contaminants that
accumulate in the organisms and sediments, and cause adverse effects to the biota (Chapman
and Wang, 2001; Oliveira et al., 2016). Estuarine pollution is a major concern in tropical
emerging countries due to the rapid change in land use, agriculture expansion, and economic
and population growth (Kennish, 2002). Estuaries will continue to degrade under increased
demands unless proper management is applied to maintain their integrity and function
(Rapport et al., 1998). For this reason, robust environmental impact studies are necessary to
evaluate the status of estuaries and improve conservation efforts.
In Brazil, estuaries are the most abundant coastal environment, covering an area of
6.696.787 ha, that is 43.7% of the available coastal habitat (MMA, 2010). Due to the
country’s long coast line (approximately 7,400 km), estuaries can be found in a variety of
climates such as humid and semi-humid climates in the south and southeast and semi-arid
regions in the north Atlantic coast (Souza and Knoppers, 2003). The climate also influences
the presence and distribution of mangroves (Schaeffer-Novelli et al., 1990) and salt marshes.
Most of the population, ports and heavy industries are located in Brazilian estuaries and bays,
and as a result, pollution is heavily concentrated in these areas (Diegues, 1999).
This chapter reviews the most commonly used approaches to estimate the effects of
contamination in Brazilian estuarine systems for the last 15 years. Our goals are to identify (i)
the main classes of contaminants studied in estuarine systems in Brazil; (ii) the most used
lines of evidence (LOEs) to estimate the effects of contaminants; (iii) the most common
experimental designs including sampling size, spatial and temporal replication; and (iv) the
major gaps in the field and suggest future directions to researchers in Brazil.

METHODS
A literature survey was conducted using the ISI Web of Science search platform for the
keywords estuar* followed by contaminant*, pollut*, ecotoxicol*, ortoxicol*. Only papers

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 Assessing the Ecological Effects of Contaminants in Estuaries in Brazil 219

conducted in Brazil and authored by Brazilian authors between 2000 and 2015 were
included in the analysis. The studies considered in this chapter must have been conducted
in an estuarine system and aimed at identifying possible effects of contaminants in the
environment. Studies conducted in mangroves were included. In this sense, papers that
conducted bioassays with estuarine organisms to estimate the effect concentration of a
selected contaminant, or only measure the concentration of a chemical in the environment
were excluded. However, papers that only measured the concentration of contaminants in
estuarine organisms were included because of the possible metabolic, energetic and long term
costs associated with bioaccumulation and thus considered as an effect per se. Papers that
evaluated a species potential as a biomonitoring tool were also included.
All papers that met these criteria were subsequently reviewed and summarized in a
database according to the following groups: (i) general information, such as the name of the
system(s), region(s) and state(s) where the study was conducted and what LOEs were used;
(ii) statistical analysis and experimental design (e.g., spatial and temporal replication, sample
size, number of reference areas and statistical methods); (iii) measured contaminants, (e.g.,
type of chemical and environmental media); (iv) species and levels of organization (e.g.,
species or group of species analyzed); and (v) bioassays (e.g., species and endpoints used in
the bioassays).
Whenever some of this information was unclear or not presented in the text, tables or
figures of a paper, it was classified as “not reported.” In order to estimate how many papers
were published in one geographic area, studies conducted in different rivers within the same
bay or estuarine complex systems were combined. For instance, studies that were conducted
in the Subaé, Jaguaripe and Paraguaçu estuaries within Todos os Santos Bay, BA, were
counted and grouped under the same bay. Studies that evaluated sites across more than one
estuarine system, including reference areas, were separated by estuarine system in the
analysis, unless the reference area was only used in bioassays. If the design was unbalanced,
sample size was estimated as the mean sample size per treatment. If the sample size was only
reported as the minimum and maximum number of samples (e.g., higher than 5 and lower
than 10) it was considered as not reported, as the mean sample size cannot be precisely
estimated.
The salinity range of the sampling stations reported in each study was recorded in order
to estimate the most frequently studied estuarine salinity regions in Brazil. Categories were
defined as oligohaline (0.5-5), mesohaline (5-18), polyhaline (18-30) and euhaline (30-40)
following the Venice System (Anon, 1959). In cases where the study was conducted in more
than one system and the salinity range was reported for both systems, they were both included
in the analysis. The number of reference sites was analyzed separately from the spatial
replicates within each system.
Six broad categories of LOEs were considered: (i) chemistry, in which contaminants
are measured and estimated in samples of water, sediments, and/or porewater; (ii)
bioaccumulation, in which the concentration of contaminants are measured in tissues of
organisms; (iii) biomarkers, including a wide range of biomarkers such as enzymatic
activities and histopathological lesions; (iv) community and/or population structure, in which
measurements are taken from in situ samples of natural populations and/or communities; (v)
bioassays, controlled laboratory experiments in which organisms are exposed to an
environmental media to assess the possible effects of contaminants in predetermined
endpoints; and (vi) manipulative field experiments. Due to its broad definition, biomarkers

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220 Marcos Krull, Denis M. S. Abessa and Francisco Barros

were further classified into biochemical biomarkers (e.g., enzymatic activities and
metallothionein), hematological parameters, histopathological lesions, imposex, and
genotoxiceffects (e.g., DNA damage and micronucleus) to quantify which are the most
commonly used in Brazil.
The contaminants measured by each study were classified in four broad classes: (i)
organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs), aliphatic
hydrocarbons (HA), benzene, polychlorinated biphenyls (PCBs) and pesticides; (ii) metals
and metalloids (such as As); (iii) nutrients, and (iv) biological, pathogens such as Escherichia
coli or fecal steroids such as coprostanol and coprostanone. Each measured element and/or
compound was recorded to evaluate the most studied among each class of contaminants.
Contaminants measured in the chemistry and bioaccumulation LOEs were pooled together in
order to estimate what are the contaminants of greater concern to ecotoxicologists studying
Brazilian estuaries.

RESULTS
Out of the 549 papers initially sampled by the literature search, only 130 papers met the
criteria for inclusion. The number of papers generally increased over time with a peak in 2013
with 24 papers (Figure 1). The 130 papers were published in 49 different journals, with eight
journals accounting for 50.8% of the total (Figure 2). Marine Pollution Bulletin was the
journal with the highest number of publications (17) and with the highest impact factor
(2.991) among the eight journals.
The studies were conducted in 49 different estuaries along the Brazilian coast with a total
of 190 samples in these systems. Santos/São Vicente Bay (São Paulo state - SP) was the most
studied system with the highest number of papers, followed by Paranaguá Bay (Paraná state -
PR) and Guanabara Bay (Rio de Janeiro state- RJ), with 30, 21 and 15 papers respectively
(Figure 3). In the Northeast region, Todos os Santos Bay (Bahia state - BA) was the most
studied system with seven papers, followed by Goiana Estuary (Pernambuco state -PE),
Itamaracá (PE) and São Marcos Bay (Maranhão state - MA) with four publications each.
Guajará Bay (Pará state - PA) was the only studied system in the North region with 4 papers.
Most studies were conducted in the Southeast region with 58 papers (44.6%). Both Northeast
and South regions had 31 papers each (23.8% each). Six papers (4.6%) were conducted in
more than one region.
More than half of the studies (84 papers) did not report the salinity range of the sampling
stations (Figure 4). Of those papers that reported the salinity range, 22 studies were conducted
in only one salinity zone (15.6%) and 24 over two zones (17%). Only four studies (2.8%)
were performed across three zones and seven (5%) along all four salinity zones.

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 Assessing the Ecological Effects of Contaminants in Estuaries in Brazil 221

Figure 1. Number of published papers per year.

Figure 2. Number of papers published in the most frequent scientific journals. The impact factors
(2015) are indicated by white circles.

Figure 3. Spatial distribution of the papers conducted in Brazilian estuarine systems. The names of the
systems with more than seven publications are shown.

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222 Marcos Krull, Denis M. S. Abessa and Francisco Barros

Figure 4. Number of papers conducted per salinity zone.

Regarding spatial replication along the estuaries, 38 studies (27.1%) did not include
spatial replication in their design (Figure 5). More than half of the systems (57.2%) had three
or less spatial replicates and 15 systems (11.4%) had ten or more replicates. Only ten studies
(7.1%) used hierarchical nested designs.
Almost half of the studies had no temporal replication in their designs (48.1%) and 93.2%
had four or less replicates over time (Figure 6). More than half of the studies (52.6%) did not
include any reference site in their design and 33.6% of the studies included only one reference
site (Figure 7). Only 5.2% of the studies had 2 or more reference sites and 5.9% used nested
designs.

Figure 5. Number of papers for each of the number of spatial replicates.

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 Assessing the Ecological Effects of Contaminants in Estuaries in Brazil 223

Figure 6. Number of papers for each of the number of temporal replicates.

Figure 7. Number of papers for each of the number of reference sites.

Figure 8. Number of papers for each of the sample sizes.

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224 Marcos Krull, Denis M. S. Abessa and Francisco Barros

Figure 9. Number of papers for each of the most common statistical methods.

Forty-nine of the sampled estuarine systems (35%) had 9 or fewer replicates per
treatment with 22 papers (15.7%) having three replicates. More than half of the papers
(43.7%) had 10 or more replicates per treatment in their design and in 29 papers (20.7%) the
number of replicates was unclear or not reported (Figure 8).
Most papers used only univariate statistical methods (59.2% or 77 papers) to analyze
their data, while 10 papers (7.7%) used only multivariate methods and 41 papers (31.5%)
used both univariate and multivariate techniques. Two papers (1.5%) were descriptive and did
not use any formal analysis. No papers performed a priori power analysis or used Bayesian
methods. Among the most used statistical methods were ANOVA (58 papers; 44.6% of all
analyzed papers), correlation (34 papers; 26.2%), or a t-test (26 papers; 20%) (Figure 9).
Non-parametric Spearman's rank correlation and parametric Pearson’s correlation were
combined because some papers did not specify which method was used, while the distinction
was clear for the parametric ANOVA and non-parametric Kruskal-Wallis tests. The number
of factors used in the ANOVA was not always clear and thus one, two and three-way
ANOVA were pooled together as well. Analysis of covariance (ANCOVA) was used in only
six papers (4.6%). Among the multivariate techniques, Principal Component Analysis (PCA)
was the most used with 24 papers (18.3%), followed by Factor Analysis (FA; 10 papers;
7.6%). Non-metric multidimensional scaling (nMDS), Cluster and BIOENV analyses were
used in seven papers each (5.3%), and Canonical Correspondence Analysis (CCA) in six
papers (4.6%).
Regarding the number of LOEs, 74 papers (56.9%) used only one line of evidence, 46
used two (35.4%), nine papers used three (6.9%) and only one paper used five LOEs (0.77%).
The most common LOE was bioaccumulation with 53 papers (40.8%), followed by chemistry
analysis with 51 papers (39.2%) and biomarkers with 44 papers (33.8%) (Figure 10).
Community structure, bioassays, and manipulative experiments were less common with 27
(20.8%), 22 (18.9%) and 3papers (2.3%), respectively.
Among the papers that used biomarkers as a line of evidence, 73.3% considered
more than one class of biomarkers. Biochemical biomarkers were the most commonly used,
being recorded in32 papers (41% of all biomarkers), of which 33% (24 papers) evaluated
enzymatic activities. Genotoxic biomarkers were measured in 16 papers (22.2%), followed by
histopatological (18.1%; 13 papers).

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 Assessing the Ecological Effects of Contaminants in Estuaries in Brazil 225

Figure 10. Number of papers for each line of evidence.

Among the classes of contaminants, metals were measured in 45 papers (34.6%),

followed by organic contaminants with 17 papers (13.1%), and 9 papers measured both
metals and organic contaminants (6.9%) (Figure 11). Studies that only measured nutrients
(4.6%) or biological contaminants (3.1%) were less frequent. Fifty-five papers (26.9%) did
not measure any contaminant in the study.
Among the classes of organic compounds, PAHs were the most common, being measured
in 19 papers (14.5%), followed by PCBs with 11 papers (8.4%), organotins (e.g., TBT, DBT,
MBT) with 5 papers (3.8%) and aliphatic hydrocarbons with 4 papers (3%). PBDE and
organochlorides were measured in three papers each (2.3%). The most measured metals were
Cu (50 papers; 38%), Zn (47 papers; 35.9%), Pb (41 papers; 31.3%), Cd (37 papers; 29.2%),
Ni and Cr (33 papers; 29.2% each). Mercury (Hg) was measured in 29 papers (22.1%),
methylmercury (MeHg) in 5 (3.8%) and the metalloid As in 12 papers (9.2%).

Figure 11. Number of papers for each of the measured contaminants.

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226 Marcos Krull, Denis M. S. Abessa and Francisco Barros

Figure 12. Number of papers for each of the general groups of studied organisms.

Regarding the media in which the contaminants were measured, 39 papers measured
contaminant concentration only in organisms (30%), 28 papers measured it only in sediments
(21.5%) and 5 papers only in water samples (3.8%). Fourteen papers (10.7%) measured the
concentration of contaminants in two different environmental media and 6 papers (4.6%)
measured it in three or more.
Fish were the most studied organisms (47 papers; 36.2%), followed by benthic
macrofauna (20 papers; 15.4%), bivalves (14 papers; 10.8%) and crustacean species (11
papers; 8.5%) (Figure 12). Only 4 papers (3.1%) studied the effects of contaminants in
mangrove plants, and no work was conducted in salt marshes. Only 2 papers (1.5%) studied
mammals (dolphins) and no paper studied birds.
Most studies were conducted with the fish species Cathorops spixii (12.3%),
Micropogonias furnieri (8.5%), Genidens genidens (5.4%), Mugil lisa (4.6%), Sciades
herzbergii (3.1%) and Centropomus parallelus (3.1%) (Figure 13). Among the bivalve
species, Crassostrea rhiziphorae (6.2%) and Perna perna (3.8%) were the most frequently
studied.

Figure 13. Number of papers for each of the most frequently studied species.

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Figure 14. Number of papers for each media used in the bioassays.

Regarding the bioassays, 12 papers (9.2%) conducted bioassays with only one species, 8
papers (6.1%) used two species, and 2papers (1.5%) used three species. The most common
species used in bioassays were the amphipod Tiburonella viscana with mortality as the
measured endpoint (11 papers; 8.4%), the sea urchin Lytechinus variegates with abnormal
larval development as the endpoint (6 papers; 4.6%), the copepod Nitokra sp. with
reproduction as the endpoint (3 papers; 2.3%).
Thirteen papers (9.9%) conducted bioassays with only one environmental media, 2
papers (1.5%) with two media and 7 papers (5.3%) with three or four media. The most
frequent media were whole sediments with 16 papers (12.2%). Due to the relative high
frequency of studies that used sediment-water interface (6.1%) we included it here as another
environmental media (Figure 14).

DISCUSSION
Although most studies were conducted in the Southeast region of Brazil, only 1.3% of the
estuarine area of the country is in this region (MMA, 2010). The least-studied North and
Northeast estuarine systems make up 77.8% and 19% of all estuarine areas, respectively
(MMA, 2010). In fact, most papers in different areas published by Brazilian authors are from
institutions in the Southeast (74.5%), followed by the South (19%), Northeast (12.2%) and
North (2.7%) (Faria et al., 2011). As discussed by Leta et al. (2006), Brazilian scientific and
technological capacity is concentrated in a relative small number of institutions, mainly in the
more wealthy states. Many estuarine systems in the south and southeast regions are heavily
industrialized and have a long history of contamination (e.g., Torres et al., 2015). However,
the low number of papers in the north and northeast region does not mean that both regions
are not impacted by pollutants. For instance, PAHs in most sediment samples of the Guajará
Bay (PA) in the North region were found at concentrations above threshold effect levels
(TEL) and in the same range of the Guanabara Bay (RJ) (Sodré et al., 2014). The northeast
region also had an intense use of organochlorine pesticides before its ban in Brazil (Oliveira

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et al., 2016). In fact, the highest concentrations of total DDTs in mussels along the Brazilian
coast were found in the northeast region (Sericano et al., 1995).

Salinity Zones

Most papers (60%) did not report the salinity range of the sampling stations (Figure 4),
thus estimates of what are the most studied salinity zones in Brazil might be biased. The
salinity was also usually reported based on single measurements during the time of sampling,
therefore the classification of the salinity zones are likely to be inaccurate. However, because
46.7% of the papers had three or fewer spatial replicates (Figure 5), it is reasonable to
conclude that most studies are conducted in just a portion of the studied estuary.
Salinity is well established as a major factor influencing the distribution of estuarine
species (e.g., Sanderset al., 1965). It also affects the osmorregulation of organisms (McLusky
and Hagerman, 1987), and the bioavailability and uptake of metals in the environment
(Chapman and Wang, 2001). Likely for these reasons, enzymatic activities such as AChE and
biomarkers of exposure such as MT can also be strongly influenced by salinity (Pfeifer et al.,
2005; Monserrat et al., 2007). Salinity can also play an important role on phenotypic
plasticity of functional traits of the mangrove Avicennia schaueriana (Arrivabene et al.,
2014). Besides, because the salinities of environmental samples are adjusted to standard
values in bioassays, toxicity could also potentially be over or underestimated among sampling
sites (Chapman and Wang, 2001). In this sense, salinity can potentially influence different
LOEs used to estimate the effects of contaminants in estuarine systems.
In many cases, the salinity gradient might also overlap the pollution gradient (e.g.,
Fitzpatrick et al., 1997) and attention is required when interpreting the obtained results. For
instance, the number of different groups of species tends to naturally decline from the
euhaline to the oligohaline salinity zone in both temperate (Dauvin, 2008) and tropical
systems (Barros et al., 2014). One could conclude that the number of species decreases with
distance from a pollutant source or that the number of species in a contaminated site in an
oligohaline zone is lower than in a reference site located downstream. In such a case, the
distinction between natural and anthropogenic stress is often unclear. This is known as the
estuarine quality paradox (Elliot and Quintino, 2007).
As discussed earlier, not only the number of species is affected by estuarine gradients, but
other LOEs might be subjected to the natural stressful conditions of the estuarine system.
Researchers have suggested that it is important to include multiple LOEs in a study to account
for the heterogeneity and variability of estuarine systems (e.g., Chapman and Wang, 2001;
Dauvin, 2008). Multiple reference areas might also be necessary to account for the spatial
heterogeneity along the salinity gradient. Thus, in most cases, the salinity gradient should not
be dismissed when designing the study and it should be reported. Effort should also be made
to collect a comprehensive salinity dataset of the studied system(s) in order to have a more
accurate estimate of the temporal and spatial fluctuation of salinity. In addition, other
gradients (e.g., temperature, pH, organic carbon, grain size) should be measured as they can
also influence the fate and effects of contaminants in estuaries. Most papers measured at least
one of these variables, thus they were not discussed in this chapter.

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Spatial and Temporal Replication

Given that studies of estuaries are subject to many confounding variables, experimental
designs aiming at evaluating the effect of contaminants need to take into account spatial
gradients and comparisons to reference sites. As discussed by Green (1993), natural
environmental heterogeneity can coincide with control versus impact locations, and spatial
replication of both contaminated and reference locations are important. In this chapter, 27.1%
of the papers had only one spatial replicate (Figure 5) and 52.6% of the papers (Figure 6) had
no reference sites in their design. Some bioaccumulation studies just evaluated if the
concentrations of contaminants in organisms where within the maximum limits for human
consumption, and in this case reference sites may be not necessary. However, if the goal is to
evaluate if the concentrations are due to a potential source of contaminants, reference sites
may be important.
Many studies (48.1%) also did not include temporal replication in their designs, and of
those that did, most looked at differences among seasons within a year (Figure 6). The main
concern with this approach is that seasons are not replicated among years and the only
inference that can be made is about the effect of season in that specific year. Thus, results of
such a design need to be carefully interpreted. There are good reasons for why season can
influence the effects of contaminants in estuaries, such as increase in contaminants runoff,
freshwater input, changes in salinity and redox potential of sediments (Chapman and Wang,
2001). How seasons affect these variables can vary among years.
For instance, Costa et al. (2009) evaluated the concentrations of Hg in tissues of the fish
Trichiurus lepturus in the Goaiana Estuary (PE) using a nested design with months nested
within seasons (dry in 2005, rainy in 2006 and dry in 2006). The data were analyzed using
two-way ANOVA considering a possible interaction of months and season instead of months
nested within season. The authors did not reject the null hypothesis of no difference in the
mean of Hg in fish tissues among months. The null hypothesis of no difference between the
dry season in 2005 and rainy season in 2006 was rejected. However, it was not rejected in the
rainy season in 2006 and the dry season in 2006. In fact, both dry seasons were considered
significantly different from each other. The authors concluded that rainfall might be the main
factor explaining the variation in Hg concentrations in fish. However, if only two seasons
were sampled, the conclusion would depend on the year of sampling.
Including replication among years can be prohibitively costly in many cases; however,
long term studies are necessary to evaluate seasonal effects. Nested designs can also help
identify the source of variation in different temporal scales. If funding is limited and
replication in the long term is infeasible, increasing sample size and including reference areas
in the design can be a better allocation of resources. Due to the importance of reference areas
in environmental impact studies, Brazilian scientists should increase efforts of mapping
reference areas at a regional and national scale.

Sample Size

Sample size is also an extremely important element of the experimental design as higher
number of samples can increase statistical power. Statistical power is the probability of
rejecting the null hypothesis 𝐻0 when it is false, in favour of an alternative hypothesis 𝐻𝐴

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230 Marcos Krull, Denis M. S. Abessa and Francisco Barros

(i.e., 1- β (Type-II error rate)). This means that an alternative hypothesis must be specified a
priori (i.e., the effect size must be defined) in order to estimate the power of the test (Green,
1993)1. In this sense, without a priori defining the hypothesized effect size and β, no
conclusions can be made about 𝐻𝐴if the p value is lower than the selected α. For this reason,
power not only depends on the sample size, but also on the Type-I error (α) and the
population effect size (Cohen, 1992). Power curves can be used to help estimate the number
of samples necessary to achieve the desirable β to detect a hypothesized effect size given an
α. Nowadays, power analysis can be easily implemented in many statistical packages such as
the R environment (R Core Team, 2015).
As is generally the case in the ecotoxicology literature, of the studies included in this
chapter none had conducted power analysis2. All papers that used frequentist inferential
statistics assumed an α of 0.05 and in only one case α was adjusted to 0.01. Some papers
stated that the adopted α was 0.05 or 0.01 which is a fundamentally flawed, but common,
practice. As discussed above, the selected α should be defined according to the number of
replicates, β and the effect size. There are many cases in the field of ecotoxicology in which
the consequences of a Type-II error are much more costly than that of a Type-I error, and the
opposite can also be true. As discussed by Gigerenzer (2014), the level of significance should
not be fixed by convention and used mechanically for either Fisher’s and Neyman-Pearson
framework. Even though descriptive and frequentist statistics were the only used approach,
other prominent methods are available such as Bayesian statistics and confidence intervals
(Hobbs and Hilborn, 2006).
In this chapter, the number of papers with sample size from three to around nine seems to
decrease and tends then to increase for ten or more replicates (Figure 8). This bimodal
distribution can be explained by the distinct designs and sampling methods used by different
LOEs. The lower number of replicates was more common in papers that used community
structure and bioassays as LOE, while the higher number of replicates was associated with
biomarkers and bioaccumulation analyses. The number of replicates per treatment was often
unclear, especially for studies conducted with fish, where usually only the total number of
caught fish is reported. In many cases, the number of organisms and the degrees of freedom
were also not reported.

Lines of Evidence

Many researchers suggest that multiple LOEs should be used when evaluating the effects
of contaminants in estuaries, especially for sediment quality assessment (e.g., Batley et al.,
2002, Torres et al., 2015). Most studies reviewed in this chapter generated only one (57.3%)
or two (35.1%) LOEs. Among the reasons to use multiple LOEs is that no single line can
reliably predict the effect of the others and there are uncertainties associated with each LOE
(Batley et al., 2002). For instance, Torres et al. (2015) might conclude based on the AVS-
SEM (Acid Volatile Sulfide/Simultaneously Extracted Metals) approach that metals are
biologically unavailable in sediments of the Santos Estuary (SP); however, high

1
Note that the concept of power and β are only applied in a Neyman–Pearson decision theory logic because, in
Fisher’snull hypothesis testing framework, noalternative hypothesis is specified (Gigerenzer, 2004).
2
Because many bioassays are standardized, it is assumed that power analyses were conducted during the
development of the protocols (ASTM, 1996; however see Crane and Newman, 2000).

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concentrations of some metals were observed in soft tissues of both transplanted and native
oysters. Krull et al. (2014) also found a lack of agreement between sediment bioassays, metal
contamination and the benthic macrofauna assemblage in the Jaguaripe Estuary (BA). Even
though it is highly advisable to use more than one LOE, the total number and particular LOEs
to be used will depend mostly on the question, logistics and funding (Chapman and Hollert,
2006).
Bioaccumulation was the most commonly used LOE (Figure 10) and bioaccumulation
studies were conducted mainly with fish and bivalves (Figures 12 and 13). Even though most
of these studies measured total length and weight, ANCOVAs were only used in six papers.
For instance, Silva et al. (2003) found a size effect using log dry weight and some metal
concentrations (e.g., Cu, Ni) in soft tissues of C. rhizophorae in the Curimatau Estuary (Rio
Grande do Norte state - RN). Some elemental concentrations can also decrease with animal
size, such as Cr and Ni in C. spixii specimens of the Paranaguá Bay (PR) (Angeli et al.,
2014). In such cases, using ANOVAs without accounting for size or weight effects (i.e.,
without adjusting the mean factor level for the effect of the covariate) can lead to biased and
incorrect results. In cases where the covariate is not significant, the statistical model can be
reduced to a single factor ANOVA3. Most studies conducted correlation analysis between
species length and the bioconcentration of contaminants, or t-tests to estimate the differences
in mean of total length among sites or seasons; however, this is insufficient as the covariate
was not formally included in the final linear model.
Length and size of the organism might also be an important covariate when analyzing
biomarkers. For instance, Flammarion et al. (2002) found that if length of the fish Leuciscus
cephalus were not included as a covariate, ChE activity would indicate that in eight sites out
of 17, the null hypothesis of no difference between reference sites and contaminated sites
would be rejected. However, after adjusting for organism size effect, the null hypothesis was
rejected in only two, notably polluted, sites. The effects of size as a covariate on fish
biomarkers were also demonstrated in other enzymatic activities such as glutathione S-
transferase (GST) (Siscar et al., 2015) and ethoxyresorufin-O-deethylase (EROD)
(Wunderlich et al., 2015). Plotting the data before running the analyses is recommended for
investigating possible effects of covariates on the response(s).
Another common approach is to measure multiple biomarkers in different tissues from
the same organisms and perform multiple statistical tests such as ANOVAs for each response
variable. Most papers (73.3%) that used biomarkers as a LOE measured more than one class
of biomarkers. As Krull et al. (2013) pointed out, these measures are not independent from
each otheras they might be correlated and multivariate analysis might be a better option to
overcome this issue. For instance, Arrivabene et al. (2014) used Linear Discriminant Analysis
and Factor Analysis to analyze anatomical and morphological parameters of mangrove leaves
and physico-chemical parameters among sites. Gusso-Choueri et al. (2015) also used Factor
Analysis to analyze metal and organic bioaccumulation and eight biomarkers in the fish C.
spixii.

3
The general ANCOVA statistical model can be written as: 𝑦𝑖𝑗 = µ + 𝛼𝑖 + β(𝑥𝑖𝑗 – 𝑥̅ ) + 𝜀𝑖𝑗 ; where 𝑦𝑖𝑗 is the
response of the ith level of the factor in the jth replicate, µ is the overall mean or the intercept, 𝛼𝑖 is the effect of
the ith treatment of the factor, β is the effect of the covariate, 𝑥̅ is the mean value of the covariate and 𝑥𝑖𝑗 is the
covariate value of the ith level of the factor and the jth replicate, and 𝜀𝑖𝑗 is the random unexplained error with
mean of 0 and variance of 𝜎𝜀2. If the covariate does not have an effect on the response variable (i.e.,
β=0) the statistical model is reduced to a single factor ANOVA.

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232 Marcos Krull, Denis M. S. Abessa and Francisco Barros

Most multivariate techniques were used to analyze biological communities or multiple

LOEs (e.g., chemistry, community structure and bioassays). Similar to a review of
multivariate methods in ecology (James and McCulloch, 1990) the most commonly used
ordination method was PCA (18.3%). Exploratory Factor Analysis (7.6%) was the second
most used ordination technique and was more common among studies that conducted
sediment quality analysis (7 out of 10 papers). The high relative frequency of Factor Analysis
(among sediment quality analysis studies) was not observed in general ecology (James and
McCulloch, 1990) and in benthic ecology papers (Carvalho et al., 2015). As discussed by
James and McCulloch (1990) both PCA and Factor Analysis are exploratory analyses which
may help understanding the patterns in the data and formulating testable hypothesis.
Bioassays and manipulative experiments were less common approaches (Figure 10). This
is probably because of the low number of developed standard bioassays with native estuarine
species in Brazil (Krull and Barros, 2012). For instance, the native sea urchin L. variegates
was the second most used species in estuaries. Regarding manipulative experiments, two
papers transplanted oysters from uncontaminated to possibly contaminated areas (Maranho et
al., 2012, Torres et al., 2015) and one paper performed a reciprocal sediment transplant
among contaminated and reference sites to look at the colonization of benthic species (Gern
and Lana, 2013).

Classes of Contaminants

Among the classes of contaminants, organics were studied much less often than metals
(Figure 11). However, this is probably not related to the fact that organic contaminants are not
a major concern in estuarine systems in Brazil. For instance, high concentrations of PAHs
were found in oyster tissues and sediments of the Santos Estuary (SP) and might be
contributing to the negative effects on the benthic community (Torres et al., 2015). The
concentrations of benzene, total phenols, TBT, and PCB in sediments and water of the São
Marcos’s Bay (MA) were above the limits set by Brazilian legislation and possibly
influencing the enzymatic activity of the fish S. herzbergii (Carvalho-Neta et al., 2012). The
biomagnification factor of PBDEs in dolphin liver in relation to scabbardfish and croaker of
the Paraíba do Sul Estuary (RJ) were similar to the ones estimated for teleost fishes and
bottlenose dolphins in Florida (Quinete et al., 2011) even though PBDEs were never
produced in Brazil (Lavandier et al., 2013).
In fact, the lower number of papers that analyzed organic contaminants can possibly be
related to the lack of facilities to conduct such analysis and associated higher costs. Dachs and
Méjanelle (2010) argue that in the last decade, organic pollutants were not being studied
around the world due to limitations in the analytical methods available and also the time and
money required for analyses. In Brazil, the first paper that measured the concentrations of
TBT in sediments was dated 2003 (Godoi, et al., 2003) even though methods had been
available for more than ten years in other parts of the world (e.g., Langston et al., 1987).
Concentrations of organochlorine pesticides (OCPs) were measured for the first time in
sediments of the Jaguaribe Estuary (Ceará state - CE) in 2016, even though this is a region of
historical high use of OCPs (Oliveira et al., 2016). In this study, the concentrations of γ-HCH
and heptachlor were judged to present high risk and DDTs presented moderate risk when
compared to sediment guidelines. Studies that evaluated the effect of metals in bioassays

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 Assessing the Ecological Effects of Contaminants in Estuaries in Brazil 233

(Nilin et al., 2013) and enzymatic activities of crabs (Davanso, 2013) of an estuary in the
same state (CE) concluded that other contaminants such as pesticides might also be affecting
the biota. However, there is insufficient data available regarding pesticide contamination in
this region.
Historically, metal analysis in estuarine sediments in Brazil can be traced from the early
eighties (e.g., Pfeiffer et al., 1980), and by the end of that decade metal contamination in
sediments was already recorded in different regions of the Brazilian coast (Pfeiffer et al.,
1988). By that time, concentrations of PAHs, PCBs, and DDT were first being measured in
sediments of the coast of Rio de Janeiro (Japenga et al., 1988) and in mussels of Todos os
Santos Bay (BA) (Tavares et al., 1988). Currently, there is considerable evidence that many
Brazilian estuaries are affected by metal contamination (e.g., Cesar et al., 2006; Hatje et al.,
2006; Souza et al., 2015) indicating that metals are a major class of contaminants in estuaries.
However, organic pollution is still growing in Brazil. As suggested by Oliveira et al. (2016),
ecotoxicology studies evaluating the effect of OCPs are urgent in mangrove and estuarine
systems of Brazil. One way of overcoming this issue is improving the network connectivity
among scientists from different institutions, states and regions so that the lack of facilities and
funding are not a major barrier driving the assessment of contaminants in estuarine systems in
Brazil.

CONCLUSION
Brazil has made great strides in addressing the impact of contaminants on estuarine
ecosystems. This chapter identified the most common patterns of publications in the field and
made some recommendations for future studies, as follows: (i) because salinity is one of main
factors affecting the distribution of organisms and the bioavailabily of contaminants in
estuaries, salinity, and other variables, need to be taken into account when selecting the
sampling sites (including the reference areas) and planning other elements of the design; (ii)
appropriate reference areas need to be included more in designs; (iii) in the absence of
sufficient temporal replication to address the study question, resources should be allocated to
other important elements of the design, such as increasing the sample size, and inclusion of
reference areas or other LOEs; (iv) conducting power analysis is necessary to select a priori
the appropriate α, β, sample size, and effect size; (v) the covariates effect needs to be
considered more when performing bioaccumulation studies; and (vi) include different LOEs
in the design when possible. Clearly stating the question and the hypotheses to be tested is
extremely important to help making decisions about the temporal and spatial scales of the
study, allocation of resources and selection of the LOEs. Currently, most studies have focused
on metal contamination and provided evidences that metal contamination is a major concern
in Brazilian estuaries. On the other hand, organic contaminants are much less studied in
Brazil even though there are many different sources and types of contaminants that could
potentially be harmful to estuarine ecosystems.

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234 Marcos Krull, Denis M. S. Abessa and Francisco Barros

ACKNOWLEDGMENTS
We are thankful to R. M. Jones and M. Newman for their comments and review on early
versions of this chapter. M. Krull was funded by the CAPES Foundation (0859/14-1) during
the tenure of this work.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 15

AVOIDANCE AND RECOLONIZATION RESPONSES OF

THE GASTROPOD OLIVELLA SEMISTRIATA EXPOSED
TO COASTAL SEDIMENTS

Cristiano V.M. Araújo1,2,3,*, David Salvatierra3,

Victoria Vera-Vera3, Luís A. Cedeño-Macías3,
Katherine Benavides3, Dayanara Macías-Mayorga3,
Matilde Moreira-Santos1 and Rui Ribeiro1
1
CFE-Centre of Functional Ecology, Department of Life Sciences,
University of Coimbra, Coimbra, Portugal
2
Institute of Marine Sciences of Andalusia (ICMAN-CSIC),
Department Ecology and Coastal Management, 11510, Puerto Real, Spain
3
Central Department of Research (DCI),
Ecuadorian Aquatic Ecotoxicology (ECUACTOX) group,
Universidad Laica Eloy Alfaro de Manabí (ULEAM), Manta, Ecuador

ABSTRACT
The spatial distribution of the snail Olivella semistriata along the Ecuadorian coast
near the city of Manta seems to be influenced by urban discharges. This observation leads
us to hypothesize that contamination might determine inhabitable areas for the snails.
Therefore, the ability of the snails to detect the local contamination and react by moving
away from contaminated sediment has been assessed. In addition, the ability of the snails
to recolonize contaminated sediment under recovery was also studied. Six sediment
samples (El Murciélago beach – reference point, El Puerto, La Poza, Río Burro, Los
Esteros, and Río Muerto) were taken and tested in two different assays: avoidance and
recolonization. Assays were performed in a non-forced exposure system in which a
contamination gradient was formed by mixing the test sample and the reference sediment.
Avoidance was more intense in the samples from Río Burro and Río Muerto. In the

*
Corresponding Author E-mail: cristiano.araujo@icman.csic.es.

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240 Cristiano V. M. Araújo, David Salvatierra, Victoria Vera-Vera et al.

recolonization assays, the reference sample was always preferred regarding all test
samples. As there is no physical barrier to prevent the displacement of the organisms
among the studied areas, the absence (visual field observation) of snails in the sediment
from Rio Burro and Rio Muerto is suggested to be due to the ability of the organisms to
avoid these areas. The present data indicate that contaminants can affect the spatial
distribution of the snails by triggering avoidance or even preventing the colonization of
contaminated areas, acting as a chemical barrier that might isolate populations.

Keywords: habitat selection, sediment, spatial distribution, toxicity

INTRODUCTION
The ability of gastropods to sense and react to environmental stimuli has been widely
documented, being differences in sediment grain size and availability and nutritional quality
of the food some of the stimuli recognized by these organisms (Barnes and Greenwood, 1978;
Forbes and Lopez, 1986, Marklevitz et al., 2008a, Araújo et al., 2015). Regarding gastropod
ability to sense contamination levels, laboratory studies have attested that they can use
different mechanisms such as retraction inside their shell (Hellou et al., 2009; Araújo et al.,
2012), floating, burying, crawling (Hampel et al., 2009; Campana et al., 2013), or grouping
(Leung et al., 2004), to reduce their contact with contaminated sediments. The gastropods
Ilyanassa obsoleta (Marklevitz et al., 2008a,b; Hellou et al., 2009) and Peringia ulvae (=
Hydrobia ulvae) (Araújo et al., 2012) have already been shown to be capable of avoiding
contaminated sediments also by moving towards less stressful areas. In a field study a change
in the population density of the snail P. ulvae was observed along an eutrophication gradient
(Lillebø et al., 1999), which could be partially explained by their avoidance behaviour to
escape from the contamination. More recently, an experimental study showed that the
gastropod P. ulvae was able to detect patches of sediment with different contamination levels
(Araújo et al., 2016). According to these results, in a contamination scenario in which
contaminated and non-contaminated sediment patches are heterogeneously distributed, local
gastropod populations could rearrange their spatial distributions by avoiding contamination.
For organisms able to detect and avoid contaminants, such behaviour might have serious
implication in their habitat selection and spatial distribution pattern (De Lange et al., 2010;
Hellou, 2011). In this sense, contaminants may play a decisive role in the habitat structure
even if no noxious physiological effect at the individual level is triggered (Ares, 2003; Hellou
et al., 2008).
Contamination can not only trigger the avoidance response of the organisms which
are able move to less stressful areas, but also prevent (re)colonization processes (Hellou,
2011; De Lange et al., 2013; Moe et al., 2013). In areas where a contamination input
is stopped and is subsequently followed by a decrease in contamination levels, it is expected
that populations gradually recolonize them, as of the moment contaminant concentrations are
no longer repulsive. Therefore, by using avoidance and recolonization experiments it is
possible, on one hand, to predict how repulsive a habitat is and, on the other hand, to assess
the potential of this area to be recolonized. The use of both avoidance and recolonization
approaches incorporates the concepts of population dynamics and disturbance ecology
in ecotoxicological studies by considering organism displacement as a response to

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 Avoidance and Recolonization Responses of the Gastropod Olivella semistriata … 241

contamination, largely contributing to understand at which temporal and spatial scales

populations are expected to recover from disturbance (Worm and Duffy, 2003; Hellou et al.,
2008; Schmitt-Jansen et al., 2008).
As deposit-feeding organisms inhabiting sediment, gastropods are suitable key organisms
to be used in sediment environmental quality assessment schemes, and due to their
responsiveness to avoid contamination, they can provide valuable information about how
habitable an area can be. The present study aimed at assessing the quality of coastal sediments
from the urban coast of Manta (Ecuador) by using as endpoint the ability of the native
gastropod Olivella semistriata to avoid contamination. Additionally, a novel approach using
experiments of recolonization was employed in order to assess the potential of sediment to be
recolonized by gastropod populations and, in the case of the occurrence of avoidance, to
predict at which contamination levels recolonization could be expected.

METHODS
Test Organisms

Individuals of the snail O. semistriata were collected from the El Murciélago beach
(Manta, Ecuador, [80° 44' 0.18'' W and 0° 56' 24.25'' S: El Murciélago beach]) during low
tide. Organisms were selected according to size (mean [n=10] 17±2 mm length) and
maintained in a 20 L aquarium with 5 L of 0.7 µm-filtered natural seawater (salinity between
26-30, pH of 7.8, alkalinity of 162 mg CaCO3 L-1, 0.25 mg L-1 of ammonium, and 0.15 mg L-
1 of nitrite) and a 3 cm layer of El Murciélago beach sediment. The culture was kept at 25°C

with a photoperiod of 12/12 h (light/dark) and renewed daily. As the main food source to
gastropods is microphytobenthos, additional food besides natural sediment from El
Murciélago beach was not provided.

Sediment Samples

Sediment samples were taken from the Ecuadorian coast near the city of Manta at six
different points (Figure 1): El Murciélago beach, El Puerto (80° 43' 15.36'' W and 0° 56'
47.07'' S), La Poza (80º 43' 9.08'' W and 0° 56' 54.21'' S), Río Burro (80° 42' 55.66'' W and 0º
56' 57.56'' S), Los Esteros (80° 42' 13.43'' W and 0° 56' 56.90'' S) and Río Muerto (80° 41'
49.42'' W and 0° 56' 52.90'' S). El Murciélago beach was the sampling point from where the
organisms for assays were collected and thus was considered as the reference point.
Superficial sediment (top 2 cm) was sampled by hand during low tide. Samples were kept at
4°C until the start of the experiments. Total metal concentrations (Al, Cd, Cu, Fe, Hg, Mn,
Ni, and Pb [inductively coupled plasma mass spectroscopy – ICP-MS, Thermo, ICAP 7000]),
total organic carbon and organic matter contents [titration; Walkley and Black, 1934], levels
of NO3, PO4, and SO4 [spectrophotometer, Hach, DR2800]), and granulometry (wet sieving)
were analysed in the sediment samples (Table 1).
A visual survey of the presence of O. semistriata in all the studied areas revealed that this
species is found in El Puerto, La Poza and Los Esteros, besides El Murciélago beach where

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242 Cristiano V. M. Araújo, David Salvatierra, Victoria Vera-Vera et al.

test organisms were collected from. Along the sampled area no organisms were found in Río
Burro and Río Muerto, areas where two important discharges of domestic and industrial
effluents occur.

Avoidance Assays

Before the assays, each tested sediment was mixed with reference sediment (El
Murciélago beach) to obtain four different contamination levels: 25, 50, 75 and 100%
(proportions of contaminated sediment). The mixture proportions were based on wet volume.
Sample mixtures were placed on rectangular trays (27.5 cm x 6 cm x 4 cm), which were
divided into five sections (5.5 x 6 cm). In each section, each of the four different proportions
of the sediment plus the reference sediment (0% of contaminated sediment) were disposed
forming a gradient of the test sediment as described in Araújo et al. (2012) such that the
reference and 100% tested sediment were at opposite extremities of the tray (Figure 2). A
control tray with only reference sediment was also prepared to test for preference/avoidance
response of organisms to any section of the tray in particular. Trays were then filled with 20
mL of seawater used in the culture of the organisms, enough to keep the sediment sufficiently
wet but without the possibility of migration of the organisms by floating. Once the trays were
prepared, five organisms were introduced in each section, totalling 25 organisms per tray.
Each tray was assayed in triplicate. After 30 min and 6 and 24 h, the distribution of the
organisms in the sections was checked by counting. Assays were performed at 25°C and in
darkness.

Figure 1. Sediment sampling points on the Ecuadorian coast near the city of Manta.

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 Avoidance and Recolonization Responses of the Gastropod Olivella semistriata … 243

Table 1. Physical-chemical characterization of the sediment samples collected from

study sites along the coastal area of Manta city, Ecuador

Samples
Parameters El Río Los Río
El Puerto La Poza
Murciélago Burro Esteros Muerto
Al (mg kg-1) 5550 5747 5774 4535 5959 3752
Cd (mg kg-1) 0.67 0.53 0.65 0.48 0.53 0.45
Cu (mg kg-1) 9.4 18.8 33.9 10.7 13.8 7.7
Fe (mg kg-1) 11015 8954 10245 7571 8668 6908
Hg (mg kg-1) <0.3 <0.3 1.3 0.4 <0.3 0.3
Mn (mg kg-1) 163.3 82.2 81.0 86.0 85.5 105.3
Ni (mg kg-1) 14.5 13.6 15.6 11.1 12.7 9.1
Pb (mg kg-1) 2.1 1.8 2.9 1.2 1.4 1.2
Zn (mg kg-1) 39.3 48.9 65.2 33.4 40.2 27.1
TOC (mg kg-1) 1400 500 5500 3500 2000 1800
OM (%) 0.25 0.08 0.95 0.60 0.36 0.31
NO3 (mg kg-1) <0.420 <0.420 <0.420 <0.420 <0.420 <0.420
PO4 (mg kg-1) 1.6 2.8 48.5 2.5 4.8 3.9
SO4 (mg kg-1) 490 430 640 230 500 330
Particle size (%)(< 2.2 17.9 34.6 5.3 14.1 6.5
75 µm)
(75 – 425 µm) 97.8 82.1 65.4 94.7 85.9 93.5
Total metal concentrations are in dry weight. TOC: total organic carbon; OM: organic matter.

Recolonization Assays

For recolonization assays, the same sediment gradient was used and the sediment
dilutions were distributed to form a toxicity gradient as previously described. In these assays,
all 25 organisms were introduced in the section containing the reference sediment (Figure 2).
As for the avoidance assays, recolonization assays solely with reference sediment were
performed to verify whether the snails were able to uniformly colonize the entire tray along
its five sections. Assays were also performed in triplicate and under the same environmental
conditions as described above. The distribution of the organisms was similarly recorded after
30 min and 6 and 24 h.

Statistical Analysis

Avoidance assays with reference sediment were used to test absence of preference/
avoidance to any section of the tray. This assumption, considered as criterion for the
validation of the results, was tested by using Yates’ chi2 test as described by Moreira-Santos
et al. (2008) with the number of expected organisms (considering a homogeneous distribution
along the five sections) and observed organisms. This statistical test was also applied to the
avoidance assays with potentially contaminated sediment samples, so that calculation of
avoidance percentage was only performed when the uniformity of the organisms’ distribution
was statistically rejected. Avoidance was calculated at the three exposure times (30 min and 6

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244 Cristiano V. M. Araújo, David Salvatierra, Victoria Vera-Vera et al.

and 24 h) from the number of expected organisms (NE). For each exposure time, NE was
calculated considering a homogenous distribution of the observed organisms, which assumes
no preference for any sediment concentration lower than that of the respective section. For
instance, for the most contaminated section (#5; 100% contaminated sediment; Figure 2), the
number of expected organisms was equal to the total number of organisms observed within
sections #1 to #5, divided by the corresponding total number of sections (i.e., 5). For the
adjacent section (#4; 75% contaminated sediment), NE was calculated considering the number
of organisms observed within sections #1 to #4, divided by the corresponding number of
sections (i.e., 4). For section #2 (less contaminated section excluding the reference sediment;
25% contaminated sediment), NE was equal to the total number of organisms observed in the
first (reference sediment) and second sections, divided by 2 (the corresponding number of
sections). The number of avoiders was determined for each sediment section by calculating
NE minus the number of observed organisms (NO) in that section: Avoiders=NE - NO. Finally,
avoidance (in %) was calculated using the formula: Avoiders/NE×100. For the calculation of
the recolonization (in %): Recolonizers=NO in each section (except the one with the reference
sediment) and Recolonization=Recolonizers/NE×100, which leads to the same result as
Recolonization=100–Avoidance. Mean avoidance and recolonization and their respective
standard errors were calculated with the three replicates. The concentration of sediment
causing the avoidance and recolonization of 50% of the population after the total exposure
time of 24 h (AC50 and RC50, respectively), and corresponding 95% confidence intervals, was
determined using the software PriProbit 1.63 (Sakuma, 1998). Finally, to better infer the
relationship between avoidance and recolonization responses under similar contamination
levels, the expected recolonization at 24 h was calculated directly from the data of the
respective avoidance assays in order to compare it with the observed recolonization obtained
in the recolonization assays. This 24 h expected recolonization was determined using the
formula: 24 h expected recolonization=100% - avoidance.

RESULTS
Reference Sediment

Results from the avoidance assay with solely reference sediment (control) aimed at
verifying the uniformity of the final distribution of the organisms along the tray in the
absence of contamination. The number of observed organisms in each section at any
observation time (30 min, 6 and 24h) was not statistically different from the number of
initially deployed organisms (Yates’ chi2 tests; p>0.05), confirming that there was no
preference/avoidance of the snails for any section in particular.

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 Avoidance and Recolonization Responses of the Gastropod Olivella semistriata … 245

Figure 2. Experimental setup of the avoidance and recolonization experiments. The order of the
sections (numbered from #1 to #5) referred to in the text goes from left to right: section #1 is at the left
extremity and section #5 is at the right extremity.

175 30 min
6h
150
% Recolonization

24 h
125 24 h expected
recolonization
100
75
50
25
0
2 3 4 5

# Section
Figure 3. Recolonization percentage of the snail Olivella semistriata for each section expected to be
colonized (as all organisms were initially placed in section #1) in the recolonization assays with
reference sediment only.

As for the recolonization assays with reference sediment, the distribution of snails along
the entire tray was influenced by exposure time (Figure 3). After 30 min, organisms mainly
colonized sections #2 and 3, the two closer to the section where they were introduced (#1).
The farthest section was colonized by 50% of the expected number of organisms after 6 h, but
after 24 h the colonization percentage increased to 100%. It was also observed that after 24 h
the variability of the recolonization response increased with the distance covered from the

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246 Cristiano V. M. Araújo, David Salvatierra, Victoria Vera-Vera et al.

reference sediment. The observed recolonization was similar to the 24 h expected

recolonization for sections #2 and #5, but lower for intermediate sections #3 and #4.

El Puerto

No avoidance was observed for any concentration of the sediment from El Puerto (graph
suppressed). Although recolonization increased with exposure time, it was less than 20% for
the two most contaminated sections. Thus, the 24 h expected recolonization was in general
overestimated except for the concentration of 25% (Figure 4).

La Poza

Avoidance was observed during the whole assay, but it was not concentration-dependent.
The most intense avoidance was observed for 25% of sediment with values around 60%,
although only after 6 h of exposure. For the other concentrations, avoidance was lower than
25%. Recolonization assays showed that only the concentration of 25% was colonized. The
24 h expected recolonization was similar to the observed recolonization percentage for the
concentration of 25% at 6 h; however, for this same concentration it was underestimated after
24 h exposure. For all the other concentrations it was overestimated throughout the entire
exposure period (Figure 5).

140
120
% Recolonization

100
80 30 min
6h
60 24 h
24 h expected
40 recolonization
20
0
25 50 75 100

% Sediment dilution
Figure 4. Recolonization percentage of the snail Olivella semistriata exposed to different dilutions of
the sediment from El Puerto.

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 Avoidance and Recolonization Responses of the Gastropod Olivella semistriata … 247

100 100
30 min
80 6h

% Recolonization
24 h 80

% Avoidance
60
30 min
40 60 6h
24 h
20 40 24 h expected
recolonization
0
20
-20
-40 0
25 50 75 100 25 50 75 100
% Sediment dilution % Sediment dilution

Figure 5. Avoidance (left) and recolonization (right) percentages of the snail Olivella semistriata
exposed to different dilutions of the sediment from La Poza.

100 60
30 min
50 6h
80

% Recolonization
24 h
% Avoidance

60 40 24 h expected
recolonization
30
40
20
20
30 min
10
0 6h
24 h 0
-20
25 50 75 100 25 50 75 100
% Sediment dilution % Sediment dilution

Figure 6. Avoidance (left) and recolonization (right) percentages of the snail Olivella semistriata
exposed to different dilutions of the sediment from Río Burro.

Río Burro

Snails avoided the sediment sample from Río Burro by more than 60% after 6 h of
exposure. Yet, for the most concentrated sediment, avoidance was slightly reduced compared
to other concentrations. With regard to recolonization, only the concentration of 25% was
recolonized at a percentage of around 40% after both 6 and 24 h exposures. The 24 h
expected recolonization was very similar to the observed recolonization for the concentrations
of 25 and 50%; however, it was overestimated for the two highest concentrations (Figure 6).

Los Esteros

During the first 30 min of exposure, organisms did not avoid the tested sediment
concentrations. Yet, after 6 h of exposure the avoidance response reached values between 25
and 50%, while at the concentration of 100% no avoidance was observed, indicating a
displacement of the organisms towards that section. This avoidance behaviour was

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248 Cristiano V. M. Araújo, David Salvatierra, Victoria Vera-Vera et al.

maintained after 24 h of exposure. After this same exposure period, a recolonization response
of 100% was observed for the concentration of 25%, while for the concentrations of 50 and
100% the recolonization percentage was around 40%. The 24 h expected recolonization was
again overestimated for the three highest concentrations and underestimated for the
concentration of 25% (Figure 7).

Río Muerto

All sediment concentrations were avoided, resulting in a 60 to 80% avoidance in the first
three concentrations (25, 50 and 75%), but for the 100% sediment concentration it decreased
to 40%. Basically, organisms only recolonized the concentration of 25%. Therefore, the
recolonization percentage predicted for the 24 h recolonization was close to the observed
recolonization for the concentrations of 25 and 50% and was again underestimated for the
highest concentrations (Figure 8).

100 30 min 30 min

140
75 6h 6h
24 h % Recolonization 120 24 h
50
% Avoidance

24 h expected
25 100 recolonization
0 80
-25 60
-50 40
-75 20
-100 0
25 50 75 100 25 50 75 100
% Sediment dilution % Sediment dilution

Figure 7. Avoidance (left) and recolonization (right) percentages of the snail Olivella semistriata
exposed to different dilutions of the sediment from Los Esteros.

100 70
30 min
60 6h
80
% Recolonization

24 h
50
% Avoidance

60 24 h expected
recolonization
40
40
30
20
20
0 30 min
6h 10
-20 24 h
0
-40
25 50 75 100 25 50 75 100

% Sediment dilution % Sediment dilution

Figure 8. Avoidance (left) and recolonization (right) percentages of the snail Olivella semistriata
exposed to different dilutions of the sediment from Río Muerto.

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AC50 and RC50 Values

AC50 values for El Puerto and Los Esteros were estimated at >100% as there was no
avoidance. For the other sediment samples, the AC50 values were <25%. With regard to
recolonization, sediment from El Puerto and Los Esteros were more intensively colonized,
with RC50 values of 55.2 and 51.4%, respectively. For La Poza, Río Burro and Río Muerto
the RC50 values were lower: 27.6, 21.8 and 26.9%, respectively. Confidence intervals were
not calculated for any of the samples.

DISCUSSION
Given the diverse avoidance and recolonization responses observed for the different
tested sediments, results of both avoidance and recolonization assays for each sediment
sample are discussed separately.

El Puerto

As no avoidance was observed, the expected recolonization was much higher than that
observed in the recolonization assays. Although the recolonization percentage clearly
increased with exposure time, it did not reach the expected value (Figure 4). A 24 h exposure
time was possibly not sufficient for the colonization of the most extreme sections. This
explanation is not contradictory to the 100% recolonization observed in the reference
sediment (El Murciélago beach) after the same exposure time. When organisms were exposed
to reference sediment in the recolonization assays there was no additional or different
stimulus (i.e., differences in the biological, chemical and physical composition of the
sediment), as the sediment present in all sections of the system was similar (El Murciélago
sediment sample). However, in the recolonization assays with the El Puerto sediment
organisms received different stimulus at each sediment concentration (e.g., El Murciélago
beach and El Puerto sediments differed in TOC, OM and particle size distribution). Therefore,
their decision to stay, return or move on might have required more time.

La Poza

Curiously, in the section with 25% of La Poza sediment there was avoidance (ca. 60%)
after 6 and 24 h but there was also more than 80% recolonization after 24 h of exposure,
whereas for the other sediment concentrations neither avoidance nor recolonization was
recorded. The absence of avoidance at the highest sediment concentrations could be related to
temporal avoidance (retraction inside their shell, a behaviour known to occur in response to
contamination; Hellou et al., 2009; Araújo et al., 2012) and, therefore, spatial avoidance
(displacement towards reference sediment) is not observed. Furthermore, as those same
concentrations are not favourable to be inhabited by the snails they were not colonized.

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250 Cristiano V. M. Araújo, David Salvatierra, Victoria Vera-Vera et al.

Another effect that could have triggered this response is the incidence of moribundity
(decrease or even loss of the ability to avoid contamination), preventing the organisms’
displacement (Gutierrez et al., 2012; Araújo et al., 2014).
The observed results also suggest the possibility that other factors than contamination
might have influenced both avoidance and recolonization responses. For instance, the organic
matter in the sediment from La Poza is much higher than that in other sediments, including
the reference El Murciélago beach sediment (see Table 1). For the lowest La Poza
concentration (25%) this content of organic matter could have been an attractive factor in the
recolonization assays since organisms moved from reference sediment with low organic
matter content. This recolonization did not increase with the increase of the organic matter
probably because the levels of other chemical elements, such as metals (see Table 1), also
increased and might have triggered an avoidance response. Possibly due to an attractive
stimulation caused by the organic matter (mainly microphytobethos, a major food source for
snails; Lopez and Levinton, 1987; Coelho et al., 2011; Araújo et al., 2015) present in the
sediment section of 25%, recolonization increased with the exposure time (% of recolonizers
increased). However, it seems not to have been sufficient to maintain the population for long,
since 60% of the snails escaped that section (Figure 5).

Río Burro

This sediment was avoided by a high percentage of the exposed snail population, even
though avoidance decreased slightly (from ca. 60 to 50%) at the highest sediment
concentration. The observed recolonization was in accordance with the avoidance response
but only for the lowest concentration (25%): avoidance of 60% and recolonization of 40%.
This was because, in general, there was no recolonization for the other sediment
concentrations. The less intense avoidance observed in that sediment concentration can be
explained by a decline in snail activity, which was indeed visually observed for organisms
recorded in the 100% section already after 6 h of exposure. In sediments triggering a strong
avoidance, organisms might initially opt for a temporary avoidance (e.g., retraction inside
their shell; Hellou et al., 2009; Araújo et al., 2012), and only after a spatial avoidance might
then occur, if moribundity does not take place.

Los Esteros

The avoidance observed was not concentration-dependent. For the first three
concentrations it varied between 25 and 50%, but for the 100% sediment concentration it was
negative (organisms moved towards that section). This result is corroborated by the trend of
the snails to colonize after 24 h of exposure the highest sediment concentration whose
colonization percentage was of 33%. Therefore, it is possible to consider that organisms do
not avoid this sediment concentration; however, the attraction to colonize it is not immediate.

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 Avoidance and Recolonization Responses of the Gastropod Olivella semistriata … 251

Río Muerto

The avoidance response observed in this sediment was similar to that occurring in the
sediment from Río Burro; snail avoidance was between 60 and 80% at the three lowest
sediment concentrations but was reduced to 40% at the 100% concentration. Equally to the
Río Burro sediment, recolonization was restricted to the first sediment concentration (25%),
suggesting the lowest avoidance in the 100% section can be explained by temporary
avoidance and/or moribundity. If the reduction in activity of the snails is deliberate and
moribundity did not occur, then a longer exposure time could trigger a spatial avoidance.

Ecological Implications

Avoidance assays with snails have only been recently employed in ecotoxicology to
assess the ability of organisms to escape from contaminated sediments, moving to most
favourable areas (Marklevitz et al., 2008a,b; Araújo et al., 2012, 2016). However, this
response deserves special attention due to the effects that it can cause on the spatial
distribution of snail populations. Additionally, in the present study, recolonization was used
to verify the ability of the organisms to move from reference towards test sediments, a
scenario likely to occur in aquatic systems under recovery. The ecological relevance of this
approach consists in predicting the contamination-triggered spatial displacement of
organisms. In fact, according to our visual observations, snails are not frequently found in all
the sampling areas of the present study: for instance, populations of O. semistriata have been
found in a very high density in the El Murciélago beach (reference point), with lower
frequency in El Puerto and Los Esteros, and sporadically in La Poza, while no records were
made in Río Burro and Río Muerto. As there is no physical barrier preventing the
displacement of the organisms between those areas (except for organisms from El Murciélago
that are isolated from all other points by a fishing port), it can therefore be hypothesized that
organisms avoid inhabiting sediments in La Poza, Río Burro and Río Muerto. Results of the
laboratory assays attested that organisms avoided sediment from Río Burro and Río Muerto,
even at low concentrations. These results seem to reflect the spatial distribution observed in
the field and the role that contamination seems to play as a chemical barrier preventing the
displacement among areas. As the ability to avoid contaminants allows organisms to escape
before effects at the individual level occur, the consequences of the avoidance response fall
on the ecosystem (e.g., pond, stream, estuary, beach) and landscape (e.g., river basin, coastal
ocean) levels by disturbing their structure and functioning.
For the recolonization assays, results were neither in accordance with avoidance
experiments nor with visual field observations, except for the reference sediment (El
Murciélago beach), in which recolonization was of 100%, as expected. It is important to
consider that characteristics of the sediment other than contamination may play an important
role in the decision of the organisms to avoid or recolonize an area (Cardoso et al., 2013).
Cardoso et al. (2013) observed that density, biomass and growth productivity of P. ulvae

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252 Cristiano V. M. Araújo, David Salvatierra, Victoria Vera-Vera et al.

increased along a mercury gradient in a shallow coastal lagoon (Ria de Aveiro, Portugal), and
that factors other than toxicity, such as resource availability, presence of refuges, etc. also
seemed to determine the spatial distribution of the organisms.
The findings of the present study allow hypothesizing that the higher the avoidance
response the lower is the expected recolonization, although the required time seems to be
sediment dependent. This pattern can also be seen by analyzing the AC50 and RC50 values,
with the first parameter being always lower than the second. Due to the design of the assays,
for the highest concentration of a sediment sample (placed in the section furthest from the
reference sediment), organisms in both avoidance and recolonization assays are not expected
to take the same time to avoid the most contaminated sample and to possibly colonize it.
Therefore, a 24 h exposure might not have been sufficient for a complete recolonization, i.e.,
the difference in the intensity of both responses can be directly related to exposure time.
Finally, it is crucial to take into account that the decision to avoid, stay or colonize a habitat
might be based on multiples factors. The intensity of the avoidance is directly related to how
repulsive is the risk (Harper et al., 2009), and thus recolonization will be faster and more
intense the more attractive the environment is.

ACKNOWLEDGMENTS
D Salvatierra, LA Cedeño-Macías and VC Vera-Vera thank the DCI (Central Department
of Research, ULEAM) for the research auxiliary fellowship. The Portuguese Foundation for
Science and Technology supported the present study through a postdoctoral fellowship
attributed to CVM Araújo and M Moreira-Santos (references SFRH/BPD/74044/2010 and
SFRH/BPD/99800/2014, respectively), funded by Programa Operacional Potencial Humano
of QREN Portugal 2007-2013 and 2014-2020, respectively, and by the Portuguese budget
through the Ministry of Education and Science. CVM Araújo is also grateful to SENESCYT
(Secretaría Nacional de Educación Superior, Ciencia, Tecnología e Innovación, Ecuador)
(PROMETEO program) and to Spanish Ministry of Economy and Competitiveness for the
Juan de la Cierva contract (IJCI-2014-19318). This study (Fugaguatox project; CUP
#91740000.0000.377799) was partially funded by the Ministry of Finance of Ecuador.
Graphical assistance was provided by FR Diz.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 16

POLLUTION IN COASTAL AREAS: AN INTEGRATED

PERSPECTIVE IN ENVIRONMENTAL RISK STUDIES

Javier García-Alonso and Delfina Castiglioni

Departamento de Ecología Teórica y Aplicada,
Centro Universitario de la Región Este, Universidad de la República,
Maldonado, Uruguay

ABSTRACT
Transitional water systems such as estuaries and subestuaries are areas of great
concern in ecotoxicology due to urban and industrial settlement, being key areas of
productivity and biogeochemical exchange between ocean and land. The ecotoxicological
risk of trace metals and organic pollutants in the environment depends on whether they
can be accumulated by organisms and this, in turn, depends on the uptake from food and
water, as well as the efflux. Chemicals are often sequestered in estuarine sediments and
thus likely to be accumulated, affecting the benthic communities directly. The surface
oxic sediments include the biofilm, and its associated biota which are ideal models to
understand the ecological risk in aquatic environments. South American estuaries along
the Atlantic coast show different levels of human impact. We reviewed the status of
environmental pollution and ecotoxicology of these estuaries and highlighted the
potential sources of novel pollutants.

Keywords: Estuaries, aquatic pollution, trace metals, POPs

POLLUTION IN ESTUARINE AREAS

Transitional water systems such as estuaries and subestuaries are highly productive
environments where biogeochemical exchanges between ocean and land occur. These
ecosystems support nursery and recruitment areas for many species (including commercial


Corresponding Author E-mail: jgalonso@cure.edu.uy.

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256 Javier García-Alonso and Delfina Castiglioni

fish) due to the presence of key prey species, such as dense populations of invertebrates
(McLusky and Elliott, 2004; Dauvin, 2008).
Estuaries are also naturally stressed areas due to the natural dynamic of mixing fresh and
marine waters. For this reason, estuarine organisms should be adapted to cope with this
complex environment. Probably most of the organisms living in transitional water systems
contain a strong defense battery to fight the naturally occurring oxidative stress. The presence
of these defense mechanisms, which are similar responses upon exposure to pollutants, imply
that these complex environments could be sources of misinterpretation if a putative toxic
response is trying to be associated with aquatic pollution. However, estuaries are areas of
great concern in ecotoxicology due to the urban and industrial settlement.
The ecotoxicological risk of trace metals and organic pollutants to the environment
depends on whether they can be accumulated by organisms, and this in turn depends on their
uptake from food and water, as well as the efflux. As estuaries are characterized by fine
grained and organic-rich sediments, they act as a sink for contaminants and are therefore
priority areas for identifying potential bioindicator species and biomarkers of pollution
(Ducrotoy, 2010). We reviewed the status of environmental pollution and ecotoxicology of
several South American estuaries and highlighted potential sources of novel pollutants.

SOURCES OF CONTAMINATION IN ESTUARINE ENVIRONMENTS

AND NEW POLLUTANTS

The most problematic chemical pollutants are the persistent, bioaccumulative and toxic
(PBTs), which include inorganic (trace metals) and some organic compounds. Inorganic
pollutants as trace metals are natural components of the Earth’s crust. Metals are released
from the erosion of rocks and appear as dissolved forms or associated with particulate
material, although they could occur in the atmosphere in particles, aerosols or gases (see
Luoma and Rainbow, 2008). Some metals have an essential biochemical function in a
metabolic pathway due to their affinity to sulfur and nitrogen, and this affinity is the one that
turns all metals into potentially toxic in high concentrations (Rainbow et al., 2006; Luoma
and Rainbow, 2008). Anthropogenic metal sources include punctual ones such as industries
and diffuse ones such as agriculture and street runoff. The recent surge of nanotechnology has
meant a rise of emerging contaminant sources. Metal based nanoparticles are produced for a
wide range of commercial and industrial products, generating a novel pollutant, with poorly
understood long-term effects on the environment (Luoma, 2008). The most used metal based
nanoparticles are composed of silver and copper, which can be internalized by organisms,
generating toxic effects in estuarine benthic communities (Khan et al., 2012; García-Alonso
et al., 2014).
The entry of metals to estuaries occurs mainly via freshwater, and upon reaching brackish
water they may suffer changes in chemical speciation that affect their partitioning and their
bioavailability.
Metals can bond to organic compounds forming organometals such as tributyltin (TBT)
and methylmercury. TBT was used as one of the main components of anti-fouling paint to
prevent the accumulation of marine organisms on vessels. This compound was slowly
released to the environment, ending up with tens of thousands of tons of TBT discharged into

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aquatic ecosystems from boat bottoms before its ban in the 1970s (Luoma and Rainbow,
2008). TBT is used as a fungicide, bactericide, insecticide, and as a preservative in textiles,
paper, leather, electrical equipment and plastics (Clark et al., 1988). During the 1970s, the
accumulation of TBT in the coast of France nearly resulted in the collapse of commercial
oyster fisheries. TBT interferes with the larval settlement and proliferation of chambers in
oyster shells of Crassosstrea gigas (Alzieu, 2000) and is also highly accumulated by aquatic
organisms (Meador, 2000). Mollusks are known to accumulate high levels of TBT. For
instance, female gastropods develop male sexual characters called imposex upon exposure
to TBT (Gibbs and Bryan., 1986). Estuarine harbors around the world are still contaminated
by TBT and imposex is still recorded in gastropods from South American estuaries
(Penchaszadeh et al., 2001).
Methylmercury is a potent toxic compound that is bioaccumulated and biomagnified
through the food web, ending up in top predators and humans. Neurotoxicity is one of the
major negative effects that this compound generates. Mercury is released by industrial
emissions and generally deposited in soil or water near the source (Renner, 2005).
Methylmercury is naturally produced by bacteria in water; therefore release of non-
bioavailable Hg elements to the environment must be avoided or reduced.
Organic pollutants are in general man-made compounds that are not natural components
of the earth’s crust, most of them containing a primary carbon structure. Among organic
pollutants, some of the most relevant are those that can persist in the environment
(UNEP/GPA, 2006). These chemicals called persistent organic pollutants (POPs) are the most
problematic chemicals since several of them are also PBTs and thus bioaccumulate and
biomagnify in the biota. There are many kinds of POPs such as plastic derivatives, surfactants
and biocides, among others. Endocrine disrupting compounds (EDCs) are POPs grouped by
the effect they generate and not by its chemical structure. EDCs interfere with the normal
endocrine function. Some plastic derivatives such as PCBs and bisphenols, as well as
detergents such as alkylphenols, act as xenoestrogens. These are EDCs that act as estradiol, a
sex steroid hormone involved in the behavior and reproductive cycle control of females, but
also in the proliferation of estrogen-dependent cancer cells. Different levels of responses to
EDCs could be useful for their detection in estuaries. Early warning effects of EDCs could be
used as the induction of detoxification enzymes (e.g., glutathione-s-transferase) (Ayoola et
al., 2011) or the expression profile of female-specific proteins in males (e.g., vitellogenin or
coriogenin) (García-Alonso et al., 2006). Endpoints such as morphological feminization or
presence of ovotestis (gonadal histology), developmental toxicity (García-Alonso et al.,
2011a), fecundity, and long term exposure recording local extinctions (Kidd et al., 2007) are
good tools to indicate the presence of EDCs in the environment.

FATE OF CONTAMINANTS IN ESTUARINE ENVIRONMENTS

As the environmental behavior of pollutants depends on the complex interaction of many
factors, any significant environmental alteration is likely to affect their distribution and fate.
In the case of transitional waters “The legacy lies in the sediments” is the title of a note made
by Rainbow and Luoma (2010), as chemicals are often sequestered in estuarine sediments and
thus likely to be accumulated and affect the associated organisms (bacterial communities of

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the biofilm and benthic infauna). In Figure 1, a conceptual scheme represents the entry of
pollutants by diffuse inputs (e.g., runoff) and punctual release (e.g., urbanisation, industries,
ships). Pollutants are trapped in sediments of intertidal mudflats and at the transitional areas
of estuaries where the salt front acts as a barrier.
Geomorphology and hydrology of the system drives the transport, precipitation and
accumulation of sediments (de Souza-Machado et al., 2016). This is why sediment analysis is
particularly useful in the detection of contamination sources and the selection of critical sites
for routine analysis, as they could evade detection if measured in water (Páez-Osuna, 2005).
There is a surface layer on the sediment called biofilm, which is rich in organic matter and
composed of small sediment grains, detritus, bacteria, fungus and micro-organisms. The
accumulation of pollutants in this interphase is probably the major source of the toxicity
process in the associated biota. Contaminants in the anoxic and compacted sediments a few
centimeters below the biofilm are probably not near the biota and do not exert any ecotoxicity
reaction.

Figure 1. Conceptual model of pollutant fate and accumulation in estuarine systems. Black arrows
denote diffuse inputs from rivers and ground-water, while white arrows denote punctual inputs from
ships, urban sewage and industrial discharge. Pollutants (small circles) tend to accumulate in the tidal
flood-plain and bottom sediments of the estuarine zone where the salt-front occurs. 1, Diffuse inputs; 2,
Subterranean water inputs; 3, Urban and Industrial inputs; 4, Fluvial transport and harbor inputs; 5,
Intertidal mudflats. Dashed area denotes the transitional water zone including the salt front. Adapted
from de Sousa-Machado et al., (2016).

The physicochemical characteristics of the environment affect the partition of

contaminants. In the case of metals, for an approximately neutral pH, metal partition between
the solid phase and solution greatly favors particulate material, which means that sediments
and this particulate material are the greatest metal reservoirs in the water body. Metals such as
Ag, Cd, Zn and Cr are assimilated more efficiently from suspended particulate material or

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from sediments with high organic matter content and, in general, more efficiently from
organic ligands than inorganic ones (Harvey and Luoma, 1985; Decho and Luoma, 1994; Lee
et al., 1998). Greater metal concentrations can be found in particles with sizes below 63 µm,
while the fine sand fraction tends to accumulate less due to the dominance of quartz
components (Kersten, 2002). Another important variable is salinity, as the stress generated by
high temperatures combined with low salinity raises the toxicity of metals (Bryan and
Langston, 1992). Also, when pH and dissolved oxygen are high (around 8.0 and 8.0 mg/L,
respectively), or even at a neutral pH, the oxidation rates are faster and therefore the
precipitation of metals rises. At low pH (5.5-6) the most important ionic metal liberation
occurs; nevertheless, low pH occurrences are very rare in natural environments (Atkinson
et al., 2007).
Organic pollutant properties in the environment differ in particular from metals. The
octanol/water coefficient classifies molecules as polar (<1) and non-polar (>1). The lipophilic
properties of a chemical drive the fate of POPs. Partition in different matrices of aquatic
systems (e.g., water column, sediment, and biota) depends on the octanol/water coefficient of
the contaminant and the sediment-water distribution of hydrophobic organic chemicals plays
a key role in their food-chain transfer. Increasing lipid content and biomagnification of POPs
are believed to be responsible for the higher bioconcentration factors that can be observed
with increasing trophic levels (Jones, 1991). Substances that bioaccumulate and biomagnify
are of great concern, as they can potentially attain toxicologically significant loads in higher
trophic-level species such as predatory fish, birds, and mammals (including humans) (Kelly et
al., 2004).

ESTUARINE BIOTA AT RISK AND ECOTOXICOLOGICAL STUDIES

Estuaries are very productive ecosystems, important nursery and recruitment areas for
terrestrial and aquatic organisms, presenting a high density of estuarine invertebrates, which
act as key prey species for higher animals (McLusky and Elliott, 2004; Dauvin, 2008). As
estuaries are characterized by transitional waters and fine grained, organic-rich sediments,
they can trap contaminants and are therefore priority areas for studies of potential
biomoitoring and bioindicator species as well as the development of pollution biomarkers
(Ducrotoy, 2010).
The naturally stressed estuarine environments are places where species which have
successfully adapted have developed different strategies to cope with this complex
environment, thus the interest in euryhaline species as models to study pollution tolerance.
Additionally, biological responses to the presence of contaminants in estuaries may have
some influence on population tolerance, survival or adaptation (e.g., detoxification activity).
Many of such responses are already used in organic and metal risk analysis of estuarine
polychaetes (e.g., Glutathione-S-transferase) (Ayoola et al., 2011; García-Alonso et al.,
2011b).
Biofilm communities and deposit-feeding organisms are ideal models to understand the
ecological risk in coastal areas. Thus, understanding the shift of bacterial communities in
impacted estuarine sediments allows the determination of a basal biological level effect.
Metagenomics is a novel tool based on high-throughput massive sequencing analysis of

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environmental samples. Advances in sequencing strategies such as pyrosequencing have

made possible a more sensitive and accurate detection of bacterial diversity, which happens
to be one to two orders of magnitude greater than previous estimates. The use of high-
resolution approaches together with a thorough analysis of environmental variables, helps to
discriminate between assemblages corresponding to the natural estuarine variability and those
shaped by anthropogenic impact (Piccini and García-Alonso, 2015).
Human impacted estuaries contain different bacterial communities in comparison to
pristine zones of the same estuary (Sun et al., 2012). It is assumed that some members of
microbial communities are always present but vary in population size depending on
environmental conditions, such as those found in estuaries (Caporaso et al., 2012). Recent
studies using metagenomics in sediments of intertidal areas of Rio de la Plata showed a clear
relationship between the prokaryotic communities and the level of impact (Piccini and
García-Alonso, 2015), becoming a useful tool for ecological risks assessment.
Several ecotoxicological studies have been performed using vertebrate model organisms
such as fish (as reviewed by Scholz et al., 2013). However, there is an increasing public
pressure to avoid the use of vertebrates. The 3Rs idea of reduction, refinement and
replacement of vertebrates should be considered for future strategies on ecological risk and
impact studies. Furthermore, many vertebrates such as fish are in constant movement and do
not reflect the exact spatial situation of the environment. Micro and macrobenthic estuarine
communities are useful organisms in ecotoxicology and more studies should be developed to
avoid the sacrifice of vertebrates.
The South American polychaete, Laeonereis acuta, that inhabits intertidal mudflats, is an
interesting model organism in ecotoxicology since it lives in close contact with the oxic
biofilm layer of the sediment. Detoxification by enzymes such as glutathione-S-transferase
(GST), super oxide dismutase (SOD), among others, have been analyzed in polychaetes
exposed to different sorts of pollutants such as metals and POPs (Geracitano et al., 2004;
Díaz-Jaramillo et al., 2016). Bioavailability of trace metals from sediments to L. acuta using a
weak (HCl) digestion has proven to be a good approach to obtain the bioavailable fraction of
metals in sediments and thus their potential toxicity in the environment (Castiglioni, 2015).

INTEGRATED APPROACHES
In order to maintain the sustainability of the surrounding environments, several
integrated approaches should be performed including managers, researchers, fishermen,
agriculturist, engineers, architects and policies makers. Latin American countries present
weak environmental legislation and poor conservation management, plus the pressure to
improve the economic situation of the regions. Due to their advantages, estuaries are
becoming attractive places for “dirty” industries to settle. Baseline studies on non-impacted
estuaries are the best start to generate the discrimination capacity when a human impact
occurs. Thus basic research should be made to support any a posteriori decisions on
environmental management, fishery politics, biodiversity and aquatic conservation policies,
and urban and socio-economical parameters.

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Figure 2. Number of “estuary” citations in Pubmed (NCBI) database as of February 2016. Comparison
among continents (A), South American countries (B) and South-American estuaries (C). 1, Rio de la
Plata; 2, Patos Lagoon; 3, Bahia Blanca; 4, Amazon; 5, Parnaiba; 6, Lenga; 7, Valdivia; 8, Orinoco; 9,
Dagua; 10, Biobio; 11, Rio Negro; 12, Paranaiba do Sul; 13, Rio Gallegos; 14, Colorado; 15,
Aconcagua; 16, Maipo; 17, Rapel.

A multidimensional approach to an environmental problem is the best way to cope with

ecological risk, being very robust disciplinary pillars the best strategy. Using a single tool to
estimate ecological risks requires several assumptions (e.g., toxicological predictions of
metals based on analytical approaches assume equal effect of the environment on its potential
toxicity), while integrative measures require fewer assumptions (e.g., adjusting the whole-
toxic effect in relationship to environmental conditions such as pH, conductivity, synergism
with organic pollutants). Interdisciplinary approaches generate synergy and as a result of this

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262 Javier García-Alonso and Delfina Castiglioni

interaction a new level of results emerges. For instance, in ecotoxicological research,

biologists, chemists and statisticians should interact in order to better interpret what is
occurring. Multivariate approaches on estuarine pollution have helped to model estuarine risk
environments (García-Alonso et al., 2011b), but further horizontal thinking is needed to
integrate the research with environmental management and policy makers.
Integrative approaches in environmental monitoring and management require
multidisciplinary research studies and therefore multivariate statistic tools. These are suitable
strategies for a sustainable management in critical areas where conservation status might be at
risk, setting fundamental bases for good models that can help to avoid environmental
weakening. New strategies to approach environmental monitoring studies are in demand
mainly including the application of multivariate statistical tools to verify the association
between pollutants, toxicity and physical variables.
Specifically for South American estuaries, no studies have been published that integrate
interdisciplinary approaches on ecological risks. Furthermore, there is a huge difference in
water quality criteria around the world, reflecting the lack of an integrative approach to
determine, for example, at which level a potential chemical would become toxic. An
international effort should be made to standardize protocols that would fit natural complex
systems such as estuaries.

STATUS OF SOUTH AMERICAN ESTUARIES

The study of contaminants in estuarine systems is of great importance as these areas are
highly productive and receive large amounts of pollutants from terrestrial drainage. In a
global context, South American estuaries appear to have relatively few ecotoxicological
studies in comparison to other regions of the world (Figure 2). Within South American
countries more studies have been performed in Brazil, followed by Argentina. The most cited
estuary is the Río de la Plata, followed by Patos Lagoon and Bahía Blanca. Several important
estuaries in South America suffer the impact of urbanization and industrialization (Muniz et
al., 2015), so we described briefly the status of some of them (Figure 3). However, many
small estuaries of the Neotropical continent have been studied, such as Santos, in Brazil (Eça
et al., 2013) and Lenga, in Chile (Diaz-Jaramillo et al., 2013), among several other small
systems.

Orinoco

The Orinoco basin covers an area of 880000 km2. The estuary comprises a huge
floodplain area including the “Delta del Orinoco Biosphere Reserve” (UNESCO, 2011). The
estuary is well preserved as it is not greatly urbanized (i.e., Guyana City) and has no diffuse
contamination inputs. Moreover, the Orinoco estuary is probably the only South American
estuary inhabited by native people, the nomadic Warao.
The Warao population reached a total of 36028 individuals by 2001, of which 83% lived
in the estuary, and 85% in traditional communities (INE, 2002). Few studies exist on human
impacts on this system and no literature was found on ecotoxicology. One of the most

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relevant impacts is the construction of a dam in the 1960s which generated a reduction of the
mangrove area (Colonnello and Medina, 1998).
Trace metal levels do not show evidence of a negative impact on the biota, even at the
discharge of red mud containing high amounts of some metals (Mora et al., 2015). Due to the
low level in relation to the sea (reaching areas with one meter below sea level), predictive
climate change models suggest that the Delta may be submerged by 2100 (Vegas-Vilarrúbia
et al., 2015).

Amazon

The Amazonia estuary receives freshwater from the biggest water basin on Earth, the
Amazon River. It covers 7050000 Km2 which represent 40% of the total surface of South
America. The Amazon Estuary, located in northern Brazil, includes also the discharge of the
Tocantins River, resulting in an annual mixture of approximately 6300 km3 of river water
carrying 93108 tons of sediments with the waters of the Atlantic Ocean (Meade et al., 1979).
New integrated approaches are being developed in order to improve the water quality of the
estuary (Monteiro et al., 2016). Trophic transfer and bioaccumulation studies of trace metals
have been performed in pearl oyster of the Amazon estuary (Vilhena et al., 2016).

Parnaíba

The Parnaíba River watershed (34400 km2) is characterized by a low industrial

development, while its estuary presents low inputs of contaminants and prevalence of diffuse
pollution sources (de Paula Filho et al., 2015). This relatively pristine transitional water
system on the Atlantic coast of South America presents an extensive mangrove, with very
high primary production in the estuary and its coastal plume. Being a unique, complex and
tropical estuary, this environment integrates an important global conservation area (MMA,
2006): since 1996 the delta was declared an Environmental Protection Area, spreading over
three states in Northeastern Brazil (Ceará, Maranhão and Piauí). Background levels of
potential toxic metals have been recently described (da Paula Filho et al., 2015), indicating
relatively low levels of these metals in the estuarine sediments.

Patos Lagoon

On the south coast of Brazil, Patos Lagoon and Rio Grande estuary system is another
huge transitional water area with the presence of urbanized regions and extensive agriculture.
It also receives the discharge of the Merin lagoon basin from an area covering the east part of
Uruguay. The Patos Lagoon estuary receives local inputs from urban (e.g., Rio Grande city),
industrial and harbor activities which have enriched the sediments with metals such as Cu, Pb,
Zn (Geracitano et al., 2004) and polycyclic aromatic hydrocarbons (PAHs) (Filho et al.,
2012). There are many ecotoxicological studies on estuarine biota, such as morphological and
physiological changes in the polychaete L. acuta (Geracitano et al., 2004; Diaz-Jaramillo et

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264 Javier García-Alonso and Delfina Castiglioni

al., 2016) and in fish such as the white croaker Micropogonias furnieri (Amado et al., 2006a)
and flounder (Amado et al., 2006b).

Río de la Plata

On the Atlantic coast, one of the biggest estuaries is the Rio de la Plata, located at 35°S.
This system is a highly productive area, sustaining valuable fisheries of Uruguay, Argentina,
and international fleets (Martinez and Retta, 2001; Acha et al., 2008). Specifically on the
Montevideo coastal area exists one of the most polluted harbors of the region (Danulat et al.,
2002; Muniz et al., 2004). Gómez et al. (2009) found that microbenthic communities of the
Río de la Plata were governed by two gradients, the first one determined by anthropic factors
and the second one by conductivity and turbidity. In the case of bacterioplankton
communities, Alonso et al. (2010) showed that bacterial abundance and diversity patterns
based on ARISA data were highest at the frontal zone, where turbid waters from Paraná
and Uruguay rivers mix with the Atlantic Ocean (Nagy et al., 2008). Regarding the
organic pollutants, there is evidence of natural and anthropogenic (petrogenic and pyrogenic)
contribution to the Uruguayan coastal portion of the Río de la Plata Estuary (Venturini et al.,
2015). Montevideo Bay, one of the most urbanized areas of the estuary, presents chronic oil
pollution with the occurrence of hydrocarbons derived from both crude petroleum and
petroleum combustion. Also, metal concentrations and biochemical markers assessed in the
area indicate that adverse effects to the biota are likely being generated (Muniz et al., 2004;
Venturini et al., 2015).

Figure 3. Map of South America showing the main Neotropical water basins associated with the most
known estuaries in term of ecotoxicological status. Dashed squares represent the approximate area of
estuarine influence in the marine environment.

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Bahía Blanca

The Bahía Blanca estuary is located in the south-eastern section of Buenos Aires
province, Argentina. It is a coastal environment with a city that exceeds 350,000 inhabitants,
whose pre-filtered effluents are directly introduced into the estuarine waters. In particular, it
presents intense anthropogenic activity at the north shoreline, including oil, chemical and
plastic factories, two commercial harbors and a fishing fleet, thus requiring regular dredging
(Ferrer et al., 2006; Arias et al., 2010). The inner part of the estuary presents low urbanized
rural lands, a tourist area and an artisanal fishing recreational port (Arias et al., 2010). A 33%
of saltmarshes ecosystem dominated by Spartina perennis have been replaced by human land
uses (Pratolongo et al., 2013) and an integrative strategy to manage the estuary threatened by
climate change and pollution has been recently proposed by researchers (Kopprio et al.,
2015).
Metals such as copper, lead and zinc that are present in surface sediments in this estuary
have concentrations comparable to those reported as natural background for similar regions.
However, cadmium concentrations were higher than those previously reported from the same
environment, and similar to concentrations reported from other industrialized areas. The same
authors addressed the potential toxicity of these elements to the native crab Neohelice
granulata (Simoneti et al., 2011). Young crabs of this species were considered potentially
dangerous agents for transfer of metals along the associated trophic network, due to their
relative elevated resistance and capacity to bioaccumulate trace metals in their tissues (Ferrer
et al., 2006). A recent assessment of POPs in surface sediments indicated that some individual
POPs exceeded the low effect range as well as the median effect range in this area (Oliva et
al., 2015). POPs monitoring has also revealed a wide recent use of some currently forbidden
insecticides such as Heptachlor and Mirex, banned in 1993 and 1999 respectively (Arias et
al., 2010).

GENERAL DISCUSSION
Although several of the South American estuarine systems are suffering some type of
human impact, few of those with an important drainage area remain pristine or have no
recorded data. In general, scarce information exists on ecotoxicological approaches to
understand the ecological risk of pollution in transitional waters of this continent. Although
European and North American macrotidal estuarine model systems as references help as a
source of basic information, Neotropical transition waters are naturally different with a wide
range of hydrological regimes that covers tropical, sub-tropical and temperate climates. For
future directions aimed at conservation status and impact assessments of South American
estuaries, baseline integrated monitoring studies to allow early prediction of human impacts
are becoming urgent.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 17

ECOTOXICOLOGY IN THE MARINE ENVIRONMENT:

BIOACCUMULATION AND BIOCONCENTRATION
FACTOR OF POLYCYCLIC AROMATIC
HYDROCARBONS

Mónica M. Zambrano1,*, Robinson F. Casanova2

and Gustavo Arencibia3
1
University of Antioquia, Academic Environmental Corporation,
Marine Sciences Headquarters, Turbo, Colombia
2
Center for Pacific Oceanographic and Hydrographic Research-Cccp, Harbour Master’s
Office, Tumaco, Colombia
3
Center of Fisheries Research-CIP, Santa Fe, Havana, Cuba

ABSTRACT
The bioaccumulation of polycyclic aromatic hydrocarbons (PAHs) was determined
for Anadara similis and A. tuberculosa (Mollusca: Bivalvia), the latter used as a marine
environmental quality bioindicator organism on the Colombian Pacific coast. For each
species a total of 120 organisms were exposed to a mixture of 16 PAHs (1.50x10-2
µg/mL) considered priority pollutants by the US Environmental Protection Agency. The
concentrations tested were those considered safe for aquatic life by the Canadian Council
of Ministers of the Environment. The exposure lasted a total of 45 days for A.
tuberculosa and 30 for A. similis (due to higher mortality rates). The accumulation of the
tested PAHs was lower for the compounds with high molecular weight (HMW) compared
to the compounds with low molecular weight. However, the bioconcentration factors for
HMW compounds were higher due to their increased stability and persistence. After
exposure, no significant differences in the levels of bioaccumulated PAHs between
species were demonstrated (Mann-Whitney U Test, p=0.24).

*
Corresponding Author E-mail: monica.zambrano@udea.edu.co.

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272 Mónica M. Zambrano, Robinson F. Casanova and Gustavo Arencibia

Keywords: bioaccumulation, bioconcentration factor, polycyclic aromatic hydrocarbons,

bivalves

INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) make up the most toxic part of petroleum and
therefore have the greatest ecotoxicological interest. Their presence in aquatic environments
makes them bioavailable to organisms, being therefore incorporated through bioaccumulation
processes and consequently biomagnified throughout the food web.
Sixteen PAHs are considered of interest as environmental contaminants by the US
Environmental Protection Agency-US EPA (Achten and Hofmann, 2009; Rey-Salgueiro et
al., 2009), the US Agency for Toxic Substances and Disease Registry (ATSDR, 2015) and the
International Agency for Research on Cancer (IARC, 1983), mainly because of their
mutagenic and carcinogenic effects (Meador et al., 1995; Potin et al., 2004). These
compounds can disperse in the water column and build up in sediments and biota (Bihari et
al., 2007). Bioaccumulation levels are a good estimate of xenobiotics in the wild, where the
sessile or sedentary organisms, with little capacity for metabolic degradation, are the most
exposed to the entry of contaminants (Fernández and Freire, 2005).
Filtering organisms such as bivalve mollusks incorporate and accumulate PAHs
compounds in their tissues at concentrations considerably higher than those in the
surrounding water (Neff, 2002). It is for this reason that bivalves have been used as
biomonitors of pollutants in aquatic environments (Chęć et al., 2008).
Biomonitoring has great importance in monitoring programs to evaluate the quality of
aquatic environments. Although in situ biomonitoring provides information regarding the
quality status of a water body, it is important to assess the accumulation of contaminants and
the effects on organisms caused by such accumulation.
Bioaccumulation of chemicals in organisms represents an important criterion for
ecotoxicological risk evaluations (Ivanciuc et al., 2006), allowing to identify the quality
of the aquatic environment. This is how the Colombian Pacific bivalve mollusk Anadara
tuberculosa has been used as a biomonitor for assessing water quality in presence of oil
derivatives. This species and A. similis were exposed to a mixture of PAHs to determine
bioaccumulation, bioconcentration factors (BCF) and differences that can make one species a
better biomonitor than another.

METHODS
Test Species

A. tuberculosa is the most exploited mollusk in Colombia; its distribution covers the area
between Lake Whales (South California) to Tumbes (Peru) (Keen, 1971). It has been used to
predict exposure, effects and susceptibility in the presence of anthropogenic substances in the
monitoring of marine pollution (such as the Mussel Watch program; Sericano et al., 1995) and

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 Ecotoxicology in the Marine Environment 273

by some countries in the American Pacific (De la Cruz, 1994; Beliaeff et al., 1997; Jara-
Marini et al., 2012).
A. similis shares the same habitat with A. tuberculosa in the mangrove ecosystem and has
a geographical distribution from Corinto (Nicaragua) to Guayaquil (Ecuador) (Keen, 1971).
Both species bury in the sediment, and features such as being filter-feeders and sessile make
them ideal in biomonitoring programs. Their economic importance constitutes 96% of the
total production of bivalves in Colombia (Instituto Colombiano de Desarrollo Rural-
INCODER, 2007).

The Test Solution

The PAHs mixture was added to the test aquaria to obtain a final concentration of
1.50x10-2 µg/mL. Concentrations of compounds in the mixture were established using the
maximum levels recommended by the Canadian Environmental Quality Guidelines of the
Canadian Council of Ministers of the Environment-CCME (2010). For some compounds that
do not have a reference level established by the CCME, a lower concentration than that
stipulated for anthracene (the most restrictive among this group within the guide) was
employed.
Acenaphthylene concentration was determined taking into account its similarity in
structure and molecular weight (MW) with acenaphthene; for chrysene was considered
the similarity in MW with pyrene. For indeno[1,2,3-cd]pyrene was established a lower
concentration with respect to the remaining compounds due to their complex structure and
high MW (Table 1).

Table 1. Composition of the PAHs mixture used in the test (µg/mL) based on the
maximum levels recommended by the Canadian Council of Ministers of the
Environment-CCME (2010) (modified from Zambrano et al., 2012a)

CCME Test
Compound
guideline concentration
Anthracene 1.2 x10-5 1.2 x10-5
Pyrene 2.5 x10-5 2.5 x10-5
Benzo[k]fluoranthene - 1.0 x10-5
Benzo[g,h,i]perylene - 1.0 x10-5
Benzo[a]pyrene 1.5 x10-5 1.5 x10-5
Benzo[b]fluoranthene - 1.0 x10-5
Benzo[a]anthracene 1.8 x10-5 1.8 x10-5
Indeno[1,2,3-cd]pyrene - 5.0 x10-6
Dibenzo[a,h]anthracene - 1.0 x10-5
Fluoranthene 4.0 x10-5 4.0 x10-5
Chrysene - 2.5 x10-5
Naphthalene 1.4 x10-3 1.1 x10-3
Acenaphthylene - 5.0 x10-3
Acenaphthene 5.8 x10-3 5.8 x10-3
Fluorene 3.0 x10-3 3.0 x10-3
Phenanthrene 4.0 x10-4 4.0 x10-4
Test concentration - 1.5 x10-2

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274 Mónica M. Zambrano, Robinson F. Casanova and Gustavo Arencibia

Bioassays

The development of bioassays was based on standard methodologies of the American

Public Health Association (APHA, 2005) and the US EPA (2000a). Live organisms were
acquired on the open market in Tumaco (Colombia) and transported to the laboratory in the
Center for Pacific Oceanographic and Hydrographic Research-Cccp where the bioassays were
performed. They were acclimatized in 250 L tanks (permanent aeration) for 10 days during
which physicochemical variables (temperature, salininity, pH and dissolved oxygen–DO)
were recorded using a WTW Multi 340i multiparameter equipment. Organisms were fed 1
L/day of 3x106 cel/mL diatomaceous cultivation (semi-intensive, not specific for a particular
species). After acclimatization, 10 organisms of each species were sacrificed in order to
determine baseline PAHslevels. The remaining organisms were moved to 20 L volumetric
glass aquariums (10 organims/aquarium). 120 organims of each species (12 aquariums/
specie) were exposed to a mixture of 16 PAHs (1.5x10-2 µg/mL) and every 5 days
accumulation of PAHs was evaluated. The PAHs mixture was totally renewed every 24 h and
food (3x106 cel/mL) was provided daily. Exposure period was of 45 days for A. tuberculosa
and, due to high mortality, only 30 days for A. similis.
Two types of controls were used, one containing only seawater and the other with the
solvent (acetone) used to prepare the mixture in a volume equal to that used to deliver the
compounds in solution (15 mL). Controls were tested in triplicate.
Aeration was not provided during due to the volatility of some compounds. The water
used to prepare the replacement solutions was aerated during several hours in order to saturate
it with DO.

Laboratory Analysis

For determination of PAHs levels, tissues (±10g; all the body) of 10 organisms
(composite samples) were homogenized and analysed by using gas chromatography and mass
spectrometry (GC/MS; gas chromatograph - Agilent Technologies 6890N Network - coupled
to a mass selective detector - MSD-Agilent Technologies 5973 Series Network) following the
methodologies described by the US EPA (1996a) and Russell et al. (2002); under the method
of selective ion monitoring (SIM) and using standard analytical high purity (Supelco
trademark) for identification and quantification. Likewise, the fats and oils content was
determined using Soxhlet extraction with ethyl ether as solvent (US EPA, 1996b). Data are
provided in dry weight.

Bioconcentration Factor (BCF)

Since BCF indicates the ratio between the concentration of a chemical in an organism and
in the water (US EPA, 2000b), calculations were performed by dividing the concentration of
PAHs in tissues by the concentration of the compound in water.

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 Ecotoxicology in the Marine Environment 275

Statistical Analysis

Statistical differences in PAHs bioaccumulation between species for each sampling

period were checked by the Mann-Whitney U Test. Data analysis were performed in SPSS
program (version 24) with a significance level of 95%.

RESULTS
Physical Chemical Variables

The physicochemical variables in the vessels during testing showed values between 23.30
and 25.70ºC for temperature, 7.91 and 8.24 for pH; 29.30 and 30.00 psu for salinity and 3.25
and 7.80 mg O2/L for DO.

Mortality

Survival of 100% occurred during the acclimation period. Throughout the test, both
species exposed to PAHs recorded a mortality reaching 43.50% in A. tuberculosa (between
2 and 29 days) and 48.50% in A. similis (between 2 and 21 days, with the highest percentage
between days 2 and 12). In the control vessels, A. tuberculosa mortality reached 33%
(between 13 and 33 days) and 53% in A. similis (between 7 and 18 days). In the treatment
containing only acetone, a mortality of 56.60% for A. tuberculosa (on days 16 and 22) and
66.66% for A. similis (between days 5 and 13) was recorded.

Fats and Oils

As for the content of oils and fats, the highest percentage in A. tuberculosa was
determinated at the start of the test and at 15 days (1.86%); the lowest percentage was found
on day 30 (0.04%) (Table 2). For A. similis the highest percentage was determined 15 days
after the start of the test (2.70%) and the lowest percentage was found at 5 days (0.08%)
(Table 3).

Bioaccumulation and BCF

The incicial PAH concentration observed before the experiment was of 1.40 µg/g in
A. tuberculosa (Table 2) and 2.00x10-2 µg/g in A. similis (Table 3). A. tuberculosa after
45 days showed a tendency to bioaccumulate in greater proportion HMW compounds (Figure
1). Acenaphthene recorded lower concentrations followed by fluorene, phenanthrene and
acenaphthylene; while the highest concentrations were recorded by the acenaphthene,
fluorene, acenaphthylene and phenanthrene.

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276 Mónica M. Zambrano, Robinson F. Casanova and Gustavo Arencibia

Figure 1. PAH concentration distribution incorporated in Anadara tuberculosa during the test (45
days).

Figure 2. PAH concentration distribution incorporated in Anadara similis during the test (30 days).

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 Ecotoxicology in the Marine Environment 277

A. similis exposure covered a period of 30 days, presenting high levels of compounds

with low molecular weight (LMW) such as acenaphthene, fluorene and acenaphthene, and
low concentrations of benzo[a]pyrene, benzo [g,h,i]perylene and dibenzo[a,h]anthracene,
compounds with HMW (Figure 2).
The BCF in A. tuberculosa indicated that indeno[1,2,3-cd]pyrene reached the
highest value (69200) after 35 days, followed by anthracene, benzo[b]fluoranthene,
benzo[k]fluoranthene, dibenzo[a,h]anthracene and benzo[g,h,i]perylene, whose maximum
values were recorded between 30 and 35 days of exposure. Lower values for the recorded
BCF of acenaphthylene ranged between 40 and 122 (Table 4).
The BCF determined in A. similis reached higher values for anthracene, followed by
byindeno[1,2,3-cd]pyrene, benzo[b]fluoranthene, chrysene, benzo[k]fluoranthene, pyrene and
benzo[a]anthracene; acenaphthylene showed the lowest BCF (Table 5). With respect to the
mixture, the last recordings were obtained at 25 (500)and 45 days (365) in A. similis and A.
tuberculosa, respectively, while the highest values recorded for A. similis (>300) were
observed after 15 days (Table 5), showing a higher affinity for these compounds in relation to
A. tuberculosa.
There were no statistical differences between the total PAHs levels accumulated in both
species (Mann-Whitney U Test;p=0.24). The dispersion in terms of incorporation was higher
in A. similis due to the differences of the concentration recorded for each compound in
particular (Figure 3).

Figure 3. Distribution of PAHs in organisms during the bioaccumulation test.

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 Table 2. PAHs concentrations and percentages of fats and oils in Anadara tuberculosa during the test
(µg/g dry weight) (modified from Zambrano et al., 2012a)

Exposure days
Compounds
0 5 10 15 20 25 30 35 40 45
Anthracene 2.00x10-2 1.02x10-1 2.18x10-1 1.14x10-1 1.93x10-1 1.12x10-1 2.94x10-1 1.55x10-1 1.94x10-1 1.95x10-1
Pyrene 2.50x10-2 6.70x10-2 1.85x10-1 9.00x10-2 1.31x10-1 9.80x10-2 1.65x10-1 8.90x10-2 1.51x10-1 1.00x10-1
Benzo[k]fluoranthene 1.60x10-3 5.70x10-2 1.10x10-1 1.14x10-1 1.63x10-1 1.22x10-1 1.72x10-1 2.26x10-1 9.90x10-2 1.33x10-1
Benzo[g,h,i]perylene 1.70x10-3 4.00x10-2 1.40x10-1 1.20x10-1 1.18x10-1 1.10x10-1 2.16x10-1 1.55x10-1 1.07x10-1 1.32x10-1
Benzo[a]pyrene 4.70x10-3 3.20x10-2 8.50x10-2 6.90x10-2 1.29x10-1 7.50x10-2 1.53x10-1 1.42x10-1 7.50x10-2 9.50x10-2
Benzo[b]fluoranthene 4.30x10-3 4.70x10-2 1.10x10-1 1.09x10-1 1.47x10-1 1.18x10-1 1.72x10-1 2.38x10-1 1.10x10-1 1.39x10-1
Benzo[a]anthracene 1.70x10-3 5.40x10-2 1.00x10-1 8.70x10-2 1.58x10-1 9.80x10-2 1.48x10-1 1.75x10-1 9.80x10-2 1.23x10-1
Indeno[1,2,3-cd]pyrene 1.90x10-2 6.50x10-2 1.00x10-1 2.05x10-1 2.50x10-1 1.73x10-1 3.08x10-1 3.46x10-1 1.45x10-1 2.24x10-1
Dibenzo[a,h]anthracene 1.10x10-2 4.10x10-2 1.37x10-1 1.07x10-1 1.12x10-1 9.60x10-2 2.20x10-1 1.68x10-1 1.13x10-1 1.39x10-1
Fluoranthene <2.00x10-4* <2.00x10-4* <2.00x10-4* <2.00x10-4* <2.00x10-4* <2.00x10-4* <2.00x10-4* <2.00x10-4* <2.00x10-4* <2.00x10-4*
Chrysene 5.00x10-4 1.02x10-1 1.41x10-1 1.61x10-1 2.73x10-1 1.73x10-1 2.48x10-1 3.32x10-1 1.50x10-1 2.13x10-1
Naphthalene <6.00x10-4* <6.00x10-4* <6.00x10-4* <6.00x10-4* <6.00x10-4* <6.00x10-4* <6.00x10-4* <6.00x10-4* <6.00x10-4* <6.00x10-4*
Acenaphthylene 1.50x10-2 3.13x10-1 2.01x10-1 5.64x10-1 5.92x10-1 3.64x10-1 3.71x10-1 5.41x10-1 5.30x10-1 6.11x10-1
Acenaphthene 1.30x10-2 7.57x10-1 3.47x10-1 1.23 1.21 8.38x10-1 7.92x10-1 1.15 1.07 1.23
Fluorene 1.40x10-2 4.40x10-1 4.82x10-1 5.75x10-1 6.29x10-1 5.06x10-1 7.04x10-1 6.37x10-1 6.11x10-1 7.31x10-1
Phenanthrene 1.20x10-2 3.24x10-1 5.09x10-1 3.60x10-1 4.07x10-1 3.55x10-1 5.95x10-1 4.44x10-1 4.25x10-1 5.23x10-1
ΣPAHs 1.40x10-1 2.44 2.86 3.91 4.51 3.23 4.56 4.80 3.87 5.48
Fats and oils (%) 4.64 0.73 0.13 1.86 1.00 1.34 0.04 1.30 1.26 1.46
* Detection limit.

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 Table 3. PAHs concentrations and percentages of fats and oils in Anadara similis during the test (µg/g dry weight)
(modified from Zambrano et al., 2012a)

Compounds Exposure days

0 5 10 15 20 25 30
Anthracene 7.00x10-4 1.14x10-1 1.44x10-1º 1.45x10-1 2.80x10-1 3.75x10-1 1.94x10-1
Pyrene 8.70x10-3 4.90x10-2 1.79x10-1 2.04x10-1 1.66x10-1 3.41x10-1 2.21x10-1
Benzo[k]fluoranthene <2.00x10-4* 1.31x10-1 9.30x10-2 1.22x10-1 4.10x10-2 1.22x10-1 1.66x10-1
Benzo[g,h,i]perylene <2.00x10-4* 8.40x10-2 5.50x10-2 6.40x10-2 1.80x10-2 8.20x10-2 1.00x10-1
Benzo[a]pyrene <2.00x10-4* 6.80x10-2 4.60x10-2 5.60x10-2 2.70x10-2 9.40x10-2 8.90x10-2
Benzo[b]fluoranthene <2.00x10-4* 9.50x10-2 9.60x10-2 1.21x10-1 5.60x10-2 1.40x10-1 1.93x10-1
Benzo[a]anthracene <4.00x10-4* 8.40x10-2 1.04x10-1 1.20x10-1 8.10x10-2 2.14x10-1 2.06x10-1
Indeno[1,2,3-cd]pyrene <6.00x10-4* 1.07x10-1 8.30x10-2 1.15x10-1 3.60x10-2 1.36x10-1 1.47x10-1
Dibenzo[a,h]anthracene <6.00x10-4* 8.30x10-2 5.90x10-2 7.70x10-2 2.40x10-2 9.60x10-2 9.60x10-2
Fluoranthene <2.00x10-4* <2.00x10-4* <2.00x10-4* <2.00x10-4* <2.00x10-4* <2.00x10-4* <2.00x10-4*
Chrysene 5.00x10-3 1.60x10-1 2.22x10-1 3.15x10-1 1.76x10-1 4.08x10-1 4.58x10-1
Naphthalene <6.00x10-4* <6.00x10-4* <6.00x10-4* <6.00x10-4* <6.00x10-4* <6.00x10-4* <6.00x10-4*
Acenaphthylene 3.40x10-3 1.35x10-1 4.00x10-2 8.14x10-1 1.04 1.09 6.45x10-1
Acenaphthene <3.00x10-4* 5.04x10-1 1.53x10-1 1.93 2.25 2.32 1.55
Fluorene 4.40x10-3 5.36x10-1 4.25x10-1 8.79x10-1 1.14 1.15 8.43x10-1
Phenanthrene <4.00x10-4* 6.00x10-1 6.78x10-1 7.02x10-1 8.65x10-1 9.30x10-1 6.69x10-1
ΣPAHs 2.00x10-2 2.75 2.38 5.66 6.27 7.50 5.58
Fats-oils (%) 0.44 0.08 1.80 2.70 1.36 0.68 1.15
* Detection limit.

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 Table 4. Bioconcentration factors for PAHs incorporated by Anadara tuberculosa during the test

Exposure days
Compounds
5 10 15 20 25 30 35 40 45
Anthracene 8500 18167 9500 16083 9333 24500 12917 16167 16250
Pyrene 2680 7400 3600 5240 3920 6600 3560 6040 4000
Benzo[k]fluoranthene 5700 11000 11400 16300 12200 17200 22600 9900 13300
Benzo[g,h,i]perylene 4000 14000 12000 11800 11000 21600 15500 10700 13200
Benzo[a]pyrene 2133 5667 4600 8600 5000 10200 9467 5000 6333
Benzo[b]fluoranthene 4700 11000 10900 14700 11800 17200 23800 11000 13900
Benzo[a]anthracene 3000 5556 4833 8778 5444 8222 9722 5444 6833
Indeno[1,2,3-cd]pyrene 13000 20000 41000 50000 34600 61600 69200 29000 44800
Dibenzo[a,h]anthracene 4100 13700 10700 11200 9600 22000 16800 11300 13900
Chrysene 4080 5640 6440 10920 6920 9920 13280 6000 8520
Acenaphthylene 63 40 113 118 73 74 108 106 122
Acenaphthene 131 60 213 209 145 126 199 185 212
Fluorene 147 161 192 210 169 235 212 204 244
Phenanthrene 810 1273 900 1018 888 1488 1110 1063 1308
PAHs mixture 163 191 261 301 215 304 320 258 365

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 Ecotoxicology in the Marine Environment 281

Table 5. Bioconcentration factors for PAHs incorporated

by Anadara similis during the test

Exposure days
Compounds
5 10 15 20 25 30
Anthracene 9500 12000 12083 23333 31250 16167
Pyrene 1960 7160 8160 6640 13640 8840
Benzo[k]fluoranthene 13000 9300 12200 4000 12200 16600
Benzo[g,h,i]perylene 8400 5500 6400 9000 8200 10000
Benzo[a]pyrene 4533 3067 3733 1800 6267 5933
Benzo[b]fluoranthene 9500 9600 12100 5600 14000 19300
Benzo[a]anthracene 4667 5778 6667 4444 11889 11444
Indeno[1,2,3-cd]pyrene 21400 16600 23000 72 27200 29400
Dibenzo[a,h]anthracene 8300 5900 7700 24 9600 9600
Chrysene 6400 8880 12600 7040 16320 18320
Acenaphthylene 27 8 163 208 219 129
Acenaphthene 87 26 333 387 400 268
Fluorene 179 142 293 381 384 281
Phenanthrene 1500 1695 1755 2163 2325 1673
PAHs mixture 183 159 378 418 500 372

DISCUSSION
According to the obtained results, each species presented different responses for each
type of pollutant. Bioaccumulation and biomagnification occur when the contaminants do not
interfeer with the life processes of the organism or when concentrations are sufficiently low to
allow tolerance, acclimatization or adaptation (Baqueiro-Cardenas et al., 2007). Bio-
accumulative contaminants are chemically hydrophobic compounds whose elimination is
very slow and difficult, so they tend to accumulate in tissues and organs such as those
consisting of fatty tissue (Fernández and Freire, 2005). Fat solubility is a determining factor
for the absorption of toxic compounds, such as PAHs that are difficult to eliminate.
The lipid content is influenced by factors such as age, species, feeding and spawning,
which changes during the year (Perugini et al., 2007). During testing it was observed that the
content of fats and oils showed variations, with an initial level that then significantly declined
and that may be related to the process of adapting to a new environment, such as when
moving from the acclimation tanks to the test vessels. Thereafter ascents and descents that are
not related to an increase or decreased concentration of of PAHs were presented.
PAHs levels absorbed by the organisms exposed to the PAHs mixture were high
considering the low exposure concentrations, which indicates the great potential of bivalves
to be used in assessment of the quality of marine environments.
PAHs with LMW were supplied at higher concentrations (for their low stability in water)
with respect to HMW, this increased is related to greater concentrations determinated in
organisms for LMW compounds.
Bioaccumulation was proportionally superior to the compounds of HMW, taking into
account the exposure levels and accumulated concentrations. This can be explained by the

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282 Mónica M. Zambrano, Robinson F. Casanova and Gustavo Arencibia

slow decline of those compounds, that remain in the water column for extended periods
(Yamada et al., 2003). LMW compounds showed an inverse behavior, due to their greater
availability and lower persistence, that allows for a greater mobility between environmental
compartments.
The persistence and the potential to bioaccumulationare higher for HMW compounds;
therefore, compounds such as benzo[a]pyrene are highly persistent and bioaccumulative,
characteristics that classify it as an aquatic pollutant of concern (Binelli and Provini, 2003).
The high accumulation observed also for LMW compounds could be not only due to the
greater exposure concentrations, but also because the solvent made them more stable in the
environment and therefore more available; PAHs are non-polar, soluble in organic solvents,
and difficult to mobilize due to their low water solubility (El-Motaium et al., 2009). However,
the bioaccumulation of most of these compounds showed large variations. Given that such
compounds canbe excreted more efficiently, the variability in the accumulation levels may
have been caused by constant absorption and excretion at short periods (Calero and
Zambrano, 1997; Albaigés, 2005; Zambrano et al., 2012b; Zambrano Ortiz, 2015).
A. similis has greater capacity to incorporate PAHs reaching 7.50 µg/g after 25 days of
exposure and a maximum of 5.48 µg/g in A. tuberculosa after 45 days, despite the fact that
the differences in terms of incorporation between species are not significant.
Since the concentration of the PAH mixture used in the tests was below the range
estimated as lethal (concentration at which a mortality of the exposed organisms has reached
50%; LC50 of 3.50x10-2 µg/mL; NOAA, 1993), the mortality observed may be associated
with factors other than the PAHs (e.g., the duration of the acclimatization period).
Regarding the commercial importance that a large number of bivalves have and due to
the fact that trail species represent an important source of food for the populations settled on
the Colombian Pacific coast, the presence of toxic compounds may represent risks for the
health of the marine environment and to human consumers. Therefore, taking into account the
regulations of the European Union (Official Journal of the European Union, 2011), it was
possible to establish that the specified concentrations in the test organisms where only safe for
human consumption prior to starting the test after several days or weeks of acclimatization, as
has been observed in others studies (Farrington et al., 1982; Lee et al., 2010; El-Gamal 2011),
because after exposure to the mixture both the level of benzo[a]pyrene (5.00x10-3 µg/g) and
the sum of indicator compounds (3.00x10-2 µg/g for the sum of chrysene, benzo[a]pyrene,
benzo[a]anthracene and benzo[b]fluoranthene as markers or indicators of toxicity) exceeded
the recomended limit.
The bioconcentration process is favored by the lipophilicity and measured through the
octanol-water partition coefficient (Kow). Polar substances have low Kow values, whereas
for hydrophobic contaminants Kow values are very high. Bioconcentration of these
compounds in water by marine organisms is directly proportional to the log Kow (Neff,
2002); LMW PHAs are generally more volatile, slightly soluble in water and less lipophilic
than HMW. These physicochemical characteristics largely determine their behavior in the
environment; therefore, bioconcentration of HMW is much higher with respect to LMW
(Pruell et al., 1986). The volatile PHAs of LMW (less than four rings) tend to be soluble in
water and have low affinity to be adsorbed to particles (Log Kow <5); PHAs with HMW are
generally insoluble in water and have a very strong affinity to be adsorbed on the surfaces of
particles suspended in air and water (log Kow>5) (López Geta et al., 2008). Generally,
substances with log Kow >6 are considered highly lipophilic (ONU, 2005).

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 Ecotoxicology in the Marine Environment 283

The PHA levels obtained allowed to determine the highest BCF in A.

tuberculosa for indeno[1,2,3-cd]pyrene followed by anthracene, benzo[b]fluoranthene,
dibenzo[a,h]anthracene and benzo[g,i,h]perylene. Both species registered the lowest BCF
with phenanthrene, acenaphthene and acenaphthylene, all with LMW and low logKow. High
concentrations recorded for LMW compounds canbe associated to the factors previously
mentioned for bioaccumulation: high concentrations of compounds used in the experiments,
and higher availability due to the use of a solvent. The BCF evidenced a high potential for
bioconcentration by obtaining readings from an order of magnitude of >3 and 4 for HMW
compounds, contrary to HMW acenaphthylene, acenaphthene and fluorene which varied
between orders of magnitude of 1 to 2, therefore classifying these compounds with a
“medium”potential to bioconcentration according to Repetto and Sanz (1995).
A. similis presented higher BCF with a maximum of 500.33 after 25 days; this is how at
15 days trial this species with 378 exceeded the maximum obtained for A. tuberculosa (365),
which was recorded after 45 days. A. similis BCF values remained higher until the end of the
exposure, a situation associated with the differences in the BCF in different species (Rezaie-
Boroon et al., 2014) as was reported by Wang (2012) for bivalves exposed to metals;
certainly for fat-soluble compounds, levels of fats and oils in organisms play a key role, these
being slightly higher in A. similis during testing.
As filter feeders, bivalves are heavily exposed to the accumulation of pollutants reaching
high BCF for metals and organic compounds in contaminated areas (Phillips, 1995). BCF is a
useful tool to evaluate risk of exposure to xenobiotics and estimate the consequences of long
term exposure tohealth to establish permissible legal levels (Repetto and Sanz, 1995). In this
context, ecotoxicology has great importance in assessing water quality before the onset of
environmental damage and in establishing safe levels of toxic compounds aimed at ensuring
the quality of ecosystems and protecting and preserving marine fauna.

CONCLUSION
The levels of PAHs incorporated by both bivalvespecies studied here suggest a potential
risk to the health of consumers regarding organisms collected in areas with direct or
indirect exposure to hydrocarbons. Even when exposed to sublethal concentrations, the
bioconcentration capacity allows them to reach PAHs levels that are unfit for human
consumption. As A. similis showed a greater tendency to bioaccumulate more PAHs and thus
present higher bioconcentration factors than A. tuberculosa, it has a potential as biomonitor
for PAHs contamination within the Colombian Pacific.

ACKNOWLEDGMENTS
The authors thank the staff of the Centre for Oceanographic and Hydrographic Research
Pacific-Cccp, Ecopetrol for the funding and support for the development of this chapter, and
Stefan Moran and Yves Pratte.

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284 Mónica M. Zambrano, Robinson F. Casanova and Gustavo Arencibia

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Yamada, M., Takada, H., Toyoda, K., Yoshida, A., Shibata, A., Nomura, H., Wada, M.,
Nishimura, M., Okamoto, K. and Ohwada, K., 2003. Study on the fate of petroleum-
derived polycyclic aromatic hydrocarbons (PAHs) and the effect of chemical dispersant
using an enclosed ecosystem, mesocosm. Mar. Pollut. Bull. 47, 105-113.
Zambrano, M., Casanova, R., Arencibia, G., Vidal, A. and Capetillo, N., 2012a. Cinética de
bioacumulación de hidrocarburos aromáticos policíclicos en Anadara similis(Adams,
1852 y Anadara tuberculosa (Sowerby, 1833). Rev. U.D.C.A. Act. and Div. Cient. 15(2),
291-302. [Kinetics of bioaccumulation of polycyclicaromatic hydrocarbonsin Anadara
similis (Adams, 1852) and Anadara tuberculous (Sowerby, 1833). Rev. U.D.C.A. Act.
and Div. Cient. 15 (2), 291-302].
Zambrano, M., Casanova, R., Prada, J., Arencibia, G., Vidal, A. and Capetillo, N., 2012b.
Bioacumulación de hidrocarburos aromáticos policíclicos en Anadara tuberculosa
(Sowerby, 1833) (Arcoida: Arcidae). Gayana 76(1), 1-9. [Bioaccumulation of polycyclic
aromatic hydrocarbons in Anadara tuberculosa (Sowerby, 1833) (Arcoida: Arcidae).
Gayana 76(1), 1-9].
Zambrano Ortiz, M.M., 2015. Evaluación de Anadara similis y Anadara tuberculosacomo
especies biomonitoras de contaminación por hidrocarburos en el Pacífico colombiano.
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Anadara similis as biomonitors species of oil pollution in the colombian Pacific. Ph.D.
Thesis. University of Havana].

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2016 Nova Science Publishers, Inc.

Chapter 18

SOIL ECOTOXICOLOGY IN ENVIRONMENTAL RISK

ASSESSMENT: A CASE STUDY IN
A METAL CONTAMINATED SITE IN BRAZIL

Júlia Carina Niemeyer1,*, Eduardo Mendes da Silva2

and José Paulo Sousa3
1Federal
University of Santa Catarina, Centre of Curitibanos,
Curitibanos, SC, Brazil
2Federal University of Bahia, Instituto de Biologia, Salvador, BA, Brazil

3University of Coimbra, Centre for Functional Ecology,

Department of Life Sciences, Coimbra, Portugal

ABSTRACT
The majority of the ecological risk assessment (ERA) studies carried out in Latin
America are based just on chemical characterization, focused on limit values of
contaminants in soils and water, usually not including biological and ecotoxicological
considerations. This chapter presents the experience of the application of a tiered ERA
framework to tropical environments, and evaluates the feasibility and usefulness of
different assessment tools to be used in different tiers within a triad approach. The case
study was carried out in an abandoned lead smelter in Santo Amaro (Bahia, Brazil). The
preliminary investigation included the problem formulation phase and the collection of
the scientific information available about the study area, that resulted in the conceptual
model and the analysis plan for the risk assessment. A tiered approach was proposed
integrating information from three lines of evidence (LoE): chemical, ecotoxicological
and ecological. Aims and actions of each phase of ERA were established to include the
ecological and ecotoxicological perspectives, focusing on the soil compartment. The
analysis plan included two tiers using the triad approach: tier 1, the screening phase, and
tier 2, the detailed risk assessment. Besides chemical analysis, the evaluations on soil
ecotoxicology included laboratory and field parameters: avoidance behavior and
reproduction of soil invertebrates; bait lamina test; vegetation structure; soil ground

* Corresponding Author E-mail: juliacarina@gmail.com.

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290 Júlia Carina Niemeyer, Eduardo Mendes da Silva and José Paulo Sousa

running invertebrates; decomposition of organic material on litter bags; and some

microbial parameters, to include ecosystem function data. The results aimed to
characterize the ecological risk, ranking sites in the study area, thus supplying
information to indicate remediation measures.

Keywords: soil ecotoxicology, contaminated sites, soil invertebrates, triad

INTRODUCTION
Relevance of ERA (Ecological Risk Assessment) in Contaminated
Sites Management

The protection of soils, its diversity and ecological functions, has become an objective of
environmental agencies around the world (Bone et al., 2010). In this context, the ecological
risk assessment (ERA) process has been recognized as a powerful tool for the decision-
making process in the management of contaminated sites or sites suspected of contamination
(USEPA, 1998; Suter et al., 2000).
ERA is a complex process of collecting, organizing and analyzing environmental data to
estimate the probability of adverse effects due to contamination, using data from different
environmental compartments collected from different lines of evidence (LoE) and different
sources of information, such as chemical analysis of contaminants, physical properties of the
environment, biological surveys, and ecotoxicity tests. Although a general trend of biological
responses can be expected in contaminated areas, the relationship between sources, exposure,
and effects on organisms are complex and often specific to a particular site, a set of
environmental conditions, and a specific receptor organism. This calls for a site-specific ERA
to support decisions about risk management and remediation (Jensen and Mesman, 2006).
In general, management actions on soil protection rest on two basic approaches: (i)
optimization of biodiversity and (ii) protection of ecosystem services (and their underlying
biological/ecological processes) essential for the survival of mankind. Soil is seen as a
multifunctional unit, supplying provisioning (food, water, fuel), regulating (soil erosion,
flood control), cultural (recreation, spiritual value, sense of place) and essentially supporting
(soil formation, nutrient cycling, oxygen from photosynthesis) services simultaneously
(Millennium Ecosystem Assessment, 2005). In this sense, an ERA process should include
indicators both of ecosystem structure and functions (Burger et al., 2007).

Main Phases of an ERA Process

Although different schemes of ERA are proposed and applied in different countries, the
components of the ERA process are similar among them, generally including the following
phases (see Figure 1):
Preliminary investigation: this phase includes the problem formulation of the assessment,
including the collection of all the scientific information available for the area; the
development of a conceptual model; and an analysis plan for the risk assessment (e.g., Pereira
et al., 2004; Weeks et al., 2004). The conceptual model is built involving what is currently

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 Soil Ecotoxicology in Environmental Risk Assessment 291

known about the site, geographical limits, source and type of contamination, historical use
and activities in the site, current pathways of exposure, and observation of perceptible risks
and ecological receptors at risk (Weeks, 2004; Jensen and Mesman, 2006; Ashton et al.,
2008).
Exploratory investigation: this is an optional phase to confirm the existence of
unacceptable contamination in the study area by performing a preliminary field sampling,
followed by chemical analysis and comparison with soil quality guidelines.
Main investigation: this phase is performed in case a contamination is confirmed and a
potential ecological risk can occur. This phase is usually done using a tiered approach where
in each tier different assessment tools belonging to different LoE are applied, including more
detailed chemical analysis, bioassays and ecological field surveys (Jensen and Mesman, 2006;
Swartjes et al., 2008). At the end of the assessment, the results are integrated to describe the
risk.
Several schemes of ERA are available in different countries, e.g., US (USEPA, 1998),
Canada (CCME, 1996), UK (Weeks, 2004). In The Netherlands, the EU country with most
experience in the application of site-specific ERA, a practical triad approach has been
developed (Rutgers and Den Besten, 2005; Mesman et al., 2007) which has been adapted and
successfully applied in Europe (Jensen and Mesman, 2006; Critto et al., 2007). In Latin
America(LA), studies involving ERA are incipient. In Brazil, the ABNT (Brazilian National
Standards Organization) started a discussion in 2014 about which methodology will be
adopted to conduct ERA processes in this country (Niva et al., 2016). The respective
guideline is under construction.

Figure 1. General scheme of an Ecological Risk Assessment process.

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292 Júlia Carina Niemeyer, Eduardo Mendes da Silva and José Paulo Sousa

The Triad Approach

Consisting of three LoE (chemical, ecotoxicological and ecological), the triad approach is
usually applied within a tiered system, i.e., information from each LoE is collected at each
tier following a stepwise cost-effective process (Jensen and Mesman, 2006). The tiered
approach is designed to be efficient in excluding extreme sites, i.e., either sites that pose no
risk to ecosystems or sites that pose a high risk and where remediation actions are needed
(Figure 2). The triad approach relies on the concept of weight-of-evidence (WoE), which is
the process of combining information from multiple LoE to reach a conclusion about an
environmental system or stressor, as such an approach minimizes the chance of false positive
and false negative conclusions (Burton et al., 2002).
Tier 1 is essentially a screening phase, aiming to produce a first spatial representation of
the risk and to determine whether a site can be excluded from higher tiers and of further
testing (either because it is unlikely to pose a risk to the relevant ecological receptors or
because a high risk is detected and there could be a need for immediate mitigation actions), or
if it needs to be further evaluated at Tier 2. Thus, the tools used in tier 1 to collect information
from each LoE should not only be able to indicate effects, but also be rapid, easy to apply and
cost-effective (Jensen and Mesman, 2006).

Figure 2. General scheme of Environmental Risk Assessment process organized in tier phases. Detail:
triad approach composed by chemical, toxicological and ecological lines of evidence.

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 Soil Ecotoxicology in Environmental Risk Assessment 293

Tier 2 (and tier 3, if necessary) is performed to reduce uncertainties about the actual risk.
In tier 2, the chemical LoE can comprise extraction techniques to assess the bioavailable
fraction of pollutants in soil. This should be complemented with information derived from
ecotoxicological tests (ecotoxicological LoE) and ecological surveys (ecological LoE)
(Jensen and Mesman, 2006). The ecotoxicological LoE usually comprises long-term studies
focusing on chronic endpoints such as reproduction and growth, and some mineralization
processes, since these effects can occur at intermediate levels of pollution. The ecological
information at tier 2 is collected to get more details about the possible impact on microbial
communities and flora and fauna populations in situ, thus assessing effects at higher
hierarchical levels of organization (Weeks et al., 2004; Critto et al., 2007).

THE STUDY CASE OF SANTO AMARO, BAHÍA, BRAZIL

An ERA process was carried out by Niemeyer et al. (2010, 2015) in the abandoned lead
smelter of Plumbum metallurgy that was operational between 1960 and 1993, located
adjacent to the urban area of Santo Amaro (BA, Brazil), about 150 mm away from Salvador,
state capital (Figure 3). The area presented a high health risk to cattle and humans due to high
levels of metals in soil and water, as well as by tailings and airborne dust from atmospheric
deposition through chimney emissions while the smelter was operational. The study aimed to
answer the following questions:

1) Does the metal contamination in the smelter area still pose some ecological risk to
the soil habitat and retention functions 17 years after the closure of the smelter? How
is the spatial extension of the risks posed by the smelter area?

Figure 3. Smelter area of Plumbum metallurgy, Santo Amaro, Bahia, Brazil.

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294 Júlia Carina Niemeyer, Eduardo Mendes da Silva and José Paulo Sousa

Figure 4. Smelter area of Plumbum metallurgy, Santo Amaro, Bahia, Brazil. A) Evidence of runoff and
erosion in a pile of furnace slag (gray material) in the smelter area during the rainy season. B) The
aspect of the smelter area in the dry season (dust and lack of vegetation).

2) Are the detected effects directly associated to the presence of the metals (direct
toxicity) or to an indirect stressor (habitat disruption)?
3) Is a tiered ERA framework suitable to be applied in contaminated sites with this
typology of contamination?
4) Which type of biological (ecotoxicological and ecological) parameters are more
sensitive to detect risk? Are they able to discriminate different levels of risk? And at
which tier should they be used?

Preliminary Investigation: Building the Conceptual Model and

Analysis Plan of an ERA Process

Based on the available information about the site, including data obtained during the site
visits (Figure 4) and the chemical pre-sampling, a conceptual model (Figure 5) and an
analysis plan for an ERA were developed.
The primary contamination source identified was soil contaminated by furnace slag
deposition or by aerial deposition (wind-blown dust or past chimney emissions). The
principal source of potential exposure to the ecological receptors is the contaminated soil,
through ingestion, cellular absorption, aerial deposition (wind-blown particles) and root
uptake. Metal contaminants can be available, posing potential risk to primary receptors such
as plants, soil invertebrates and soil microbial communities. In addition, other species can be

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 Soil Ecotoxicology in Environmental Risk Assessment 295

linked to contaminants through the terrestrial food chain, such as invertebrates feeding on
plants, and vertebrates, such as birds (seed-, plant-eating and invertebrate-feeding species),
small mammals, amphibians, reptiles and raptor species.

Table 1. Assessment and measurement endpoints.

Adapted from Weeks et al. (2004)

Receptor Relevance for the Assessment endpoints Measurement endpoints

ecosystem
functioning
Plant Food and habitat Habitat function that
Determination of vegetation cover in situ
community supply for animal sustains plant (1).
species. germination, growth,Plant toxicity test with
Maintenance of soil biomass and species monocotyledonous and dicotyledonous
structure. richness. species (2).
Supply of nutrients. Determination of vegetation richness
species in situ (2).
Soil invertebrate Food supply for Habitat function that Avoidance behavior tests with
community and animal species. sustains diverse and earthworms and springtails (1).
activity Predation of active invertebrate Feeding activity evaluated by bait lamina
microfauna. populations. test in situ (1)
Decomposition of Reproduction tests with earthworms,
organic material. springtails and enchytraeids (2).
Maintenance of soil Composition of soil surface dwelling
structure. macroarthropod community collected
with pitfall traps (richness, diversity
index, differences in community
composition) (2).
Decomposition rate of organic material in
litter bags in situ (2).
Soil microbial Nutrient supply to Habitat function that Soil basal respiration (1).
community support plant sustains viable and Soil microbial biomass of carbon and
growth. functional microbial nitrogen (2).
Important in populations. Microbial enzymatic activity
maintaining (dehydrogenase, acid phosphatase and
microagregate soil asparaginase) (2).
structure. Soil nitrification and ammonification rate
(2).
Organic material breakdown in litter bags
in situ (2).
Microorga- Primary production. Retention function of soil Vibrio fischeri (bacteria) luminescence
nisms and algae Recycling of to evaluate risks to test (1).
nutrients. aquatic receptors (in this Algae growth test (2).
case particularly via
groundwater
contamination).
Aquatic Aquatic food web. Retention function of soil Cladoceran lethal tests (1).
invertebrates to evaluate risks to Cladoceran reproduction tests (2).
aquatic receptors (in this
case particularly via
groundwater
contamination).
(1) Tier 1; (2) Tier 2.

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 Leaching

Runoff
SOIL SURFACE WATER

AMPHIBIANS
Dermal absorption Ingestion; dermal
REPTILES absorption

Ingestion
Celular MICROORGANISMS Cellular
MICROORGANISMS
absorption absorption
Ingestion Ingestion GROUNDWATER
Absorption PLANTS AQUATIC Ingestion;
INVERTEBRATES absorption
Ingestion Ingestion
Ingestion; dermal
Ingestion; dermal TERRESTRIAL FISHES absorption
absorption INVERTEBRATES
Ingestion Ingestion
Ingestion Ingestion
Ingestion; dermal
BIRDS
absorption
BIRDS
Ingestion
MAMMALS Ingestion; dermal
absorption
CATTLE
Ingestion Ingestion

HUMANS Ingestion; dermal

absorption

Figure 5. Conceptual model for risk assessment in the contaminated area in Santo Amaro, Bahia, Brazil. Environmental compartments are ellipses, exposure
pathways are arrows and ecological receptors are represented by rectangles. The soil is the main source of contaminants (tail deposits and aerial deposition).
Receptors in bold were those considered for evaluation in the study. Niemeyer (2012).

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 Soil Ecotoxicology in Environmental Risk Assessment 297

The analysis plan for the site-specific risk assessment was focused on the soil
compartment, addressing indirectly the risk to groundwater and freshwater systems. Direct
effects on non-soil invertebrates, vertebrates and water systems were not covered by this
study. A summary of the assessment and measurement endpoints is shown in Table 1. Details
about sampling strategy are described by Niemeyer et al. (2010). Due to the heterogeneity of
the soil among the different sampling points, it was necessary to work on a multi-reference
basis. Therefore, soils were assembled into three groups based on a Factorial analysis. Each
group differed mainly in terms of texture, organic matter content and pH. To find matching
reference soils, soil from several points in the surroundings of the area were screened,
analyzed for metals and soil properties, and three reference soils (the best possible for each
identified group of soils) were selected at 9 km (Ref. 1) and 3 km (Refs. 2 and 3) from the
site. Details on this process (grouping of soils and finding the reference soils) can be found in
Niemeyer et al. (2010).
The analysis plan included two tiers. In tier 1 (screening), the chemical LoE comprised of
the calculation of the toxic pressure (Rutgers and Jensen, 2011) based on the comparison of
the total concentrations of metals of the study site with soil screening levels, HC50cor (Dutch
HC50EC50 values; Rutgers et al., 2008) corrected for sampling site-specific differences, taking
into account the organic matter and the clay content of each soil. The ecological information
at tier 1 was collected through a quick vegetation survey and by assessing easy measureable
functional parameters, such as soil respiration (Jensen and Mesman, 2006) and soil faunal
feeding activity using bait lamina sticks (Von Törne, 1990). Regarding the ecotoxicological
LoE, short-term cost-effective bioassays evaluating both the habitat and retention functions of
the soil were carried out. The latter was evaluated using soil extracts (eluates) in tests with a
cladoceran species (Daphnia magna acute test) and with the luminescent bacteria Vibrio
fischeri (Van Gestel et al., 2001; Loureiro et al., 2005). Soil samples were used to evaluate
the loss of habitat function through avoidance tests with earthworms and collembolans (Natal-
da-Luz et al., 2004). Details on this phase can be found in Niemeyer et al. (2010).
Tier 2 (detailed assessment) was performed to reduce uncertainties about the actual risk
shown by tier 1. The chemical LoE comprised the calculation of the toxic pressure based on
total metals in habitat function (as done in tier 1) and the analysis of extractable metals using
0.01 M CaCl2 solution to assess the soil retention function (mainly with the aim of evaluating
potential of ground-water contamination). The ecotoxicological LoE comprised standardized
chronic tests with Collembola (ISO, 1999) and Oligochaeta (ISO 1998, 2004), which were
performed to evaluate sub-lethal effects of soil matrix on reproduction of soil invertebrates.
Effects towards plants were evaluated by performing plant growth tests using standard species
(one monocotyledonous and one dicotyledonous species) following ISO 11269-2 (ISO,
2005). In addition, soil extracts (eluates) were used to perform widely established tests with
cladocerans (OECD, 2008) and microalgae (OECD, 1984) to evaluate the retention function
of soil, thus assessing the indirect risk to the aquatic compartment (mainly groundwater)
(Jensen and Mesman, 2006; Chelinho et al., 2009).
Regarding the ecological LoE at tier 2, information was collected to obtain more
details about the possible impact on selected ecological receptors. Changes in diversity and
community composition of plants (by field survey), soil surface dwelling invertebrates
(pitfall trapping), as well as several functional processes were evaluated. Among them,
microbiological soil-quality indicators considered in this study were microbial biomass,

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298 Júlia Carina Niemeyer, Eduardo Mendes da Silva and José Paulo Sousa

substrate-induced respiration, enzymatic activity and nutrient transformations. These are

proxies for important processes related to soil fertility and can be used as bioindicators of soil
stressed by contamination (Castaldi et al., 2004; Gulser and Erdogan, 2008), or to indicate
suitable management and restoration practices (Nogueira et al., 2006).The ecological
evaluation was complemented with the assessment of effects on organic matter (litter)
decomposition in litter bags, a functional parameter by excellence, which can be used as
indicative of negative effects on the soil microbial community, soil fauna or both (Knacker et
al., 2003; Römbke et al., 2003).
For the data analysis differences among contaminated soils and the respective reference
soil in ecotoxicity tests were in general evaluated by one-way analysis of variance (ANOVA),
followed by one-tailed Dunnet’s test when necessary. Prior to all analysis, normality and
homoscedasticity were checked via the Shapiro-Wilk’s test and Bartlet test, respectively.
When homoscedasticity was not fulfilled, an equivalent non-parametric test was used, namely
the Kruskal-Wallis ANOVA followed by Dunn’s multiple comparisons test. Ecological data
were analyzed using ANOVA, t-tests, or ANOSIM approaches according to the parameters.
Such analyses are described in Niemeyer et al. (2010, 2012a,b, 2015).
All parameters measured for each LoE were used for risk calculations where risk values
were expressed in a scale ranging from zero (“no risk”) to one (“highest risk”) assuming that
the risk value of reference soils is zero. For each sampling point, risk values were calculated
following three steps, according to calculations presented by Jensen and Mesman (2006): (1)
scale the results (between 0 and 1) of each test/parameter within each LoE; (2) integrate all
scaled information of all parameters within each LoE; (3) integrate the information from the
three LoEs and calculate the integrated risk. In the present study, the integrated risks to the
soil habitat and retention function were calculated separately. More details about risk
calculations are presented in Niemeyer et al. (2010, 2015).

Results from Tier 1 and Tier 2

Chemical LoE from Tier 1 pointed to high risk due to metal concentrations, mainly Pb,
Cd, Cu and Zn, in superficial soil from the smelter area and surrounds. Despite the low
extractability of metals in CaCl2 0,01M solution, high risk levels were indicated by
ecotoxicity tests and ecological evaluations at the site. Avoidance tests indicated that
earthworms and collembolans avoided contaminated sites when compared to reference soils.
The lack of these groups of organisms can affect important soil functions related to nutrient
cycling, structure and fertility of soil, besides impacts on food chain level (Römbke et al.,
2005). Eluates evaluated by aquatic tests with cladocera and bacteria showed a compromised
retention function at sites corresponding to residue deposits, which point to some risk of
migration to surface and groundwaters. The low vegetation cover inside the smelter area and
low feeding activity of soil fauna in bait lamina test evidenced impact to plants and soil
invertebrates on the site. In general, integrated risk of Tier 1 pointed to high risk values inside
the smelter area (more details in Niemeyeret al., 2010).
Results of the tier 2 evaluation confirmed the high risk levels in the smelter area already
indicated in tier 1, and associated with tailing deposits, but with a further reduction of
uncertainties. Locations outside the smelter area demonstrated lower or acceptable risks. As

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in tier 1, the low toxicity in eluate tests indicated high adsorption of metals in soil, probably
favored by content and type of clay, ageing and neutral pH, and consequently negligible risk
due to the high retention capacity in most sampling points, except for residue deposits.
Moreover, the results indicate that the current cover of the tailing deposits failed to restore the
site by not creating appropriate conditions for the establishment of plant (revegetation),
microbial and animal communities inside the area. So, besides the direct effects of metal
contamination, also indirect effects are visible due to the presence of these contaminants,
compromising the functioning of the ecosystem inside the smelter area. Detailed results are
presented and discussed in Niemeyer et al. (2012ab, 2015).

Recommendations for the Studied Site

High risk values in habitat function inside the smelter area indicate the need to proceed
with some remediation action, such as an improved encapsulation of tailing deposits and
recovery of the vegetation (Figure 6). These actions could not only improve soil conditions
and ecosystem functioning, but could mainly avoid the transport of contaminants to other
environmental compartments, namely via dust dispersal to outside the area, or via surface
runoff to the existing temporary ponds and the Subaé river.

Figure 6. Suggested actions and expected ecological improvements according to the Environmental
Risk Assessment results.

The improvement of the vegetation cover can be effective in providing the necessary
surface stability to prevent wind-blow of contaminated soil particles, preventing erosion, and
reducing water pollution by interception of a substantial proportion of incident precipitation
(Tordoff et al., 2000; Wong, 2003). Furthermore, the choice of appropriate vegetation is
crucial to remediate the adverse physical and chemical properties of the site and to reestablish
ecosystem functioning (Wong, 2003), besides the aesthetical improvement of the site.
Organic matter, soil nutrients and species diversity generally increase with community
development during succession (Wang et al., 2011). It is important to consider the use of
locally adapted species which are tolerant not only of physical and chemical conditions of

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tailings, but also to the climatic conditions of the site. Furthermore, plant species with
different traits can increment the heterogeneity of soil habitats (Podgaiski and Rodrigues,
2010), and can thus support soil communities that demand different requirements of food and
shelters (Wardle et al., 2004).

Sensitivity of Ecotoxicological and Ecological Parameters for Risk

Assessment

Aiming at evaluating how sensitive and cost-effective the different ecotoxicological and
ecological parameters in the risk assessment could be, a sensitivity analysis was conducted
taking into account not only the ability of each parameter to detect differences between
contaminated and non-contaminated points (outside the affected area), but also their ability to
detect a gradient of contamination. For this purpose, individual parameters were analyzed
based on observed significant differences in comparison to reference points (using ANOVA).
For soil fauna abundance and taxonomic richness, the sensitivity analysis was based on
significant differences between sampling points outside the area (non or low contamination)
and points inside the area (high contamination): high (p<0.001); medium (p<0.01) and low
(p<0.05) sensitivity. Ability to differentiate the level of contamination was based on
significant correlations with metal loadings (Widianarko index): high (p<0.001); medium
(p<0.01); low (p<0.05). The time necessary to obtain the parameter was also estimated.
Results are presented in Tables 2 and 3.

Table 2. Valuation of the sensitivity of each ecotoxicological parameter assessed at the

smelter area. grey - parameters selected based on sensitivity criteria
(see text for details)

Organism Category Parameter Significant Ability to Days needed to Use in

group response in differentiate the obtain the ERA
contaminated level of parameter (time (Tier)
sites (1) contamination (2) of exposure)
Soil inverte- Habitat Reproduction of
High High 56 2
brates function for Eisenia andrei
soil Reproduction of
invertebrates Enchytraeus High High 28 2
crypticus
Reproduction of No
No 28 2
Folsomia candida differences
Plants Habitat Growth of Avena
Low Medium 14-21 2
function for sativa
plants Dry weight of A.
No No 14-21 2
sativa
Growth of
No No 14-21 2
Brassica rapa
Dry weight of B. No to
No 14-21 2
rapa medium
(1) Information based on observed significant differences against reference points (ANOVA): high (p<0.001); medium
(p<0.01); low (p<0.05).
(2) Information based on significant correlations with metal loadings (Widianarko index): high (p<0.001); medium
(p<0.01); low (p<0.05).

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Table 3. Summary of the sensitivity of each ecological parameter assessed at the smelter
area. Light grey - parameters derived from multivariate analysis based on individual
parameters from the corresponding organism group; Dark grey - parameters selected
based on sensitivity criteria (see text for details)

Organism Category Parameter Significant Ability to Days needed to Use in

group response in differentiate obtain the ERA
contaminated thelevel of parameter (Tier)
sites (1) contamination (2) (estimated
number of
working days)
Community Microbial
Low to high High 8 (4) 1-2
activity respiration
Community Microbial biomass
Low to high High 2 (2) 1-2
structure C
Community Dehydrogenase
Low to medium No 1 (1) 1-2
activity activity
Micro- Community Acid phosphatase
Low to high Medium 1 (1) 1-2
organisms activity activity
Biological
Nitrification rate Low to high Medium 21 (3) 1-2
process
Multivariate
analysis with all
Medium 1-2
microbial
parameters
Community Feeding activity
High Medium 14 (4) 1
activity (bait lamina)
Community Group
Abundance No 2
structure dependent; low
Community (Morpho) Species Group
No 2
structure richness (3) dependent; low
Community Shannon diversity
No differences No 2
Inverte- structure index (3)
37 (30)
brates Community Pielou evenness
No differences No 2
structure index (3)
Community Margalef richness
No differences No 2
structure index (3)
Community Berger-Parker index
No differences No 2
structure (3)
Community Changes in species
High 2
structure composition (3)
Community % of vegetation
High Low 1 (1) 1
structure cover
Community
Plants Species richness (3) No differences No 2 (2) 1-2
structure
Community Changes in species
High 2
structure composition (3)
Microorga-
Ecosystem Litter breakdown
nisms and High Low 140 (25) 2
function (decay rate)
invertebrates
(1) For individual parameters, information based on observed significant differences against reference points (ANOVA);
for soil fauna abundance and taxonomic richness and for integrated multivariate analysis (ANOSIM), based on
significant differences between sampling points outside the area (non or low contamination) from points inside the
area (high contamination); high (p<0.001); medium (p<0.01); low (p<0.05).
(2) Information based on significant correlations with metal loadings (Widianarko index): high (p<0.001); medium
(p<0.01); low (p<0.05).
(3) Parameters that require specific taxonomic knowledge.

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302 Júlia Carina Niemeyer, Eduardo Mendes da Silva and José Paulo Sousa

The ability of the avoidance behavior to detect toxicity within a short test period and at
low cost makes this test suitable for use in decision processes. However, some care should be
taken in the choice of reference soils (similar in properties except for the contamination
level). If finding matching reference soils becomes a difficult task, models are available to
correct for the influence of soil properties (Chelinho et al., 2011).
The results demonstrated the high sensitivity of oligochaeta reproduction tests to evaluate
the contaminated sites; however, results obtained with Folsomia candida were not sufficient
for an adequate assessment of metal contaminated soils. As soil invertebrate species are
affected in a different way, it is recommended for a suitable evaluation of the risk the use of
several species from different ecological groups, representing distinct routes of exposure to
contaminants.
The microbial community was highly impaired by metal contamination. However, only
basal respiration, microbial biomass (C), acid phosphatase activity and nitrification presented
a high to medium capacity to distinguish the level of soil contamination. Since the first two
parameters were highly correlated, assessing only one is enough to give information relative
to microbial activity. Bacterial parameters related to community structure (not assessed in this
study) and bacterial growth/biomass were highly rated by Critto et al. (2007) as parameters to
assess in all triad tiers, mainly due to their rapidity and low cost. Based on these findings only
one parameter seems not to be sufficient to give information about general microbial activity,
and it should be complemented with other parameters related to microbial genetic diversity
(e.g., DGGE) or metabolic diversity (e.g., Biolog), in addition to other specific activity
parameters if processes involving particular nutrients are of interest.
The high sensitivity of feeding activity of soil fauna, allied to the fact that several studies
showed the relation between bait-lamina data and abundance of several microarthropod
groups and lumbricids (Birkhofer et al., 2011), make the bait-lamina test a definitive
parameter to include in the ecological LoE in site specific assessments. Due to its ease of use
and practicability, allowing to process the information from a large number of sampling
points over a short time, it is a parameter to use in tier 1 of a triad approach. Regarding
abundance and normal biodiversity descriptors of surface dwelling arthropods, results did not
show very promising results in distinguishing different levels of contamination. However,
community composition could be a more promising parameter. Although not performed in
this study, identification of the family and/or trophic group, including the use of vulnerability
traits, could be more useful to better decipher the true risks to this group of organisms.
Another aspect, also not contemplated in this study, is to sample not only surface dwelling
organisms but true soil dwellers that could have a completely different response.
Vegetation cover and changes in plant composition were able to detect differences
between points inside and outside the smelter area. However, their ability to detect gradients
of contamination was not met. Critto et al. (2007) presented a low rank for vegetation survey
related parameters in tier 1, mainly due to their cost. However, in higher tiers (tiers 2 and 3)
these parameters presented higher ranking mainly related to their site specific relevance.
Plant litter decomposition showed a high sensitivity to contamination and derived habitat
disruption, but presented a low capacity to differentiate the level of contamination. In this
case, it gave a similar information as the bait lamina test (r=0.83, p<0.01), thus not being a
priority parameter to integrate in a tiered scheme (also due to the long time needed to obtain
results).

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FINAL CONSIDERATIONS
According to Rutgers and Jensen (2011), although many tools for a triad approach in
ERA are available, the increasing number of triad-based risk assessment will demand for
improved, new, standardized, robust and cost-effective tools. This is specially applied to Latin
America, where more adequate experimentation design should be pursued to provide more
realistic scenarios taking into consideration native species and environmental conditions (such
as temperature and soil types). As pointed out by Filser et al. (2008), ecotoxicologists should
make better use of basic ecology when establishing new tests or risk assessment schemes and
convince regulatory authorities of the necessity of such an approach. Experience can be
acquired by testing the triad basic approach in practical situations, at a number of
characteristic sites, aiming to provide important information to help the regular use of the
ERA schemes in supporting site restoration and reclamation decisions in Brazil and Latin
America.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2016 Nova Science Publishers, Inc.

Chapter 19

ECOTOXICOLOGICAL ASSESSMENT OF DREDGED

SEDIMENTS FROM GUANABARA AND SEPETIBA BAYS
(RIO DE JANEIRO, BRAZIL)
USING BIOASSAYS WITH EARTHWORMS

Ricardo G. Cesar1, , Ana P.C. Rodrigues2,

Bianca C.R.C. da Rocha3,4, Tácio M. de Campos4,
Christiane N. Monte2, Simone Dealtry4,
Zuleica C. Castilhos2,3 and Wilson T.V. Machado2
1
Federal University of Rio de Janeiro, UFRJ,
Department of Geography, Cidade Universitária, Rio de Janeiro, RJ, Brazil
2
Fluminense Federal University, UFF, Department of Geochemistry,
Outeiro São João Baptista, s/n Centro, Niterói, RJ, Brazil
3
Centre for Mineral Technology, CETEM/MCTI,
Sustainable Development Service, Cidade Universitária,
Rio de Janeiro, RJ, Brazil
4
Pontifical Catholic University of Rio de Janeiro, PUC-Rio,
Department of Civil Engineering, Gávea, Rio de Janeiro, RJ, Brazil

ABSTRACT
The limits for metal concentrations in soil and terrestrial disposal of dredged
sediments established by Brazilian law are based on Canadian and Dutch legislations. To
evaluate the adequacy of such threshold values, dredged sediments were collected from
polluted aquatic systems of Rio de Janeiro state: Saco do Engenho River (Sepetiba Bay),
Meriti River (Guanabara Bay) and São Francisco River (Sepetiba Bay). The sediments
were mixed with an artificial soil in proportions between 0% (pure artificial soil) and
30% for performing acute bioassays with Eisenia andrei. The earthworm median lethal


Corresponding Author E-mail: ricardogc.geo@gmail.com

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310 Ricardo G. Cesar, Ana P.C. Rodrigues, Bianca C.R.C. da Rocha et al.

concentration (LC50) for the Saco do Engenho River was 16.26% while for Meriti River
it was 17.46%. No significant mortality was detected for the sediment from São
Francisco River. The highest toxicity observed for the sediment from Saco do
EngenhoRiver is in agreement with the higher metal levels among the sediments studied.
The organisms increased their biomass when exposed to low dosages of sediments from
Saco do Engenho River and Meriti River, while those exposed to sediment from São
Francisco increased their biomass with increasing sediment dose. The bioconcentration
factors (BCF) for zinc and copper generally decreased with increasing concentrations of
sediment, indicating the existence of internal regulating processes. The BCFs for lead,
chromium and cadmium were <0.3. The highest BCF values were found for nickel in
mixtures with the sediment from Saco do Engenho River. The bioassays performed were
adequate for a preliminary evaluation of the damages associated with disposal of dredged
sediments in soil systems.

Keywords: bioconcentration factor, Eisenia andrei,ecotoxicity, acute tests, metals

INTRODUCTION
Over the last decades large amounts of domestic and industrial wastes have been
discharged into aquatic systems of South America without an adequate treatment. This
situation is particularly critical along the coastal area of Rio de Janeiro state (RJ; southeastern
Brazil), especially in the Guanabara and Sepetiba bays where concentrations of metals,
hydrocarbons, pathogenic microorganisms and domestic wastes are abnormally high (Junior
et al., 2002, Machado et al., 2002, Cesar et al., 2014). In the RJ state, the dredging of
contaminated sediments is often required to keep these water-ways navigable and to avoid
their eutrophication (Cesar et al., 2015a). Given the fact that the transport of dredged
sediments is relatively expensive (about 50-60% of total costs of the dredging), the disposal
of these sediments in adjacent soils is a common practice in Brazil and can cause serious
damages to soil ecosystems (Cesar et al., 2014, 2015b).
The limits for metal concentrations established by Brazilian legislation for terrestrial
disposal of dredged sediments is based on Canadian and Dutch guidelines, which were
idealized for temperate environments and dredging of ports areas. Thus, the application of
these values in tropical regions (e.g., in Brazil) should not be recommended. Furthermore, the
dredging of estuarine and fluvial systems (and not only port areas) of RJ state is also required
to attend for safe practice of water sports and fishing for human consumption, which
include the dredging of sediments and decontamination of water and bottom sediments from
Guanabara Bay.
Most studies on metal contamination in tropical soils and bottom sediments have been
traditionally based on total chemical analysis, which generally includes selective metal
analysis and geochemical speciation (DePaula and Mozeto, 2001; Gleyzes et al., 2002; Cesar
et al., 2011). Even though those analyses are extremely relevant, they are not sufficient to
predict metal ecotoxicity and to prevent damages on soil ecosystems if not complemented
with bioassays using representative soil organisms. In this context, earthworms have been
widely used as test organisms in bioassays, since they are abundant in both tropical and
temperate soils, ingest large amounts of soil and are sensitive to contaminants (Hinton and
Veiga, 2002; Nahmani et al., 2007). Moreover, the determination of metal concentrations

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 Ecotoxicological Assessment of Dredged Sediments … 311

in tissues of surviving organisms from bioassays provides relevant information on their

bioavailability levels in the soil, which may be related to the occurrence of adverse effects on
soil biodiversity (Straalen et al., 2005).
The acute toxicity test with Eisenia andrei made part of a ring test to define a test battery
to evaluate the ecotoxicity of three representative waste types (Moser and Römbke, 2009).
Those assays were performed by different European laboratories, and showed that the acute
bioassay with E. andrei is an adequate test for characterizing the risks of waste disposal in
terrestrial systems (Moser and Römbke, 2009). Cesar et al. (2014) studied the ecotoxicity of
dredged sediments from Cunha Channel (Guanabara Bay basin, RJ) using acute bioassays
with E. andrei and showed that this assay may be a robust method for evaluating the effects
of the dredged sediment to soil fauna.
The present chapter aims to make a preliminary ecotoxicological evaluation of dredged
sediments from Guanabara and Sepetiba bays (RJ) using survival, biomass changes and
bioaccumulation of metals as endpoints. To attain this purpose, acute bioassays with E.
andrei were applied in artificial soils treated with different doses of dredged sediments from
Guanabara and Sepetiba bays. The working hypotheses are: (i) the acute bioassay with E.
andrei is an appropriate method to evaluate the ecotoxicity of dredged sediments from
Guanabara and Sepetiba bays to soil biota; (ii) the current threshold metal concentrations
defined by Brazilian legislation are not adequate to prevent noxious effects on soil organisms;
and (iii) the organic matter provided by the sediment is a potential source of food for the test
organisms.

METHODS
Sampling of Dredged Sediments

Three samples of dredged sediment were obtained from three different collecting stations
(one sample from each collecting point) in September 2012 by using a Van Venn Grab
Sampler. Two sampling points are located at Sepetiba Bay (mouths of Saco do Engenho
River and São Francisco River), while the other one is located at Guanabara Bay(mouth of
Meriti River) (Figure 1). The samples were dried at 40°C and ground for ecotoxicological
evaluation.
The basins of Guanabara and Sepetiba bays are characterized by high concentrations of
metals, petroleum hydrocarbons and domestic wastes in sediments, water and biota (Machado
et al., 2002; Bidone and Lacerda, 2004; Silva et al., 2007; Silveira et al., 2010; Rodrigues et
al., 2011). Meriti River basin receives high amounts of domestic wastes and effluents
containing heavy metals, especially mercury. The mouth of Meriti River is often silted which
disturbs natural water circulation in the Guanabara Bay. The Saco do Engenho River has been
receiving high amounts of heavy metals (lead, cadmium, zinc and copper) due to an old (and
nowadays not active) metallurgical industry located at its adjacent areas. The mouth of Saco
do Engenho River is often dredged to support the activities of the Sepetiba Port. The São
Francisco River basin contains high amounts of domestic wastes and some of its tributaries
drain industrialized areas. Its mouth is also frequently silted.

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Figure 1. Location of sampling sites for dredged sediment in the Guanabara and Sepetiba bays (Rio de
Janeiro, Brazil). ME: Mouth of Meriti River; SE: Mouth of Saco do Engenho River; SF: Mouth of São
Francisco River.

Acute Toxicity Test with Eisenia andrei

The acute earthworm test was performed according to the ASTM (2004) protocol with
some adaptations. Five replicates per test concentration were prepared, each one consisting of
a cylindrical plastic box (3.8 cm diameter and 6.4 cm height) with 500 g (dry weight
equivalent) of uncontaminated soils or mixtures of soil and dredged sediment. Ten adult
organisms (previously acclimated in the natural soils for more than one day, washed with
distilled water and weighed) were added to each replicate. Earthworms with an average
weight of 0.548±0.18 g (average ± standard deviation, n=300) were used in the test. Moisture
content of test mixtures was previously adjusted to 50% of its water-holding capacity. The
test was performed under constant illumination and controlled temperature (20±2ºC).
Additional replicates with pure tropical artificial soil were also prepared. The artificial soil
was prepared according to Garcia (2004) consisting of 70% of quartz sand, 20% of kaolin and
10% of coconut shells dust. After 14 days of exposure, mortality and biomass change of the
surviving earthworms were determined. After being counted, washed and weighted, surviving
organisms were maintained in moistened absorbent paper for 24 h to purge the gut before
being stored for metal content determination. The validity criterion of acute tests assumed
earthworm mortality lower than 10% in replicates with pure artificial soil.

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Chemical Characterization

Organic carbon contents were quantified by elementary analysis, using a LECO SNS-
2000 equipment. The total concentrations of Al, Cd, Cu, Cr, Fe, Ni, Pb, Zn and Mn in the
dredged sediment and test mixtures were determined through the solubilisation of 1 g of
homogenized sample in 40 ml of an acid mixture of HF:HCl:HClO4 (2:1:1). The
concentration of metals in soil extracts was determined by inductively coupled plasma -
atomic emission spectrometry (ICP-AES; Horiba JobinYvon, Ultima 2). Vanadium (V)
concentrations were obtained by using the same procedure used for metals. Detection limits
were 0.4 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.01 mg/kg, 0.4 mg/kg, 0.01 mg/kg, 0.2 mg/kg, 0.2
mg/kg, 1.4 mg/kg and 0.4 mg/kg, for Al, Cd, Cu, Cr, Fe, Ni, Pb, V, Zn and Mn, respectively.
Metal concentration obtained in the sediments was compared to the threshold
values established by Brazilian law for dredged sediment disposal (CONAMA 454): the
limits of low (Level I) and high (Level II) probability of adverse effects on biota. Metal
concentrations determined in mixtures of soil and sediment were compared to the limits
defined by Brazilian law for soil quality (CONAMA 420 and CETESB 2005): the reference
limit (soil geochemical background for Sao Paulo state), prevention limit (based on the risks
on soil ecological receptors) and intervention limit (based on the risks on human health).
After being frozen, lyophilized and triturated, total metal concentrations in tissues of
surviving earthworms were determined by using the same procedures applied to the dredged
sediment. Metal bioavailability levels were evaluated with the bioconcentration factor (BCF),
a ratio between total metal content in the organisms and total metal content in the soil. When
earthworm metal concentration was lower than the detection limit, the BCF was determined
according to its respective detection limit value.
Total metal concentrations in the dredged sediment and test mixtures were compared to
the limits established by Brazilian legislation for terrestrial disposal of estuarine sediments
(CONAMA, 2012) and Brazilian soil quality (CONAMA, 2009). Metal concentrations
measured in the dredged sediment were used to calculate the Geoaccumulation Indices
(IGEOs) for each metal, using background values determined by other researchers in
sediment cores collected in Guanabara and Sepetiba bays (Tables 1 and 2). When such values
were not available, the values determined in standard shale (Müller, 1979) were used and a
factor of 1.5 was applied to account for regional differences. Fe, Al and Mn are generally
abundant metals in tropical soils and, due to this reason, the IGEO is not applicable to such
elements. Vanadium (V) is not a metal, a thus the application of IGEO is also unfeasible.
Because of that, Fe, Al, Mn and V backgrounds are not mentioned in the Table 1. The IGEO
can be calculated applying the following equation, where Me means metal concentration
obtained in the field, while NBNME means the background value for a specific metal:

IGEO=Log2 Me / NBNMe x 1.5 (1)

Statistical Analysis

Earthworm weight change and mortality found in the test mixtures of soil and sediment
were compared to that in the respective controls (uncontaminated soils - 0%) by one-way

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analysis of variance (ANOVA) followed by Dunnett’s post hoc test. The number of surviving
earthworms in the acute toxicity tests was used to calculate the earthworm median lethal
concentration (LC50) through Probit analysis (using the software PriProbit 1.63; Sakuma,
1998).

Table 1. Background metal concentrations obtained in sediment cores from Sepetiba

bay (Gomes et al., 2009), Guanabara bay (Monteiro et al., 2012)
and in the standard shale (Turekian and Wedepohl, 1961).
*=metal concentration obtained in the standard shale

Metal Sepetiba bay (mg/kg) Guanabara bay (mg/kg)

Cd 0.34 0.30*
Cr 90* 90*
Cu 8.04 2
Ni 8.32 60
Pb 20 15
Zn 54 6.1

Table 2. Geoaccumulation Indexes (IGEO) of heavy metals in sediments of theRhine

River(Germany). Adapted from Müller (1979)

Metal accumulation in the

Intensity of pollution IGEO class
sediment (IGEO)
Very strongly polluted >5 6
Strongly to very strongly polluted >4-5 5
Strongly polluted >3-4 4
Moderate to strongly polluted >2-3 3
Moderately polluted >1-2 2
Low to moderately polluted >0-1 1
Practically non-polluted <0 0

RESULTS
Dredged Sediment and Test Mixtures

The organic matter contents measured in the dredged sediments were 1.80, 4.90 and
0.93%, for the month of Saco do Engenho River (Sepetiba bay), Meriti River (Guanabara
bay) and São FranciscoRiver (Sepetiba bay), respectively (Table 3). According to the current
Brazilian legislation (CONAMA, 2004), the only sediment whose Cd concentration was
above the second level (high probability of adverse effects on biota) was the Saco de
Engenho. On the other hand,Cd concentrations were lower than the detection limits for the
other sediments. In the Saco do Engenho River, Zn, Cd and Pb concentrations were extremely
high. In the Meriti River, all heavy metal concentrations were above maximum values
established by the Brazilian law (generally higher than the first level), except Cd. On the
other hand, in the São Francisco River, Zn was the only metal whose concentration was above
the Brazilian threshold concentration (higher than the first level).

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The estimated IGEOs indicated absence of Cr pollution (class 0) for all tested sediments
(Table 3). In the Saco do Engenho River, the sediment was “strongly to very strongly”
polluted by Cd and Zn (class 5), while the Ni pollution was classified as “moderate to
strongly polluted” (class 3). The sediment of the Meriti River was “very strongly polluted”
(class 6) by Zn and Cu, and “moderate to strongly polluted” (class 3) by Pb. The sediment
from the São Francisco River showed the lowest values of IGEO classes, and the highest one
was observed for Zn (class 2; “low to moderately” polluted) (Table 3).
For the sediments from Saco do Engenho River and São Francisco River, the pH of the
test mixtures increased with increasing sediment dosage in soil (Tables 4 and 5). However,
water holding capacity (WHC) decreased for high dosages of sediment application. For the
Meriti River, the pH decreased with the increasing doses of sediment, while the WHC
increased with the increasing sediment dose in soil (Table 6).

Table 3. Metal concentrations (mg/kg), organic matter content (OM; %) and IGEO
classes obtained for the dredged sediments collected in Sepetiba and Guanabara bays

CONAMA 454 Saco do Engenho Meriti River São Francisco River

(Brazilian law) River (Sepetiba Bay) (Guanabara Bay) (Sepetiba Bay)
Metal
Level
Level IIb mg/kg IGEO mg/kg IGEO mg/kg IGEO
Ia
Cd 1.2 7.2 7.2** 5 <0.1 0 <0.1 0
Cr 81 370 133* 0 111* 0 43.5 0
Cu 34 270 15 1 111* 6 11.1 1
Ni 20.9 51.6 58.4** 3 42.9* 0 13 1
Pb 46.7 278 36.8 1 90.9* 3 30.2 1
V - - <0.2 - <0.2 - <0.2 -
Zn 150 410 1200** 5 926** 6 206* 2
OM (%) - - 1.8 - 4.9 - 0.93 -
Al (%) - - 8.2 - 9.4 - 8.2 -
Fe (%) - - 4.9 - 3.9 - 3.4 -
Mn (%) - - 275 - 196 - 290 -
a – limit of low probability of adverse effects on biota; b – limit of high probability of adverse effects
on biota (CONAMA 2012). */**=value higher than CONAMA level I/II (CONAMA 454),
respectively. CONAMA 454: threshold values established by Brazilian legislation for disposal of
dredged sediments.

Metal concentrations measured in the test mixtures were lower than the intervention
limits (the limit of occurrence of adverse effects on human health considering agricultural
land uses) defined by Brazilian law (CETESB, 2005; CONAMA, 2012) for all the sediments
(Tables 4, 5 and 6). In the Saco do Engenho River, the concentrations of Zn and Cd were
higher than the prevention limits for ecological receptors in some test mixtures (18, 24 and
30%) (Table 4). In fact, the concentrations of Zn and Cd were always higher than the
reference limits (CETESB, 2005; soil geochemical background for São Paulo state) in all test
mixtures (Table 4). In the Meriti and São Francisco Rivers, metal concentrations determined
in the test mixtures were generally lower than the prevention limits (Table 5). The exception
was Zn for the highest dose of sediment application from the Meriti River. In this respect, in
the Meriti River the concentrations of Zn and Pb were also always higher than the reference
limits for all test mixtures, while in the São FranciscoRiver such concentrations were higher
than the reference limits only in some test mixtures (Table 6).

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Earthworm Acute Toxicity Test

The validity criterion of the acute earthworm test was fulfilled since no mortality was
found in replicates with pure artificial soil (0%). The mortality observed in the mixtures of
artificial soil and sediments from Saco do Engenho River and Meriti River showed dose-
response relationships for treatments of both sediments (Figure 2). No mortality was found
for the treatments using the sediment from the São Francisco River. Mortality found in
mixtures with the sediment from Saco do Engenho River and Meriti River was significantly
higher than that in the control for concentrations ≥15% and 18%, respectively (Figure 2). The
estimated values of LC50 (and 95% confidence interval) were 16.26% (15.72-16.78) and
17.46% (16.71-17.68), for the Saco de Engenho and Meriti River, respectively. The biomass
loss was higher in mixtures with the sediment from Saco do Engenho River compared to the
other sediments. The organisms increased their biomass levels at low doses of sediment
application from Meriti River (6 and 12%), followed by a biomass reduction at higher doses
(18%). The organisms exposed to mixtures with the sediment from São Francisco River
significantly increased their biomass levels (Figure 2).

Figure 2. Mortality (diamonds) and biomass change (columns) (average % ± standard deviation; n=4) of
Eisenia andrei exposed to mixtures of artificial soil with dredged sediments from the mouth of Saco do
Engenho River (Sepetiba bay), Meriti River (Guanabara bay) and São Francisco River (Sepetiba bay). *
Biomass change significantly different from control (p≤0.05). + Mortality significantly different from control
(p≤0.05).

Earthworm Bioconcentration Factors

Cr, Cd and Pb concentrations measured in the earthworms exposed to the test mixtures
were the lowest among the measured metals and their BCF values were <0.3 for mixtures of
both test soils (Table 7). The earthworm Pb concentrations were lower than the Pb detection
limits in all test mixtures. BCF values of Ni were also <1 in mixtures with the sediments from
Meriti and São Francisco, while such values were >1 for some test mixtures with the
sediment collected in the Saco do Engenho River. BCF values of Cu were generally <1,
except for the 30% treatment with the sediment from São Francisco River, whose values
increased with the increased dredged sediment dose (Table 7). On the other hand, Cu
concentrations and its BCF values decreased with the increasing sediment dose from Saco do

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 Ecotoxicological Assessment of Dredged Sediments … 317

Engenho River. Earthworm Zn concentrations were the highest among the measured metals
with BCFs >1 for the all sediments studied, decreasing with the increased dredged sediment
dose for the sediments collected in the Meriti River and Saco do Engenho River. The
treatments with the sediment from São Francisco increased their BCF values of Zn with
increased dredged sediment dose (Table 7).

Table 4. Water holding capacity (WHC), pH and metal

concentrations (mg/kg) determined in mixtures of artificial
soil with dredged sediments from Saco do Engenho River
(Sepetiba bay, Rio de Janeiro). 0%: pure artificial soil

Doses WHC
pH Cr Cu Ni Pb Zn Cd
(%) (%)
0 84.2 5.74 7.20 - 2.90 13.00 48.60 0.100
6 82.4 5.88 14.75 2.05 6.23 14.43 117.68* 0.526*
12 78.8 6.37 22.30 3.10 9.56 15.86 186.77* 0.952*
15 82.0 6.72 26.07 3.62 11.23 16.57 221.31* 1.165*
18 79.4 6.73 29.84 4.14 15.62* 18.45* 312.50** 1.378**
24 76.1 6.46 37.39 5.19 16.22* 18.71* 324.94** 1.804**
30 73.9 7.01 44.94* 6.24 19.55* 20.14* 394.02** 2.230**
Reference* 40 35 13 17 60 <0.5
Prevention** 75 60 30 72 300 1.3
Intervention*** 150 200 70 180 450 3.0
* – pedogeochemical background levels of the São Paulo state, Brazil (CETESB, 2005); ** – limit of
occurrence of adverse effects on soil ecological receptors; *** – limit of occurrence of adverse effects
on human health (CETESB, 2005; CONAMA, 2009).

Table 5. Water holding capacity (WHC), pH and metal

concentrations (mg/kg) determined in mixtures of artificial
soil with dredged sediments from São Francisco River
(Sepetiba Bay, Rio de Janeiro). 0%: pure artificial soil

Doses (%) WHC (%) pH Cr Cu Ni Pb Zn

0 75.7 5.64 7.20 6.80 2.90 13.00 48.60
6 79.2 5.67 9.38 7.06 3.51 14.03 58.04
12 74.5 5.80 11.56 7.32 4.11 15.06 67.49*
18 76.0 6.00 13.73 7.57 5.21 16.94 84.68*
24 73.5 6.04 15.91 7.83 5.32 17.13* 86.38*
30 72.9 6.11 18.09 8.09 5.93 18.16* 95.82*
Reference* 40 35 13 17 60
Prevention** 75 60 30 72 300
Intervention*** 150 200 70 180 450
* – values representing pedogeochemical background of the São Paulo State, Brazil (CETESB, 2005); ** –
represent the limit of occurrence of adverse effects on soil ecological receptors; and *** – represent the
limit of occurrence of adverse effects on human health (CETESB, 2005; CONAMA, 2009).

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Table 6. Water holding capacity (WHC), pH and metal concentrations (mg/kg)

determined in mixtures of artificial soil with dredged sediments from Meriti River
(Guanabara bay, Rio de Janeiro). 0%: pure artificial soil

WHC
Doses (%) pH Cr Cu Ni Pb Zn
(%)
0 74.8 5.85 7.20 6.80 2.90 13.00 48.60
6 83.3 5.13 13.43 13.05 5.30 17.67* 101.24*
12 85.1 4.87 19.66 19.30 7.70 22.35* 153.89*
18 89.0 4.93 25.88 25.56 12.07 30.85* 249.70*
24 92.1 4.96 32.11 31.81 12.50 31.70* 259.18*
30 91.1 4.60 38.34 38.06* 14.90* 36.37* 311.82**
Reference* 40 35 13 17 60
Prevention** 75 60 30 72 300
Intervention*** 150 200 70 180 450
* – values representing pedogeochemical background of the São Paulo State, Brazil (CETESB, 2005);
** – represent the limit of occurrence of adverse effects on soil ecological receptors; and *** –
represent the limit of occurrence of adverse effects on human health (CETESB, 2005; CONAMA,
2009).

DISCUSSION
The mortality and biomass loss observed in the acute earthworm tests agree with the
results of the metal determination. The highest metal concentrations and the highest toxicity
(expressed in LC50 values) were detected for the sediment collected in the Saco do Engenho
River, followed by Meriti River. The highest organic matter content in the sediment from
Meriti River (4.9%) was an important aspect to toxicity, since organic matter plays an
important role in the reduction of metal mobility and bioavailability by decreasing metal
concentration in pore water (Vijver et al., 2005; Lukkari et al., 2006). This fact may also
explain the lower toxicity levels found for the sediment from Meriti River compared to the
sediment from Saco do Engenho River. Last but not least, metal contents measured in the
sediment collected in the São Francisco River were the lowest among the sediments studied
and their low IGEO classes also support the absence of earthworm mortality and of biomass
loss found in all the treatments.
Metal concentrations in the pure sediments were generally higher than the limits for the
first level of contamination (low probability of adverse effects on biota), and some metals
were even higher than the limits established for a high probability of adverse effects on biota
(CONAMA, 2004), especially the sediments from Saco do Engenho River and Meriti River.
Those levels of contamination were confirmed by the toxicity found in the bioassays.

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 Table 7. Bioconcentration factors (BCF) measured in three composite samples (average, n=4) obtained from the surviving
earthworms (40 individuals in treatments without mortality) after 14 d of exposure in mixtures of dredged sediment with
artificial soil. SE: Saco do Engenho River (Sepetiba Bay); ME: Meriti River (Guanabara Bay); SF: São Francisco River
(Sepetiba Bay); UD: unavailable data. BCF of Cd was not calculated for MR and SFR because the concentrations in the
sediment were lower than the detection limits

Dredged Cr Cu Ni Pb Zn Cd
sediment
SE MR SF SE MR SF SE MR SF SE MR SF SE MR SF SE
dose (%)
0 0.24 0.03 0.03 0.79 0.37 0.37 0.05 0.10 0.10 0.15 0.15 0.02 1.58 1.87 1.89 0.10
6 0.12 0.10 0.02 0.75 0.38 0.38 0.03 0.06 0.09 0.14 0.11 0.01 0.51 0.92 1.72 0.19
12 0.22 0.20 0.02 0.67 0.36 0.40 2.30 0.04 0.07 0.13 0.09 0.01 1.09 1.04 1.52 0.11
15 0.20 UD UD 0.51 UD UD 1.30 UD UD 0.12 UD UD 0.87 UD UD 0.09
18 0.19 0.25 0.02 0.48 0.33 0.55 1.24 0.22 0.06 0.11 0.09 0.01 0.25 0.79 1.66 0.07
24 UD UD 0.03 UD UD 0.51 UD UD 0.08 UD UD 0.01 UD UD 1.94 UD
30 UD UD 0.02 UD UD 1.00 UD UD 0.28 UD UD 0.01 UD UD 1.68 UD

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Zn and Cd determination in test mixtures with the sediments from Saco do Engenho
River and Meriti River revealed that only the respective doses of ≥18 and 30% were higher
than prevention limits (for ecological risk). However, significant mortality levels and biomass
loss were found for lower dosages of sediment application onto soil. Therefore, toxicity data
are not in agreement with the fact that metal concentrations in the test mixtures were lower
than metal limits established by CETESB (2005) and CONAMA (2009) for soil quality. In
this respect, other tests with tropical local soils are highly recommended to confirm such
results, since soil properties play a crucial role in the toxicity and bioavailability of heavy
metals.
Cesar et al. (2015a), when performing chronic bioassays with E. andrei using mixtures of
artificial soils and dredged sediments from Cunha Channel (Guanabara bay), foundmuch
lower toxicity (e.g., no mortality for 20% dose) compared to the sediments collected in the
Saco do Engenho River and Meriti River. Cesar et al. (2013), when studying the ecotoxicity
of dredged sediments from rivers highly contaminated by domestic wastes in Belford Roxo
Municipality (Guanabara Bay basin, RJ), also found much lower toxicity levels, since even
the pure sediment did not cause significant mortality in acute bioassays with E. andrei. Such
observations highlight the high toxicity of the sediments collected in the Saco do Engenho
River and Meriti River, even compared to other dredged sediments from RJ state.
The bioassay with the sediment collected in the Saco do Engenho River and Meriti River
showed that the organisms increased their biomass when exposed to low doses of dredged
sediment (6 and 12%), while the earthworms exposed to the mixtures with the sediment from
São Francisco increased their biomass in all test mixtures. The organic matter content found
in the sediment of the Meriti River (4.9%; mainly from domestic wastes) was high and
suggests that the organisms apparently recognized it as potential source of food. Carbonell et
al. (2009) and Cesar et al. (2012, 2014), when performing bioassays with E. andrei using
residues containing high organic matter contents from domestic wastes, also detected an
increase of earthworm biomass. However, the organic matter concentrations in the other
sediments (from Saco do Engenho River and São Francisco River) are low (<1%) and do not
explain such increase of biomass, especially in the test mixtures with the sediment from the
São Francisco River. On the other hand, it is important to note that these aquatic systems are
often impacted by domestic wastes, which usually contain high concentrations of hormones
(whose concentrations were not quantified for this chapter). Some of these compounds are
endocrine disruptors and can induce earthworm biomass changes. Cesar et al. (2015a) studied
the ecotoxicity of mixtures of an artificial soil with dredged sediment from Cunha Channel
(Guanabara Bay) using chronic bioassays with E. andrei, and suggested that endocrine
disrupters could play an important role in the biomass changes.
The increase of pH with the increasing dose of sediment from Saco do Engenho River
and São Francisco River is most probably due to the presence of carbonated minerals in the
sediment, that are common in estuarine sediments. On the other hand, an opposite behavior
was detected for the sediment from Meriti River, i.e., reduction of pH and increase of WHC
values with the increasing dose of sediment. Meriti River sediment is much more impacted by
domestic wastes compared to the other sediments, which explains its high organic matter
content (4.9%) and the decrease of pH due to anaerobic conditions. It is thus important to
highlight that the variations of pH may influence the oxidation and geochemical availability
of metals in the environment (Peijnenburg and Jager, 2003).

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For Ni, while the BCFs estimated with sediments from Meriti and São Francisco Rivers
were <0.3, the BCFs with the sediment from Saco do Engenho River were >1 (12, 15 and
18% doses). This fact evidences that Ni uptake happened only in mixtures with the sediment
from Saco do Engenho River, which agrees with the respective LC50 that is lower than that
estimated for the other sediments. On the other hand, the BCFs found for Zn and Cu at higher
doses of sediment reflect the constant Zn and Cu concentration in earthworms and no
tendency of increased BCF with increasing Zn concentration in soil, except for Cu with
sediment from São Francisco, where the BCFs increased with the increasing sediment dose.
This is possibly due to the ability of the earthworms to internally regulate these metals, since
these elements are essential for their physiology (Lukkari et al., 2005). In this respect, Zn
plays an important role in the cell metabolism, and in the development, growth and
regeneration of some tissues, while Cu participates in the transport of substances among cells
and tissues (Lukkari et al., 2005).
Pb, Cr and Cd are highly toxic metals and well-known for their ability to cause serious
noxious effects on biota, even at low concentrations (Katz et al., 1993, Paoliello et al., 2002).
Given the fact that the earthworm BCFs found for Pb and Cd in the present study were low,
apparently those metals were not highly available to the test organisms, not occurring in
bioaccumulable forms (e.g., ionic forms and/or bound to geochemical supports).

CONCLUSION
The bioassays performed were appropriate for a preliminary assessment of the acute
damages to soil biota associated with the disposal of dredged sediment in soil. The most toxic
sediment was the one collected in the Saco do Engenho River (Sepetiba bay), followed by
Meriti River (Guanabara Bay) and São FranciscoRiver (Sepetiba Bay). Furthermore, some
toxic metals (e.g., Ni) were highly bioavailable for the earthworms when dredged sediments
were mixed with artificial soil. More ecotoxicological tests (e.g., reproduction tests)
using other test species (e.g., collembolans, predatory mites, etc.) are required to confirm
and further investigate these findings. Also, bioassays using local soils are extremely
recommended, since soil properties play a crucial role in metal and dredged sediment toxicity
(Cesar et al., 2014). The data obtained suggest that the limits established by Brazilian law are
not adequate to prevent the occurrence of adverse effects on soil fauna. The development of
metal threshold values that reflect the variety of Brazilian tropical soils and biomes is also
urgent and will more effectively support decision-makers in actions of environmental control
and soil biodiversity preservation.

ACKNOWLEDGMENTS
The authors would like to thank the colleagues from the Mineral Analysis Group
(COAM) of the Centre for Mineral Technology (CETEM/MCT) for their help in the chemical
determinations. Ricardo Cesar was supported by CAPES (Coordination for the Improvement
of Higher Level -or Education- Personnel – Postdoctoral grant).

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 20

SOIL ECOTOXICOLOGY:
CHILEAN STUDIES WITH SOILS POLLUTED
BY COPPER MINING

Alexander Neaman1,*, Rosanna Ginocchio2 and Carolina Yáñez3

1
Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso,
Quillota, Chile
2
Facultad de Agronomía e Ingeniería Forestal (FAIF) and
Center of Applied Ecology and Sustainability (CAPES),
Pontificia Universidad Católica de Chile, Santiago, Chile
3
Instituto de Biología, Pontificia Universidad Católica de Valparaíso,
Valparaíso, Chile

ABSTRACT
Chile has had a long mining history and is currently considered the most important
copper (Cu) producer in the world. The environmental problems historically associated
with Cu mining are widely known in north and central areas of the country, particularly
in relation to the contamination of agricultural soils by metals and metalloids. Copper is
the dominant contaminant in polluted soils of Cu mining areas in Chile. Arsenic is also
present in polluted soils as it is a common secondary element found in Cu ores. In this
chapter, we discuss the importance of using field-contaminated soils, instead of metal-
spiked soils, in ecotoxicological studies. We also derive thresholds of Cu toxicity to
ryegrass and arsenic toxicity to Eisenia fetida. Finally, we discuss the necessity of future
studies using toxicity tests with microbial properties/processes.

Keywords: copper, arsenic, ryegrass, Eisenia fetida, microorganisms

*
Corresponding Author E-mail: alexander.neaman@pucv.cl.

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326 Alexander Neaman, Rosanna Ginocchio and Carolina Yáñez

SCOPE OF THE CHAPTER

Soil ecotoxicology studies the effects of chemicals on organisms in the soil environment
(van Gestel, 2012). The field of soil ecotoxicology is very wide and includes studies with
different pollutants (organic and inorganic), biological receptors, and soil types. Sensitivity of
organisms to metals is species-dependent (Bierkens et al., 1998) and reactivity of organisms
to metals varies according to soil type (Carporale and Violante, 2015). Therefore, several
ecotoxicity tests need to be performed in order to give a clear view of toxic effects of metals
in soils for organisms (ISO 17402, 2008; ISO 17616, 2008). In this chapter, we limit the
discussion on Chilean studies with soils polluted by Cu mining (inorganic pollutants), and
studies on toxicity tests with ryegrass (plant) and Eisenia fetida (earthworm). We also discuss
the necessity of future studies using toxicity tests with microbial properties/processes.

RELEVANCE OF THE CHAPTER

Chile has had a long mining history and is currently considered the most important
copper producer in the world (Comisión Chilena del Cobre, www.cochilco.cl). The
environmental problems historically associated with copper mining are widely known in
north and central areas of the country, particularly in relation to the contamination of
agricultural soils by metals, such as copper (Cu), zinc (Zn), lead (Pb), and cadmium (Cd), and
metalloids such as arsenic (As; henceforth referred to as “metal” for convenience) (González
et al., 1984; Ginocchio, 2000; De Gregori et al., 2003). Copper is the dominant contaminant
in polluted soils of Cu mining areas in Chile (Goecke et al., 2011). Arsenic is also present in
polluted soils as it is a common secondary element found in Cu ores (O'Neill, 1995). Copper
is an essential micronutrient to all organisms that becomes toxic above a certain threshold,
while As is non-essential and potentially toxic for all organisms, particularly for animals
(McBride, 1994; Adriano, 2001).
Currently, Chile does not have any legislation on the maximum permissible
concentrations of metals in soils nor regulations to protect soil quality. However, the
government has developed in the last decades a national policy for evaluating how likely is
that the human health and the environment may be impacted in suspected contaminated sites,
as a result of exposure to environmental stressors such as metals (CONAMA, 2009). The
main emphasis of the policy was human health protection but ecological risk assessment was
afterwards incorporated for crop and wildlife protection. In this context and for the proper
implementation of the policy, we consider that it is important to distinguish between soils
where metals are present but do not represent an ecological risk (i.e., high total metal
concentrations but low metal bioavailability) from those that, at similar total metal
concentrations, do represent significant ecological risks (i.e., high metal bioavailability). It is
also critical to keep track that background soil metal levels in Chile would be much higher
than elsewhere given the underlying geochemistry of the Andes range (Badilla-Ohlbaum et
al., 2001).
The concept of metal bioavailability has been extensively discussed in the literature
(Ginocchio et al., 2006, 2009; Carporale and Violante, 2015) and will be not developed here.
However, it is relevant to consider that metal bioavailability may be assessed in two

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 Soil Ecotoxicology 327

complementary ways (ISO 17402, 2008): (1) biological methods which expose organisms to
metal enriched soils in order to monitor effects, and (2) chemical extraction methods which
determine different fractions of a contaminant in the soil. These analytically-determined
values need to be correlated with biological effects (Adriano, 2001). If a correlation between
the resulting chemical values and effects has been demonstrated, soil quality criteria might be
obtained (Sauvé et al., 1998).

USE OF METAL-SPIKED SOILS VERSUS

FIELD-CONTAMINATED SOILS
Early versions of the protocols of soil quality tests with plants and earthworms proposed
the use of artificial soils (made of peat, clay, and sand) or natural non-contaminated soils
spiked with solutions of metals at increasing concentrations (ISO 11268-2, 1998; ISO 11269-
2, 2005; OECD 208, 2006; OECD 222, 2004). However, it is well known that soil solubility
of metals, and thus their potential toxicity, is greater in artificially-contaminated soils in
comparison to field-collected soils (Spurgeon and Hopkin, 1995; McBride et al., 2009;
Smolders et al., 2009; Hamels et al., 2014). This difference in solubility is explained by aging
processes in soils, which are very slow and occur during several years (Ma et al., 2006;
Martínez and McBride, 2000). Thus, metal-spiked soils cannot adequately represent real
environmental contamination conditions (Davies et al., 2003). As a result, the latest version of
the protocols of soil quality tests with plants and earthworms (ISO 11268-2, 2012; ISO
11269-2, 2012) also consider field-collected soils, instead of artificially-contaminated soils.
We therefore argue that metal-spiked soils have been relevant for understanding basic
scientific phenomenon but have limited relevance from an environmental point of view.
Accordingly, we emphasize the importance of using field-collected soils for plant- and
animal-based toxicity tests. However, the use of field-collected soils presents several
difficulties. First, in areas near Cu mining activities in Chile, soils have high concentrations of
a mixture of several metals (Cu, Pb, Zn, Cd and As, among others) (De Gregori et al., 2003;
Ginocchio et al., 2004). In this case, it might be difficult to distinguish between the effects
of different metals on organism responses. Second, the intrinsic physical-chemical
characteristics of the soil - such as pH, salinity, texture and organic matter content - are
known to affect metal uptake and toxicity for plants (Adriano, 2001; McBride, 1994; Rooney
et al., 2006) and earthworms (Ma et al., 1983; Spurgeon et al., 2006; Owojori et al., 2008,
2009a). Finally, distribution of earthworm populations is known to be affected by soil organic
matter (Jordan et al., 1999; Leroy et al., 2008; Fonte et al., 2009; Monroy et al., 2011),
salinity (Valckx et al., 2009), texture (Klok et al., 2007; Holmstrup et al., 2011), and pH
(Klok et al., 2007); however, less attention has been paid to the effects of soil chemical and
physical properties on earthworm responses in bioassays. Likewise, differences in soil
nutrient availability and soil chemical and/or physical properties may also affect plant
responses, in addition to metal toxicity (Ginocchio et al., 2009; Verdejo et al., 2016).
Despite these arguments, in this chapter we present evidence that detailed
characterization of soil properties and of metal concentrations in plant/earthworm tissues
would allow discrimination between confounding factors and metal-toxicity factors, and thus
better estimation of metal toxicity thresholds for plants and earthworms.

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USE OF SOILS SPIKED WITH CU-RICH SOLID MINE WASTES

Chemical forms of metal contaminants released to the environment by mining operations
may be very different from metal salts used for soil spiking in standard organism toxicity
tests. In one of our studies (Ginocchio et al., 2006), we determined Cu solubility and
phytotoxicity using soils spiked with different types of Cu-rich solid mine wastes (i.e.,
tailings, slags, smelter dust) and Cu-concentrate. A Cu salt (copper sulfate, CuSO4) was
also included for comparison. We found that mine materials pose different levels of risk to
plants at the same total Cu concentration due to their different chemical/mineralogical
characteristics. The following general tendency for Cu solubility and possible phytotoxicity,
from high to low, was found: CuSO4, smelter dust, acidic tailing sand >> sulfidic Cu ore, Cu
concentrate, smelter slag. This tendency shows Cu salt-based toxicity tests represent the
worst-case scenario and may be only useful for testing acute toxicity effects of Cu in soil
organisms but not exemplifies real environmental effects. It also represents a short-term
contamination event since no aging was considered in the study of Ginocchio et al., (2006).
With regards to long-term contamination, we performed another study (Mondaca et al., 2015)
on Cu solubility and partitioning in two areas polluted with different Cu-rich mine wastes
(smelter dust versus tailings sand). We argued that the type of Cu-rich mine waste might not
have a major influence on soil chemical degradation once it has had a long time to equilibrate
in the field.
Finally, in an additional study of our group (Ginocchio et al., 2009), we spiked soils with
two Cu-rich mine wastes (oxidized tailings and smelter dust) in order to determine the
relevance of confounding factors on metal toxicity, such as secondary soil acidification.
Addition of these mine wastes resulted in soil acidification and thus high concentrations of
soluble Cu and Zn. Neutralization of experimental soil mixtures with buffers resulted in
marked decrease in soluble Cu and Zn, and significantly reduced metal toxicity in barley
(Hordeum vulgare L.). Thus, we stress the importance of considering confounding effects on
derivation of phytotoxicity thresholds when using short-term laboratory tests.

THRESHOLDS OF CU PHYTOTOXICITY
In this chapter, we present results of one of our studies (Verdejo et al., 2015) on
emergence and early growth (21 days) of perennial ryegrass (Lolium perenne L.) in
agricultural soils historically contaminated by mining activities (smelting or metal processing)
in central Chile (Puchuncaví valley and Aconcagua river basin). Ryegrass is recommended by
the ISO and OECD methods for testing toxicity of compounds in soils (ISO 11269-2, 2005;
OECD 208, 2006) and has been used often as a toxicity bioindicator for metals in soils
contaminated by mining activities (Arienzo et al., 2004; Stuckey et al., 2009; Goecke et al.,
2011). When using field-collected soils rather than artificially-contaminated soils for metal
phytotoxicity tests, other soil characteristics relevant for plant growth are also expected to
vary, thus posing confounding effects in test results. In the study of Verdejo et al., (2015),
selected soils showed variation in metal contents and in a range of other soil characteristics,
such as nutrient availability (Table 1). Results of the study demonstrated, however, that
ryegrass responses were unaffected by soil nutrient availability, suggesting that ryegrass is a

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 Soil Ecotoxicology 329

good bioindicator of Cu toxicity in contaminated soils with different nutrient availability. In

contrast, other crops (such as lettuce, maize and tomato) have limited applicability for metal
toxicity assessment in metal-contaminated soils with different nutrient availability, due to
sensitivity of their responses to nutrient deficiencies (Verdejo et al., 2016).
In terms of Cu toxicity, the study found that total Cu content in soils was the best
predictor of ryegrass responses. The effects of Pb, Zn, and As on plant responses were not
significant, suggesting that Cu is a metal of prime concern for plant growth in soils exposed
to Cu mining activities in central Chile. Shoot length of ryegrass was found as a robust
response variable for metal toxicity assessment in selected contaminated soils. Therefore,
effective concentration (EC) values (EC10, EC25 and EC50) of total soil Cu were estimated for
shoot length response variable (Table 2). The EC10, EC25 and EC50 values for Cu shoot
concentrations in ryegrass were also estimated, using shoot length as response variable (Table
2). Derived values are higher than the Cu concentrations considered normal for ryegrass (11
mg kg-1) (Davis and Beckett, 1978). Similarly, the EC10 found in the study (22 mg kg-1) is
very comparable to the lowest observed effect concentration of foliar Cu of 21 mg kg-1
reported for ryegrass by Davis and Beckett (1978).

Table 1. General physicochemical properties (mean and range values; n=27 samples)
of soils historically contaminated by mining activities in central Chile
(Puchuncaví valley and Aconcagua river basin). Based on Verdejo et al. (2015)

Soil characteristic Mean Range

Total metal (mg kg-1) Cu 418 82 - 1295
Pb 46 25 - 97
Zn 160 86 - 345
Soluble metal (mg L-1) Cu 0.22 0.04 - 0.71
As 0.04 0.002 - 0.18
Texture (%) Sand 54 21 - 95
Clay 18 5 - 37
Silt 28 0 - 43
General pH 7.0 5.7 - 7.6
pCu2+ 8.6 6.8 - 9.8
OM (%) 3.1 0.7 - 5.8
EC (dS m-1) 2.6 0.7 - 10
-1
Nutrient availability (mg kg ) N 33 4 - 134
P 48 8 - 123
K 302 78 - 1143
pCu2+: negative logarithm of the activity of free Cu 2+; OM: soil organic matter; EC: electrical
conductivity of saturated paste extract.

In the study of Verdejo et al. (2015), the derived 95% confidence intervals for EC10, EC25
and EC50 values of total soil Cu were rather widespread (Table 2), not allowing a robust
assessment of metal toxicity for agricultural crops, based on total soil Cu concentrations.
Therefore, plant tests might need to be performed for metal toxicity assessment. These results
are in agreement with other studies (i.e., Hamels et al., 2014). Another point that should be
taken into consideration is that the 50% inhibition represents a drastic impact on agricultural

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330 Alexander Neaman, Rosanna Ginocchio and Carolina Yáñez

productivity. Although this might be deemed acceptable for an industrial site, it would
certainly not be acceptable from an agricultural perspective where even a 10% yield reduction
would generate serious financial difficulties.

Table 2. Effective concentration (EC10, EC25 and EC50) of total Cu (mg kg-1) content in
soils and in plant tissues for shoot length in ryegrass; 95% confidence intervals in
parenthesis. Based on Verdejo et al. (2015)

Matrix Shoot length

EC10 EC25 EC50
Soil 327 (94 - 559) 735 (575 - 896) 1144 (874 - 1413)
Tissue 22 (16 - 28) 31 (27 - 35) 39 (32 - 47)

EFFECT OF DIFFERENT CU FRACTIONS ON PLANT RESPONSES

Generally, it is considered that total metal content is not a good indicator of soil metal
toxicity (McBride, 1994; Adriano, 2001) compared to metal soluble fractions (Kabata-
Pendias, 2004; McBride et al., 2009). Furthermore, the free Cu2+ ion is considered the main
bioavailable form of copper in soils and the best indicator of copper phytotoxicity (Oliver et
al., 2004; Sauvé et al., 1998). However, other ions, principally H+, Ca2+ and Mg2+ according
to the Terrestrial Biotic Ligand (Thakali et al., 2006), and soil components, such as organic
matter and dissolved organic carbon (i.e., Ginocchio et al., 2009), compete with Cu2+ and,
therefore, affect its toxicity. Toxicity is correlated only to the fraction of the total biotic ligand
sites occupied by Cu2+. For this reason, soluble copper concentrations and/or free Cu2+
activities in a soil extract are not suitable indexes for Cu phytotoxicity (Zhao et al., 2006) and
Cu uptake by plants (Zhang et al., 2001).
Consistent with these arguments, total Cu concentration was a better predictor of ryegrass
responses (Table 3), in comparison to soluble Cu and activity of free Cu2+ (pCu2+) (Verdejo et
al., 2015). Likewise, the shoot Cu concentration was correlated with total Cu concentration in
soils, while soluble copper and pCu2+ were not significant in explaining shoot Cu
concentration. These findings are consistent with results of another study on Cu uptake
by vegetable crops grown in Chilean metal-polluted soils (Ginocchio et al., 2002). We
demonstrated that Cu concentration in plant tissues depends not only on the availability of
free Cu ions in soil solution but also on other soil Cu pools that supply the element to the soil
solution.

Table 3. Determination coefficients (R2) of regressions between different Cu fractions in

the soil and ryegrass responses (p < 0.05).
Based on Verdejo et al. (2015)

Shoot length Root length Dry shoot mass Dry root mass
CuT CuS pCu2+ CuT CuS pCu2+ CuT CuS pCu2+ CuT CuS pCu2+
0.58 0.24 ns 0.40 0.40 0.33 0.34 ns ns 0.35 ns ns
CuT: total soil Cu concentration; CuS: soluble Cu; pCu 2+: -log[Cu2+]; ns: not significant.

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 Soil Ecotoxicology 331

The findings of our studies are consistent with arguments of Zhang et al. (2001), that
resupply from the solid phase due to local depletion is the dominant controlling process for
Cu uptake by plants. In other words, the process of Cu uptake by roots depends on the
buffering capacity of the soil to resupply pCu2+ (Sauvé, 2002; Zhao et al., 2006). For these
reasons, Cu uptake by ryegrass and Cu toxicity to ryegrass in the study of Verdejo et al.
(2015) depended on the total soil Cu pool that was capable of supplying Cu to the soil
solution at the same time as plant roots locally depleted the ions through active uptake.
The technique of diffusive gradients in thin films (DGT) has shown to be better at
predicting Cu phytotoxicity and Cu uptake by plants, in comparison to soluble copper
concentrations and/or free Cu2+ activities in a soil extract (Zhang et al., 2001; Zhao et al.,
2006). It has been demonstrated that plant bioavailability of Cu in soil depends on Cu
speciation, interactions with protective ions (particularly H+, Ca2+ and Mg2+), and the
resupply from the solid phase. Those studies concluded that the DGT measurement provides
an integrated measurement of both intensity (free activities of Cu2+ and H+) and resupply. One
can argue that the DGT-measured Cu would be a better predictor of plant responses, in
comparison to total soil Cu, but a controversy is still discussed in the literature (i.e., Hamels
et al., 2014). We have not used the DGT technique in our studies yet.

THRESHOLDS OF ARSENIC TOXICITY TO EISENIA FETIDA

As a result of the key-ecological importance of earthworms (Blouin et al., 2013; Edwards
and Bohlen, 1996), they have been adopted as indicator organisms for the assessment of soil
quality (ISO 11268-2, 2012; OECD 222, 2004). These tests use the cosmopolitan earthworm
E. fetida because it can be easily cultivated in the laboratory (OECD 207, 1984; OECD 222,
2004).
In this chapter, we present results by Bustos et al. (2015) on earthworm reproduction of
E. fetida in soils affected by Cu mining activities (smelting or metal processing) in central
Chile (Table 4). Results of this study indicated that As was a metal of prime concern for E.
fetida, while Cu exhibited a secondary effect, contrary to what one could expect in soils
affected by Cu mining activities. Perhaps this difference is due to the fact that the assimilation
(and thus toxicity) of Cu by E. fetida can be regulated by homeostatic mechanisms of Cu
excretion (Spurgeon and Hopkin, 1999). In contrast, no elimination of As by E. fetida has
been reported (Lee and Kim, 2013). As a consequence, the bioconcentration factor for As was
considerably higher than that for Cu (Table 4), which may imply higher toxicity. Lee and
Kim (2013) reported no elimination of As by E. fetida during its exposure to clean soil,
probably due to a formation of As-thiol complexes in earthworm tissues. This absence of an
elimination pattern of As is similar to other non-essential metals (Cd and Pb), while rapid
excretion is known for Cu and other essential metals (Spurgeon and Hopkin, 1999).
We were able to estimate the EC10 [8 (-6-21) mg kg-1], EC25 [14 (7-22) mg kg-1] and EC50
[22 (17-26) mg kg-1] of total soil As for responses of cocoon production. Lee and Kim (2013)
reported that As(III) showed higher toxicity to E. fetida than As(V). In the studied soils of
central Chile, As(V) and As(III) represented 75±12% and 12±6% of the total soil As (Vargas
et al., 2015). Thus, derived thresholds correspond mainly to As(V). Based on our literature
review, we noted a lack of data on thresholds of As toxicity to E. fetida in field-collected

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332 Alexander Neaman, Rosanna Ginocchio and Carolina Yáñez

soils. Thus, findings of the present study provide new data for estimating thresholds of As
toxicity to E. fetida.

Table 4. Bioconcentration factor (BCF) of Cu and As by Eisenia fetida and E. andrei in

field-contaminated soils. Average and range of values are shown

Earthworm pH BCFCu BCFAs Reference

E. fetida 6.97 0.15 3.21 Bustos et al., (2015)a
(5.70–7.57) (0.04–0.40) (1.43–5.87)
E. andrei 6.49 0.43 0.76 Janssen et al.,
(5.26–7.17) (0.15–1.25) (0.12–3.31) (1997)b
a
For soils with electrical conductivity ≤ 0.29 dS m (measured in 1:5 soil:water extract), which
-1

corresponds to EC50 of salt toxicity for E. fetida (Owojori and Reinecke, 2009).
b
For a sub-sample of soil with pH > 5, to avoid direct effects of low pH, as suggested by Janssen et al.,
(1997).

Results of the present study differed from previously-reported thresholds of As toxicity

for E. fetida in artificially-contaminated soils. Specifically, Lee and Kim (2009) reported a
LC50 of 5.9 mg kg-1 for total soil As, in a spiked soil, for an exposure time of 4 weeks (as
used in the present study), while there were no lethal effects observed at this concentration in
the present study. Similarly, Fischer and Koszorus (1992) reported a LC10 of 50 mg kg-1 of
added potassium arsenate, expressed on wet basis (70% of water content). This value is
equivalent to 85 mg kg-1 of potassium arsenate expressed on dry basis, which in turn is
equivalent to 25 mg kg-1 of total soil As expressed on dry basis. However, there were no
lethal effects observed at this concentration in the present study which is based on field-
collected soils and without spiked As. Moreover, the exposure time was of 8 weeks in the
study of Fischer and Koszorus (1992), and the values of lethal concentration increase with
decreasing exposure time (Lee and Kim, 2009). Thus, one would expect a higher value of
LC10 for an exposure of 4 weeks used in the present study, making even greater the
discrepancy between the results of the present study and the findings of Fischer and Koszorus
(1992). Likewise, Lock and Janssen (2002) reported EC50 of 10.8 mg kg-1 for total soil As,
using cocoon production as a response variable, while a considerably higher value (22 mg kg-
1) was found in the present study for the same response variable.

Based on a direct determination by electron probe microanalysis (Ávila et al., 2007), iron
oxides and Cu sulfides are the As-bearing phases in agricultural soils affected by mining
activities in the Aconcagua river basin, central Chile. On the other hand, potassium/sodium
arsenate was added in the above-mentioned studies with artificially-contaminated soils. Thus,
the discrepancy between the previously-reported thresholds of As toxicity to E. fetida in
artificially-contaminated soils with those reported in the present study could be explained
by differences in solubility of As-bearing phases. Findings of the present study indicate
that metal-spiked soils cannot adequately represent real environmental conditions and,
consequently, have limited relevance from an environmental point of view (i.e., Hamels
et al., 2014).
We were also able to estimate the EC10 [38 (24-53) mg kg-1], EC25 [47 (38-56) mg kg-1]
and EC50 [57 (51-62) mg kg-1] of earthworm tissue As for cocoon production. Thresholds of
earthworm tissue As obtained in the present study are consistent with previously-reported

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thresholds of tissue As in E. fetida: least-observed effect concentrations of 18 mg kg-1 (Lock

and Janssen, 2002), non-lethal concentrations of 80 mg kg-1 (Leduc et al., 2008) and near
the lethal concentrations of 900 mg kg-1 (Fischer and Koszorus, 1992). Likewise, we were
able to determine a no-observed effect As concentration in tissue As of E. fetida of 24 mg kg-1
(95% confidence interval of -2–49 mg kg-1). The previously-reported no-observed effect
concentration of 10 mg kg-1 (Lock and Janssen, 2002) is within the 95% confidence interval
of our study. Thus, findings of the study of Bustos et al., (2015) provide new data for
establishing thresholds of As bioaccumulation by E. fetida.
In the study of Bustos et al., (2015), soil electrical conductivity was another toxicity
factor, besides metal toxicity. These results are consistent with other reports of reproduction
sensitivity of E. fetida to salts (Owojori and Reinecke, 2009; Owojori et al., 2009b). Multiple
regressions revealed that the interaction between As and soil electrical conductivity was not
statistically significant (p > 0.05), thus suggesting that these two toxicity factors are
independent of each other. Thus, the challenge of interpreting results of the study of Bustos et
al., (2015) was in discernment between metal and salt toxicity factors. Therefore, the
earthworm reproduction test might have a limited applicability in soils with high electrical
conductivity because salinity-induced toxicity will hinder the interpretation of results. In
order to isolate the effect of soil salinity on earthworm reproduction, one could consider only
soils with electrical conductivity ≤ 0.29 dS m-1 (measured in 1:5 soil:water extract), which
corresponds to the reported EC50 of salt toxicity for E. fetida (Owojori and Reinecke, 2009).

NECESSITY OF FUTURE STUDIES WITH MICROBIAL

PROPERTIES/PROCESSES
Soil microbial properties are being increasingly used as indicators of soil quality since
they provide a direct measure of soil functioning (Garbisu et al., 2011). Also, there is strong
evidence that soil microbes are more sensitive to metals than animals or plants (Sauvé et al.,
1998; Giller et al., 1999).
We are currently comparing the sensitivity of different microbial properties/processes to
metals in Chilean agricultural soils polluted with metals. We will determine the most suitable
microbial properties/processes to monitor the quality of metal-polluted soils. We will also
determine threshold limits of metal concentrations corresponding to X% of inhibition of
biological response (i.e., microbial number and biomass, microbial diversity, nitrification rate,
nitrogen mineralization rate, and soil bacterial diversity).

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 21

INTER-LABORATORY CALIBRATION OF
THE OSTRACODTOXKIT F ASSAY USING
THE CRUSTACEAN HETEROCYPRIS INCONGRUENS
ON PAH-CONTAMINATED SOILS

Belgis Chial Z.1,2,, Guido Persoone2, Doris Hirmann3

and Andreas P. Loibner3
1
Departamento de Biología Marina y Limnología. Universidad de Panamá,
Ciudad Panamá, Panamá
2
Laboratory of Environmental Toxicology and Aquatic Ecology.
Ghent University, Ghent, Belgium
3
Department of Environmental Biotechnology, Institute for Agrobiotechnology,
Tulln, Austria

ABSTRACT
A short inter-laboratory calibration exercise was conducted by the Laboratory of
Environmental Toxicology and Aquatic Ecology (LETAE), Ghent University with the
participation of the Department of Marine Biology and Limnology, University of Panama
and the Institute for Agrobiotechnology in Tulln, Austria. The purpose was to identify the
precision and repeatability of the Ostracodtoxkit F assay using the species Heterocypris
incongruens, when PAH-contaminated soils have to be assessed. Six soil samples were
collected from a known site polluted with polyaromatic hydrocarbons (PAHs) in Austria
and were packaged, homogenized and shipped to each laboratory. In each laboratory the
Ostracodtoxkit F assay was performed and an additional bioassay named Lumistox
inhibition assay based on bacteria was performed in Austria. Chemical analysis in the
soils were also performed in order to determine the concentration for 13 PAHs. All
laboratories reached a successful hatching of ostracod cysts as well as successful validity
conditions of the Ostracodtoxkit F assay. The outcome for the lethality of the Ostracods
exercise showed that the intra-laboratory precision (coefficient of variation) varied


Corresponding Author E-mail: belgis.chial@gmail.com.

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340 Belgis Chial Z., Guido Persoone, Doris Hirmann et al.

between 0 and 19% and the inter-laboratory precision between 0 and 11%.
Reproducibility was attained when both laboratories found similar results for mortality
(r=0.98; p<0.05). The Ostracodtoxkit assay showed to be sensitive to soil contaminated
with PHAs, and in particular to acenaphthene according to the statistical correlation
(r=0.82; p=0.046). These results were also found at a similar statistical significance with
the second bioassay, the Lumistox assay.

Keywords: maintenance free tests; ostracod Heterocypris incongruens; soil; Lumistox;

polyaromatic hydrocarbons

INTRODUCTION
The solid phase sediment tests with aquatic invertebrates are complex, time-consuming
and costly. The search for a user-friendly alternative in the Laboratory of Environmental
Toxicology and Aquatic Ecology (LETAE, Belgium) eventually led to the development of a
new “cyst-based” free microbiotest (independent of the year-round culture costs) with the
ostracod Heterocypris incongruens. The new 6 days microbiotest is based on two endpoints:
mortality and growth inhibition. The measurement of sublethal effects is particularly useful in
samples in which no mortality is detected.
A first study was hence undertaken on 16 soil samples obtained from former zinc
smelters in Flanders, Belgium and from sites on which contaminated river sludges had been
spread. The sensitivity of the 6 days H. incongruens microbiotest (Chial and Persoone, 2002)
was compared to the 28 days reproduction inhibition assay with the springtail Folsomia
candida (Lock and Janssen, 2001). The outcome of the investigations revealed that for most
samples the ostracod assays were substantially more sensitive than the springtail tests.
Leachates were subsequently prepared with the same soils to determine to what extent the
detected toxicity was due to the “soluble” compounds that partitioned to the leachates or to
the chemicals that remained absorbed to the soil particles after leaching. The ostracod tests
performed on both the solid and liquid fractions indicated that the mortality of the organisms
was still very high in leached-soil contact tests, whereas it was low in most leachates (Chial
and Persoone, 2002). The latter findings clearly confirm the necessity of direct contact tests
for contaminated soils, as has already been demonstrated for sediments (Burton et al., 1996;
Vangheluwe et al., 2000).
This chapter reports on a short laboratory intercalibration exercise carried out by LETAE
in Ghent Univesity and the Institute for Agrobiotechnology in Tulln, Austria, with the
Ostracodtoxkit F assay on PAH-contaminated soils. Finally, the sensitivity of this assay was
compared with the acute Lumistox luminescent bacteria test.

METHODS
The soil used was excavated from a historically PAH contaminated site, a former gas
plant, in the east of Vienna, Austria. The contamination was determined for five different soil
samples coded as WGalpha, WGbeta, BZ 10, TA 14-16 and TA 4-6. The soil samples were
collected, homogenized and packaged. One part of the sample was tested in Austria and the

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 Inter-Laboratory Calibration of the Ostracodtoxkit … 341

other in Belgium. The inter-calibration exercise consisted of undertaking a whole

soil bioassay with the Ostracodtoxkit using the specie H. incongruens on this soil
contaminated with PAHs in both mentioned laboratories. The test was run twice for each soil
sample in each country. The second inter-calibration exercise, run in Austria was performed 2
months after the first one due to the lack of material. In addition to the Ostracodtoxkit
bioassay, a second available bioassay named Lumistox inhibition assay was performed
in Austria without repetition. The concentrations of acenaphthene, fluorene, phenanthrene,
anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene,
benzo(k)fluoranthene, benzo(a)pyrene, benzo(g,h,I) perylene and indeno(1,2,3-c,d)pyrene
PAH known to be present, were determined in the soil.

Ostracodtoxkit Bioassay

The 6-d solid phase ostracod microbiotests with H. incongruens were applied in
accordance with the methodology described in Chial and Persoone (2002), which is the basis
for the Standard Operational Procedure of the ostracod microbiotest (Ostracodtoxkit FTM,
2001). The method consists of adding freshly hatched ostracod neonates to multiwell plates
(10 organisms per test well in 6 replicates) with 2 ml synthetic freshwater (US EPA, 1991),
300 µL soil and 3x107 algal cells (Raphidocelis subcapitata renamed Pseudokirchneriella
subcapitata) as complementary food. Calibrated reference sand is used as a control sediment
(Ostracodtoxkit FTM, 2001).
The 300 µL soil samples were obtained as follows: in a plastic test tube filled with the
sediment sample wetted previously with synthetic freshwater, a large-mouth syringe without
the plunger was inserted and pushed until reaching the bottom. The syringe was pulled out
with the sample and then the plunger inserted. The plunger was then pushed down until it
reached the sediment core in the syringe tube, and the sediment core was compacted. For
very smooth sediments, the syringe had to use the plunger to suck up the sediment by pulling
the plunger. Finally, 300 µL was dispensed into each testing well to expose the ostracods.
The same procedure was repeated for the replicates of the reference sediments. A reference
soil, Ifa-acker from Austria (Department of Environmental Biotechnology, Institute for
agrobiotechnology, Konrad Lorenz Str. 20, A-3430 Tulln, Austria) was also tested. This
second reference soil was used in order to identify other possible sources of control sediment.
After 6 days incubation at 25 °C in darkness, the ostracods were retrieved from the
substrate (soil in this case) and the mean percentages of mortality and growth inhibition were
determined. The percentage of growth inhibition in the test samples is calculated with the
formula: % growth inhibition=100 – (Linc test sed/Linc control sed) x 100, in which “Linc
test sed” is the mean growth (length increment) of the organisms in the test sample after 6
days (i.e., their length at the end minus the length at the start, in µm), and “Linc control sed”
the mean growth of the organisms in the control sediment (calibrated reference sand) after 6
days.
The validity criterion of the ostracod assays is a minimum survival of 80% and a
minimum length increment of 400 µm of the test organisms in the control sediment.

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Lumistox Inhibition Assay

The Lumistox test was conducted with the soil elutriates and according to the manual
procedure using the Luminometer, reagents and Aliivibrio fischeri (formerly known as Vibrio
fischeri) luminescent bacteria (DANETV Test Environmental Technology Verification
Program, 2011).

Chemical Analysis of PAHs

To perform the PHA analysis, an elutriate from each soil was obtained (soil:distilled
water=1:3.5). Liquid chromatography was performed with an HP 1050LC (Hewlett-Packard,
USA) equipped with an HP1100 series three-dimensional fluorescence detector. Injection
volume was set to 20 µl for standards and samples on a 1050 HP autosampler. For separation,
an ODS Hypersil guard column (20x4 mm, particle size 5 µm) followed by a C/18 Grace
Vydac separation column (250x4.6 mm, particle size 5 µm) was used. The column was heated
at 26 °C and operated with an eluent flow rate of 1.5 mL/min. The eluent gradient profile was
set up as follows: 50% acetonitrile (ACN)/50% Milli-Q- water for 2.5 min, followed by a
linear gradient of 9.5 min up to 90% acetonitrile and a linear gradient of 8 min up to 100%
acetonitrile, held for 2.5 min. Subsequently, a linear gradient (2.5 min) was set back to initial
conditions for column conditioning before the next run. The 3d.FLD was operated at four
different excitation/emissions wavelength pairs: excitation was set to 260 nm, emission
wavelengths were 350, 420, 440 and 500 nm, respectively.

Data Treatment

In order to compare the data obtained by both laboratories for each soil sample, the mean
percentages of mortality and inhibition growth were calculated. The intra and inter-laboratory
variability (precision) was estimated by means of the coefficient of variation (CV).
The degree of correspondence between the assays and the PAHs was subsequently
determined by general Pearson pairwise product-moment correlations between percentages
of mortality and growth inhibition, and the concentration of the PAHs to estimate the
reproducibility of the test regarding the level of significance.

RESULTS
In both laboratories the ostracods hatched properly from cysts within 52 h and the
validity conditions of the Ostracodtoxkit F assay were reached (control mortality <20% and a
minimum mean growth increment of 400 µm).
Table 1 displays the concentration of PAHs, mortalities and growth inhibition for each
soil sample. The PAHs determined in elutriates, displaying a differential concentration of the
PAHs where the maximum amount is observed in the soil WG alpha. However, acenaphthene

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 Inter-Laboratory Calibration of the Ostracodtoxkit … 343

increased accordingly with the known contamination of the soil samples: WG alpha=WG beta
< BZ 10 < TA 14-16 < TA 4-6.
The two Ostracodtoxkit F assay repetitions and the Lumistox assay were performed with
each soil sample in both laboratories (Belgium and Austria) showed that the Ostracodtoxkit F
as well as the Lumistox assay detected three soil samples (BZ 10, TA 4-6 and TA 14-16)
presenting a high toxicity range for mortality and inhibition endpoint (between 91 and 100%)
among the laboratories. The coefficient of variation of the studied endpoints was 0% for all
Ostracodtoxkit results, while it was between 2.6 and 3.7% for acute Lumistox and all other
Ostracodtoxkit results. The mortalities detected in the other soils (Ifa-acker, WG beta and
WG alpha) were in the range considered non toxic (11 to 19%), with the exception of the soil
sample WG alpha when tested with the Ostracod assay in Austria, in which case the mortality
was 32%.

Table 1. Concentration of poliaromatics hydrocarbons (PAHs) in soils coded and results

of the bioassays obtained in each country with the Lumistox acute
and Ostracodtoxkit test

Soils
Reference
PAHs
WG alpha WG beta BZ10 TA 14-16 TA 4-6 soil (Ifa-
acker)
Acenaphthene (2 rings) 0 0 16 54 27 0
Fluorene (3 rings) 51 10 20 70 29 0
Phenanthrene (3 rings) 219 27 40 150 55 0
Anthracene (3 rings) 171 12 10 39 36 0
Fluoranthene (4 rings) 909 74 8 42 54 0
Pyrene (4 rings) 771 78 4 29 31 0
Benzo(a)anthracene (4 rings) 408 34 1 5 9 0
Chrysene (4 rings) 342 31 1 6 9 0
Benzo(b)fluoranthene (5 rings) 281 30 0 2 4 0
Benzo(k)fluoranthene (5 rings) 190 20 0 1 2 0
Benzo(a)pyrene (5 rings) 398 41 0 1 4 0
Dibenz(a,h)anthracene (5 rings) 0 0 0 0 0 0
Benzo(g,h,i)perylene (6 rings) 163 18 0 0 3 0
Indeno(1,2,3-c,d) pyrene (6 rings) 299 30 0 1 3 0
Sum of PAHs 4202 405 101 400 266 0
Bioassays by country and endpoint Results
Lumistox acute-Austria (% inhibition) 13.8 12.3 93.7 91 93.5 0
Ostracods-Austria (% mortality to
32 12 100 100 100 17
calibrated reference sand)
Ostracods-Austria (% growth
51.8 34.8 100 100 100 0
inhibition to reference soil Ifa-acker)
Ostracods-Austria (% growth
inhibition to calibrated reference 79 67 100 100 100 54
sand)
Ostracods-Belgium (% Mortality) 20 17 100 100 100 11
Ostracods-Belgium (% growth
58 47 100 100 100 47
inhibition)

Mortality data obtained in Belgium indicates that the coefficient of variation between
repetitions for five (Ifa-acker, WG beta, BZ 10, TA 4-6 and TA 14-16) of the six soil samples

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344 Belgis Chial Z., Guido Persoone, Doris Hirmann et al.

ranged from 0 to 7% and one soil sample (WG alpha) reached 64% CV. In Austria, the CV
between repetitions ranged from 0 to 19% for all soil samples.
The inter-laboratory coefficient of variation between the mean mortalities ranged from 0
to 11% for five of the six soil samples, and 49% for one soil sample (WG alpha).
Growth inhibition data obtained after six days in each laboratory is presented in Table 1.
The Ostracodtoxkit F assay showed growth inhibition in all soil samples tested (Ifa-acker,
WG beta and WG alpha) in both laboratories for which no significant mortalities (less than
20%) were observed. The growth inhibition obtained in Belgium ranged from 47 to 58% with
a CV between repetitions from 4 to 10%. In Austria, the growth inhibition ranged from 54 to
79% and was not possible to estimate the CV because only a single data was recorded. In both
laboratories the lowest inhibition corresponded to the Ifa-acker soil in which PAH
concentrations were not detected.
The interlaboratory variability for growth inhibition obtained in Belgium and the single
value recorded in Austria ranged from 6 to 17%, being the lowest value for Ifa-acker soil
obtained in Austria.
The correlation analysis between mortality found in both laboratories with the
Ostracodtoxkit F assay indicates good correspondence (r=0.98; p<0.05), as well as with the
acenaphthene concentrations (r=0.82; p=0.046) and not with other PAHs. A similar
correlation was found with the same PAH and the Lumistox inhibition assay data (r=0.80;
p=0.055).

DISCUSSION
The low intra-laboratory variability observed for the Ostracodtoxkit F assay indicates a
reasonably good precision when mortality was the endpoint considered, according to the
commonly “~30 %” acceptable variation of toxicity (USEPA, 1991; EPS, 1995). A possible
weak point could be the apparent high variability results observed in the soil sample WG
alpha performed in Belgium. This variability could be due to the soil heterogenity since it was
not observed in Austria.
The inter-laboratory variability also showed very good precision, with the exception of
soil sample WG alpha. This variability for WG alpha caused by an increasing mortality in the
second repetition of the soil assay performed in Austria might be due to the fact that it was
performed ca. 2 months after the first test. The storage conditions and time could have
influenced the physical-chemical parameters in such a way that it increased the availability of
the PHAs, resulting in a higher toxicity response. This toxicity could be due to the differential
solubility of PHAs, reported in previous studies (Guzzella, 1998; Jennings et al., 2001; Scherr
et al., 2008).
Moreover, the inter-laboratory precision could be verified when taking into consideration
that the reference field soil, Ifa-acker, had an intra-laboratory CV lower than 10%, while the
inter-laboratory precision was 11%. Besides, good accuracy of the assay was apparent
because the results did not produce false positives for this natural, non-polluted soil.
Substantial information can be found in the literature regarding the factors affecting intra
and inter-laboratory test results (Dorn et al., 1987; Persoone et al., 1993; EPS, 1995; Burton
et al., 1996). The success of the present study was due to the two most important factors:
the analyst experience and test organism health were maintained homogeneous in both

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 Inter-Laboratory Calibration of the Ostracodtoxkit … 345

laboratories since the operators were previously trained and the organisms were obtained
from the same cysts batch. Therefore, any significant variability is most probably due to test
conditions and soil storage, i.e., handling conditions.
The significant statistical correlation found between the mortality endpoints produced
in the soils once the Ostracodtoxkit F assay was performed in both countries is indicative
of its good reproducibility (r=0.98; p<0.05). Furthermore, the toxicity response of the
Ostracodtoxkit F could also be corroborated when a significant statistical correlation was
found with the light inhibition signals obtained when the Lumistox assay was performed
(r=0.997; p<0.05).
The Ostracodtoxkit F assay demostrates a sensitivity to PHAs, in particular to
acenaphthene according to the significant correlation found. The correlation with acenaphtene
might be indicating the known chemical-species toxicity dependence or the differential
availability of PAHs (Guzzella, 1998; Jennings et al., 2001; Scherr et al., 2008).
Although the additive toxicity of PAHs does not seem to play an important role in the
toxicity caused to the ostracods and bioluminisce bacteria, final conclusions have to be drawn
with caution because the final PAH concentrations were not measured in the supernatant nor
were the organisms exposed separately to each PAH concentration. The growth inhibition
(calculated on the basis of the calibrated reference sand), identified with the Ostracodtoxkit F
assay for these soils indicated a fairly acceptable intra-laboratory precision, thus the
operational procedures should now be standardized in order to obtain comparable results. It
seems that the Ifa-acker soil itself presents a growth inhibition to the ostracods, producing
interlaboratory variability when taken as the reference soil to estimate the growth inhibition.
Considerations are taken by the University of Panama through the Department of Marine
Biology and Limnology to continue this research and implement it in Panama in order to
evaluate sediment and soil pollution from different sources. This and other bioassays will
complement the traditional chemical analysis performed until now in the country and obtain a
more realistic evaluation of pollutant effects on wildlife. The adoption of this approach in our
region will add a more scientific support to protect biodiversity, while generating new toxicity
information from tropical regions.

CONCLUSION
Reasonably good intra and inter-laboratory precision has been demonstrated by the
Ostracodtoxkit F assay for soils contaminated with PAHs.
The new ostracod solid phase sediment microbiotest showed also to be applicable to
detect soils contaminated with PAHs on the basis of lethality and growth inhibition.
Growth inhibition of ostracods seems to be an important alternative to detect toxicity
using the ostracod toxicity test when other than acenaphthen PAH has to be detected in soil.
More knowledge of the sensitivity of the ostracod assay to the PAHs could be obtained if
PAH concentrations are also determined in the supernatant of the test during the assay.
Analyst experience and the use of similarly aged and healthy organisms obtained from
the dormant stage (cyst) probably contributed to the success of this short inter-laboratory
intercalibration.

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346 Belgis Chial Z., Guido Persoone, Doris Hirmann et al.

REFERENCES
Burton, A.G. Jr., Arnold, W.R., Ausley, L.W., Black, J.A., DeGraeve, G.M., Fulk F.A.,
Heltshe, J.F., Peltier, W.H., Plet, J.J., Rodgers, J.H. Jr., 1996. Effluent toxicity test
variability: Discussion synopsis. In D.R. Grothe, K.L. Dickson, D.K. Reed-Judkins (Eds),
Whole effluent toxicity testing: An evaluation of methods and prediction of receiving
system Impacts. Conference Proceedings, SETAC Press, Pensacola, Florida, pp. 131-156.
Chial, Z.B., Persoone, G., 2002. Cyst-based toxicity tests XV - Application of the ostracod
solid phase microbiotest for toxicity monitoring of contaminated soils. Environ. Toxicol.
18, 347-352.
Dorn, P.B., Rodgers, J.H. Jr., Jop, K.M., Raia, J.C., Dickson, K.L., 1987. Hexavalent
chromium as a reference toxicant in effluent toxicity tests. Environ. Toxicol. Chem. 6,
435-444.
EPS (Environmental protection series), 1995. Guidance document on measurement of toxicity
test precision using control sediments spiked with a reference toxicant. Report EPS
1/RM/30. Canada.
Guzzella, L., 1998. Comparison of test procedures for sediment toxicity evaluation with
Vibrio fischeri bacteria. Chemosphere 37, 2895-2909.
Jennings, V.L.K., Rayner-Brandes, M.H., Bird, D.J., 2001. Assessing chemical toxicity with
the bioluminescent photobacterium (Vibrio fischeri): a comparison of three commercial
systems. Wat. Res. 35, 3448-3456.
Lock, K., Janssen, C.R., 2001. Ecotoxicity of zinc in spiked artificial soils versus
contaminated field soils. Environ. Sci. Technol. 35, 4295-4300.
Ostracodtoxkit FTM, 2001. Chronic « direct contact » Toxicity Test for Freshwater Sediments.
Standard Operational Procedure. Creasel, Deinze, Belgium., pp. 18.
Persoone, G., Blaise, C., Snell, T., Janssen C., Van Steertegem, M., 1993. Cyst-based toxicity
tests: II.-Report on an international intercalibration exercise with three cost-effective
Toxkits, Zeitschrift für Angewandte Zoologie 79, 17-36.
Scherr, K., Hasinger, M., Castaldini, F., Loibner, A.P., 2008. Polycyclic aromatic
Hydrocarbon degradation. 4th Bioremediation Conference. CHANIA, CRETE, Greece.
US EPA - US Environmental Protection Agency, 1991. Technical Support Document for
Freshwater Quality Based Toxic Control. EPA/505/2.90.001 EPA, Washington DC,
USA. pp. 145.
DANETV Test Environmental Technology Verification Report, 2011. Advanced Monitoring
Systems Center. Process Document for U.S. EPA ETV AMS Center, DHI DANETV
Water Centre and ETV Canada. Joint Verification of the HACH-LANGE GmbH
LUMIStox 300 Bench Top Luminometer and ECLOX Handheld Luminometer. Battelle.
Vangheluwe, M.L., Janssen, C.R., Van Sprang, P.A., 2000. Selection of bioassays for
sediment toxicity screening. In G. Persoone, C. Janssen, W. De Coen (Eds), New
microbiotests for routine toxicity screening and biomonitoring. Kluwer Academic/
Plenum Publishers, pp. 449-458.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 22

MINING IN VENEZUELA: ITS EFFECTS ON

THE ENVIRONMENT AND HUMAN HEALTH

Antonio Machado-Allison*
Academia de Ciencias Físicas, Matemáticas y Naturales, Instituto de Zoología y Ecología
Tropical, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela

ABSTRACT
Factors that affect water quality and river habitats of the Orinoco River Basin have
been a source of constant concern and their study has become a priority for the
Venezuelan scientific community. Due to economic pressures, the old and isolated
artisanal gold mining has exploded uncontrollably, become more industrialized and has
extended to almost all river basins of the Venezuelan Guiana Shield. Precious mineral
prospection and extraction has become a serious threat to aquatic ecosystems by
destroying the surrounding protective forests and riverbanks, by polluting the water with
mercury and other heavy metals that upon entering the food chain cause serious
deleterious health effects to local inhabitants. The diminishing quantity and quality of
food, as well as the health problems due to mercurial toxicity have created drastic
changes in the social structure of these communities. We summarized studies made on
mercurial contamination in fish and how it is being transferred to humans. Finally, their
overall impact on the native indigenous population is presented. The actions controlling
gold extraction and other minerals cannot be limited to a single country and require their
consistent and vigorous application within all nations of the Amazonian, Guianian and
Orinoquian river basins.

Keywords: Guiana Shield, gold, biodiversity, human health

*
Corresponding Author E-mail: antonio.machado@ciens.ucv.ve.

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348 Antonio Machado-Allison

INTRODUCTION
The conservation and environmental services offered by the biodiversity in tropical areas
in favor of human health has been a matter of recent consideration in academic, economic,
social and political grounds. The action of natural or anthropic factors as well as the
responses, norms and necessary regulations are equally considered in workshops, symposia
and scientific papers (Lasso et al., 2010, 2011; Machado-Allison et al., 2011; Machado-
Allison, 2015). An unlocked wealth remains hidden within the tropics’ biodiversity
considering its enormous potential for new food production, pharmacological derivatives,
green-energy and industrial derivatives. Equally important is to understand how this
immensely varied biodiversity maintains a delicate balance in terrestrial and aquatic
ecosystems, in order to avoid major perturbations, and providing the most abundant and un-
intervened sources of clean air and potable water necessary to maintain several native human
groups as well as a rich wildlife. The world’s tropical regions will be the basis for global
sustainable development in the 21st century and beyond (Machado-Allison, 2015).
Venezuela is located at the northern tip of South America and constitutes an area of great
biotic importance. One of its geographical features is the Guiana Shield, dated from the Pre-
Cambrian (1.5-2 x 103 million years of age), and is one of the most ancient geological rock
formations on Earth. Numerous processes of biotic and abiotic evolution have produced an
amalgam of organic ecosystems and communities, many of them unique. The water bodies
and their aquatic communities do not escape this fabulous history, which continues to evolve
to this day (Machado-Allison, 2014).
In contrast to this natural history, the social and economic reality is quite different.
Despite the country’s immense wealth in resources (petroleum, gas, iron, hydroelectric
power, fertile land, agriculture), a significant inequality gap persists with many living in
extreme poverty and some dying of hunger. This has created a social structure and
environmental deterioration that has reached levels never seen in the past. More recently, the
development of the Orinoco Bituminous Belt and oil operations alongside the Orinoco River
in addition to the artisanal and industrial exploitation of gold (such as Mining Arch) in some
of its headwaters have created a substantial threat to Venezuela’s main aquatic environments.
The purpose of this chapter is to discuss some of the gold extraction operations occurring
in Venezuela and their impact on the environment and local human populations. At the end
we discuss the importance of guiding human activities that allow both environmental
sustainability and a closure of the social inequality gap.

GEOGRAPHIC AREA: GENERAL CHARACTERISTICS

The Venezuelan “Guiana” includes an area occupied by Amazonas, Bolívar and Delta
Amacuro states located to the south of the Orinoco River and extends approximately 620,000
km². Contained within this area are the headwaters of numerous rivers (Aro, Autana, Caroní,
Caura, Cuchivero, Cuyuní, Paragua, Ventuari) and the Orinoco itself. Within this basin
there are “clear,” “white” and “black” rivers (Sioli, 1965, 1975), which have significant
heterogeneity in their physical-chemical and biotic characteristics. Besides representing the
main source of drinkable and irrigation water for most residents living on their banks, they

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 Mining in Venezuela 349

also constitute one of the greatest hydro-electric reserves of the world with an estimated
generation capacity that will save 750000 barrels of oil per day once all projects are
completed (Caroni and Paragua rivers).
The landscape is covered by an exuberant and luxurious tropical wet evergreen forest,
which grows on nutrient poor soil. Tree growth is very slow and nutrient extraction is
critically dependent at root level of complex biological associations called microrhizae. The
lowland forest is broken by high mountain “tepuis” on top of which special and unique plant
communities have been able to adapt and survive. In between and at different altitudes there
is a variation of evergreen, deciduous forest and grasslands. Over nine thousand plant species
have been described from these different biota including grasses, ferns, shrubs, evergreen and
deciduous trees, “palm tree morichales,” bromeliads, orchids, and other highly endemic flora
living on of the “tepuis” (Hubber and Alarcon, 1988; Hubber, 1990; Fernández et al., 2010).
Importantly, the vegetation that lives in close association to water bodies is the main source of
organic nutrients and food (seeds, fruits, insects) for many aquatic organisms such as the
fruit-eating piranhas Pygopristis sp. (Marrero et al., 1997; Machado-Allison et al., 2010).
Terrestrial fauna is immensely diverse and constitutes the last and only wild reserve
for a great number of endangered species such tapirs, jaguars, dears, toucans, monkeys and
macaws (Aguilera et al., 2003; Rodríguez and Rojas-Suárez, 2008). The aquatic fauna is
abundant and equally varied, containing an important list of fishery species (for food
production or aquarium enthusiasts) and that is the primary source of protein for the human
communities in the area.
Humans have been present in these areas for over 6-9 thousand years. At least 15
autochthonous tribes live in Venezuelan Guayana (Figure 1), and generally reside close to
large water bodies (rivers and lakes), where they obtain resources for housing, wood for
canoes, fiber for hammocks, food and water.
Recognizing its biological, anthropological and geological importance, several regions
have been assigned as “Áreas Bajo Regimen de Administración Especial” (ABRAE) (Special
Protection Areas) and are protected by national environmental laws. These include some of
the world’s largest national parks (Parima-Tapirapeco 39000 km2, Canaima 30000 km2, La
Neblina 13600 km2, Duida-Marahuaca 3800 km2, Jaua-Sarisariñama 3300 km2, Yapacana
3200 km2), and several important biosphere and forest reserves. All tepuys are also
considered under protection (Bevilacqua et al., 2006).

MERCURY: THE PROBLEM

Conflicting interests have not allowed a consensus among international, national and
regional governmental and non-governmental agencies, indigenous communities, research
groups and economic groups with regards to the sustainable use of the resources of the
Venezuelan Guayana, including the exploitation of precious minerals (gold and diamonds)
and how to mitigate its effect unto the environment. Table 1 summarizes some of the most
important developments in the last 30 years (in addition see notes from Machado-Allison,
2014).

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350 Antonio Machado-Allison

Figure 1. Distribution of local indigenous communities in Venezuelan Guayana: 1. Panare, 2. Hoti, 3.

Piaroa, 4. Puinave, 5. Piapoco, 6. Baniva, 7. Kurripaco, 8. Bare, 9. Yanomami, 10. Yekuana, 11.
Sanema, 12. Niamn, 13. Pemon, 14. Kapon, 15. Kariña.

Despite severe environmental disasters, the loss of wildlife and threats to public health,
uncontrolled mineral extraction continues and has recently shown even more worrisome
figures (USEPA, 1984, 1989; Nico and Taphorn, 1994; Veiga et al., 1995; Veiga, 1997;
Machado-Allison et al., 2000; Machado-Allison, 2005; Farina et al., 2009; Trujillo et al.,
2010; Milano, 2014; SPDA, 2014). In general, “artisanal” gold extraction is characterized by:

 The use of elemental metallic Hg for gold separation from sediments and
amalgamation. Mercury is later evaporated by heating and released into the
atmosphere.
 Approximately 45% of this Hg enters directly into the water column where
microorganisms convert it into highly toxic methyl-mercury (MeHg). Sediments are
then dragged by white water currents, favoring its dispersion.
 The other 55% of evaporated Hg remains in the atmosphere in the form of ethyl-
mercury, being latent up to 24 months in dry zones, but rapidly dropping into the soil
in regions with high rainfall (Veiga, 1997).
 MeHg is quickly incorporated into the food chain (Figure 2), accumulating mostly in
top predators such as: fish, aquatic reptiles, birds and mammals (Rosas and Lehti,
1996; Gutleb et al., 1997). This causes serious effects on diversity as predators may
die off and is a high risk to people that ingest this contaminated food (e.g., fish and
reptiles) (USEPA, 1984, 1989; Fréry et al., 2001; Porto et al., 2005; Castilhos et al.,
2006; Trujillo et al., 2010.

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 Mining in Venezuela 351

Table 1. Historical reports and confused data in studies performed in the Caroni (1)
and Cuyuni (2) River Basins (see Figure 1)

Source Area Conclusion

Norodin and 1 The river system is fragile; great impact due to removal of soil and large
Falcon (1989) amount of sediments in water resulting in change of transparency;
removal of vegetation; mining activities must be regulated.
PLEXUS 1 69% of miners and 37% of the nearby population were found
(1990) contaminated with Hg.
Bermúdez 1 Levels of Hg in water below 1.0 µg/L; main river channel with low
(1993) productivity; material removed from the bottom with low environmental
impact; carnivore fish with less than 0.5 µg/kg of Hg.
CVG (1995) 1 Sediments samples showed a variation from 0.0052 to 7.02 µg/g.
Lower Caroni River with 7.01 Hg/g (source of potable domestic water
system). Yuruari River with 0.0133 Hg/g.
Water values below 2 µg/L; sediments above 20 µg/L; fish contaminated
but values below 500 µg/kg; Hg was 45 to 80 times higher compared to a
non-contaminated river.
Water with <1 µg Hg/L; sediments and suspended soils (>0.5 µg Hg/g);
fish contaminated but below 0.5 µg Hg/g except in those coming from
Amarillo Creek.
Beaches close to mining camps with 36 µg/g of Hg; rivers with 350 times
higher than the background natural concentration; fish with Hg but not
reaching dangerous limits.
CVG (1995) 2 Water and sediment samples with high mercury concentrations of 13.1
µg/L and 42.2 µg/g, respectively.
2 High Hg in sediments downstream from mining site. Hg in fish above 0.5
µg/g.
Machado- 2 High sedimentations and forest destruction in Amarillo Creek and Cuyuni
Allison et al. River; elimination of fish, macroinvertebrates and water plants in both
(2000) rivers; modification of river bottom and changes in water quality; high
impact on river banks and main channel over several kilometers.

DAMAGES TO ENVIRONMENT AND BIODIVERSITY

Machado-Allison et al. (2000) produced one of the first reports showing the
environmental and biodiversity deterioration produced in the Amarillo Creek part of the
Cuyuní river basin in Venezuela, which is the main tributary of the Esequibo River in
Guyana. This study revealed how the devastation of the Amarillo Creek (near Km 88/Las
Claritas, Bolivar State) was affecting fish diversity both up and downstream from its
discharge into the Cuyuní River (Table 2 and Figure 3).
Furthermore, Nico and Taphorn (1994), Farina et al. (2009), Trujillo et al. (2010), and
Milano (2014) showed Hg present in fishes captured in the Orinoco River Basin and made a
public statement of it to be considered a problem of national concern. Table 3 depicts the
summary on the data obtained from these studies.

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352 Antonio Machado-Allison

Modified from: Machado-Allison, 2015.

Figure 2. Incorporation and accumulation of MeHg in the food chain.

Table 2. Ecological parameters taken from the Cuyuní River

Parameters Field Stations

Downstream (1-6) Upstream (7-12)
1 2 3 4 5 6 7 8 9 10 11 12*
Temperature (C) 24.0 24.0 24.0 24.0 26.5 26.5 26.5 26.5 26.5 26.5 25.0 26.5
Transparency (cm) 50 50 10 10 20 10 0 10 5 20 >50 0
pH 5-6 5-6 6-7 6-7 5-6 5-6 7 5-6 7 5-6 5-6 >7
Aquatic vegetation (%) 100 100 25 25 25 0 0 0 0 25 25 0
Terrestrial vegetation (%) 100 100 50 50 50 25 25 50 25 50 75 0
N species 87 30 9 15 42 17 14 21 16 9 51 0
N specimens 1762 180 103 175 348 64 74 165 58 187 531 0
Diversity (D) 40.0 15.4 2.7 3.6 18.8 8.6 7.7 10.5 11.1 7.2 5.1 0
Equitativity (E’) 0.79 0.80 0.53 0.39 0.77 0.78 0.84 0.79 0.90 0.67 0.79 0
Source: Machado et al., 2000.
*
Amarillo Creek.

HUMAN HEALTH
For the last 25 years, contamination by Hg has become an increasingly worrisome public
health problem. A significant number of studies have revealed high levels of Hg in blood
samples of people living in these mining zones (Romero et al., 1987; Herrero et al., 2004;
Silva et al., 2004; Veiga et al., 2004; Alvarez and Rojas, 2006; Carrasquero-Duran, 2006;
Milano, 2014). These authors found that Hg concentrations in samples coming from miners
working in “El Callao” are the highest in the world (>2.7 µg/g). It is not surprising that there
has been a steady increase in the incidence of severe neurological degenerative disease among
miners and their families.

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 Mining in Venezuela 353

Table 3. Mercury level in carnivorous fish tissue from the Cuyuní River

Species Hgtotal (µg/kg) MeHg (µg kg) MeHg HQ*

(µg/100 g)
Plagioscion squamosissimus 4105.7 3900.5 390.1 55.7
Electrophorus electricus 3935.8 3739.0 373.9 53.4
Cynodon septenarius 2352.1 2234.4 223.4 31.9
Cynopotamus essequibensis 2158.4 2050.5 205.0 29.3
Pimelodus ornatus 1769.3 1680.8 168.1 24.0
Ageneiosus inermis 1618.8 1537.9 153.8 22.0
Acestrorhynchus microlepis 1456.2 1383.4 138.3 19.8
Serrasalmus rhombeus 968.6 920.2 92.0 13.1
Crenicichla lenticulata 785.3 746.1 74.6 10.7
Crenicichla johanna 527.5 501.1 50.1 7.2
Hoplias macrophthalmus 493.0 468.3 46.8 6.7
Synbranchus marmoratus 221.1 210.0 21.0 3.0
Sternopygus macrurus 203.3 193.1 19.3 2.8
Acestrorhynchus falcatus 184.2 175.0 17.5 2.5
*
HQ: Hazard Quotient (USEPA 1989).
Modified from Farina et al., 2009.

Source: Machado-Allison et al., 2000.

Figure 3. Relative number of fish species (up and downstream) from Amarillo Creek, Cuyuní River.

Hair and urine samples obtained from indigenous communities of Brazil and Venezuela
living along some of these rivers have also shown toxic concentrations of mercury. Their
levels tend to be higher than those obtained from miners coming from the same locality,
especially if fish is their main source of protein (Rojas, 2010). Fetuses and children of all ages
are particularly susceptible to Hg, presenting birth defects and impaired neurological
development (WHO, 1990). Among children from native communities we have found values
from 0.07 to 2.7 µg/g (normal is <0.01 µg/g). Close to 92% of women in reproductive age
examined along the Caura River (Sanema and Yek’uana tribes) had levels higher than 0.5
µg/g, that are considered a high-risk exposure. Importantly, some of these effects can be
detected as far as 200 km from the source of pollution. Disturbances in fertility and child

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354 Antonio Machado-Allison

development can cause a significant impact on demographic growth for these populations,
completely disrupting social structures and even existence for some of these tribes.
In parallel, Milano (2014) has made several observations of the mining communities of
Las Claritas (1996), Santo Domingo (1996), El Manteco (2002), El Callao (2005), and the
Low Caroní River (2007). They found that 68% of children and 87% of women showed
mercurial exposure with higher concentrations than those considered safe by the WHO.
Miners, on the other hand, had highly variable exposure with 25 to 84% having abnormal
values. However, these percentages went up to 90% if miners were directly involved in the
gold refining process. Their studies also confirmed the correlation between the incidence of
different neurological diseases with the degree of exposure and toxicity.

LEGAL AND INSTITUTIONAL ASPECTS

The legal framework related to mineral extraction in Venezuela is quite extensive and is
even mentioned in several articles of the national Constitution (1999). These include a series
of national laws, executive decrees and regulations that protect the environment and human
indigenous rights, and regulate the rational exploitation of mineral resources. The most
important laws are: Environmental Penal (Criminal) Law, Criminal Code, Water Law,
Indigenous Population Rights Law and different Mining Laws.
In Milano (2014) we can find a recent chronology with regards to different gold mining
regulatory agencies and institutions that until 1999 were under the central control of the
Ministry of Energy and Mines and the Environmental and Natural Resources Ministry. These
include:

 1999, Decree 3091: allowing the mining metal giant Guayana Venezuelan
Corporation (CVG) to provide permits for gold exploration and exploitation,
bypassing those that were originally given by the Environmental and Natural
Resources Ministry (MARNR).
 2001, Decree 1234: Official Gazette 37155; The National Institute of Geology and
Mining is created and is responsible for research and scientific expertise in: geology,
mineral resources, geo-physics, geo-chemistry, and geo-technology.
 2005, creation of the Popular Ministry for Basic Industries and Mining (MPPIBAM)
that incorporates the CVG and takes over policy-making and control of the mining
sectors.
 2011, Decree 8683: declaration of all gold mines within Venezuela by the national
government as “Public Domain,” reserving its exclusive right for their exploitation.
This operational task is now assigned to the Venezuelan Petroleum Company
(PDVSA) replacing the MPPIBAM.
 2013, Mining Venezuela Corporation (CVM) is created as part of PDVSA-Industrial,
to control, organize and develop gold mining.

However, Milano (2014) states that “…nevertheless all manifestations of ‘the state’ done
through codes, laws, decrees and other normative (regulation) with goals to avoid, contain
and minimize the negative impacts of mining activity have achieved inefficient results.”

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 Mining in Venezuela 355

Table 4. Summary of proposed actions by Red Ara (2013)

Area Actions
Public Health - Develop programs to allow fast determination of contamination or
intoxication.
- Guarantee permanent medical attention.
- Establish a permanent educative program on environmental and
human health risks due to mercury contamination
- Establish programs allowing capacity building in all sectors related
to public health and environmental management
- Establish actions that guarantee production and intake of
uncontaminated food in human rural indigenous settlements
- Develop epidemiological evaluations.
Social and - Promote consulting processes and participation in decision-making
Development actions with all the actors
Management - Promote permanent programs of social action and economic support
for a better quality of life. Close surveillance of human rights.
- Establish dialog processes to show development alternatives.
- Establish effective measures to control illegal activities.
Environment - Train miners in a better use of techniques of low environmental
impact.
- Enforce Venezuelan environmental legislation.
- Establish continuous monitoring programs of the mercury levels in
the environment.
- Establish continuous programs for evaluating contamination,
degradation and mitigation of soils.
- Promote research programs directed to establish new techniques
friendly with the environment.
-Integrate international/cooperative programs with neighboring
countries.
- Promote programs of adequate mercury disposal, bioremediation
and restoration of degraded areas including rivers.
- Promote interchange of scientific information and knowledge.

DISCUSSION AND RECOMMENDATIONS

In order to achieve a reasonable balance between economic development and nature, it is
necessary to understand how environments and ecosystems behave, from the biotic, abiotic
and social points of view. It is also important to determine their significance in terms of
natural and economical resources and prioritize their real benefits for both local and broader
human communities. Every effort must be made to maintain aquatic ecosystems as a
renewable and sustainable resource. It is naive to think that future economic development in
tropical countries will spare casualties and avoid damage to the aquatic ecosystems without
the loss of aquatic floral and faunal species. However, what we need is to encourage the use
of modern techniques avoiding the unnecessary extension and irreversibility of environmental
impacts and allowing the recovery of an ecosystem once mineral extraction is finished.

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356 Antonio Machado-Allison

All countries have the moral obligation to be responsible and preserve their aquatic
resources. The maintenance of high quality, pollutant free water may be critical for the
survival of human settlements downstream. Even though rivers flow in only one direction, an
environmental impact can spread in all directions.
As previously mentioned, the environmental contamination by mercury is a dynamic
process that affects the entire ecosystem and has accumulating effects in upper echelons of
the food chain. Many of the top predators such as the great catfishes of the genera
Brachyplatystoma and Pseudoplatystoma (that accumulate Hg) happen to be migratory fish. It
is thus not surprising that mercurial exposure can be detected far away from its source of
origin due to the consumption of these “prize catches.”
Unfortunately, despite multiple and well-documented evidence of environmental
deterioration, hazardous exposure to local population and serious threats to public health, the
policy-makers and regulatory agencies have done very little to reduce the use of mercury for
gold processing. On the contrary, data indicates that its use is at a historical high (Veiga et al.,
2004).
We subscribe the very important efforts from different NGOs such as the Red ARA (Red
Organizaciones Ambientales No Gubernamentales de Venezuela – Network of Environmental
NGOs of Venezuela) that continue to bring these issues to the forefront of discussion by
organizing meetings, workshops and connecting with the mass media. One of these
workshops, “Mercury Contamination in the Venezuelan Guiana Shield: A Proposal and
Dialog for Action” (Red Ara, 2013) calls for important action plans that could be
implemented (Table 4).

CONCLUSION
Recognizing the importance of this environmental problem, several international
organizations such as the United Nations-Development Program and United Nations-
Development Bank, the World Wildlife Fund (WWF) and members for the Convention on
Biological Diversity met in May 2014 to engage in a conversation of all nations within the
Guiana Shield. The “Guiana Shield Action Plan to Facilitate Biodiversity, Corridors, Achieve
Aichi Targets” calls for governments (Brazil, Colombia, Guyana, Suriname, French Guayana
and Venezuela) and partner organizations to:

1. Support national efforts to better manage and monitor small and medium-scale gold
mining, including best practices for biodiversity conservation and water resources
management.
2. Encourage the further development of trans-boundary protected areas in the Guiana
Shield.
3. Create synergies with existing global and regional platforms in order to take
advantage of efficiencies of scales, efforts and momentum.
4. Develop and strengthen linkages with collaborative connectivity projects.
5. Organize a technical database that allows identifying and prioritizing trans-boundary
corridors

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 Mining in Venezuela 357

6. Strengthen and facilitate academic research (university research cooperation and

other research institutions) into a more cooperative science.

Gold mining and the “sustainable development” of other resources within ecosystems of
the Guiana Shield should not be considered a utopian dream. However, it does require a
conscious and collective effort to understand and maintain their delicate balance. The real
wealth present in these forests and rivers is not the value of the metals and diamonds that are
found once in a lifetime, but in the biodiversity present in them that could represent the next
revolutionary food, drug and energy source that could save mankind in this century and
beyond.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 23

ENVIRONMENTAL RISK OF LEAD ACCUMULATION

IN CROPS IRRIGATED WITH WATER FROM
THE MANTARO RIVER, JAUJA SECTOR, PERU

Edith Orellana1 and Raymundo Erazo2,*

1
Universidad Nacional del Centro del Perú,
Facultad de Ciencias Forestales y del Ambiente, Huancayo, Peru
2
Universidad Nacional Mayor de San Marcos,
Facultad de Ingeniería Química, Lima, Peru

ABSTRACT
Lead is an environmental pollutant highly toxic to human health. This chapter deals
with the hazard index and environmental risk of lead accumulation in soil and
agricultural products irrigated with water from the Mantaro River on the left bank of the
Mantaro Valley in Jauja, the Junín region, Peru. The methodology applied was that
recommended by the Environmental Protection Agency (EPA) and the Environmental
Ministry of Peru (MINAM). Soil, water, corn and potato samples were analyzed and 35
people who are frequently exposed to lead contamination were interviewed including
males and females from 4 to 83 years old. The results indicate that lead concentrations in
corn and potato exceeded the maximum allowed levels by between 5 and 30 times. The
hazard index between 0.1163 and 0.1186 corresponds to a low risk to the health of the
population exposed. The environmental risk level for agricultural areas irrigated by the
Mantaro River was high (0.67). Overall, the probability of damage to humans and the
environment and at a socio-economic level due to contaminated food intake and lead
accumulation in agricultural soils and groundwater was very high.

Keywords: environmental risk, hazard index, lead accumulation, Mantaro River, Peru

*
Corresponding Author E-mail: rerazoe@unmsm.edu.pe.

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364 Edith Orellana and Raymundo Erazo

INTRODUCTION
High levels of heavy metals in irrigation water are a problem for agriculture, biodiversity
and human health. Heavy metals and metalloids in irrigation water are dangerous due to their
non-biodegradable nature. Consequently, they can accumulate in agricultural soils and exert
toxicity on different crops (Garcia and Dorronsoro, 2005).
The water resource in the Mantaro Valley of the Junín Region, Peru, is becoming scarce,
and there are 25 important districts in the region where the irrigation of agricultural soils
depends on the Mantaro River. In the study area (Jauja sector, province of Jauja, Junín
region), farmers have been irrigating their agricultural crops (corn, potatoes, beans, carrots,
artichokes) and pasture land with water from the Mantaro River for more than 70 years, with
high contents of heavy metals (Cr, Cu, Fe, Hg, Pb, Cd, Ni, Zn) and metalloids (As). These
have originated from the different mining operations in the region and the La Oroya
metallurgical plant (Proyecto Mantaro Revive, 2008). As a result, the soils present high
concentrations of these elements, which cause a deterioration of the quality of the soil and
aquifers (Porta et al., 2003). There are more than 17,060 irrigation users that irrigate 7,754
acres of soil (36% of the total surface irrigated; Ministerio de Agricultura, 2011a) with
contaminated water. In 2005, research conducted by Saint Louis University in Missouri and
the Archdiocese of Huancayo determined that in the provinces of Oroya and Concepción
(near the province of Jauja), about 25% of the child population of Concepción from zero to
six years old exceeded the 10 µg/dl limit of lead in the blood (Saint Louis Missouri
University, 2005).
Lead is a high-density heavy metal (Manahan, 2013) that persists in the soil for from
1,000 to 3,000 years (Duran, 2010), is assimilated by organisms, is transferred through the
food chain and represents a threat to human health and ecosystems (Calderon and Maldonado,
2008). The adsorption of these toxic substances by soil particles (Duffus, 1983) affects the
soil quality due to their high persistence and difficult degradation (Porta et al., 2003; Duran,
2010). According to the Agency for Toxic Substances and Disease Registry (ATSDR), the
World Health Organization (WHO), the Environmental Protection Agency (EPA) and the
International Agency for Research on Cancer (IARC), lead is considered a dangerous and
highly toxic substance, and they have determined that it is probably carcinogenic for humans.
Lead exposure causes neurological, renal, endocrinal, gastrointestinal, cardiovascular,
reproductive and developmental damage, which in some cases, is irreversible (ATSDR,
2007). The portion of lead that is not excreted is distributed via the blood to soft and mineral
tissues (bones and teeth): in adults, 95% of the body burden is stored in bones (Saldívar
Osorio et al., 1997; World Health Organization, 2000) with a half-life of 10 to 20 years
(ATSDR, 2007).
Evans et al., (2003) and Zuck and Ize (2010) define risk as the probability that negative
consequences (physical, chemical, biological and cultural) may occur due to exposure to a
hazard. The probability of a risk is expressed in values between 0 and 1: the closer the value
is to 1 the higher the certainty that there is a risk (Tyler Miller, 2002). Although the
probability theory plays an important role in making decisions, future events cannot be
predicted with absolute certainty (Beck, 1998).

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The exposure to heavy metals by inhalation, dermal contact or ingestion is associated

with different effects on the population’s health. The risk assessment methodology constitutes
a useful tool in the decision making related to public health (Hernández, 2012).
The present chapter presents an estimation of the hazard index and environmental risk
due to the accumulation of lead in the soil and agricultural products irrigated with water
contaminated by flooding upstream of the Mantaro River water basin.

METHODS
Study Area

The study area of approximately 13.50 km2 is located on the left bank of the Mantaro
River, province of Jauja, Department of Junín, located between 3370 and 3350 m.a.s.l., 40 km
from the city of Huancayo. The irrigation system starts in “Siclachaca,” 250 m north of Stuart
Bridge at the entrance of the Mantaro valley with a river flow of 400 m3/s in the rainy season
and of 100 m3/s in times of low water (Ministerio de Agricultura, 2011a).
The water samples were collected in August (2013) at six points in each one of the
secondary channels of the irrigation main channel: Sausa, Ataura, Huamalí, The Mantaro, San
Lorenzo and Apata. Each sample was placed in 1 L plastic bottles with nitric acid and sent in
a thermal box (cooler) (Ministerio de Agricultura, 2011b) to the laboratory of soil, plant,
water and fertilizer analysis of the Universidad Nacional Agraria La Molina.
Twelve soil samples, six from corn crops and six from potato crops, were taken from the
six sampling sites in September (2013). Six to nine 1 kg soil sub-samples were collected from
a depth of 0-30 cm using an auger and according to the square grid sampling technique. The
sub-samples were then pooled and homogenized to form a composite sample (MINAM,
2014). In the laboratory, each composite soil sample was dried, sifted and stored in 500 g
polyethylene bags until further analysis.
Six corn (variety “cuzqueado”) samples were collected in December (2013). Each corn
sample was composed of 16 corncobs taken at random from each plot; they were peeled and
500 g of corn grains were dehydrated in a drying oven in the laboratory and placed in plastic
containers. Six potato (variety “yungay”) samples were selected in March (2014). Each
sample was randomly composed of six units, washed and dried, then separated into 500 g of
fresh potato and placed in plastic containers. Subsequently the potato samples were
dehydrated in a drying oven in the laboratory. The collection of soil samples, corn and
potatoes were performed by farmers who agreed to the collection at the six sampling sites.
Soil and agricultural crop samples originated from the same selected plot.
Irrigation users from 115 families were interviewed in the six towns to know about their
irrigation practices, the frequency of corn and potato intake, economic activity and income.
The population at risk was represented by children, adults and some older adults (35 people in
total) that consumed corn and potato on a daily basis.

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Risk Assessment Methodology

The methodology used to estimate the hazard index on the population’s health was based
on the US Environmental Protection Agency (EPA, 1998), and the environmental risk
assessment procedure that was established by the Environmental Ministry of Peru according
to the Spanish standard UNE 150008:2008 (MINAM, 2010; 2011).
The hazard index for the population’s health was studied in four stages:

i. Hazard Identification
Lead concentrations in the irrigation water, soil and agricultural products (corn and
potato) were determined via the atomic absorption spectrophotometer according to 3111B:
direct method of air flame – acetylene (APHA, AWWA, WPCF, 1992).
The lead reference levels in water and soil were those established by the Environmental
Quality Standards (EQS) of the Environment Ministry of Peru: EQS of 0.05 mg/L for
vegetable irrigation and animal drinking water and of 70 mg/kg for agricultural soil
(MINAM, 2008; 2013). The permissible levels of lead in agricultural products were those
recommended by the Codex Alimentarius Commission: 0.10 mg/kg for potato and 0.20
mg/kg for corn (Codex Alimentarius Commission, 2013).

ii. Exposure Assessment via the Average Daily Dose (ADD)

The ADDs were calculated for each agricultural product and individually for each person
studied, using the following expression:

𝐶𝑖 ∗ 𝐼𝑖
𝐴𝐷𝐷 (mg/kg/day) =
𝑊𝑖

where Ci is the lead concentration in the food (mg/kg), Ii the daily food (corn or potato) intake
(kg/day), and Wi the human body weight (kg).
Corn and potato ADD were compared between age groups and genders via analysis of
variance with a significance level of p=0.05. The means comparison of corn and potato ADD
according to gender and age groups were performed using Tukey’s test. Data processing was
performed using the statistical software R (Kuhnert and Venables, 2005; De Mendiburu,
2009).

iii. Risk Characterization via the Hazard Index (HI) or Endangerment

Coefficient (HQ)
The HI was calculated from ADD data for each location using the following expression:

𝐴𝐷𝐷 (mg/kg/day)
𝐻𝐼 = 𝑅𝑓𝐷 (mg/kg(day)

where RfD is the theoretical reference dose.

If the HI is greater than 1, then the metal ADD exceeds the RfD, which indicates that
there is a potential risk associated with the metal. The HI is a conservative index and is
related to small responses of the organism when exposed to a metal (Teuschler et al., 1999).
The probabilistic hazard index (HIp) was calculated with the data of probabilistic ADD

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(ADDp) by applying 10000 Monte-Carlo simulations (Rodríguez, 2011) to the lead

concentration data using the statistical software R (Kuhnert and Venables, 2005; De
Mendiburu, 2009).

iv. Risk Assessment

The following 4-step procedure was adopted (MINAM, 2011):

a) Hazard Identification
The risk factors derived from the lead accumulation in agricultural soils were identified.
Additionally, the effects on the human and natural environments, as well as on the socio-
economic conditions were considered:

 Natural environment indicators (water, soil and crops).

 Human environment indicators (population health, affected population).
 Socio-economic indicators (economic activities such as income and crop yield)

b) Occurrence Probability Estimation

The probability of the occurrence of risks that were threatening the health of the
population were estimated as well as the quality of the environment and the socio-economic
environment as a result of exposure to lead. The intake frequency of corn and potato, the
irrigation frequency of crops, the frequency of low crop yields and low prices were identified.
Values of probability of occurrence for the three environments were assigned as those
established by the MINAM (2011): very probable (5), highly probable (4), probable (3),
slightly probable (2) and very slightly probable (1).

c) Gravity Estimation
This consisted in estimating the damage that the risk scenarios may cause. The gravity
was applied to the values suggested by the MINAM (2011) with the following expression:

 Human environment gravity=amount + 2 dangerousness + extension + affected

population
 Natural environment gravity=amount + 2 dangerousness + extension +
environmental quality
 Socio-economic environment gravity=amount + 2 dangerousness + extension +
productive capital

The “amount” refers to the quantity of pollutant (lead) in the risk scenario;
“dangerousness” refers to the lead toxicity level; “extension” refers to the area affected by the
pollutant; “affected population” refers to the number of people affected by the contaminant;
“environment quality” refers to the damage that the pollutant causes to natural resources;
and “productive capital” is related to the soil’s productivity level. The gravity assessment
assigned to each one of the aforementioned components is from 1 to 5. The resulting gravity
estimation levels (and values) are: critical (5), severe (4), moderate (3), mild (2) and not
relevant (1).

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d) Environmental Risk Assessment

The environmental risk was determined by multiplying the given value of the occurrence
probability by the gravity value.

RESULTS
Lead Concentrations at Sampling Sites

The concentration of lead in water from the Mantaro River exceeded the Environmental
Quality Standard for crop irrigation water and animal drinking water in the six sampling sites.
The agricultural soils of Huamali and Apata had lead concentrations above the national
reference value, while the lead content in corn and potato samples exceeded permissible
levels recommended by the Codex Alimentarius Commission of the Food and Agriculture
Organization of the United Nations and World Health Organization at all six sampling sites
(Table 1).

Assessment of Lead Exposure per Inhabitant

There were significant differences in the food intake between males and females
regarding corn (F=6.19, p=0.018) and potato (F=9.33, p=0.005), with males consuming more
than females (Figure 1). Significant differences in food consumption according to age groups
for corn (F=28.19, p=0.000) and potato (F=35.42, p=0.000) were also observed. The Tukey
test indicated that seniors, aged above 50 years old, registered a higher consumption of corn
(150.0 g/day in females and 236 g/day in males) and potato (200 g/day in females and 290
g/day in males). The corn and potato intake in children was lower than adults (56 g/day of
corn in girls and 56 g/day in boys and 80 g/day of potato in girls and 78 g/day in boys).

Table 1. Lead concentration in water, soil, corn and potato irrigated with water from
the Mantaro River

Sampling site Lead concentration

Irrigation water Agricultural soil Corn Potato
(mg/L) (mg/kg) (mg/kg) (mg/kg)
Sausa 0.117 46.41 0.80 0.50
Ataura 0.115 55.50 1.00 0.50
Huamalí 0.115 78.94 1.85 2.50
El Mantaro 0.114 67.88 5.00 1.60
San Lorenzo 0.106 42.06 3.95 2.85
Apata 0.082 73.13 4.35 1.85
Reference level 0.050a 70.00b 0.20c 0.10c
aEnvironmental Quality Standards (EQS) for crop irrigation water and animal drinking water (D. S. N° 002-

2008-MINAM, Peruvian standard, 2008); bEnvironmental Quality Standards (EQS) for agricultural soil
(D. S. N° 002-2013-MINAM, Peruvian standard 2013); cPermissible maximum level of lead for human
consumption (Food and Agriculture Organization of the United Nations (FAO) and World Health
Organization (WHO): Codex Alimentarius, CODEX STAN 193-1995, 2013).

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Figure 1. Daily corn and potato intake according to the age and gender of inhabitants of all six sampling
sites (A: <13 years old; B: from 13 to 50 years old; C: >50 years old). Black dots indicate outliers of
daily corn and potato intake in females and males.

Figure 2. Average Daily Dose of lead (ADD; mg/kg/day) in female and male inhabitants consuming
corn and potatoes from the six sampling sites irrigated with water from the Mantaro River. Black
horizontal lines indicate 1/8 maximum allowed level for corn (0.2 mg/kg) and 1/5 maximum allowed
level for potato (0.1 mg/kg) (CODEX STAN 193-1995, 2013). Outliers of ADD of potato in female of
San Lorenzo are indicated with a thick dark vertical bar.

Figure 3. Lead Hazard Index (HI) in females and males from the six sampling sites regarding the daily
consumption of corn and potato irrigated with water from the Mantaro River (1/4 HI (1)). Outliers of HI
in females of San Lorenzo are indicated with a thick dark vertical bar.

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Figure 4. Observed and probabilistic data for the General Hazard Index (HI) for lead.

Average Daily Dose of Lead from Diet

The corn and potato ADD were lower than the maximum recommended level (0.1
mg/kg/day for potato and 0.2 mg/kg/day for corn; Figure 2). There were no significant
differences in ADD between males and females regarding corn (F=3.002, p=0.093) and potato
(F=2,973, p=0.095). The age groups showed no significant differences in ADD for corn
(F=1,725, p=0.195) and potato (F=0.200, p=0.820).

Human Risk Characterization

The hazard index (HI) was below one in the males and females studied form all the
sampling sites (Figure 3). Males presented higher deterministic and probabilistic HI values
than females. The Huamali, El Mantaro, San Lorenzo and Apata sampling sites presented the
highest HI values, but still below the maximum allowed level.
The HI probabilistic values were lower than one (the maximum reference level) and the
result of 40% of the HI probabilistic data was between 0.1291 and 0.1642 (Figure 4).

Environmental Risk Assessment

The daily intake of corn and potato determined a risk occurrence probability of highly
probable (4) in the human environment. The crop irrigation frequency and lead concentration
in the soil and aquifers determined an occurrence probability for the natural environment of
highly probable (4). The decrease in crop yield and the low prices of the agricultural products
determined an occurrence probability of highly probable (4) in the socio-economic
environment (Table 2).
The gravity of the damage identified in the study area was critical (5) in the human
environment and severe (4) in the natural and socio-economic environment. This outcome
was influenced by the high lead concentration in corn and potatoes i.e., lead toxicity, in more
than 1000 acres of corn crops and 350 acres of potato crops. 750 farmers were affected and

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more than two natural resources (water, soil, crops) were affected as well as some moderately
productive soils (Table 2).

DISCUSSION
Hazard Index

Water from the Mantaro River has been used to irrigate agricultural fields in the Sausa,
Ataura, Huamali, Mantaro, San Lorenzo and Apata localities of the Jauja sector, every 15 or
20 days for more than 70 years, representing a potential hazard to population health and the
natural ecosystems (Chávez et al., 2011). Agriculture is the main economic activity of the
population studied. Irrigation water presented high levels of lead concentrations (considered a
highly dangerous substance; Díaz-Barriga, 1999; ATSDR, 2007), among other heavy metals,
because of the mining and metallurgical activity in the upper and middle basin of the Mantaro
river (Proyecto Mantaro Revive, 2008).
The concentrations of lead in the irrigation water exceeded what is acceptable, according
to water quality standards, at the six sampling sites, in agreement with data previously
reported by Goetendia and Ruiz (1989), Tello (1996), Proyecto Mantaro Revive (2008) and
Turco (2009).
The Huamali and Apata sites presented values for lead accumulation values in the
agricultural soil above the maximum recommended level, constituting a hazard for human
health and the environment (Porta et al., 2003).

Table 2. Probability, gravity and environmental risk to human, natural and socio-
economic environments, in different lead (Pb) contamination scenarios

Scenario Probability Gravity Environmental

risk (%)
Human environment
Irrigation of agricultural areas with heavy 4 5 80
metals (Pb in corn and potatoes)
Irrigation of agricultural areas with heavy 4 4 64
metals (Pb in soil and water)
Human environment average risk (%) 72
Natural environment
Irrigation of agricultural areas with heavy 4 4 64
metals (Pb in soil)
Irrigation of agricultural areas with heavy 4 4 64
metals (Pb in groundwater)
Natural environment average risk (%) 64
Socio-economic environment
Low income 4 4 64
Low crop yield 4 4 64
Socio-economic environment average risk (%) 64
Average risk characterization (%) 67

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Contaminants may remain for a long time in soil. In the long run, this permanence is
serious in the case of inorganic contaminants such as lead, which cannot be degraded
(ATSDR, 2007; Calderon and Maldonado, 2008). In temperate soils lead remains present for
1,000 to 3,000 years depending on humidity and temperature (Duran, 2010). In general heavy
metals in soil may experience different outcomes: be retained in the soil, be absorbed by
plants and incorporated into trophic chains, or enter the atmosphere by volatilization and
move to surface water or groundwater (García and Dorronsoro, 2005).
The lead concentration found in corn and potatoes exceeded the permitted maximum
levels (as recommended by the Codex Alimentarius of the World Health Organization and
Food and Agricultural Organization (2013) by between 5 to 30 times. The long-term
persistence of lead in the soil contributes to the progressive accumulation and transfer to the
food chain, thus constituting a danger to human health and natural ecosystems (ATSDR,
2007) at the six sampling sites studied.
More than 30% of the population interviewed was exposed to lead through corn and
potato consumption. Males consume more than females and individuals above 13 years of age
recorded a higher intake compared to the group aged below 13.
The lead ADD in the population studied is influenced by the variation of lead
concentration in the agricultural products, the ingestion rate and the body weight of the
individuals. The deterministic and probabilistic ADD was lower than the maximum allowed
level in male and female inhabitants in the three age groups in the six towns. Males showed
higher values of ADD (Figure 2). Furthermore, these values are below the RfD, these results
match the assessment of the health risk constituted by consuming drinking water
contaminated with heavy metals performed by Hernández (2012).
The HI for lead intake calculated deterministically and probabilistically in males and
females is less than one, which represents a minimal risk to the health of the population
exposed to it, in comparison to the RfD, in six locations of Jauja (Figure 3), with a range that
varies between 0.1163 and 0.1186 of probabilistic HI. The probabilistic maximum values of
lead HI distribution are, in general, higher than the deterministically calculated HI, where
the maximum value of 0.2553 (in males) represents a low level of risk to human health
due to lead exposure. The observed and simulated hazard indexes were higher in males and
females from Huamali, Mantaro, San Lorenzo and Apata probably due to the higher lead
concentration in corn and potatoes, although they were below the theoretical reference levels.
These results coincide with those reported by Hernández (2012) who found a hazard ratio of
below one concerning the consumption of drinking water, which is considered a minimal or
not dangerous risk.
The Environmental Protection Agency (EPA, 2001) points out that some effects on
human health, such as alterations to enzyme levels in the blood and neurological development
deficiencies in children can occur at blood lead levels as low as those established only as
threshold levels in studies concerning exposure to lead. The EPA also points out that current
knowledge about lead pharmacokinetics indicates that the risk values obtained by standard
procedures cannot indicate the potential risk, because of the difficulty which exists in
accounting for the pre-existing levels of lead in the body: the metal bioaccumulates in the
bones with a half-life of 10 to 20 years (Saldívar Osorio et al., 1997; World Health
Organization, 2000) and is influenced by other variables such as age, health, nutritional status
and any pre-existing levels of lead in the mother during gestation and lactation. It is therefore

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generally considered that the current information on theoretical thresholds is inadequate to

decide the reference values for lead.

Environmental Risk

According to the risk assessment scale proposed by the MINAM (2011) the risk level
estimated for the human environment is 72% (Table 2), which is characterized as high. This is
explained by the high lead concentrations in foods such as corn and potatoes that exceed the
maximum allowed levels suggested by Codex Alimentarius Commission (2013) by 5 to 30
times. Lead is immobilized in bones and does not contribute to the immediate toxicity, but it
is a potential hazard (Saldívar Osorio et al., 1997; Rojas, 2002). Recent scientific research has
discovered that damage caused by lead occurs at concentrations much lower than it was
previously thought. Another form of exposure to lead for farmers in the six towns of Jauja is
the dermal contact with the contaminant in the soil and irrigation water during agricultural
activities such as: irrigation, planting, weeding and harvesting (potato). In general, heavy
metals are considered very dangerous because of their high toxicity, and high tendency to be
bioaccumulated (ATSDR, 2007).
The risk level for the natural environment is 64%, which is characterized as high; this is
attributed to the lead concentrations in the soil and aquifers that affect their quality. The
availability of organic matter containing nitrogen, phosphorus and potassium is low in the
soils and there are also slight problems of salinity further, the pH varies from slightly acidic
to mildly alkaline, the texture of the soils are sandy loam and clay loam. Soils with a high
organic matter content lack elements such as Pb, Cu and Zn. Although the sandy nature of the
soil favors infiltration by heavy metals they do not tend to be adsorbed and retained. Heavy
metals do not usually become fixed in such soils, which results in their going underground
and contaminating the groundwater (Calderon and Maldonado, 2008).
Absorption and accumulation of lead in agricultural products diminishes their quality
thus precluding the production of crops for the purpose of export. This limits the destination
of agricultural production to local and national markets and for self-consumption. The low
income of farmers (more than 50% of the population interviewed have a monthly income of
less than $300.00), the decrease in crop production in recent years and the lack of alternative
crops for export are factors that have contributed to an increase in the rate of poverty in the
region.
The overall average environmental risk estimated for the agricultural areas studied and
irrigated with water from the Mantaro river containing lead on the left bank of the province of
Jauja is high. This is attributed to the presence of lead which: affects population health, soil
fertility, the biogeochemical cycle, degrades the ecological function of the soil, decreases the
crop yield and changes the composition of the agricultural products.
Thus, it is recommended that the Local Water Authority of Junín build sedimentation
pools in “Siclachaca” treated with Sphagnum spp., to reduce lead concentration in the
irrigation water of the Mantaro River.

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CONCLUSION
Lead concentrations in agricultural products (corn and potatoes) exceeded the maximum
allowed level by between five and thirty times. In Huamali and Apata the lead concentrations
in soil exceeded the limits established by the environmental quality standard for soil. The
population studied is exposed to lead by its daily intake of corn and potatoes, with average
daily doses in females and males below the maximum recommended levels.
The HI of the population exposed to lead through the consumption of agricultural
products irrigated with the water from the Mantaro River, corresponds to a low risk, it does
not represent a significant risk for the individuals studied.
The environmental risk level estimated from the accumulation of lead in soils and
agricultural products was high on sites that are irrigated with water from the Mantaro River.
The likelihood of damage to humans, the natural environment and the socio-economic
environmental was highly likely through the intake of food with lead levels that exceeded the
maximum allowed level by lead accumulation in soil and aquifers. The severity of damage to
the human environment was critical.

ACKNOWLEDGMENTS
The authors would like to thank the National University of the Center of Perú for the
support given to the research and the users (farmers) of the irrigation channel of the left bank
of the Mantaro River, Jauja sector, for their support with study samples and with providing
information during the data collection stage.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 24

HUMAN HEALTH RISKS DUE TO AIR MERCURY

EXPOSURE PRODUCED BY ARTISANAL
AND SMALL-SCALE GOLD MINING ACTIVITIES
IN PORTOVELO, ECUADOR

María Fernanda Rivera-Velásquez1*,

Patricio Colón Velásquez-López2, Victor Hugo Gonzalez-Carrasco2
and Carmine Fallico3
1
Universidad Nacional de Chimborazo, Facultad de Ingeniería, Riobamba, Ecuador
2
Universidad Técnica de Machala, Centro de Investigaciones, Machala, Ecuador
3
Universitá della Calabria, Dipartimento di Ingegneria. Civile, Rende, Italy

ABSTRACT
Ecuador’s main gold mining activities, located in the southern region, are
characterized by large veins of ore that have been exploited since the colonial era.
Mercury pollution generated during amalgamation and cyanidation in artisanal and small-
scale gold mining processing centres threatens human health. The aim of this chapter is to
assess the effects of mercury exposure on health in terms of toxic risk (hazard
quotient,HI) in the El Pache (Portovelo district) mining community. This chapter presents
data on atmospheric mercury concentrations from sampled areas around processing plants
and when miners burn gold-mercury amalgams and zinc shavings after cyanidation.
Sampled individuals were from the mining community living and working around
processing centres, made up of administrative staff, miners, women, and children, who
are exposed to mercury by air inhalation. Samples were taken in 2007, 2008, 2011, and
2012 during winter and summer. The most important specific conditions considered for
the calculation of risk were concentration at the source, concentration at the point of
exposure, target identification, pathways and exposure modes, and exposure time. Results
show that human health risk due to mercury exceeds the limits of acceptability (HQ 1).

*
Corresponding Author E-mail: mariarivera@unach.edu.ec.

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378 M. Rivera-Velásquez, P. Velásquez-López, V. Gonzalez-Carrasco, et al.

Keywords: mining activities, mercury contamination, risk analysis, inhalation, hazard

quotient

INTRODUCTION
In Latin America, mining activity dates back to precolonial times. It continued to be very
important during colonization and has been developed up to the present. In Ecuador, artisanal
and small-scale gold mining has had a great effect across the country. Gold potential is
mainly located in the provinces of Zamora, Azuay, and El Oro, in the Zaruma-Portovelo,
Nambija, and Ponce Enriquez deposits, respectively (Sacher and Acosta, 2012). The
Portovelo mining district is the main gold mining processing centre in Ecuador.
In the district there are around 90 processing plants, which process around 4000 tonnes of
ore per day. Approximately every year, 3000 miners visit the processing centres of the
Portovelo mining district to rent equipment to recover the gold. Almost 40% of the processing
centres are located in El Pache. Although ore processing and gold recovery is the main
activity in El Pache, there are other types of business, such as gold buyers, mineral
laboratories, restaurants, car washers, mining shops, and neighborhood stores.
Artisanal miners manipulate mercury to achieve the amalgamation process for gold
recovery. Artisanal and small-scale gold mining is by itself the largest source of mercury
pollution worldwide, with effects that can last several years even after the completion of
mining activities. These activities have left a legacy of soil and water contamination of heavy
metals, which pose a risk to the health of surrounding populations. The importance of
studying the ecological health risk of heavy metals, including mercury, lies in their relation to
the consumption of irrigated crops and fish, because of the risk of bioaccumulation and
biomagnification in the food chain (Shiowatana et al., 2001). Mercury accumulation in water,
soil, and sediments presents a danger to living beings.
The use, transport, marketing, and health risks for people related to mercury have become
topics of discussion in the social policy agenda of national governmental authorities seeking
measures to minimize emissions and mitigate the noxious environmental consequences. Since
January 2013, Ecuador’s Ministry of Environment (MAE) implemented the Zero Mercury
Plan, in which the main objective was the gradual reduction of mercury utilization in various
processes.
In January 2011 the MAE’s Ministerial Agreement No. 003 defined the “List
of Hazardous Chemicals of Severely Restricted Use” through which formulation,
manufacturing, marketing, storage, use, and possession of mercury were restricted all over the
country. On July 16, 2013, the Mining Act was amended with the prohibition of using
mercury in mining operations, and the Transitional Law was enacted. In order to eradicate the
use of mercury, these actions enforced upon natural or legal persons both national and foreign
and holders of mining rights that they must apply alternative methods to eliminate mercury
progressively in mineral recovery processes for a period of two years after the date of the Act.
In April 2014 the regulations governing the procedures and requirements to obtain
authorization for the transfer and use of mercury were implemented.

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 Human Health Risks Due to Air Mercury Exposure Produced by Artisanal… 379

Figure 1. Selected sampling sites along the processing centres road in El Pache, Portovelo, southeastern
Ecuador.

Figure 2. Mercury exposure scenario for the El Pache community in the Portovelo district.

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380 M. Rivera-Velásquez, P. Velásquez-López, V. Gonzalez-Carrasco, et al.

This chapter analyzes the human health risk by quantifying the toxicological damage due
to exposure to atmospheric mercury generated in the main mining processing centre of
Ecuador, El Pache, Portovelo, where miners use mercury for gold recovery.

METHODS
Study Area and Monitoring

The assessment was carried out strategically in the El Pache area, at the Portovelo gold
mining town (Figure 1). In Portovelo there are around 90 processing centres for gold
extraction. About 30 processing centres are located in the El Pache mining community. For
monitoring purposes, nine sites were selected along a 3 km stretch of the main road in front of
the processing centres. Air mercury concentrations were monitored in the summer (dry) and
winter (rainy) seasons in different years: in September, October, and November (summer) for
2007 and 2011, and in January and February (winter) for 2008 and 2012. A total of 603
samples were collected, 306 in the dry season and 297 in the rainy season. Specifically, 270
samples were taken in 2007, 36 samples in 2011, 270 in 2008, and 27 in 2012. Total mercury
concentrations in ambient air were determined using the LUMEX portable atomic absorption
spectrophotometer (RA-915+) with an optical length of 9.6 m and a detection limit of 1.0
ng/m3.
Figure 2 describes the exposure scenario considered in the El Pache mining community
case study. Two groups were assessed for risk assessment. The first group corresponded to
the floating population (occasional visitor, administrative staff, and miners); and the second
group corresponded to the community settled in the town—men, women, and children ages
one to six years. ‘Occasional visitors’ are individuals entering the area sporadically and
staying a limited amount of time. The miners at times visit the processing centres for ore
processing that involves burning mercury. The settled population consists of processing
centre administrators working in the contaminated environment and the population living in
the area. The workers of the processing centres are exposed to gaseous elemental mercury and
gold dust.

Quantitative Estimating of the Noncarcinogenic Hazard

The average daily dose (ADD; mg/kg/day) from inhalation of mercury was calculated as
follows, in equation 1:

ADD=(C*IR*F) / BW (1)

where C is the concentration at the point of exposure (mg/m3), IR is the intake rate from the
contaminated medium (m3/day), F is the exposure factor (unitless), and BW is the body
weight (kg).
The criterion used to select the representative concentration at the point of exposure is
given by the precautionary principle. For this reason we used the so-called upper confidence

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 Human Health Risks Due to Air Mercury Exposure Produced by Artisanal… 381

limit (UCL). This procedure calculates the representative concentration through a data set
obtained in situ. This value represents a highly conservative estimate of the true value of the
average. To calculate statistical significance we used EPA’s Pro Upper Confidence Level
software (USEPA, 2013). The exposure factor was calculated via equation 2:

F=(EF*ED) / AT (2)

where EF is the frequency of exposure (day/year), ED is the exposure duration (years), and
AT is the average time (ED x 365 days/year). The exposure factor gives the dose averaged
over the exposure period. Site-specific exposure values, namely the parameters that contribute
to the determination of F, are published in the EPA’s Exposure Factors Handbook (USEPA,
2011). The key to calculating the most accurate exposure dose is to identify values that relate
specifically to the exposure situation being assessed. If site-specific information is not
available, several conservative exposure assumptions can be applied. In order to more
accurately calculate the dose of exposure related to a pollutant, it is important to gather proper
data from the studied site.
Moreover, one must take into account that the dose-response relation describes the effect
on an organism caused by the amount and the specific route of exposure to an agent
(delivered dose) after a certain exposure time (Crump et al., 1976; Rivera-Velásquez et al.,
2013). As the dose increases, the response tends also to increase. At low doses there may be
no response, i.e., a body has a certain degree of tolerance upon exposure to a particular
substance. A tolerable dose exists below which no adverse effects on human health are
present for noncarcinogens (Davis and Svendsgaard, 1990; Daniels et al., 2000; Steenland
and Deddens, 2004).
A reference dose (RfD) is a daily oral intake rate that is estimated to pose no appreciable
risk of adverse health effects. From the RfD, the HQ assessment is calculated as follows in
equation 3:

HQ=ADD/RfD (3)

where HQ is the hazard quotient (unitless) and RfD is the reference dose (mg/kg/day). The
standards for human protection defined by various international agencies provide the
following acceptability criterion (APAT, 2008; USEPA, 2011): HQ (exposure to one or more
substances) <1.0.

Health Risks Due to Air Mercury Contamination Produced by

Mining Activities

The HQ was calculated for each sampling period. To calculate the representative
concentration, the ADD (equation 1) was used for the whole series of samples corresponding
to 603 in situ samples (306 in summer and 297 in winter). The approach taken to determine
this value was 95% UCL. Two UCL values were calculated using ProUCL 5.0 software
(USEPA, 2013).

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382 M. Rivera-Velásquez, P. Velásquez-López, V. Gonzalez-Carrasco, et al.

The HQ was calculated by the deterministic method (Rivera-Velasquez et al., 2013) and
evaluated assigning a single value in correspondence to the receptors considered.
For the HQ assessment, two different groups (and subgroups) of receptors were
considered: the floating population (occasional visitors, miners, and administrative staff) and
the settled community (men, women, children). The first group corresponds to people who
visit the site for work or as casual visitors but that do not live in the area. The second group
refers to people who conduct their entire daily activities within the studied area. The BW and
IR values, considered in the HQ equation, were assigned according to the EPA database
(USEPA, 2000, 2011). The other input values were obtained assuming site-specific
characteristics, such as ED and EF.

RESULTS
Initially the concentration data obtained from the examined samples was subjected to a
careful statistical analysis. Table 1 shows the mean, standard deviation, minimum, and
maximum values of mercury concentration for each sampling point and each year: 2007
(n=270), 2008 (n=36), 2011 (n=270), and 2012 (n=27).
For the HQ model, mercury concentrations were computed using 95% UCL criteria
corresponding to 0.004302 mg/m3 and 0.006428 mg/m3 for winter and summer seasons,
respectively. For mercury inhalation, the RfD used in all cases was 8.571x10-5 mg/kg/day
(Table 2).
Figure 3 shows the histogram of the HQ values for the six receptors considered. The
histogram shows that the reference HQ is exceeded for almost all receptors with the exception
of the nonresident people (floating population) in the winter season.

Figure 3. HQ values for the dry (winter) and rainy (summer) seasons for the El Pache mining
community.

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 Table 1. Values for mercury air concentration (ng/m3) detected
in the El Pache zone

Site Winter 2007 Winter 2011

Mean SD Min Max Mean SD Min Max
Site 1 1051 1306 198 5520 905 945 353 2317
Site 2 1029 798 168 3110 935 578 444 1746
Site 3 1003 448 301 1740 1165 448 683 1715
Site 4 2608 2840 584 10150 2300 2229 1038 5639
Site 5 8139 10581 766 35216 7550 8012 1491 19307
Site 6 10844 20616 891 111105 9636 7662 2033 19940
Site 7 10061 11090 960 48600 9991 5906 2254 16521
Site 8 2042 1758 665 6871 1820 1415 901 3919
Site 9 1242 603 211 2100 1142 486 665 1680
Mean ± SD For winter: 4202±8935
Summer 2008 Summer 2012
Mean SD Min Max Mean SD Min Max
Site 1 320 143 120 773 320 41 280 362
Site 2 633 571 110 2185 633 495 181 1162
Site 3 450 129 148 692 451 32 428 488
Site 4 474 166 158 771 474 50 421 520
Site 5 1254 1705 298 5898 1254 1243 434 2684
Site 6 12815 19549 399 50000 12815 20795 591 36826
Site 7 4383 5621 580 22416 4383 5167 931 10323
Site 8 490 191 150 861 489 204 312 712
Site 9 391 130 193 587 391 149 220 494
Mean ± SD For summer: 2357±7693
SD: standard deviation; Min: minimum; Max: maximum.

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Table 2. Hazard quotient and related parameters for floating and settled population in
the El Pache mining community

Parameter Floating population Settled community

Occasional Miners Administrative staff Men Women Children
visitors (1-6 years)
IR (m3/day) 15.2 15.2 15.2 15.2 11.3 10
EF (years) 24 104 240 350 350 350
BW (kg) 70 70 70 70 65 15
ED (years) 3 8 8 24 24 5
AT (years) 3 8 8 24 24 5
HQ (dry) 0.72 3.11 7.17 10.45 8.37 32.09
HQ (rainy) 1.07 4.64 10.71 15.62 12.50 47.94
IR: intake rate; EF: exposure frequency; BW: body weight; ED: exposure duration; AT: averaging time;
HQ: hazard quotient. For calculation of HQ the value of AT is equal to ED.

DISCUSSION
Some studies indicate mercury as a carcinogen, although there is no definitive evidence.
The EPA considers mercury as Class D, “Not Classifiable as to Human Carcinogenicity”
(Burger and Gochfeld, 2012). The gold mining operation sets a good example of the possible
exposure pathways for air mercury contamination. In the present case study, the risk from
mercury in air was quantified via the HQ.
The values of Table 1 show that the highest concentrations of mercury were detected for
sites 6 and 7, where more processing centres and gold shops are located. This means that
mercury vapor comes from amalgamation and cyanidation gold processing and the amalgam
refining process in gold shops (Velasquez-Lopez et al., 2011).
Moreover, seasonal variation of mercury concentration in the air was recorded during the
study. In fact, as highlighted in Table 1, mean mercury concentrations were greater in
summer (2357 ± 7693 ng/m3; n=297) than in the winter season (4202 ± 8935 ng/m3; n=306).
Variation among the values in each season reveals the variation in mercury concentration
among different sites. This variation demonstrates the existence of specific points of mercury
emission within the study area. In Ecuador, the rainy season is characterized by having the
highest temperatures, rain, and evaporation. The results of the study show that in the summer
season, characterized by a dry environment and high temperatures, concentrations of mercury
were higher than in the winter season.
Taking into account the assumption of the toxicological analysis, and placing particular
emphasis on the EF and ED, in the floating population the values of these parameters are
lower than in those of the community settled in the town. This leads to lower HQ values in
the floating population in comparison to those of the settled community (Table 2).
The children are the most vulnerable target (settled community), largely exceeding the
maximum recommended HQ in both summer and winter seasons. The least prone group is
that of the “occasional visitors,” considering that the maximum recommended HQ was
only slightly exceeded in the summer. In increasing order of vulnerability are nonresidents,
miners, administrative staff, men, women, and children (Table 2). Other studies carried out in

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 Human Health Risks Due to Air Mercury Exposure Produced by Artisanal… 385

Portovelo reported mercury values around 1500 ng/m3 in exhaled air from the breath of
miners after the amalgamation process (Velasquez-Lopez et al., 2010; Gonzalez-Carrasco et
al., 2011). This confirms that miners can uptake mercury directly when they are exposed to
mercury vapors. Those miners are highly exposed, but temporarily, and the residents are more
constantly exposed but to lower concentrations of mercury in the air that remain after burning
mercury-contaminated products such as amalgams. Therefore, risk assessment can vary from
one group of people to another according to their working conditions and the time of
exposure.
Risk assessment for women and children is often calculated according to the sensitivity
of the developing fetus and young children (ATSDR, 1999). The exposure limit established
by the EPA to protect human health (0.2 ppm) probably does not protect the fetal and the
neonatal brain (Landrigan et al., 2006). Mercury has neurobehavioral effects in adults and
neurodevelopmental effects on the fetus (APAT, 2008; Burger and Gochfeld, 2012).
Mercury volatility increases with increasing ambient temperature. The equilibrium
concentration in the air is 2.0 mg/m3 at 25° C, increasing volatility eight times with increasing
temperature from 20° C to 50° C (Mantyla and Wright et al., 1976). From this scenario, the
risks due to mercury exposure in the rainy (summer) season should be of concern: the highest
value recorded was 50,000 ng/m3 in the dry (winter) season and more than double that
(111,105 ng/m3) in the summer. Other studies reported similar concentrations in Portovelo
(Velasquez-Lopez et al., 2010) and artisanal gold mining communities in Colombia (Cordy et
al., 2011).
According to topography, wind speed and direction, mercury can remain in the same site
or it can travel long distances from the source of contamination (Hylander et al., 1994),
increasing or reducing the risk on site. Therefore, risk due to mercury exposure will be
determined by the time of exposure, its concentration, the topography of an area, and
meteorological factors in the contaminated area. In this study it has been shown that many
seasonal factors, such as temperature and rainfall, are very important environmental
indicators of mercury pollution conditions.
It is believed that mercury concentration in the air will decrease, because the Ecuadorian
government has recently enforced its elimination. However, it is known that miners are still
using mercury and burning amalgams in their homes. Until complete mercury elimination and
a new work model operates, it is important to emphasize the need to implement a monitoring
campaign for mercury in the air, in an attempt to understand mercury dispersion and its
behavior in the environment.
The recommended limit for public exposure to inorganic mercury vapors is 1.0 ng/m³
(WHO, 2000). In environments where workers are subjected to long-term exposure to
mercury vapors, the LOAEL (lowest-observed-adverse-effect-level) might be around 15,000-
30,000 ng/m³ (WHO, 2000). The recommended health-based exposure limit for metallic
mercury is 25,000 ng/m³ for long-term exposure—the time weighted average (TWA)
concentration for an 8-hour day and a 40-hour work week during which workers can be
repeatedly exposed without adverse effect. The normal atmospheric levels of mercury in rural
areas are about 2-4 ng/m³, and in urban areas about 10-20 ng/m³ (Veiga and Baker, 2004). It
has been reported that the usual levels of mercury in the air in El Pache, Portovelo are around
10,000 ng/m³ in the dry season and around 5000 ng/m³ in the rainy season (Gonzalez-
Carrasco et al., 2011), and recent policy action of the government of Ecuador towards

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386 M. F. Rivera-Velásquez, P. C. Velásquez-López, V. H. Gonzalez-Carrasco et al.

mercury elimination could have diminished such levels. A continuing monitoring of air
mercury contamination in El Pache, Portovelo is suggested.
To protect the health of miners, processing plant workers, and the settled community,
the authorities should recommend implementation of safety procedures and continuous
monitoring of mercury levels, atmospheric conditions, and the health of the exposed
population. Implementation of more frequent monitoring and decision making that
emphasizes early detection and treatment of toxicological signs would help to guarantee safe
occupational levels of mercury. The creation of educational programs on hazard management
and risky behaviors in the contaminated environment is important. These recommendations
can be implemented satisfactorily through a mercury reduction and elimination program.
Although this study has focused on the risk of mercury from air contamination, it is also
necessary to know the concentrations of mercury in the soil and in biota in the area. Intensive
monitoring of populations at high risk from mercury poisoning in artisanal and small-scale
gold mining communities is strongly recommended in order to provide evidence of adherence
to existing regulatory criteria. The human health risk assessment methodology facilitates
conducting of assessments at a contaminated site responsive to local and federal governments.

CONCLUSIONS
The survey carried out in the community of El Pache indicated that artisanal and small-
scale gold mining activities present a high risk to human health, owing to pollution caused
by mercury used for these activities. This community has been exposed to mercury levels
above the recommended limits. New campaigns for sampling and monitoring are needed to
confirm atmospheric mercury concentration and behavior over time, in order to know the
effectiveness of the implemented governmental or nongovernmental measures.
From the results obtained, with only one exception (nonresident people in winter), the
current concentrations of mercury vapor in samples of air collected in the El Pache district
exceeded the maximum recommended levels of HQ=1, with relevant influence on the health
of the surrounding community. Children are the most vulnerable group, with values three to
four times higher in comparison to other exposed groups. The risk assessment in this study
provides semiquantitative information to determine the potential damage to humans and
provides an early warning of potential harm in order to help responsible public decision
makers.
The high values of air mercury demonstrate urgency for implementing adequate practices
regulating gold mining activities, in particular concerning artisanal and small-scale gold
miners and owners of processing plants. Alongside government efforts to eradicate mercury,
permanent environmental monitoring including health assessment of exposed people will
increase awareness of mercury pollution not only in Portovelo but also in other regions where
artisanal and small-scale gold mining operates. In order to prevent damage to the health of the
community of El Pache, further studies should be performed to determine the point source,
exposure pathways, concentration in all forms, and the concentration in the food web.

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 Human Health Risks Due to Air Mercury Exposure Produced by Artisanal… 387

ACKNOWLEDGMENTS
This chapter was based on work supported by the Sustainable Managing Environmental
Health Risk, Ecuador project financed by the Canadian International Development Agency
and the University of British Columbia. The authors express special gratitude to the reviewers
for their comments and suggestions to publish this manuscript.

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Estadísticos sobre la Minería Industrial en el Ecuador. Quito, Ecuador: Universidad
Politécnica Salesiana Press.

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Shiowatana, J., McLaren, R. G., Chanmekha, N., Samphao, A., 2001. Fractionation of arsenic
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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 25

ENVIRONMENTAL IMPACT OF AGRICULTURAL

ACTIVITIES ON HUMAN AND WILDLIFE
POPULATIONS FROM
PALIZADA, CAMPECHE, MEXICO

Jaime Rendón von Osten1,*, Lucia Guilhermino2,3

and Amadeu M. V. M. Soares4
1
Instituto EPOMEX, Universidad Autónoma de Campeche,
Campeche, Mexico
2
ICBAS – Institute of Biomedical Sciences of Abel Salazar, University of Porto,
Department of Population Studies, Laboratory of Ecotoxicology, Porto, Portugal
3
CIIMAR/CIMAR-LA – Interdisciplinary Centre of Marine and Environmental Research,
University of Porto, Research Group on Ecotoxicology, Stress Ecology and
Environmental Health, Porto, Portugal
4
Departamento de Biología and CESAM, Universidade de Aveiro, Aveiro, Portugal

ABSTRACT
The main impacts of agricultural activity are land use change and the use of
dangerous pesticides. Approximately 25 million pesticide poisonings occur annually
among agricultural workers in Southern countries. The state of Campeche is located in
Southeastern Mexico. Here, more than 200,000 hectares are dedicated to agricultural
activity. Many pesticides are employed on rice crops grown in the Palizada River system,
mainly carbofuran, chlorpyrifos, glyphosate, monocrotophos, metomil, edifenfos,
benomyl, among others. In order to assess the impact of agricultural activities on human
and animal populations from the Palizada River system, mosquito fish (Gambusia
yucatana) and black-bellied whistling duck (Dendrocygna autumnalis) were chosen as
bioindicators for the analysis of biomarkers of exposure and effect to pesticides. Farmers
and agricultural workers from the communities of El Juncal, Ich-Ek, Suc-Tuc and
Castamay were also evaluated because they are exposed to pesticides during application.

*
Corresponding Author E-mail: jarendon1@gmail.com.

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390 Jaime Rendón von Oste, Lucia Guilhermino and Amadeu M. V. M. Soares

The integration of information generated in wild fish and exposed in situ reflected a
concordance between percentages of acetylcholinesterase (AChE) activity inhibition
during the pesticide application period. Results indicate that whistling ducks are exposed
to pesticides (inhibition above 30% of AChE activity), at least during the season of
intensive application. All agricultural workers are exposed to dangerous pesticides, and
farmers from Suc-Tuc and Ich-Ek have a significant inhibition of AChE activity. It is
recommended the use of pesticides that are less aggressive to the environment and human
health. Further field studies incorporating a systematic monitoring of wildlife populations
and investigation of population parameters are particularly important for local pesticide
management.

Keywords: pesticides, aquatic ecosystem, biomarkers, agriculture, Mexico

INTRODUCTION
Agricultural activity has had an adverse effect not only on the physical environment but
also on human and wildlife health, mainly due to habitat loss and alteration, and pesticide
exposure (Tilman et al., 2011).
In 1984, the United Nations Environment Programme (UNEP), in conjunction with
several national authorities and international organizations, prepared a list of priority
chemicals/pollutants to focus on major environmental problems. Pesticides were included in
this list because they enter in the environment in significant amounts as a result of human
activities, widely distributed and in some regions they represent potential threats to
ecosystems as well as to human health due to their toxicity and/or persistence.
Mexico is a great consumer of pesticides; the use of agrochemical in 2008 was around
93,000 tons (23% insecticides; 33% herbicides; 43% fungicides) (SEMARNAT, 2010). More
than 1500 active ingredients have been registered as pesticides. Formulators mix these
compounds with one or more of some 900 “inert” ingredients to create approximately 50,000
commercial products registered for use (PAHO, 1993).
In the majority of Southern countries, such as Mexico, insecticide use predominates, with
a correspondingly higher level of acute risk. Although the use of pesticides in Southern
countries is small in comparison to their use in Northern countries, it is nonetheless
substantial and continuously growing. Pesticide use is particularly intense where export crops
predominate.
As xenobiotics, pesticides suffer biotransformation in the body and thus chemical
detection is not easy. Biomarkers are therefore determined to confirm and assess the exposure
of individuals or populations, or the deleterious effect to a particular substance, providing a
link between external exposures and internal dosimetry. Among the biomarkers used to assess
the environmental exposure to pesticides are acetylcholinesterase (AChE, EC 3.1.1.7),
glutathione-S-transferase (GST, EC 2.5.1.18) and lactate dehydrogenase (LDH, EC 1.1.1.28).
The fluvial lagoon system of the Palizada River (FLSPR; 18º19′04″/18º30′13″N;
91º44′36″/91º51′31″W) is located in the southwestern part of Terminos Lagoon in the State of
Campeche, Mexico (Figure 1). The drainage basin is about 2460 km2 (Benitez and Barcenas,
1996). With at least 64 fish species and four primary plant communities, Palizada River
system (PRS) is one of the most important ecological lagoon-estuarine ecosystems in the
southern Gulf of Mexico (Fuentes-Yaco et al., 2001).

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 Environmental Impact of Agricultural Activities on Human … 391

Three climatic seasons exist in the FLSPR: the rainy season (from June to September),
the “nortes” season (from October to January) and the dry season (from February to May).
The rainy season is characterized by strong and intensive rain (mean of the season: 157.75
mm); the “nortes” season is characterized by a cold wind from the North (mean of the season:
68.7 mm) (Fuentes-Yaco et al., 2001); and the dry season is characterized by a relatively low
rainfall, with a season mean of 24.1 mm.
In the study area, there are two main human activities with potential negative
environmental impact: agriculture and cattle ranching. The most important activity is
agriculture, with about 10,000 hectares of rice fields of which 5,000 are continuously irrigated
by several small channels. At the beginning of the 90s, herbicides such as propanil, 2,4-D and
molinate were identified as the pesticides most employed by farmers (Benitez and Barcenas,
1996). At present, carbofuran, chlorpyrifos, glyphosate, propanil, amines, monocrotophos,
metomyl, edifenphos and benomylare intensively applied in these agriculture fields.
In this chapter the impact of the agricultural activity and the pesticide use in the FLPRS,
and on local human populations is presented. To attain this general goal, four specific
objectives were considered:

Figure 1. Map of fluvial-lagoon system of Palizada River (FLSPR) with indication of sampling sites
(black triangles).

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392 Jaime Rendón von Oste, Lucia Guilhermino and Amadeu M. V. M. Soares

1) Study the effect of land cover on the FLSPR water quality.

2) Develop and validate an integrated approach (including a biomonitoring study, in situ
assays and water quality parameters) with Gambusia yucatana to diagnose
environmental contamination by chemicals.
3) Diagnose the exposure of wild duck populations (Dendrocygna autumnalis) to
anticholinesterase pesticides.
4) Assess the effects of pesticides used in agriculture on farmers.

METHODS
Fieldwork was carried out every one or two months, during two years. Samples were
collected in nine sites in the FLSPR watershed (Figure 1). Each sampling site was
georeferenced in UTM and briefly characterized according to its ecological and vegetable
characteristics (Table 1).

Table 1. Sampling sites and its georeference in UTM

Site Location Longitude Latitude Characteristics

1 Area of immediate 627,319.85 2,044,602.11 Mouth of the Palizada River and
influence of the Terminos connected to Terminos Lagoon. High
Lagoon salinity, surrounded by mangrove.
Fishing activity.
2 San Francisco Lagoon 630,894.40 2,039,422.24 Lagoon without direct fluvial inflow with
submerged hydrophytes and surrounded
by mangrove. Poor fishing activity.
3 Este Lagoon 627,061.84 2,038,340.87 Lagoon with fluvial inflow without
submerged hydrophytes. Surrounded by
unforested wetland. Intense fishing
activity.
4 Palizada River 623,377.28 2,037,007.77 Influence of wastes from Palizada town.
Surrounded by unforested wetland.
Intense fishing activity.
5 Confluence of El Vapor 622,065.40 2,035,867.28 Unforested wetland. Surrounded by
Lagoon and Palizada unforested wetland. Fishing activity
River
6 El Vapor Lagoon 623,685.85 2,030,189.96 Lagoon with fluvial inflow with very
scarce submerged hydrophytes.
Surrounded by unforested wetland.
Fishing activity.
7 Confluence of Marentes 623,544.47 2,024,307.84 Water body with unforested wetlands.
and Las Piñas streams
8 Marentes stream 626,932.26 2,020,713.36 Influence of incipient cattle ranching
activity, surrounded by grassland. Scarce
fishing activity.
9 Las Piñas stream 626,607.70 2,012,150.33 Strong agricultural activity in the area
(rice crop). Scarce fishing activity.

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 Environmental Impact of Agricultural Activities on Human … 393

Land Cover Changes in the FLSPR Basin (1980 – 2000)

The watershed boundaries of FLSPR were established using topographic information and
1:50,000 scale maps (INEGI, 2000). The FLSPR basin is a geographic area of land bounded
by topographic features and land altitude that drains waters to a shared destination. This area
is a large temporally flooded plain; therefore, the division of the basin was traced according to
the drainage or crestlines that mark the hydrologic map. In order to determine the land cover
change, differences between data of each land use cover type reported in the National Forest
Inventory 2000 and data from 1980 reported by Benítez et al. (1995) were calculated in
Arc/View V.3.2 (ESRI, 1999).

Variables Analyzed and Analytical Methods

Sixteen water parameters were determined: temperature, dissolved oxygen (DO), pH,
redox, salinity, conductivity, depth, transparency, hydrogen sulfide, ammonium, nitrites,
nitrates, phosphorus, silicates, sulphates and hardness.
The indexes presented in the results section were calculated with the values obtained
during the whole sampling period. A Water Quality Index (WQI) (Rendón-von Osten et al.,
2006) was obtained using five of the mentioned variables as follows:

WQI=(pH + Dissolved Oxygen + Ammoniun + Nitrites + Phosphorus)/5,

where 5 is the number of variables considered for index calculation and has been introduced
to obtain a WQI value ranging from 0.0 to 1.0.

In Situ Assays

For the in situ assays, mosquito fish were cultured under controlled conditions in a
container filled with 1,000 L of local well water (renewed every 15 d) with submerged
vegetation. Containers were maintained in outdoor facilities with natural photoperiod and
temperature. These fish were considered as control in order to compare the results of each
studied biomarker.
In situ assays were performed using test chambers (Rendón-von Osten et al., 2006) with
ten G. yucatana fish in each chamber. Chambers with fish were exposed during 21 days in
three sampling sites: Palizada River (site 5), Marentes stream (site 8) and Las Piñas stream
(site 9) (Figure 1).
After 21 days, mortality was recorded and the surviving fish were weighed, body size
measured and sacrificed by decapitation. Decapitation is an acceptable method, provided the
procedure is performed quickly and accurately (Nickum et al., 2004). Immediately after
decapitation of the animals, head, gills and a piece of dorsal muscle were removed and
prepared for biochemical determinations. Physical and chemical water parameters were
monitored at the beginning and at the end of the in situ study as described by Rendón von
Osten et al. (2006).
In situ assays were performed in each of the three selected sites in different periods
according to climatic seasons: at the beginning and at the end of the dry season (February and

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394 Jaime Rendón von Oste, Lucia Guilhermino and Amadeu M. V. M. Soares

May 2000), during the rainy season (July 2000) and during the “nortes” season (November
2000). In 2001, fish were exposed in February (beginning of the dry season) and in August
(middle of the rainy season) to complete the information obtained in the previous year.

Biomonitoring

A total of 310 native G. yucatana were evaluated, being 232 male and 78 female. Of
these, 95 fish were from the stream in the area with cattle ranching activity (site 8), 116 from
the stream near the agricultural area (site 9) and 99 from the Palizada River (site 5) (Figure 1).
Immediately after decapitation of the animals, head, gills and a piece of dorsal muscle were
removed and prepared for biochemical determinations.

Black-Bellied Whistling Duck Sample Collection

To assess the impact of rice crop pesticide used on natural populations of D. autumnalis,
26 ducks were sampled during a period of intensive agrochemical use (November) and 23
ducks during a period with presumably no use (July). All the ducks used were obtained
from local inhabitants who hunted them for meat consumption. After decapitation, the
forebrain was immediately removed and put in ice-cold phosphate buffer until analysis of
acetylcholinesterase (AChE). Duck sex, body weight, body length, and other morphometric
parameters were recorded. To obtain information about the range of AChE brain activity in
non-exposed black-bellied whistling ducks, seven adult individuals from a local duck farm
were used as reference group (data from Rendón von Osten et al., 2005). These ducks were
considered as references because the duck farm is located 110 km from the agricultural area.

Biochemical Determinations in Mosquito Fish and Ducks

Activity of AChE in mosquito fish muscle and duck brains was determined by the
method described by Ellman et al. (1961). Gill GST activity was determined by the method of
Habig et al. (1974). The activity of LDH in muscle was determined by the method of Vassault
(1983). All the methods were adapted to microplate. A Lab system Multiskan EX microplate
reader was used for all determinations.

Human Blood Sample Collection and Analysis of Erythrocyte AChE

Blood samples from a fingertip of farmers and agricultural workers were taken during
the period of insecticide application. Samples were taken immediately after the end of the
workday. In order to minimize the bias of AChE reactivation from carbamate exposure, a
Test-Mate OP field Kit was used to determine erythrocyte cholinesterase levels immediately
after blood sampling (Rendón von Osten et al., 2004).

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 Environmental Impact of Agricultural Activities on Human … 395

A questionnaire about the possible symptoms of pesticide poisoning experienced by

farmers was performed at the end of the workday in which several variables were considered
(socio-demographic variables such as education levels; unsafe pesticide practices such as
eating and smoking in the field, type of poisoning and symptoms classified as mild,
moderated and severe). The interview included the communities of El Juncal, Ich-Ek, Suc-
Tuc and Castamay.

Data Analysis

Analysis of variance (ANOVA) was used to compare different treatments in the

experiments with selective inhibitors and pesticides, as well as to compare AChE activity
among reference ducks, birds collected during the application period, and ducks captured
when no pesticides were applied in the field. If ANOVA indicated statistical significant
differences among treatments, Tukey’s HSD multiple comparisons test was carried out to
identify significantly different treatments. Student t-test was used to compare AChE activity
between males and females.

RESULTS
At present, the surface of FLSRP dedicated to agricultural activity is 30,075 ha (12.2% of
the water basin surface). At the beginning of the 80s, the agricultural activity in FLSPR
occupied only 2,450 ha (1.0%). At the beginning of the 90s the entire agricultural surface was
dedicated to rice, totaling 20,580 ha (8.4% of the water basin surface) (Benítez et al., 1995).
The main change in the vegetation cover was due to agriculture, which had an increment
of more than 1,200% during a period of 20 years. Reduced vegetation covers, possibly
changed to agricultural areas, were mangrove, popal-tular and forest, that decreased from
169,050 ha in 1980 to 145,158 ha in 2000, a reduction of almost 16%. In 2000, the natural
vegetation present in the basin occupied 28.5% of its surface, while an area of 169,050 ha
(36% unforested wetland, 25% forest, 4.0% mangrove and 4.0% of disturbed forests) was
reported in the 80s and an area of 142,100 ha (approximately 58.0% of basin surface) was
reported for the 90s by Benítez et al. (1995).

Water Quality in the FLSPR

Of the obtained data records, 27.8% failed to fulfill DO requirement, 74.1% were above
ammonium limit value, 9.3% of the data were above nitrite limit concentration and all the
records were above the phosphorous limit according to the DOEU (2006).
The WQIs ranged from 0.49 (July 2000) to 0.70 (March 2000) in Marentes, from 0.44
(March 2000) to 0.73 (November 2000) in Las Piñas, and from 0.54 (September 2001) to 1.00
(May 2001) in Palizada.
Statistically significant differences among sites were found (F8,162=3.82; p < 0.0001). The
lowest WQI value (WQI=0.57), which was significantly different from all the others, was

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396 Jaime Rendón von Oste, Lucia Guilhermino and Amadeu M. V. M. Soares

determined for site 8, under the influence of cattle ranching activity. The low WQI is mainly
due to low DO values. From the remaining sites, the lowest WQI was calculated for site 9
(WQI=0.71), located in the agricultural area. However, differences among these sites were
not statistically significant.
Significant differences in WQI mean values were found among different months
(F13, 112=12.31; p < 0.0001). The lowest values were found in June 2000 (WQI=0.59) and
September 2001 (WQI=0.58), in the rainy season. OD and nitrites were the variables that
influenced the WQI behavior.
During the 1999-2000 climatic cycle, WQI was higher during the rainy season with
exception of site 8 which had in the three seasons a very low value. However, during the
2000-2001 cycle the highest WQI values were found in the dry season, with the exception of
site 8 that had low WQI values in all the seasons.
Mortality of mosquito fish recorded in each site during the in situ assays is shown in
Figure 2 and weight alterations are shown in Figure 3. In February and May 2000 (dry
season), no mortality occurred in fish exposed in the Palizada River. In addition, these fish
gained 6% in body weight. After this period, mortality was 60% in July 2000 (rainy season),
30% in November 2000 (nortes season) and 100% in August 2001(rainy season). Surviving
fish had a loss of body weight of 12% in July and of 20% in November. In the site with cattle
ranching activity, 20% of mortality was found in February 2000 (nortes season), with a loss
of 22% in the body weight, and 100% mortality was observed in all the other months. In the
site with agricultural activity, the mortality was always equal to or higher than 80%, except in
November 2000 where a low mortality (10%) and only an 8% reduction of body weight were
found.

Figure 2. Percentage of mortality of Gambusia yucatana recorded per month in in situ assays at each
sampling site.

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 Environmental Impact of Agricultural Activities on Human … 397

Figure 3. Monthly Gambusia yucatana weight variations in in situ assays at each sampling site.

Figure 4. Percentage of AChE muscle activity measured in Gambusia yucatana that survived until the
end of the 21-day in situ assay. Values above the columns represent the percentage of AChE inhibition.

Figure 4 shows the percentage of AChE activity in fish that survived the 21-day exposure
relatively to the activity determined in correspondent time periods in fish from natural
populations that had been maintained in the laboratory.
In Palizada River, only in November did the percentage of AChE activity in fish not
show a decrease, while during May and July, and March and September fish exposed in the
Palizada River showed an AChE depression.

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398 Jaime Rendón von Oste, Lucia Guilhermino and Amadeu M. V. M. Soares

Figure 5. Percentage of GST gill activity measured in Gambusia yucatana that survived until the end of
the 21-day in situ assay. Symbol > means the percentage of GST activation.

Figure 6. Percentage of LDH muscle activity measured in Gambusia yucatana that survived until the
end of the 21-day in situ assay. Symbol < means the percentage of LDH inhibition.

Figures 5 and 6 show the percentage of gill GST and muscle LDH activity in fish
exposed in situ relatively to the activities determined in laboratory animals. GST activities of
fish exposed in the Palizada River were similar to control animals in November 2000 and
September 2001, and a slight increase of activity was observed in March (about 17%). A high
inhibition of LDH activity (above 78%) was found in November in fish exposed in both Las
Piñas stream and Palizada River. Almost full inhibition was observed in fish exposed in the
Palizada River in March and about 50% of inhibition was found in animals exposed in this
site in September.

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 Environmental Impact of Agricultural Activities on Human … 399

In general, fish from the Palizada River showed a pattern of AChE inhibition similar to
that of fish from the Las Piñas stream, having in the most part of the months a higher
depression (Figure 7).
In the case of GST, activity pattern variation from March 2001 until September 2001 was
similar for all the sampling sites (Figure 8). Inhibition of GST activity relatively to laboratory
fish was 8% in the site with agriculture activity and an induction of GST (41%) was observed
in the Palizada River in March; an inhibition was recorded from 13 to 34% in May, and 20%
in all sites in September.

Figure 7. Temporal variation of percentage of AChE activity in muscle of native Gambusia yucatana.
Symbol < means the percentage of AChE inhibition.

Figure 8. Temporal variation of percentage of GST activity in gill of Gambusia yucatana from three
sampling sites. Symbol > means the percentage of GST activation.

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Figure 9. Percentage of temporal variation of LDH activity in muscle of Gambusia yucatana. Symbol <
means the percentage of LDH inhibition.

Figure 10. Brain cholinesterase activity in reference ducks and in wild ducks (Dendrocygna
autumnalis) during pesticide application and when no pesticide was applied. Symbol < means the
percentage of AChE inhibition.

The pattern of fish LDH variation from November 2000 until September 2001 was
similar for all the sampling sites (Figure 9). Inhibition of LDH activity relative to laboratory
animals (control) was from 51 to 63% in November, 82 to 94% in March, 65 to 76% in May
and 13 to 31% in September.
In relation to the monitoring of ducks, significant differences in AChE activity (F2,
54=6.21, p=0.003) were found among reference ducks (ducks reared on a farm), ducks
collected during the season of intensive pesticide use (November), and ducks collected during
the period without pesticide application (July) (Figure 10). Compared to the reference group,

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 Environmental Impact of Agricultural Activities on Human … 401

ducks captured during the pesticide application period showed a decrease of 34.2% of AChE
activity.
According to all the farmers interviewed (n=121), the main mild symptoms associated
with pesticide exposure were headache (15.7%), physical weakness (10.7%), nausea (8.3%)
and low appetite (8.3%). Moderate symptoms most often reported were sweating (4.1%) and
tiredness (4.1%). Tremor (2.5%) was the primary severe symptom reported.
In Ich-Ek, 22 farmers with AChE activity levels indicating poisoning due to pesticide
exposure were found, threeof them with values compatible to severe poisoning. In addition, a
case of poisoning in El Juncal, three in Castamay and four in Suc-Tuc were found. However,
in the period of blood sample collection, none of the subjects from any of the four
communities reported symptoms of pesticide poisoning.

DISCUSSION
The results indicated that during the last 20 years, in the FLSPR, the land use change has
been drastic, mainly in agriculture cover that increased more than 1,000%; other land covers
decreased in different percentages: unforested wetland decreased by nearly 25% and the
grassland about 20%. It has been demonstrated that an increase of agricultural activity has an
important environmental impact due to the use of agrochemicals, such as fertilizers and
pesticides, and soil erosion (Neumann et al., 2002).
Land use can influence the water quality. Indeed, the results obtained indicate negative
correlations between agriculture cover, pH and DO and sulphates concentrations. When the
WQI was applied to the PRS, the results showed a clear differentiation between sites with
diverse human activities. From the nine sampling sites studied in the PRS, Marentes
(WQI=0.58) and Las Piñas streams (WQI=0.65) had a significantly lower WQI average than
the remaining sites (WQI=0.68-0.73); these results indicate a lower water quality in sites
associated with cattle ranching and agricultural activities, respectively. Water DO was the
variable with a high weight in the WQI and showed a seasonal variability. In the region,
between-year variations in the precipitation exist. It was observed that water quality depends
on many environmental factors, but it is necessary to take into account the climatic seasons of
the region.
Regarding the in situ assays, 100% of mortality was observed in fish exposed in Marentes
stream (cattle ranching activity) in all months except February 2000, when only 20% of
mortality was found. The concentration of DO in this site was always lower than 3.5 mg/L.
Parameters such as nitrites, nitrates and ammonia for fish mortality could contribute to this
process, because even low concentrations of nitrites during short-term exposures can be toxic
to fish (Martinez and Souza, 2002). Furthermore, a decrease in water DO may increase
ammonia toxicity (Randall and Tsui, 2002). In some periods, in Marentes stream, a locally
well-known phenomenon called the “barbasco” was observed. Barbasco is a group of natural
rotenoids present in a tree of the genus Lonchocarpus. Rotenoids are potent fish killers
(Metcalf and Müeller, 2000), and may have caused or contributed to the mortality of fish at
the beginning of the rainy season.
In the Las Piñas stream, mortality was equal or above 80% in all months with the
exception of November. AChE inhibitions lower than 20% were found in July and September

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but not in February and May. Considerable LDH and GST inhibitions were also found in
November. Although the results of these parameters should be analyzed with precaution due
to the low number of surviving fish, it is important to note that GST inhibition may be caused
by carbofuran, which is applied in this zone, because in vivo gill GST inhibition was observed
in G. yucatana.
The chambers used here allowed the survival of G. yucatana during 21 days in sites with
low DO concentrations. In addition to mortality, it was possible to use other indicative
parameters, namely weight variation, AChE, LDH and GST activities. The caging assays may
overestimate the effects of contaminants in wild fish since these are able to move and avoid or
reduce the exposure, while fish in closed chambers cannot.
LDH activity was found to be positively correlated with pH and ortophosphates and
negatively correlated with salinity, ammonium, DO, and redox. Our results are in good
agreement with Wu and Lam (1997) who found that LDH activity was significantly higher in
mussels from sites with lower DO values. Gill GST activity was found to be positively and
highly correlated with nitrates. Therefore, particular care with this parameter should be taken
when measuring GST in field populations, because positive correlations of this enzyme with
pH and negative correlations with sulfide and redox were also found. Sturm et al. (2000)
reported for three-spined stickleback (Gasterosteus aculeatus) this same correlation,
particularly with pH.
In the Las Piñas stream, where pesticides are continuously used, an AChE inhibition
higher than 20% was found in February, March and July 2000 and September 2001,
indicating exposure to anticholinesterase pesticides (Figure 4). Farmers apply insecticides
incorrectly, mainly where the fields are continuously irrigated. In some conditions
organophosphate pesticides may persist for considerable periods of time in soil and water
(Bondarenko and Gana, 2004). A previous study in the area of Palizada river reported that
chlorpyrifos residues in water were about 20 pg/L in April 2000 (Carvalho et al., 2009). This
is a concerning situation because chlorpyrifos is applied mainly from August to October and
was detected in April in several sites of Palizada River system. The induction of gill GST
activity (Figure 8) and the inhibition of muscle LDH (Figure 9) found in fish collected in this
month also suggest the presence of pollutants in the area.
AChE inhibition higher than 40% was found in March 2000 (Figure 7) but no inhibition
was observed in March 2001 which confirms that farmers apply pesticides in different time
periods from year to year, mainly according to climatic conditions, such as precipitation.
In Las Piñas stream, in November, when the WQI showed the highest value calculated
during the period of the study, a low mortality of fish (10%) was recorded in the in situ assay,
despite the LDH inhibition found in surviving fish. As a whole, these results suggest the
presence of pollutants other than anticholinesterase pesticides. In this area, -DDT (531 pg/L)
and -PCBs (4250 pg/L) have been found (Carvalho et al., 2009). PCBs are nucleophilic
agents and GST participates in their detoxification (PCB 153) (Machala et al., 1998; Boon et
al., 1989).
In relation to the biomonitoring of wild ducks, compared to reference ducks, a decrease
of 34% in AChE activity was found in ducks captured in August during the pesticide
application period, clearly above the 20% inhibition that has been used to diagnose exposure
to anticholinesterase agents (Figure 10). On the other hand, no significant differences in

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 Environmental Impact of Agricultural Activities on Human … 403

AChE activity were found between reference animals and ducks collected in July, when no
pesticides were applied, indicating probably recovery of AChE levels in wild ducks.
The results suggest that wild D. autumnalis are exposed to pesticides at least during the
season of intensive application. Inhibition above 30% of AChE activity found in wild ducks
during the pesticide application period may indicate that effects on the neurological function
have been induced. Therefore, it is likely that other physiological functions have been
affected during the period when ducks experience low levels of AChE.
The results obtained from the interview to farmers indicated a considerable variation in
education levels across the four communities studied. The non-use of protective clothing
among the four communities is common and this behavior seems to be related with the local
social connotation of the use of protective clothing as a weakness in the workplace (Tinoco
and Halperin, 1997). Eating in the fields can be a route of pesticide exposure in farmers
because over 30% of the farmers confirmed that they do this.

Figure 11. AChE activity in farmers from four communities of Campeche, Mexico. Symbol < means
the percentage of AChE inhibition.

Twenty five per cent of El Juncal and Suc-Tuc farmers showed symptoms of mild
poisoning that seem to be associated with carbofuran and methamidophos, respectively.
According to pesticide use symptoms probably compatible with acute organophosphate and
carbamate poisoning, mainly headache, weakness, nausea, sweating and tiredness were found
in the communities.
The lower AChE activity levels in Ich-Ek and Suc-Tuc farmers relatively to the other
groups seem to be due mainly to the use of organophosphate pesticides in these sites (Figure
11), since in El Juncal carbofuran, a carbamate insecticide, is intensively used and AChE
activity was not significantly affected.
Depending on the pesticide used in a region, erythrocyte AChE inhibition provides a
good biomarker of exposure to OP in field studies with human populations. However, for
carbamate exposure this biomarker needs to be used with care because this enzyme can be
reactivated in a short period of time (Nigg and Knaak, 2000).

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404 Jaime Rendón von Oste, Lucia Guilhermino and Amadeu M. V. M. Soares

CONCLUSION
In the FLSPR, the main change in land use occurred in the last 20 years and the
agricultural area increased from 2,450.0 ha in the 80s to 30,075 ha in 2000. The
implementation of a WQI in the Fluvio Lagoon System of Palizada River was useful to
differentiate the environmental impact on sites with several human activities. Sites close to
cattle ranching and agricultural activities have much lower WQI than other sites with
different land use covers.
The in situ assays were able to discriminate levels of water contamination both in time
and space, thus chambers and cabinets are suitable to in situ exposure. Biomonitoring studies
with autochthonous organisms (G. yucatana) using enzymatic biomarkers present the
advantage of diagnosing the exposure of natural populations to environmental contaminants
or the effects of pollutants in wild populations.
The biomonitoring in the terrestrial compartment via the quantification of the inhibition
of AChE activity in wild black-bellied whistling ducks (D. autumnalis) suggests that these
organisms are exposed to pesticides at least during the season of intensive application. Due to
the abundance and its widespread occurrence in wetlands, D. autumnalis can be used as a
bioindicator for field studies incorporating a systematic monitoring of wild populations and
investigating also population parameters in order to avoid irreversible adverse effects.
In farmers from the four localities studied, the main pesticides involved in any type of
symptoms related to pesticide poisoning were carbofuran, methamidophos, methomyl,
monocrotophos and methyl parathion. Erythrocyte AChE inhibition in farmers exposed to
pesticides is a good biomarker of exposure to OP in field studies.
It was demonstrated that an integrated approach using in situ assays (based on mortality,
growth and biomarkers), WQI and biomonitoring studies with aquatic and terrestrial
organisms are of great utility to assess the effects of anthropogenic activities on the
environment. The current pesticides used in the agricultural activity of the Palizada River
system have a negative impact on human and animal populations from Palizada (Campeche,
Mexico). The use of biomarkers in different ecological levels in the tropics is a cost-effective
tool for an integrated risk assessment of wetlands.

ACKNOWLEDGMENTS
The whole study was supported by International Co-operation (INCO) of the European
Community (TROCA-WET Project) contract: ERBIC18CT980264. Authors wish to thank
Rodolfo Dzul, Martín Memije and Alejandro Ortíz from EPOMEX for their technical
assistants in field and laboratory work.

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(Eds), Methods of enzymatic analysis. Vol. III. Academic Press, NY pp 118-126.
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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 26

BIOACCUMULATION OF MERCURY IN YELLOWFIN

TUNA (THUNNUS ALBACARES) FROM ECUADOR
AND OTHER WORLDWIDE REGIONS:
A COMPARATIVE STUDY

Luis A. Cedeño-Macias1, David Villarreal1,2,Fernando R. Diz1

and Dayanara Macías-Mayorga1,3,*
1
Departamento Central de Investigación (DCI), Ecuadorian AquaticEcotoxicology
(ECUACTOX) Group, Universidad Laica Eloy Alfaro de Manabi
(ULEAM), Manta, Ecuador
2
Facultad de Ciencias del Mar, Universidad Laica Eloy Alfaro de Manabi
(ULEAM), Manta, Ecuador
3
Facultad de Ciencias Agropecuarias, Universidad Laica Eloy Alfaro de Manabi
(ULEAM), Manta, Ecuador

ABSTRACT
Metals have been recognized as one of the main causes of pollution due to their
persistence, toxicity and continuous accumulation in the environment. Metals can be
incorporated into the trophic web and be bioaccumulated by organisms causing
deleterious effects. Studies of metal accumulation in fish have had a significant increase,
due to the risk of contaminated fish consumption by humans. Mercury (Hg) is one of the
potentially toxic metals known to beaccumulated via the intake of contaminated fish
found at the top of the trophic web. The present chapter aims at comparing reported Hg
levels in yellowfin tuna Thunnus albacares collected or marketed in different regions of
the Pacific Ocean and among other regions of the world. The collected information
indicates that the highest levels of Hg found in individuals from the Eastern Pacific
Ocean landed in Manta City, Ecuador. This can mean that Hg levels in yellowfin tuna
vary depending on fish size, and the Pacific region where it is collected, given that

*
Corresponding Author E-mail: dayanara.macias@gmail.com.

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408 Luis A. Cedeño-Macias, David Villarreal, Fernando R. Diz et al.

specimens collected in this area are larger in size. Despite registering the highest levels of
Hg in muscle tissue for this area, the yellowfin tuna from this region can be consumed
with moderation.

Keywords: Thunnus albacares, mercury, bioaccumulation, human health

INTRODUCTION
Mercury (Hg) contamination is a problem of worldwide concern, since it is a
persistent contaminant and is widely distributed in the environment, having either natural or
anthropogenic sources (Ordiano-Flores et al., 2012). This metal usually enters the
environment in an inorganic form as a result of natural processes (i.e., weathering and/or
meteorization of rocks, degasification of the earth’s crust, terrestrial and submarine
volcanism), and from human activities (e.g., electric and measurement appliance industry,
gold extraction, iron and steel industry, chloride-alkali production, fossil fuel burning, and
production of fungicides made with Hg) (Wiener et al., 2003).
The World Health Organization (WHO) considers Hg among the top 10 chemicals of
“major public health concern.” The main form of Hg to which human populations are
generally exposed to is methylmercury (MeHg). Once in the environment, Hg can be
transformed by bacteria into MeHg, which then bioaccumulates in tissues of fish at the top of
the aquatic food web (WHO, 2013). In human beings, MeHg exposure occurs predominantly
via the consumption of seafood (including freshwater and marine varieties, and marine
mammals) (WHO, 2013). MeHg is a particularly harmful neurotoxin to fetal brain
development (UNEP, 2008). Extensive research has demonstrated relationship between
exposure in utero and developmental neurotoxicity (e.g., deficits in fine motor skills,
language and memory) among populations that consume seafood regularly (CTEM, 2000).
It has been widely recognized that fish ingestion is the main source of Hg exposure in
humans. In the case of top predators such as large pelagic fishes, when exposed to metal
contamination the risks tend to be exacerbated as many heavy metals tend to accumulate
in their bodies at concentrations higher than those found in the environment and in their
prey (Damiano et al., 2011; Onsanit et al., 2012). For commercially important fish, the
bioaccumulation of metals can have serious implications for human health (Kojadinovic et al.,
2006). Such studies have been used to develop health-based reference doses below which no
appreciable risk of harm is thought to occur, including the provisional tolerable weekly intake
(PTWI), established by the Joint Expert Committee on Food Additives (JECFA) of the Food
and Agriculture Organization (FAO) and WHO (UNEP, 2008).
In Ecuador, fishing and fishery products are highly important economic resources and are
considered the second most important activity in the country, accounting for almost 12% of
total trade (Pro Ecuador, 2013). One of the most important harbors of Ecuador is located in
the city of Manta (INP, 2015), known as the tuna capital of the country. The yellowfin tuna
(Thunnus albacares) is consumed in high quantities and is a commercially relevant resource
for national and international markets: Ecuador’s tuna catch can reach up to 200,000 tons per
year (IATTC, 2014). Given that this species is a top predator, the exposure and accumulation
of metals via the food web tend to be intensified and could become a risk to human health
(Storelli et al., 2005; Kojadinovic et al., 2007).

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 Bioaccumulation of Mercury inYellowfin Tuna (Thunnus albacares) … 409

The present chapter aims at comparing reported Hg levels in yellowfin tuna T. albacares
collected in Ecuador, with yellowfin tuna collected or marketed in different regions of the
Pacific Ocean and among other regions of the world.

COMPARISON OF MERCURY LEVELS REPORTED FOR

THUNNUS ALBACARES IN DIFFERENT SITES OF THE PACIFIC OCEAN
Data from all regions of the Pacific Ocean and other oceans around the world were
considered in the current review. Regarding the Pacific Ocean, data from four different
countries were considered in the chapter: Ecuador, Mexico, USA and Japan. We classified the
data according to the different regions of the Pacific Ocean: Equatorial Eastern Pacific,
Central Equatorial Pacific, North Eastern Pacific, and North Western Pacific. For all other
regions of the Pacific there is a lack of data published. Only mean data of Hg determined in
muscle tissue of T. albacares during the last ten years were considered, discarding studies in
which only a few individuals were studied or those with historical data. Mean size data were
also considered when available, due to evidence regarding the relation between Hg levels in
muscle tissue and size (Cal et al., 2007; Drevnick et al., 2015).
Data from all regions of the Pacific Ocean demonstrate the tendency of the species T.
albacares to accumulate high Hg concentrations in muscle tissue (Al-Busaidi et al., 2011)
(Figure 1). In general, concentrations of Hg in the muscle tissue reported from the different
regions of the Pacific Ocean presented levels below 0.5 ppm, far from the permissible intake
limit of 1 ppm established by the European Commission Regulation-EC (2006). Only
reported values from the T. albacares landed in the port of Manta (Ecuador) presented values
above this permissible intake limit (Araújo and Cedeño-Macias, 2016); however, the reported
values presented a wide range of deviation. The latter may be due to the wide range of sizes
of the individuals sampled. It is therefore possible that the high values of Hg reported by
Araújo and Cedeño-Macias (2016) in Ecuador correspond to the largest T. albacares
individuals for the Pacific Ocean. This corroborates previous evidence indicating that the
bioaccumulation of Hg in muscle tissue is correlated to the size of this species (Cal et al.,
2007; Drevnick et al., 2015). An important number of studies performed in the Pacific Ocean
did not report data of fish size (Figure 1) which makes it difficult to compare the ratio of
bioaccumulation depending on the size range. We therefore recommend standardizing the
method of collecting data such that all reports present not only the Hg values, but also the size
and weight of animals sampled.

WORLDWIDE COMPARISON OF MERCURY LEVELS REPORTED FOR

THUNNUS ALBACARES IN MANTA (ECUADOR)
Figure 2 compares the mean values of Hg in muscle tissue of T. albacares reported in
Manta (Ecuador) (Araújo and Cedeño-Macias, 2016) to those reported in other regions of the
world. Kojadinovic et al. (2007) reported high values of Hg (1.15 ppm) for the Reunion
Island Region in the Indic Ocean. These values of Hg are similar to those reported by Araújo
and Cedeño-Macias (2016) for the Pacific Ocean, being these two sites where Hg levels were

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410 Luis A. Cedeño-Macias, David Villarreal, Fernando R. Diz et al.

found to be above the permissible intake limit established by the European Commission
Regulation-EC (2006). Mean Hg values reported in the Atlantic Ocean for this species are
similar among sites: 0.35 ppm (Besada et al., 2006); 0.30 ppm (Teffer et al., 2014); 0.25 ppm
(Adams, 2014). In the Indic Ocean, the lowest mean value of Hg (0.30 ppm) was reported by
Jinadasa et al., (2014), in the Sri Lanka region. Thus, the consumption of yellowfin tuna, in
general, is safe for all the ports of the world considered here.
T. albacares fishery is a highly important economic resource worldwide, so Hg
contamination in this species is an issue of special concern for human health. The risk
associated to metal intake on a weekly basis was therefore estimated for each region of the
world by calculating the Maximum Quantity for Weekly Consumption of T. albacares muscle
(MQWC; grams per week; Araújo and Cedeño-Macias, 2016) via the following equation:
MQWC=PTWI* BW/[Hg], where PTWI is the Provisional Tolerable Weekly Intake of 5 µg
kg-1, recommended by FAO/WHO (2010), BW is the Body Weight considered as 70 kg
(Araújo and Cedeño-Macias, 2016), and [Hg] is the mean metal concentration determined in
fish muscle. In order to calculate a value of PTWI for an average body weight of 70 kg, the
mean value of Hg concentration was substituted by the Permissible Intake Limit (PIL) of 1
ppm established by the European Commission Regulation-EC (2006). This value obtained is
350 g of muscle per week (PTWI=70 kg* 5 µg kg-1/1 mg kg-1), and it was considered as a
reference value to compare the rest of data from around the world(Figure 3).
Most of the data reviewed in the current chapter are under the PIL of 1 ppm established
by the European Commission Regulation-EC (2006), as shown in Figures 1 and 2. Thus,
consumption of T. albacares from most regions of the world is not considered a threat to
human health. In order to consider the maximum quantities suggested for consumption per
week by an adult of 70 kg, we only included data under 800 g per week, given that countries
presenting high consumption of yellowfin tuna as Mexico, the average national consumption
is only 200 g per week (Ordiano-Flores et al., 2011). Furthermore, values over 800 g per
week are far enough from the 350 g per week recommended by FAO/WHO (2010) as PTWI
value. In this way, the weekly quantity of the yellowfin tuna marketed in the Equatorial
Eastern Pacific Ocean (landed in Mexico and Ecuador) and in the Western Indian Ocean
(landed in Madagascar) considered safe for consumption according to the present study was
less than that recommended by FAO/WHO (2010). Nevertheless, according to mean Hg
values, the quantity per week for consumption in these regions is over the average national
consumption in Mexico, thus even for these places, the moderate consumption under these
limits offered in Figure 3 could be considered safe for human health. It should be noted that
values of safe consumption for special populations such as children or pregnant women were
not considered in the current chapter. Nevertheless, permissible limits should be considered as
a suggested value and not as an absolute one.
In any case, T. albacares is known to migrate over large distances, thus metal levels
found in muscle tissue might integrate contamination from several different areas and not
only from the site of capture (Kojadinovic et al., 2007; Adams, 2009; Chen et al., 2014).
Future monitoring studies in order to determine the levels of Hg in T. albacares from the
Eastern Equatorial Pacific Ocean and the Western Indian Ocean are recommended, in order to
identify the sources of these high levels.

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 Bioaccumulation of Mercury inYellowfin Tuna (Thunnus albacares) … 411

0.8
2.5

Hg concentration (ppm)

Hg concentration (ppm)
0.6 2.0
j
p 1.5
0.4 b
c
e
0.3 a g m 1.0
d f h n
0.2
o 0.5
0.1 i k
l
0.0 0.0
ND 0 60 80 100 120 140 160
Fish length (cm)

Figure 1. Average levels of Hg bioaccumulated in Thunnus albacares and respective fish length (when
available) reported in the literature for different regions of the Pacific Ocean. Black dots correspond to
the left Y axis, and x marks correspond to the right Y axis. ND means data of fish length not available.
Vertical lines of each data represent the standard deviation for mean values of Hg (when available) and
horizontal lines represent the standard deviation for mean size values (when available). a: Kaneko and
Ralston, 2007; b: Yamashita et al., 2009; c: Drevnick et al., 2015; d, g, h and i: Ferris and Essington,
2011; e: Hisamichi et al., 2010; f: Boush and Thieleke, 1983; j and m: Ordiano-Flores et al., 2011; k:
Ordiano-Flores et al., 2012; l: García-Hernández et al., 2007; n: Burger et al., 2011; o: Cal et al., 2007;
p: Araújo and Cedeño-Macias, 2016.

3.0
Hg concentration (ppm)

2.0

1.5 j
h
1.0 d
m
i k
0.5 b
a e
f g l
0.0 c

ND 0 80 100 120 140 160 180

Fish length (cm)

Figure 2. Average levels (black dots) of Hg bioaccumulated in Thunnus albacares and respective fish
length reported in the literature for different regions of the world. ND means data of fish length not
available. Vertical lines of each data means standard deviation for mean values of Hg (when available),
horizontal lines mean standard deviation for mean size values (when available). Dashed line at 1 ppm is
the Permissible Intake Limit of 1 ppm established by the European Commission Regulation-EC (2006).
a: Burger and Gochfeld, 2011; b: Besada et al., 2006; c: Al-Busaidi et al., 2011; d: Bosch et al., 2015; e:
Cal et al., 2007; f: Adams, 2014; g: Teffer et al., 2014; h: Kojadinovic et al., 2007 ; i: Kojadinovic et al.,
2007; j: Araújo and Cedeño-Macias, 2016; k: Kojadinovic et al., 2006; l: Jinadasa et al., 2014, m:
Kojadinovic et al., 2006.

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412 Luis A. Cedeño-Macias, David Villarreal, Fernando R. Diz et al.

800

MQWC (g per week)

600

400

200

0
or xic
o
xic
o
nti
c
ca
r ue
ad tla as biq
-E cu c -Me c -Me . A a g am
fic ifi ifi . E a d o z
i c c S -M
ac Pa Pa ian
-M
.P E. E. ian
q .E E q. N. . Ind . Ind
E W W

Regions

Figure 3. Maximum quantity for weekly consumption (MQWC, in g per week) of Thunnus albacares
(yellowfin tuna) muscle by a 70 kg adult, calculated individually for each of the different regions
considered: Equatorial Eastern Pacific-Ecuador (Araújo and Cedeño-Macias, 2016); Equatorial Eastern
Pacific-Mexico and North Eastern Pacific (Ordiano-Flores et al., 2011); South Eastern Atlantic (Bosch
et al., 2015); Western Indian-Madagascar and Western Indian-Mozambique (Kodajinovic et al., 2007).
Dashed line at 350 g per week means the reference value recommended by FAO/WHO (2010).

CONCLUSION
In general, T. albacares landed in different regions of the Pacific Ocean can be safely
consumed given that the reported Hg levels in muscle tissue are under the permissible intake
limit of 1 ppm established by the European Commission Regulation-EC (2006). Only T.
albacares landed in Ecuador (Araújo and Cerdeño-Macias, 2016) and in Madagascar
(Kojadinovic et al., 2007) presented levels of Hg above this permissible intake limit, although
it could be considered as safe for human consumption considering the provisional tolerable
weekly intake (PTWI) value recommended by FAO/WHO. Future studies regarding
bioaccumulation of Hg in Eastern Equatorial Pacific T. albacares landed in Ecuador are
suggested in order to identify the reasons of the high levels of Hg found in this species.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 27

LICHENS AND AIR QUALITY IN LATIN AMERICA

Jesús Hernández1,*, Raiza Fernandez2, Mariana Cárdenas Henao3,

Christopher Franco4, Henry Gomez2, Antonio Roschman-González5,
Zully Benzo6 and Federico Galarraga2
1
Instituto Experimental JardínBotánico Dr. Tobías Lasser, Jardín Botánico de Caracas.
Universidad Central de Venezuela (UCV), Caracas, Venezuela
2
Centro de Geoquímica, Instituto de Ciencias de la Tierra,
Facultad de Ciencias, UCV, Caracas, Venezuela
3
Postgrado en Ecología, Instituto de Zoología y Ecología Tropical,
Facultad de Ciencias, UCV, Caracas, Venezuela
4
Universidad Católica Andrés Bello (UCAB), Caracas, Venezuela
5
Centro de Microscopía Electrónica, Facultad de Ciencias, UCV, Caracas, Venezuela
6
Laboratorio de Química Analítica, Centro de Química, Instituto Venezolano de
Investigaciones Científicas (IVIC), Caracas, Venezuela

ABSTRACT
Lichens can be used as bioindicators/biomonitors for research and control of air
pollution. They are valid instruments for assessing air quality affected by emissions from
mobile or fixed sources. In this context, this chapter gathers information about the
progress of research on the use of lichens as biomonitors of air quality in Latin America
(with emphasis on Venezuela). This review discusses the most important lines in the state
of knowledge in this field, evaluating the methodological applications (sampling,
physical and chemical treatment, instrumental analysis techniques and future prospects)
of lichens, and their advantages/disadvantages compared to conventional research
methods. The review is divided in three parts, summarizing the methodologies and
experiences of using lichens as bioindicators/biomonitors, with special emphasis on the
use of lichen physiology, their ability to accumulate heavy metals and polycyclic
aromatic hydrocarbons (PAHs) over time and the analysis of lichen community, using
Index of Atmospheric Purity and the Environmental Classification Factor.

* Corresponding Author E-mail: jeshernandezm@gmail.com.

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418 Jesús Hernández, Raiza Fernandez, Mariana Cárdenas Henao et al.

Keywords: bioindicators, biomonitors, Latin America, heavy metals, PAHs, IAP

INTRODUCTION: LICHENS AS BIOINDICATORS AND BIOMONITORS

Bioindicators are organisms that manifest particular symptoms in response to
environmental changes, generally determined in a quantitative way. On the other hand,
biomonitors refers to such organisms, their distribution or population studied over time and
compared with standard values or base-line surveys, taking into account the deviations from
the expected behavior (Hawksworth et al., 2005). Even though both concepts are different, in
this chapter the word bioindicators will be used referring to both unless the term biomonitors
is specified.
Since Grindon (1859) observed the gradual disappearance of lichens in Manchester
(England), and then Nylander (1866) warned that lichens of the Jardin du Luxembourg
(France) were disappearing probably due to air pollution, much research has been done
concerning the use of lichenized fungi as bioindicators of air pollution.
Lichens are by definition a symbiosis of two organisms, a fungus (denominated
mycobiont) and a photosynthetic organism (photobiont), which can be either a green algae or
a cyanobacteria (Nash, 2008). Unlike higher plants, lichens have no roots or a well-defined
cuticle, which emphasizes its applicability in monitoring purposes. These organisms are
strongly dependent on dry depositions as moist mineral nutrients on the thallus surface. In
addition, the lichen surface and structure facilitate interception and retention of particles
(Szczepaniak and Biziuk, 2003).
Vareschi’s publication (1953) is the first study on the use of lichens as bioindicators of
air pollution in Latin America. By that time, the correlation between the presence of lichens
and levels of air pollution was starting to be accepted worldwide. Since then, Latin America
has demonstrated interest, and various institutions have committed to develop animportant
amount of research on the subject.
This chapter gathers information about the progress of research on the use of lichens as
bioindicators and biomonitors of air quality in Latin America with emphasis in Venezuela,
evaluating lichen methodological applications and their advantages/disadvantages compared
to conventional research methods.

PHYSIOLOGICAL RESPONSE OF LICHENS TO POLLUTANTS: WHAT

HAS BEEN DONE IN LATIN AMERICA?
Because of their particular biological conditions, lichens are affected by numerous
factors, especially atmospheric pollution whose physiological effects have been widely
studied. Indeed, using lichen physiological responses as indicators of pollution has been a
major resource for the assessment of air quality. Despite the fact that Latin America is one of
the regions with less effort on this subject, in the last 20 years research teams from Argentina,
Mexico and Brazil have been working to elucidate how lichen physiological parameters are
influenced by pollutants.

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 Lichens and Air Quality in Latin America 419

Since 1994 in Argentina, studies involving transplanted lichens of the genera

Canomaculina (González and Pignata, 2000; González et al., 2003), Parmotrema (Cañas et
al., 1997), Punctelia (González and Pignata, 1994, 1997; González et al., 2003), Ramalina
(Levin and Pignata, 1995; González et al., 1996, 1998, 2003; Pignata et al., 2004; Rodriguez
et al., 2007; Bermudez et al., 2009) and Usnea (Carreras et al., 1998, 2005; Carreras and
Pignata, 2002, 2007; Rodriguez et al., 2007; González et al., 2012), have been used to
quantify and compare the effects of pollutants, especially heavy metals, produced from
industrial activity and traffic. In Brazil, Parmotrema tinctorum and Teloschistes exilis have
been used to assess how mutagenic and cytotoxic agents cause morphophysiological
alterations in lichen thalli (Käffer et al., 2012). On the other hand, in Mexico the influence of
chronic air pollution on tolerance and production of protective secondary metabolites in
Ramalina asahinae and Parmotrema stuppeum, using in situ and experimental reactivity data
have been studied (Valencia-Islas et al., 2007).
Most of these studies emphasize on measuring parameters such as chlorophyll and
phaeophytin degradation, malondialdehyde (MDA), sulphur concentration, conjugated dienes,
soluble proteins, phaeophytina/chlorophyll a, chlorophyll b/chlorophyll a and fresh/dry
weight ratios; even some pollution indices have been calculated. These physiological
parameters are usually compared with pollutant levels in the environment, accumulation of
heavy metals, suspended particles and meteorological conditions, proving lichen efficiency as
indicators of human health (Carreras and Pignata, 2001; Carreras et al., 2009a,b).
The general results of these studies showed that an increase of heavy metal
concentrations negatively affects photosynthetic and non-photosynthetic pigment
concentrations, as well as photosynthetic processes, synthesis of chlorophyll molecules and
degradation of secondary metabolites. Also membrane integrity and selective permeability is
affected by pollutants due to modifications of membrane lipids and protein structure. Other
effects are reduction of percentage of live cell, carbon monoxide and chlorophyll values, as
well as an increase of plasmolyzed cells, antioxidant activity, antiradical power of secondary
metabolites and morpho-anatomical changes (e.g., chlorosis, necrosis, and medulla
exposition).

LICHENS AS BIOMONITORS OF HEAVY METALS AND PAHS

Lichens incorporate environmental pollutants such as heavy metals and polycyclic
aromatic hydrocarbons (PAHs), among others, and can be used as indicators of the
bioavailability of such substances over time, allowing in some cases to compare pollution
levels in different geographic areas (Conti and Cecchetti, 2001). In this sense, several
countries (Argentina, Slovenia, Spain, USA, France, Italy, Portugal, among others) have
continued to develop research in order to examine in detail the potential of lichens as
bioindicators of the presence of heavy metals and PAHs in air (Jeran et al., 2002; Carreras et
al., 2009a). Countries such as Brazil and Venezuela have recently joined the study, especially
in urban areas (Saiki et al., 2006; Fernandez et al., 2011; Gomez et al., 2013a).

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HEAVY METALS
A large number of studies have used lichens as bioindicators of heavy metals (e.g.,
Branquinho et al., 1999; Conti and Cecchetii, 2001; Carreras and Pignata, 2002; Jeran et al.,
2002; Scerbo et al., 2002; Garty et al., 2003; Loppi et al., 2004; Tuncel et al., 2004;
Williamson et al., 2004; Monnet et al., 2006; Bergamaschi et al., 2007; Brunialti and Frati,
2007; Basile et al., 2008; Godinho et al., 2008; Sorbo et al., 2009). Latin America has not
been left behind in this area of research (Marcano et al., 1996; Carreras and Pignata, 2002,
2007; Carreras et al., 2005; Quijada, 2006; Bermúdez et al., 2009; Käffer et al., 2012;
Hurtado et al., 2013).
The accumulation of heavy metals in lichen thalli is one of the aspects of lichen biology
that has been most studied in Latin America (Carreras and Pignata, 2001; Carreras et al.,
2009a,b) and worldwide. It usually depends on many factors such as morphology, ion
exchange properties, type of reproduction, etc. (Carreras et al., 2009a,b). Therefore, the
degree of tolerance to heavy metals is characteristic of each lichen species. A well-
documented phenomenon is their ability to tolerate high elemental levels thanks to a number
of mechanisms to avoid toxicity, although only a few have been thoroughly studied for some
elements.
Research on the detection of atmospheric heavy metals using lichens in Latin America
follows a similar methodology worldwide. The most important contributions from Latin
America in this field have been the effects of heavy metals on the physiology of the
lichenized fungi (e.g., Carreras and Pignata, 2001; Carreras et al., 2009a,b). For this reason
more emphasis will be given to the use of lichens as biomonitors of PAH.

POLYCYCLIC AROMATIC HYDROCARBONS

Unlike the large number of studies which use lichens as bioindicators of heavy metals,
little research has been reportedon their use as bioindicators of PAHs. PAHs are a group of
organic compounds known for their carcinogenic, mutagenic and teratogenic effects. They are
a particular family of corresponding hydrocarbon compounds containing a number of benzene
rings fused by two or more carbon atoms. At an international level, up to 2013 there are less
than 30 publications relating to the determination of this group of compounds in lichens
(Gomez et al., 2013a), being eight pioneers in this research: Spain, Italy, India, Canada,
Poland, Portugal, Sweden and Venezuela.

Sampling

In Venezuela, studies have taken place only in urban areas, taking as criteria for selecting
sampling stations different levels of traffic flow on the main streets and avenues or
proximities to main highways. Motor vehicles are one of the main sources of PAHs
(Fernandez et al., 2011).
In passive monitoring, lichens are collected directly from where they are growing in the
study area. This is the most common method until now, because most of these studies are

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 Lichens and Air Quality in Latin America 421

preliminary assessments in which one of the objectives is to test the feasibility of using
lichens as biomonitors of PAHs in the selected locations (Fernandez et al., 2011). Only one
study in Latin America (Gomez et al., 2013b) has transplanted lichens from their natural
ecosystem (reference locality) to an urban area.
Height at which the lichens are sampled or transplanted plays an important role. This
generally varies between 1.0-3.5 m above the ground (Fernandez et al., 2011), seeking to
avoid the influence of soil particles that probably also contains PAHs. Another reason is that
lichens located just above the floor could be overprotected from atmospheric depositions by
the existence of weeds.
In active monitoring (transplantation method), procedures should be standardized very
well as many other factors can affect the response of lichens to pollutants in an area where
lichens usually do not grow (Gomez et al., 2013b). In this sense, it is advisable to protect
them once transplanted from direct solar radiation. It should be ensured that the exposed
surface area of the biomonitors is as large and homogeneous as possible in all the studied
locations (Gomez et al., 2013b).

Lichen Species Frequently Used

Foliose and fruticose lichens are the most used in environmental studies (Owczarek et al.,
2001; Migaszewski et al., 2002; Guidotti et al., 2003, 2009; Blasco et al., 2006, 2007, 2008;
Domeño et al., 2006; Naeth and Wilkinson, 2008; Augusto et al., 2009; Shukla and Upreti,
2009; Shukla et al., 2010; Fernandez et al., 2011; Gomezet al., 2013a) mainly due to the fact
that they are easily removed from the substrate, compared to crustose lichens, which grow
much more attached to their phorophyte. 24 different species of lichens have been used for
PAH biomonitoring. In Venezuela only two species have been tested: Pyxine cocois (Sw.)
Nyl. And Parmotrema sancti-angelii (Lynge) Hale (Fernandez et al., 2011; Gomez et al.,
2013b).

Physical Treatment of the Samples

In the period of time between sampling and the physical and chemical processing in the
laboratory, samples should be properly preserved at low temperature in order to avoid the
maximum loss of the more volatile PAHs. They should also be protected from solar radiation
to prevent photolysis of the compounds of interest. Different methods have been used to
maintain these conditions. In some studies, lichens are kept wrapped in aluminum foil and at
low temperatures, while in others the samples are kept at -20 °C in amber glass bottles until
analysis (Fernandez et al., 2011).
The physical treatment of the samples has so far not been standardized, hence each author
reports different steps for this stage of the analysis. Guidotti et al. (2003, 2009) dried the
samples in an oven at 40 °C for 48 h; samples were cleaned by removing foreign materials
such as dust, leaf and bark remnants, insects and pebbles using a microscope. They were then
pulverized in an agate mortar; this procedure was also followed by Fernandez et al. (2011)
and Gomez et al. (2013b), who additionally employed liquid nitrogen to pulverize the
samples. Fernandez et al. (2011) rinsed the dried lichens using deionized water and gentle

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422 Jesús Hernández, Raiza Fernandez, Mariana Cárdenas Henao et al.

stirring. However, Gomez et al. (2013b) recommends not following this step since some
significant PAHs may be lost in the process.
Despite there being no standardized protocol for the physical treatment of the samples,
most authors include sample cleaning, pulverization or homogenization and drying of the
samples. This is because the presence of materials other than the lichen thalli, sample
heterogeneity and humidity can introduce a significant variance and/or error in the dry weight
and the determination of PAHs.

Extraction

The extraction process is essentially based on placing the sample in contact with a
suitable solvent. The mixture of the solid sample with the liquid solvent is then subjected to a
series of treatments of varying intensity, ranging from simple stirring to critical pressure and
temperature conditions, depending on the strength of the interaction between the analyte and
the matrix (Blasco, 2008).
The conventional extraction method for PAHs in environmental samples has been the use
of the Soxhlet extractor (Naeth and Wilkinson, 2008; Augusto et al., 2009; Shukla and Upreti,
2009; Shukla et al., 2010), taking as main reference the 3540 method of the United States-
Environmental Protection Agency (US-EPA) (16-24 h, 300 mL of solvent).
Gomez et al. (2013b) and Fernandez et al. (2011) used a static ultrasound assisted
extraction technique. The choice for this was made taking into account the advantages it
offers in terms of time and amount of solvent used, compared to conventional extraction
methods (Soxhlet). Fernandez et al. (2011) used 30 mL of cyclohexane: dichloromethane (4:1
v/v) as a solvent for 2 g of sample, employing an ultrasonic bath at room temperature for 30
min and performing the method twice with the same residual solid, subsequently combining
the extracts. Gomez et al. (2013b) succeeded in reducing extraction time to 15 min by using a
mixture of hexane:dichloromethane (3:2 v/v) as a solvent, for the same amount of solid and
solvent employed by Fernandez et al. (2011).
The most significant contribution in terms of extraction time, quantity of solvent and
sample mass required for quantification of PAHs was provided by Domeño et al. (2006), who
in order to optimize the treatment step of the sample, compared three different organic
extraction methods: DSASE (Dynamic sonication-Assisted Extraction Method), Soxhlet
extraction and static ultrasound assisted extraction. The same amount of sample (0.2 g of
lichen) and solvent (hexane) were used. They found that all three techniques have similar
recovery rates for the 16 PAHs listed by the US-EPA in the priority pollutant list (US-EPA,
2000), and that the main advantage provided by the DSASE technique is in terms of time and
solvent consumption. This solvent comparison was also done by Gomez et al. (2013b),
agreeing with Domeño et al. (2006) in that hexane is more efficient in extracting the
compounds of interest (aromatic), besides being much more selective than dichloromethane,
which contributes to reducing interference at the moment of instrumental analysis.

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 Lichens and Air Quality in Latin America 423

Purification or Cleaning of the Aromatic Fraction

For the cleaning or purification step of PAHs, Gomez et al. (2013b) and Fernandez et al.
(2011) used the method 3630 of the US-EPA (1996), which consists of preparing a silica gel
column to purify nonpolar substances. These authors performed the purification or separation
of the PAH from other organic compounds using activated silica gel as the stationary phase.
This purification technique is by far the most efficient in terms of time and low consumption
of solvent in comparison to the conventional method of column chromatography.

Instrumental Analysis Techniques

The characterization and quantification of PAH is performed by HPLC, coupled

with fluorescence detector and/or UV-visible, and gas chromatography-mass spectrometry
(GC/MS). Both techniques allow separation of a large number of molecules with similar
structural features, which makes them ideal in this type of analysis. PAHs commonly
quantified are the US-EPAs selection of 16 major pollutants: naphthalene, acenaphthylene,
acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo
[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]
anthracene, benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene.
Augusto et al. (2009), Shukla and Upreti (2009), Shukla et al. (2010), Fernandez et al.
(2011), and Gomez et al. (2013b), used HPLC for the quantification of PAHs, based on the
US-EPA 8310 method (Bursey, 1991).
Fernandez et al. (2011) and Gomez et al. (2013b) used the UV-visible detector for the
quantification of all studied PAHs, selecting a wavelength of 254 nm as has been used by
many others (Shukla and Upreti, 2009; Shukla et al., 2010). They also used a wavelength of
208 nm, because low molecular weight PAHs generally exhibit greater sensitivity at 208
nm and higher molecular weight at 254 nm. However, in the case of fluoranthene,
dibenzo[a,h]anthracene and benzo[g,h,i]perylene, sensitivity was higher at 208 nm. It is
important to say that detection by UV-visible has numerous disadvantages in terms of
selectivity and sensitivity and in not being able to discriminate matrix interferences,
especially in complex matrices such as lichens. Moreover, the fluorescence detector is more
selective (many matrix components are not fluorescent) and sensitive. Gomez et al. (2013b)
used an excitation wavelength of 340 nm and emission of 425 nm and Fernandez et al. (2011)
an excitation wavelength of 375 nm and emission of 425 nm. However, not all commonly
studied PAHs are fluorescent (naphthalene, acenaphthylene, acenaphthene, fluorene,
phenanthrene, chrysene and indeno[1,2,3-cd]pyrene are not fluorescent), so Augustus et al.
(2009), Gomez et al. (2013b) and Fernandez et al. (2011) combined both detectors (UV-
visible and fluorescence) for quantification of PAHs.
Gomez et al. (2013b) mention that one of the main advantages shown by HPLC with
respect to GC/MS is analysis time, taking longer with GC/MS. On the other hand, greater
selectivity and better resolution of the peaks is shown by GC/MS, facilitating the elucidation
of isomers with much more efficiency, compared to the HPLC. Additionally, GC/MS allows
the identification of PAHs that do not fluoresce from the ones that do using only one detector.
In this sense, Gomez et al. (2013b) recommends instrumental techniques used jointly for this
type of studies.

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424 Jesús Hernández, Raiza Fernandez, Mariana Cárdenas Henao et al.

Comparison between the Cumulative Capacity of Different Species of

Lichens and Other Environmental Samples

Foliose lichens show the highest levels of accumulation of PAH (Gomez et al., 2013a). It
can also be said that more accumulation is seen in passive sampling than in lichens that were
transplanted from a reference site to the study area, which may be related directly with the
length of time during which these organisms have been exposed to the atmosphere being
studied, as well as their adaptation to it (Gomez et al., 2013a).
Most studies indicate that lichens tend to have a profile of PAHs rich in compounds of 2,
3 and 4 rings associated with the gas phase, in contrast to compounds of 5 and 6 rings,
associated with atmospheric particles (Owczarek et al., 2001; Migaszewski et al., 2002;
Blasco et al., 2006, 2008; Augusto et al., 2009; Guidotti et al., 2003, 2009; Shukla and Upreti,
2009; Shukla et al., 2010; Fernandez et al., 2011). However, Blasco (2008), Shukla et al.
(2010), and Gomez et al. (2013b) reveal that the profile will depend in many cases on the
morphology of the lichen employed, noting that fruticose and crustose-squamulose lichen
morphologies tend to preferably accumulate PAH associated with the gas phase, while those
with foliose morphology tend to retain on their surface more of those PAHs associated to
particles (monitoring the full spectrum of PAHs). This aspect has implications on choosing
the appropriate species according to the specific objective of the study. In this sense, the most
resistant lichens to air pollution will allow a monitoring of a more toxic mixture of PAHs.
Blasco et al. (2006) found that the concentrations of PAHs in atmospheric particles are 2
orders of magnitude higher than concentrations of PAHs in lichens. Blasco et al. (2006) and
Augusto et al. (2009) agree that in both profiles (atmospheric particles and lichens) 4-ring
PAHs are the most abundant. Blasco et al. (2006) conclude that there is a good correlation
between the levels of PAHs found in lichens and in samples of air at different sampling
stations.
Blasco et al. (2006) and Augusto et al. (2009) indicate a high content of 2 and 3 ring
PAHsin lichens, while atmospheric particles have a higher content of 5- and 6-ring PAHs,
which is directly related to 5- and 6-rings PAHs that are found in atmospheric particles, while
the 2 and 3 ringsin the gas phase. Shukla et al. (2010) and Gomez et al. (2013b) indicate that
the secondary metabolite biosynthesis by lichens (commonly depsids and depsidones) and
other lichen substances consisting of -OH radicals-rich rings provide the hydroxy group for
adduct formation. This would explain the high accumulation of compounds with 2 and 3 rings
in lichens. The lichens used by Fernandez et al. (2011) and Gomez et al. (2013b) were P.
cocois and P. sancti-angeli. P. cocois contains mainly lichexantone, which comes from the
same acetate-polymalonate pathway as depsids and depsidones, and also contains –OH
radicals. P. sancti-angeli contains gyrophoric acid which is a depside. Thus, both species
concur with the assumption of Shukla et al. (2010) and Gomez et al. (2013b).
Blasco et al. (2006) claim that both sampling techniques (biomonitoring and conventional
monitoring without organisms) provide complementary information because lichens record
information of air pollution for prolonged periods (in an integrated manner), whereas
sampling of particles gives information generated during the sampling period (specific
moment in time).
Blasco et al. (2008) determined the existence of a correlation between the Index of
Atmospheric Purity (IAP) and PAH concentration. The combination of the two methods
indicates that low values of the IAP and therefore little lichen biota, may be associated with

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the presence of mixtures of highly toxic PAHs (phenanthrene, fluoranthene, pyrene,

dibenzo[a,h]anthracene and benzo[g,hi]perylene).

Future Prospects for This Kind of Environmental Studies

Although the last decade has shown much progress in the study of lichens as biomonitors
of atmospheric PAH, there are still important issues to be clarified and researched. Some of
these aspects can be listed as follows:

1. Standardize as rigorously as possible the sampling stage, with special emphasis on

the care that should be taken in transplanting lichens from natural ecosystems to a
contaminated area (active monitoring sampling).
2. Standardize methodologies for extraction and purification of PAHs in lichen matrices
internationally.
3. Develop a certified reference material for PAHs in lichens.
4. Optimize conditions for separation and quantification of PAHs in lichens using
HPLC and GC/MS techniques.
5. Conduct studies leading to the selection of at least one PAH pollution tracer.
6. Establish standards at regional, national and local scales for maximum permissible
limits for such compounds in lichens.

BACK TO BASICS: USING LICHEN COMMUNITIES AS ECOLOGICAL

INDICATORS OF ATMOSPHERIC QUALITY
A Latin American Perspective

In the preceding sections of this chapter, we explore the chemical and physiological way
to evaluate how pollutants affect the inner performance and chemical behavior of lichen
species. Contaminants not only affect the physiological processes within the individual, but
also affect how individuals respond to the environment and thus affecting the species’ local
distribution. Therefore, the presence of pollutants can influence the composition of lichen
communities. For this reason the study of lichen ecology and community composition is a
well-used resource in assessment of atmospheric quality.
The lichen ecological approach on atmospheric quality assessment has two different
lines. First, the observational and qualitative framework, which uses richness data,
qualitatively established indicator species and general descriptions of lichen community.
Second, the quantitative approach, based on a numerical index of air quality and statistical
proceedings that allow a quantitative way of indicator species establishment and community
composition evaluation (van Haluwyn and van Herk, 2002). The second line includes the
characterization of lichen communities as indicators of some particular condition or pollution
level, instead of one single indicator species.
LeBlanc and De Sloover (1970) formalized the first mathematical approach to calculate
an IAP, in the form of a linear model that takes intoaccount the major factors that vary with

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the influence of pollution: lichen richness, a scale of frequency and cover and the sensitivity
degree of each lichen species. This quantitative method has been widely used despite the
major disadvantage of reduced precision due to the use of a scale of lichen frequency and
cover data. Naturally, it has been modified several times in order to contextualize its
application to each case (Nimis and Purvis, 2002). Nonetheless, the original model gives a
good estimation of pollution levels.
In the tropics these kinds of studies were neglected for a long time. Nevertheless, it is
noteworthy that even before the mathematical development of IAP, Vareschi (1953) used the
richness, frequency and morphology of lichens to assess the influence of forests and urban
parks on the air quality of Caracas, Venezuela. Twenty years later, Vareschi and Moreno
(1973) made the same assessment once again in Caracas.
After the mid-80's other countries in Latin America began to apply this methodology:
García and Rubiano (1984) and Rubiano (1986, 1987) made the first approach in Colombia.
Other countries such as Argentina (González and Pignata, 1994) and Peru (Tovar and
Aguinaga, 1994) began to consider lichens as a tool to assess the air pollution in the early
90's. The second half of the 90's saw an explosion in the use of lichens as bioindicators in
Latin America, with a particular emphasis on chemicals and physiological protocols. The
ecological approach was forgotten, although a few papers were published on this subject
(Marcano et al., 1996; Estrabou, 1998).
In the early 2000's, the ecological approach was again prevalent with a high
diversification of methods. Not only the richness and cover of lichen thalli were used, but also
the type of reproductive structures and the morphological changes detected on the thalli
(Monge-Nájera et al., 2002; Rubiano, 2002; Estrabou et al., 2004; García, 2004; Canseco et
al., 2006; Rubiano and Chaparro, 2006; Santoni and Litjeroff, 2006; Anze et al., 2007;
Martins et al., 2008; Riquelme, 2008; Calvelo et al., 2009; Litjeroff et al., 2009; Jaramillo and
Botero, 2010), although some of them still remain as unpublished research.
In the last six years, there has been an increase of research in ecological assessment with
the use of lichen communities as bioindicators of pollution in Latin America (e.g., Darré,
2011; Estrabouet al., 2011; Käffer et al.,2011; Neurhor et al., 2011, 2013; Simijacaet al.,
2011, 2014; Quispeet al., 2013; Valois and Mosquera, 2014, Díaz et al., 2016), with an
improvement in the use of statistical tools such as multivariate analysis to establish indicator
species of every pollution level. One of the most important advances was the proposal of a
new index to evaluate atmospheric quality. In order to increase the sensitivity for the
detection and categorization of pollution zones, Käffer et al. (2011) proposed a mathematical
factor that complements IAP. The environmental classification factor (ECF) assigns scales of
cover to different growth forms, assuming a major sensitive scale of fructicose lichens, while
crustose lichens seem to be more tolerant; the foliose lichens are somewhat moderately
sensitive (Wetmore, 1981; Käffer et al.,2011).
The general sampling methods applied in Latin America vary between studies and are
usually according to the particular conditions of each case. Although the most used index
(i.e., IAP) only requires lichen richness and a scale of cover and frequency, most researchers
prefer to measure lichen cover precisely, because it provides better elements for statistical
treatment. Trees are the most typical substrate studied and free sampling, grids of different
sizes and shapes, horizontal or vertical orientation rubber bands, have been applied. In almost
every study developed in Latin America, the sampling unit is the tree and the number of
sampling units varies according to every study. On the other hand, the ecological approaches

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of bioindication studies in Latin America have been assessed with both quantitative (e.g.,
IAP) and qualitative methods, with a major emphasis on richness and its negative correlation
with pollution.
The pool of results is much more diverse than the methods applied, since every locality
shows a different lichen community composition and different abiotic/biotic conditions. Even
tolerant/sensitive taxonomic species identity, richness and the ranges of IAP vary between
studies. In fact, the general idea of the sensitivity level of growth forms is in discussion, such
that in some unpolluted areas great richness of crustose lichens was detected (Rubiano, 1987)
and in other polluted areas fructicose lichens represented tolerant taxa (Rubiano, 1986).
This suggests that none of these parameters/factors are standard indicators. Composition
of neighboring lichen communities, dispersion/colonization/establishment capability of
species, lichen growth rate and, of course, different limiting factors besides pollution (e.g.,
latitude, altitude) are responsible for the overall species composition. This means that the
complexity of the application of lichen community ecology to bioindication analysis can only
be reliable if we have full understanding of every context, especially in tropical regions (van
Haluwyn and van Herk, 2002).
However, two common patterns emerge. First, whatever the context is, it has a general
decrease of lichen richness in polluted areas. Second, Physciaceae (e.g., Hyperphyscia,
Dirinaria and Pyxine) and Parmeliaceae (e.g., Canoparmelia and Parmotrema) are the most
common and richest families in polluted areas, especially in those with high influence of air
pollution produced by intense traffic flow and industries. On the other hand, sensitive species
are very different between studies.

Biomonitoring of Air Pollution in Venezuela: The Ecological Assessment

Despite that Venezuela was the pioneer country in the use of lichen communities as
indicators of air quality in Latin America (Vareschi, 1953; Vareschi and Moreno, 1973), these
methods were almost forgotten for more than 40 years, because of the emphasis on chemical
evaluations. Even though in the last years great development has been made in the fields
of taxonomy and ecology of lichens in the country, there is a major gap in national
biomonitoring studies since the 70s.
The first two approaches of this nature done in Venezuela were exclusively based on
richness analysis. Vareschi (1953) categorized Caracas into three zone types: some isolated
areas with high air pollution (lichen deserts), a large area of low air pollution (almost the
entire city) and normal, peripheral areas with a high level of air purity and exuberant lichens.
Nevertheless, Vareschi and Moreno (1973) warned about the spread of lichen deserts to
almost the entire city just 20 years after the first assessment. Since then, only chemical
approaches have been made.
Currently, the application of lichen community ecology to biomonitoring of air pollution
is a priority objective of Venezuelan researchers. In this context, the first national
bioindication network was created, aiming at standardizing the protocols of sampling and
monitoring air quality in Venezuela. At present, this project is being applied in Caracas, with
more than 130 monitoring stations (and other cities such as San Cristóbal and Mérida), and
will start in other cities such as Puerto Ordaz, Barquisimeto, Maracaibo, Maracay, and

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428 Jesús Hernández, Raiza Fernandez, Mariana Cárdenas Henao et al.

Porlamar. The general purpose of this national network includes standardizing the following
factors:

1. Substrate: species and general conditions. Identify those tree species with high
frequency and good general conditions that allow the colonization and establishment
of lichen communities, limiting the study to individuals with circumference higher
than 40 cm at breast height.
2. The protocol on measuring richness and cover. It is recommended the use of the
rectangular grid protocol (30 × 20 cm). The grid should be located between 1.5 and
1.8 m from the tree base and in the zone with highest lichen richness and cover.
3. Use of quantitative methods. The project emphasizes on the use of IAP (LeBlanc and
De Sloover, 1970) and ECF (Käffer et al., 2011), using the mean cover percentage
and frequency to assign the scales of cover and frequency.

Finally, the national network of bioindication intends to facilitate the general analysis of
the local projects and visualization of national data in a free-access online national data base,
providing online tools designed to calculate each index for atmospheric quality assess.

ACKNOWLEDGMENTS
We would like to give special acknowledgements to our students Melesio Quijada, María
Gabriela Réquiz, José Gregorio Díaz, Erika Arguello, Wilbert Hurtado, Maria Fernanda
Cabezas, Estefanía Mora, Miguel Bermudez, Ángel Lacruz and our students in San Cristóbal
and Mérida, that have worked very hard on their special projects and have contributed to the
state of knowledge in this area for Venezuela and Latin America.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 28

LICHENS AS BIOINDICATORS OF
AIR QUALITY IN MINING AREAS OF
LATIN AMERICA, WITH SPECIAL REFERENCE TO
CATAMARCA, ARGENTINA

Martha S. Cañas1,*, Raquel C. Jasan2 and Rita R. Plá2

1Faculty
of Technology and Applied Sciences – Research and Transference Centre of
Catamarca (CITCA, CONICET – UNCA),
National University of Catamarca, Catamarca, Argentina
2Departament of Nuclear Chemistry, Ezeiza Atomic Centre,

Argentine National Atomic Energy Commission, Buenos Aires, Argentina

ABSTRACT
The lichens Parmotrema austrosinense and Canomaculina consors were
transplanted to a site within an open-pit mining project and to three localities potentially
affected by mining emissions in the Western region of Catamarca, Argentina. In order to
contribute to the interpretation of chemical response of these species to mining airborne
pollutants and to analyse the feasibility of their use in air quality biomonitoring
programmes, results of multielemental determinations by instrumental neutron activation
analysis in the thalli are presented. The observed quali- and quantitative interspecific
differences in the accumulative response of the transplanted lichens could be interpreted
using the exposed/control ratio (EC ratio). The elemental accumulation of transplanted
thalli could relate to mining airborne pollution only in P. austrosinense. Therefore, this
species is the most suitable for biomonitoring air quality in areas with open-pit mines and
environmental characteristics as those of Western Catamarca. In both species, the
multielemental accumulation of the thalli reflected the geochemical characteristics of
each transplantation site and local and regional environmental dynamics. In this regard,
the results presented here contribute to establish environmental quality baselines for this
region, which is in the process of exploiting its natural resources.

*
Corresponding Author E-mail: marthacanas@tecno.unca.edu.ar.

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438 Martha S. Cañas, Raquel C. Jasan and Rita R. Plá

Keywords: air pollution, biomonitoring, lichen, multielemental accumulation, open-pit

mining

INTRODUCTION
Mining is one of the primary production activities that has a major impact on the
environmental quality of a region and particularly on air quality (Moretton et al., 1996;
Boamponsem et al., 2010). This is due to the extraction process itself, as well as crushing,
grinding and deposits of sterile waste, among other associated activities. In particular, open-
pit mining involves the removal of a large amount of rock materials, which can possibly be
dispersed in an area beyond the mine itself.
Even though some mining projects have automatic continuous monitoring of atmospheric
particulate matter levels, it is difficult to extend this methodology to areas that could
be affected by pollution, due to economic and technical reasons, since simultaneous
measurements of various pollutants at different points requires many sampling stations.
An alternative to traditional air quality monitoring methods, with low cost and relatively
fast implementation and execution, is the use of lichens, which have been proposed as
biomonitors both of accumulation and response (Arndt et al., 1995). Lichens can be used as
accumulative biomonitors due to their ability to absorb and/or retain various elements from
the atmosphere, combined with their longevity and resistance to environmental stress.
Biomonitoring assumes that the concentration of trace elements in lichens reflects the
average concentration of particulate matter in the air, and both wet and dry deposition during
a certain period (Steinnes, 1989). Furthermore, the metal content in certain lichen species
allows to infer the proportion of these elements in the environment (Pignata et al., 2007).
As sensitive or reacting biomonitors, lichens function as integrators of stress caused by
air pollutants and as preventive alarm systems. Due to the sensitivity of some species to
certain substances or their combinations, biological response is indicative of air quality and
provides information that cannot be obtained by conventional physical-chemical methods.
Thus the biomonitor informs about the presence of certain substances in the air, and the effect
of pollutants at a biotic level of the ecosystem.
At present, monitoring with lichens is widespread (Carreras et al., 2009; Koz et al., 2010;
Loppi and Nascimbene, 2010), and there are various studies on the use of lichens as
bioindicators of air quality in mining areas (Naeth and Wilkinson, 2008; Boamponsem et al.,
2010; Sondergaard et al., 2010). In Latin America, this kind of studies are scarce and consist
of floristic studies and analysis of heavy metals in lichens from some mining areas of Peru
(Díaz Rivas Plata, 2006), list of species in areas of mining exploration in Ecuador (Carrasco
Merchán and Guzmán Cárdenas, 2011), active monitoring in a mine of Venezuela (Hurtado et
al., 2013), among others.
In Argentina, there have been studies of air quality related to mining areas in Catamarca
province. For the last two decades approximately, mining has become the main economic
resource for this province and the activity is mainly centred in the western region with
prevalence of metalliferous ores extraction. In this region, within the Yacimientos Mineros
Agua de Dionisio (YMAD) district, one of the most important open-pit mining projects of the
country and of South America is placed.

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 Lichens as Bioindicators of Air Quality in Mining Areas 439

Due to lichen scarcity in this semiarid region, active monitoring has been the chosen
methodology for air quality studies, by transplanting lichens collected far away from
the mining area, in slightly anthropized areas. The first researches were conducted in Belén
(approximately 60 km southwest of the mine), with Parmotrema austrosinense (Zahlbr.) Hale
as an accumulative and sensitive bioindicator. Within a study area of about 600 km2, and in
consecutive periods of time, lichen bags were transplanted to 29 monitoring sites, remaining
exposed to the weather during three months. The concentrations of 25 elements were
determined in the thalli by instrumental neutron activation analysis. Photosynthetic pigments,
malondialdehyde (MDA) and sulphur were also quantified, and the pollution index (PI)
proposed by González and Pignata (1994) was calculated. Preliminary results of this research
were published by Mohaded Aybar et al. (2006) and Palomeque et al. (2006).
Regarding physiological parameters, photosynthetic pigments, defined by Cañas (2001)
as biomarkers of air quality in urban-industrial areas, were those that better explained the
variability of physiological data obtained in transplants to Belén during the study time. Also
chlorophyll b/chlorophyll a and phaeophytin a/chlorophyll a ratios proved to be good
explanatory variables (Ocampo et al., 2009). In general, pigments showed certain regularity in
the response of this species transplanted during different periods of the year (Palomeque,
2008; Ocampo et al., 2009). Other parameters, such as sulphur content and MDA, depended
on the exposure period (Mohaded Aybar et al., 2008a).
In general, P. austrosinense showed low elemental accumulation when transplanted to
the study area in Belén. Nevertheless, a differential behaviour could be detected for this
species, both for individual element retention and for multielemental accumulation at
transplant sites. From the elemental analysis of P. austrosinense, it could be inferred that
elements from soil and rock have an influence over air quality in the study zone.
Multielemental composition of this species reflected natural geochemical associations, which
are a characteristic of different lithologic formations present in the study area, as well as in
the region where it is located. No emission sources other than natural ones have been
detected, including wind blown soil and sediments from nearby areas, although they could be
associated to overexploitation by shepherding or land clearing for agricultural purposes. No
effect from the mine has been detected. This could be due to its location NE from the study
area, while prevailing winds blow mainly from the SE (Palomeque, 2008; Jasan et al., 2011).
P. austrosinense showed a certain level of stress related to the total multielemental
accumulation. In transplant sites where the accumulation in thalli was high, values of PI (in
which parameters such as MDA content and phaeophytin a/chlorophyll a ratio are related
with sulphur content as an accumulation parameter) were the highest (Palomeque, 2008). Due
to these results, the author recommended P. austrosinense as a useful species for
biomonitoring of airborne dust pollution, in areas with environmental characteristics similar
to those of the study region.
Between October 2006 and January 2007, an air quality study with transplanted lichens
was conducted within the Farallón Negro Volcanic Complex and its area of influence. The
study established four transplant sites: Bajo de la Alumbrera mine and three localities
potentially affected by mining emissions (Amanao, Andalgalá and Hualfin). In this case, P.
austrosinense and Canomaculina consors (Nyl.) Elix& Hale were used as biomonitors. On
the basis of physiological parameters, interspecific differences in lichen response to mining
airborne pollution were established. For P. austrosinense, a higher stress level was detected in
thalli that were transplanted within the mine boundaries; while for C. consors, the stress level

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440 Martha S. Cañas, Raquel C. Jasan and Rita R. Plá

was higher in thalli transplanted on the outskirts of Andalgalá city, far away from the mine
(Mohaded Aybar et al., 2008b). Other parameters such as sulphur content and the PI, were
accurate solely in evaluating air quality related to mining activity, with P. austrosinense, as
for C. consors they did not show differences between transplant sites (Mohaded Aybar et al.,
2010).
In this chapter, the results obtained from the multielemental composition analysis in P.
austrosinense and C. consors samples are shown. These samples correspond to the previously
mentioned study of air quality in Farallón Negro Volcanic Complex. The aims were to
contribute to the interpretation of these species chemical response to mining airborne
pollutants and thus to analyse the feasibility of their use in air quality biomonitoring
programmes in Western Catamarca mining area. Methodological aspects and results of the
research presented here could be considered for the evaluation of air quality by lichens in
other mining regions of Latin America with similar environmental, geochemical and
exploitation characteristics to those of this Argentine province.

METHODS
Study Area

The study area is located at the Central-Western region of Catamarca Province, in the
extreme North of the morphostructural unit of the North-Western Pampean Ranges (Caminos,
1979). The area is characterized by the presence of narrow valleys and wide closed
intermountain depressions (bolsones), alternating with uplifted basement blocks whose
western slopes are usually steeper than the eastern ones. Geologically, it consists of a
precambrian igneous-metamorphic basement, covered mainly by quaternary alluvial deposits.
The climate is warm arid; precipitations are very scarce with only about 150 to 300 mm per
year, mostlyin summer (about 60-70% of the total precipitation); prevailing winds blow from
NE. Phytogeographically, it is located in the Monte Phytogeographic Province in the
Chaqueño Domain of the Neotropical region (Morlans, 1995).
In the region, there are important Au, Ag, Cu and Mo deposits currently in operation. The
first large scale mining operation of Argentina - Bajo de la Alumbrera - is located here. It is
an open-pit copper-gold (Cu-Au) mine, which corresponds to a porphyry-type deposit
included in Farallón Negro Volcanic Complex (Gutiérrez et al., 2006).This mine has been
operating since 1997.
The study area is sparsely populated: the most important locality is Andalgalá (11,411
inhabitants), the third largest city of the province. There are also little villages such as
Amanao (48 inhabitants) and Hualfín (993 inhabitants; INDEC, 2001). At these two
localities, on the outskirts of Andalgalá and within the mining project, four transplant sites
were established for this study (Figure 1). The three towns are located in intermountain
depressions: Amanao and Andalgalá at the North end of the Bolsón de Pipanaco and Hualfin
in the Hualfin Valley. Given their relative proximity to Bajo la Alumbrera, these human
settlements are considered environmentally vulnerable. For this reason, they were chosen as
monitoring sites for this study. Location data of each site are shown in Table 1.

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 Lichens as Bioindicators of Air Quality in Mining Areas 441

Figure 1. Location of the four sites to where lichens were transplanted in Farallón Negro Volcanic
Complex and its area of influence, Catamarca, Argentina: Bajo de la Alumbrera mining project (La
Alumbrera), Amanao, Andalgalá and Hualfín.

Sampling Procedure

In October 2006, thalli of the lichens P. austrosinense and C. consors were collected
from a clean site near Coneta (central valley of Catamarca). Part of this freshly picked
material was also analysed to obtain a baseline level with which to compare the transplant
analysis results. Lichen bags were prepared and transplanted, following the methodology
described by González and Pignata (1994). At each transplant site, three bags were exposed
for four months. It is important to note that this study constitutes the first one in which C.
consors is evaluated as a possible bioindicator of air quality in Catamarca province.

Table 1.Location of transplant sites in Farallón Negro Volcanic Complex

and its area of influence, Catamarca, Argentina

Transplant site Latitude Longitude Altitude Distance to the

(m.a.s.l.) open pit (km)
Bajo de la Alumbrera 27º 18' 00'' S 66º 35' 10'' W 2,406 3
Amanao 27º 32' 04'' S 66º 30' 10'' W 1,173 25
Andalgalá 27º 33' 52'' S 66º 19' 23'' W 1,123 39
Hualfín 27º 13' 28'' S 66º 49' 52'' W 1,845 25

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Multielemental Analysis

Instrumental Neutron Activation Analysis (INAA) was used to determine elemental

composition. Samples were ground in a Spex Centi Prep 6750 cryogenic mill and lyophilized
for 24 h. About 300 mg of lyophilized material was pelletized and wrapped-up in aluminium
foil for irradiation, together with two certified reference materials, NIST SRM 1633b Coal
Fly Ash and IAEA Lichen 336, for calibration purposes. Irradiations were done at the RA-3
reactor (thermal flux 3 x 1013 cm-2 s-1, 8 MW) of the Argentine National Atomic Energy
Commission (Ezeiza Atomic Centre) for 4 h. Two measurements were performed after 7 and
30 day-decay using GeHP detectors (30% efficiency, 1.9 keV resolution for 1332.5 keV 60Co
peak), coupled to an Ortec 919E multichannel buffer module. Concentrations of As, Ba, Br,
Ce, Co, Cr, Cs, Eu, Fe, Hf, La, Lu, Na, Nd, Rb, Sb, Sc, Se, Sm, Ta, Tb, Th, U, Yb and Zn
were calculated using a software developed at the NAA laboratory. GBW07405 soil was used
as control sample and the results obtained are summarized in Table 2. For all determined
elements, experimental values showed good agreement with those in the material’s certificate.
One way ANOVA was used to compare the elemental concentration of the two species in
baseline conditions. A two way ANOVA was applied to transplanted sample data, being the
factors species and transplant site.

Table 2. Quality control results (μg g-1) obtained for the analysis of GBW07405 soil by
Neutron Activation Analysis (NAA)

Element Certified Experimental

As 412 ± 24 407 ± 12
Ba 296 ± 40 316 ± 100
Ce 91 ± 15 97.9 ± 4.5
Co 12 ± 2 12.40 ± 0.39
Cr 118 ± 10 117 ± 6
Cs 15 ± 2 15.0 ± 0.8
Eu 0.82 ± 0.06 0.86 ± 0.16
Fe 9.0E+04 ± 2.0E+03 8.6E+04 ± 1.1E+03
Hf 8.1 ± 1.7 7.71 ± 0.98
La 36 ± 6 37.4 ± 0.5
Lu 0.42 ± 0.07 0.423 ± 0.047
Na 890 ± 30 886 ± 96
Rb 117 ± 9 121 ± 9
Sb 35 ± 7 34.7 ± 0.4
Sc 17 ± 2 17.4 ± 0.1
Sm 4.0 ± 0.6 4.54 ± 0.22
Ta 1.8 ± 0.3 2.03 ± 0.45
Tb 0.7 ± 0.2 0.755 ± 0.071
Th 23 ± 2 23.4 ± 0.6
U 6.5 ± 1.1 7.07 ± 0.97
Yb 2.8 ± 0.5 2.60 ± 0.55
Zn 494 ± 39 493 ± 35
n=10 (replicates).

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 Lichens as Bioindicators of Air Quality in Mining Areas 443

Data Analysis

For lichens transplanted to each site, the ratio between the concentration of each element
after exposure and that of the baseline sample prior to exposure (exposed/control ratio, EC
ratio) was calculated according to Frati et al. (2005). EC ratios were explained by means of a
5-class interpretative scale, based on the deviation of the EC ratio from “normal” conditions,
which refers to “natural” baseline concentrations of trace elements in lichens growing in
unpolluted environments. Normal concentrations of trace elements show rather large
variations due to the influence of many different factors (Markert, 1992), even in lichens
collected at the same remote area. Metal accumulation depends not only on environmental
availability of elements, but also on organism characteristics (species, age, state of health,
type of reproduction, etc.) and on other parameters such as temperature, moisture availability,
and substrate characteristics (Baker, 1983). Therefore, Frati et al. (2005) assumed a deviation
of± 25% of EC ratio equal to 1, when considering that an element concentration is at normal
levels in an exposed sample. As a rule of thumb, this deviation should account for natural
fluctuations in trace element concentrations in the biomonitor (Loppi et al., 2002).
On the basis of previous observations (Palomeque, 2008) and of those performed in this
study, the deviation proposed is considered reasonable for P. austrosinense and C. consors in
natural conditions, hence a 5-class interpretative scale has been chosen for the explanation of
EC ratios in both species. According to Frati et al. (2005), the scale is as follows: (a) 0≤ EC
ratio ≤0.25 (severe loss); (b) 0.25< EC ratio ≤0.75 (loss); (c) 0.75< EC ratio ≤1.25 (normal);
(d) 1.25< EC ratio ≤1.75 (accumulation); (e) 1.75< EC ratio (severe accumulation).

RESULTS
Concentrations of elements analysed in both transplanted and baseline samples of P.
austrosinense and C. consors are shown in Table 3. Similar concentrations were found in
both species for most elements analysed, although higher contents of Br and Se were recorded
in P. austrosinense and of Ba and Yb in C. consors.
In samples transplanted to the study area (Table 4), a significant interaction between
species and transplant site was found for As, Ce, Cs, Eu, Fe, La, Lu, Rb, Sc, Se, Sm, Tb, and
Yb. Cobalt showed the effect of the transplant site, regardless of species, and its content was
significantly higher in samples transplanted to Bajo de la Alumbrera than in those of Amanao
and Andalgalá. Bromine and U showed the individual effect of the two main factors. Bromine
content was significantly higher in samples transplanted to Bajo de la Alumbrera than in those
of Andalgalá and Hualfín. As could be observed in baseline samples, Br content in P.
austrosinense was significantly higher than that in C. consors. Uranium content in samples
transplanted to Amanao was significantly higher with respect to those of Bajo de la
Alumbrera and Hualfín. Although U content did not show significant differences between
species in baseline samples, it was higher in transplants of C. consors than in those of P.
austrosinense. Chromium and Nd contents were significantly higher in P. austrosinense, as
were those of Hf and Na in C. consors, regardless of the transplant site.

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Table 3. Elemental concentration (mg kg-1, dry weight) in Parmotrema austrosinense and
Canomaculina consors transplanted to Farallón Negro Volcanic Complex and its area of
influence (Catamarca, Argentina) and in baseline material. Baseline values in bold are
significantly different at p<0.05 (ANOVA)

P. austrosinense C. consors
Range Baseline Range Baseline
C.V. C.V.
(min. - max.) value (min. - max.) value
As 1.6 - 2.3 0.12 1.5 1.5 - 2.5 0.15 1.6
Ba 17 - 33 0.19 17 17 - 30 0.18 23
Br 3.5- 9.5 0.39 4.6 2.6 - 4.3 0.15 3.1
Ce 4.1 - 7.9 0.21 4.6 4.3 - 6.1 0.11 4.4
Co 0.54 - 1.69 0.39 0.54 0.61 - 1.03 0.17 0.62
Cr 6.7 - 48.1 0.40 30.0 8.0 - 19.3 0.29 27.1
Cs 0.50 - 0.71 0.12 0.49 0.54 - 0.80 0.13 0.56
Eu 0.07 - 0.14 0.21 0.08 0.08 - 0.12 0.13 0.08
Fe 1306 - 2263 0.18 1415 1485 - 2271 0.14 1610
Hf 0.14 - 0.28 0.21 0.18 0.19 - 0.33 0.20 0.22
La 2.0 - 3.2 0.14 2.1 2.1 - 3.2 0.11 2.1
Lu 0.026 - 0.037 0.12 0.024 0.027 - 0.043 0.11 0.027
Na 379 - 589 0.13 530 450 - 855 0.23 529
Nd 2.4 - 4.3 0.19 2,1 1.7 - 3.7 0.32 2.0
Rb 4.6 - 9.2 0.24 7.6 6.1 - 11.8 0.18 6.9
Sb 0.097 - 0.501 0.64 0.108 0.137 - 0.301 0.26 0.116
Sc 0.45 - 0.75 0.17 0.48 0.52 - 0.79 0.13 0.55
Se 0.24 - 0.55 0.24 0.34 0.21 - 0.36 0.16 0.26
Sm 0.47 - 0.77 0.18 0.43 0.36 - 0.79 0.22 0.47
Ta 0.03 - 0.73 1.66 0.06 0.06 - 0.10 0.21 0.05
Tb 0.055 - 0.136 0.31 0.059 0.040 - 0.070 0.14 0.053
Th 0.55 - 0.80 0.12 0.53 0.57 - 0.94 0.16 0.61
U 0.14 - 0.44 0.37 0.19 0.23 - 0.43 0.20 0.19
Yb 0.14 - 0.25 0.18 0.15 0.15 - 0.25 0.13 0.17
Zn 22 - 132 0.64 21 26 - 82 0.37 22

The elemental accumulation in thalli transplanted to the study area was estimated by
means of the EC ratio. For P. austrosinense, results shown below had been already partially
published by Mohaded Aybar et al. (2012). As shown in Table 5, for this species, Bajo de la
Alumbrera showed higher average multielemental accumulation in transplanted thalli than
other sites. Concentrations of Cr, Na, and Rb showed normal levels in all transplant sites,
although a slight Rb loss was detected in lichens exposed in Hualfín and a slight Cr loss in
those exposed in Andalgalá. Neodymium and Zn showed accumulation in thalli transplanted
to all sites. The accumulation of Nd was severe in Amanao and Hualfín, while for Zn, it was
severe in Bajo de la Alumbrera, Amanao, and Andalgalá. For other elements, concentrations
increased in more restricted areas, such as Eu with high EC ratio values only for Bajo de la
Alumbrera, and Hf and Se showing accumulation only in Amanao. The remaining analysed
elements presented certain specific accumulation patterns. Thus, Ba and Sb showed an
increase in thalli transplanted to Bajo de la Alumbrera, Amanao, and Andalgalá; As, Br, Ce,

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Cs, Co, Fe, La, Lu, Sc, Sm, Ta, and Yb did the same for transplants to Bajo de la Alumbrera
and Amanao, Tb and Th in thalli transplanted to the latter two sites and Hualfín, and U in
those transplanted to Amanao and Andalgalá.
For C. consors, the average multielemental accumulation was higher in thalli transplanted
to Hualfín, although its value was similar to the accumulation observed in Andalgalá and
Amanao. In all transplant sites, Ba, Br, Ce, Fe, Hf, Sc, and Tb concentrations were found at
normal levels; while thalli showed loss of Cr and accumulation of Sb, Ta, and Zn. Zinc
accumulation was severe in Amanao. Some elements showed specific patterns of
accumulation, such as La, Lu, Nd, Sm and Yb, whose EC ratio values were high only in
transplants to Andalgalá and Hualfín; and as As, Cs, Rb and U, whose EC ratio values were
high in Amanao, Andalgalá, and Hualfín. Other elements showed an increase in more
restricted areas, such as Co and Na whose EC ratio values were high only in thalli
transplanted to Bajo de la Alumbrera; Eu and Se that showed accumulation only in Hualfín,
and Th, whose accumulation was observed only in Andalgalá.

Table 4. Comparison of element concentration mean values ( ± standard deviation) (in

mg kg-1, dry weight) and its significance, between lichen species
and among transplant sites

Factors As Ba Br Ce Co
Species
P. austrosinense 1.9 ± 0.2 23 ± 3 5.8 ± 1.8 5.9 ± 1.3 0.79 ± 0.26
C. consors 2.2 ± 0.3 26 ± 4 3.3 ± 0.3 5.5 ± 0.5 0.80 ± 0.10
Site
Alumbrera 1.8 ± 0.1 25 ± 2 5.6 ± 2.6 a 6.1 ± 1.7 1.01 ± 0.22 a
Amanao 2.1 ± 0.1 23 ± 1 5.2 ± 3.0 ab 5.9 ± 1.1 0.74 ± 0.01 b
Andalgalá 2.1 ± 0.3 25 ± 4 3.8 ± 1.0 b 5.2 ± 0.4 0.64 ± 0.06 b
Hualfín 2.1 ± 0.6 25 ± 7 3.6 ± 0.4 b 5.5 ± 1.0 0.78 ± .18 ab
ANOVA
Effects
Species ** ns *** ns ns
Site ** ns * ns *
Species x Site *** ns ns *** ns
Factors Cr Cs Eu Fe Hf
Species
P. austrosinense 26.7 ± 7.7 0.62 ± 0.07 0.10 ± 0.02 1749 ± 324 0.21 ± 0.04
C. consors 13.4 ± 1.8 0.74 ± 0.13 0.10 ± 0.01 1995 ± 273 0.27 ± 0.04
Site
Alumbrera 19.8 ± 9.5 0.63 ± 0.05 0.12 ± 0.02 2047 ± 105 0.23 ± 0.01
Amanao 24.9 ± 17.8 0.70 ± 0.02 0.10 ± 0.01 1882 ± 52 0.26 ± 0.01
Andalgalá 18.0 ± 2.6 0.64 ± 0.12 0.09 ± 0.01 1610 ± 235 0.20 ± 0.06
Hualfín 17.5 ± 7.5 0.73 ± 0.26 0.10 ± 0.02 1951 ± 618 0.26 ± 0.09
ANOVA
Effects
Species *** ** ns * **
Site ns ns ** ns ns
Species x Site ns ** ** * ns

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Table 4. (Continued)

Factors La Lu Na Nd Rb
Species
P. austrosinense 2.6 ± 0.4 0.033 ± 0.004 480 ± 38 3.3 ± 0.5 6.7 ± 1.4
C. consors 2.8 ± 0.3 0.036 ± 0.003 671 ± 47 2.6 ± 0.5 9.8 ± 1.6
Site
Alumbrera 2.8 ± 0.4 0.034 ± 0.003 578 ± 159 2.6 ± 0.9 7.9 ± 0.1
Amanao 2.8 ± 0.1 0.035 ± 0.001 605 ± 96 3.0 ± 1.0 8.8 ± 1.5
Andalgalá 2.6 ± 0.4 0.033 ± 0.006 536 ± 99 2.8 ± 0.1 7.7 ± 2.8
Hualfín 2.8 ± 0.5 0.035 ± 0.006 583 ± 186 3.4 ± 0.3 8.5 ± 4.7
ANOVA
Effects
Species ns ns ** ** ***
Site ns ns ns ns ns
Species x Site ** ** ns ns **
Factors Sb Sc Se Sm Ta
Species
P. austrosinense 0.184 ± 0.059 0.60 ± 0.11 0.36 ± 0.07 0.61 ± 0.11 0.12 ± 0.12
C. consors 0.179 ± 0.020 0.69 ± 0.08 0.30 ± 0.06 0.60 ± 0.10 0.08 ± 0.01
Site
Alumbrera 0.208 ± 0.030 0.70 ± 0.04 0.30 ± 0.08 0.60 ± 0.16 0.18 ± 0.16
Amanao 0.154 ± 0.004 0.66 ± 0.00 0.36 ± 0.13 0.62 ± 0.10 0.08 ± 0.01
Andalgalá 0.218 ± 0.028 0.56 ± 0.09 0.31 ± 0.03 0.60 ± 0.11 0.06 ± 0.02
Hualfín 0.148 ± 0.047 0.67 ± 0.20 0.34 ± 0.07 0.59 ± 0.12 0.08 ± 0.03
ANOVA
Effects
Species ns * * ns ns
Site ns ns ns ns ns
Species x Site ns * ** *** ns
Factors Tb Th U Yb Zn
Species
P. austrosinense 0.087 ± 0.023 0.70 ± 0.06 0.27 ± 0.09 0.19 ± 0.03 59 ± 28
C. consors 0.065 ± 0.015 0.78 ± 0.13 0.32 ± 0.03 0.21 ± 0.02 48 ± 12
Site
Alumbrera 0.088 ± 0.040 0.68 ± 0.06 0.25 ± 0.04 b 0.20 ± 0.02 68 ± 40
Amanao 0.073 ± 0.026 0.76 ± 0.01 0.36 ± 0.05 a 0.21 ± 0.01 55 ± 13
Andalgalá 0.061 ± 0.002 0.71 ± 0.11 0.31 ± 0.07 ab 0.19 ± 0.04 50 ± 16
Hualfín 0.083 ± 0.006 0.81 ± 0.19 0.25 ± 0.08 b 0.20 ± 0.06 39 ± 11
ANOVA
Effects
Species ** ns * * ns
Site ns ns * ns ns
Species x Site * ns ns ** ns
ANOVA results: ns=not significant; * p<0.05; ** p<0.01; ***p<0.001. Values in the same column
followed by the same letter do not differ at p<0.05 (LSD Test).

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Table 5.EC ratio for Parmotrema austrosinense and Canomaculina consors transplanted
to four sites in Farallón Negro Volcanic Complex and its area of influence: Bajo de la
Alumbrera (Al), Amanao (Am), Andalgalá (And), Hualfín (Hu). Accumulation in light
grey; severe accumulation in dark grey; means were calculated from the four
sampling sites results

P. austrosinense C. consors
Element
Al Am And Hu Mean Al Am And Hu Mean
As 1.26 1.50 1.25 1.17 1.29 1.04 1.29 1.43 1.46 1.30
Ba 1.50 1.30 1.26 1.15 1.30 0.97 1.00 1.21 1.16 1.08
Br 1.62 1.60 0.99 0.85 1.26 1.15 0.97 0.99 1.00 1.03
Ce 1.60 1.47 1.08 1.05 1.30 1.09 1.15 1.24 1.20 1.17
Co 2.18 1.37 1.12 1.24 1.48 1.33 1.19 1.08 1.21 1.20
Cr 0.88 1.25 0.66 0.76 0.89 0.46 0.45 0.59 0.60 0.52
Cs 1.36 1.40 1.14 1.12 1.25 1.03 1.26 1.27 1.32 1.22
Eu 1.55 1.24 0.97 1.03 1.20 1.25 1.23 1.19 1.33 1.25
Fe 1.50 1.36 1.02 1.07 1.24 1.18 1.13 1.09 1.15 1.14
Hf 1.19 1.44 0.88 1.07 1.14 1.07 1.21 1.08 1.12 1.12
La 1.48 1.39 1.09 1.18 1.28 1.15 1.24 1.33 1.30 1.25
Lu 1.47 1.50 1.20 1.25 1.35 1.11 1.19 1.31 1.30 1.23
Na 0.88 1.01 0.88 0.85 0.90 1.26 1.25 1.13 1.13 1.19
Nd 1.55 1.80 1.30 1.76 1.60 0.93 1.15 1.38 1.73 1.30
Rb 1.06 1.02 0.76 0.68 0.88 1.10 1.41 1.38 1.49 1.34
Sb 2.12 1.44 2.19 1.06 1.70 1.53 1.28 1.68 1.51 1.50
Sc 1.53 1.38 1.04 1.22 1.29 1.19 1.18 1.15 1.17 1.17
Se 1.06 1.34 0.99 0.87 1.06 0.93 1.01 1.11 1.38 1.11
Sm 1.67 1.59 1.22 1.17 1.41 0.99 1.15 1.42 1.43 1.25
Ta 5.09 1.35 0.76 0.91 2.03 1.47 1.70 1.54 1.51 1.55
Tb 1.97 1.53 1.04 1.33 1.47 1.06 1.00 1.09 1.15 1.07
Th 1.36 1.43 1.19 1.27 1.31 1.01 1.21 1.26 1.20 1.17
U 1.13 2.00 1.30 0.96 1.35 1.40 1.67 1.81 1.28 1.54
Yb 1.42 1.40 1.05 1.05 1.23 1.10 1.18 1.29 1.31 1.22
Zn 4.66 2.21 2.92 1.52 2.83 1.75 2.89 1.72 1.87 2.06
Mean 1.72 1.45 1.17 1.10 1.36 1.14 1.25 1.27 1.29 1.24

DISCUSSION
In general, concentration values detected in P. austrosinense have the same order of
magnitude as those registered for this species transplanted to an area of Department of Belén,
during the spring of 2005 (Jasan et al., 2011). Only Ce, Cs, Hf, Na, Rb, and Th contents
decreased in the mining area respect tothose in Belén. There are no multielemental
composition data for C. consors in Catamarca, due to the fact that the present study is the first
one in which this species is evaluated as a possible bioindicator of air quality in the province.
In lichens transplanted to the mining area, and for 14 of the 25 elements analysed, a
species-dependent spatial variation of concentration was observed (Table 4). Other elements
such as Cr, Nd, Hf, Na, and U presented significantly different concentrations between

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species, regardless of transplanting site, even when their concentrations were similar in
baseline samples. This might indicate a differential response of P. austrosinense and C.
consors to being transplanted from their natural environment to the study area. Only Br, Co,
and U showed a similar spatial variation of concentration in both species.
Interspecific differences in element concentration variation in exposed thalli with respect
to baseline levels can be better interpreted by analysing elemental accumulation. Inorganic
substances may occur in lichens as 1) particles (adsorbed onto the thallus surface or within
intercellular spaces), 2) ions bound to extra- or intracellular exchange sites, and 3) soluble
intracellular ions (Bargagli and Mikhailova, 2002). Elemental concentrations, as determined
in this study, represent the sum of those fractions, and any variation in the elemental content
of transplanted thalli can be considered as an accumulation or a loss with respect to the
baseline level.EC ratio, which reports the enrichment degree of transplanted samples with
respect to baseline samples, was calculated for determination of accumulation in lichen thalli,
at different exposure sites.
In this sense, Cr (whose concentration, together with that of Nd, was significantly higher
in P. austrosinense than in C. consors) showed EC values that correspond to concentration at
normal levels for P. austrosinense (with the exception of thalli transplanted to Andalgalá);
and EC ratios that also correspond to a Cr loss in thalli of C. consors (Table 5). Moreover, Nd
enrichment was higher in P. austrosinense than in the other species, which accumulated this
element in only two of the four exposure sites.
Regarding those elements with higher concentrations in C. consors than in P.
austrosinense, EC ratio showed a slight Na accumulation in thalli of C. consors transplanted
to Bajo de la Alumbrera, whilst the content of this element in P. austrosinense was at normal
level in all transplant sites. Moreover, U accumulation was observed in thalli of C. consors
transplanted to Amanao, Andalgalá, and Hualfín, and only at the first two sites for P.
austrosinense. Considering EC ratio, the higher Hf content in transplants of C. consors in
comparison to the other species, was not due to its accumulation, but to a higher
concentration (even though not significant) in the original baseline material. In fact, P.
austrosinense showed accumulation of this element in thalli transplanted to Amanao.
In general, elemental enrichment in P. austrosinense was higher than in C. consors, due
to both the number of accumulated elements and the number of sites where a severe
accumulation was determined. Thus, P. austrosinense showed a severe accumulation in the
four transplant sites, whilst C. consorsin only two. In addition, P. austrosinense showed a
severe accumulation for seven elements (Co, Nd, Sb, Ta, Tb, U, Zn), and C. consors for only
U and Zn (Table 5).
Certain elements such as Nd and Zn in P. austrosinense, and Sb, Ta and Zn in C. consors
showed accumulation for all the exposure sites. The remaining elements, either presented EC
ratios that correspond to concentrations at normal levels, or below normal (Cr, Na and Rb in
P. austrosinense; Ba, Br, Ce, Cr, Fe, Hf, Sc, Tb in C. consors); or an accumulation restricted
to certain transplant sites in the study area.
Regarding the accumulation at each transplant site (Table 5), P. austrosinense
accumulated a higher number of elements in thalli exposed at Bajo de la Alumbrera and
Amanao (19 and 21 elements, with severe accumulation of 5 and 3 elements in each site,
respectively), followed by Andalgalá and Hualfín (5 and 4 elements, with severe
accumulation of 2 and 1 element in each site, respectively). On the contrary, in C. consors a
higher accumulation was observed in Hualfín and Andalgalá (14 and 13 elements,

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respectively; with severe accumulation of one element in the latter site), followed by Amanao
and Bajo de la Alumbrera (7 and 5 elements respectively; with severe accumulation of one
element in Amanao). Although for both species, high EC ratios were observed for Co, Sb, Ta,
and Zn in thalli transplanted to Bajo de la Alumbrera. These EC ratios only corresponded to a
severe accumulation in P. austrosinense.
It is necessary to clarify that the designation “severe” refers to neither the toxicity of the
different elements towards lichen, nor to a comparison with air quality standards; but it refers
to how marked the accumulation of some elements results, in thalli transplanted to certain
sites.
A similar behaviour, with regard to the accumulation degree of each species at different
transplant sites, is observed when considering the average EC ratios for each species (Table
5). P. austrosinense showed a higher average EC ratio in Bajo de la Alumbrera, followed by
Amanao, Andalgalá, and Hualfín. This suggests a distance gradient with respect to the mining
exploitation area itself. For Hualfin, these results could be related to the NW site location
with respect to the mine. Although the region has NE prevailing winds, for the months during
which this study was conducted, NW winds were very frequent. Similar results were reported
by Mohaded Aybar et al. (2010) for sulphur content in these samples, since they observed a
concentration gradient in thalli transplanted to Bajo de la Alumbrera>Amanao>Andalgalá,
with significantly higher contents of this element in Bajo de la Alumbrera and Hualfín with
respect to Andalgalá. These authors also observed higher values of PI in Bajo de la
Alumbrera and Hualfín with respect to the other transplant sites. Given that PI has shown to
be a good parameter for detecting the effect produced by airborne particulate matter in Belén
(Palomeque, 2008), this could be indicating high levels of stress in lichens exposed to these
kinds of pollutants at these sites.
Apart from the mining activity itself, atmospheric pollution in Bajo de la Alumbrera may
originate from mobile sources (medium and heavy vehicles) within the mining project. In
Hualfín, atmospheric pollution could have its origin from these mobile sources, given that
National Route 40, along which heavy traffic moves to and from mining exploitations,
crosses this town. Sulphur accumulation related to high levels of vehicular traffic has been
previously observed in lichens in Argentina (Levin and Pignata, 1995; González et al., 1996;
González et al., 2003). On the other hand, in an air monitoring study with automatic samplers
and for the same period of lichen transplant, Herrero (2008) observed in Los Nacimientos,
near Hualfín, moderately heavy traffic of trucks and cars on National Route 40. This road was
then still under construction at this place, being a major source of particulate matter. The
report stated that concentration values of respirable particulate matter (PM 10) were above the
national standard reference (Ley Nacional Nº 24,585, 1995) in Bajo de la Alumbrera and Los
Nacimientos, but not in Amanao and Andalgalá.
As can be seen in Table 5, the average EC ratio in C consors was higher in Hualfín,
which may be related to particulate matter generated during road repairing. According to
chemical-physiological analyses conducted on these samples (Mohaded Aybar et al., 2010),
no significant differences were detected in sulphur content and PI of thalli transplanted to the
four exposure sites. Considering the photosynthetic pigments content, Mohaded Aybar et al.
(2008b) observed a certain stress level in thalli of C. consors transplanted to Andalgalá. Due
to the fact that this transplanting site was located in the outskirts of the city, it appeared
unlikely that the lichen response had been caused by the presence of urban pollutants. In this

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study and using photosynthetic pigments as biomarkers, a higher stress level was detected in
P. austrosinense thalli transplanted to Bajo de la Alumbrera.
With the aim of determining the degree of correspondence between the elemental
enrichment of lichens and the geochemical characteristics of different transplant areas,
multielemental geochemical data of stream sediments reported by the Argentine Mining
Geological Service - SEGEMAR (Ferpozzi et al., 2002) were considered. According to
these data and taking into account only the elements considered in this study, Co, Fe, Sb, Sc,
and Zn are abundant in Bajo de la Alumbrera area, which coincides with the EC ratio value
of these elements obtained for P. austrosinense transplanted to this site. Particularly,
geochemical data correspond to the EC ratios for Co, Sb and Zn (elements with EC ratios
values above 1.75). C. consors also accumulated these three elements in thalli transplanted to
Bajo de la Alumbrera.
Zn is characteristic of hydrothermal ore deposits such as Bajo de la Alumbrera and the
rest of the ore deposits of Farallón Negro Volcanic complex. The high Zn, Co and Sb
enrichments observed in P. austrosinense and in a lower degree in C. consors transplanted to
Bajo de la Alumbrera could be also related to local pyrite oxidation phenomena in both
natural state (ore deposit) as well as from tailings generated in the mining operation. On this
site and by using automatic samplers, Herrero (2008) detected abnormal concentrations of Zn
in atmospheric dusts, possibly correlated with (in other order of magnitude) geochemical
anomalies of mineralized areas. The agreement of these findings with the results of the
present study indicate that P. austrosinense may be an appropriate active bioindicator for this
area.
P. austrosinense also showed severe accumulation of Ta and Tb. In contrast to Zn, Ta is
very scarce in mineralized zones of Farallón Negro-Bajo de la Alumbrera; for this reason
their accumulation in thalli transplanted to this area might indicate an origin from nearby
granitic areas by physical dispersion phenomena. On the other hand, C. consors showed Ta
and Na accumulation in thalli transplanted to Bajo de la Alumbrera. In this area, the largest
Na availability consists of sulphate outcrops that fill fractures that affect the ore deposit. Na -
Ba - K is a characteristic association in hydrothermal mineralizations, which are abundant in
the region. Normally, they are also present in granitoid rocks.
For the Amanao area, geochemical data indicate an abundance mainly of Sb and Sc. The
area where the town is located is extremely anomalous in Sb and several other elements, with
the addition of certain elements from Quebrada de Vis Vis and Quebrada de Amanao ravines,
both located to the North. In thalli transplanted to this site, P. austrosinense accumulated Sb
and Sc, together with Hf and Se (which only showed an accumulation in this site for this
species), Zn and rare earth elements. A severe accumulation of Zn, together with Nd and U,
which are typical of the granitic domain of Quebrada de Amanao, was observed. Meanwhile,
C. consors also accumulated Sb together with As, Cs, Rb, Ta, U and Zn, and the EC ratio of
this last element was above 1.75. For both lichen species, Zn accumulation in Amanao might
be explained by the presence of high contents of soluble sulphates in stream sediments, due to
the contribution of some watercourses originating in strongly mineralized areas still
unexploited.
For the Andalgalá zone, geochemical data show an abundance of As, Cs, Sb, Sm, Ta, Tb,
Th, U, Yb and Zn; as well as an abundance of other elements to the north of this area, such as
other rare earths (Ce, La, Lu, Nd) and U. P. austrosinense showed an accumulation of Ba, Nd
and U in thalli transplanted to this site as well as an accumulation of Sb and Zn with EC ratio

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 Lichens as Bioindicators of Air Quality in Mining Areas 451

values above 1.75. C. consors showed accumulation of some of those abundant elements in
that same area. Severe U accumulation resulted particularly important in thalli of this species
transplanted to Andalgalá. In this site, EC ratio values obtained for U in both species could be
reflecting the presence of important uranium sources (still unexploited) in the region where
the study area is located. Radioactive granites present in the region have U and Th associated
to rare earth elements such as Sm. On the other hand, Zn accumulation in thalli correspond to
the high natural geochemical values of Zn in stream sediments in Andalgalá, which possibly
come from the dispersion produced by Candado River, originated from copper porphyry
deposits also unexploited. It is not excluded that, in this area, Zn can have an anthropogenic
origin, apart from its natural origin. This element is present in certain agrochemicals used in
fumigations of fruit trees, which are very abundant near the city of Andalgalá.
For the Hualfín zone, geochemical data show abundance of As, Cs, Rb, and Sb, with
important concentrations of other elements such as Hf, Sc, Ta, U, Zn, and rare earth elements
in nearby zones. For P. austrosinense, an accumulation of Nd, Tb, Th, and Zn was observed
in thalli transplanted to this site, and the EC ratio of Nd was above 1.75. Due to the presence
in the area of recent carbonate hydrothermal activity, the exclusive Nd accumulation in thalli
transplanted to Hualfín would seem to indicate a selective accumulation of this element in P.
austrosinense. In previous studies (Palomeque, 2008; Jasan et al., 2011), a similar behaviour
of this species regarding Ta and Zn has been found. For this reason, it is likely that EC ratio
values obtained in the present work are determined by geochemical characteristics of the
transplant areas as well as by accumulation mechanisms of this lichen species.
C. consors accumulated As, Cs, Rb, and Sb in thalli transplanted to Hualfín; and Ta, U,
Zn and rare earth elements (Eu, La, Lu, Nd, Se, Sm, Yb) in accordance with geochemical
abundance of these elements in the area. Pyrite and arsenopyrite, containing As, are very
abundant in the study area. In the West of Catamarca, As origin is attributed to the presence
of volcanic ashes in the soil that have been carried from volcanic zones by aeolian forces; as
well as to igneous, sedimentary and metamorphic rock meteorization, that can release arsenic
compounds (arsenites, arsenates or arsenic trioxide) of great mobility which are able to
migrate long distances from the source. For the province of Catamarca, it cannot be dismissed
the possibility that As can be mobilised from different anthropogenic processes, such as
mineral beneficiation that can lead to arsenic contamination phenomena (Vilches et al., 2005).
It is noteworthy that according to geochemical data, Cr is a slightly abundant element in
the study area, which matched EC ratio values obtained in thalli of both lichen species
transplanted in all exposure sites.

CONCLUSION
P. austrosinense and C. consors exhibit a differential response of multielemental
accumulation due to their transplant to different sites within the study area. These quali- and
quantitative interspecific differences could be interpreted using the EC ratio.
P. austrosinense was the species with a higher elemental enrichment in general terms,
particularly in Bajo de la Alumbrera and in Amanao, which is located to the South of the
mine. Elemental accumulation in thalli transplanted to these sites can be related to mining
airborne pollution.

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452 Martha S. Cañas, Raquel C. Jasan and Rita R. Plá

In C. consors, elemental accumulation was higher in thalli transplanted to Hualfín and

Andalgalá. This can be related to emission sources other than open-pit mining exploitation
present in the study area.
In both species, multielemental accumulation in thalli reflected the geochemical
characteristics of each transplanting site, and somehow, also the local and regional
environmental dynamics. Therefore, it is considered that P. austrosinense and C. consors are
suitable for detecting variations of air quality, which might be produced due to natural or
anthropogenic factors in the region. In this regard, the results of this study contribute to
establish environmental quality baselines for a region that is in the process of exploiting its
natural resources.
Even though P. austrosinense and C. consors allowed determining for the first time the
atmospheric availability of different elements in the study area, it will be necessary to inquire
about a possible selective biogeochemical behaviour of each species. That will allow a better
interpretation of results obtained in air quality studies by means of these species as
biomonitors.
Results presented here allow concluding that, of the two species, P. austrosinense is the
most suitable one for biomonitoring air quality in areas with open-pit mines and
environmental characteristics similar to those of the Western region of Catamarca.

ACKNOWLEDGMENTS
This study was supported by the Secretaría de Ciencia y Tecnología de la Universidad
Nacional de Catamarca (SECyT). We thank E.H. Peralta for his invaluable collaboration in
the geochemical discussions. Thanks are extended to Walter D. Acosta for English technical
assistance.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 29

DYNAMICS OF PRIORITY POLLUTANTS AND

ADEQUACY OF WASTEWATER TREATMENTS IN
THE LAKE MARACAIBO BASIN (VENEZUELA)

Julio César Marín-Leal1,*, Sedolfo José Carrasquero-Ferrer1,

María Carolina Pire-Sierra2 and
Elisabeth Hedwig Behling de Calmón1
1Departamento
de Ingeniería Sanitaria y Ambiental (DISA),
Escuela de Ingeniería Civil, Facultad de Ingeniería, La Universidad del Zulia,
Estado Zulia, Venezuela
2Programa de Ingeniería Agroindustrial, Decanato de Agronomía, Universidad

Centroccidental Lisandro Alvarado (UCLA),

Estado Lara, Venezuela

ABSTRACT
The origin and dynamics of priority contaminants in Lake Maracaibo (Venezuela)
will be discussed in this chapter, as well as the potential of different treatment
technologies for domestic and industrial effluents discharged in this basin which has
received little or no pretreatment. These wastewaters serve as a major source of pollution
to this aquatic ecosystem, coupled with other anthropogenic activities such as extraction
and transportation of oil, agriculture, tanneries, petrochemical and food industry, etc.
Ecotoxicological studies with native organisms are scarce and only in recent years have a
few been made using some bacteria, ciliate protozoa and bivalve mollusks, showing high
resistance to heavy metals. Maracaibo Lake's current trophic level indispensably drives
the enforcement of recovery plans and environmental quality monitoring, these geared
towards ensuring the survival of the native species and to perpetuate the existence of an
alternate source of water for the human population.

*
Corresponding Author E-mail: jmarin@fing.luz.edu.ve.

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458 J. César Marín-Leal, S. José Carrasquero-Ferrer, M. Carolina Pire-Sierra et al.

Keywords: biotechnology, contaminant mobility, eutrophication, organic matter, wastewater

treatment

INTRODUCTION
Watershed of Lake Maracaibo

Lake Maracaibo is a partially mixed hypertrophic tropical estuary, located in Western

Venezuela (Zulia). It consists of four different water bodies: Maracaibo Lake, Maracaibo
Strait, Tablazo Bay and Gulf of Venezuela, in addition to its 135 tributaries (Figure 1) (Parra-
Pardi, 1979; Rodríguez, 2000).

73°00’ 72°00’ 71°00’

Republic of Colombia Gulf of

Venezuela
Sinamaica
Limón River Lagoon
11°00’ Socuy Reservoir El Moján Matícora River
El Tablazo
Guasare River Santa Cruz Bay Ancón de Iturrez
Punta Vigía
Tulé Reservoir Punta Palmas Norte
Socuy River Cachirí River
Lake Strait

Maracaibo Pueblo Viejo

Punta de Leiva River 0 20 40 Km
Palmar River Punta Palmas Sur
Cabimas
Cd. Ojeda Burro Negro
Lagunillas Reservoir
Barranquitas Machango River
Apón River Machango Reservoir
10°00’ Bachaquero

Negro River Misoa River

Lake Maracaibo Agua Viva Reservoir

Aricuaisá River Carache River

Motatán River
Santa Ana River

Catatumbo River Torondoy River

Castan River

09°00’
Tarra River Tucaní River Caribbean Sea

Zulia River Escalante River VENEZUELA

Chama River

Onia Reservoir Colombia

Brazil

Figure 1. Map of Lake Maracaibo showing its main tributaries (normal font), reservoirs (italic font),
coastal cities and villages (bold font).

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 Dynamics of Priority Pollutants and Adequacy of Wastewater Treatments … 459

The lake’s watershed has an area of approximately 121,823 km2; of which 12,400 km2
correspond to the lake and 1,120 km2 to Maracaibo Strait and Tablazo Bay that connect it
with the Gulf of Venezuela. It is located between 70°30' and 73°24' West longitude and
between 8°22' and 11°51' North latitude. This lake has four basic limnological features: i)
hypolimnetic cone in the center; ii) counterclockwise movement of water masses from
epilimnion around the cone; iii) influence of Catatumbo river; and iv) intrusion of seawater.
Between 1953 and 1963, a navigation channel was dredged across the strait (100.6 km) to
connect the Gulf of Venezuela with Lake Maracaibo and allow the entry of large ships (Parra-
Pardi, 1979). This channel influences the salt wedge and therefore the distribution of nutrients
and salinity in the system.
This basin has the largest oil reserves in South America and one of the largest in the
world, with a total amount of 33 billion barrels extracted between 1914 and 1995, most of
which come from the bottom of the lake. Besides oil exploitation, many other activities take
place in the basin, including the petrochemical industry, gas processing, transportation of
hydrocarbons, exploitation and transportation of coal in opencast mines, metal-mechanical
industries, tanneries, pharmaceutical factories, food companies like slaughterhouses and
shrimp, among others (Rodríguez, 2000).
The main contributor of organic matter and inorganic nutrients is represented by the
direct discharge of domestic wastewater into the lake (33,724 kg/d) containing fecal material,
detergents rich in phosphates and nitrogenous substances, of which the highest proportion is
discharged untreated into the Maracaibo Strait, from the cities of Maracaibo, Cabimas and
Ciudad Ojeda (Rodríguez, 2000). The development of agriculture and livestock in the
Northwest and South Lake (over 83,843 hectares) also represents an important source of
organic and inorganic compounds, including pesticides, metals, nitrogen and phosphorus
compounds (Rodríguez, 2000; Meléndez et al., 2005).

Adequacy of Wastewaters according to Venezuelan Discharge Regulations

Lake Maracaibo receives large amounts of wastewaters from domestic and industrial
sources without treatment, infringing the Venezuelan legislation for quality control of water
bodies and discharge of liquid effluents, which include permissible limits for: biochemical
oxygen demand (BOD5), chemical oxygen demand (COD), total suspended solids (TSS),
nitrogen, phosphorus, metals, hydrocarbons, pesticides, coliform bacteria, among others
(Decreto 883, 1995).
In order to propose alternatives for the treatment of wastewaters discharged into the lake,
numerous studies have been made to adapt the quality of the final effluent to Venezuelan law
(Decreto 883, 1995). Some of the technologies that have been evaluated are: upflow
anaerobic sludge blanket reactors (UASB), rotating biological contactors (RBC), sequential
batch reactors (SBR), constructed wetlands, adsorption with activated carbon, microbial
consortia, coagulation-flocculation, among others.
In this chapter, previous studies of different researchers are described and discussed,
concerning: i) the origin and dynamics of priority pollutants in Lake Maracaibo; and ii)
treatment of domestic and industrial wastewaters conducted with effluents generated in the
watershed of Lake Maracaibo, aimed to counteract both the progress of the eutrophication

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process as well as of the modification of ecological dynamics to native communities. Recent

reports of ecotoxicological tests with autochthonous species are also presented.

ANALYSIS OF PRIORITY POLLUTANTS AND NUTRIENTS

Carbon, Nitrogen and Phosphorus

Numerous studies have analysed C, N, P contents in the water column and sediments of
Lake Maracaibo basin, including tributary rivers. Determinations have included both organic
and inorganic forms, by applying standard methods (Parra-Pardi, 1979; APHA et al., 1998;
Gardner et al., 1998; Ledo et al., 2003; Rivas et al., 2005, 2009; Torres et al., 2010; Polo et
al., 2014).
Mejías et al. (2014) applied water quality indices to describe the trophic state and
environmental condition of Lake Maracaibo, specifically in its coastal zone. They used data
from 42 sampling stations around the Lake during rainy and dry periods in 2012. The indices
were: index of the National Sanitation Foundation-USA (INSF), weighted arithmetic water
quality index (WQIWA), geometric-weight multiplicative water quality index (WQIGWM),
unweighted arithmetic water quality index (WQIUWA) and unweight geometric-multiplicative
water quality index (WQIUWGM). The authors assessed index applicability by comparing
contamination degree via principal component analysis (PCA).

Pesticides

Twelve organochlorine pesticides - α-BHC (α-benzene hexachloride), β-BHC, γ-BHC

(lindane), heptachlor, aldrin, endosulfan, DDE (dichlorodiphenyldichloroethylene), dieldrin,
TDE (1,1-dichloro-2,2-bis(4-chlorophenyl)ethane), endrin, DDT (1,1,1-trichloro-2,2-bis(4-
chlorophenyl) ethane) and metoxichloride - were analysed in samples of water, sediment
(Urdaneta, 1989) and muscle fatty tissue of 14 fish species (Urdaneta et al., 1994) from
pisciculture ponds located in the Limón river basin (Guasare and Socuy rivers) by gas
chromatography with electron capture detector (Urdaneta, 1989; Urdaneta et al., 1994).
Moreover, Sánchez et al. (2005) determined glyphosate concentrations in 144 water
samples of the Catatumbo river basin (six stations in Tarra, Socuavo, Zulia and Catatumbo
rivers), using a liquid-liquid extraction with dichloromethane and ion chromatograph
equipped with a conductivity detector.

Hydrocarbons and Phenols

Table 1 describes the analytical methods used to determine concentrations of

hydrocarbons and phenols in environmental samples from Lake Maracaibo.

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Table 1. Analytical methods and other study variables for hydrocarbons and phenols in
Lake Maracaibo

Parameter Method Sample Location in Lake Reference

Maracaibo
n-alkanes, methylnaphthalenes, Gas chromatography Water, fish North, center and Templeton et
total saturate hydrocarbons, and 24-hour median and shrimp South al. (1975)
total aromatic hydrocarbons tolerance limit (TLm24)
and toxicity
Total hydrocarbons; 1,2,4, Extraction with carbon Water Samples taken 50 López and
trimethylbenzene; 1,3,5 tetrachloride, analysis and 250 m from Márquez (1991)
trimethylbenzene; by infrared spectrometry the shore in 7 sites
diphenylmethane; anthracene and gas chromatography
and pyrene
Phenol; o; m; and p-cresol; 2- Liquid-solid extraction Water 38 samples taken Burger (2000)
isopropylphenol; o and p- with graphitized carbon at different depths
ethylphenol; 2,3; 2,4; 2,5; 2,6; cartridges and gas in 13 stations
3,4 and 3,5-xylenol; and 2,3,5- chromatography
trimethylphenol
16 polycyclic aromatic Liquid-solid extraction Water 48 superficial Ulacio et al.
hydrocarbons disks C18 (500 mg samples in 5 (2000)
octadecylsilane) and gas stations
chromatography
Benzene, toluene, CS2 extraction and gas Sediment 2 samples from Faría et al.
ethylbenzene, m-xylene and o- chromatography 9°51´17" N (2005)
xylene latitude and
71°31´41" W
longitude)
Total hydrocarbons Standard gravimetric Water Port of Toas Alburgue
method island (Tablazo (2015)
Bay)

Metals

The content of heavy metals in Lake Maracaibo has been determined in samples of water,
sediment and biota (aquatic birds, fish, macrophyte Lemna spp. and bivalve Polymesoda
solida), using the techniques of atomic absorption spectrometry with graphite furnace and
cold vapour, and inductively coupled plasma mass spectrometry (ICP-MS) (Prieto and
García, 1988; Barco, 1989; Urdaneta, 1995; de Bautista et al., 1999; Hermoso and Márquez,
2005; Pirela and Casler, 2005; Ávila et al., 2007, 2010; Rojas, 2012; Marín et al., 2014). The
most studied metals were cadmium (Cd), chromium (Cr), vanadium (V), lead (Pb), mercury
(Hg), copper (Cu) and nickel (Ni).

ASSESSMENT OF WASTEWATER TREATMENTS

The sequential batch reactor (SBR), rotating biological contactors (RBC), constructed
wetlands and upflow anaerobic reactors (UASB) were evaluated at laboratory or pilot scale

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for the treatment of different types of wastewater (Behling et al., 2003, 2008, 2012a; Vera et
al., 2010; Díaz et al., 2012a; Carrasquero et al., 2015). These researches were carried out
using domestic and industrial wastewater effluents considered pollutants to Lake Maracaibo.
The parameters measured to determine the efficiency of treatment systems were defined
according to standard methods (APHA et al., 1998).
Microalgae and autochthonous bacteria were isolated from water samples of Lake
Maracaibo and from oil pit located in the eastern shore of this ecosystem, and were tested
individually, in mixed cultures (coexistence of different species) and microbial consortia
(dependent association between different species) regarding their ability to degrade
hydrocarbons. For this, feasibility and treatability assays were performed in order to establish
bacterial efficiency in nutrient and hydrocarbon fraction removal (analysis of saturates,
aromatics, resins and asphaltenes, SARA) (Araujo et al., 2002; Díaz et al., 2012b, 2013;
Alburgue, 2015).
The physicochemical treatments have also been evaluated regarding removal efficiency
of priority pollutants in Lake Maracaibo, such as organic matter, nutrients, heavy metals
and hydrocarbons (Rojas et al., 2008). The processes include coagulation-flocculation
(Carrasquero et al., 2014a; Pardo et al., 2014) and adsorption (Contreras et al., 2008).

ECOTOXICOLOGICAL BIOASSAYS
Data on ecotoxicological bioassays with organisms of Lake Maracaibo are scarce or
nonexistent. Tests with heavy metals in bacteria isolated from macrophyte Lemna spp. (Díaz
et al., 2007; Marín et al., 2011), pelagic bacteria (Castro et al., 2015) and ciliated protozoa
(Rincón, 2014) have been carried out in recent years. The minimum inhibitory concentration
(MIC) (bacteria), inhibitory concentration for 50% of population (IC50, 24 h) (bacteria) and
lethal concentration for 50% organisms (LC50, 1 h) (protozoa) were determined.
Rojas (2012) performed acute and chronic toxicity bioassays with the bivalve mollusc P.
solida exposed to metals Cd2+, Cr6+, Pb2+ and V5+, determining incorporation and elimination
rates of metals and individual LC50 (96 h) values.

PHYSICOCHEMICAL CHARACTERISTICS OF
SURFACE WATER
A physicochemical characterization of surface water in the different zones of Lake
Maracaibo is presented in Table 2. These features are influenced by the input of freshwater
from the South (tributaries) and the entry of seawater forming a salt wedge on the North (Gulf
of Venezuela). The dissolved oxygen concentrations decrease from North to South, due to
intensification of organic matter oxidation processes entering from natural and anthropogenic
sources.

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Carbon, Nitrogen and Phosphorus

Studies have shown that the high productivity of Lake Maracaibo coupled with the
elevated level of nutrients in local areas of high biological production and discharges have
contributed to the acceleration of the eutrophication process. The N/P ratio is low (<5),
resulting from an excess of P in the system (Parra-Pardi, 1979; Gardner et al., 1998; Polo et
al., 2014).
The accumulation of N (5,471.86 mg/kg dry weight) and P (2,505.51 mg/kg dry weight)
in sediment from thecentre of the lakeand mobility of nutrients across the sediment-water
interface are relatively high. This highlights the importance of sediments for nutrient
dynamics in this ecosystem, based on dissolved oxygen levels of the hypolimnetic cone (Ledo
et al., 2003; Torres et al., 2010). Tributary rivers contribute significantly to nutrient loading
and eutrophication of the lake (Rivas et al., 2005, 2009).
Water quality indices applied to Lake Maracaibo explain the trophic state and
environmental condition of the water in the coastal area. The temporal-spatial gradient shows
high variability between sampling sites and seasonal periods (dry and wet), as well as
influence of local phenomena on water quality (Mejías et al., 2014). The PCA indicated that
INSF responds better regarding lake characteristics; however, other indices used (WQIWA,
WQIGWM, WQIUWA and WQIUWGM) can also describe the water condition when applied
together, becoming a versatile tool to know about ecological state or environmental condition
of this water body.

Pesticides

The presence, mobility and persistence of pesticides in Lake Maracaibo basin have been
poorly studied. The literature reflects scarce current data for these contaminants in waters of
tributaries or adjacent zones to vegetable cultivation plots in the Northern and Southern areas
of the lake.

Table 2. Physicochemical characteristics of surface waters from Lake Maracaibo

Area Tempe- pH Dissolved Salinity Electric Reference

rature oxygen (psu) conductivity
(°C) (mg/L) (mohm/cm)
Gulf of 26-27 30-34 Rodríguez (2000)
Venezuela
Tablazo Bay 30.7 ± 8.20- 7.8-10.9b 8.13- a
Polo et al.
3.1a 8.72b 13.09b (2014),
bRojas (2012)

Lake Strait 31.3 ± 7.95- 6.01-10.5b 2.92- aPolo et al.

1.2a 8.81b 5.77b (2014),

bRojas (2012)

Lake 30.19- 7.09- 4.38-8.22 3.8-4.4 7,500- Gardner et al.

31.17 8.20 8,540 (1998)
Tributary 28.8 ± 6.28- 0.161 ± 0.083 Rivas et al. (2005)
rivers 1.84 8.04

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An analysis of organochlorine pesticides made between 1987 and 1992 in water,

sediment (Urdaneta, 1989) and fishes (Urdaneta et al., 1994) of aquaculture ponds in Limón
river prairie (Northwest basin area) showed the following concentrations: between 0.0001
(DDE) and 0.9000 (α-BHC) μg/L in water, between 0.0002 (endrin and dieldrin) and 0.4123
(α-BHC) mg/kg in sediment (Urdaneta, 1989) and in cultured fish fat tissue between 0.0058
μg/g of aldrin in Pimelodus clarias and 2.8729 μg/g of metoxichloride in Roeboides dayi
(Urdaneta et al., 1994).
Recently, Sánchez et al. (2005) did not detect the presence of glyphosate (herbicide) in
the Catatumbo river basin (detection limit: 1.24 µg/L), suggesting that the basin was not
affected by its use for eradication of poppy crops in the neighboring Republic of Colombia
for the sampling period.
Monitoring of the presence and persistence of pesticides in water and sediments of Lake
Maracaibo needs to be carried out. This is of particular importance regarding rivers running
through lands of vegetable cultivation and animal husbandry in the Northern and Southern
areas of the basin, in order to detect their incorporation into aquatic and benthic food chains,
and possible ecological consequences for the ecosystem.

Hydrocarbons and Phenols

A two-year study conducted by Templeton et al. (1975) showed low concentrations of

hydrocarbons in Lake Maracaibo water (<1 mg/L) and no detectable accumulation of
petroleum in muscle tissue of selected commercial fish (<1 µg/g; lake’s center). Nonetheless,
in the South area of the lake, hydrocarbon concentrations were high in some species: 38.4
µg/g in Prochilodus reticulates and 11.3 µg/g in Anodus laticeps, probably as a result of
differences in diet and metabolism of these species. Toxicity studies with fish and shrimp in
vivo, also developed by Templeton et al. (1975), indicated that relatively high concentrations
of oil are required to cause mortality, with TLm24 (24-hour median tolerance limit) of 15,000
mg/L for shrimp Penaeus schmitti and of 2,400 mg/L for fish Mugil curema, using Tía Juana
medium crude oil (Tía Juana geological formation).
In surface waters of the Northeastern area of the lake, between El Hornito and Los
Coquitos sectors, López and Márquez (1991) reported concentrations of total hydrocarbons
between 1.22 and 9.97 mg/L. These values are 1.54 times higher than those found in 1973 for
this same area, which could be related to increase in oil production operations. The presence
of 1,2,4-trimethyl benzene, 1,3,5-trimethyl benzene, biphenyl methane, anthracene and
pyrene was also detected.
Surface waters from Port of Toas Island (Tablazo Bay) exhibited a total hydrocarbon
concentration of 6,211.67 ± 2,972.83 mg/L (Alburgue, 2015). This value exceeds the
permissible limit of 20 mg/L established by Venezuelan law for discharge to a marine-coastal
environment (Decreto 883, 1995), which can seriously affect biological processes of the lake.
Ulacio et al. (2000) reported concentrations of polycyclic aromatic hydrocarbons between 0.4
and 4.4 µg/L for water samples from five sampling stations.
Regarding the content of phenols, a high presence of methylphenols was found in water
samples from the coast of Maracaibo city (66-212 µg/L), with concentrations of phenol 84 ±
15; 2-isopropylphenol 59 ± 15; 3,4-xylenol 42 ± 14 and o-cresol 17 ± 8 µg/L (Burger, 2000)
above those allowed by Venezuelan law (2 µg/L, Decreto 883, 1995).

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Air Aquatic birds

Hg: 2,09±2,07
(mg/kg, Pirela and Casler, 2005)

Water column Fish of Catatumbo river

Macrophyte Lemna spp. V: 0.13-2.62, Cr: 0.07-33.99,
V: 2.573±3.177, V: 0.73-15.73, Cd: 0.12-1.02, Pb: 1.35-9.89,
Cr: 6.738±8.156, Cr: 10.57-50.83, Ni: 0.25-170.08, Cu: 1.22-49.33,
Cd: 0.054±0.109, Pb: 1.46-37.97, Hg: 0.06-2.62
Pb: 1.448±2.495 Ni: 8.20-68.59 (mg/kg, Hermoso and
(μg/L, Rojas, 2012) (μg/kg, Ávila et al., 2007) Márquez, 2005)
Sediment
V: 1.29-121.20, Cr: 3.98-98.28, Bivalve Polymesoda solida
Cd: 0.46-7.90, Pb: 17.8-143.9, V: 0.95-0.50, Cr: 3.04-1.56,
Ni: 16.76-177.62, Cu: 2.31-75.46 Cd: 0.03-0.05, Pb: 2.23-2.25
(mg/kg, Ávila et al., 2010) (mg/kg, Rojas, 2012)

Figure 2. Current data regarding heavy metal concentrations in water, sediment and biota of Lake
Maracaibo and fish of Catatumbo river.

In the case of sediments, Faría et al. (2005) did not detect residues of aromatic
hydrocarbons in samples from the lake center, indicating possible interference during analysis
due to the presence of moisture in the samples as a result of extraction with CS2 (insoluble in
water). Detection limits were: 260 (benzene), 92 (toluene), 90 (ethylbenzene), 120 (m-xylene)
and 87 µg/kg (o-xylene).

Metals

Historically, metal concentrations in Lake Maracaibo have been very low, becoming
undetectable during the 1970s and ‘80s. However, Prieto and García (1988) found 17.5-70.8
ngHg/L in proximity to Tablazo Petrochemical Complex. Barco (1989) reported mean
concentrations of 93.10 ± 4.96 μgPb/L and 0.53-17.67 μgHg/L for the Lake Strait, while de
Bautista et al. (1999) found 2.8 (surface) to 19.76 mgHg/L (bottom) in this area (Figure 2).
The presence of heavy metals in sediment and biota has been reported in several studies
(Barco, 1989; Urdaneta, 1995; Pirela and Casler, 2005; Hermoso and Márquez, 2005; Ávila et
al., 2007, 2010; Rojas, 2012; Marín et al., 2014), showing that their concentrations increase
through the aquatic food web (Figure 2).
The dynamics of heavy metals in Lake Maracaibo show increasing levels from North
(Tablazo Bay) to South (Lake) as a result of metal load provided by tributaries.
Concentrations in sediments meet the Canadian sediment quality guidelines for protection of
aquatic life (CCME, 1999). However, the content of metals found in the clam P. solida
(maximum 1.267 mgCd/kg dry weight and 38.412 mgPb/kg dry weight; Marín et al., 2014) is
not considered acceptable for human consumption, according to the maximum tolerable
weekly limit ingestion per body weight, established by the Food and Agriculture Organization
of the United Nations (WHO/FAO, 1999).

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Table 3. Characteristics and removal percentages of some effluents treated by

sequential batch reactor (SBR)

Operational HRT CRT VOL

Wastewater Co (mg/L) % removal Reference
sequence (h) (d) (kgCOD/m3d)
BOD: 92
BOD: 130
COD: 85 Cárdenas et
Domestic An/Ae/Ax 24 20 0.331 COD: 276
TKN: 52 al. (2012)
TKN: 99 TP: 8
TP: 67
COD: 12,784 COD: 88
An/Ae/Ax/ Angulo et
Dairy factory 24 20 15.34 TKN: 99 TKN: 90
Ae/Ax al. (2015)
TP: 37 TP: 57
COD: 530
COD: 83 Díaz et al.
Shrimp industry Ae/Ax 10 15 1.271 TKN: 68
TKN: 78 (2012a)
TP: 8
COD: 16,606 COD: 95
Carrasquero
Slaughterhouse An/Ae/Ax 15 25 26.569 TKN: 646 TKN: 90
et al. (2015)
TP: 11 TP: 44
SCOD: 65
SCOD: 794
Phenol:
Phenol: 1.47 Díaz et al.
MOPW Ae 15 15 1.27 87.5
TH: 104 (2005a)
TH: 77
O+G: 148
O+G: 56
SCOD: 1065
Phenol: 19.3 SCOD: 88 Díaz et al.
LOPW Ae 16 15 1.598
TKN: 34 Phenol: 97 (2005b)
TP: 1.07
SCOD: 307
Phenol: 2.7 SCOD: 63 Díaz et al.
HOPW Ae 16 15 0.46
TKN: 10 Phenol: 83 (2005b)
TP: 2.68
COD: 47
COD: 27,148
Tannery (soak TKN: 30 Pire et al.
Ax/Ae 12 15 54.926 TKN: 2,696
liquour + dyeing) TP: 65 (2010a)
TP: 21 Cr: 14
Cr: 62
COD: 6,024 COD: 47
Tannery
TKN: 644 TKN: 30 Pire et al.
(Tanning+ Ax/Ae 12 15 12.048
TP: 13 TP: 75 (2010b)
dyeing)
Cr: 174 Cr: 79
BOD: 880
Carrasquero
COD: 1,254 BOD: 83
Food industry Ae 6 15 3.520 et al.
TN: 14 COD: 92
(2014b)
TP: 8
COD: 2,008
COD: 49 Carrasquero
TKN: 229
Tannery Ae/Ax 15.6 25 3.089 TKN: 63 et al.
TP: 7,7
TP: 28 (2014c)
Cr: 2,3
HRT: Hydraulic retention time; CRT: Cellular retention time; VOL: Volumetric organic loading; C o: Initial
concentrations; SCOD: Soluble chemical oxygen demand; O+G: Oil and grease; TH: Total
hydrocarbons; COD: Chemical oxygen demand; BOD: Biochemical oxygen demand; TP: Total
phosphorus; TN: Total nitrogen; TKN: Total Kjeldahl nitrogen; MOPW: Medium oil production water;
LOPW: Light oil production water; HOPW: Heavy oil production water; An: Anaerobic condition; Ae:
Aerobic condition; Ax: Anoxic condition.

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Table 4. Characteristics and removal percentages of some effluents treated by rotating biological
contactors (RBC), upflow anaerobic sludge blanket (UASB) combined with RBC and anaerobic
rotating biological contactors (AnRBC)

Wastewater Reactor HRT (h) OL (g/m2d) Initial % removal Reference

concentration
(mg/L)
Slaughterhouse RBC 7.7 COD: 92
12.3 88 Behling et
24 COD: 12,250
18.4 86 al. (2003)
24.6 77
Shrimp industry RBC 24 4.0 COD: 74.5
16 2.3 71.9
Behling et
12 6.7 COD: 973 92.3
al. (2008)
8 6.3 73.3
6 16.4 44.6
Synthetic ww RBC
24 COD: 1,000-2,000 COD: 97-99
(glucose)
Behling et
Synthetic ww COD/Phenol COD: 97.5
al. (2005)
(glucose+ 12 1,000/6 Phenol: 99.7
phenol) 1,000/20
Synthetic ww RBC 24 6.10 COD: 96.25
COD: 1,096 Behling et
(sucrose+urea) (3-stage) 12 11.68* TN: 62.95
TN: 226.6 al. (2012b)
6 23.97
Synthetic ww UASB+ UASB RBC UASB RBC COD: 4,097.8 COD: 95.84
(glucose+ RBC 10.12 18.97 9.42 8.38 TKN: 200 TKN: 80.71 Behling et
phenol) 10.12 18.97 9.73 5.5 PO34-: 40.6 PO34-: 81.03 al. (2012a)
15.15 28.41 6.55 3.45 Phenol: 17.6 Phenol: 99.6
Synthetic dairy AnRBC Continuous†
industry reactor:
Behling et
(sucrose+milk) 24 24.67 COD: 2,500 COD: 82.7 Batch†
al. (2013)
reactor: COD:
93.6
* Best treatment. Synthetic ww: Wastewater prepared in the laboratory; HRT: Hydraulic retention time; OL:
Organic load; RBC: Rotating biological contactors; UASB: Upflow anaerobic sludge blanket; AnRBC:
Anaerobic rotating biological contactors; COD: Chemical oxygen demand; BOD: Biochemical oxygen
demand; TP: Total phosphorus; TN: Total nitrogen; TKN: Total Kjeldahl nitrogen; PO43-:
Orthophosphate; †: Flow regime.

PERFORMANCE OF WASTEWATER TREATMENTS

Sequential Batch Reactor (SBR)

SBR have been successfully used in the treatment of domestic and industrial wastewater,
demonstrating the flexibility of SBR systems to adapt to effluents with different
physicochemical characteristics (Table 3). The SBR system promotes the removal of nutrients
using an anaerobic/aerobic/anoxic (An/Ae/Ax) sequence in the treatment, resulting in
removal of carbon, nitrification, denitrification, anaerobic phosphorus release and uptake of
excess phosphorus during the aerobic phase. Cárdenas et al. (2012) used this sequence for

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simultaneous nutrient removal from domestic sewage of the city of Maracaibo, yielding an
effluent that meets the current Venezuelan regulatory standards of discharges to water bodies.
Waters associated with oil exploitation represent one of the main pollutants of Lake
Maracaibo. During oil production, a certain amount of effluent known as production water
(OPW) is coproduced. Díaz et al. (2005a,b) used laboratory reactors for treatment of light oil
production water (LOPW), medium oil production waters (MOPW) and heavy oil production
water (HOPW). They concluded that OPW with total phenol concentrations between 1.40 ±
0.27 and 19.36 ± 2.02 mg/L can be successfully treated by SBR reactors (Table 3).
SBR have also been used successfully to simultaneously remove nutrients and organic
matter from effluents of cattle slaughtering processes (Carrasquero et al., 2015) and from
shrimp processing (Díaz et al., 2012a).
Pire et al. (2010a,b) removed COD (chemical oxygen demand), TKN (total Kjeldahl
nitrogen) and TP (total phosphorus) from tannery effluent with a chromium concentration of
174 mg/L. These researchers showed that the strongest removal of compounds occurred in the
early hours of treatment, which was attributed to the anoxic conditions that allowed the
decomposition of structures of the complex organic matter. Besides, Carrasquero et al.
(2014c) successfully treated the mixed settled effluents produced in a tannery. The
operational sequence Ae/Ax in combination with a cell retention time of 25 days allows for
the best efficiencies for the simultaneous nutrient and COD removal.
From effluents with nutrient deficit, such as those from vegetable processing industries,
average removal values for COD and BOD of 80 and 92%, respectively, were obtained.
These removals were achieved without needing to add macronutrients (nitrogen and
phosphorus) to the wastewater (Carrasquero et al., 2014b).

Rotating Biological Contactors (RBC)

RBC has been tested at laboratory scale showing high efficiency, low cost, and ease of
operation and maintenance (Table 4). Behling et al. (2003, 2008) evaluated the behavior of a
RBC for different types of wastewater. In the case of effluents from meat and shrimp
industries the system was able to remove a high amount of organic matter. Particularly for the
shrimp, effluent, COD and pH were reduced to levels that comply with the limits established
in the Venezuelan regulations. In another study, RBC achieved 97% removal of phenolic
compounds (Behling et al., 2005).
Moreover, Weffer (2010) and Behling et al. (2012a) evaluated the performance of a
combined system treatment (UASB+RBC) using a synthetic effluent (glucose+phenol), and
they obtained a high overall efficiency of phenol degradation (99.6%) and removal of organic
matter (95.8%). Weffer (2010) concluded that the UASB followed a first-order kinetics
Monod model and RBC followed the Kincannon and Stover model.
Behling et al. (2012b) studied the influence of organic load (OL) on efficiency of the
aerobic 3-stage RBC to treat a synthetic industrial effluent: the greater removal of C and N
was reached in the first stage of reactors, meeting the Venezuelan discharge limit for COD
(<350 mg/L). The authors observed that nitrification increased the concentration of inorganic
nitrogen in the final effluent. At the same time, Marín et al. (2012) found that the abundance
of bacteria was affected by both organic loads and the position in the reactor chambers. They

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also noted simultaneous occurrence of the nitrification-denitrification processes, evidenced by

the detection of both bacterial groups in the biofilm.
Finally, Behling et al. (2013) conducted tests in an anaerobic RBC (AnRBC) using a
synthetic effluent with similar characteristics to that generated in the dairy industry
(sucrose+milk). Significant removals of COD were achieved, but without meeting the
discharge limits in the Venezuelan regulation. Remaining organic matter content in the
AnRBC effluent was due to the contribution of suspended solids; they therefore
recommended the installation of a secondary settler after the reactor.

Constructed Wetlands

Constructed wetlands have been tested successfully for the treatment of domestic,
industrial (water deriving from oil production) and mining wastewater, removing
contaminants from the effluents through different ways (absorption, adsorption,
sedimentation). For instance, Vera et al. (2010) evaluated the removal of nutrients and
organic matter from domestic sewage using a free water surface (FWS) wetland planted with
Typha dominguensis, and found that it was more efficient in removing organic matter in
relation to the control (without emergent vegetation); nevertheless, the nutrient removal was
higher in the control treatment. Results showed the important role of this macrophyte during
removal of suspended solids and organic matter. However, more studies are needed to
determine the importance of the macrophyte in nutrient removal (Table 5). On the other hand,
Vera et al. (2010) determined that eight weeks of growth were the optimal time for T.
dominguensis pruning.
In another study, Núñez et al. (2007) observed the synergistic effect of the combination
of macrophytes (Lemma sp. and T. dominguensis) on removal of TN and COD over the
system that operated without vegetation (control). They also observed the important activity
of bacteria and algae during nutrient removal from wastewater.
Araujo et al. (2014) evaluated the influence of surface coverage of the FWS wetland with
Pistia sp. on the removal efficiency of nutrients from domestic effluent. They obtained the
largest removals of TN and TP when the wetland surface was completely covered with
macrophytes. However, in the treatment where no coverage with Pistia sp. was used, a
significant removal of nutrients was obtained due to the presence of the microalgae Chlorella
sp. (401.2x104 cells/mL).
On the other hand, Blanco-Fontalvo (2008, 2011), treating effluents from oil production
industry in a SSF wetland, obtained higher removal of phenols, hydrocarbons, COD, SS, Cl-,
S2- and metals (Cr, Cu, Ni, Pb and Zn) when using the combination of macrophytes (Table 5).
The results suggest that SSF wetlands are efficient in removing contaminants from this kind
of effluents, and such efficiency depends on the macrophytes used.
Simultaneously, Paz-Palacios (2008) and Paz et al. (2012) evaluated the efficiency of
pilot-scale FWS wetlands for the treatment of wastewater from oil production. The removal
of TP, hydrocarbons, S2-, phenol, and TN showed no significant differences between the
wetland planted with Cyperus luzulae, C. fertile, C. ligularis, Paspalum sp. and T.
dominguensis, and the control (without emergent vegetation). Particularly, they could
not demonstrate the influence of these macrophytes in the removal of S2- and phenolic
compounds.

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470 J. César Marín-Leal, S. José Carrasquero-Ferrer, M. Carolina Pire-Sierra et al.

Marín et al. (2006) observed a marked trend towards the reduction of the content of
heavy metals (Pb, Zn, Fe, Al and Ni) as the hydraulic retention time (HRT) was increased
during the treatment of coal mine drainages in wetlands. Sediment resuspension from the pit,
secretion by plants and atmospheric deposition material mechanisms could be related to the
mobility of metals.

Table 5. Characteristics and removal percentages of some effluents treated by different

types of constructed wetlands: free water surface (FWS) and subsurface flow (SSF)

Wastewater Wetland Organism % removal Reference

Domestic FWS Pistia sp. TN: 100
TP: 77.7 Araujo et
Chlorella sp. TN: 97 al. (2014)
TP: 70.7
Domestic FWS Typha dominguensis COD: 30.3-30.5
BOD: 34.6-31.6
TSS: 43.9-52.4 Vera et al.
VSS: 42.9-60.8 (2010)
NH4+: 45-55
TKN: 48-50
Domestic FWS T. dominguensis + COD: 42
Lemma sp. TKN: 84
Núñez et
N-NH4+: 93.4
al. (2007)
N-NO2-: 54
N-NO3-: 41
Water from oil SSF T. dominguensis + Phenol: 90.69 Blanco-
production OL=29.4 Paspalum sp. Hydrocarbons: 76 Fontalvo
g/m2d Cyperus luzulae, COD: 31.8 (2008)
HRT: 7d C. fertile, TSS: 50.8
C. ligularis Cr: 35.5, Cu: 47.4, Ni:
52.6, Pb: 52.9, Zn: 27.0
Water from oil SSF T. dominguensis, C. Cu: 58 Blanco-
production luzulae, C. feraz, C. Pb: 53 Fontalvo et
Ligularis al. (2011)
Water from oil FWS C. luzulae, C. feraz, C. TP: 11 Paz-
production HRT: 7 d ligularis, Paspalum Hydrocarbons: 68 Palacios
sp., S2-: 66 (2008)
T. dominguensis Phenol: 57
TN: 72
Cr: 91, Ni: 20
Cu: 91, Zn: 28
Pb: 7
Water from oil FWS C. luzulae, S2-: 62-66 Paz et al.
production HRT: 7 d C. ligularis Phenol: 61-71 (2012)
C. feraz, Paspalum
sp.,
T. dominguensis
Coal mine SSF T. dominguensis SO42- > 80 Marín et al.
HRT: 1, 3, 5, (2007)
7, 9 d
HRT: Hydraulic retention time; OL: Organic load; COD: Chemical oxygen demand; BOD: Biochemical
oxygen demand; TP: Total phosphorus; TN: Total nitrogen; TKN: Total Kjeldahl nitrogen; N-NH4+:
Ammoniacal nitrogen; N-NO2-: Nitrite; N-NO3-: Nitrate; TSS: Total suspended solid; VSS: Volatile
suspended solid, S2-: Sulfides; SO42-: Sulfates.

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 Dynamics of Priority Pollutants and Adequacy of Wastewater Treatments … 471

In a parallel study, Marín et al. (2007) evaluated the behavior of sulfates, which are one
of the main problems in the liquid effluents of the coal industry. The results showed that
concentrations of SCOD, TKN and TP remained below the permissible limit set by the
Venezuelan regulations. The variability of the parameters studied is determined by the type of
support material employed in the wetlands and HRT. The system was highly efficient in
reducing the sulfate content in a biological way.

Anaerobic Biological Treatment Systems

Recent research has demonstrated the applicability of anaerobic biological treatment

system of wastewater containing high organic loads and toxic or inhibitory compounds,
simulating the wastewater discharged in the Lake Maracaibo basin (Table 6). Caldera et al.
(2007) demonstrated the efficiency of a UASB reactor treating slaughterhouse effluents.
Besides, Gutiérrez et al. (2007) evaluated the treatability of water oil production in batch
reactors, showing that the biodegradability of LOPW under thermophilic conditions presented
no difference compared with mesophilic conditions (78 and 80%, respectively); however,
results were different for MOPW and HOPW, where the increase in temperature improved
biodegradability.
Other biological treatments have been evaluated for domestic effluents discharged in the
basin of Lake Maracaibo. Cárdenas et al. (2002) used aerated ponds for the treatment of
municipal wastewater from the city of Maracaibo. They demonstrated the efficiency of this
treatment in tropical weather (removal of 91% SCOD, with a HRT of one day).

Microbial Consortia and Native Microorganisms

Native bacteria and microalgae isolated from surface waters of Lake Maracaibo have
demonstrated high efficiency in hydrocarbons removal. Fertilized and aerated cultures of
bacteria from the lake were able to remove up to 89% of total hydrocarbons present in
lubricating oil (initial concentration of 7,000 mg/L) (Araujo et al., 2002), while microalgae
mixed cultures of Coenochloris sp. and Chlorococcum sp., isolated from an oil pit, in
consortium with its associated bacteria, efficiently degraded kerosene to 68% at an initial
concentration of 0.5%, and to between 43 and 57% with kerosene initially at 3% (Díaz et al.,
2012b), highlighting the remediation potential of the established microbial consortium.
Kerosene (1%) hydrocarbons were removed up to 60% in 30 days by mixed cultures of
bacteria Yersinia rohdei, Pantoea agglomerans, Sphingobacterium thalpophilum and
Actinobacillus capsulatus, isolated from an oil pit in the Zulia State. S. thalpophilum and A.
capsulatus were equally efficient in total hydrocarbons removal in axenic cultures (Díaz et
al., 2013). Thus, microbial consortia and native microorganisms can be efficiently used in the
biological treatment of hydrocarbon contaminated water before being discharged into Lake
Maracaibo.

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472 J. César Marín-Leal, S. José Carrasquero-Ferrer, M. Carolina Pire-Sierra et al.

Table 6. Characteristics and removal percentages of some effluents treated by anaerobic

reactors: double chamber anaerobic reactor (DCAR), bacth reactor (BR),
upflow anaerobic sludge blanket (UASB)

Wastewater Reactor HRT VOL T Co % Biogas Reference

(h) (kgCOD/ (mg/L) removal (L/d)
m3d)
Synthetic ww DCAR 12-2 0.9-6.0 M COD: 467-508 COD: 69-85 0.89- Rincón et
(glucose) 2.56 al. (2013)
Synthetic ww BR 24 1.0-3.0 M COD: 1,000- COD: 86-16 0.2- Martínez
(glucose+Ni) 3,000 4.5 et al.
Ni: 20-50 (2012)
Synthetic ww UASB 24 0.9-5.6 M COD: 903-5,596 COD: 82 2-6 Vacca et
(glucose+ Phenol: 20 Phenol: 34 al. (2008)
phenol) 2-chlorophenol:
77
2-nitrophenol: 55
2,4
dinitrophenol: 77
Tannery UASB 24 1.0-3.0 M COD: 1,000- COD: 92-94 0.31- Behling et
3,000 0.85 al. (2004)
Slaugtherhouse UASB 12 17 M COD: 8,500 COD: 87 15.5
Caldera et
al. (2007)
LOPW MOPW BR 24 1.28 8.64 Tm SCOD: 1,280 SCOD: 71 - Gutiérrez
HOPW 9.33 SCOD: 864 SCOD: 60 et al.
SCOD: 933 SCOD: 62 (2007)
LOPW UASB 24-6 1.4-5.6 Tm SCOD: 1,400 SCOD: 79-53 - Gutiérrez
M SCOD: 75-40 et al.
(2006)
Clarifier BR 24 0.55-0.84 M SCOD: 20-60 - Mas y
(a mix of SCOD: 553-848 Naphthalene: Rubí et al.
LOPW+MOPW+ Naphthalene: 99.5 (2011)
HOPW) 7.91 Acenaphthylene:
Acenaphthylene: 0
1.25
Shrimp industry UASB 32- 1.43-6.1 M COD: 1,902- COD: 50-83 0.34- Pérez et
11 2,849 3.38 al. (1999)
DCAR: Double chamber anaerobic reactor; Synthetic ww: Wastewater prepared in the laboratory; HRT: Hydraulic
retention time; VOL: Volumetric organic loading, Co: Initial concentrations; T: Temperature; BR: Batch reactor;
COD: Chemical oxygen demand, UASB: Upflow anaerobic sludge blanket; SCOD: Soluble chemical oxygen
demand; M: Mesophilic; Tm: Termophilic; MOPW: Medium oil production water; LOPW: Light oil production
water; HOPW: Heavy oil production water.

Physicochemical Treatment of Effluents

Carrasquero et al. (2014a) used chitosan (obtained from crab waste) as a coagulant for
treating wastewater from the flour processing industry. They found removals of 98.9 and
80.2% of turbidity and COD, respectively. Besides, the ability of chitosan to form complexes
with metal ions was explored by Pardo et al. (2014), obtaining a 45% removal of chromium
during the pretreatment of tannery effluent.

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 Dynamics of Priority Pollutants and Adequacy of Wastewater Treatments … 473

Activated carbon prepared from sugarcane bagasse has a high surface area and a porous
structure suitable for adsorption of complex organic compounds during wastewater treatment,
being able to remove 2-chlorophenol, 2-nitrophenol and 2,4-dimethylphenol: removal
percentages obtained from the adsorption of these compounds on activated carbon obtained
from sugar cane bagasse were 49.8, 66.3 and 57.6%, respectively (Contreras et al., 2008).

RECENT ECOTOXICOLOGICAL BIOASSAYS

Ciliated protozoa Euplotes sp., Uronema sp. and Loxodes sp. (Rincón, 2014), bacterial
strains (Castro et al., 2015) and the plant Lemna ssp. (Díaz et al., 2007; Marín et al., 2011)
from Lake Maracaibo, showed high resistance to heavy metals, with very high MIC, LC50
andIC50 values, particularly in bacteria (to the order of 108 mg/L), as a result of pollution
prevailing in the ecosystem (Castro et al., 2015).
Toxicity bioassays with bacteria indicated that the most toxic metal was Cr6+, with the
following sensitivity pattern Cr6+ > Cd2+ > Cr3+ > Ni2+ > Pb2+ (Castro et al., 2015). In the case
of ciliated protozoa, results showed that Euplotes sp. was the most sensitive to Cr6+ and Cr3+,
while Loxodes sp. exhibited major susceptibility to Cd2+ and V5+ (the sensitivity order to the
tested metals was Cd2+ > V5+ > Cr3+ > Cr6+). Euplotes sp. exhibited the greatest response to
all the tested heavy metals, showing LC50 of 14.35 mgCr6+/L, 4.82 mgCr3+/L, 0.82 mgCd2+/L
and 2.13 mgV5+/L, indicating that this species is a potential bioindicator of metal
contamination in Lake Maracaibo (Rincón, 2014).
The concentrations of heavy metals in soft tissues of P. solida (Cr > Pb > V > Cd) did not
correlate with levels found in water and sediment. Toxicity tests showed LC50 (96 h) values of
15.94 mgCr6+/L, 77.79 mgPb2+/L, 128.25 mgCd2+/L and 401.96 mgV5+/L. Bioassays of
incorporation and elimination of Cr6+ and V5+ were characterized by a rapid uptake and
increased sensibility to Cr6+. Chromium (VI) accumulation was higher but V5+ was retained
for a longer time (Rojas, 2012; Rojas et al., 2015). Thus, P. solida is also recommended as
bioindicator for heavy metal pollution in Lake Maracaibo estuarine system due to its ability to
incorporate, tolerate and accumulate metals in concentrations higher and proportional to
exposure levels.

CONCLUSION
The hydrodynamic conditions of Lake Maracaibo basin are particularly complex due to
the simultaneous interaction of different factors such as tides, winds, stratification, saline
intrusion, and hydrologic and hydraulic effects. A high accumulation of C, N, P and heavy
metals in bottom sediments has been identified, which, depending on oxygenation conditions
of the water-sediment interface, maybe retained in the water column. Bioaccumulation
processes and high resistance to heavy metals have also been identified in the biota of this
aquatic system.
Treatment technologies tested at laboratory and pilot scale seem to be viable alternatives
for depuration of wastewaters discharged into Lake Maracaibo.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 30

IMPORTANCE OF BACTERIAL BIOFILM FOR ZN

RETENTION IN THE SEDIMENT-WATER INTERFACE:
THE ROLE OF BACTERIAL ZN RESISTANCE

Wilson Machado1,*, Elisamara Sabadini-Santos1,

Katia N. Suzuki2, Edimar C. Machado3,
Alfredo V. B. Bellido4, Ricardo E. Santelli5,
Ricardo T. Lopes6 and Mirian A. C. Crapez7
1
Departamento de Geoquímica, Instituto de Química,
Universidade Federal Fluminense, Niterói, RJ, Brazil
2
Programa de Pós-graduação em Geoquímica,
Universidade Federal Fluminense, Niterói, RJ, Brazil
3
Instituto Federal de Educação, Ciência e Tecnologia
do Rio de Janeiro, Nilópolis, RJ, Brazil
4
Departamento de Físico-Química, Instituto de Química,
Universidade Federal Fluminense, Niterói, RJ, Brazil
5
Instituto de Química, Universidade Federal do Rio de Janeiro,
Cidade Universitária, Rio de Janeiro, RJ, Brazil
6
Laboratório de Instrumentação Nuclear, COPPE,
Universidade Federal do Rio de Janeiro, RJ, Brazil
7
Programa de Pós-Graduação em Biologia Marinha,
Universidade Federal Fluminense, Niterói, RJ, Brazil

ABSTRACT
Bacterially-mediated processes have a pivotal role in determining the cycling of trace
elements in sedimentary environments, while the potential role of microbial biofilms
developed under heavily-contaminated conditions on this cycling is less known.
Microcosm experiments were performed to test the hypothesis that metal resistance

* Corresponding Author E-mail: wmachado@geoq.uff.br.

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482 Wilson Machado, Elisamara Sabadini-Santos, Katia N. Suzuki et al.

induced by exposure to elevated metal levels can result in the production of bacterial
biofilms more able to affect the zinc (Zn) cycling in the sediment-water interface of a
tidal creek that drains a mangrove ecosystem. After 4 h of flooding by tidal water with
high Zn concentration (50 mg L-1), creek sediments with Zn-resistant biofilm showed
significantly different HCl-soluble Zn concentrations (62% higher) and inventories (56%
higher) than control sediments. A significant correlation between HCl-soluble Zn and Fe
occurred for most data, with the exception of results from the uppermost sediment layers
treated with Zn-resistant biofilm, evidencing that this treatment overwhelmed the general
positive effect of Fe on Zn concentration. A positive feedback between enhanced Zn
resistance and Zn sorption capacity of sediments was observed. This finding may
contribute to a better comprehension of the Zn cycling at sites in which Zn resistance is
developed and sites that receive Zn-resistant biofilm associated to suspended particulate
matter transported from nearby contaminated areas. Since the short flooding time (4
hours) simulated in these experiments evidenced a significant biofilm effect on Zn
accumulation, longer periods of evaluation are recommended to test the duration of the
observed effect (e.g., throughout complete tidal cycles).

Keywords: bacterial biofilm, zinc resistance, sorption, sediments, microcosm experiments

INTRODUCTION
Bacterially-mediated processes have a pivotal role in determining the cycling of trace
elements in sedimentary environments, driving diagenetic reactions that produce and degrade
geochemical compounds (Shaw et al., 1990; Borch et al., 2010), and affecting sediment
physical properties, such as sediment particles cohesion (Decho, 2000). The assemblages of
bacterial populations attached by extracellular polymeric substances (EPS) to aquatic system
substrates (i.e., bacterial biofilms) can constitute important trace element-binding surfaces in
such systems, considering the metal sorption by bacterial surfaces (Fein et al., 2001; Guiné et
al., 2006) and by the EPS secreted by bacterial consortia (González et al., 2010; Kenney and
Fein, 2011). When a dissolved or paticulate substance transported by the water phase meets a
biofilm, it encounters a heterogeneous hydrogel with very different sorption sites, including:
charged groups (e.g., -COO-, -SH-, etc.), apolar groups (e.g., aromatics, alifatics, etc.), cell
walls, cytoplasmic membrans and cytoplasms; while the EPS consist of polysaccharides,
proteins and lipids (Flemming and Leis, 2002). The EPS composition seems to be not
constant, but is influenced by growth conditions and environmental stress (Flemming and
Leis, 2002).
The biofilm sorption of trace metals has been widely evidenced in sediments, soils,
natural waters and wastewater (Borrok et al., 2004; Toner et al., 2005; Hockin and Gadd,
2006; Ha et al., 2010). This biofilm role can occur in different ways, for instance, by ion
exchange reactions, complexation and precipitation, as passive binding (e.g., electrostatic
interactions, ion exchanges, precipitations and complexations with biofilm components
already present and not formed in response to the bound substance) or active binding (i.e.,
through the excretion of binding, chelating or precipitating substances in response to the
presence of the dissolved substance) processes (Flemming and Leis, 2002). Biofilm coatings
can often enhance metal sorption by sandy sediments, actively participating in binding
dissolved metals from both overlying and pore water (Schlekat et al., 1998), while elevated
metal exposure can induce bacterial toxicity and increased EPS production to cope with such

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 Importance of Bacterial Biofilm for Zn Retention … 483

effect, resulting in increased metal immobilization by EPS (Yang et al., 2013). Therefore,
considering that metal resistance is the ability of microorganisms to continue growing when
exposed to the considered metal (Harrison et al., 2007), it may be hypothesized that the metal
sorption by sediments can be significantly affected by biofilms with amplified metal
resistance.
Possible effects of bacterial biofilm with amplified Zn resistance on the sorption of Zn
from overlaying water by tidal creek sediments from the Itacuruça mangrove ecosystem
(Sepetiba Bay, SE Brazil) were evaluated in microcosm experiments. Although it is known
that biofilms can affect the metal sorption capacity of sandy sediments (Schlekat et al., 1998;
Yang et al., 2013), evaluations of this biofilm role in intertidal tropical ecosystems were not
found in the literature. Moreover, enhanced metal sorption capacity by the influence of
bacterial biofim with improved metal resistance (in response to previous metal exposure) may
be an additional factor affecting metal accumulation in these environments.

METHODS
Zinc-resistant biofilm was obtained after a treatment of surface sediments (0–3 cm depth)
collected from the mouth of the Saco do Engenho creek (Figure 1), Sepetiba Bay
(22°55′12′′S, 43°49′6′′W). This creek is recognized as the major source of Zn to the bay due
to past drainage from a large Zn smelt plant refuse pile (Barcellos et al., 1991; Molisani et al.,
2004). Zinc emissions from this source were estimated as 3,660 t yr-1until 1997, when the
plant was closed (Molisani et al., 2004).
The procedures adopted to obtain the bacterial biofilm with amplified Zn resistance were
those described by Pennafirme et al. (2015), which employed the same exposure material used in
this work. Resistant bacteria were isolated from Saco do Engenho creek sediments and
multiplied in a sterilized cultivation media of filtered sea water (<0.45 μm) with bacto-
peptone (2 g L-1), urea (2 g L-1), glucose (6 g L-1) and Zn (50 mg L-1), during 30 days. In
April 2013, surface sediments and surface tidal water were collected in the tidal creek that
drains a mangrove forest dominated by the red mangrove (Rhizophora mangle L.) located in
Itacuruçá, Sepetiba Bay (22°55′19′′S, 43°53′09′′W) to proceed with the experiments. There
are previous evidences that after the sorption of 65Zn from overlying water by sandy
sediments from Itacuruçá creek, this radiotracer presented a partial release back to overlying
water, while adjacent mangrove-colonized, muddy sediments presented more efficient 65Zn
retention (Machado et al., 2008). In order to test the potential role of Zn-resistant biofiolm in
improving the Zn sorption from tidal water, such sediments with lower Zn sorption capacity
were chosen for the present study. From the studied sampling site (Figure 1), surface
sediments (0–5 cm depth) were collected using a plastic spatula and stored in a plastic bag
under refrigeration until processing in laboratory, while surface (~5 cm depth) tidal water was
sampled at the same moment during an ebbing tide, by using a 25-L plastic container. The
experiments were performed ~2 h after the sampling.
Plexiglas tubes were used to contain experimental microcosms (Figure 2), which were
composed of 6 cm height sediment columns covered by 120 mL of local tidal water spiked
with Zn at a concentration of 50 mg L-1. The experimental apparatus was adapted from
Petersen et al. (1998), according to Machado et al. (2008). Since natural variability in

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484 Wilson Machado, Elisamara Sabadini-Santos, Katia N. Suzuki et al.

sediment composition and benthic organisms activity can affect trace element sorption from
overlying water (e.g., Suzuki et al., 2013), sampled sediments were homogenised prior to tube
preparation to improve data comparability.

22º55’00”

SE
N n d
sla

C
aI
d eir
Ma Sampling
1
2
site6
uaí 3
Itag or 5
a rb

22º56’00”
H 4 7
8
9 12
11
10

Sepetiba Bay 500 m

43º50’00” 43º49’00”

Figure 1. Location of Saco do Engenho creek (SEC), Sepetiba Bay, Brazil, and the study sampling site
at the SEC mouth.

One set of triplicate cores received 1 mL of culture medium containing Zn resistant

biofilm (with 1.13 × 109 cells cm-3), while the other set of other triplicate cores were used as
controls. After a 12-h acclimation period, all cores were slowly covered (minimizing
sediment re-suspension) with Zn-spiked tidal water to start the experiments, which were
carried out at room temperature (~26°C). Tidal water pH (8.0) and salinity (28) were
measured using a Hanna pH meter and a Shibuya refractometer, respectively. Oxygen
saturation of overlying water was induced by air pumping. After 4 h (simulating a short
flooding period), the overlying water was removed and the sediments were extruded and
sliced at 2 cm depth intervals. After agitation of sediments in 1 mol L-1 HCl for 16 h (Huerta-
Diaz and Morse, 1990), reactive Zn (ZnHCl) concentrations in sediments were determined,
considering that this extraction has been employed to estimate potential metal bioavailability
for benthic organisms (e.g., Sokolowski et al., 2007; Sabadini-Santos et al., 2014a,b). This
procedure extracts metals associated to carbonates, Fe and Mn (oxy)hydroxides, labile
organic matter and metal sulfides, besides metal adsorbed to the surface of sediment particles.
Possible associations of Zn with organic matter and reactive Fe (FeHCl) and Mn (MnHCl)
contents were evaluated. Metal concentrations were determined in duplicates by ICP OES,
showing reproducibility better than 10%. Organic matter contents were estimated by the loss-
on-ignition (LOI) method (450°C, 24 h). Dry sediment density was estimated by weighing a
known volume of wet sediment after drying at 50°C for 72 h, ranging from 1.59 to 1.72 g cm-
3, which is consistent with the coarse nature of sediments from this site (generally showing

over 60% sand; Silva et al., 2003). Inventories of ZnHCl were calculated as the product
between ZnHCl concentrations (mg kg-1), sediment density (g cm-3) and sediment depth
intervals (cm). Bacterial cell number was determined by epifluorescent microscopy, using
acridine orange method and UV-radiation (Kepner and Pratt, 1994). A Mann-Whitney U
test was used to compare results from the Zn-resistant biofilm treatment with the control
data, while a Pearson correlation analysis was performed to evaluate possible statistical

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 Importance of Bacterial Biofilm for Zn Retention … 485

associations between the variables. A significance level of 0.05 was accepted for all statistical
tests.

Tubing for air

pumping
Sealing
tape

10 cm
water
column

6 cm
sediment
column

4.4 cm i.d.

Figure 2. Used experimental apparatus, as modified from Petersen et al. (1998). “i.d.”=inner diameter.
Note the presence of a tubing for air pumping and a smaller tubing communicating the “head space”
with the atmosphere.

RESULTS
The evaluated concentrations of potential geochemical carriers (LOI, FeHCl and MnHCl) of
trace metals did not present statistically significant differences between control sediments and
sediments with Zn-resistant biofilm in the different depth intervals (Figure 3a). An exception
was FeHCl in the uppermost depth interval, which was significantly lower in sediments treated
with Zn-resistant biofilm.
Concentrations and inventories of ZnHCl did not present significant differences between
control sediments and sediments with Zn-resistant biofilm at different depth intervals, with
the exception of significantly higher values observed in the uppermost layers of sediment
cores treated with Zn-resistant biofilm (62% and 56% higher in average, respectively) than in
control sediments (0-2 cm depth data; Figure 3b). Although there was a significant correlation
between ZnHCl and FeHCl for most data (r=0.61; p<0.05), data from the uppermost layers
treated with Zn-resistant biofilm did not follow this trend (Figure 4), which evidenced that
this treatment overwhelmed the general positive effect of FeHCl on ZnHCl concentration. This
explains the higher ZnHCl accumulation under conditions of lower FeHCl levels found in the
uppermost layers treated with Zn-resistant biofilm. Therefore, a change in the geochemical
controls on ZnHCl retention within sediments was evidenced, in response to Zn-resistant
biofilm effects on surface layers. On the other hand, there was no significant correlation of

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486 Wilson Machado, Elisamara Sabadini-Santos, Katia N. Suzuki et al.

ZnHCl with the very low contents of LOI (essentially below 0.8%) and MnHCl (essentially
below 9 mg kg-1) observed in the studied sediments (r=-0.19 and -0.39, respectively).

Figure 3. Depth variability of (a) LOI, FeHCl and MnHCl contents, and (b) ZnHCl contents, ZnHCl
inventories and ZnHCl/FeHCl ratios in control sediments (grey bars) and sediments with Zn-resistant
biofilm (white bars). Bars represent mean values. Error bars represent the ranges of triplicate analyses.
Depth intervals with significant differences between control sediments and sediments with Zn-resistant
biofilm are indicated by an asterisk (Mann-Whitney U test, p< 0.05).

Figure 4. Relationship between ZnHCl and FeHCl levels. The white symbol represents the average of
duplicate data from used sediments before the experiment. Black symbols represent surface layers with
Zn-resistant biofilm. Grey symbols represent all other data (irrespective of depth and treatment)
obtained at the end of the experiment, for which correlation analysis trendline and results are presented.

Considering the reactive (HCl-extractable) Fe compounds as major binding phases for

trace metals (e.g., Fe (oxy)hydroxides), Morse et al. (1993) used a ZnHCl/FeHCl ratio to
evaluate anthropogenic ZnHCl enrichments in Galveston Bay (USA) sediments. In an
analogous approach, ZnHCl/FeHCl ratios were used in the present study to evaluate the extent in
which Zn-resistant biofilm affected ZnHCl accumulation, in addition to the evaluation of
absolute ZnHCl concentration differences. Figure 3b demonstrates that ZnHCl/FeHCl ratios
presented the same trend observed for ZnHCl concentrations. There was a significant

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 Importance of Bacterial Biofilm for Zn Retention … 487

difference in the uppermost depth intervals for these ratios, corresponding to an average value
2.2-times higher in the Zn-resistant biofilm treatment, in comparison with the control
sediment data. In fact, most ZnHCl/FeHCl ratios were essentially near those found in the
sediments before starting the experiments (averaging 7.4 × 10-3), whereas ratios ranging from
17.3 to 24.0 × 10-3 were observed for the uppermost layers treated with Zn-resistant biofilm
(Figure 3b).
There was no statistically significant change (as indicated by Mann-Whitney U tests) in
the bacterial cells number in relation to control sediments at the end of the experiment (Table
1). This observation evidenced that higher Zn sorption by bacterial consortia adapted to high
Zn exposure and/or enhanced binding of Zn to EPS secreted by this adapted consortia
occurred without dependence on changes in bacterial cell number.

Table 1. Depth variability of bacterial cell number (No. cm-3)

in sediments after 4 h experiments

Control Zn-resistant biofilm addition

Depth (cm)
Mean Minimum Maximum Mean Minimum Maximum
0-2 2.86 × 108 2.77 × 108 2.96 × 108 2.97 × 108 2.74 × 108 3.31 × 108
2-4 2.14 × 108 1.67 × 108 2.41 × 108 1.85 × 10 8 1.73 × 108 2.10 × 108
4-6 2.05 × 108 1.75 × 108 2.30 × 108 2.78 × 108 1.93 × 108 3.43 × 108

DISCUSSION
Although the used extraction procedure can extract metals adsorbed to sediment particles
and bound to carbonate, hydrous aluminosilicates and amorphous and crystalline
oxyhydroxides (Huerta-Diaz and Morse, 1990), a predominance of FeHCl oxidized phases is
expected, considering that oxygenated tidal water generally covers the sediments from the
Itacuruçá creek (Lacerda et al., 1988), which are coarse-grained and periodically exposed to
air. Since Fe-reducing bacteria are ubiquitous in coastal sediments (Borch et al., 2010), a
possible explanation for the observed decrease in FeHCl contents within the uppermost depth
interval is an enhanced consumption of the Fe(III) previously accumulated, used as a terminal
electron acceptor in the bacterial respiration after Zn-resistant biofilm addition.
The observed Zn sorption trends by Saco do Engenho creek sediments are probably a net
result of cell-EPS-mineral aggregate effects (Hao et al., 2013). The results were significantly
affected by a short (4 h) time period under Zn-resistant biofilm influence, as evidenced by
evaluating ZnHCl and FeHCl concentrations and concentration ratios. This indicates that even a
single, short flooding period can be sufficient to promote such significant changes in the
reactive Zn behavior for the evaluated sandy sedimentary environment. While Machado et al.
(2008) evidenced experimentally that Zn sorption by these sediments during the tidal flooding
may be a reversible process, it was observed that the Zn-resistant biofilm influence may
contribute to limiting this reversibility. The results, therefore, supported the hypothesis that a
metal-resistant biofilm coating can often enhance the metal sorption capacity of sandy
sediments, as found for Zn. Longer periods of evaluation are necessary, however, to test the
duration of the observed effect (e.g., throughout complete tidal cycles).

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It is well known that coupled effects of living and nonliving organic matter, metal (Fe,
Mn and Al) oxides, and clay minerals can control the binding of trace metals to aquatic
sediments (e.g., Chapman et al., 1998), but the form and extent in which biofilms can affect
the involved mechanisms deserve further attention. A positive feedback between enhanced Zn
resistance and Zn sorption capacity of studied sediments is suggested by the observed results.
This may contribute to a better comprehension on the Zn cycling across sediment-water
interface in sites in which Zn resistance development occurs and sites that receive bacterial
biofilm with enhanced Zn resistance transported from adjacent areas (e.g., attached to
particulate matter released from contaminated sites).
Considering the known ubiquity of biofilms in the environment (e.g., Costerton et al.,
1995), the metal-binding properties of biofilms (e.g., Decho, 2000) and the results from the
present study, the suggested role of biofilms with enhanced Zn resistance can be an important
mechanistic aspect involved in determining Zn cycling near the sediment-water interface in
contaminated coastal zones. The relatively rapid effect observed after Zn-resistant biofilm
addition to the sediment surface, often affecting the role of FeHCl compounds as binding
phases for ZnHCl, evidences that such an effect may be significant along with typical tidal
flooding periods. This experimental data suggests that potential cumulative effects of Zn-
resistant biofilm on Zn accumulation during larger time periods deserve investigation to
evaluate to what extent the suggested feedback mechanism can affect the Zn sorption capacity
of sediments.
The implications of these processes in determining the Zn cycling across the sediment-
water interface could also be related to the protective role that the biofilms may have
towards microphytobenthos communities against metal toxicity, since EPS-imbedded mature
microbial biofilm consortia act as biogeochemical barriers (Ivorra et al., 2000). These
microphytobenthos communities may also have pivotal functions in the trace metal
biogeochemistry in coastal systems, e.g., by affecting the accumulation of these elements
within the sediments (Harbison, 1986). However, the concurrent ability of biofilms to trap
metals may be strongly dependent on the historical exposure to these elements, as exemplified
here for Zn.
Coastal microbial mats are naturally able to favor trace metal accumulation within thin
uppermost sediment layers, although a large knowledge gap exists (e.g., Glavaš et al., 2015).
This is also the case in relation to polluted systems, in which the metal toxicity to the benthic
bacterial consortia can possibly affect this role. However, there is consistent evidence in the
literature that the sensitivity of benthic bacteria derived from sites previously exposed to trace
metals is lower than that observed for bacteria derived from clean conditions, which may
result in a change in the microbial community towards a metal-resistant consortia (e.g.,
Admiraal et al., 1999). This evolution has, therefore, important implications for the metal
cycling in the colonized environments.
However, the replacement of sensitive microorganisms by resistant species may have
strong ecological implications, since the community adaptation to a pollutant can often result
in failure of the maintenance of ecological functions (van Beelen and Doelman, 1997). For
instance, a number of specific geochemical and mineralization reactions mediated by
microbial consortia are often performed by specialized bacteria that cannot be easily replaced
by other species, while bacterial toxicity can affect biomass production and taxonomic
diversity (van Beelen and Doelman, 1997). Consequently, these potential losses of ecological
roles from bacterial communities may often affect other processes involved in determining

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the sedimentary cycling of the toxic trace elements (as positive or negative feedbacks in
relation to metal accumulation by biofilms).
Biofilm formation is a useful strategy used by microorganisms to survive an exposure to
adverse conditions, such as contact with various toxic elements (Harrison et al., 2007).
Multiple impacts are frequent in the coastal zone that can receive refuses from multiple
anthropogenic sources, making difficult the prediction of metal behavior derived from
experiments carried out with a single pollutant. For instance, there is more information
regarding benthic microalgal responses to concurrent exposure to multiple contaminants,
including toxic metals (e.g., Ivorra et al., 2002). Since this multiple-contaminant exposure is a
situation likely observed in many coastal systems at a worldwide scale, evaluations on the
role of biofilm resistance in the trace metal cycling in response to multiple-contaminant
exposure are recommended to clarify potential synergisms and antagonisms for different
elements. Further research is requested for assessing the net effects of the discussed processes
for different environmental conditions and trace elements.

CONCLUSION
Experiments on the potential effects of bacterial biofilm with amplified Zn resistance on
the Zn sorption by intertidal sandy sediments from Sepetiba Bay (SE Brazil) showed that
significantly different HCl-soluble Zn concentrations (62% higher) and inventories (56%
higher) were induced after 4 hours of Zn-resistant biofilm addition. This treatment was
evidenced as a factor affecting the geochemical controls on Zn retention, since a significant
correlation between ZnHCl and FeHCl was observed for data from most sediment layers, with
the exception of data from the uppermost layers treated with Zn-resistant biofilm. A positive
feedback between enhanced Zn resistance and Zn sorption capacity was suggested, which
may contribute towards a better comprehension of the Zn cycling across sediment-water
interfaces at sites where Zn resistance development occurs and sites that receive bacterial
biofilm with enhanced Zn resistance transported from adjacent areas. These findings imply an
important role of bacterial biofilm resistance to determine biofilm ability in affecting the Zn
behavior, since biofilms may act as biogeochemical barriers at the sediment-water interface.
However, the experiments were performed under nearly-steady conditions, and the extent to
which strong natural disturbances (e.g., intense benthic fauna bioturbation, storm-induced
resuspension, etc.) and anthropogenic disturbances (e.g., dredging, trawling, etc.) of
sediments can affect this biofilm role remains unknown. Further research on these aspects
of the bacterial biofilm role in the water-sediment interface are necessary in order to
elucidade potentially different responses for different trace elements, according to variable
environmental conditions, considering longer evaluation periods (e.g., throughout tidal
cycles).

ACKNOWLEDGMENTS
The authors thank the Rio de Janeiro State Research Foundation (FAPERJ) and the
Brazilian Research Council (CNPq) for the financial support. K.N. Suzuki thanks a grant

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490 Wilson Machado, Elisamara Sabadini-Santos, Katia N. Suzuki et al.

from the Brazilian Ministry of Science (PNPD/CAPES). We thank the careful work of two
anonimous reviewers.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 31

OVERVIEW OF THE OCCURRENCE, EFFECTS AND

RISKS OF REGULATED AND EMERGING
CONTAMINANTS TO FRESHWATER ORGANISMS IN
LATIN AMERICAN WATERSHEDS

Diana Míguez1,2,*
1
Water Program, Environmental Projects, R&D&I Division,
Technological Laboratory of Uruguay (LATU), Montevideo, Uruguay
2
Latitud Research Foundation of LATU, Montevideo, Uruguay

ABSTRACT
This chapter offers an overview of the occurrence and effects of both regulated and
emerging contaminants to freshwater organisms in Latin American watersheds, the
sentinel wild fish that have been proposed, and the ecological risk assessment
methodology in use. Freshwater ecosystems in Latin America are threatened by natural
events and human activities including overpopulation, dams, deforestation,
agrochemicals use, inefficient wastewater treatments and offshoring and outsourcing of
large production companies. Many of the thousands of existing chemicals are emerging
contaminants, making it necessary to estimate the overall effects of their mixtures
through ecotoxicity testing. Moreover, under the influence of climate change, the
complexity of the analysis of effects increases. If the end-points of bioassays are
developmental, reproductive or other sub-lethal effects, special considerations must be
taken to derive dose-response relationships, as they act at very low doses and via not
completely elucidated mechanisms of action. Even though some investigations in Latin
America have described the problem, there is still scarcity in databases for the Southern
Hemisphere to substantiate risk assessments. The ecoregion approach is proposed to
assess the risk at the Latin American level. A case study is presented concerning an
integrated risk assessment of endocrine disruptors in the Uruguay River as an example of
multiple stressors released from multiple sources. Tools included in vitro screens,

*
Corresponding Author E-mail: dmiguez@latu.org.uy.

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494 Diana Míguez

biomarkers of endocrine disruption, tissue residues, biomonitoring, and bioassays with

wild fish and standard species.

Keywords: Latin America, ecotoxicity, risk assessment, endocrine disruptors, emerging

contaminants

INTRODUCTION
This chapter reviews the occurrence and effects caused by exposure of freshwater species
to contaminants and ecological risk assessment (ERA) methodologies used to evaluate the
risk of different stressors in selected watersheds in South America and the Caribbean.
Freshwater ecosystems are increasingly threatened by human activities and natural
events. Therefore, there is a need to implement measures to ensure environmental
sustainability (Iscan, 2004). The hazards to ecosystem health should be identified, and the
impact of chemicals on organisms and on the interactions of organisms with each other and
with their environment (Bilgen and Sarıkaya, 2015). Risk management measures should then
aim at decreasing the continuous inputs of contaminants to diminish the probability of
harming aquatic organisms, in order to guarantee the survival of future generations (Smital,
2008).
An ERA produces a risk characterization by combining both the exposure profiles and
the exposure-effect characterization, to identify a level at which harmful effects occur on
plants and animals. The systematic steps for performing an ERA are outlined in Figure 1.

Figure 1. Ecological Risk Assessment framework (adapted from USEPA, 2015a).

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In part due to offshoring and outsourcing of South America locations to large production
companies (Crook, 2015), the situation has changed from pristine conditions to somewhat
altered conditions. Moreover, under the influence of climate change, ecosystems are subject
to variations that add complexity to the analysis of the stressor effects (Giarratani et al.,
2013). The potentially existing chemicals in the environment add up to more than eighty
thousand man-made substances, but only about 200 priority substances have been regulated
(Dudgeon, 2014). Many of these substances are “emerging compounds”, meaning “any
synthetic or naturally occurring chemical or any microorganism that is not commonly
monitored in the environment, but has the potential to enter the environment and cause known
or suspected adverse ecological and/or human health effects” (USEPA, 2015b). Their effects
are still not completely known, making it necessary to estimate the overall effects of a
mixture of chemicals through ecotoxicity testing.

METHODS
A proposal for a systematic methodological approach in the case of regional stressors
would be to focus on the main shared river basins. However, another one could be to divide
the continent into ecoregions to consider the differences in wildlife receptors. Therefore, the
threats, hazards, and risks in the main freshwater basins, as well as some of the more
prevalent pollutants and the most used species found in the literature for exposure and effects
assessment in some ecoregions of the continent are described.

Scope

Geographical Scope
The focus of this chapter is Latin America, a highly diverse subcontinent, in terms of
landscape, culture and biological species.

Wildlife and Human Receptors

The influence of biodiversity loss, contaminated fish through bioconcentration and
biomagnification, and deteriorated water quality, eventually also affect human receptors. This
implies that some biomarkers that are common to humans and animals can be used in order to
be more conservative regarding any receptor within the environment. The ERA is turned into
an integrated risk assessment, to include both.

Hazard Identification

Anthropic Activities and Threats in Latin American Watercourses and Ecoregions

Anthropic pressures displace creatures from their natural pristine environments due to
constructions or cultures feeding an ever increasing population. Domestic, agricultural and
industrial sources produce multifactorial stressors that combine to produce a cocktail of
substances that eventually enter freshwater aquatic environments.

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496 Diana Míguez

Some of the threats are common to the different ecoregions, while others are more critical
due to the high biodiversity, or the presence of endangered species of fauna and flora. The
geographical description within the following information was extracted from the Freshwater
Ecoregions of the World online database (FEOW, 2015).

Deforestation, River Transportation, Pollution and Mining

Deforestation implies threats to biodiversity in several ecoregions such as the North
Andean Pacific Slopes – Atrato River, or the Amazonas River Basin and Rainforest - because
of logging for timber and pastures for cattle grazing. The Paraná/Paranaíba inner forests are
also experiencing similar effects.
The Uruguay River has several dams diverting the watercourses that affect the natural
habitats, and natural forests are being converted to eucalyptus plantations for pulp production,
soy crops and large scale pulp mills. The Paraná River also presents dams and dykes, while
hunting, urban expansion, and pollution represent threats to wildlife. Similar problems are
found along the delta region of the Orinoco River, because of oil extraction and overhunting.
The São Francisco River suffers the effects of urban expansion, timber extraction, and of
pollution by agrochemicals (Britto et al., 2015). The Northeastern Mata Atlântica suffers from
urban expansion, while the Paraíba do Sul from illegal timber extraction and forest
conversion into agricultural lands.
The Chaco region is altered due to cattle grazing. The Pantanal is the largest wetland in
South America, and the largest in the world not substantially modified by humans, but
pesticide runoff in the Paraguay River Basin and gold mining are threats. The temperate
grasslands of the Pampas have amphibian populations at risk from the increasing use of
agrochemicals in soy fields (Lajmanovich et al., 2010).

Megacities
According to the World Meteorological Organization (WMO, 2015), in 2010, 83% of the
population of South America resided in cities, of which 20% inhabited the largest cities, some
of them megacities of more than 10 million people (Buenos Aires, Rio de Janeiro, Sao Paulo,
and Belo Horizonte) or near that value (Bogota, Lima and Santiago). The increase of built
environments leads to the displacement of terrestrial and aquatic natural ecosystems.

Climate Change
The impact of pollution enhanced by climate change is one of the main stressors to
aquatic biota. Changes in fish susceptibility to abrupt changes in temperature have been
linked to the presence of pollutants that affect their immunity and decrease their ability to
overcome adverse environmental conditions, exacerbating fish mortality events. Climate
change combined with pollution can also increase the threats to freshwater ecosystem goods
and services (Munang et al., 2010).

Transgenic Crops and Pesticide Use

The world´s population in 2015 estimated by the United Nations was 7,349.472
inhabitants. It is projected to increase by more than one billion people within the next 15
years, reaching 8.5 billion in 2030, and further to 9.7 billion in 2050 and 11.2 billion by 2100
(United Nations, 2015). This implies that more resources will be needed to feed the

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population, more land used for crops and more water abstracted for drinking water production
and other uses. Land use has drifted from native woods to cereals and in Argentina, Brazil,
Bolivia, Paraguay, and Uruguay to genetically modified soybean cultivations able to
withstand the herbicide glyphosate, other herbicides, co-adjuvant surfactants, and
insecticides. Several investigations show that glyphosate cause toxicity in frogs (Lajmanovich
et al., 2010), in fish (Rocha et al., 2015) and in periphyton communities (Vera et al., 2010).

Eutrophication, Cyanobacterial Blooms and Floating Plants

Excess phosphates and nitrates produce eutrophication, leading to cyanobacteria
overgrowth under certain conditions of light and temperature. Microcystis aeruginosa
produces microcystin-LR and about 80 other toxins. The increase in frequency of algal
blooming events is due to agricultural, municipal and industrial inputs of nutrients, and
nitrogen fixation from the air. Losses in ecosystem services (such as tourism) and in water
quality can threaten human health via intake of contaminated drinking water, fish or
mollusks. Macrophytes can also be a problem, as is the case of Maracaibo Lake that suffers
from overgrowth of Lemna obscura that blocks light passage and kills other plants and algae
(González, 2004).

Urban and Industrial Discharges and Challenges to Conventional Wastewater

Treatment Processes
The presence of increasing amounts of conventional and emergent contaminants in
effluents requires enhanced treatments. Some cities do not have secondary or tertiary
treatment to treat the organic matter, nutrients and recalcitrant compounds that human
activities produce.

RESULTS
Exposure Assessment

Regulated Contaminants
In Latin America, most countries routinely perform surveillance monitoring to control the
compliance to environmental regulations, with different established limits among each nation.

i. Heavy metals
Fernández Severini et al. (2011) found lead (Pb) in dissolved and particulate phases
and in zooplankton from the Bahía Blanca estuary, Argentina, in samples collected
between March and December 2005. They linked the origin to industrial and sewage
discharges. Mean concentrations of dissolved Pb were 2.15 ± 0.46 μg/L and of
particulate Pb 13.52 ± 3.07 μg/g dry weight (dw). In copepods the concentration was
13.38 ± 4.41 μg/g dw, while in mysids it was 9.81 ± 1.89. In the Negro River,
Uruguay, the reported median concentrations of Pb in sediments were 7.7 + 3.8 μg/g
at Baygorria site, and less than the quantitation limit (<5 μg/g) at Bonete site
(Míguez et al., 2012).

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ii. Pesticides and herbicides

Endosulfan, a highly toxic persistent organochlorine pesticide, was shown to
bioaccumulate in fish and sediments (Boleda et al., 2011; Míguez, 2013).
Campanha et al. (2015) investigated pyrethroid residues and toxicological effects in
Jenynsia multidentata, correlating integrated biomarkers with total pesticide levels.
At higher temperatures the survival rate of fish larvae of Odontesthes bonariensis,
“pejerrey”, exposed to cypermethrin decreased (Carriquiriborde et al., 2009). Its
metabolites were identified in the bile of the before-mentioned fish (Carriquiriborde
et al., 2012). Hunt et al. (2016) determined 17 insecticides in sediments from 53
streams in soy production regions (Argentina in 2011-2014, Paraguay and Brazil in
2013) during peak application periods, finding toxicity towards Hyalella azteca.
Glyphosate, one of the most used herbicides, causes skeletal malformations in
zebrafish and affects human placental cells, even at concentrations below regulatory
levels (Mesnage et al., 2015).
iii. Persistent organic pollutants (POPs)
POPs can exert toxic effects on human and environmental health because of their
potential to bioaccumulate and biomagnify along the food chain. There are regulated
compounds, such as dioxins, furans, PCBs, and PAHs, and unregulated chemicals,
which lay within the category described in the next subchapter. For example, studies
carried out in several Chilean lakes, revealed that sediment PCB concentrations
ranged from 1.2 ± 1 to 64 ± 30 ng/g dw (Pozo et al., 2007).

Emerging Contaminants
Not many references on the occurrence of emergent contaminants in South America exist
given that the analytical techniques required to determining their concentrations are complex
and still too expensive for some countries.

i. Endocrine disruptors
These chemicals do not share a similar molecular structure, but act on the
homeostasis and endocrine systems, causing reproductive, immunological, or
developmental effects (Diamanti-Kandarakis et al., 2009). Studies carried out at a
location on the Uruguay River, subject to multiple stressors from agricultural,
industrial (pulp mill), and domestic municipal discharges, showed the presence of
several of the prioritized chemicals with potential or demonstrated endocrine
disruptive action (Míguez, 2013). Later on, their occurrence was also detected in the
Santa Lucía Basin, as pharmaceuticals such as ibuprofen resisted depuration within a
wastewater treatment plant. Natural estrogens and the synthetic estrogen
ethynilestradiol used in contraceptive pills were also present, as well as nonylphenol
(NP) and its ethoxylates; NP was found at the final discharge site, which justifies the
application of a tertiary treatment (Astigarraga, 2015). In another project carried out
from September 2012 to September 2014 in the Uruguay River and Plate River,
microcystin-LR and other non-regulated toxins were found in these watercourses
(Kruk et al., 2015).
Given that the sources of water abstraction and of contamination inputs can influence
drinking water quality, it is important to determine the existing compounds in these
matrixes. Surface drinking water supplies receive large amounts of raw sewage

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inputs, introducing emerging contaminants that could reach consumers. In Campinas,

Brazil, endocrine disruptors were found in tap water samples, namely bisphenol A
quantified at a concentration of 0.16 ± 0.03 µg/L (Sodré et al., 2010).
ii. Stimulants and drugs of abuse
Stimulants and drugs of abuse have also been recently detected in environmental
samples. Sodré et al. (2010) found caffeine at a concentration of 0.22 ± 0.06 µg/L in
drinking water supplied to Campinas, Brazil. In South American countries and in
Panamá, Rosa et al. (2011) reviewed the concentrations of drugs of abuse in drinking
water at levels below 1 ng/L. The most prevalent compounds, cocaine and its main
metabolite, were quantified at 0.6 and 4.5 ng/L, respectively; caffeine and nicotine
were found at frequencies of 81 and 88% and at concentrations of 38 and 40 ng/L,
respectively.
iii. Pharmaceuticals and personal care products
Pharmaceuticals and personal care products (PPCPs) are yet another category of
chemicals of the emerging contaminants group. Pharmaceuticals, hormones, and
triclosan were analyzed in surface water in São Paulo State quantifying caffeine,
paracetamol, and atenolol at 12.96x104, 3.42 x 104 and 0.82x104 ng/L, respectively,
while hormones estrone and 17-β-estradiol were detected at levels up to 14.8 ng/L
(Campanha et al., 2015). Some of the most commonly prescribed pharmaceuticals
(atenolol, carbamazepine and diclofenac, among others) were detected in the Suquía
River basin (Córdoba, Argentina), and found to bioaccumulate in Gambusia affinis, a
widely distributed fish species inhabiting the river basin (Valdés et al., 2014).
iv. Emerging hydrophobic organic pollutants
Among this class, brominated flame retardants (BFRs) such as polybrominated
diphenyl ethers (PBDEs) bioaccumulate and biomagnify. The European Union has
banned many of them but due to their persistence they can still pose a risk to human
and environmental receptors (European Food Safety Agency, 2015). BFRs were in
higher concentrations than UV filters (UV-F) in Chilean and Colombian sediments:
BFRs at up to 2.43 and 143 ng/g dw of PBDEs in Chile and Colombia, respectively,
and UV-F at up to 2.96 and 54.4 ng/g dw in Chile and Colombia, respectively (Baron
et al., 2015).

Effects Assessment

Even when the international framework for risk assessments is broadly applicable to
Latin America, it needs further refinement in terms of extrapolation from the Northern
Hemisphere to local conditions and native species (Carriquiriborde et al., 2014), and from
single species and single compound toxicity tests to multiple species and mixtures of
contaminants.

Acute Toxicity
Ecotoxicological information considered for regulatory measures is in general obtained
via acute toxicity testing. As an example of investigations that used this end-point, the
crustacean Daphnia similis experienced high toxicity after exposure to the wastewater

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treatment plant of a hair care products company that produces shampoos, conditioners,
moisturizers and hair vitamins in Brazil (Dias de Melo et al., 2013).

Sub-Lethal Effects
Giusto et al. (2012) exposed Hyalella curvispina amphipods in chronic ten-day bioassays,
finding a decrease in survival and growth at 11.25 mg Cd/L in liquid phase and at 5.6 mg
Cd/Kg dw in solid phase of samples of water and sediments taken from the Pampasic region
of Argentina. Also in Argentina, Carriquiriborde et al. (2009) found effects on growth after
exposing specimens of the fish species Odontesthes bonariensis in the laboratory to pyretroid
pesticides. In Uruguay, in the Negro River the highest sub-lethal toxicity experienced by H.
curvispina was found with samples from the Baygorria site (83%) by comparison to Bonete
site (94%), showing a possible link to the increased concentration of lead in the clay fraction
(Míguez et al., 2012). In Brazil, avoidance, a behavioural sub-lethal effect, was observed in
fish exposed to a commercial formulation containing 300 g/L of pyrimethanil collected from
outdoor mesocosm systems (Araújo et al., 2014).

Chronic, Carcinogenetic, Developmental, Immunotoxic, Neurotoxic and

Reproductive Effects
Other potential effects of metals relate to cancer, developmental and reproduction effects.
Genotoxicity was observed in model organisms exposed to environmentally relevant
concentrations of cadmium (Pereira et al., 2016). Neurotoxicity of organophosphate
pesticides such as azinphos-methyl has been studied in the native snail Chilina gibbosa,
deeming it applicable as sentinel species for Argentina and Chile (Bianco et al., 2014). Castro
et al. (2015) found that azinphos-methyl affected cell viability and phagocytic activity in
hemocytes of this freshwater snail.

40
Percentage of affected embryos

35
30 Terata
Lethality
25 Lethality + terata
20
15
10
5
00
6.25
1 12.5
2 25
3 50
4 100
5 6

Concentration (%)

Figure 2. Non-monotonic kinetics of larval spine malformations of Pimephales promelas exposed to

sediment elutriates. The photo on the right illustrates a typical example of spinal-cord malformation in
an exposed embryo; arrow indicates the vertebral malformation observed. Sediments were dredged
from a site close to industrial effluents into the lower Uruguay River, Uruguay; n=8 (Míguez et al.,
2011; Míguez, 2013).

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Aquatic frogs can also be subject to reproductive effects. The genus Telmatobius (T.
ceiorum, T. laticeps and T. pisanoi) has historically been present in streams of Tucumán
Province, Argentina, but T. pisanoi populations have declined since 1968 from 1-36
specimens, to 0-3 in 2005 (Barrionuevo and Ponssa, 2008).
Non-monotonic responses are paradoxical kinetic behaviors, meaning that the highest
effects are seen at the lowest tested concentrations. In laboratory embryo development tests
with the reference fish species Pimephales promelas exposed to elutriates of sediments
dredged near the outfall of a pulp mill, the kinetics followed non-monotonic behavior
regarding bone alterations (Míguez et al., 2011). Lethality in samples from the upstream
Nuevo Berlín site was 21.5% while for those from the Fray Bentos site it was 7.5%. While
the upstream site showed no developmental anomalies in exposed embryos, the sample
elutriate from Fray Bentos lead to 3.3% spinal-cord malformations (Figure 2).

Endocrine Disruption
Endocrine disruption was observed in carp fish exposed to sediments of industrial areas
of the Uruguay River (Rivas-Rivera et al., 2014). Endocrine disruptive effects were also
observed using P. promelas fish and Astyanax fasciatus as a sentinel wild fish from a large
river (Míguez, 2013). This characid fish, widely distributed from Central America to the Río
de la Plata Basin of western Uruguay, has been used before to evaluate hepatic porphyrins
(Carrasco-Letelier et al., 2006). Other fish species successfully tested as sentinel are
Cichlasoma dimerus (Teleostei, Perciformes), “chanchita”, a South American fish of streams
of the Paraná River basin, the Paraguay River drainage in Brazil, Bolivia and Paraguay, and
the Paraná River drainage of Argentina (FishBase, 2015), Odontesthes bonarienses (Table 1),
and Cnesterodon decemmaculatus. The responses of the latter were compared to those of P.
promelas in effluents and reference toxicants, being in agreement for effluents and potassium
dichromate, but not for sodium dodecyl sulfate (Saona et al., 2015).

Table 1. Effects experienced by native model fish species in Latin America

Chemical(s) and/or sample

Species Effects Reference
origin
Environmental pollutants, Astyanax fasciatus Endocrine disruption Míguez (2013)
Lower Uruguay River Basin
Furnas Reservoir, Grande A. fasciatus Endocrine disruption Prado et al. (2011,
River and Paraguay-Paraná 2014)
basin
Endosulfan Cichlasoma Gonadal steroidogenesis disruption Piazza et al., 2015
dimerus
Endosulfan C. dimerus Effects in GnRH cells Da Cuña
et al., 2013
17β-Estradiol (E2) and C. dimerus Decrease in sperm motility, Meijide et al.,
synthetic 17α-ethynilestradiol fertilization, and embryo and larval 2016
(EE2) survival
17β-estradiol C. dimerus Vitellogenin in male fish and other Moncaut, 2003
endocrine disruptive effects
Octylphenol C. dimerus Non monotonic kinetics Genovese
et al., 2014
Estradiol and ethynilestradiol Odontesthes Estrogenicity and low sperm quality, Gárriz et al., 2015
bonariensis fertilization and embryo-larval
survival
GnRH: Gonadotropin-releasing hormone.

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Frameworks for Exposure and Effects Assessment

i. Caged fish in the river and laboratory exposure assays

In Chile, Chiang et al. (2015) exposed male and female juvenile rainbow trout to
pulp mill effluents in laboratory experiments, both showing significantly higher
levels of plasma vitellogenin (Vtg) than the control. The concentrations of Vtg
were comparatively higher in fish exposed to Eucalyptus globulus than to Pinus
radiata-based effluent. Moreover, male fish showed intersex characteristics in
all exposure assays. Furthermore, similar estrogenic responses were observed in
caged fish in the river.
ii. Tiered approach for exposure and effects assessment
An integrated risk assessment of endocrine disruptors in the lower Uruguay River
was carried out by Míguez (2013). The experimental design of the exposure and
effects assessment relied on a tiered approach that combined both in vitro and in vivo
tests, chemical analyses, molecular biomarkers and lab exposure of standard species
and field studies with a native fish (Figure 3).

Figure 3. Tiered methodology for exposure assessment of endocrine disruptors in the Uruguay River
(Míguez, 2013). Adsorbable organic halogens (AOX), extractable organic halogens (EOX),
nonylphenol (NP), nonylphenol ethoxylates (NPEO), total organic carbon (TOC), biochemical oxygen
demand (BOD), total nitrogen (TN), total phosphorus (TP), temperature (T), estrogen receptor (ER),
androgen receptor (AR).

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 Overview of the Occurrence, Effects and Risks of Regulated and Emerging … 503

The first tier assessed the river health status by measuring general water quality
parameters, while effects were assessed by developing acute toxicity tests with crustaceans,
fish, amphipods and bioluminescent bacteria. The second tier analyzed AOX and phenols in
river water and effluent and EOX in sediments, while effects were evaluated via chronic
toxicity tests for reproduction with the cladocer crustacean Ceriodaphnia dubia. In the third
tier, the specificity increased to measure the occurrence of endocrine disruptors and to
determine endocrine disruption effects. Firstly, luciferase tests ER-CALUX® and AR-
CALUX® were applied to screen the presence of ER- and AR-binding compounds in the
watershed, which resulted positive. In vivo analyses were then performed in the laboratory to
assess the effects in fish gonads at molecular, tissue and functional levels: P. promelas were
exposed to both pulp mill effluent and stream water subject to municipal wastewater
discharges during 21 days each. A set of biomarkers of estrogenicity by PCR, vitellogenin
(Vtg) by ELISA, histological analysis of the gonads and fecundity (egg counts) were then
measured. Moreover, field biomonitoring with more than 1000 specimens of the native fish A.
fasciatus was carried out at sites located upstream and downstream from the pulp mill and
city discharges. The occurrence of prioritized chemicals was determined in the water
catchment to then determine their bioaccumulation in fish, crustaceans and other organisms,
including microorganisms, plants and additional invertebrates. Complex mixtures related to
anthropic activities were found in sediments, water and biota. Among the pesticides,
endosulfan was also found in fish, and the herbicide glyphosate in sediments.

Risk Estimation and Risk Characterization

From a holistic stand point, the harm to wildlife and human beings living in the
watershed should be evaluated in all dimensions and compartments, with a basin approach,
taking into account the water cycle and food web interactions. In a study performed by
Míguez (2013), a decrease in fecundity was observed after exposure of P. promelas to an
industrial effluent, but no intersex appeared in this case nor in assays with samples of a
stream receiving municipal discharges. The estrogenicity of the latter was demonstrated by
the results of estradiol, zona radiata biomarker, estrogen receptor 1 and IGF-I expressions in
the exposed fish in the laboratory, along with the positive results of the luciferase receptor-
binding screen ERCALUX®, and the occurrence of estrogens and nonylphenol. On the other
hand, anti-estrogenicity or androgenicity was suggested to be caused by the industrial
effluent. Biomonitoring of wild A. fasciatus revealed changes in gonad size at sites near the
outlet from a pulp mill, and differences in condition factor in areas influenced by municipal
wastewater. The risks of endocrine disruption to humans via fish and water ingestion were
characterized as low, and from low to moderate to freshwater biota. However, hotspots of
higher risk were identified for risk management.

CONCLUSION
There are no complete databases that are ecologically relevant to Southern Hemisphere
ecosystems for the development of risk assessments that support informed risk management

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decisions. Unlike the Northern Hemisphere, there are currently no protocols describing which
species to use to assess the risks of contamination of Southern Hemisphere water bodies, in
part due to the scarcity of data reporting the responses of native species. The experiments
described in this chapter concerning wild fish can be used to derive internationally
standardized guidelines through comparison of the responses among standard model fish and
native fish, to build databases and derive sound risk assessments.
Threats to freshwater ecosystems do exist nowadays in Latin America and may act
additively, synergistically or antagonistically. Natural conditions such as climate change
and extreme events (drought or flooding) are not the only causative agents as human
interventions, overpopulation and overconsumption of natural resources (e.g., for crop
production or production of luxury items) are some of the most influential factors that have
altered many pristine areas around the world. At the macro scale, the ecoregion approach
could be applied to the study of Latin America ecosystems, within the framework of
integrated water resources management (IWRM), coupled with a study of drivers, pressures
and ecosystem services that consider every water use. On the other hand, at a detailed scale,
ecologically relevant bioindicators could be chosen by joint research teams to enlarge
databases, for which increased international collaboration is needed. Further research must be
done to determine the effects of not only regulated pollutants but also of emerging
contaminants, including the most recent target agents such as nanoparticles, microplastics and
endocrine disruptors.

ACKNOWLEDGMENTS
The Uruguay River research was performed as part of the requirements to fulfil the PhD
degree granted to the author by Cranfield University, School of Applied Sciences, Cranfield
Water Science Institute, Cranfield, UK. The experimental part was carried out mostly at
laboratories of both the Technological Laboratory of Uruguay, Montevideo, Uruguay, at
Unidad Fray Bentos, and at Cranfield University.

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Last updated 30th September 2015. Available: http:// www.epa.gov/ risk/ conducting-
ecological-risk-assessment. Accessed: 31/ 1/2016.
U.S. Environmental Protection Agency, (2015b). TSCA Chemical Substance Inventory.
Office of Chemical Safety and Pollution Prevention. Basic Information. Available:
http://www.epa.gov/oppt/existingchemicals/pubs/tscainventory/basic.html. Accessed:
19/08/2015.
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Economy and Social Affairs. Population Division. Available: http:// esa.un.org/unpd/
wpp/Download/Standard/Population/. Accessed: 3/1/2016.
Valdés, M. E., Amé, M. V., Bistoni, M. A. and Wunderlin, D. A. (2014). Occurrence and
bioaccumulation of pharmaceuticals in a fish species inhabiting the Suquía River basin
(Córdoba, Argentina). Sci. Total Environ., 472, 389-396.
Vera, M. S., Lagomarsino, L., Sylvester, M., Pérez, G. L., Rodríguez, P., Mugni, H., Sinistro,
R., Ferraro, M., Bonetto, C., Zagarese, H. and Pizarro, H. (2010). New evidences of
Roundup® (glyphosate formulation) impact on the periphyton community and the water
quality of freshwater ecosystems. Ecotoxicology, 19(4), 710-721.
WMO - World Meteorological Organizaton, (2015). Bulletin: The impact of climate change:
migration and cities in South America. Available: https://www.wmo.int/bulletin/es/
content/impact-climate-change-migration-and-cities-south-america. Accessed: 1/1/2016.

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 32

NANOMATERIALS AS CECS:
FROM ENVIRONMENTAL INTERACTION
TO REGULATORY EFFORTS FOR A SAFE
USE IN LATIN AMERICA

Pamela Zuniga Fallas1,2,*, Jaime F. Quesada-Kimzey2,3

and Ricardo A. Morales Vargas1,4
1
Health Technologies School, Faculty of Medicine,
Universidad de Costa Rica, Montes de Oca, Costa Rica
2
Fundación Technoinnovation, Curridabat, Costa Rica
3
Chemistry School, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica
4
Regulatory Unit for Environmental Health Services, Directorate for Human
Environment Protection, Ministry of Health, San José, Costa Rica

ABSTRACT
The term “contaminant of emerging concern” (CEC) is applied to non-regulated
anthropogenic material recently found in the environment that may represent
environmental or public health risks. In the present chapter, general aspects regarding
nanomaterials (NMs) are addressed, from their role as CECs to the potential deleterious
effects on organisms and the environment. Five NMs are considered as examples: silver
nanoparticles representing metallic NMs, ceria and titanium dioxide representing metallic
oxides, and carbon nanotubes and fullerenes representing carbon NMs. An overview of
the processes determining the lifetime of nanoparticles in the environment is given. A
perception survey was conducted amongst Costa Rican high-ranking authorities from the
environmental and health sectors, and researchers from public universities as
representatives of the academic sector. The results show that even though awareness
regarding the relevance of CECs exists, the participants of the survey perceive an
important lack of regulatory tools and of effective governmental actions. An overview of
the current situation of regulatory efforts and accomplishments regarding NMs in

* Corresponding Author E-mail: pzuniga.nanotec@gmail.com, pzuniga@technoinnovation.org.

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Argentina, Brazil, Costa Rica and Mexico is given, showing advances mostly in Mexico
and Brazil, where alliances with European counterparts seems to have helped.

Keywords: CEC, ENM, nanomaterial, regulation, Latin America

INTRODUCTION
The term CECs was a much-needed precision of the still commonly used terms
“emerging contaminants” and “emerging pollutants.” CECs can be applied to non-regulated
anthropogenic material recently found in the environment that may represent environmental
or public health risks. This term comprises a wide variety of substances and materials; some
are intentionally produced, like pesticides, antibiotics, drugs, plasticizers, some nanomaterials
(NMs), and some are unintentionally produced (PCDF/Ds, PAHs, some aerosols, some NMs).
Their importance is increasing in the scientific community, because of their bioaccumulation,
biomagnification, widespread exposure, potential persistence, and environmental and health
implications. Some CECs are suspected or well known carcinogens or endocrine disrupters,
and there are others for which effects are still to be discovered.
CECs can be classified based on how long they have been known as contaminants (Sauvé
and Desrosiers, 2014). Some are in fact new, emerging substances or materials, which are
recognised as contaminants. Other CECs such as arsenic, although considered contaminants
for centuries, are subject of renewed interest. Engineered nanomaterials (ENMs) have made
some significant contributions to CECs, mostly as new materials with previously unsuspected
interactions with the environment and organisms. These interactions arise mostly from a
combination of size and chemical composition at the surface. This chapter deals with NMs,
namely ENMs, in view of their potential as CECs. Our approach aimed to help safeguard the
exploitation of this technology by promoting the questioning and environmental health
assessment methodologies. The safe use of NMs is the prevailing objective while mindfulof
preventing governmental overregulation or negative public perception.

Nanomaterials General Aspects

NMs are the materials occurring naturally in the size range of 1 to 100 nm (frequently
called nanoscale) while ENM are intentionally manufactured materials, containing highly
uniform structures with at least one critical dimension in the nanoscale. According to the US
National Nanotechnology Initiative (NNI), NT is allowing the development of modern
advances in different fields of knowledge including chemistry, physics, biology, medicine,
and materials engineering (NNI, 2000). NM represent a bridge between atomic or molecular
structures and bulk materials, due to the large surface area per unit of volume, and the
quantum effects that appear at the nanometer scale, which consequently lead to a significant
improvement of particular intrinsic properties of the substance. The potential market value for
NT-related products between 2011 and 2015 was estimated to be up to US$1 trillion per
annum (Navarro et al., 2008).
Variations in NM specific surface area can render these materials much more reactive,
and thus more toxic than their larger homologues (Ozin and Arsenault, 2005; WHO, 2011).

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Even substances known to be chemically passive can become reactive at the nanoscale (e.g.,
gold, silica), posing unsuspected risks if assessed traditionally (WHO, 2011). This can be
enhanced by the potential of ENM particles to translocate body barriers, penetrate cell
membranes, accumulate in mitochondria, and deposit in specific organs.
Numerous reports have suggested that due to their smaller size, ENM can easily bypass
water treatment plants, air pollution control equipment, or be released during handling of
waste (European Commission, 2011; OECD, 2016); nonetheless, current research has shown
considerable variability on ENM removal efficiency (Andersen et al., 2014). It has thus
become necessary to develop new assessment methods for treating these materials (see
Ozin and Arsenault, 2005; Ponce, 2010; OECD, 2012; Savolainen, 2013), and assessing the
risk of NMs in penetrating various animal tissue membranes (Savolainen, 2013) and
bioaccumulating or causing damage to tissues or DNA. Individual experts have called for
such regulation of risk assessment procedures for NMs (El Universal, 2011), while at the
same time suggesting that a balance must be struck between technology developments and
protecting the public (Foladori and Záyago, 2011).

Nanotechnology and the Environment

There are various ways in which NT can improve environmental technology and provide
benefits to the environment. For example, by contributing with products that enhance
energetic efficiency, improve energy storage, provide potable water or reduce waste.
In the case of NT, the environment will be increasingly exposed to affectations by
pollution from manufacture, use, and disposal of ENM. For instance, a three-fold increase has
been identified for Europe since 2006, especially after considering the number of products
containing nanoparticulate materials, including consumer products (Wijnhoven, 2011).
Currently, there are a widespread variety of regulations in place in manufacturing
countries, especially in the European Union, including those regarding consumer and
environmental protection. NT has also been spurred in Latin American countries such as
Brazil, Argentina, Chile, Mexico, Cuba, and Costa Rica. These advances are considered
promising from a quality of life standpoint. Despite this, very few Latin American countries
have regulatory frameworks that specifically address the right-to-know, as a mechanism for
assuring consumer knowledge of ENM content and the consequent risks in products
containing them (Foladori, 2006). Moreover, nano-content is not necessarily disclosed
appropriately to manufacturers and consumers. It is even less reflected in waste handling
regulations regarding content of discarded products or in specific regulatory and collection
schemes for this special waste (OECD, 2016).
Differences in toxicity between bulk materials and particles having at least one dimension
in the nanoscale have shed some light on modes of ecotoxic action of NMs. The factors that
can influence the ecotoxicity of NMs can be divided into two types: those concerning the NM
itself, and those concerning the medium and the associated ecological receptors.
The size of a NM (regularly the only parameter considered), the surface properties of the
particles, and the physicochemical environmental conditions, such as pH, temperature, redox
conditions, ionic strength, and natural organic matter, affect its mode of interaction with
the environment. Knowledge regarding physicochemical interactions of NMs with the

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environment has been increasing; meanwhile their potential impact may be affecting
biogeochemical cycles due to considerable mobility and toxicity (OECD, 2016).
The level at which most of the current research on ENM has been performed ranges from
effects on unicellular organisms - assessing microbial toxicity in order to extrapolate the
observed effects of chemicals to organisms of higher levels of organization – (Mortimer,
2011; Elsaesser and Vyvyan, 2012), to that of whole multicellular organisms, in order to
compare the susceptibility of organisms at various trophic levels.
Research has shown that ENMs may lead to unforeseen health risks due to their ability to
bypass protective biological mechanisms, including the blood brain barrier, or negatively
affect the environment due to their antibacterial properties (OECD, 2016). Owing to the risks
and uncertainties, a precautionary approach must be followed and countries should embark on
a “mission” to experiment with novel schemes of extended producer responsibility and right-
to-know activities, hopefully reflected in regulatory instruments.

BIOAVAILABILITY AND CELLULAR UPTAKE OF ENM

The bioavailability of an ENM affects dose and exposure duration and is generally
determined by physicochemical factors of the material, amount and stability of the substance,
and receptor sensitivity. Most inorganic ENMs such as TiO2, Ag, CeO2, ZnO, and quantum
dots are likely to be found in the environment due to their increasing production and
application in consumer products. According to Gottschalk et al. (2013), in studies referring
to the environmental concentration of these ENM, the most studied material was nano-TiO2,
followed by nano-Ag fullerenes and CeO2. Furthermore, nano-Ag and TiO2 lead the list of the
most ingested NMs (Woodrow Wilson International Center, 2006; Illuminato et al., 2007).
It should be considered that physicochemical properties may affect the potential toxicity of
NMs. For this there are several extensive lists of properties that are applicable to NMs, such
as the list proposed by the Organisation for Economic Co-operation and Development
(OECD, 2010).
Herein, environmental, health-relevant results for five ENMs are presented. Considering
that particle composition is probably the element that plays a primary role in the cytotoxic
effects of different nanoparticles (NP), the main difference among the five selected ENMs
(CeO2, fullerenes, Ag, TiO2, and CNTs) is the composition. These ENMs will be named
criteria NMs, as they represent substances of generalized use of a certain category, sharing
characteristics that influence their intrinsic hazard. First, inorganic ENMs (CeO2, Ag and
TiO2) will be addressed, followed by the carbon ENM products (fullerenes and carbon
nanotubes).

Inorganic Criteria ENMs

Metallic ENMs
Nano-sized silver is the most used and representative ENM for this category. Different
forms, including NPs, silver wires and oxides, are currently being widely used for their
antimicrobial activity in fabrics and personal care and medical products.

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Human oral and pulmonary administration of AgNPs has shown silver bioavailability,
being spleen, liver, and kidney the main target organs for deposition after systemic
availability. Moreover, exposure to ionic silver and AgNPs has been shown to interfere with
gene expression (SCENIHR, 2014).
For the aquatic ecosystem, tests with zebrafish embryos have reported decreased hatching
rates, weak heart beats, edema and abnormal notochords after exposure to 10-20 nm Ag-NPs
for 48 h in concentrations of 10-20 μg/L (Yeo and Pak, 2008).
Regarding prokaryotic cells, it has been found that Ag-NPs affect bacterioplankton
metabolism, production, community composition and the emergence of rare bacteria
phylotypes (Das et al., 2012).

Metal Oxides
Due to its diverse range of applications, CeO2 is a widely used material, representative of
metal oxides, similarly to TiO2. Current applications of nano-CeO2 range from catalyst to
insulator on silicon substrates and as electrolyte material of solid oxide fuel cells. Ceria NPs
have attracted attention for the study of their ecological and health effects. In vitro tests have
suggested that nano-CeO2 could be a potential exogenous source of reactive oxygen species
(ROS) (Park et al., 2008). Cell death and tissular damage of lung epithelial cells when
exposed to different sizes of nano-CeO2 (from 15 to 45 nm) have been reported (Park et al.,
2008).
Oxidative stress damage and ROS accumulation was observed in Caenorhabditis elegans
in vivo testsat different concentrations (1-100 nM), causing significant decrease of nematode
mean lifespan upon exposure with low and environmentally relevant concentrations such as 1
nM. This decrease was presumably due to oxidative damage induced by ROS accumulation
(Zhang et al., 2011).
Fine and nanosized TiO2 powders have been extensively manufactured worldwide. Their
application ranges from pigments and food additives to their use as photocatalysts. The latter
has allowed the use of TiO2 in the oxidation of organic compounds, especially in water
decontamination and the production of H2 as a fuel using solar energy. As a result of the wide
range of applications, there are also several exposure pathways, making dermal penetration,
ingestion and intravenous injection possible. However, toxicity studies have primarily
focused on dermal penetration and inhalation (Zuniga and Quesada, 2015).
TiO2 particles larger than NPs are generally considered to be biologically inert for living
beings. However, cytotoxicity effects for TiO2 have been found, although very low as
compared with other materials (ZrO2, Al2O3, Si3N4) (Yamamoto et al., 2003).
Although size alone is not an effective predictor of cytotoxicity, it is the only parameter
considered in NM cytotoxicity studies. However, this simple general aspect is lacking in
product labels regarding the specification of the properties of commercial TiO2 powders
(Zuniga and Quesada, 2015). This increases the risk for workers and consumers upon
exposure, and precludes the establishment of appropriate security measures.
Another especially important parameter for cytoxicity studies of this material is the
crystalline structure. For photocatalytic applications, the anatase structure is more effective
than the rutile (although some applications may require mixtures and proper toxicity
analysis). An aspect of significant importance is that paints are the main end use market of
TiO2,(ICIS, 2010), conferring risks mainly due to inhalatory and dermal exposure. The
average retention time of TiO2NP in rat lungs was of 541 days, 4.62 times longer than the

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average time for 250 nm-sized NPs. Studies in rats exposed to TiO2 rutile structure fine
powders, at a dose of 12.5 mg/kg, showed systemic translocation. Higher accumulation was
found for 80 nm-sized NPs, which remained mainly in the liver; the 25 nm NPs and fine
particles were not retained in the liver but found to be accumulated in lung tissue, kidney and
spleen (Wang et al., 2007).

Carbon ENM Products

This category basically includes allotropes of carbon such as fullerenes, carbon nanotubes
(CNTs), and some others with a specific targeted market such as carbon black, hydrochar and
graphene wires. A special feature that must be considered is the wide range of materials of
this group, an aspect that hinders establishing comparisons and the development of
appropriate regulations in order to mitigate potential risks. Manufacture of these materials is
also occurring in increasing amounts for different applications, ranging from cosmetics to
drug delivery, as is the case of the C60 fullerene.

Fullerenes
This material (also called buckyballs, short for buckminsterfullerene) presents a
molecular structure with sixty carbon atoms. Some of the market available variations of this
ENM are fullerols and carboxyfullerenes, more soluble derivatized molecules that imply an
increasing potential of interaction with organisms.
Water-soluble fullerene (nC60) had shown to induce lipid peroxidation (LPO) in brain
of juvenile largemouth bass (Oberdörster, 2004). Daphnia magna exposure for 21 days to
2.5 and 5 ppm of nC60 caused a delay in molting and a decrease in offspring production.
Furthermore, tetrahydrofuran-solubilized-fullerenes effects on two fish species were
compared to water-stirred fullerenes. A 100% fish mortality was found between 6 and 18 h
exposure in tetrahydrofuran-solubilized-fullerenes, while in water-stirred fullerenes no
physical effects after 48 h were detected (Zhu et al., 2006).
Tests on mice have shown suppression of P450-cytochrome levels (Ueng, 1997) and
blood-brain-barrier penetration (Yamago et al., 1995).
Bactericidal activity was observed in prokaryotic cells exposed to aqueous-C60
suspensions, metallofullerenes and carboxyfullerenes (Kamat, 2000; Tsao et al., 2002; Lyon
et al., 2005), while oxidative eukaryotic cell damage was caused by hydroxylated fullerenes
(Tsao et al., 2002).

Carbon Nanotubes
CNTs are arrangements of carbon atoms in a crystalline tubular graphene form. The main
categorization considers the number of layers or tubes within a tube, distinguishing single and
multi-walled carbon nanotubes.
It is questioned if there are analogous mechanisms to other fibrous particles, such as
asbestos, which could enable CNT lung tissue penetration. Features such as biopersistence,
ratio between width and height, and the fibrogenic character of CNTs are important
characteristics that may influence the occurrence of adverse health effects. Some studies have

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confirmed CNT asbestos-like or even more harmful effects, e.g., greater fibrotic potency of
single-walled CNT (SWCNT) on mice 1 year after aspiration (Shvedova et al., 2013).
The in vitro and in vivo exposure to CNTs revealed the potential of this ENM to cause
DNA strand breakages, oxidative damage, mitotic spindle formation and mutations (Darne, et
al., 2014). Another study found SWCNT to be the ENM to induce most breakage in DNA
strands in fibroblasts, compared to carbon black, silicon dioxide (SiO2), and zinc oxide (ZnO)
NPs (Yang et al., 2009).
It has been found that multi-walled CNT (MWCNT) was internalized in vacuoles of
human epidermal cells. Furthermore, down-regulation of membrane scaffold protein and cell
cycle inhibition has also been observed (Montero-Riviere et al., 2005).
Differences in bioactivity between SWCNT and MWCNT are apparent, as well as other
differences regarding relevant physicochemical properties that may affect their potential
toxicity (Oberdörster et al., 2015). For CNTs, the most relevant properties are: the synthesis
method, surface properties, impurities, density, and shape. In terms of their toxicological
effects, characteristics such as shape, length, width, morphology and dispersion are
considered determinant, as has been observed in asbestos and glass fiber toxicological
analysis (Broaddus et al., 2011).
Metallic NP such as Fe, Co, Ni, and Mo are commonly used as catalysts for CNT
growing in a synthesis process (Hoyos-Palacio et al., 2014). Morphological effects, efficiency
of the synthesis process and toxicity are aspects that can be influenced by the selected
catalyst. Iron, one of the most common catalyst-substrates for CNT production (Dundar and
Karatepe, 2011), has been found to interfere with bacterial and fungal cell reproductive
processes (Chaves et al., 2013). The latter was seemingly due to nanotube endocytosis
mediated by siderophores, which are iron-sequestering molecules produced by the
microorganisms. Fungal and microbial cell saturation with siderophore containing nanotubes
were observed after 30 min, leading to cell death because of interference with electron
transport (Chaves et al., 2013).

Hydrochar
Hydrothermal carbonization (HTC) is an emerging technology that promises to be widely
implemented in coming years for the treatment of wastewaters highly loaded with suspended
and dissolved organic matter (Quesada et al., 2016; Zuniga and Quesada, 2016), as well as of
very moist biomassic waste materials (Libra et al., 2011; Sevilla et al., 2011; Xiao et al.,
2012). Hydrochar, the solid product of this process, is comparable to charcoal in some
macroscopic aspects. The wastewater from the HTC process contains some soluble organic
compounds and all the soluble mineral components of the original waste material (Libra et al.,
2011); uses as a foliar fertilizer or directly on the soil have been mentioned, but release
into water bodies may become frequent. In the wastewater from the HTC process,
nanoparticulatehydrochar can remain suspended and therefore may be also released (Quesada
et al., 2016).

SINK PROCESSES AND PERSISTENCE MECHANISMS

ENMs can be released via several pathways to the environment: transformation of
the ENM into another material by dissolution or by chemical reactions; or permanent

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immobilization by being irreversibly aggregated or deposited and/or incorporated into

sediment and thus being transformed into another material. The combination of both
processes can also occur, when aggregated NPs, usually in sediments, are subjected to
changing physicochemical conditions leading to dissolution or reaction.
Experimentation with pure ENMs and environmentally unrealistic concentrations or
settings have been criticized (Lowry, 2008; Olson and Gurian, 2012; Gottschalk et al., 2013;
Judy and Bertsch, 2014; Mitrano et al., 2015). Risk-assessment (Wiesner et al., 2009) and
life-cycle approaches (Mitrano et al., 2015) have been proposed along with some
prioritization mechanisms (Wagner et al., 2014).
Generally, the environmental impact of an ENM is considered to be determined by the
material’s persistence. Thus our major concern here is focused on ENMs that may lack
dramatic acute effects, but do show some degree of persistence.

Processes Affecting Released ENMs

ENMs can be expected to form part of a product that is distributed, used, and eventually
disposed of in waste streams. A pure ENM will reach a waste stream as a result of an
accidental or exceptional event. However, it will usually be subject to processes occurring in
the waste stream, after which it will undergo further processes at the final receptor site. Even
though experimentation with pure ENMs has been questioned as to its relevancy regarding
real scenarios, it has led to knowledge concerning the primary processes that NP can undergo
in different media.

Processesin the Atmosphere

The fate of nanoparticulate materials emitted to the atmosphere is predictable from the
aerosol science standpoint, with major concerns regarding exposure near the source, for
example of workers, and regarding interactions of the particles with the environment after
deposition (Klaine et al., 2008; Lin et al., 2010). Particles in the atmosphere, whether solid or
liquid, have been subject of very relevant studies for decades. NPs in the atmosphere undergo
a series of processes, ending up deposited in water bodies or on solid surfaces, with variable
degree of transformation.
Atmospheric deposition of particles can be dry or wet; particles and aggregates may
collide with surfaces and be trapped, or be carried in a depositing water droplet to a surface.
Particles collide with each other to form aggregates. Gravitational deposition or sedimentation
becomes important for diameters above 10 µm. Atmospheric particles and aggregates can be
trapped by collision with water droplets, but they can also nucleate atmospheric water
droplets and undergo cloud processing. Water droplets may evaporate leaving behind
modified particles, usually conglomerates, or they may be removed by rainout. Atmospheric
solid particles in the accumulation mode, with diameters from about 50 nm to about 2 µm,
which may be single or agglomerates, have longer atmospheric lifetimes, in the order of
weeks, as opposed to other size ranges which last minutes or days at most (Finlayson-Pitts
and Pitts, 2000; Seinfeld and Pandis, 2006).

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Processes in the Condensed Phases

ENMs released to the environment will eventually reach a water body or the vadose zone
(Berkowitz et al., 2008; Wagner et al., 2014), and will thus be exposed to processes taking
place in water. Longer life times can lead to their presence in underground water.
Composition and arrangement at the surface of the NPs determine their behaviour in aqueous
media, where the most relevant processes they may undergo are:

 Dissolution and reaction

Dissolution and reaction of ENM in aqueous media lead to their transformation into other
materials, occasionally new NMs. Dissolution of a material is less likely near saturation, a
condition to be rarely expected in ecosystems. When dissolved species are formed, either due
to dissolution or to reaction, as in the cases of some AgNP oxidizing to Ag+ or ZnO
dissolving in water, these species will have their own interactions with the surroundings; for
example, Ag+ may react with Cl- to form AgCl or may undergo uptake into living tissue,
among other possibilities. Transformation by dissolution can also lead to formation of other
crystalline phases with the same composition, occasionally with similar sizes, or to the
formation of an outer layer with a different composition (Judy and Bertsch, 2014).
Reactivity and solubility of the base materials, combined with composition of the media,
are determinant factors. The kinetics are determined by exposed surface area, presence of
species that can adsorb onto the particles or else stabilize the reaction products (chiefly
dissolved organic matter (DOM)), ionic strength, pH and concentrations of other reactants
(Aiken et al., 2011; Wagner et al., 2014). Coatings on NPs determine their solubility and
reactivity, especially if chemically bonded to the surface, often rendering them inert and thus
extending their lifetime (Jarvie and King, 2010).

 Aggregation and deposition

NPs suspended in aqueous media form colloids and may stay in a colloidal state for
considerable time spans, even in soil-pore water (Whitley et al., 2013). Aggregation among
suspended NPs is driven by Brownian motion, differential settling and fluid motion (Wagner
et al., 2014). The primary tool to understand and predict aggregation behavior of colloids
is the DLVO theory (named after those that progressively developed it: Boris Derjaguin,
Lev Landau, Evert Verwey and Theodor Overbeek). The DLVO theory is a quantitative
description for stable aqueous dispersions, which uses van der Waals (vdW) and Coulombic
forces to predict the aggregation or else repulsion of suspended particles.
It applies well to most cases of colloids, including bare ENMs (Hotze et al., 2010;
Hartmann et al., 2014; Wagner et al., 2014) though not if the particles are coated with layers
that stabilize them via steric and electrosteric interactions. Additionally, when such outer
layers are chemically bonded, persistence increases.
The DLVO theory predicts highest aggregation rates when the charge on colloidal
particles due to the electrical double layer (EDL) approaches zero. Charge is determined by
the interactions at the particles’ surface and can be varied by changing pH, ionic strength, or
by ions with high charge density. The destabilization of colloids produced by ions with higher
charge density can be predicted as well. As known by experience (consider river deltas), high

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salinity and especially higher valence cations (consider water treatment plants) lead to colloid
destabilization. Aggregation of particles of the same material (homoaggregation) is the
simplest case and the most often considered in published studies. In real settings,
heteroaggregation (different materials) is several orders of magnitude more likely than
homoaggregation or deposition to surrounding surfaces. Particles of different materials show
different charges in the same conditions, so opposite electrostatic charges may occur leading
to destabilization and aggregation (Hartmann et al., 2014; Wagner et al., 2014).
Hydrophobic interactions, steric repulsion, polymer bridging and magnetic and hydration
effects can also influence aggregation/deposition, and are termed non-DLVO processes.
Coatings on NPs determine their colloidal behavior as well as solubility and reactivity,
especially if chemically bound to the surface, often enhancing their mobility far beyond that
of bare NPs, due to non-DLVO processes. The behavior of NPs below 30 nm has been shown
to diverge from DLVO predictions as well, showing longer lifetimes (Wagner et al., 2014;
Hartmann et al., 2014).
Aggregates in a colloid tend to grow and eventually depose, and NPs become thus
immobilized. Forces between aggregated particles are often strong enough to avoid that the
aggregate redisperses (peptization). Judy et al. (2012) hypothesized that the formation of
aggregates should reduce the bioavailability of the ENMs, for which confirmatory evidence
was produced. Notwithstanding, aggregates may in specific circumstances undergo
peptization, whereby previously immobilized ENPs can again become available.

 Adsorption

Additional to the EDL, which is adsorbed on the surface of a particle, nonionic solutes
may be adsorbed onto a suspended particle’s surface (physisorption) and driven mostly by
vdW forces. Generally, longer molecules with more interacting sites can adsorb more
effectively than shorter ones. Molecules adsorbed onto NPs change their interaction with the
surroundings, often increasing their colloidal stability by solvation and by conferring them a
negative charge, or by electrosteric stabilization (Hartmann et al., 2014). Adsorbing
molecules may be long enough to produce bridging among particles, leading to aggregation,
as flocculants do (Hartmann et al., 2014; Wagner et al., 2014).

 Interactions with dissolved organic matter (DOM) and other dissolved species

DOM comprises a variety of organic compounds including small to macro-molecules,

among them polymeric substances, proteins, humic and fulvic acids, carboxy-compounds,
amines, and thiols, all with a great variety of heteroatoms lending them excellent water
solubility and, at the same time, excellent possibilities to establish electrostatic as well as
vdW interactions (Aiken et al., 2011; Lowry et al., 2012). NPs with non-polar surfaces as
CNTs, as well as those with polar interactions, are commonly stabilized by DOM in the
colloidal state. DOM can form a coating, stabilizing them as colloids; these coated particles
may not behave according to the DLVO theory. For example, coagulation induced by Ca2+
will be inhibited in the presence of DOM, which is not predicted by DLVO theory (Hartmann
et al., 2014; Wagner et al., 2014). With high concentrations of cations, the DOM can be
desorbed from the surface, if not chemically bonded to the surface of the NPs, facilitating
destabilization and eventually aggregation of the NPs.

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 Interaction with the soil and other porous media

In the case of the soil media, interaction with sediments and their constituents, especially
those with high specific surface areas such as clays and organic matter, may bind NM,
changing their bioavailability as compared to that of their suspensions in pure water (NPCA,
2008; Rana and Kalaichelvan, 2013). NPs in porous media such as soils may persist as
colloids for considerable timespans, but can also depose onto the surfaces. As surface area
increases, the likelihood of deposition to the surface also increases and that of aggregation
decreases (Wagner et al., 2014). Deposition is driven by the same interactions as aggregation,
but physical entrainment due to pore size may also occur, as well as size exclusion from
pores, in spite of colloidal stability.

 Uptake by organisms

While ENM are known to undergo uptake by organisms such as plants and worms
(Nyberg et al., 2008; Tourinho et al., 2012; Hartmann et al., 2014), where biotransformation
may take place, trophic transfer, bioaccumulation and biomagnification have also been shown
to occur. Uptake by plant roots has been proven to depend on factors that are extrinsic (pH,
ionic strength, temperature, surface of the NP, irrigation, and so on) as well as intrinsic (cell
wall pore size, root exudates, hydraulic conductivity) to the plants (Judy and Bertsch, 2014;
Sharma et al., 2014). Although uptake of ENMs into organisms could be assumed to be
at the end of their lifetime in the environment, trophic transfer, bioaccumulation and
biomagnification may actually prolong it (Judy and Bertsch, 2014). As the research hereby
implied can undoubtedly take significant efforts for each ENM studied, it is well worth
considering an approach that includes an initial screening step for persistence regarding those
materials more likely to reach the environment, before assessing uptake and subsequent
processes in organisms, as proposed by Olson and Gurian (2012). Subsequently, attention
should be given first to ENMs with persistent coatings and those lacking a natural equivalent
(Wagner et al., 2014).

The End of the Lifetime of a Released ENM

ENMs released to the environment should reach an end to their lifetimes as soon as
possible, causing minimum impact. In general, when not by dissolution/reaction, ENMs will
be led to the end of their lifetimes by aggregation, deposition, sedimentation and subsequent
processes, which may involve dissolution/reaction or simply permanent immobilization.

PERCEPTION SURVEY TO COSTA RICAN AUTHORITIES

As a mean to preliminarily identify the needs, weaknesses and positioning of the local
authorities regarding ENM and their potential as CECs, some results of a survey undertaken
in Costa Rica are herein presented. The study was performed in order to pursue the safe use of
this promising technology, while protecting public health. The survey aimed at contrasting the

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perception of national authorities from the health, environment, and academic sector on four
key issues that are described below.
High-ranking authorities were selected as respondents representing three different sectors
(environmental, health and academic sectors) and no volunteers were considered. In all cases,
the survey was developed indicating the specific interest in CECs but not specifically in NMs,
to avoid bias. Although nine categories of pollutants were included in the survey form as
answer options of potential CECs, only the section on NMs was considered here.

Knowledge About the Term CECs

Most of the surveyed authorities (93.3%) ensured to know the concept, and when asked
to give a definition for the term. The major similarities in the concepts were regarding their
novelty (35.7%), implications of toxicity or harm for living beings (28.5%), and the absence
of regulation or standards (21.4%).

Extent of Agreement Regarding Current Local Affectation of the

Environment and Public Health by CECs

Respondents were asked to indicate their perception of the current local affectation by
CECs to the environment and to the public health. First, the surveyed authorities were invited
to express their extent of agreement with the phrase: Currently in Costa Rica, CECs affect
local environmental conditions. The total of the surveyed high-ranking authorities (n=15)
chose to express their extent of agreement by selecting the option“totally agree” in a closed-
ended question.
The perception on the affectation of public health due to CECs was evaluated with the
phrase: Currently in Costa Rica, CECs affect the health of the country’s inhabitants. The
results are illustrated in Figure 1.
A significant variation of opinion was found within and between sectors with respect to
public health affectation. None of the surveyed authorities selected the options “Totally
disagree” and “Moderately disagree,” although both of these were available options along
with the other three reflected in figure 1. The academic sector was the only group where most
respondents totally agreed with the statement. However, it was also in this group where more
than two options were selected as an answer. Generally, the health sector tended to show
more moderate positions than those found in the other two groups.
Surveyed authorities were asked to express their opinion on the importance that should be
given to CECs considering the contrasts that were exposed above. The latter was performed
by giving a qualification of 1 to 10 in which 10 was the most important. Mean values for the
given qualifications are illustrated in Figure 2.
Differences between perceptions of the same and different public sectors related to the
influence of CECs on current local affectations are a key element for environmental health
policy development.

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Figure 1. Extent of agreement of each of the three surveyed groups with current local affectation of the
population health by CECs; n=15.

Figure 2. Agreement of each of the three surveyed groups on the extent towhich the CECs deserve
attention in contrast to other affectations; n=15.

Perception of ENM as CECs

The results regarding the choice of substances, from the given pollutants list, that
respondents considered ENM to be CECs or not, are shown in Figure 3. Previous to this
question, an official definition of CECs was read to all respondents, regardless of the answer
given to having knowledge or not of what is a CEC. Furthermore, the authorities were
informed of the existence of a one-page glossary available for their consultation from that
moment, in which the definition of ENM was included.

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Figure 3. Selection of ENM as CECs from the given pollutants list by the three groups of respondents;
n=15.

The perceptions were different between the three sectors. Most of the respondents from
the environmental and academic sector do consider ENM as a CEC, contrary to what the
health authorities did. However, the coefficient of variation of the answers of the health sector
(%CV=34) was the highest in comparison to that of the other two sectors (%CV=47) which
had the same deviation.
Furthermore, respondents were asked to express their opinion on the importance that
should be given to the contaminants included in the same pollutant list provided, by giving a
qualification from 1 (the most important) to 10 (the less important). The values resulting for
ENM on the list of each authority are exposed in Table 1.

Table 1. Qualification of importance given to ENM by respondents

among a pollutant given list; n=15

Surveyed sector Minimum Maximum Mean Coefficient of variation (%)

Environmental 1 8 5.0 55
Academic 3 7 5.2 30
Health 4 9 7.2 34

ENM were given a higher mean qualification, and thus importance, by respondents of the
health sector followed by the academic sector. The environmental sector presented the
smallest mean value, minimum qualification, and also a higher coefficient of variation
(%CV=55), than in the other two groups and than in the entirety of the results (%CV=40).
This shows that although the authorities seem to be the ones who consider it slightly less
important to give attention to ENM, this opinion is variable within the authorities of this
sector compared to their counterparts in the health sector as well as the surveyed researchers.

Difficulties and Weaknesses in Addressing CECs

Authorities were asked to indicate if they considered that Costa Rica is having difficulties
for an effective response and action in situations that may be associated with the CECs. With

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the exception of the health sector, the different authorities perceived unfavourable conditions.
Respondents who considered that such a situation is occurring, were asked to detail which are
the weaknesses. The answers were mainly linked to lack of information about specific risks
and difficulties for proper generation and systematization. Lack of political interest and of
knowledge in the population was also mentioned.
An important aspect is that this answer was given considering CECs under general terms
which could represent a scenario even less favorable to ENM.
This preliminary survey suggests that a thorough review of current regulatory schemes
for registration/import of products including ENM into and within Latin America is
necessary, including defining the main product types and risk assessment methods.

STATE OF LEGISLATION AND REGULATORY SCHEMES IN

ARGENTINA, BRAZIL, MEXICO AND COSTA RICA- MINIMIZING THE
CONSEQUENCES OF UNFORESEEN HEALTH RISKS
Regulation of nanotechnologies in Latin America has lagged behind its technological
development. While consensus on defining nanotechnology and nanomaterials (NMs) has
been convergent (OJEU, 2011), there is still no general recognition or consensus of the issues
to be regulated world-wide. It is evident that health issues seem more urgent than
environmental concerns. However, the risk of liberating NMs to the environment eventually
becomes a health issue, as they can be reincorporated into the food chain (WHO, 2011).
Regional initiatives prompted by WHO (2011) concern for health and food safety and the
current growth and use of nanotechnologies, purport a wide array of areas that need to be
regulated: development of nanostructures to enhance or modify food textures, solubilization
or encapsulation of nutrients, nanosensors to detect food degradation, improved food
containers, use of nanofiltration to remove unwanted ingredients, use of nanochemicals to
improve production or application of human and veterinary drugs, pesticides and other
agrochemicals. A very complete assessment of regulatory needs has been undertaken by the
European Commission (2011), covering existing environmental regulation and anticipating
future trends and existing gaps. The impact of these products on the post-market phase must
also be addressed, since they currently end up in the general waste stream. It is important to
analyze the impact at recycling facilities, incinerators, landfills, wastewater treatment plants
and other waste handling operations, in view of Best Available Technologies (BATs), which
have shown limited success as effective barriers for their liberation to the environment. NMs
can end up in waste streams (ordinary solid and hazardous waste, wastewater, gas emissions)
as well as drinking water, due to their ubiquitous presence in various consumer products,
ranging from cosmetics to automobiles (European Commission, 2011):

“This understanding drew on possible environmental exposure pathways for specific

nanomaterials and for nanomaterials in general and on data on the possible hazards associated
with specific nanomaterials that were identified in the literature. Although a wide range of
possible exposure pathways were identified, concrete evidence of releases was only found to
support some of these pathways, notably releases of treated wastewaters into surface waters
and into soil through sewage sludge and treated effluent from sewage plants. For other

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pathways, either the very limited number of studies or the complete lack of studies made the
identification of possible exposure more speculative (Conclusions, p.203).”

However, the only solid piece of legislation to disclose ENMs content in consumer
products is linked to the European Union's initiative to inform the public of these substances
in cosmetics (Savolainen, 2013), through the Cosmetics Legislation (Regulation No.
1223/2009), which for products manufactured after July 2013 requires labelling the presence
of ENM. According to Savolainen (2013), no specific mention of ENMs is made in the
Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) legislation
(OJEU, 2008), the broad definition of “substance” therein contained can be interpreted to
include ENMs. As a result, the European Chemicals Agency (ECHA) has updated its
guidance on safety data sheets in order to include information requirements and safety
assessment of NMs. The EU has updated its biocidal products regulation (EU 528/2012) so
that approval of an active substance will not include the NM form, unless NM have been
specifically assessed for their risk to human and environmental health. Other regulatory
initiatives include plastic materials (EU 10/2011) and other articles that may come in contact
with food. In addition, a number of concrete decisions on ‘ecolabelling’ contain specific
considerations regarding NMs (2011/383/EU, 2011/381/EU). France has initiated a registry of
NMs, and other countries in Europe may follow suit (OECD, 2014).
OECD's work in this area is performed through a Working Party on Manufactured
Nanomaterials, periodic meetings with country advances and round-tables, and its testing
program. This program includes: a) NMs information/identification, b) physical-chemical
properties, c) environmental fate, d) environmental toxicology, e) mammalian toxicology and
f) material safety. The Recommendation of the Council on the Safety Testing and Assessment
of Manufactured Nanomaterials (OECD, 2013), provides a strong incentive for OECD
members to adhere to the organization's tools and guidelines for testing of ENMs, which
include recommendations for sample preparation and dosimetry, exposure measurement and
risk assessment. Other topics researched by OECD include exposure assessment and
mitigation, workplace exposure assessment, guidelines for personal protective equipment in
the workplace, specific guidelines for laboratory exposure assessment and control, lifecycle
assessment of NMs, genotoxicity of NMs, methods and models for exposure assessment, and
development of interspecies variability factors for human health risk evaluation. OECD is a
Europe-based economic organization, which also includes countries such as the USA,
Mexico, Chile, Australia, South Korea and Japan, and which for the topic of NMs partners
with the Inter-Organisation Programme for the Sound Management of Chemicals (IOMC),
FAO, United Nations Institute for Training and Research (UNITAR) and WHO. It is to be
seen whether other regions of the world, such as Latin American countries, some of which are
discussed below, are able to take advantage of these developments.

Regional Initiatives in the Americas

Initiatives in the Americas include the Strategic Approach to International Chemicals

Management, henceforth the Strategic Approach to International Chemicals Management
(SAICM) (Foladori et al., 2013), UNITAR (2011) and work pursued by the Inter-American
Development Bank (IADB, 2015), all of which are fostering partnerships between experts and

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creation of professional and government networks, which point towards regulatory programs
in the mid and long term.
The SAICM work is conducted through a series of technical workshops geared to foster
knowledge on the “state-of-the-art” in nanosciences and the risks posed by substances or
waste containing NMs, and has partnered in joint activities with UNITAR and OECD.
SAICM aims to work with the Latin American and Caribbean countries and has proposed a
life-cycle approach (cradle-to-grave) to chemicals’ management, thus encompassing health
and environmental issues related to nano-containing consumer products. Proceedings from a
2011 SAICM Workshop in Panama, acknowledge that the use of NMs has increased in the
last decades, while there is a lack of evidence on risks posed by these materials, to which
must be added the lack of national and international regulatory instruments of such
substances. SAICM also recognizes that these substances may harbor important risks to
vulnerable groups, such as children (including embryos) and the elderly, and thus calls for the
formation of expert groups and capacity building to analyze such risks while appealing to
manufacturers to disclose the nano-content of their consumer products through appropriate
registration and labeling schemes (Foladori et al., 2013). SAICM also calls for integration of
this precautionary principle into public policies related to chemicals’ management and
scientific/technical information exchange, including consultation to workers’ associations,
and limiting of waste transfers to countries that lack the capacity to manage them adequately
(Foladori et al., 2013).
UNITAR has specifically addressed capacity building more directly, and has developed
an 85-page manual with the Swiss Government (UNITAR, 2011), alerting on public health,
environment and worker´s health, as areas to be covered when developing national programs
on NT. The manual further goes into governance based on current national legislation and
international agreements, priority setting, and keeping these priorities at a manageable level
through adequate coordinating mechanisms and capacity building. UNITAR has partnered
with the Latin American Network on Nanomaterials and Society (RELANS), which has
achieved a considerable number of joint publications with other international and national
networks (Invernizzi, 2015).
The IADB cites the following countries as having important activities in nanoscience:
Brazil, Mexico, Argentina, Colombia, Chile, Venezuela, Peru, Uruguay, Dominican Republic,
Costa Rica, Cuba, Guatemala, El Salvador, Ecuador and Panama (Foladori and Invernizzi,
2012), while only Brazil is currently part of NanoReg, an international endeavor, sponsored
by a group of European countries and which also includes Australia, Canada, the Republic of
Korea, the USA and Japan.
Prominent environmental and awareness-raising organizations have also voiced their
concerns at a regional level, promoting workshops and meetings aimed at analyzing the risks
of technology deployment, promoting consumer right-to-know schemes and the application of
the precautionary principle when regulating NMs (ETC Group, 2008, 2010). Although
advances in the regulatory field have been limited, countries such as Argentina, Brazil,
Mexico and Costa Rica have prompted efforts in this area.

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Argentina

Current efforts in Argentina have been made, but there is to date no specific regulation of
NT that includes risk assessment to health and the environment. Work has been done by
ANMAT (National Authority for Medicine, Food and Medical Technology), through an
“Observatory,” with the aim of promoting information exchange and networking, and in the
longer term to have an adequate diagnostic of scientific and regulatory issues (ANMAT,
2015; Cid Pharma Packaging, 2012). The Ministry of Science, Technology and Productive
Innovation, through Law 26.338 of 2007 (a modification of the Law of Ministries) is
responsible for the promotion of NT. This has been implemented through the Fundación de
Nanotecnología created through Decree Nº 380/05 (Infoleg, 2015) under the supervision of
the Ministry of Economy and Production.
Although criticized by the Comité Nacional de Ética en la Ciencia y la Tecnología for
allowing loopholes in the handling of national budgets (CECTE, 2005), the Decree
establishes clear responsibilities and functions for this Foundation, which is focused only on
NT promotion, capacity building and market creation, with no mention of risk assessment or
regulation, nor research in these key areas. Other efforts have been made to establish a
General Framework Law for Development of Micro and Nanotechnologies (Argentinian
Senate and Congress, n.d) proposed to Congress, based on Law 25.467, General Framework
Law for Science, Technology and Productive Innovation, in an effort to repeal Decree Nº
380/05. Nonetheless, this proposal offered no guidance on risk management and regulation of
nanotechnologies in the health and environmental areas, and was never enacted (Foladori,
2006).

Brazil

Similarly to Argentina, Brazil has had important developments in national networks

since the year 2000 and experts have joined international and regional networks such as
SAICM or RELANS (Foladori, 2006; Foladori et al., 2013; Rocha, 2014). Brazil, however,
has granted greater attention to toxicology research and workers’ health (Arcury, 2010).
Through legislation enacted by the Ministry of Science, Technology and Innovation (Law
510 of 2012), Brazil created an Inter-Ministerial Committee on Nanotechnology (CIN),
encompassing 10 Ministries, in charge of the Brazilian Nanotechnology Program (PBN)
and a Network of Strategic (government) and Associate (university) Laboratories for
Nanotechnology (SisNano). Aims of the CIN include management of the Nanotechnology
Program, integrating financial resources, promoting networks, international cooperation and
program evaluation. Toxicology research has been fostered, and there are networks in: aquatic
toxicology of NPs, cytotoxicity and genotoxicity of nanostructured composites, toxicology of
NPs in the oil and gas industry, occupational and environmental nanotoxicology (regulation
and risk evaluation), toxicity of NPs in biological systems, and toxicity of materials applied to
medicine and agriculture using in vivo and in vitro methods (Rocha, 2014). Although health
and environmental areas are considered in terms of toxicology research, and a firm
governmental basis exists for possible regulation of technologies, there is no formal
regulatory mechanism for NM.

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Brazil partners with NanoReg, 14 European countries with the common goal of
establishing a firm basis for regulatory testing of NMs. This, as well as an official decision to
join regulatory efforts in Europe (Nanosafety Cluster), and aligning its environmental health
and safety programs in NT with those of Europe and the USA, will undoubtedly promote
North-South exchange and will allow harmonization of standards and mutual data acceptance
(Rocha, 2014). Brazil has recognized international efforts for health and safety regulation,
and its highly developed scientific networks will benefit from insertion into global initiatives
such as the Nanosafety Cluster and NanoReg.
Brazilian NT efforts are “ahead of the game” in terms of anticipating ill-effects of
technology deployment. Nonetheless, a project for legislation in 2005 calling for policies and
a fund for work in nano-safety (including establishing a multisector National Technical
Commission), as well as a project voted in 2013, pushing for disclosure of NM content in
food, cosmetics and pharmaceuticals, were never enacted (Portal Brasil, 2013). The Inter-
Ministerial Committee on Nanotechnology, based on a single piece of legislation that created
it, is steering Brazil´s work on NT.

Mexico

Since the year 2000, Mexico´s Special Program on Science and Technology, as part of
the National Development Plan 2001-2006, gave a strategic positioning to NT development.
A specific program for NT was drafted, a steering committee was instituted, and a National
Network for Nanoscience and Nanotechnology (NNNN) was created in 2009 (Foladori et al.,
2013). Funding, however, has not been strongly supported by the government and most
researchers and enterprises have been financed by partnering with larger firms abroad
(Foladori, 2006). This, however, has not hampered participation of scientists in the network,
which by 2010 totalled 500 researchers and 60 universities (Foladori and Zayago, 2011).
Although solid regulations have not been in place in Mexico, commercial exchange with
the US, under NAFTA, has facilitated adherence of Mexican policy makers to US guidelines
for NT and NM regulation.
Recently, and based on an OECD survey comparing NT policies in 24 countries, the
Mexican case was studied (Foladori et al., 2015). This survey concludes that Mexico does not
have a national strategy for NT development; even though it considered its importance since
2001. Additionally, Mexico has not promoted public participation in NT; yet it does have
institutionalized mechanisms to encourage participation on other topics in the scientific
agenda. Scientific policy does not include exposure and risk of manufactured NPs, even
though Mexico has access to OECD Decisions and Recommendations, which must be
reflected in policy and regulation. Mexico participates in SAICM but has scarcely translated
its recommendations or decisions into legislation. Not until 2013 was a Mexican Technical
Committee on NMs constituted, based on the Federal Law on Metrology and Standardization.
The Committee established its rules of operation, and is empowered to create standards for
NT. It actively participates in setting international standards for NT through the International
Organization for Standardization (ISO) within the Technical Committee ISO/TC 229
Nanotechnologies (Foladori et al., 2015).

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Costa Rica

Costa Rica has envisioned NT as a long-term strategy and has considered it, along with
biotechnology, a cornerstone for development towards 2050 (Estrategia Siglo XXI, 2006;
MICITT, 2015), including it in its National Plan for Science, Technology and Innovation
2015-2021. In 2011, the official newspaper La Gaceta published Decree 36567, by which
research in NT and its applications, as performed in institutions of the Science and
Technology Sector and public universities, is declared of public interest; research is
considered an instrument of economic and social development (La Gaceta, 2011).
Additionally, the decree calls all public and private institutions to contribute, according to
their means and regulatory frameworks, economic, logistic and technical resources, to
improve scientific research in NT and its applications. The preamble to the decree recognizes
work already initiated by the Ministry of Science and Technology (MICyT), the Rectory
Council for Public Universities (CONARE), the National Center for High-Tech (CENAT)
and the National Laboratory for Nanotechnology (LANOTEC), and places NT as an
emerging and convergent technology that can foster scientific development plans considered
within the National Development Plan 2011-2014 (La Gaceta, 2011). This, however, is the
only regulatory instrument which, although visionary, does not provide sound funding for
research activities within a coordinated national plan for nanotechnology, as other countries
in the Americas have managed to propose. In spite of these constraints, the National Plan for
Science, Technology and Innovation 2015-2021 considers that Costa Rica should promote
work in nanomedicine, nanopharmaceuticals, nano-biotechnology, nano-microelectronics,
environmental applications of NT, development of nano and microsensors, nanocatalysts;
without disregarding intellectual property, ethical, and social aspects of NT. The
aforementioned comments in the National Plan, proposed by the LANOTEC director, end
with a recommendation to draft, alongside this National Plan, a technical report that charters
the route for development and sustainability of NT in Costa Rica.

CONCLUSION
While the concern of this chapter is the potential of the ENM as contaminants,
there are several benefits (environmental technology, remediation, medical and commercial
applications) that humanity can obtain from NT products, if developed in a sustainable way.
As the fields of applications of ENM and products containing them grow, it is pivotal to
give more attention to the field of nanotoxicology from an ecosystemic point of view.
Research on the presence of CECs, their risk and their quantification in the environment,
should be of special interest for Latin America, which possesses the richest diversity of
species and ecoregions in the world and holds one third of the world’s renewable water
resources and close to 30% of the world’s total runoff (ECLAC, 2002).
Latin America has lagged in providing populations with proper regulation for NMs. In
these fields many NGOs have raised concerns and called for risk assessment and control.
Though inter-governmental initiatives such as SAICM and OECD are providing some
capacity building efforts, most countries have started this technological journey by providing
merely a technology-development and promotion infrastructure, with varying degrees of
success.

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Common Regulatory Approaches in Leading Countries

The countries portrayed in this brief overview of regulatory capacities show varying
strategies in response to their specific political and scientific context. A common response has
been the promotion of policies for NT development, network creation, alliances and public
participation at the national and international levels (spurred by regional and international
organizations). This response, however, has not been enough to promote strong NT regulation
and protect consumers. In most countries one concludes that awareness of the risks is not
strong enough to overcome industries’ fear of regulation by the legislative/executive branches
of the government.

Closing the Gap

Latin American countries aim to partner with international agencies or intergovernmental

initiatives, share knowledge and build capacities for both technology development and risk
assessment, with a sound foundation on toxicology, perhaps the “new nano-toxicology.” This
knowledge will create risk awareness and set the basis for its reflection in regulatory
instruments, avoiding “re-inventing the wheel” and optimizing resources worldwide. These
regulatory means will be focused on the worker and the population’s right-to-know,
particularly with regard to food products, cosmetics, pharmaceuticals, chemicals and
agrochemicals, coupled with the precautionary principle. All must be based on a sound risk
assessment throughout the life-cycle, focused on size-dependent properties, and the
recognition that assessment of the micro and larger homologues bears little, if any,
relationship to the nano-scale version.
Some topics that the countries in Latin America should consider in their legislation
include governance and technology innovation, regulatory authorities´ responsibilities,
definitions of NMs, material testing (physical-chemical, ecotoxicology and human health
testing -acute and/or chronic when appropriate-, metrology), manufacturing reports by
industry, disclosure of NM content in products of environmental or health concern, workers’
protection in laboratories, scale-up plants and industry, life-cycle risk assessment, and
mechanisms for information exchange between experts and authorities.
Even though this work has been limited thus far, development of knowledge and
regulations in the Americas seems promising, and the growing networks of experts will
certainly play an important role in bridging this gap.

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Complimentary Contributor Copy
In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 33

PHARMACEUTICAL AND PERSONAL CARE

PRODUCTS (PPCPS) IN THE ENVIRONMENT:
LATIN AMERICAN OCCURRENCES, ADVERSE
EFFECTS AND PERSPECTIVES

Luciane Alves Maranho1,*

and Camilo Dias Seabra Pereira1,2
1
Departamento de Ciências do Mar,
Universidade Federal de São Paulo, Santos, São Paulo, Brazil
2
Departamento de Ecotoxicologia,
Universidade Santa Cecília, Santos, São Paulo, Brazil

ABSTRACT
This chapter presents a review on pharmaceuticals and personal care products
(PPCPs) occurrence and adverse effects performed by researchers in Latin America.
Recent data confirm that PPCPs occur widely in freshwater, soils and marine and coastal
environments in Brazil, Argentina, Colombia, Chile and Mexico. Unfortunately, Latin
America has a common and very serious environmental health problem: the failure or
absence of wastewater treatment plants. In contrast, the Latin American pharmaceutical
market represents approximately 25% of global pharmaceutical sales. Therefore, studies
regarding occurrence and assessment of potential risk to biota need further efforts.
Because PPCPs are bioactive compounds being designed to interact with specific
physiological pathways at low doses, it is important to focus on modes of action and to
address the molecular, cellular and individual levels on species from Latin America. In
addition, new polices on environmental risk assessment, waste management, and
publiceducation must be enforced locally.

Keywords: personal care products, pharmaceuticals, Latin America

* Corresponding Author E-mail: lmaranho@gmail.com.

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538 Luciane Alves Maranho and Camilo Dias Seabra Pereira

INTRODUCTION
In recent decades, the impact of chemical pollution has focused almost exclusively on the
conventional "priority" pollutants, such as metals, polycyclic aromatic hydrocarbons (PAHs),
polychlorinated biphenyls (PCBs) and pesticides, especially those that cause acute toxicity,
considered carcinogenic and persistent in the environment (Daughton and Ternes, 1999).
Pharmaceuticals are considered emerging pollutants, and include different kinds of substances
varying in physicochemical and pharmacological properties, modes of action, fate in the
environment and degradation. Since the human body does not completely metabolize
pharmaceuticals they are excreted slightly transformed or even unchanged, and end up in
water systems. For these reasons, the study of the behaviour and possible effects of these
compounds in the environment is complex. A vast amount of information has been generated
during the last 20 years in Europe, North America, and Asia regarding the environmental
occurrence and possible adverse effects of pharmaceuticals on biota. The European and North
American legislation include guidelines for assessing environmental risk caused by exposure
to pharmaceutical substances. In these guidelines, environmental risks are recommended as
part of the approval process of new substances. It is especially important to determine the
issue of pharmaceutical compounds to the environment and ecotoxicological assessment of
possible adverse effects. Unfortunately, information available for Latin America is still very
limited.
Pharmaceuticals and personal care products (PPCPs) have been recently considered an
environmental problem. This issue is of increasing scientific concern (Kümmerer et al., 2010;
Heberer, 2002), represented by the growing number of scientific papers on concentrations of
pharmaceuticals in environmental matrices (Pereira et al., 2016). Studies on possible adverse
effects on the biota also increased in recent years, focusing mainly on the acute toxicity of
pharmaceuticals (Santos et al., 2010). The main studies are on growth, reproduction and
mortality (Santos et al., 2010). However, few research groups have developed studies to
understand more deeply the bioavailability of these compounds and possible chronic effects
on aquatic communities. In addition, the existing literature is restricted almost entirely to
works developed in temperate environments.
In Latin America, there is a rich diversity of ecosystems which may be differently
affected by emergent pollutants such as PPCPs. Latin America houses the richest biodiversity
in the world, including biomes such as mangroves, pampas, tropical forests, deserts, cerrado,
among others. Focusing on aquatic environments, few studies have been performed to
determine the environmental concentration of pharmaceutical products. This lack of
knowledge may be due to the fact that environmental toxicology and chemistry in Latin
America is a relatively young discipline (Carriquiriborde and Bainy, 2012). A quick analysis
of studies published in 2011 by researchers based in Argentina, Brazil and Chile, the three
countries of the Latin-American branch of the Society of Environmental Toxicology and
Chemistry (SETAC-LA) with the greatest number of contributions, revealed subtle
differences in the relative development of environmental disciplines (Carriquiriborde and
Bainy, 2012). In Brazil, 2.1% of major environmental issues tackled by researchers were
about PPCPs, while Chilean researchers published 3.4% of scientific work on PPCPs
(Carriquiriborde and Bainy, 2012). Beek et al. (2016) also elucidated that peer-reviewed
publication of environmental data in universities located in developing countries such as

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 Pharmaceutical and Personal Care Products (PPCPs) … 539

Brazil is relatively uncommon as a result of language barriers, publication costs and limited
time frames for graduation procedures.
The world consumption of PPCPs has risen drastically in the last decade, also increasing
the excretion of their metabolites in their original form. Pharmaceutical consumption is often
associated with longevity and stressful lives, particularly in urban areas. Advances in
medicine, the significant growth of chemistry and pharmaceutical companies, and changes in
social conditions have all led to the more widespread use of pharmaceuticals and
psychotropic substances (Borova et al., 2014). Between 2000 and 2010, the consumption of
antibiotic drugs increased by 36% (Van Boeckel et al., 2014). Brazil, Russia, India, China and
South Africa accounted for 76% of this increase.
Latin America is a diverse, multi-coloured and dynamic region, boasting the highest life
expectancy among developing regions (Valverde, 2014). Unfortunately, Latin America has a
common and very serious environmental health problem: the failure or absence of wastewater
treatment plants (WWTPs). The Latin American pharmaceutical market represents
approximately 25% of global pharmaceutical sales (Valverde, 2014). In consequence, high
pharmaceutical concentrations are expected in Latin American environmental matrices. The
constant release of PPCPs elucidates the importance and urgency of the determination of
environmental concentrations of different PPCPs, as well as the potential environmental
effects, including acute and chronic toxicity, and bioaccumulation data.

PPCPS CONCENTRATIONS FOUND IN ENVIRONMENTAL MATRICES IN

LATIN AMERICA
Sewage outfalls, WWTPs and wastewater without proper treatment are the main sources
of PPCPs to the environment. Even in places supported by WWTPs, it is known that
conventional WWTPs are not able to entirely degrade complex organic molecules to reduce
their toxicity and improve their biodegradability. For this reason new technologies, i.e.,
advanced oxidation processes, are being developed to handle this demand (Borghi and Palma,
2014).
Most of the efforts to assess PPCPs occurrence and potential impacts on non-target
organisms in aquatic environments have focused on freshwater rivers and streams heavily
impacted by wastewater effluent (Pereira et al., 2016). These observations were mainly made
in Brazil (São Paulo, Rio de Janeiro and MatoGrosso do Sulstates), Argentina, Colombia and
Mexico. Concentrations varied from ng·L-1 to μg·L-1, indicating significant inputs of PPCPs
into these regions.
The first studies about PPCPs occurrence in Brazil were in the state of Rio de Janeiro
published by Ternes et al. (1999) and Stumpf et al. (1999). The first article was about the
quantification of estrogens in sewage samples varying from 0.021 to 0.040 μg·L-1(Ternes et
al., 1999). Stumpf et al. (1999) published about the detection of pharmaceutical residues such
as lipid regulators, anti-inflammatories and some metabolites in raw sewage, treated waste
water and river water. The median concentrations in the effluents of sewage treatment plants
(STPs) of most pharmaceuticals investigated ranged from 0.1 to 1 μg·L-1. After the STP,
median concentrations ranged from between 0.02 and 0.04 μg·L-1 n river water, whereas the

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maximum values were observed to be up to 0.5 μg·L-1. Caldas et al. (2013) also found 18
PPCPs and 33 pesticides in surface and drinking waters in the state of Rio de Janeiro.
Sodré et al. (2010) evidenced the occurrence of PPCPs in drinking water of the city
of Campinas, Brazil. Seasonality was observed, since some pharmaceuticals were detected
only in one season. Stigmasterol showed the highest average concentration (0.34 ± 0.13
μg·L-1), followed by cholesterol (0.27 ± 0.07 μg·L-1), caffeine (0.22 ± 0.06 μg·L-1) and
bisphenol A (0.16 ± 0.03 μg·L-1) (Sodré et al., 2010). Acetaminophen, acetylsalicylic acid,
diclofenac, ibuprofen, caffeine, 17β-estradiol, estrone, progesterone, 17α-ethynylestradiol,
levonorgestrel, diethylphthalate, dibutylphthalate, 4-octylphenol, 4-nonylphenol and
bisphenol were detected in surface waters from the same region, in the Atibaia watershed
(São Paulo state, Brazil) (Montagner and Jardim, 2011). The lowest concentration determined
was 0.096 μg·L-1for diclofenac, whereas caffeine showed concentrations as high as 127 μg·L-
1 (Montagner and Jardim, 2011). Antibiotics amoxicillin, ampicillin, cefalexin (CEF),

ciprofloxacin (CIP), norfloxacin (NOR), sulfamethoxazole, tetracycline (TET) and

trimethoprim were quantified also in surface water samples from the Atibaia watershed
(Locatelli et al., 2011). In the Atibaia River, sample concentrations ranged from 29 ng·L-1 for
CEF to 0.5 ng·L-1 for NOR. In a sewage-affected stream, however, concentrations up to 2.422
ng·L-1 CEF were found.
A preliminary risk assessment of Triclosan (TCS) was conducted for Brazilian surface
waters in accordance to an approach for the prioritization of organic toxicants (von der Ohe et
al., 2011), which has been used to assess the priority of TCS in Europe (von der Ohe et al.,
2012). Of the 71 samples analysed, 32 contained TCS at concentrations above the limit of
quantification, ranging from 2.2 to 66 ng·L-1, and with six out of seven sites exceeding the
lowest “Predicted no-effect concentrations” (PNEC) (Montagner et al., 2014).
The occurrence as well as the spatial and temporal variations of some commonly
prescribed pharmaceuticals was determined in water samples from the Suquía River basin
(Córdoba, Argentina) (Valdés et al., 2014). Eight out of 15 pharmaceuticals studied were
found in river waters: ciprofloxacin, enalapril, estrone, dihydrotestosterone, oxcarbazepine,
carbamazepine, atenolol and diclofenac. Among all compounds analysed, atenolol,
carbamazepine and diclofenac were the most frequently detected (reaching high levels of
μgL−1).
Estrogens have been identified as the main contributors to the estrogenic activity of STP
effluents. They are considered as endocrine disrupting chemicals (EDCs) that can interfere
with the normal functioning of the endocrine system in wildlife and humans. Valdés et al.
(2015) investigated for the first time the concentrations of estrone (E1), 17β-estradiol (E2)
and 17α-ethinylestradiol (EE2) in sewage effluents and receiving waters of the “Rio de la
Plata” estuary and neighbouring areas (Argentina). E2 and EE2 showed concentrations
ranging between 122 and 631 ng·L-1 and between 65 and 187 ng·L-1 in effluents, while these
estrogens were detected at 369 ng·L-1and 43 ng·L-1 in surface waters, respectively.
Bertin et al. (2011) measured the levels of one synthetic (EE2) and three natural (estrone,
17β-estradiol, estriol) estrogens in coastal sediment samples collected from nine locations of
central-southern Chile. Steroid estrogens were found in every sample. Remarkably high levels
of 17α-ethinylestradiol were detected, reaching up to 48.14 ng·g−1 dry weight. The studied
coastal region houses a large variety of fish and seafood of commercial value such as flatfish,
hake, red and pink cusk-eels, drumfish, crabs, mussels, abalone and clams (Sernapesca,
2008). Thus, the contamination of marine sediments by steroid estrogens should not be

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neglected, particularly in areas which host edible fish and seafood, as this may turn out to be a
significant health issue for humans (Bertin et al., 2011).
The first study on the presence and distribution of PPCPs in Colombia was published by
Gracia-Lor et al. (2012). Seventy three surface water samples (rivers, lakes and reservoirs and
effluent wastewater) showed that the large majority of the target analytes were present in the
samples analysed. Target analytes were two anti-inflammatories (ibuprofen, diclofenac), a
lipid regulator agent (clofibric acid), two angiotensin II antagonists (losartan, ibersartan), two
antiepileptic drugs (gabapentin, carbamazepine) and a diuretic (furosemide). Among personal
care products, four preservatives (methyparaben, ethylparaben, propilparaben, butylparaben)
and five UV filters were included (benzophenone, benzophenone-1, benzophenone-2,
benzophenone-3, benzophenone-4).
Hernández et al. (2015) made alargescreening in Colombia of around 1,000 emerging
contaminants, focused on PPCPs, illicit drugs and their metabolites. This survey was made in
urban wastewaters (both influent and effluent) and surface waters from the area of Bogotá.
The main compounds detected were acetaminophen/paracetamol, carbamazepine and its
dihydro-dihydroxylatedmetabolite, clarithromycin, diclofenac, ibuprofen, gemfibrozil,
lincomycin, losartan, valsartan, the two metabolites of metamizole (4-acetamido-antipyrine
and 4-formylamino-antipyrine), sucralose, cocaine and its main metabolite benzoylecgonine.
Also caffeine, the sweetener saccharin, two hydroxylated metabolites of losartan and
lidocaine were identified in the samples analysed.
In Mexico, environmental studies concerning pharmaceuticals are mainly focused on
soils irrigated with wastewater. One of the main objectives of Siemens et al. (2008) was to
characterize the flow of pharmaceutically active substances through the Mezquital Valley
wastewater irrigation system (Mexico City, Mexico). Trimethoprim, erythromycin, naproxen,
ibuprofen and diclofenac in sewage equalled or exceeded the US FDA (US Food and Drug
Administration, US Department of Health and Human Services, 1998) action limit of 1 mg·L-
1 for detailed environmental risk assessment (ERA).

Gibson et al. (2010) reported the persistence and leaching potential of a group of acidic
pharmaceuticals, carbamazepine, and three endocrine disruptors in soils from the Tula Valley
in Mexico, one of the largest irrigation districts in the world that uses untreated wastewater.
Ibuprofen (742-1,406 ng·L-1), naproxen (7,267-13,589 ng·L-1) and diclofenac (2,052-4,824
ng·L-1) were consistently present while clofibric acid, gemfibrozil and ketoprofen were
below limits of detection (LOD) in all samples (100, 50 and 50 ng·L-1, respectively) and
were not considered further. Concentrations of ciprofloxacin, enrofloxacin, sulfamethoxazole,
trimethoprim, clarithromycin, carbamazepine, bezafibrate, naproxen and diclofenac, as well
as the occurrence of Enterococcus spp. and sul and qnr resistance genes in soil irrigated with
wastewater were observed by Dalkmann et al. (2012).
There is a lack of knowledge about PPCP concentrations and adverse effects in marine
environments. The most recent study concerning the occurrence of pharmaceuticals, illicit
drugs (cocaine) and its metabolite (benzoylecgonine) in the marine environment was
published by Pereira et al. (2016). Pharmaceuticals and cocaine were assessed in a subtropical
coastal zone (Santos, São Paulo state, Brazil) in five sampling sites around the sewage outfall.
Acetaminophen (17.4-34.6 ng·L−1), caffeine (84.4-648.9 ng·L−1), diclofenac (19.4 ng·L−1),
ibuprofen (326.1-2094.4 ng·L−1), losartan (11.8-32 ng·L−1) and valsartan (10.8-75 ng·L−1)
were quantified in seawater samples. Ibuprofen showed the highest concentrations in the
order of μg·L−1. Cocaine and benzoylecgonine were both quantified in all of the samples

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analysed at concentrations that ranged from 12.6 to 537 ng·L−1 and 4.6 to 20.8 ng·L−1,
respectively.
Beretta et al. (2014) detected PPCPs in marine sediment from Todos os Santos Bay
(Bahia, Brazil). The highest concentrations were found for the fragrances galaxolide (52.5
ng·g−1) and tonalide (27.9 ng·g−1), followed by caffeine (23.4 ng·g−1) and pharmaceuticals
ibuprofen (14.3 ng·g−1), atenolol (9.84 ng·g−1), carbamazepine (4.81 ng·g−1), erythromycin
(2.29 ng·g−1), diclofenac (1.06 ng·g−1) and diazepam (0.71 ng·g−1).
Elorriaga et al. (2013) published the first survey of pharmaceuticals in municipal
wastewaters discharging into fresh and estuarine waters from areas with varying degrees of
urbanization of Argentina. Pharmaceutical concentrations were in μg·L-1 which may be
considered higher than the average concentration of such compounds in the environment that
are normally at then ng·L-1 level. Concentrations were within the range of 0.9-4.2 μg·L-1and
0.4-13.0 μg·L-1 for caffeine and ibuprofen, and at the lower levels of 0.2-2.3 μg·L-1, 0.2-1.7
μg·L-1 and 0.03-1.2 μg·L-1 for carbamazepine, atenolol and diclofenac, respectively.

ADVERSE EFFECTS ON BIOTA AND ENVIRONMENTAL

RISK ASSESSMENT
Pharmaceuticals represent a versatile group of chemical compounds. In general,
chemicals can be harmful and toxic because of many different mechanisms. For example,
chemicals can bind to molecules such as hormones, DNA and RNA, lipid membranes and
proteins. Recent studies have been performed in Latin America concerning adverse effects of
PPCPs on biota. Godoy et al. (2015) assessed the ecotoxicity of propranolol hydrochloride
and losartan potassium, both individually and combined, by using the Lemna minor growth
inhibition test. The risks associated to the effects of both pharmaceuticals on non-target
organisms were quantified through the measured environmental concentration (MEC) /
predicted-no-effect concentration (PNEC) ratios. For propranolol, the total frond area was the
most sensitive endpoint (effect concentration - EC50=77.3 mg·L-1), while for losartan there
was no statistically significant difference between the endpoints. Losartan is only slightly
more toxic than propranolol. Both concentration addition and independent action models
overestimated the mixture toxicity of the pharmaceuticals at all the effect concentration levels
evaluated. The joint action of both pharmaceuticals showed an antagonistic interaction for L.
minor. Derived risk quotient (RQ) assumed lower values for propranolol than for losartan.
The MEC/PNEC ratios showed that propranolol may pose a risk for the most sensitive
aquatic species, while acceptable risks posed by losartan were estimated for most of aquatic
matrices.
TCS (Triclosan- 5-chloro-2-(2,4-dichlorophenoxy) phenol) is an antibacterial compound
widely employed in PPCPs. Cortez et al. (2012) observed effects on reproduction
(fertilization and embryo-larval development) and physiological stress (lysosomal membrane
stability - LMS) in the marine sentinel organism Perna perna. The mean inhibition
concentrations for fertilization (IC50=0.490 mg·L-1) and embryo-larval development
(IC50=0.135 mg·L-1) were above environmental relevant concentrations (ng·L-1) given by
previous studies. On the other hand, a significant reduction of LMS was found at 12 ng·L-1,

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demonstrating the current risk of the continuous introduction of TCS into aquatic
environments.
Borghi and Palma (2014) draw attention to the fact that there are other compounds in
addition to the bioactive substance of the drugs, which can also cause adverse effects to biota.
One example is the antibiotic tetracycline which has high world consumption, representing a
human consumption of about 23 kg·day-1 in Brazil in 2007. At the moment, research is being
made to develop new tetracycline that incorporate metals (Hg, Cd, Re, Pt, Pd) to their
structures in order to increase their bactericidal effect.
Chronic adverse effects due to the antidepressant fluoxetine (FLX, 0.54 μg·L-1) and the
synthetic estrogen, 17 α-ethinylestradiol (EE2, 5 ng·L-1), alone and in combination, were
observed in malegoldfish Carassius auratus after 14-days of exposure (Assis et al., 2013).
The results showed an increase in estrogen receptor alpha (esr1) and vitellogenin (VTG) gene
expression by 1.9-2.4 fold in the FLX and EE2 groups.There was a significant increase in the
number of plasma proteins that were related to endocrine system disorders in the FLX and
FLX plus EE2 groups. The level of VTG protein was increased in the plasma from goldfish
exposed to EE2, FLX, and FLX plus EE2. This study demonstrates that low concentrations of
FLX and EE2 in a simple mixture produce strong estrogen-like effects in male goldfish.
Ribas et al. (2014) published a work about toxic effects of non-steroidal anti-
inflammatory drugs (NSAIDs) diclofenac (0.2, 2, 20, 200 and 2000 ng·mL-1), ibuprofen (0.1,
1, 10, 100, and 1000 ng·mL-1) and acetaminophen (0.025, 0.25, 2.5, 25 and 250 ng·mL-1)
evaluated on primary culture of monocytic lineage of Hoplias malabaricus anterior
kidney. NSAIDs influenced lipopolysaccharide (LPS)-induced nitric oxideproduction and
caused DNA damage in monocytic cells, suggesting that these substances can induce
immunosuppression and genotoxicity.
Effects of diclofenac and dexamethasone on hematological parameters and immune
response were observed also in the fish species H. malabaricus after trophic exposure (Ribas
et al., 2016). Intraperitoneal inoculation with diclofenac (0, 0.2, 2 and 20 μg·kg-1) or
dexamethasone (0.03, 0.3, and 3 μg·kg-1) was performed in 20 doses. Although some fish
responses were variable for different compounds, the results suggested that trophic exposure
to diclofenac and dexamethasone can lead to hematological changes and immunotoxic effects.
The gonad and liver from the same fish were collected to calculate gonadosomatic
and hepatosomatic indices (Guiloski et al., 2015). Antioxidant enzyme activity and
biotransformation were also evaluated in liver and gonads. Both diclofenac and
dexamethasone reduced the levels of testosterone, causing impairment in reproduction.
Guiloski et al. (2015) suggested that diclofenac and dexamethasone caused oxidative stress
and reduced testosterone levels with negative impacts in fish.
Biochemical, genetic and hematological biomarkers were assessed in the liver, kidney,
and blood of juvenile Rhamdia quelen fish exposed to diclofenac for 96h at concentrations of
0.2, 2, and 20 μg·L−1. No oxidative stress was observed in liver. In kidney, the superoxide
dismutase activity increased in all concentrations, suggesting an alteration in the hydrogen
peroxide production. Diclofenac exposure increased the red blood cells number at
concentrations of 0.2 and 2 μg·L−1, and monocytes and neutrophils at 2 and 20 μg·L−1,
respectively. Ghelfi et al. (2016) suggested that acute exposure to diclofenac caused
hematologic and renal enzymatic alterations.
The first report on pharmaceuticals in superficial waters of Argentina as well as the first
report on the bioaccumulation of atenolol in whole body fish was reported by Valdés et al.

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(2014). The bioconcentration of carbamazepine and atenolol was studied under controlled
conditions in Gambusia affinis, a widely distributed fish species inhabiting the Suquía River
basin (Córdoba, Argentina). Estimated bioconcentration factors (BCFs) were: 0.13 and 0.08
μg·kg−1 upon exposure to 100 and 1000 μg·L−1 atenolol in water, respectively; while BCFs
were 0.7 and 0.9 μg·kg−1 when exposed to 10 and 100 μg·L−1 carbamazepine, respectively.
Estrogen concentrations are able to disrupt the endocrine system of aquatic vertebrates
(Sumpter and Jobling, 2013). E2 concentrations as low as 50 ng·L-1 were effective in
impairing the expression of brain aromatase and the sexual behaviour of the South American
fish Jenynsia multidentata (Guyón et al., 2012) and EE2, fiftytimes more potent, was able to
disrupt the expression of the gonadal aromatase andthe sex ratio of Odontesthes bonariensis
(Pérez et al., 2012), an emblematic species of the “Pampas” streams and shallow lakes.
According to Valdés et al. (2014), the measured concentration of estrogens in the receiving
waters of the ‘‘Pampas’’ region with low dilution capacity, would represent a risk for the
reproductive successes of local fish, and probably other aquatic organisms.
Magdaleno et al. (2014) performed toxicity and genotoxicity assessment with effluents
from a STP serving around 10 million inhabitants in Buenos Aires (Argentina). Toxicity
assays showed that 55% of the wastewatersamples from a public hospitalwere toxic to the
green algae Pseudokirchneriella subcapitata and genotoxicity for 40% of the samples tested
with the Allium cepa. Nevertheless, the sample fromthe STP was not genotoxic to A. cepa but
toxic to the algae, showing that sewage treatment was not totally effective.
González-González et al. (2014) exposed common carp Cyprinus carpio to water from
Madín Reservoir (Mexico) for 96 h. Water samples contained contaminants such as metals
and NSAIDs. Results showed that contaminants (metals and NSAIDs) present in the water
induced oxidative stress.

PERSPECTIVES
Environmental risk and impacts of PPCPs have been largely assessed via acute toxicity
assays, which are likely to be underestimating real impacts, since such compounds have been
found at low concentrations in surface waters (ng·L-1), but are being continually released into
ecosystems. This steady flow contributes to their persistence and potential adverse effects
(Alygizakis et al., 2016; Andreu et al., 2016). Nowadays, it is well known that these bioactive
compounds might cause mainly chronic toxicity with serious consequences for the
environment. Chronic exposures and specific mode of action of PPCPs need to be considered
as major aspects for future scientific investigation in Latin America.
Knowledge on the transformation of pharmaceuticals in WWTPs and aquatic ecosystems
is rather fragmented. Several studies have assessed attenuation (net balance between removal
and release to and from the water column) of pharmaceuticals in either WWTPs or freshwater
ecosystems, but none have assessed attenuation of pharmaceuticals and their transformation
products including both systems within the studied system boundaries and using the same
approach (Aymerich et al., 2016). The conventional WWTPs are not able to promote
significant PPCP load reduction. Thus, the main step to minimize the PPCPs contamination
should be the development ofeffective treatments for wastewater discharges. Implementation

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of mitigation actions andthe development of environmental tools to monitor PPCPs

contamination and effects are also crucial to preserve aquatic biodiversity.
Concerning specifically illicit drugs and their breakdown products, occurrences in
regions of production and use and areas with insufficient wastewater treatment are not well
studied (Rosi-Marshall et al., 2015). Several studies have found cocaine and its metabolites in
wastewater, surface water, sediment and organisms, and adverse effects have been observed
(Zuccato et al., 2008; Binelli et al., 2012; Klosterhaus et al., 2013; Pereira et al., 2016). These
substances must be considered of environmental concern since illicit drugs are a growing
social and public health problem. The most recent World Drug Report estimates that the
highest rates of cocaine use (including crack cocaine) occur in North, Central, and South
America (UNODC, 2014). Illicit drug use has increased dramatically in the past decades in
developing countries of South America (Johnson et al., 2013). The consumption and
trafficking of cocaine in South America has become more prominent, particularly in Brazil,
due to factors such as geographic location and the increasing populations clustered in urban
centres.
New polices on ERA and environmental monitoring are necessary. According to
Aymerich et al. (2016), for instance, the anti-inflammatory diclofenac, the synthetic hormone
ethinylestradiol (EE2) and the antibiotics erythromycin, clarithromycin andazithromycin have
recently been included in the ‘Watch List’ of priority substances under the European Water
Framework Directive (Decision 2015/495/EU). In addition, in 2015 the US Environmental
Protection Agency included pharmaceuticals such as erythromycin and EE2 in the Water
Contaminant Candidate List 4, a list of contaminants that are currently not subject to any
proposed or promulgated national primary drinking water regulation but are known or
anticipated to exist in public water systems. Developing countries in Latin America should
follow such examples to reduce the amount of PPCPs waste released into aquatic
environments, since the populationsare growing, aging, and becoming more concentrated in
metropolitan areas.
Public education must be enforced and should include both health professionals
(prescriptions) and consumers (release) in order to reduce PPCPs in aquatic ecosystems.

ACKNOWLEDGMENTS
Camilo Dias Seabra Pereira thanks Conselho Nacional de Desenvolvimento Científico e
Tecnológico - CNPq for financing the Project intitled “Avaliação do risco ambiental de
compostos farmacêuticos no ambiente marinho” (Project #481358/2012-9) and for the
productivity fellowship (Project #307074/2013-7). Camilo Dias Seabra Pereira also thanks
São Paulo Research Foundation (FAPESP)for financing the project intitled “Estudo
ecotoxicológico e avaliação do risco ambiental de drogas ilícitas em ecossistemas marinhos?”
(Project #2015/17329-0). Luciane Alves Maranho thanks CNPq for the Postdoctoral
fellowship (Project #166122/2015-7).

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In: Ecotoxicology in Latin America ISBN: 978-1-53610-600-8
Editors: C. V. M. Araújo and C. Shinn © 2017 Nova Science Publishers, Inc.

Chapter 34

RESTORATION AND CONSERVATION ACTIONS:

CHILEAN STUDIES ON PHYTOREMEDIATION OF
METAL-POLLUTED, ACIDIC SOILS

Alexander Neaman1,*, Rosanna Ginocchio2 and Carolina Yáñez3

1Escuela
de Agronomía, Pontificia Universidad
Católica de Valparaíso, Quillota, Chile
2Departamento de Ecosistemas y Medio Ambiente (DEMA),

Center of Applied Ecology and Sustainability (CAPES),

Pontificia Universidad Católica de Chile, Santiago, Chile
3Instituto de Biología, Pontificia Universidad Católica de Valparaíso,

Valparaíso, Chile

ABSTRACT
The topic of restoration and conservation actions is very broad. In this chapter, we
limit the discussion to phytoremediation of soils polluted by copper mining, focusing on
two remediation options: phytostabilization and phytoextraction. We present Chilean
studies for each of these two options of remediation of metal-polluted, acidic soils in a
site located in proximity of a copper smelter. Specifically, the Puchuncaví Valley, located
on the coast of Central Chile, received strong atmospheric depositions from the Ventanas
smelter from 1964 to 1999. The soils of the valley are currently acidic, with scarce
vegetation. They are severely eroded and contain high total concentration of metals, such
as Cu, Zn, Pb, and Cd, and metalloids, such as arsenic. Currently, a large-scale
remediation is needed to reverse the strong historic degradation of polluted soils around
the Ventanas industrial complex. In this chapter, we demonstrate that phytostabilization
is a technique that could be effective for large-scale remediation of the metal-polluted,
acidic soils of the Puchuncaví Valley. On the other hand, we conclude that the
phytoextraction technique is unfeasible because of the long time necessary for cleaning
up soil metals with plants, besides other restrictions.

*
Corresponding Author E-mail: alexander.neaman@pucv.cl.

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552 Alexander Neaman, Rosanna Ginocchio and Carolina Yáñez

Keywords: phytostabilization, phytoextraction, revegetation, earthworms, microorganisms

IN SITU METAL IMMOBILIZATION AND PHYTOSTABILIZATION

The in situ immobilization of metals in polluted topsoils has been suggested as a cost-
effective method of soil remediation (Mench et al., 2000; US EPA, 2007). A plant-based
approach for in situ metal immobilization in polluted soils is phytostabilization.
Phytostabilization has been defined as the use of metal-tolerant plants (metallophytes) that
accumulate metals in underground tissues (excluder metallophytes). Excluder metallophytes
contribute to in situ metal immobilization through root absorption and sorption (Berti and
Cunningham, 1999; Bouwman and Vangronsveld, 2004) and the exudation of organic
compounds that chelate metal ions in the rhizosphere (Meier et al., 2012). Metal accumulation
in roots and immobilization in the rhizosphere stabilize polluted soils and reduce or eliminate
the risk of movement of toxic elements from the contaminated soil to the environment (e.g.,
Macnair et al., 2000). Thus, metals are not removed from the soil but are reduced to less-
soluble forms that are not available for biological actions, such as root uptake and
phytotoxicity in less tolerant and/or ecologically or commercially relevant species
(Vangronsveld and Cunningham, 1998; Macnair et al., 2000).
Phytostabilization can be used in combination with organic and/or inorganic soil
amendments, being then named aided phytostabilization (Mench et al., 2010). It consists of
reducing metal concentrations in the soil solution through the incorporation of organic and/or
inorganic soil amendments into polluted topsoil. Sorption and/or precipitation reactions
induced by soil amendments decrease the concentration of bioavailable metals in the soil. Soil
amendments also improve other soil limiting conditions for plant establishment and growth
derived from soil degradation, such as macronutrient deficiencies, microbial malfunctions,
and physical limiting conditions (US EPA, 2007). Aided phytostabilization thus allows
revegetation and ecosystem remediation on contaminated sites. Vegetation recovery reduces
or even prevents the dispersal of soil contaminants via wind and water erosion and improves
the esthetical value of formerly bare areas (Vangronsveld and Cunningham, 1998; Macnair et
al., 2000).

ACTIVE VERSUS PASSIVE REVEGETATION OF

METAL-POLLUTED SOILS
Two options have been used for restoring plant covers in degraded soils once plant-
growth restrictions have been removed from the site (e.g., by the use of soil amendments).
The first, active revegetation, employs seeding and/or outplanting of plant species (Arienzo et
al., 2004; Clemente et al., 2005; Alvarenga et al., 2008). The second, passive revegetation,
consists in the spontaneous development of plants from the natural soil seed bank, seeds
dispersed by natural vectors, and/or vegetative reproduction of remaining vegetation (Álvarez
et al., 2003; Madejón et al., 2006; Conesa et al., 2007). Both practices have been extensively
studied and used in the field of ecological restoration (e.g., Van Andel and Aronson, 2012;

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 Restoration and Conservation Actions 553

Zahawi et al., 2014) but not in the field of aided phytostabilization (e.g., Velayoudon et al.,
2014), where plants have been actively introduced.
A field study performed by our work team (Córdova et al., 2011) compared aided versus
passive revegetation in a metal-polluted, acidic site in the Puchuncaví Valley from the
approach of aided phytostabilization. Our results showed that plant cover and aboveground
biomass were the same under active and passive revegetation regimes, suggesting that plant
cultivation was unnecessary. Likewise, under the assisted revegetation regime, both the
aboveground biomass and plant cover of all spontaneous species were significantly higher
than those of cultivated species (Table 1). We concluded that the remaining native soil seed
bank of the study site may be sufficient to recover the plant cover and biomass production of
original vegetation, after application of proper soil amendments. Stimulation of spontaneous
recolonization from remaining propagules though proper soil management practices would
reduce the costs that are inherent of cultivation practices.

Table 1. Plant cover and aboveground biomass of cultivated and the accompanying
spontaneous species under aided phytostabilization, seven months after soil amendment
application. Based on Córdova et al. (2011)

Plant species type Plant cover (%) Aboveground biomass

(kg ha-1, dry weight)
Cultivated 9±8a 246 ± 369 a
Accompanying spontaneous 55 ± 17 b 1387 ± 1083 b
Average data and standard deviation (n=6) are shown. Different letters in the same column indicate
significant differences among the plant species type (p<0.05).

Table 2. Chemical characteristics of topsoil (0-5 cm) in experimental plots five months
after amendment application. Average data and standard deviation are shown (n=3).
Based on Ulriksen et al. (2012)

Characteristic Treatment
Control Lime Compost Lime+compost
pH 4.5 ± 0.1 a 8.1 ± 0.0 b 7.3 ± 0.1 b 8.0 ± 0.1 b
pCu2+ 4.4 ± 0.2 a 9.1 ± 0.7 b 9.4 ± 0.8 b 9.8 ± 0.7 b
-1
Cuex,mg kg 33 ± 16 a 0.3 ± 0.2 b 1.4 ± 0.6 b 1.5 ± 0.5 b
OM, % 1.0 ± 0.3 a 0.8 ± 0.3 a 2.8 ± 1.0 b 3.0 ± 0.5 b
N, mg kg-1 15 ± 1.0 a 13 ± 2.4 a 42 ± 12 b 42 ± 5.4 b
P, mg kg-1 30 ± 3.6 a 29 ± 6.6 a 114 ± 33 b 117 ± 17 b
K, mg kg-1 0.4 ± 0.0 a 0.3 ± 0.0 a 1.9 ± 0.2 b 1.6 ± 0.2 b
WHC, % 33 ± 0.6 a 33 ± 0.6 a 39 ± 2.4 b 39 ± 0.6 b
Different letters in the same row indicate significant differences (p<0.05). pCu 2+: -log[Cu2+], where
[Cu2+] is activity of Cu2+ ion in the soil solution; a higher value of pCu2+ implies lower activity of
Cu2+ ion. Cuex: exchangeable copper determined in an extract of 0.1 M KNO3 (at a 1:2.5 soil:liquid
ratio). OM: organic matter. WHC: water holding capacity.

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LIME AND COMPOST PROMOTE PLANT RECOLONIZATION OF

METAL-POLLUTED, ACIDIC SOILS
In this chapter, we present field results on the use of lime and compost in promoting plant
recolonization of metal-polluted, acidic soils in our study site at the Puchuncaví Valley
(Córdova et al., 2011; Ulriksen et al., 2012). We found that the application of lime and/or
compost decreased the Cu2+ ion activity in the soil solution and the exchangeable Cu in the
soil, showing effective Cu immobilization in the topsoil (Table 2). Likewise, incorporation of
compost into degraded soil significantly increased the organic matter content, concentration
of available nutrients (NPK), and the water-holding capacity of the soil (Table 2).
Lime application had no effect on plant productivity in comparison to unamended
control, while application of either compost or lime+compost increased plant cover and
aboveground biomass (Table 3). The regression analyses revealed that plant productivity (in
terms of either the aboveground biomass or plant cover) significantly and positively
responded to the nutrient availability and water-holding capacity of the soil (Córdova et al.,
2011; Ulriksen et al., 2012). Other soil chemical characteristics, including total concentrations
of Cu, Zn, Pb, and As, organic matter content, pCu2+, soluble and exchangeable Cu, and pH,
did not significantly correlate (p>0.05) to plant response variables. These findings are in
agreement with the study of Ginocchio (2000), who determined the soil characteristics that
best explained the changes observed in the plant species richness and abundance in the
Puchuncaví Valley. This author reported that the low nitrogen availability was the main soil
stress factor limiting plant establishment and growth, whereas soil acidification and soil Cu
enrichment were the second and third stress factors, respectively.
Although the Cu2+ activity and the exchangeable Cu were markedly lower in the
amended soils than in the unamended control, the shoot Cu concentration was similar among
most combinations of plant species and amendments (Table 4). Thus, the Cu accumulation in
the aboveground tissues of the Loliums pp. and Eschscholzia californica- dominant plant
species in the study area - did not depend solely on Cu availability in the soil. These species -
not responsive to the soil Cu concentrations, with constant Cu concentrations in the
aboveground tissues - would be classified as excluders according to the classification of
Baker (1981). However, to confirm these phenotypes in the present study, it would have been
necessary to determine the Cu concentrations in the underground tissues.
In general, a concentration of 20 mg kg-1 is considered the threshold Cu concentration in
plants, not considering highly sensitive and tolerant species (Kabata-Pendias and Pendias,
1992). However, the same authors reported that the majority of plant species can accumulate
more than 20 mg kg-1 of Cu when growing in soils with elevated Cu concentrations. In
accordance with this observation, shoot Cu concentrations of Lolium spp. were slightly higher
and those of E. californica were considerably higher than the threshold value of 20 mg kg-1
(Table 4). Likewise, the shoot concentrations of Zn, Pb, and As were above the background
values for the shoots of plants growing in unpolluted soils (Benton, 1983; Kabata-Pendias and
Pendias, 1992; Adriano, 2001; Römheld, 2012; Alloway, 2013) (Table 4). Nevertheless,
plants were able to complete their life cycles: they flowered and produced seeds even when
growing on the unamended control soil. It may therefore be stated that the plant development
in all of the treatments was independent of elevated soil metal concentrations.

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Table 3. Plant cover and aboveground biomass of treatments at the end of the first plant
growing season. Average data and standard deviation
(n=3) are shown. Based on Ulriksen et al. (2012)

Treatment Plant cover, % Aboveground biomass, kg ha-1

Control 36 ± 22 a 337 ± 247 a
Lime 56 ± 16 a 582 ± 28 a
Compost 95 ± 8.1 b 2516 ± 377 b
Lime + compost 99 ± 2.3 b 2630 ± 767 b
Different letters in the same column indicate significant differences between the treatments (p<0.05).

Table 4. Shoot concentration of Cu, Zn, Pb, and As in dominant plant species of the
study area. Average data and standard deviation (n=3) are shown. There were no
significant differences between treatments with regards to shoot metal concentration of
the same species. Based on Ulriksen et al. (2012)

Species Treatment Total concentration, mg kg-1

Cu Zn Pb As
Lolium spp. Control 28 ± 10 69 ± 17 7.6 ± 3.1 6.4 ± 1.0
Lime 30 ± 2.1 65 ± 2.5 11 ± 0.7 7.6 ± 0.8
Compost 29 ± 9.6 71 ± 21 10 ± 3.8 7.0 ± 0.6
Lime+compost 27 ± 8.1 60 ± 2.8 9.2 ± 3.1 5.2 ± 0.5
Eschscholzia Control 46 ± 9.5 57 ± 11 7.2 ± 3.7 5.0 ± 2.1
californica Lime 73 ± 36 64 ± 6.2 11 ± 5.7 4.5 ± 0.9
Compost 70 ± 11 70 ± 14 12 ± 3.3 4.8 ± 1.7
Lime+compost 71 ± 22 41 ± 9.0 8.8 ± 1.9 5.3 ± 0.1

EARTHWORMS AS BIOINDICATORS FOR ASSESSMENT OF

THE EFFECTIVENESS OF IN SITU METAL IMMOBILIZATION

The effectiveness of in situ soil immobilization treatments can be assessed by toxicity

tests/assays with organisms. In one of our field studies (Neaman et al., 2012), we used
earthworms as bioindicators for assessing the effectiveness of lime and compost treatments
for in situ immobilization of trace elements in polluted soils of the Puchuncaví Valley. Lime
application had no effect on earthworm reproduction in comparison to the unamended
control, whereas application of compost increased cocoon and juvenile production (Table 5).

Table 5. Results of chronic toxicity tests with earthworms for different treatments.
Number of cocoons and number of juveniles were used as endpoints.
Based on Neaman et al. (2012)

Treatment Cocoons Juveniles

Control 3.1 ± 3.6 a 5.0 ± 5.3 a
Lime 3.0 ± 2.8 a 5.1 ± 3.8 a
Compost 11.2 ± 6.7 b 16.7 ± 12.8 b
Lime+compost 4.8 ± 4.5 a 15.3 ± 10.7 b
Average data and standard deviation are shown (n=12). Different letters in the same column indicate
significant differences among the treatments (p<0.05).

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Table 6. Richness*, measured as number of ribotypes, of bacterial community in

experimental soils based on T-RFLP and DGGE analysis. Based on Rojas (2013)

Treatments T-RFLP (MspI) T-RFLP (HhaI) DGGE

Control 72 ± 11 a 89 ± 11 a 22 ± 0.5 a
Lime 115 ± 8.1 b 138 ± 6.3 b 24 ± 1.1 ab
Compost 146 ± 4.0 c 182 ± 4.6 c 29 ± 1.2 c
Lime+compost 162 ± 3.3 d 195 ± 2.3 d 25 ± 0.6 b
T-RFLP (MspI): Terminal restriction fragment length polymorphism; enzyme used MspI.
T-RFLP (HhaI): Terminal restriction fragment length polymorphism; enzyme used HhaI.
DGGE: Denaturing gradient gel electrophoresis.
* Richness is equal to the number of T-RFLP peaks or DGGE bands.
Values given for T-RFLP are the means of five replicates ± standard deviation and those for DGGE are
the means of three replicates ± standard deviation. Values of the same letter in a column are not
significantly different at p<0.05.

Spatial variability of soil properties exists within treatments in experimental field plots.
This allowed the identification of which soil properties were actually having an impact on
earthworm reproduction. For both cocoon and juvenile production, soil organic matter was a
positive factor, i.e., more soil organic matter increased cocoon or juvenile production. The
toxicity (negative) factor was total soil As. However, total Cu and total As were well
correlated (R2=0.80, p<0.001), hence some trends could have been masked. Thus, compost
treatment was effective in improving the quality of soils of the Puchuncaví Valley, increasing
earthworm reproduction.

MICROORGANISMS AS BIOINDICATORS FOR ASSESSMENT OF

THE EFFECTIVENESS OF IN SITU METAL IMMOBILIZATION

Soil microbial properties are being increasingly used as indicators of soil quality since
they provide a direct measure of soil functioning (Garbisu et al., 2011). Furthermore, there is
strong evidence that soil microbes are more sensitive to metals than animals or plants (Sauvé
et al., 1998; Giller et al., 1999). This is another reason to use soil microbial properties as
indicators of soil quality.
In one of our field studies (Rojas, 2013), we assessed the effectiveness of the above-
mentioned lime and compost treatments by using bacterial community richness and diversity
as bioindicators of soil quality. Results indicated that the soil bacterial community responded
to lime and compost treatments, changing their structure and increasing their richness and
diversity (Tables 6 and 7), and thus improving soil quality. It has been reported that
differences in diversity and composition of soil bacterial communities could largely be
explained by a single variable, soil pH, with a higher bacterial diversity in neutral soils
compared to acidic soils (Fierer and Jackson, 2006). Indeed, amendments used in our study
increased soil pH, and this variable was significantly correlated to the Shannon diversity
index, calculated using T-RFLP (MspI) profile (Table 8). Likewise, the Shannon diversity
index was significantly correlated to Cu bioavailability indices (exchangeable Cu and pCu2+).

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 Restoration and Conservation Actions 557

On the other hand, bacterial community richness was a less sensitive bioindicator, being only
marginally correlated (p<0.1) to activity of the Cu2+ ion in the soil solution (Table 8).

Table 7. Shannon indices of diversity (H’)* of bacterial community in experimental soils

based on T-RFLP and DGGE analysis. Based on Rojas (2013)

Treatments T-RFLP T-RFLP DGGE

(MspI) (HhaI)
Control 3.4 ± 0.03 b 3.4 ± 0.04 c 3.0 ± 0.03 c
Lime 3.6 ± 0.10 a 3.8 ± 0.03 b 3.2 ± 0.04 b
Compost 3.6 ± 0.30 a 3.7 ± 0.09 a 3.5 ± 0.09 a
Lime+compost 3.6 ± 0.06 a 3.6 ± 0.10 a 3.6 ± 0.09 a
* Shannon index (H’) was calculated as follows: H’=-Σpiln(pi)=−Σ(ni/N)ln(ni/N), where pi is the
relative abundance of a given T-RFLP peak, ni is the intensity of band i as judged by its peak
height and N is the sum of all peak heights in a given DGGE profile.
Values given for T-RFLP are the means of five replicates ± standard deviation and those for DGGE are
the means of three replicates ± standard deviation. Values of the same letter in a column are not
significantly different at p<0.05.

Table 8. Pearson correlation coefficients between richness/diversity of bacterial

community and soil properties of experimental soils

Variable Richness (number of ribotypes) Shannon indices of diversity

T-RFLP T-RFLP DGGE T-RFLP T-RFLP DGGE
(MspI) (HhaI) (MspI) (HhaI)
pH ns ns ns 0.978* ns ns
OM, % ns ns ns ns ns 0.928**
pCu2+ 0.917** 0.908** ns 0.994* ns ns
Cuex,mg kg-1 ns ns ns -0.999* ns ns
*=p<0.05; **=p<0.1; ns=not significant (p>0.1); OM=organic matter. pCu2+: -log[Cu2+], where [Cu2+]
is activity of Cu2+ ion in the soil solution; a higher value of pCu2+ implies lower activity of Cu2+
ion. Cuex=exchangeable Cu.

PHYTOEXTRACTION OF SOIL COPPER BY OENOTHERA PICENSIS

Phytoextraction is a soil remediation technique that uses plants, preferably native species,
for the extraction of metals from polluted soils. This technique requires plants that are tolerant
to relatively high concentrations of metals, and also capable of absorbing and accumulating
large quantities of these elements within their aerial biomass. Ideally, such species are
hyperaccumulators, capable of accumulating over 1000 mg kg-1 of Cu, Pb, As and Zn (Baker
and Brooks, 1989).
To identify hyperaccumulator plants representative of the Chilean conditions, González
et al. (2008) carried out a survey of plant diversity in the area affected by the emissions of the
Ventanas smelter (92-872 mg kg-1 of total soil Cu) and in the area near a smelter slug pile
(533-2790 mg kg-1 of total soil Cu). Copper concentrations in shoots of identified plants were
determined. The species with the highest accumulation of copper (614 mg kg-1) was

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558 Alexander Neaman, Rosanna Ginocchio and Carolina Yáñez

Oenothera picensis (Fragrant Evening Primrose). However, no hyperaccumulator species

were found, i.e., species showing more than 1000 mg Cu kg-1 dry tissues. However, the
population of O. picensis was considered as a good candidate for phytoremediation initiatives
in Chile, because it is an easy to propagate perennial herb with high biomass production.
As we mentioned above, the success of the phytoextraction process is based on the
capacity of the selected plant species to extract the metal of interest from the soil and
translocate it to its aerial biomass (Chen et al., 2003). Metal bioavailability in a given soil (the
fraction which is available to be absorbed by an organism) (National Research Council, 2003)
is also an important factor, as it often happens that high total concentrations of such metals
are found in a soil, and yet, the bioavailable fraction is low. Under such conditions, plants do
not absorb much metal, even when they have the capacity for increased accumulation (Ernst,
1996). In this situation, chelates can be used to increase metal bioavailability in soil. In a
laboratory study, González et al. (2011) evaluated the effects of the biodegradable chelate
MGDA (methylglycinediacetic acid) on Cu extraction by O. picensis and on leaching of Cu
through the soil profile. They used an ex situ experiment with soil columns of varying
heights. They found that Cu extraction by O. picensis was increased 14 times by the addition
of 6 to 10 mmol plant-1 of MGDA. Although the application of MGDA did increase
the leaching of Cu, this leaching was drastically diminished with depth, making it unlikely
to contaminate groundwater. Thus, application of MGDA may be an effective and
environmentally safe way to improve Cu extraction by O. picensis in these soils.
In another study, González et al. (2014) reported the effect of MGDA on Cu
phytoextraction by O. picensis under field conditions. In the chelate treatment, Cu
phytoextraction was 2.6 ± 2.1 mg plant-1, which is equivalent to 212 ± 171 g ha-1 year-1 at a
planting rate of 8 plants m-2. The Cu phytoextraction by O. picensis was greater than that by
sunflower (59 g ha-1) (Kolbas et al., 2011), but considerably smaller than that by Elsholtzia
splendens (550-720 g ha-1) (Jiang et al., 2004). The Cu phytoextraction by O. picensisis
expected to be improved by increasing planting density.
The time necessary for phytoextraction of metals from the soil is the drawback of the
phytoextraction technique. This time can be estimated by a simple calculation. Assuming soil
bulk density of 1200 kg m-3, total Cu concentration in the soil of 1000 mg kg-1, and metal
accumulation depth of 5 cm (Ulriksen et al., 2012), there is 600,000 g of total Cu in one
hectare of soil. Assuming hypothetical high Cu extraction rate of 1000 g ha-1 year-1, 300 years
will be required to reduce total Cu concentration in the soil by half. Although the application
of biodegradable chelates reduces the time necessary for phytoextraction of metal from the
soil, the required time is still impractically long, making the phytoextraction technique
unfeasible due to lack of Cu hyperaccumulator species in Chile and worldwide. On the other
hand, as demonstrated above, phytostabilization is a technique that could be effective for large-
scale remediation of the metal-polluted, acidic soils of the Puchuncaví Valley.

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 INDEX

Acute Toxicity, 10, 21, 25, 97, 98, 99, 100, 146, 165,
# 167, 169, 180, 181, 196, 311, 312, 314, 316, 328,
336, 499, 503, 534, 535, 538, 544
17α-ethinylestradiol (EE2), 501, 540, 543, 544, 545,
adaptation, 19, 36, 70, 124, 206, 259, 281, 312, 424,
549
488
17β-estradiol (E2), 501, 507, 540, 544, 549
Adélie penguin, 105, 107, 109, 110, 111, 112, 113,
24h-EC50, 90, 91
114, 115, 116, 117
48h-EC50, 90, 91, 92, 93, 94, 95
administrators, 380
adsorption, 59, 195, 299, 364, 459, 462, 469, 473,
A 490
adults, 27, 28, 107, 113, 140, 364, 365, 368, 385
A. fasciatus, 501, 503 adverse conditions, 489
abatement, 361 adverse effects, 3, 74, 138, 141, 151, 218, 264, 290,
abstraction, 498 311, 313, 314, 315, 317, 318, 321, 381, 404, 535,
AC50, 244, 249, 252 537, 538, 541, 542, 543, 544, 545
access, ix, 3, 151, 179, 428, 527 aerobic bacteria, 175
accident prevention, 155 aerosols, 256, 510
acclimatization, 122, 274, 281, 282 Ag, 76, 141, 145, 258, 268, 440, 512, 513, 517, 534
accommodation, 54 agencies, 89, 151, 290, 349, 381, 529
accounting, 220, 231, 372, 408 aggregation, 517, 518, 519, 531
acetaminophen, 541, 543 aggression, 18
acetone, 274, 275 aging process, 327
acetonitrile, 342 AgNPs, 513
acetylcholine, 24 agriculture, x, xii, 17, 18, 20, 24, 141, 143, 148, 164,
acetylcholinesterase, 20, 21, 24, 83, 237, 390, 394, 166, 167, 171, 172, 173, 174, 175, 179, 180, 204,
405, 406 206, 207, 218, 256, 263, 348, 364, 368, 371, 390,
acid, 4, 21, 24, 25, 86, 108, 113, 116, 117, 131, 172, 391, 392, 395, 399, 401, 406, 408, 414, 457, 459,
185, 200, 253, 254, 295, 302, 313, 365, 424, 540, 465, 526, 534
541, 558, 559, 560 Air Mercury, vii, 377, 380, 381, 384, 386, 387
Acid mine drainage, 4, 139 air pollution, 218, 417, 418, 419, 424, 426, 427, 429,
activated carbon, 459, 473 430, 431, 432, 433, 434, 438, 453, 454, 455, 456,
active bioindicator, 450 511, 534
active monitoring, 421, 425, 438, 439 air quality, 417, 418, 425, 426, 427, 429, 430, 432,
activity level, 401, 403 433, 434, 435, 437, 438, 439, 440, 441, 447, 449,
452, 454, 455, 456
air temperature, 204

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air toxics, 429 aquatic animals, v, 73

alcohols, 85 aquatic ecosystem, 2, 17, 20, 21, 22, 36, 52, 100,
algae, 53, 57, 58, 59, 60, 63, 64, 68, 69, 70, 165, 295, 135, 140, 141, 142, 143, 151, 153, 164, 168, 170,
469, 490, 497, 532, 544 171, 174, 175, 177, 178, 184, 206, 257, 286, 347,
alkalinity, 142, 241 348, 355, 358, 359, 390, 405, 457, 505, 513, 544,
alternative hypothesis, 229 545, 548
aluminium, 442 aquatic habitats, 3
amalgam, 140, 348, 384 aquatic life, 24, 25, 31, 271, 465, 475
Amazon, 139, 140, 152, 157, 158, 161, 261, 263, aquatic pollution, xvii, 35, 37, 38, 163, 199, 255, 256
268, 269, 270, 360, 361 aquatic systems, 88, 96, 139, 140, 156, 184, 251,
Amazon River, 140, 263, 268 259, 309, 310, 320
ambient air, 380 aquifers, 138, 141, 149, 151, 364, 370, 373, 374, 387
amines, 391, 518 Argentina, v, viii, x, xii, xv, xix, xxi, xxii, xxiv, xxv,
amino acids, 119, 120, 125 xxvi, 23, 25, 26, 28, 31, 32, 33, 34, 35, 105, 117,
ammonia, 10, 401 127, 139, 140, 141, 142, 144, 146, 147, 148, 150,
ammonium, 241, 393, 395, 402 152, 156, 158, 160, 161, 262, 264, 265, 267, 268,
amphibia, 138, 156, 496 269, 418, 419, 426, 429, 430, 431, 432, 433, 434,
amphibians, 4, 5, 14, 18, 171, 295 437,438, 440, 441, 444, 449, 452, 453, 454, 455,
Anadara similis, 271, 276, 279, 281, 287 456, 497, 498, 499, 500, 501, 505, 506, 508, 510,
Anadara tuberculosa, 272, 276, 278, 280, 284, 287 511, 523, 525, 526, 531, 537, 538, 539, 540, 542,
anaerobic sludge, 459, 467, 472 543, 544, 547, 548, 549
anatase, 513 arithmetic, 460
ancestors, 139 aromatic compounds, 285
ANCOVAs, 231 aromatic hydrocarbons, 38, 49, 50, 52, 53, 272, 284,
androgen, 74, 502 286, 432, 461, 465
androgenicity, 503 aromatic rings, 44
angiotensin II, 541 aromatics, 462, 482
animal husbandry, 464 arsenic, 101, 140, 141, 151, 153, 157, 159, 160, 234,
annealing, 76, 77, 78 325, 326, 331, 334, 336, 338, 388, 413, 414, 451,
ANOVA, 62, 90, 217, 224, 229, 231, 298, 300, 301, 456, 510, 551, 560
313, 395, 442, 444, 445, 446 arthropods, 96, 302
anoxia, 124, 135 aryl hydrocarbon receptor, 74
Antarctic Peninsula, vi, 105, 106, 107, 112, 114, 116 asbestos, 106, 514, 515, 534
anthropic pressures, 201, 495 ascorbic acid, 114
antibiotic, 172, 175, 539, 543, 549 Asia, 269, 538
antibody, 86 aspiration, 515
antidepressant, 543 assays, 14, 31, 35, 83, 98, 100, 122, 124, 239, 241,
antiepileptic drugs, 541 242, 243, 245, 246, 249, 250, 251, 252, 311, 340,
anti-inflammatories, 539, 541 341, 342, 392, 393, 396, 397, 401, 402, 404, 462,
anti-inflammatory drugs, 548 502, 503, 544, 555
antimony, 334 assessment, 2, 9, 14, 20, 21, 22, 32, 33, 34, 36, 38,
antioxidant, 29, 57, 59, 62, 63, 65, 67, 68, 70, 71, 52, 60, 62, 68, 71, 99, 100, 102, 103, 125, 132,
105, 106, 107, 112, 113, 114, 115, 117, 124, 134, 135, 139, 157, 160, 161, 165, 174, 175, 176, 178,
135, 419 181, 184, 200, 202, 230, 233, 234, 236, 241, 253,
Antioxidant blood status, 105 260, 265, 266, 269, 270, 281, 284, 289, 290, 291,
antisense, 76 297, 298, 302, 303, 304, 305, 306, 321, 322, 329,
appetite, 401 331, 335, 358, 361, 367, 372, 380, 381, 385, 386,
aquaculture, 20, 172, 175, 177, 203, 214, 218, 285, 418, 425, 426, 427, 430, 434, 456, 493, 495, 502,
464 505, 506, 508, 510, 511, 516, 523, 524, 529, 533,
aquaria, 3, 273 537, 538, 544, 546, 560
aquarium, 75, 76, 77, 241, 274, 349 assets, 303

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 Index 565

assimilation, 268, 269, 331 bilirubin, 114

Astyanax fasciatus, 501, 505, 507 bioaccumulation, vii, viii, ix, xii, 24, 34, 35, 60, 102,
atmosphere, 140, 142, 186, 256, 350, 372, 424, 438, 124, 125, 138, 151, 154, 160, 217, 219, 220, 224,
485, 516, 531 229, 230, 231, 233, 237, 263, 268, 270, 271, 272,
atmospheric deposition, 293, 421, 428, 470, 551 275, 277, 281, 282, 283, 284, 285, 287, 311, 322,
atomic emission spectrometry, 313 333, 335, 378, 407, 408, 409, 412, 413, 414, 428,
atrophy, 50, 83 430, 473, 478, 503, 508, 510, 519, 539, 543, 549
attractive stimulation, 250 bioassays, vii, 10, 20, 22, 23, 24, 25, 26, 27, 28, 29,
Austria, x, xii, xiv, xvii, 237, 339, 340, 341, 343, 30, 31, 33, 75, 76, 77, 99, 119, 121, 122, 154,
344, 361, 388 163, 175, 219, 224, 227, 228, 230, 231, 232, 274,
authorities, 138, 303, 378, 386, 390, 509, 520, 521, 291, 297, 304, 306, 309, 310, 311, 318, 320, 321,
522, 523, 529 322, 327, 334, 335, 343, 345, 346, 462, 473, 493,
authority, 522 500, 508
automobiles, 523 bioavailability, 24, 52, 59, 68, 101, 146, 183, 184,
average daily dose, 366, 369, 370, 374, 380 186, 187, 194, 195, 196, 197, 198, 199, 200, 228,
avian, 113, 116 254, 256, 260, 266, 311, 313, 318, 320, 322, 323,
avoidance, vi, 2, 3, 4, 5, 6, 7, 8, 9, 10, 16, 17, 165, 324, 326, 331, 333, 335, 337, 338, 419, 484, 490,
239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 507, 512, 513, 518, 519, 532, 538, 556, 558, 559,
249, 250, 251, 252, 254, 289, 295, 297, 298, 302, 560
304, 305, 337, 500, 504 biochemistry, 19
awareness, 386, 509, 525, 529 Bioconcentration, vii, 138, 151, 231, 259, 271, 272,
Azinphos-methyl, 23, 24, 25, 27, 28, 29, 30, 31, 32, 274, 280, 281, 282, 283, 285, 310, 313, 316, 319,
33, 34, 35, 500, 505 331, 332, 495, 544
bioconcentration factors (BCFs), 259, 271, 272, 280,
281, 283, 285, 310, 316, 317, 319, 321, 544
B biodegradability, 471, 476, 539
biodegradation, 476
bacteria, 59, 89, 120, 172, 213, 257, 258, 268, 295,
biodiversity, ix, xi, 5, 6, 13, 19, 58, 138, 151, 158,
297, 298, 339, 340, 342, 345, 346, 408, 457, 459,
161, 170, 171, 203, 213, 238, 254, 260, 290, 302,
462, 468, 469, 471, 473, 483, 487, 488, 490, 491,
305, 311, 321, 345, 347, 348, 351, 356, 357, 358,
503, 513
359, 361, 364, 429, 432, 495, 496, 506, 538, 545
Bahía Blanca, 144, 147, 158, 160, 262, 265, 266,
Biofilm, viii, 255, 258, 259, 260, 269, 469, 481, 482,
267, 269, 497
483, 484, 485, 486, 487, 488, 489, 491, 492
bait lamina test, 289, 295, 298, 302
biogeography, 358, 559
ban, 227, 257
bioindicators, viii, xii, 21, 26, 34, 51, 84, 98, 100,
banks, 39, 121, 125, 203, 348, 351
298, 323, 389, 417, 418, 419, 420, 426, 428, 429,
barriers, 7, 488, 489, 511, 523
430, 431, 432, 433, 434, 437, 438, 453, 454, 504,
base, 58, 97, 131, 191, 196, 418, 428, 517
555, 556
basic research, 260
biological behavior, 535
Bayesian methods, 224
biological markers, 25, 34
behavioral change, 25
biological processes, 119, 464
behaviors, 386, 501
biological responses, 25, 36, 259, 290
Belgium, x, xii, xix, 339, 340, 341, 343, 344, 346
biological systems, 25, 526
benefits, 203, 355, 511, 528
biomagnification, 138, 151, 159, 232, 259, 268, 281,
benthic diatoms, 28
378, 414, 495, 510, 519
Benthic Macroinvertebrates, 96, 177
biomarkers, v, vi, ix, xii, 13, 14, 15, 17, 18, 19, 20,
benzene, 220, 232, 420, 460, 464, 465
21, 22, 23, 24, 25, 26, 30, 31, 32, 33, 34, 35, 38,
benzo(a)pyrene, 341
52, 59, 60, 73, 74, 75, 78, 83, 84, 85, 86, 106,
B-esterases, 33, 506
107, 112, 115, 116, 119, 125, 128, 131, 132, 133,
BHC, 171, 460, 464
134, 135, 157, 163, 165, 168, 169, 175, 180, 217,
bias, xi, 394, 520
219, 224,228, 230, 231, 234, 235, 236, 256, 259,
bile, 498, 505

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566 Index

266, 389, 390, 404, 405, 406, 439, 450, 494, 495, 312, 317, 318, 322, 323, 324, 353, 356, 418, 419,
498, 502, 503, 507, 543, 547 432, 434, 452, 481, 483, 484, 489, 491, 492, 497,
biomass, 7, 70, 153, 155, 251, 286, 295, 297, 301, 498, 499, 500, 501, 505, 510, 511, 523, 525, 526,
302, 310, 311, 312, 316, 318, 320, 333, 488, 491, 527, 530, 537, 538, 539, 540, 541, 542, 543, 545,
532, 534, 535, 553, 554, 555, 557, 558 546, 548, 549
biomolecules, 58 breakdown, 181, 295, 301, 304, 545
biomonitoring, ix, 26, 31, 36, 54, 58, 59, 60, 61, 62, breeding, 28, 31, 113, 116
68, 71, 98, 120, 121, 134, 135, 153, 219, 237, brominated flame retardants, 499
238, 272, 273, 346, 392, 394, 402, 404, 406, 421, brown algae, 58, 59, 60, 63, 64, 68, 69, 70
424, 427, 429, 431, 433, 434, 437, 438, 439, 440, Brown Seaweeds, v, 57, 58, 59, 60
452, 453, 454, 494, 503 Brownian motion, 517
biomonitors, 34, 100, 134, 272, 287, 417, 418, 419, bulk materials, 510, 511
420, 421, 425, 429, 430, 432, 433, 438, 439, 452, burn, 377
453, 455, 456 buyers, 378
Biomphalaria straminea, 23, 26, 27, 30, 32, 36, 505
bioremediation, 306, 355
biosphere, xi, 270, 349
C
biosynthesis, 150, 424
C. dubia, 87, 89, 90, 91, 92, 93, 95, 173
biotechnology, 60, 458, 528
C. silvestrii, 87, 88, 89, 91, 92, 93, 95
biotic, ix, x, 7, 120, 170, 199, 330, 337, 348, 355,
Ca2+, 330, 331, 518
427, 438
cabinets, 404
biotic factor, 121
cacao, 168
birds, 107, 112, 113, 116, 117, 138, 171, 178, 226,
cadmium, 9, 33, 38, 54, 69, 98, 99, 101, 112, 119,
259, 295, 350, 395, 461, 477
120, 131, 132, 133, 134, 135, 143, 160, 183, 184,
bisphenol, 499, 540
185, 194, 196, 199, 265, 310, 311, 323, 326, 336,
bivalves, vi, 16, 18, 34, 38, 41, 50, 54, 117, 119, 121,
413, 414, 461, 491, 500, 506, 507
124, 125, 127, 128, 130, 131, 132, 133, 226, 231,
caffeine, 499, 540, 541, 542
267, 268, 272, 273, 282, 283, 286
Caged fish, 15, 502
Black Sea region, 454
calcium, 69, 99, 102
blood, 16, 52, 105, 106, 113, 115, 323, 352, 364,
calibration, 93, 339, 341, 442
372, 394, 401, 512, 514, 543, 547
CAM, 144
boat, 257
Cambrian, 348
body size, 39, 393
campaigns, 386
body weight, 42, 372, 380, 384, 394, 396, 410, 465
canals, 170
Bolivia, 139, 141, 142, 155, 428, 429, 430, 497, 501
cancer, 257, 500
bonding, 197
candida, xv, 300, 302, 304, 336, 337, 340
bones, 159, 364, 372, 373, 501
candidates, 18, 68, 100
Bosnia, 28, 31, 32
Canomaculina consors (Nyl.) Elix& Hale, 439
brain, 16, 85, 86, 116, 169, 385, 394, 406, 408, 512,
capacity building, 214, 355, 525, 526, 528
514, 533, 544
carbamazepine, 499, 540, 541, 542, 544
brain activity, 394
carbon atoms, 420, 514
branching, 31
carbon monoxide, 419
Brazil, vi, vii, x, xii, xiii, xiv, xv, xvi, xvii, xviii, xxi,
carbon nanotubes, 509, 512, 514, 530, 533
xxii, xxiii, xxiv, xxv, xxvi, xxvii, xxviii, 34, 87,
carbon tetrachloride, 461
89, 95, 96, 97, 98, 99, 100, 101, 102, 124, 125,
carbonization, 515, 532, 534, 535
128, 139, 140, 141, 142, 144, 145, 146, 149, 150,
carboxylesterases, 27, 32
152, 153, 154, 155, 156, 157, 158, 159, 160, 183,
carcinogen, 384
184, 185, 186, 189, 190, 191, 194, 195, 197, 198,
Carcinogenetic, 500
199, 200, 217, 218, 219, 220, 227, 228, 232, 233,
Caribbean, 18, 21, 122, 124, 127, 128, 137, 138, 139,
234, 235, 236, 237, 238, 262, 263, 266, 267, 270,
143, 151, 156, 157, 159, 161, 163, 166, 168, 170,
289, 291, 293, 294, 296, 303, 304, 305, 309, 310,
177, 181, 494, 525, 530, 531

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 Index 567

Caribbean countries, 525 Chile, x, xii, xiii, xv, xvi, xx, xxvii, 28, 35, 37, 38,
carob, 203 42, 46, 48, 50, 51, 52, 53, 57, 58, 60, 61, 62, 66,
cascades, 120 67, 68, 69, 70, 139, 140, 141, 150, 154, 157, 161,
case study, xii, 98, 100, 101, 183, 198, 253, 266, 205, 262, 267, 325, 326, 327, 328, 329, 331, 332,
285, 289, 304, 305, 380, 384, 387, 453, 493 334, 335, 338, 433, 499, 500, 502, 505, 511, 524,
catalase (CAT), 14, 15, 17, 20, 29, 57, 59, 61, 63, 65, 525, 530, 537, 538, 540, 546, 548, 551, 558, 560
67, 68, 105, 107, 109, 112, 113, 115, 134, 165, Chilina gibbosa, 23, 28, 29, 30, 32, 33, 500, 505
169, 172 China, 54, 539
catalysis, 113 Chironomus xanthus, 87, 89, 90, 94, 97, 98, 99, 101
catalyst, 513, 515, 530 chitosan, 472
catastrophes, 201 chlorinated hydrocarbons, 153
catchments, 181 chloroform, 3
categorization, 426, 514 chlorophyll, 59, 70, 165, 177, 253, 419, 431, 439,
category b, 514 453
category d, 498 cholesterol, 540
catfish, 98, 140, 234, 547 cholinesterase, 14, 20, 21, 22, 27, 30, 32, 33, 34,
cattle, 293, 391, 392, 394, 396, 401, 404, 468, 496 165, 168, 180, 235, 394, 400, 405, 406, 505
causality, 19, 33 Choromytilus chorus, 37, 38, 40, 42, 43, 44, 45, 46,
CCA, 142, 224 47, 48, 53, 54
cDNA, 76 chromatography, 166, 235, 274, 286, 342, 423, 460,
CECs, viii, 509, 510, 519, 520, 521, 522, 523, 528 461, 547
cell culture, 548 chromium, 11, 33, 99, 100, 131, 236, 237, 253, 310,
cell cycle, 515 323, 346, 461, 468, 472, 478
cell death, 60, 515 Chronic, 6, 10, 21, 32, 33, 55, 75, 76, 77, 95, 97, 99,
cell membranes, 511 101, 102, 140, 146, 165, 167, 168, 169, 180, 254,
cell metabolism, 321 264, 293, 297, 306, 320, 346, 419, 434, 462, 500,
cellular homeostasis, 67 503, 505, 507, 508, 529, 538, 539, 543, 544, 555
Census, 179, 454 Cichlasoma dimerus, 501, 506, 507
Central American Institute for Studies on Toxic cilia, 49
Substances (IRET), xviii, xix, xxii, xxiii, xxvii, circulation, 311
13, 163, 164, 165, 166, 174, 175, 176, 177 cities, 58, 201, 204, 208, 213, 214, 387, 427, 458,
central nervous system, 132 459, 496, 497, 508
CeO2, 512, 513, 535 classes, 67, 75, 217, 218, 220, 225, 232, 315, 318
ceramic, 535 classification, 180, 203, 228, 234, 306, 426, 554
ceria, 509, 513 clay minerals, 488
Ceriodaphnia cornuta, 87, 89, 92, 93, 98, 101 clean air, 348
Ceriodaphnia dubia, 88, 99, 101, 503 cleaning, 149, 422, 551
cerium, 533 cleanup, 434
certificate, 442 climate, x, 88, 146, 151, 159, 167, 203, 206, 218,
chain transfer, 259 263, 265, 440, 493, 495, 496, 504, 507, 508, 534
challenges, xii, 254, 268, 530 Climate Change, 146, 174, 206, 254, 263, 265, 493,
charge density, 517 495, 496, 504, 508, 534
chelates, 558 climates, 218, 265
chemical characteristics, 183, 185, 327, 554 closure, 293, 348
chemical degradation, 328 clothing, 403
chemical properties, x, 299, 306, 524 CMC, 549
chemical reactions, 515 Cnesterodon decemmaculatus, 501, 508
child development, 354 CO2, 108, 534
children, 323, 353, 354, 365, 368, 372, 377, 380, coal, 139, 430, 459, 470, 471
381, 384, 385, 410, 525 coastal ecosystems, 58, 208, 218
coastal region, 50, 198, 204, 236, 322, 490, 540

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568 Index

coastal sediments, vi, 58, 200, 239, 241, 487 497, 498, 499, 503, 518, 537, 538, 540, 541, 542,
coastal system, 183, 184, 234, 237, 488, 489 543, 544
coastal zone, vi, ix, 126, 127, 129, 143, 183, 201, comprehension, 185, 482, 488, 489
202, 203, 460, 488, 489, 541, 548 computer, xxix
coatings, 482, 491, 519 computing, 237, 375
cocaine, 499, 541, 545, 546, 548 concentration ratios, 487
cocoon, 331, 332, 555, 556 conceptual model, 267, 289, 290, 294
coding, 65 concordance, 390
coefficient of variation, 94, 339, 342, 343, 344, 522 condensation, 146
coffee, 164, 535 Condensed Phases, 517
collaboration, xii, 174, 218, 452, 504 conditioning, 342
Colombia, x, xii, xiv, xxv, xxvi, 18, 35, 37, 139, 141, conductivity, 2, 90, 189, 194, 261, 264, 333, 393,
142, 145, 149, 150, 152, 271, 272, 273, 274, 356, 460, 463, 519
358, 359, 361, 375, 385, 387, 426, 430, 433, 434, conference, 20
464, 499, 525, 537, 539, 541, 547 confinement, 121, 122
colonization, 7, 139, 165, 232, 240, 245, 249, 250, conflict, 139
378, 427, 428, 561 confounding variables, 229
combined effect, 184, 199 Congress, 176, 177, 178, 454, 456, 526, 530
combustion, 149, 264 conjugated dienes, 419
commerce, 157 conjugation, 15
commercial, 75, 76, 77, 84, 121, 125, 211, 255, 256, connectivity, 233, 356, 505
257, 265, 282, 346, 361, 390, 412, 414, 464, 500, consensus, 138, 349, 523
507, 513, 527, 528, 533, 540, 547, 560 conservation, xii, 86, 116, 156, 168, 174, 218, 235,
communication, 153 260, 262, 263, 265, 270, 348, 356, 358, 359, 361,
communities, ix, 2, 5, 33, 58, 59, 97, 125, 134, 138, 551
140, 163, 165, 167, 170, 172, 175, 176, 177, 197, consolidation, x
202, 219, 230, 232, 234, 253, 254, 255, 256, 257, constituents, xi, 97, 519
259, 260, 262, 264, 268, 270, 295, 297, 299, 300, Constitution, 354
302, 306, 334, 347, 348, 349, 350, 353, 354, 355, constructed wetlands, 459, 461, 470
358, 377, 379, 380, 381, 382, 384, 385, 386, 389, construction, 204, 209, 210, 263, 266, 291, 449
390, 395, 401, 403, 417, 425, 426, 427, 428, 429, consulting, 355
430, 432, 435, 460, 488, 497, 508, 510, 513, 530, consumers, 282, 283, 499, 511, 513, 529, 545
538, 556, 557, 559, 560 consumption, 15, 41, 46, 47, 50, 121, 139, 140, 150,
compaction, 335 153, 168, 229, 282, 283, 310, 356, 368, 369, 372,
comparative analysis, 413 373, 374, 378, 394, 407, 408, 410, 412, 422, 423,
competitiveness, 6 465, 487, 539, 543, 545
complement, 4, 91, 345 containers, 106, 365, 523
complementary DNA, 132 contaminated food, 350, 363
complexity, 7, 14, 58, 60, 174, 184, 427, 493, 495 contaminated sites, 172, 231, 290, 294, 298, 300,
compliance, 497 301, 302, 303, 304, 306, 326, 488, 552
composites, 526 contaminated soil, 286, 294, 298, 299, 302, 307, 323,
composition, 166, 183, 249, 295, 297, 301, 302, 373, 325, 327, 328, 329, 332, 333, 335, 336, 337, 339,
425, 427, 439, 440, 442, 447, 454, 455, 482, 484, 340, 346, 552, 559, 560
510, 512, 513, 517, 535, 556 contaminated water, 151, 364, 471
compost, 553, 554, 555, 556, 557, 559, 560, 561 contamination gradient, 3, 4, 9, 54, 239, 252, 504
compounds, 2, 18, 23, 24, 25, 38, 42, 43, 48, 49, 50, Contamination of aquifers, 138, 149
52, 59, 66, 68, 73, 74, 75, 83, 84, 86, 96, 102, Continental, 208
141, 142, 146, 153, 167, 172, 183, 196, 235, 257, contingency, 203, 204
271, 272, 273, 274, 275, 277, 281, 282, 283, 285, control condition, 67
320, 328, 340, 390, 413, 420, 421, 422, 424, 425, control-charts, 89
432, 451, 459, 468, 471, 473, 482, 486, 488, 495, controversial, 125

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 Index 569

convention, 230 Cyanobacteria, 96, 138, 149, 150, 152, 153, 154,
Convention on Biological Diversity, 356 156, 159, 160, 161, 418, 497
cooperation, 139, 214, 357, 526 Cyanobacterial Blooms, 154, 497, 506
copper, v, vii, 3, 4, 5, 6, 7, 8, 9, 10, 11, 53, 57, 58, Cyanobacterial toxins, 138, 149, 160
59, 68, 69, 70, 71, 100, 101, 119, 122, 130, 132, cycles, 28, 76, 77, 78, 119, 125, 130, 200, 290, 298,
133, 141, 143, 154, 155, 184, 198, 252, 253, 256, 476, 481, 482, 487, 488, 489, 512
265, 310, 311, 322, 323, 325, 326, 328, 330, 331, Cylindrospermosin, 150
334, 335, 336, 337, 338, 413, 432, 440, 451, 461, cyst, 340, 346
490, 551, 553, 557, 560 cysteine, 119, 120, 125
coral reefs, 143 cytochrome, 14, 58, 70, 134, 165, 514, 534
corn, 363, 364, 365, 366, 367, 368, 369, 370, 371, cytochrome P450, 14, 165, 534
372, 373, 374 cytoplasm, 50
correlation, 18, 37, 42, 47, 49, 113, 127, 187, 191, cytotoxic agents, 419
195, 217, 224, 231, 327, 340, 344, 345, 354, 402, cytotoxicity, 513, 526, 535
418, 424, 427, 482, 484, 485, 486, 489, 557
correlation analysis, 187, 231, 344, 484, 486
correlation coefficient, 49, 557
D
correlations, 190, 191, 195, 196, 300, 301, 342, 401,
D. similis, 87, 88, 89, 90, 91, 95
402
dairy industry, 467, 469
cortex, 120, 132, 133
damages, 6, 310, 321
cosmetics, 506, 514, 523, 524, 527, 529
danger, 372, 378
cost, 68, 88, 117, 121, 149, 175, 292, 297, 300, 302,
Daphnia laevis, 87, 89, 91, 92, 98, 101
303, 346, 404, 438, 468, 552
data analysis, 90, 99, 298
Costa Rica, vi, x, xii, xviii, xix, xx, xxii, xxiii, xxv,
data collection, 374
xxvi, xxvii, 13, 14, 16, 17, 18, 19, 20, 21, 22, 142,
data set, 380
143, 159, 163, 164, 165, 166, 167, 172, 173, 174,
database, 88, 124, 164, 173, 179, 219, 261, 356, 361,
175, 176, 177, 178, 179, 180, 181, 284, 432, 433,
382, 496, 506
509, 511, 519, 520, 522, 523, 525, 528, 530
DCI, xvi, xvii, xviii, xxii, xxviii, 1, 239, 252, 407
cotton, 181
DDT, 43, 53, 106, 142, 167, 171, 233, 238, 402, 460
Council of Ministers, 271, 273, 284, 303, 475
decay, 301, 442
covering, 209, 218, 263, 523
decision makers, x, 386
crabs, 53, 233, 265, 540
decision-making process, 290
Croatia, 284
decomposition, 290, 298, 302, 304, 336, 468
crocodile, 15, 144, 145, 153, 159
decontamination, 310, 513, 559
crops, 18, 25, 141, 143, 166, 167, 170, 171, 172,
defects, 353
174, 206, 207, 329, 330, 364, 365, 367, 370, 373,
defence, 57, 59, 67
378, 389, 390, 464, 496, 497, 507
defense mechanisms, 256
cross-fertilization, 27
deficiencies, 50, 143, 329, 372, 552, 560
crude oil, 8, 143, 159, 464, 476
deficit, 213, 468
crust, 256, 257, 324, 408
deforestation, xi, 493
crystalline, 487, 513, 514, 517
degradation, xi, 50, 138, 143, 170, 206, 207, 236,
CSA, 200
272, 346, 355, 364, 419, 431, 453, 468, 523, 538,
Cuba, x, xii, xix, 141, 145, 146, 148, 154, 155, 160,
551, 552
271, 511, 525
degraded area, 355
cultivation, 168, 274, 285, 463, 464, 483, 553
DEHP, 85, 86
cultural heritage, 203
Delta, 262, 263, 266, 267, 348
culture, x, 38, 61, 86, 241, 242, 340, 484, 495, 543
denaturation, 76, 77, 78
cure, xx, 255
Dendrocygna autumnalis, 389, 392, 400, 406
cuticle, 418
denitrification, 467, 469
cyanide, 139, 140
Denmark, 388
Department of Energy, 286

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570 Index

Department of Health and Human Services, 387 218, 221, 228, 230, 233, 237, 239, 240, 242, 243,
depolarization, 69 244, 245, 249, 251, 252, 257, 259, 267, 268, 272,
deposition, 157, 185, 218, 294, 296, 430, 434, 438, 273, 276, 327, 337, 360, 372, 418, 425, 430, 432,
452, 453, 513, 516, 517, 518, 519 459, 474, 535, 541, 546, 559
deposits, 139, 149, 296, 298, 299, 378, 438, 440, diuretic, 541
450, 451, 453 diversification, 426
depression, 95, 397, 399 diversity, xii, 58, 69, 119, 134, 137, 138, 165, 167,
depth, 39, 76, 124, 185, 186, 191, 193, 197, 365, 168, 169, 170, 234, 260, 264, 266, 269, 290, 295,
393, 483, 484, 485, 486, 487, 558 297, 299, 301, 302, 333, 350, 351, 430, 454, 488,
deregulation, 161 528, 538, 556, 557, 559
derivatives, 24, 141, 257, 272, 348, 532 DLVO theory, 517, 518
desorption, 198 DNA, 70, 75, 115, 120, 124, 131, 133, 161, 220,
destruction, 204, 205, 209, 210, 211, 351 511, 515, 542, 543
detectable, 464 DNA damage, 161, 220, 543
detection, 17, 25, 43, 46, 59, 86, 150, 154, 169, 187, DOI, 21, 175, 178, 179, 180
235, 257, 258, 260, 284, 313, 314, 316, 319, 380, dominance, 259
386, 390, 420, 423, 426, 431, 453, 456, 464, 469, Dominican Republic, 525
507, 539, 541 dosage, 315
detergents, 257, 459 dose-response relationship, 120, 316, 493
detoxification, 113, 120, 131, 151, 257, 259, 402 down-regulation, 515
developed countries, 14 draft, 528
developing countries, 538, 545 drainage, 4, 141, 169, 170, 185, 207, 214, 262, 265,
Developmental, 121, 126, 257, 364, 408, 493, 498, 390, 393, 483, 501
500, 501, 505 dream, 357
deviation, 91, 92, 409, 411, 443, 522, 556, 557 dredged sediment, vii, 185, 309, 310, 311, 312, 313,
dialysis, 57, 62, 152 314, 315, 316, 317, 318, 319, 320, 321, 322
diamonds, 316, 349, 357 Dredging activities, vi, 183, 184, 196
diet, 33, 41, 53, 113, 464 drinking water, 141, 148, 150, 151, 174, 366, 368,
diffusion, 196, 201, 492 372, 497, 498, 499, 508, 523, 540, 545, 548
digestion, 37, 38, 187, 200, 260, 434 drought, 27, 201, 202, 206, 207, 504
dioxin, 22, 180 drug delivery, 514
direct measure, 333, 556 drugs, 499, 505, 510, 523, 539, 541, 543, 545, 546,
directives, 533 547, 548, 549
disaster, 284 drugs of abuse, 499, 505, 546
discharges, x, 4, 10, 129, 137, 138, 143, 151, 160, drying, 365, 422, 484
203, 207, 212, 239, 242, 463, 468, 497, 498, 503, dumping, 211
504, 544 dusts, 450
disclosure, 527, 529 dyeing, 466
discrimination, 260, 327 dykes, 496
diseases, 51, 206 dynamism, 143
dispersion, 5, 28, 139, 205, 277, 350, 385, 427, 450,
451, 515
displacement, 3, 201, 204, 240, 247, 249, 250, 251,
E
496
early warning, 13, 14, 19, 26, 31, 59, 214, 253, 386,
dissolved organic matter (DOM), 303, 304, 515, 517,
506
518, 529
earthquakes, 148, 205
dissolved oxygen, 2, 10, 121, 149, 185, 259, 266,
earthworms, vii, 253, 295, 297, 298, 304, 305, 306,
274, 393, 462, 463
309, 310, 312, 313, 314, 316, 319, 320, 321, 322,
distilled water, 312, 342
323, 327, 331, 334, 335, 336, 337, 552, 555, 556,
distribution, ix, 6, 7, 9, 10, 18, 34, 49, 51, 88, 102,
560
113, 116, 121, 122, 149, 155, 195, 196, 199, 208,
EC ratio, 437, 443, 444, 445, 447, 448, 449, 450, 451

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 Index 571

ecological information, 293, 297 election, 453

ecological processes, 266, 290 electrical conductivity, 185, 190, 329, 332, 333, 337
ecological relevance, 2, 19, 32, 38, 51, 251, 334, 559 electricity, 206
ecological restoration, 552 electrolyte, 513
ecological risk, ix, xi, 6, 7, 20, 52, 75, 98, 139, 143, electron, 39, 58, 59, 332, 460, 487, 515
160, 165, 184, 199, 255, 259, 260, 261, 262, 265, electrophoresis, 15, 76, 556
289, 290, 291, 293, 303, 304, 305, 306, 307, 320, ELISA, 15, 503
326, 375, 493, 494, 507, 508 elongation, 337
ecological roles, 488 elucidation, 174, 423
ecology, x, 2, 9, 33, 36, 53, 116, 166, 232, 234, 236, emergency, 155, 204
240, 303, 358, 360, 425, 427, 505, 559 Emerging Contaminants, viii, ix, 156, 493, 494, 498,
economic activity, 140, 207, 365, 371 499, 504, 505, 508, 510, 541, 548
economic development, 355 Emerging hydrophobic organic pollutants, 499
economic indicator, 367 emission, 187, 342, 384, 423, 429, 431, 434, 439,
economic losses, 201, 202 452, 456
economic resources, 408 emitters, 214
ecoregion, 203, 493, 504 encapsulation, 299, 523
Ecoregions, 203, 495, 496, 506, 528 encoding, 64, 67, 70
ecosystem, ix, 2, 3, 5, 6, 7, 21, 36, 37, 38, 50, 51, 57, endangered, 349, 496
59, 68, 88, 135, 163, 167, 170, 172, 174, 185, endangered species, 349, 496
201, 202, 203, 210, 215, 237, 238, 251, 265, 269, endemic chironomid, 96
273, 285, 287, 290, 295, 299, 303, 334, 355, 356, endocrine disrupting chemicals (EDCs), 74, 75, 85,
357, 390, 405, 420, 438, 457, 462, 463, 464, 473, 86, 257, 540
482, 483, 491, 492, 494, 496, 497, 504, 507, 513, endocrine disruptors, xii, 14, 15, 21, 73, 74, 84, 85,
546, 552 86, 143, 320, 493, 494, 498, 499, 501, 502, 503,
ecotoxicity, 33, 98, 100, 156, 188, 258, 290, 298, 504, 507, 508, 541, 547, 548
310, 311, 320, 326, 346, 493, 494, 495, 507, 511, endocrine system, 83, 498, 540, 543, 544
532, 533, 542 energy, 37, 38, 42, 47, 49, 50, 51, 53, 83, 124, 127,
ECs, 520 128, 157, 160, 161, 199, 203, 303, 348, 357, 454,
Ecuador, vii, viii, ix, x, xii, xv, xvi, xvii, xviii, xxi, 511, 513
xxii, xxiii, xxv, xxvii, xxviii, 1, 8, 141, 142, 174, enforcement, 457
239, 241, 243, 252, 273, 377, 378, 379, 384, 385, engineering, x, 510
387, 388, 407, 408, 409, 410, 412, 414, 438, 453, England, 60, 61, 307, 418
525 ENM, 510, 511, 512, 514, 515, 516, 517, 519, 521,
EDCs, 74, 75, 85, 257, 540 522, 523, 524, 528
edema, 513 ENSO, 203, 214
education, 99, 214, 395, 403, 545 environmental change, 38, 418, 431, 506
educational programs, 386 environmental characteristics, 437, 439, 452
Effects Assessment, 495, 499, 502 environmental classification factor (ECF), 417, 426,
effluents, xii, 4, 10, 69, 16, 22, 86, 98, 106, 146, 154, 428
161, 166, 167, 172, 242, 265, 311, 346, 457, 459, environmental conditions, 15, 59, 60, 61, 62, 121,
462, 466, 467, 468, 469, 470, 471, 472, 497, 500, 122, 133, 140, 234, 243, 260, 261, 290, 303, 332,
501, 502, 503, 504, 505, 523, 539, 540, 541, 544, 489, 496, 511, 520, 534
549 environmental contamination, 21, 284, 327, 356,
egg, 27, 28, 29, 171, 503 392, 406
Eisenia fetida, 304, 322, 325, 326, 331, 332, 334, environmental control, 268, 321
335, 336, 337, 560 environmental degradation, 177
El Murciélago beach, 239, 241, 242, 249, 250, 251 environmental effects, 165, 328, 359, 539
El Niño, 201, 202, 203, 204, 209, 213, 215 environmental factors, 125, 130, 133, 401
El Salvador, 70, 141, 142, 156, 157, 212, 213, 525, environmental impact, xi, 21, 58, 70, 174, 180, 218,
548 229, 235, 351, 355, 356, 391, 401, 404, 516, 546

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572 Index

environmental issues, 148, 152, 156, 525, 538 ERA, 165, 289, 290, 291, 293, 294, 300, 301, 303,
environmental management, 22, 260, 262, 355 494, 495, 541, 545
environmental movement, 161 erosion, 10, 141, 201, 202, 209, 210, 211, 213, 214,
environmental organizations, x 216, 256, 290, 294, 299, 401, 552
environmental pollutants, 18, 115, 419, 501, 546 erythrocytes, 105, 107, 110, 111
environmental protection, 174, 511 ester, 25, 142, 161
Environmental Protection Act, 454 estriol, 540
Environmental Protection Agency, 43, 74, 102, 146, estrogen, 73, 74, 75, 78, 79, 80, 82, 83, 84, 85, 86,
161, 181, 200, 271, 272, 286, 287, 306, 346, 361, 257, 266, 268, 498, 502, 503, 506, 543
363, 364, 366, 372, 375, 388, 422, 434, 508, 529, estrogenic genes, 73, 78
531, 545, 561 estrogenicity, 501, 503, 505
environmental quality, ix, 2, 95, 132, 213, 236, 241, estrone (E1), 499, 540, 549
253, 267, 271, 273, 286, 323, 366, 367, 368, 374, Estuaries, vi, 54, 151, 217, 218, 219, 220, 222, 228,
437, 438, 452, 457 229, 230, 232, 233, 234, 235, 236, 237, 255, 256,
environmental quality baselines, 437, 452 257, 258, 259, 260, 261, 262, 264, 265, 266, 267,
environmental regulations, 497 268
environmental risk, vi, vii, ix, x, 2, 35, 143, 148, 183, estuarine environments, 259
198, 200, 201, 203, 208, 255, 289, 292, 299, 305, estuarine gradients, 228
363, 365, 366, 368, 370, 371, 373, 374, 388, 537, estuarine systems, 217, 218, 221, 224, 227, 228, 232,
538, 541, 544 233, 258, 262, 265
environmental risk assessment (ERA), vii, ix, x, 2, etals, 256, 407
35, 165, 289, 290, 291, 292, 293, 294, 299, 300, ethanol, 75, 108
301, 303, 305, 366, 368, 370, 377, 494, 495, 497, ethyl acetate, 108
531, 537, 541, 545 eucalyptus, 496
environmental services, 348 eukaryotic, 514, 532
environmental stimuli, 240 eukaryotic cell, 514
environmental stress, xii, 34, 52, 59, 73, 120, 326, Europe, xi, 88, 267, 291, 376, 388, 511, 524, 527,
438, 482 534, 538, 540
environmental sustainability, 157, 348, 494 European Commission, 178, 409, 410, 411, 412, 413,
environmental technology, 511, 528 511, 523, 530
environmental threats, 203 European Community, 404
environmental variables, 260 European market, 284, 535
enzymatic activity, 50, 232, 295, 298 European Union, 22, 175, 282, 286, 499, 511, 524,
enzyme, 15, 22, 24, 30, 34, 59, 105, 107, 108, 109, 533, 534
110, 112, 113, 114, 134, 169, 172, 372, 402, 403, eutrophication, 138, 142, 149, 151, 152, 155, 160,
432, 543, 556 240, 254, 310, 458, 459, 463, 497
enzyme-linked immunosorbent assay, 15 evaporation, 384
EPA, 24, 25, 26, 35, 48, 52, 74, 85, 161, 173, 187, evidence, 18, 31, 59, 138, 154, 217, 218, 224, 225,
272, 274, 286, 287, 306, 341, 346, 361, 363, 364, 233, 234, 263, 264, 289, 290, 292, 303, 327, 333,
366, 372, 375, 380, 381, 382, 384, 385, 388, 422, 356, 384, 386, 409, 488, 518, 523, 525, 556
423, 434, 552, 561 evolution, 19, 69, 116, 131, 322, 348, 488
EPC, 475 EXAFS, 490
epidemiology, 388 excitation, 342, 423
epinephrine, 117 exclusion, 59, 61, 62, 63, 68, 70, 519
epithelial cells, 49, 513 excretion, 37, 38, 113, 124, 282, 331, 337, 482, 535,
epithelium, 53 539
EPS, 99, 165, 178, 344, 346, 482, 487, 488 execution, 438
equilibrium, 385 executive branch, 529
equipment, 76, 77, 78, 106, 257, 274, 313, 378, 511, exercise, 339, 340, 341, 346, 375
524 exopolysaccharides, 490
exotic species, 88, 96

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 Index 573

experimental condition, 51, 53, 115 465, 493, 495, 496, 497, 498, 499, 500, 501, 502,
experimental designs, 75, 217, 218, 219, 229, 502 503, 504, 506, 507, 508, 514, 540, 543, 544, 546,
expertise, 354 547, 548, 549
exploitation, 134, 151, 201, 208, 209, 348, 349, 354, fisheries, 203, 207, 209, 214, 218, 257, 264
440, 449, 452, 454, 459, 468, 510 fishing, 38, 125, 202, 207, 237, 251, 265, 310, 392,
exports, 141 408, 414
Exposure Assessment, 34, 366, 497, 502, 524 fitness, 50, 59, 106, 165
extinction, 218 flame, 235, 366
extraction, 39, 70, 140, 143, 149, 159, 166, 176, 187, flexibility, 467
195, 196, 203, 208, 274, 293, 323, 327, 347, 348, Floating Plants, 497
349, 350, 354, 355, 380, 388, 408, 422, 425, 430, flocculation, 459, 462
438, 457, 460, 461, 465, 484, 487, 496, 557, 558 flooding, 24, 201, 202, 204, 205, 206, 365, 482, 484,
extreme poverty, 348 487, 488, 504
exudate, 560 flora, 293, 304, 335, 349, 431, 453, 496
flotation, 478
flour, 472
F fluctuations, 203, 443
fluid, 133, 492, 517
factories, 265, 459
fluorescence, 342, 423
false negative, 292
fluoxetine, 543, 546
false positive, 292, 344
Food and Drug Administration (FDA), 541, 549
families, 21, 180, 352, 365, 427
food chain, 153, 295, 298, 347, 350, 352, 356, 364,
farmers, 206, 364, 365, 370, 373, 374, 390, 391, 392,
372, 378, 464, 498, 523
394, 395, 401, 402, 403, 404, 406
food industry, 457
farmland, 204
food intake, 368
farms, 172, 180, 203
food production, 348, 349
fat, 283, 464
food products, 529
fauna, 28, 215, 235, 266, 283, 298, 300, 301, 302,
food safety, 214, 523, 534
303, 311, 321, 323, 349, 359, 360, 489, 496, 559
food security, 285
fear, 529
food web, 26, 140, 203, 254, 257, 272, 295, 386,
federal government, 386
408, 465, 503
Federal Register, 375
force, 128
fertility, 122, 298, 353, 373
forced exposure, 1, 2, 3, 4, 5, 6, 7, 239
fertilization, 501, 506, 542
forebrain, 394
fertilizers, 143, 401
forest fire, 204
fetal development, 74
formation, 21, 70, 107, 108, 117, 120, 140, 149, 150,
fiber, 349, 515, 530
187, 206, 290, 331, 424, 464, 489, 515, 517, 518,
fibroblasts, 515
525
filters, 41, 499, 504, 541
formula, 41, 187, 244, 341
filtration, 53, 121
fouling, 70, 256
financial, 51, 198, 330, 489, 526
Fracking, 148, 149
financial resources, 526
fractures, 450
financial support, 51, 198, 489
Frameworks for Exposure, 502
fish, 3, 4, 6, 9, 10, 14, 15, 16, 17, 18, 20, 21, 22, 33,
France, 257, 305, 336, 418, 419, 524
38, 52, 73, 75, 77, 79, 81, 82, 83, 84, 86, 90, 102,
freedom, 230
107, 121, 133, 140, 141, 149, 151, 152, 153, 156,
freshwater, ix, x, xi, 4, 9, 10, 14, 23, 24, 26, 28, 31,
157, 161, 165, 168, 169, 172, 180, 205, 213, 226,
32, 33, 34, 35, 60, 71, 95, 98, 99, 100, 102, 133,
229, 230, 231, 232, 235, 236, 237, 238, 254,
137, 138, 142, 146, 149, 150, 151, 152, 156, 157,
256,259, 260, 264, 268, 323, 347, 350, 351, 353,
160, 163, 165, 170, 174, 229, 253, 256, 263, 287,
356, 357, 358, 359, 360, 361, 378, 389, 390, 393,
297, 341, 360, 408, 462, 493, 494, 495, 496, 500,
394, 396, 397, 398, 399, 400, 401, 402, 405, 406,
407, 408, 409, 410, 411, 413, 414, 460, 461, 464,

Complimentary Contributor Copy

574 Index

503, 504, 505, 506, 507, 508, 537, 539, 544, 547, glutathione peroxidase (GPx), 105, 107, 109, 110,
548 113, 114, 115, 118
Freshwater ecosystems, vi, ix, 98, 137, 138, 151, glutathione S-transferase (GST), 14, 15, 16, 17, 21,
163, 165, 493, 494, 504, 508, 544 29, 83, 105, 107, 109, 110, 113, 114, 115, 117,
freshwater species, 14, 494 165, 168, 169, 172, 231, 235, 260, 390, 394, 398,
frost, 204 399, 402, 405
fruits, 349 Glyphosate, 142, 168, 170, 389, 391, 460, 464, 497,
FTIR, 490 498, 503, 507, 508
fullerenes, 509, 512, 514, 532, 533, 534, 535 GnRH, 501, 507
function values, 30 gold, vii, 53, 139, 140, 141, 144, 155, 156, 157, 161,
funding, 214, 229, 231, 233, 283, 528 269, 322, 347, 348, 349, 350, 354, 356, 357, 358,
fungi, 258, 286, 418, 420, 532 361, 377, 378, 379, 380, 384, 385, 386, 387, 388,
408, 452, 496, 511, 532, 546
gonads, 76, 77, 78, 79, 80, 81, 82, 84, 86, 127, 503,
G 507, 543
goods and services, 218, 496
Gambusia affinis, 499, 544
governance, 525, 529
Gambusia yucatana, 75, 86, 389, 392, 396, 397, 398,
governments, 212, 356
399, 400
GPA, 257, 270
gametogenesis, 127
graduate students, x
Garimpos, 139
grain size, 183, 187, 191, 195, 228, 240
gastropods, v, vi, 23, 24, 26, 28, 31, 32, 33, 34, 36,
grants, 32
38, 54, 134, 239, 240, 241, 253, 257, 505
granulometry, 241
GDP, 202, 206
graph, 108, 246
gel, 15, 422, 434, 556
graphite, 461
gene expression, v, 14, 58, 73, 74, 75, 76, 77, 78, 83,
grass, 77, 303, 349
85, 86, 120, 131, 506, 513, 543, 548
grasslands, 349, 496
gene promoter, 120
gravity, 367, 368, 370, 371
genetic diversity, 302
grazing, 69, 207, 496
genetics, 560
Greece, 346
genome, 60, 69, 75
green alga, 179, 418, 544
genomics, 74
grids, 426
Gentoo penguin, 105, 107, 109, 110, 113, 114, 115
gross domestic product, 202
genus, 17, 96, 98, 100, 401, 501, 505
groundwater, xi, 34, 141, 148, 166, 167, 204, 207,
geochemical data, 450, 451, 453
295, 297, 363, 371, 372, 373, 558
geography, 506
grouping, 240, 297
geology, 354
growth, 6, 7, 9, 27, 31, 37, 38, 42, 47, 50, 51, 52, 53,
Germany, 22, 181, 314, 323, 549
54, 55, 58, 59, 60, 61, 62, 74, 83, 85, 121, 122,
germination, 165, 295
124, 139, 153, 165, 168, 169, 171, 206, 251, 293,
gestation, 372
295, 302, 304, 305, 321, 328, 335, 340, 341, 342,
GHG, 158
343, 344, 345, 349, 354, 404, 426, 427, 432, 469,
gill, 6, 51, 235, 398, 399, 402, 547
482, 500, 505, 523, 535, 538, 539, 542, 552, 554
GIS, 405
growth factor, 85
gland, 115, 124, 135, 235
growth hormone, 85
global climate change, 174, 254
growth rate, 60, 61, 121, 124, 139, 427
global demand, 139
Guanabara Bay, 159, 184, 197, 220, 227, 309, 310,
globalization, 146
311, 314, 315, 316, 318, 319, 320, 321, 322, 323,
glucose, 467, 468, 472, 483
324
glutathione, 14, 15, 29, 57, 59, 61, 64, 65, 66, 67, 70,
Guatemala, 18, 141, 142, 146, 148, 156, 161, 525
83, 105, 107, 117, 122, 129, 132, 165, 231, 235,
Guiana Shield, 347, 348, 356, 357
257, 260, 390, 405
guidance, 161, 303, 335, 524, 526

Complimentary Contributor Copy

 Index 575

guidelines, 24, 35, 39, 117, 188, 193, 198, 232, 234, herbicides, 9, 142, 167, 168, 170, 171, 179, 181, 390,
284, 291, 305, 310, 376, 465, 475, 504, 524, 527, 391, 497, 498
538 hermaphrodite, 28
Gulf of Mexico, 125, 143, 159, 390, 405 heterogeneity, 228, 229, 297, 300, 348, 422
Guyana, 139, 145, 149, 156, 262, 351, 356, 359 hexachlorobenzene, 167
hexane, 422
highlands, 202, 204, 205, 453
H highways, 420
histogram, 382
H. curvispina, 500
histology, 257
habitat, xi, 2, 6, 7, 8, 9, 58, 70, 71, 74, 148, 165, 170,
historical data, 409
218, 240, 252, 253, 273, 293, 294, 295, 297, 298,
history, x, 61, 63, 66, 67, 68, 121, 130, 227, 325,
299, 302, 390, 432
326, 348, 359, 430, 491
habitat selection, 2, 7, 9, 240, 253
homeostasis, 74, 124, 131, 235, 498
habitats, ix, x, 5, 7, 26, 27, 31, 60, 131, 203, 218,
homes, 385
234, 300, 347
homogeneity, 62
hair, 140, 157, 500
Honduras, 18, 141, 142
half-life, 25, 364, 372
Hong Kong, 36, 54, 134
halogens, 502
hormone, 74, 257, 501, 545
HAP, 430
hormones, 74, 75, 83, 117, 141, 320, 499, 542
harbors, 257, 264, 265, 408
host, 28, 541
hardness, 90, 393
hotel, 143
hardwood forest, 335
hotspots, 503
harmful effects, 174, 494, 515
House, 117
harmonization, 527
housing, 349
harvesting, 373
human actions, 214
Hawaii, 414
human activity, 112, 115, 209
hazard index, 363, 365, 366, 369, 370, 371, 372
human body, 366, 538
hazardous substances, 284
human health, vii, xii, 2, 85, 121, 140, 151, 156, 176,
hazardous waste, 523
179, 213, 284, 287, 313, 315, 317, 318, 326, 347,
hazards, 20, 172, 175, 177, 494, 495, 523
348, 352, 355, 357, 359, 361, 363, 364, 371, 372,
headache, 401, 403
377, 379, 381, 385, 386, 387, 390, 408, 410, 419,
health and environmental effects, 85
430, 453, 495, 497, 524, 529
health effects, 157, 158, 347, 381, 513, 514
human resources, 214
health problems, 207, 347
human rights, 355
health risks, 378, 512
humidity, 372, 422
health status, 413, 503
hunting, 106, 496
heart rate, 114, 116
Hyalella azteca, 88, 98, 102, 165, 178, 498
heat shock protein, 67
Hyalella curvispina, 500, 506
heavy metals, xii, 26, 38, 84, 106, 112, 116, 121,
hydrazine, 108
122, 125, 132, 135, 155, 157, 160, 184, 198, 199,
hydrocarbons, vii, xii, 2, 5, 37, 38, 39, 42, 44, 45, 46,
206, 266, 269, 284, 287, 311, 314, 320, 322, 323,
47, 48, 49, 50, 51, 52, 53, 54, 73, 82, 84, 106,
334, 336, 347, 364, 365, 371, 372, 373, 378, 408,
141, 143, 146, 147, 148, 153, 157, 158, 160, 220,
413, 417, 419, 420, 429, 431, 433, 438, 453, 454,
225, 238, 263, 264, 267, 269, 271, 272, 283, 284,
456, 457, 461, 462, 465, 470, 473, 474, 497, 546,
285, 286, 287, 310, 311, 323, 339, 340, 343, 417,
559
419, 420, 430, 431, 432, 433, 434, 459, 460, 461,
heavy oil, 468
462, 464, 465, 466, 469, 470, 471, 479, 538
height, 205, 312, 428, 483, 514, 557
hydrochar, 514, 515
hemodialysis, 149, 156
hydroelectric power, 348
hemoglobin, 113
hydrogen, 58, 64, 108, 124, 393, 543
hepatocytes, 84
hydrogen peroxide, 58, 64, 108, 124, 543
herbicide, 100, 464, 497, 503, 508

Complimentary Contributor Copy

576 Index

hydrogen sulfide, 393 inductor, 120, 122

hydrolysis, 24, 50 Industrial contamination, 116
hydrothermal activity, 451 industrial emissions, 257
hyperplasia, 6 industrial revolution, 532
hypothesis, 229, 230, 231, 232, 236, 481, 487 industrial wastes, 146, 196, 310, 490
hypothesis test, 230, 236 industrialization, xi, 49, 143, 262, 432
hypoxia, 406 industries, x, xi, 38, 88, 129, 138, 139, 140, 141,
143, 146, 148, 174, 204, 207, 211, 218, 256, 258,
260, 311, 408, 427, 431, 453, 459, 466, 467, 468,
I 469, 471, 472, 477, 506, 526 529
inequality, 348
IAP, 417, 424, 425, 426, 427, 428
information exchange, 525, 526, 529
ibuprofen, 498, 540, 541, 542, 543
infrastructure, 143, 211, 214, 528
Iceland, 53
ingest, 310, 350
ideal, 26, 121, 174, 255, 259, 273, 423
ingestion, 41, 47, 140, 294, 365, 372, 408, 465, 503,
identification, 7, 13, 32, 59, 154, 269, 274, 302, 423,
513
430, 506, 524, 556
ingredients, 167, 390, 523
identity, 427
inhibition, 16, 17, 20, 21, 24, 27, 29, 30, 33, 34, 59,
illumination, 312
70, 165, 168, 169, 172, 180, 305, 329, 333, 339,
immobilization, 483, 516, 519, 552, 554, 555, 560
340, 341, 342, 343, 344, 345, 390, 397, 398, 399,
immune activation, 117
400, 402, 403, 404, 515, 542
immune response, 6, 11, 33, 505, 543
inhibitor, 30, 76, 120
immune system, 30
injury, 59
immunity, 496
inoculation, 543
immunoglobulin, 114
insecticide, 9, 25, 32, 171, 257, 390, 394, 403
immunosuppression, 543
insects, 9, 349, 421
Immunotoxic, 500, 543
insertion, 527
impact assessment, 155, 265
institutions, 202, 211, 227, 233, 354, 418, 528
improvements, 299
instrumental neutron activation analysis, 437, 439,
impurities, 515
442
in transition, 256, 265
insulin, 74, 85
in utero, 408
integration, 5, 6, 151, 204, 390, 525
in vitro, 83, 84, 86, 115, 178, 493, 502, 515, 526,
integrators, 438
530
integrity, xi, 163, 218, 419
in vivo, 70, 75, 76, 77, 83, 402, 464, 502, 513, 515,
intellectual property, 528
526
Inter-American Development Bank, 156, 157, 524
inbreeding, 7
interface, 199, 227, 463, 473, 476, 482, 488, 489
incidence, 250, 352, 354
interference, 422, 465, 515
income, 365, 367, 371, 373
inter-laboratory calibration, vii, 339
incubation time, 41
International Atomic Energy Agency, 176
Index of Atmospheric Purity, 417, 424
international standards, 167, 412, 527
India, 420, 434, 539
internship, 20
indirect effect, 299
interphase, 258
individuals, 1, 7, 39, 42, 43, 44, 45, 47, 49, 50, 51,
intersex, 502, 503, 505
52, 59, 60, 74, 110, 113, 124, 125, 169, 173, 262,
intervention, 313, 315
319, 372, 374, 377, 380, 390, 394, 407, 409, 425,
intoxication, 150, 152, 355
428
invertebrates, 9, 32, 34, 35, 50, 88, 89, 95, 98, 102,
Indonesia, 357
120, 121, 124, 130, 131, 134, 168, 181, 256, 259,
inducer, 128
266, 267, 287, 289, 290, 294, 295, 297, 298, 300,
induction, 16, 17, 26, 57, 58, 59, 69, 70, 83, 84, 85,
301, 303, 322, 324, 340, 503, 534
120, 122, 125, 131, 133, 168, 172, 257, 266, 399,
investments, 139
402

Complimentary Contributor Copy

 Index 577

ionization, 154 landfills, 213, 214, 523

ions, 120, 330, 331, 448, 517 landings, 121, 125, 205, 414
IPCS, 52 landscape, x, 3, 7, 22, 148, 157, 203, 251, 253, 349,
iron, 101, 186, 194, 197, 199, 332, 348, 408, 515, 495
530 language barrier, 539
irradiation, 442 larvae, 4, 10, 11, 21, 90, 93, 98, 175, 180, 498
irrigation, 141, 174, 203, 204, 206, 207, 214, 348, larval development, 227, 542
364, 365, 366, 367, 368, 370, 371, 373, 374, 505, larval stages, 27
519, 541, 548 laws, 349, 354
islands, 159 LC-MS, 152, 154
isomers, 43, 423 leaching, 24, 340, 541, 547, 558
isotope, 413, 414 lead accumulation, vii, 33, 363, 366, 367, 368, 371,
Israel, 430 373, 374, 506
issues, ix, xii, 34, 85, 148, 151, 174, 185, 204, 236, lead content, 368
253, 323, 356, 425, 520, 523, 526, 531, 533 leakage, 148
Italy, x, xii, 55, 286, 377, 419, 420, 429, 431, 432, leaks, 139
434, 453 legislation, 25, 140, 188, 191, 193, 195, 232, 260,
306, 310, 311, 313, 314, 315, 326, 355, 455, 459,
524, 525, 526, 527, 529, 538
J legume, 303
Lemna, 165, 173, 178, 461, 462, 473, 474, 476, 477,
Japan, 85, 409, 524, 525
497, 506, 542, 547
Jenynsia multidentata, 498, 544
lending, 518
Jordan, 327, 335
Lepidoptera, 35
juveniles, 9, 27, 122, 130, 131, 506, 507, 555
lesions, 219
liberation, 259, 523
K Lichen bags, 439, 441
Lichens, viii, 417, 418, 419, 420, 421, 423, 424, 425,
K+, 132 426, 427, 428, 429, 430, 431, 432, 433, 434, 435,
keratinocytes, 532 437, 438, 439, 440, 441, 443, 444, 447, 448, 449,
kerosene, 471 450, 452, 453, 454, 455, 456
kidney, 116, 120, 133, 513, 514, 543, 548 life cycle, 31, 88, 90, 97, 119, 125, 127, 213, 532,
kinetics, 268, 337, 468, 500, 501, 517 554
Korea, 525 life expectancy, 539
life sciences, 2
lifetime, 206, 357, 509, 517, 519
L
ligand, 199, 330, 337
La Niña, 201, 203, 205, 214, 215 light, 2, 106, 112, 116, 117, 121, 241, 345, 447, 468,
labeling, 525, 535 476, 497, 511
Laboratory for Ecotoxicological Studies (ECOTOX), linear model, 108, 231, 425
15, 152, 166, 173, 174, 181, 267, 414, 429, 507, Lines of Evidence, 217, 218, 230, 289, 290, 292
547, 548 lipid peroxidation (LPO), 14, 15, 16, 17, 64, 66, 68,
laboratory studies, 240 105, 107, 108, 110, 113, 114, 116, 122, 131, 165,
laboratory tests, 18, 89, 122, 168, 171, 184, 328 169, 172, 514
lactate dehydrogenase, 390, 406 lipid peroxides, 117
lactation, 372 lipids, 114, 419, 482
Lake Maracaibo, viii, 457, 458, 459, 460, 461, 462, liquefied natural gas, 209
463, 464, 465, 468, 471, 473, 476, 477, 478 liquid chromatography, 154, 546, 548
lakes, 28, 97, 101, 142, 152, 156, 157, 158, 349, 498, liquid phase, 500
541 liquids, 148, 209
laminar, 3

Complimentary Contributor Copy

578 Index

liver, 75, 78, 79, 83, 84, 86, 116, 117, 149, 152, 169, marine environment, 52, 116, 125, 134, 264, 270,
232, 405, 507, 513, 514, 543 271, 281, 282, 284, 286, 406, 541
livestock, 167, 177, 204, 206, 459 marine fish, 157, 412, 414
local authorities, 519 marine species, 35
local conditions, 499 marketing, 378
local government, 212, 214 marsh, 286
logging, 496 Maryland, 52, 286, 406
logistics, 231 mass, 27, 29, 39, 120, 133, 154, 241, 274, 286, 330,
longevity, 101, 438, 539 356, 422, 423, 461, 546, 547, 548
low risk, 206, 363, 374 mass spectrometry, 154, 274, 286, 423, 461, 546,
low temperatures, 421 547, 548
lower lip, 68 materials, 106, 143, 185, 188, 199, 328, 335, 375,
LSD, 446 421, 422, 438, 442, 510, 511, 513, 514, 515, 516,
lubricating oil, 471 517, 518, 519, 524, 525, 526, 559
luciferase, 503 matrix, 42, 47, 297, 323, 422, 423
LUMEX, 380 matrixes, 498
luminescence, 295 matter, 129, 139, 196, 197, 250, 299, 318, 320, 327,
Luo, 133, 559 334, 348, 373, 387, 435, 438, 449, 469, 482, 484,
Luxemburg, 433 488, 491, 506, 556, 559
lysozyme, 133 maximum allowed level, 363, 369, 370, 372, 373,
374
Maximum quantity for weekly consumption, 412
M measured environmental concentration (MEC), 542
measurement, 2, 15, 25, 37, 38, 39, 41, 42, 51, 53,
macroalgae, 58, 59, 70
108, 117, 219, 228, 235, 295, 297, 331, 338, 340,
macronutrients, 468
346, 408, 438, 442, 524
magnesium, 59
meat, 204, 394, 468
magnitude, 4, 44, 188, 205, 260, 283, 424, 447, 450,
MEC, 542
518
media, 149, 217, 219, 226, 227, 376, 483, 491, 516,
majority, 3, 59, 139, 169, 170, 213, 289, 390, 541,
517, 519
554
median, 10, 99, 254, 265, 309, 314, 461, 464, 497,
malondialdehyde, 419, 439, 455
539
mammals, 120, 138, 213, 226, 259, 295, 350, 408
medical, 355, 512, 528
management, x, xii, 21, 101, 102, 117, 156, 160,
medicine, 510, 526, 539
163, 166, 174, 179, 180, 185, 188, 201, 202, 204,
Mediterranean, 198, 237, 303, 413, 414, 546
212, 214, 218, 235, 260, 262, 268, 269, 285, 290,
medulla, 419
298, 334, 356, 386, 390, 494, 504, 505, 507, 525,
Megacities, 496
526, 553
melatonin, 116
manganese, 186, 194, 199
melon, 170
mangrove, 153, 185, 196, 199, 203, 226, 228, 231,
melting, 139, 146, 202
233, 234, 235, 237, 263, 273, 323, 392, 395, 482,
membranes, 511, 542
483, 491, 492
memory, 408
mangrove forests, 203
mercury, vii, viii, xxviii, 7, 9, 34, 119, 131, 139, 140,
mangroves, 218, 219, 538
143, 152, 153, 154, 156, 157, 158, 159, 161, 225,
Manta, xvi, xvii, xviii, xxi, xxii, xxviii, 1, 239, 241,
235, 252, 253, 257, 269, 311, 322, 323, 347, 349,
242, 243, 407, 408, 409
350, 351, 353, 355, 356, 357, 358, 360, 361, 377,
Mantaro River, vii, 363, 364, 365, 368, 369, 371,
378, 379, 380, 381, 382, 383, 384, 385, 386, 387,
373, 374
388, 407, 408, 409, 412, 413, 414, 415, 461, 477,
mantle, 127
492
manufacturing, 378, 511, 529
metabolic responses, 57, 59, 66
manure, 334
mapping, 131, 166, 229, 506

Complimentary Contributor Copy

 Index 579

metabolism, 15, 58, 59, 60, 65, 68, 74, 124, 267, 161, 185, 195, 206, 285, 322, 323, 325, 326, 327,
464, 513 328, 329, 331, 332, 334, 338, 347, 348, 351, 352,
metabolites, 25, 43, 75, 149, 170, 171, 419, 434, 498, 354, 356, 357, 358, 359, 360, 361, 364, 371, 377,
539, 541, 545, 546, 547 378, 379, 380, 381, 382, 384, 385, 386, 387, 388,
Metagenomics, 259, 260 435, 437, 438, 439, 440, 441, 447, 449, 450, 451,
metal ions, 118, 120, 472, 552 452, 453, 454, 455, 456, 469, 496, 551, 559, 560
metal nanoparticles, 143 Ministry of Education, 8, 252
metal oxides, 513 mission, 512
metal resistance, 481, 483 Missouri, 364, 376
metal salts, 328 misuse, 19, 20, 21
Metal toxicity, 133, 138, 199, 253, 327, 328, 329, mitochondria, 58, 511
330, 333, 334, 488, 559 mixing, 239, 256
Metallothioneins, vi, 59, 70, 119, 120, 123, 124, 126, MMA, 218, 227, 236, 263, 269
127, 130, 131, 132, 133, 134, 135, 153 mobilization of metal, 184
metallurgy, 140, 293, 294 model system, 265
meter, 205, 263, 484 modelling, 375
methodology, 39, 41, 75, 90, 95, 199, 291, 341, 363, models, 59, 99, 122, 166, 200, 253, 255, 259, 262,
365, 366, 386, 420, 426, 429, 438, 439, 441, 493, 263, 302, 337, 524, 542
502 modifications, 108, 188, 419
methylation, 140 moisture, 443, 465
methylmercury, 157, 225, 256, 257, 358, 408, 413 molecular biology, 15, 19, 74
metropolitan areas, 545 molecular mass, 119
Mexico, vii, x, xii, xix, xx, xxiv, xxvi, xxviii, 18, 53, molecular structure, 498, 510, 514
73, 75, 100, 125, 137, 139, 141, 142, 143, 144, molecular weight, 49, 113, 271, 273, 277, 423
145, 146, 149, 150, 151, 152, 153, 154, 155, 158, molecules, 59, 120, 124, 130, 259, 419, 423, 514,
159, 160, 161, 178, 389, 390, 403, 404, 405, 406, 515, 518, 530, 539, 542
409, 410, 412, 413, 414, 418, 419, 434, 510, 511, mollusks, 33, 35, 133, 134, 257, 272, 285, 457, 497
524, 525, 527, 530, 531, 537, 539, 541, 544, 547, momentum, 356
548 Montenegro, 548
Mg2+, 59, 330, 331 moribundity, 5, 250, 251
mice, 149, 514, 515, 534 morphology, 420, 424, 426, 430, 515
microbial community, 172, 175, 178, 260, 293, 294, morphometric, 394
295, 298, 302, 488 mortality, 1, 2, 4, 5, 6, 8, 50, 150, 157, 168, 169,
Microcosm, 481, 482, 483 171, 172, 206, 227, 252, 271, 274, 275, 282, 309,
Microcystins, 149, 150, 152, 153, 156, 160, 161 312, 313, 316, 318, 319, 320, 340, 341, 342, 343,
microelectronics, 528 344, 345, 387, 393, 396, 401, 402, 404, 464, 496,
micronucleus, 220, 360 514, 538
micronutrients, 452, 559 mosaic, 69
microorganisms, 310, 325, 350, 471, 483, 488, 489, motor skills, 408
495, 503, 515, 552, 556 Mozambique, 412
microscope, 421 MQWC, 410, 412
microscopy, 484, 491 mRNA, 75, 76, 77, 78, 118
migration, 196, 204, 242, 298, 508 MT isoforms, 119, 124
mine soil, 559 MTI, 120
mineral resources, 354 MTs, 119, 120, 121, 122, 123, 124, 125, 126, 127,
mineralization, 293, 333, 488 128, 130, 131
miners, 58, 351, 352, 353, 354, 355, 377, 378, 379, mucus, 507
380, 381, 384, 385, 386, 388 multicellular organisms, 512
minimum inhibitory concentration, 462 multidimensional, 224, 261
mining, vii, viii, xi, xii, 58, 69, 70, 129, 137, 138, multielemental accumulation, 437, 438, 439, 444,
139, 140, 141, 143, 146, 155, 156, 157, 159, 160, 445, 451, 452

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580 Index

multiples, 252 negative effects, 28, 50, 59, 112, 114, 196, 232, 257,
multivariate analysis, 231, 301, 426 298
Multivariate approaches, 262 negative relation, 125
multiwalled carbon nanotubes, 530 neglect, ix
Municipal discharges, 137, 138, 498, 503 nematode, 513, 559
muscles, 83, 413 neonates, 341
muscular tissue, 133 nervous system, 169
mussels, 16, 17, 22, 39, 41, 42, 47, 49, 50, 51, 52, Netherlands, 21, 270, 285, 291, 304, 306, 506, 559
53, 54, 55, 228, 233, 284, 286, 402, 456, 540, 548 networking, 526
mutations, 515, 560 neurological disease, 354
Neurotoxic, 27, 29, 30, 31, 32, 33, 140, 169, 408,
500
N neurotransmitter, 24
neutral, 90, 258, 299, 556
Na+, 132
neutrophils, 543
NaCl, 90, 95, 101
New England, 414
NAFTA, 527
NGOs, 356, 528
nanofibers, 533
Nicaragua, 141, 142, 144, 145, 273
Nanomaterials, viii, xii, 509, 510, 523, 524, 525,
nickel, 11, 155, 236, 310, 334, 461
529, 530, 531, 532, 533, 534, 535
nicotine, 499
nanomedicine, 528
Nile, 83
nanometer, 510
nitrates, 170, 393, 401, 402, 497
nanometer scale, 510
nitric oxide, 543
nanoparticles, 2, 143, 256, 267, 504, 509, 512, 529,
nitrification, 295, 302, 333, 467, 468
530, 531, 532, 533, 534, 535
nitrite, 241, 395, 405
nanostructures, 523
nitrogen, 113, 141, 142, 153, 256, 295, 333, 373,
nanotechnology, 143, 256, 268, 523, 528, 526, 531,
421, 459, 466, 467, 468, 470, 497, 502, 554
533, 534, 535
nitrogen fixation, 497
nanotube, 515, 532
NM, 108, 342, 423, 510, 511, 513, 514, 516, 518,
naphthalene, 423
519, 524, 526, 527, 529
National Academy of Sciences, 453
NM cytotoxicity, 513
national parks, 349
NMs, 509, 510, 511, 512, 517, 520, 523, 524, 525,
national policy, 326
527, 528, 529
National Research Council, 25, 413, 558, 560
N-N, 470
national strategy, 527
NOAA, 282, 286
native cladocerans, 87
non-forced exposure system, 6, 7, 239
native species, 13, 16, 18, 19, 24, 26, 30, 31, 34, 88,
non-polar, 259, 518
89, 95, 96, 99, 122, 163, 166, 173, 174, 303, 457,
non-steroidal anti-inflammatory drugs, 543
499, 504, 557
North America, xi, 127, 151, 180, 265, 538
NATO, 285
NPs in the atmosphere, 516
natural assemblages, 70
NRC, 25, 34
natural catastrophes, 201
NSAIDs, 543, 544
natural compound, 103
NT, 510, 511, 525, 526, 527, 528, 529, 533
natural disasters, 201
nucleotide sequence, 124
natural disturbance, 489
nucleotides, 124
natural gas, 148, 201, 209
null hypothesis, 217, 229, 231
natural habitats, 31, 496
nutrients, 2, 143, 149, 171, 201, 202, 220, 225, 290,
natural resources, 367, 371, 437, 452, 504
295, 298, 299, 302, 327, 328, 349, 418, 459, 462,
nausea, 401, 403
463, 467, 468, 469, 497, 491, 523, 554, 560
necrosis, 419
nutrition, 560
negative consequences, 364
nutritional status, 372

Complimentary Contributor Copy

 Index 581

ornamental plants, 168

O ostracod cysts, 339
Ostracodtoxkit F assay, vii, 339, 340, 342, 343, 344,
obstacles, 149
345
oceans, 409
outsourcing, 493, 495
octopus, 205
ovaries, 84
Odontesthes bonariensis, 498, 500, 501, 505, 506,
overlap, 228
544, 548
overpopulation, 493, 504
ODS, 342
ox, 28, 487
OECD, 88, 165, 180, 297, 305, 327, 328, 331, 334,
oxidation, 114, 151, 160, 197, 259, 320, 450, 462,
336, 337, 511, 512, 524, 525, 527, 528, 531, 533
513, 539
offshoring, 493, 495
oxidation rate, 259
Oil and Natural Gas, 201, 208
oxidative damage, 57, 59, 64, 71, 105, 106, 107, 111,
oil production, 161, 202, 464, 466, 468, 469, 470,
112, 113, 114, 115, 116, 117, 135, 513, 515
471, 472
oxidative stress, 22, 28, 58, 59, 61, 62, 63, 67, 106,
oil spill, 106, 208, 218, 284, 285
114, 115, 116, 117, 122, 124, 256, 267, 513, 533,
oilseed, 560
535, 543, 544, 547
oligomers, 59, 62
oxide nanoparticles, 533
Olivella semistriata, vi, 239, 241, 245, 246, 247, 248
oxygen, 2, 41, 47, 50, 52, 59, 132, 139, 185, 290,
one dimension, 511
459, 463, 466, 467, 468, 470, 472, 502, 532
oogenesis, 85
oxygen consumption, 47, 50, 52, 132
open-pit mining, 437, 438, 452
oysters, 53, 134, 153, 154, 198, 231, 232, 237, 257,
operations, 114, 117, 328, 335, 348, 364, 378, 464,
263, 270
523
opportunities, 139, 156, 203, 530
optimization, 4, 16, 20, 290 P
ores, 141, 325, 326, 438
organic chemicals, 24, 52, 259 P. promelas, 173, 501, 503
organic compounds, 96, 142, 200, 225, 256, 283, p53, 535
286, 420, 423, 473, 513, 515, 518, 552 Pacific, 18, 122, 163, 166, 170, 172, 203, 204, 271,
organic matter, 2, 52, 140, 170, 195, 196, 197, 241, 272, 273, 274, 282, 283, 287, 407, 409, 410, 411,
243, 250, 254, 258, 259, 297, 298, 299, 304, 305, 412, 413, 414, 496
311, 314, 315, 318, 320, 327, 329, 330, 334, 335, PAHs, 37, 39, 42, 43, 44, 46, 47, 49, 50, 51, 160,
336, 373, 458, 459, 462, 468, 469, 484, 488, 491, 220, 225, 227, 232, 233, 263, 271, 272, 273, 274,
497, 511, 515, 517, 518, 519, 529, 553, 554, 556, 275, 277, 278, 279, 280, 281, 282, 283, 286, 287,
557 339, 341, 342, 343, 344, 345, 417, 419, 420, 421,
organic solvents, 282 422, 423, 424, 425, 429, 430, 498, 510, 538
organism, 5, 6, 7, 13, 25, 26, 29, 38, 50, 51, 59, 60, paints, 268, 513
73, 74, 84, 88, 90, 95, 97, 101, 121, 122, 125, palm oil, 170
128, 130, 231, 240, 260, 271, 274, 281, 290, 301, PAN, 173, 180
327, 328, 345, 366, 381, 418, 443, 542, 558 Panama, x, xii, xiv, 18, 142, 161, 339, 345, 525
Organization for Economic Cooperation and Paraguay, 139, 141, 142, 149, 160, 496, 497, 498,
Development, 305, 533 501
organize, 354 parallel, xi, 4, 66, 354, 471
Organochlorine, 37, 43, 84, 146, 153, 159, 171, 178, parasites, 51, 138
179, 181, 227, 232, 236, 361, 405, 460, 464, 498 Parmotrema austrosinense (Zahlbr.) Hale, 439
organochlorine compounds, 84, 153 participants, x, 509
organochlorine pesticides, 37, 159, 181, 227, 232, partition, 50, 258, 282
405, 460, 464 passive monitoring, 420
organs, 33, 84, 132, 149, 281, 511 pastures, 170, 171, 204, 364, 496
Orinoco, 127, 128, 261, 262, 266, 269, 270, 347, pathogens, 220
348, 351, 358, 359, 361, 496

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582 Index

pathways, 70, 73, 131, 268, 291, 296, 377, 384, 386, phosphate, 107, 108, 149, 394, 406, 459, 497
513, 515, 523, 537 phosphoenolpyruvate, 134
Patos Lagoon, 144, 261, 262, 263, 266, 267 phosphorous, 395
PCA, 224, 232, 460, 463, 519 phosphorus, 24, 168, 373, 393, 459, 466, 467, 468,
PCBs, 225, 233, 236, 238, 257, 402, 498 470, 502
PCM, 212 phosphorylation, 50, 83, 120, 534
peat, 327 photolysis, 421
peptides, 158 photosynthesis, 59, 70, 290
permeability, 59, 419 phycocyanin, 153
peroxidation, 15, 68, 129, 131 physical environment, 390
perseverance, xxix physical properties, 290, 327, 482
Persistent Organic Pollutants, 146, 155, 257, 498 physicochemical characteristics, 258, 282, 467
personal communication, 127 physico-chemical parameters, 231
Peru, vii, x, xii, xviii, xxv, 60, 139, 141, 149, 174, physicochemical properties, 329, 512, 515
202, 203, 204, 205, 206, 208, 209, 210, 212, 213, physics, 354, 510, 534
214, 215, 272, 285, 358, 363, 364, 366, 375, 376, physiological mechanisms, 18
426, 433, 438, 453, 525 physiological responses, 13, 14, 37, 38, 42, 47, 49,
perylene, 49, 273, 277, 278, 279, 280, 281, 283, 341, 50, 52, 54, 55, 106, 121, 132, 418
343, 423, 425 physiology, 69, 114, 116, 321, 417, 420
pesticides, xi, xii, 9, 13, 15, 16, 17, 18, 19, 20, 21, phytoextraction, 551, 552, 557, 558, 560
22, 23, 24, 25, 27, 31, 37, 74, 86, 89, 95, 98, 138, phytoplankton, 125, 128, 149, 153, 156, 158, 165,
141, 142, 143, 155, 156, 157, 159, 161, 163, 164, 169, 181, 270
165, 167, 168, 169, 170, 171, 172, 173, 174, 175, phytoremediation, 551, 558, 559
176, 177, 179, 180, 181, 220, 227, 233, 303, 304, phytostabilization, 551, 552, 553, 558, 559, 560
323, 389, 390, 391, 392, 395, 401, 402, 403, 404, pigmentation, 128
405, 406, 459, 460, 463, 464, 498, 500, 503, 505, Pimephales promelas, 173, 500, 501, 508
510, 523, 538, 540, 546 pipeline, 208
pests, 24, 206 plankton, 145, 158
Petrochemical contamination, 138 plant establishment, 552, 554
petroleum, 31, 49, 50, 51, 52, 54, 73, 106, 138, 157, plant growth, 295, 297, 328, 329
208, 264, 269, 272, 284, 286, 287, 311, 348, 464 plants, 27, 58, 106, 141, 146, 151, 161, 168, 173,
Petroleum, 146, 354 209, 214, 226, 294, 297, 298, 300, 304, 327, 328,
pH, 2, 9, 25, 90, 107, 108, 122, 185, 186, 188, 190, 330, 331, 333, 334, 335, 337, 338, 351, 372, 377,
193, 194, 196, 228, 241, 258, 261, 266, 274, 275, 378, 386, 418, 431, 452, 470, 494, 497, 503, 511,
297, 299, 315, 317, 318, 320, 327, 329, 332, 334, 518, 519, 523, 529, 532, 537, 539, 549, 551, 552,
336, 337, 352, 373, 393, 401, 402, 463, 468, 484, 554, 556, 557, 558, 559, 560
511, 517, 519, 553, 554, 556, 557 plasma proteins, 543
phaeophytin, 177, 419, 439 plasticity, 7, 228
pharmaceuticals, 2, 143, 498, 499, 508, 527, 529, plastics, 257
537, 538, 539, 540, 541, 542, 543, 544, 545, 546, platform, xii, 133, 134, 218
547, 548, 549 playing, 203
pharmaceuticals and personal care products (PPCPs), PM, 449, 454
viii, 499, 537, 538, 539, 540, 541, 542, 544, 545, point of origin, 202
546, 547 Poland, 420, 432
pharmacokinetics, 372 polar, xi, 202, 259, 282, 518
PHAs, 282, 340, 344, 345 policy, 262, 326, 354, 356, 385, 520, 527, 533
phenol, 117, 464, 467, 468, 469, 472, 542 politics, 260
phenolic compounds, 59, 61, 62, 65, 68, 468, 469, polyacrylamide, 15
479 polybrominated diphenyl ethers, 236, 237, 499
phenotypes, 554 polychaetes, 121, 259, 260, 267
Philadelphia, 32

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 Index 583

polychlorinated biphenyls, 38, 146, 154, 220, 237, preference, 2, 3, 7, 8, 10, 197, 242, 243, 244, 252,
285, 405, 507, 538 254, 323
polychlorinated biphenyls (PCBs), 38, 146, 154, 220, preparation, 39, 84, 202, 484, 524
225, 233, 236, 237, 238, 257, 285, 402, 405, 498, present value, 49
507, 538 preservation, x, 257, 321
polycyclic aromatic hydrocarbons (PAHs), vii, 5, 37, prevention, 151, 160, 201, 202, 313, 315, 320
39, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, principal component analysis, 460
146, 147, 148, 158, 160, 220, 225, 227, 232, 233, principles, 100, 115
263, 267, 269, 271, 272, 273, 274, 275, 277, 278, priority pollutants, viii, 34, 271, 457, 459, 460, 462
279, 280, 281, 282, 283, 284, 285, 286, 287, 323, PriProbit, 244, 314
339, 341, 342, 343, 344, 345, 417, 419, 420, 421, private sector, 202
422, 423, 424, 425, 429, 430, 431, 433, 434, 461, probability, 142, 188, 204, 229, 290, 313, 314, 315,
464, 498, 510, 538 318, 363, 364, 367, 368, 370, 494
polymerase chain reaction (PCR), 15, 75, 76, 77, 78, probe, 185, 332
85, 152, 154, 503 process, x, 7, 19, 53, 65, 95, 108, 113, 140, 149, 151,
polymerization, 57, 61, 62, 64 195, 202, 258, 281, 282, 290, 291, 292, 293, 294,
Polymesoda solida, 461, 477, 478 297, 301, 302, 331, 346, 354, 356, 378, 384, 385,
polysaccharides, 59, 482, 491 401, 421, 422, 437, 438, 452, 460, 463, 487, 515,
polyunsaturated fatty acids, 113 538, 558
ponds, 97, 212, 299, 460, 464, 471, 475, 477 producers, 58, 159, 168
pools, 330, 373 professionals, xii, 545
POPs, 146, 255, 257, 259, 260, 265, 284, 498 progesterone, 540
population, ix, xi, 3, 4, 5, 6, 9, 10, 27, 28, 37, 42, 43, project, xxix, 21, 115, 140, 175, 181, 202, 204, 212,
44, 49, 50, 58, 59, 60, 64, 69, 70, 98, 99, 107, 237, 252, 387, 427, 428, 437, 440, 441, 449, 498,
116, 121, 138, 143, 149, 151, 156, 166, 206, 207, 527, 545
212, 213, 218, 219, 230, 240, 244, 250, 253, 254, prokaryotic cell, 513, 514
259, 260, 262, 268, 334, 336, 347, 351, 356, 363, proliferation, 151, 206, 257
364, 365, 366, 367, 371, 372, 373, 374, 380, 381, proline, 120
382, 384, 386, 390, 404, 418, 457, 462, 495, 496, promoter, 120
508, 521, 523, 529, 558 propranolol, 542, 547
porous media, 492, 519 protected areas, 114, 171, 235, 356
porphyrins, 501 protection, 14, 19, 24, 25, 31, 106, 124, 132, 287,
portfolio, xv 290, 304, 326, 346, 349, 381, 465, 475, 529
Portugal, x, xii, xiii, xv, xvi, xx, xxii, xxiv, xxvii, 1, protective role, 488
7, 8, 22, 181, 197, 199, 239, 252, 254, 289, 304, protein oxidation (PO), 14, 105, 107, 108, 110, 113,
305, 389, 419, 420, 430 114, 124, 125, 127, 165, 196, 201, 232, 234, 249,
positive correlation, 113, 402 250, 252, 321, 322, 395, 438, 450, 451, 488, 514
positive feedback, 482, 488, 489 protein sequence, 124
potassium, 87, 90, 91, 92, 93, 94, 95, 96, 99, 107, protein structure, 419
108, 332, 373, 501, 542, 547 proteins, 16, 24, 59, 75, 114, 116, 117, 119, 120,
potassium dichromate, 87, 90, 91, 92, 95, 501 124, 125, 235, 257, 419, 431, 453, 482, 518, 542
potato, 168, 363, 365, 366, 367, 368, 369, 370, 372, proteomics, 70
373 prototype, 61
poverty, 373 provisional tolerable weekly intake (PTWI), 408,
precious minerals, 349 410, 412
precipitation, 128, 164, 168, 258, 259, 299, 401, 402, pruning, 469
440, 482, 552 public health, x, 24, 154, 160, 350, 352, 355, 356,
predation, 6, 51 361, 365, 408, 509, 510, 519, 520, 525, 545
predators, 6, 257, 350, 356, 408, 412, 413, 414 public interest, 528
Predicted no-effect concentrations (PNEC), 540, 542 public sector, 520
publishing, 287

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584 Index

pulp, 4, 10, 22, 496, 498, 501, 502, 503, 505 recovery processes, 378
pumps, 269 recreation, 148, 203, 209, 290
pure water, 519 recreational, 149, 212, 218, 265
purification, 39, 422, 425 recycling, 65, 214, 523
purity, 274, 427 red blood cells, 543
pyrite, 139, 184, 450, 491 red mud, 263
pyrolysis, 532 reduced glutathione (GSH), 15, 29, 57, 59, 61, 62,
63, 64, 65, 66, 68, 105, 107, 108, 109, 111, 113,
114
Q redundancy, 431, 453
reference sites, 170, 217, 219, 222, 223, 229, 231,
Q-PCR, 75, 76, 77, 78
232
qualifications, 520
reference toxicants, 87, 90, 501, 508
quality control, 103, 459
regeneration, 204, 321, 535
quality of life, 355, 511
regions of the world, 262, 407, 409, 410, 411, 524
quantification, 18, 20, 76, 77, 78, 132, 206, 274, 404,
Registry, 272, 364, 387
422, 423, 425, 528, 539, 540
regression, 40, 129, 554
quantum dots, 512
regression line, 129
quartz, 259, 312
regrowth, 560
questioning, 510
Regulated Contaminants, 497
questionnaire, 395
regulations, 67, 74, 83, 137, 159, 180, 201, 202, 212,
267, 282, 286, 326, 348, 354, 378, 405, 409, 410,
R 411, 412, 413, 459, 468, 469, 471, 497, 510, 511,
514, 515, 520, 523, 524, 526, 527, 528, 529, 533,
radiation, 421 545
radicals, 117, 118, 424 regulatory agencies, 88, 354, 356
radula, 26 regulatory framework, 14, 511, 528
rainfall, 94, 202, 204, 229, 350, 385, 391 regulatory requirements, 303
rainforest, 181 rehabilitation, ix
rare earth elements, 450, 451 relevance, 2, 19, 32, 38, 51, 197, 251, 302, 324, 327,
RBC, 459, 461, 467, 468, 469, 474, 475, 476, 479 328, 332, 334, 509, 559
RC50, 244, 249, 252 relief, 106
reactants, 517 remediation, 89, 151, 158, 184, 198, 290, 292, 299,
reactions, 10, 76, 197, 482, 488, 552 304, 306, 471, 490, 528, 551, 552, 557, 558, 561
reactive oxygen species (ROS), 58, 59, 64, 66, 68, remote sensing, 166
106, 112, 114, 513, 532 renewable energy, 203
reactivity, 326, 419, 434, 517, 518, 531 rent, 378
reagents, 342 repetitions, 343, 344
realism, 122 replication, 217, 218, 219, 222, 229, 233
reality, 348, 360, 361 representativeness, 88
receptors, 6, 73, 74, 78, 79, 80, 81, 82, 83, 85, 86, reproduction, 6, 24, 33, 38, 50, 58, 74, 83, 85, 88, 96,
166, 290, 291, 292, 294, 295, 296, 297, 313, 315, 106, 167, 168, 169, 227, 254, 289, 293, 295, 297,
317, 318, 326, 381, 382, 495, 499, 502, 503, 506, 302, 304, 321, 331, 333, 335, 336, 340, 420, 430,
511, 512, 516, 543 443, 500, 503, 538, 542, 543, 555, 556, 560
recognition, 523, 529 reproductive age, 84, 353
recolonization, vi, ix, 235, 239, 240, 241, 243, 244, reproductive cycles, 119, 130
245, 246, 247, 248, 249, 250, 251, 252, 553, 554 Reproductive Effects, 500, 501
recommendations, 20, 233, 386, 524, 527 reproductive mechanism, 125
recovery, ix, 26, 29, 34, 36, 55, 139, 140, 236, 239, reptile, 76
251, 299, 355, 378, 379, 403, 422, 457, 552 repulsion, 7, 517, 518
recovery plan, 457 requirements, 26, 50, 62, 89, 300, 378, 395, 504, 524

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 Index 585

research institutions, 357 304, 305, 306, 307, 320, 326, 328, 350, 353, 357,
researchers, ix, x, xii, xxix, 19, 121, 163, 174, 218, 358, 363, 364, 365, 366, 367, 368, 370, 371, 372,
230, 260, 265, 313, 426, 427, 459, 468, 509, 522, 373, 374, 375, 376, 377, 378, 379, 380, 381, 384,
527, 537, 538 385, 386, 387, 388, 390, 404, 407, 408, 410, 414,
reserves, 50, 148, 204, 349, 459 430, 453, 490, 493, 494, 495, 496, 499, 502, 503,
residuals, 195, 532 505, 507, 508, 511, 513, 523, 524, 526, 527, 528,
residues, 18, 21, 34, 58, 95, 140, 152, 166, 167, 168, 529, 533, 535, 537, 538, 540, 541, 542, 543, 544,
169, 170, 171, 176, 177, 178, 180, 187, 253, 287, 548, 549, 552, 558
298, 299, 320, 402, 405, 465, 494, 498, 539, 547, river basins, 347, 495
549 RNA, 22, 67, 70, 75, 76, 77, 78, 124, 131, 133, 180,
resilience, 253 542
resins, 462 room temperature, 186, 422, 484
resistance, 69, 124, 142, 155, 265, 438, 457, 473, root, 116, 165, 168, 169, 172, 178, 235, 237, 294,
481, 482, 483, 488, 489, 491, 541, 547 330, 331, 337, 349, 418, 519, 552, 560
resolution, 10, 188, 260, 423, 442 routes, 7, 124, 130, 302, 405
resource availability, 7, 252 Royal Society, 533
resources, xi, 14, 101, 137, 138, 139, 148, 151, 152, RT-PCR, 15, 75, 76, 77, 78
155, 202, 203, 205, 229, 233, 284, 285, 348, 349, rubber, 426
355, 356, 357, 496, 528, 529, 531 runoff, 17, 24, 94, 100, 172, 175, 229, 256, 258, 294,
respiration, 16, 17, 37, 38, 122, 172, 295, 297, 298, 299, 496, 528
301, 302, 487 rural areas, 212, 385
response, 3, 4, 6, 7, 9, 10, 16, 17, 18, 19, 20, 22, 25, Russia, 435, 539
28, 30, 35, 47, 50, 54, 57, 69, 71, 73, 80, 83, 84, rutile, 513
100, 105, 113, 114, 120, 121, 122, 125, 131, 167, ryegrass, 325, 326, 328, 329, 330, 331, 334, 559
176, 185, 186, 197, 231, 237, 240, 242, 245, 247,
249, 250, 251, 252, 253, 254, 256, 300, 301, 302,
329, 332, 333, 336, 344, 345, 381, 387, 388, 418,
S
421, 429, 431, 437, 438, 439, 440, 448, 449, 451,
saccharin, 541
453, 454, 455, 473, 482, 483, 485, 489, 492, 504,
safety, 121, 140, 151, 386, 524, 527, 533
505, 506, 508, 522, 529, 554, 559, 560
salinity, 39, 53, 54, 60, 88, 121, 122, 132, 133, 185,
responsiveness, 241
199, 207, 217, 219, 220, 222, 228, 229, 233, 236,
restaurants, 378
237, 241, 259, 266, 268, 275, 327, 333, 337, 373,
restoration, xii, 298, 303, 307, 355, 551, 561
392, 393, 402, 459, 484, 518
restriction fragment length polymorphis, 556
Salinity Zones, 220, 228
restrictions, 551, 552
Salmonella, 165
resuspension, 183, 184, 185, 186, 187, 188, 189,
salts, 333
190, 193, 194, 195, 196, 197, 198, 199, 200, 470,
saltwater, 173, 413
489
Sample Size, 219, 223, 229, 230, 233
revaluation, 101
samplings, 38
revegetation, 299, 306, 333, 552, 553, 559
San Salvador, 157
Rhodophyta, 70
SARA, 462
rice field, 172, 181, 391
saturated hydrocarbons, 479
Río de la Plata, 262, 264, 266, 269, 270, 501, 506,
saturation, 185, 484, 515, 517
549
scaling, 224
risk, ix, x, xi, xii, 2, 4, 7, 20, 22, 31, 35, 52, 54, 59,
scarcity, xi, 149, 439, 493, 504
75, 85, 88, 96, 97, 98, 100, 137, 138, 139, 140,
scholarship, 51
143, 146, 153, 156, 157, 158, 165, 168, 170, 177,
school, 204
179, 181, 197, 199, 201, 202, 204, 206, 208, 209,
science, 2, 85, 269, 304, 357, 516
212, 213, 214, 232, 252, 253, 255, 256, 259, 260,
scientific papers, 348, 538
261, 262, 265, 269, 272, 283, 284, 289, 290, 291,
scope, 37, 42, 51, 52, 53, 55
292, 293, 294, 296, 297, 298, 299, 300, 302, 303,

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586 Index

Scope for Growth (SFG), 37, 38, 42, 47, 48, 50, 51, shoot, 329, 330, 554, 555, 557
52, 53, 54, 55 shoreline, 265
SDS, 93, 95 shores, 58, 127
sea level, 202, 263, 270 showing, 29, 50, 93, 124, 140, 196, 209, 264, 277,
seafood, 408, 540 351, 444, 457, 458, 465, 468, 471, 473, 484, 500,
seasonal changes, 133 502, 510, 518, 544, 554, 558
seasonal factors, 385 shrimp, 4, 6, 9, 10, 11, 14, 196, 237, 459, 461, 464,
seasonal variation, 119, 121, 125, 127, 128, 130, 468, 477
131, 132, 384, 548 shrubs, 349
seasonality, 164, 238 Si3N4, 513
secretion, 74, 470 signal transduction, 58, 131
security, 513, 532 signals, 13, 19, 59, 83, 120, 345
sediment toxicity, 6, 10, 26, 32, 87, 89, 90, 97, 98, significance level, 187, 275, 366, 485
99, 200, 253, 321, 346 signs, 14, 27, 30, 33, 57, 386
Sediment Transport, 206 silica, 422, 511
sedimentation, 209, 210, 373, 469, 516, 519 silicon, 513, 515
seed, 165, 266, 295 silver, 86, 139, 256, 267, 268, 509, 512, 513, 530,
seeding, 552 534, 535
selectivity, 423 silver nanoparticles, 267, 509, 530, 534, 535
selenium, 413, 414, 415 simulation, 3
self-fertilization, 27 simulations, 367
Senate, 526, 530 SiO2, 515
sensitivity, xi, 8, 10, 13, 18, 19, 27, 32, 34, 38, 51, skin, 74
84, 87, 88, 89, 90, 91, 93, 95, 96, 100, 101, 102, slag, 294, 328
103, 170, 173, 300, 301, 302, 329, 333, 334, 340, sludge, 102, 200, 284, 322, 504, 523, 534, 561
345, 385, 406, 423, 426, 427, 438, 473, 488, 491, smoking, 395
512 snail, 7, 26, 32, 33, 34, 54, 239, 240, 241, 245, 246,
sentinel species, 18, 24, 31, 500 247, 248, 250, 251, 252, 253, 254, 500, 505
Sepetiba bay, vii, 183, 184, 185, 186, 188, 189, 190, SNS, 313
191, 195, 196, 197, 198, 199, 200, 309, 310, 311, SO42, 470
312, 313, 314, 315, 316, 317, 319, 321, 483, 484, social conflicts, 206
489, 491 social development, 202, 528
septic tank, 504 social policy, 378
sequencing, 259, 474, 475 social structure, 347, 348, 354
serine, 120 society, 151
serum, 108, 116 Society of Environmental Toxicology and Chemistry
services, 174, 203, 213, 238, 290, 334, 497, 504 (SETAC-LA), xii, 135, 337, 476, 538
settlements, 143, 355, 356, 357, 440 sodium, 87, 90, 91, 92, 93, 95, 99, 332, 501
severe stress, 50 sodium chloride, 87, 90, 91, 92, 93, 95
sewage, xii, 151, 211, 212, 218, 258, 284, 322, 468, sodium dodecyl sulfate (SDS) , 87, 90, 91, 92, 93,
469, 475, 497, 498, 508, 523, 534, 539, 540, 541, 95, 501
544, 548, 549 software, 90, 99, 244, 314, 366, 367, 380, 381, 442
sex, 17, 86, 121, 257, 394, 505, 544 soil ecotoxicology, vii, ix, 289, 290, 304, 305, 325,
sex ratio, 544 326, 338
sex steroid, 257 soil erosion, 290, 401
sexual behaviour, 544 soil invertebrates, 289, 290, 294, 297, 298, 300, 303,
sexual development, 74 322, 324, 534
Shale gas, 157 soil particles, 340, 364, 421
shallow lakes, 544 soil pollution, 345
shape, 515, 535 Soil Salinization, 207
shelter, 58 soil seed bank, 552, 553

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 Index 587

soil type, 303, 326, 337 state, 8, 35, 106, 107, 115, 118, 122, 125, 126, 130,
solid oxide fuel cells, 513 135, 149, 161, 176, 217, 219, 220, 231, 232, 285,
solid phase, 166, 176, 258, 331, 340, 341, 345, 346, 293, 309, 310, 313, 315, 317, 320, 338, 354, 389,
500 417, 428, 431, 443, 450, 463, 517, 518, 525, 539,
solid surfaces, 516 540, 541, 549
solid waste, 201, 212, 213, 214, 561 statistical power, 229, 234
solubility, 25, 281, 282, 327, 328, 332, 336, 344, statistics, 99, 118, 230, 235
517, 518, 560 steel, 38, 146, 408
solution, 76, 90, 108, 139, 186, 197, 210, 258, 274, sterile, 107, 165, 438
297, 298, 330, 331, 338, 552, 553, 554, 557 steroids, 220
solvation, 518 stimulation, 7, 84, 168, 249, 250, 499
solvents, 24, 141 stock, 75
sorption, 191, 196, 197, 198, 405, 482, 483, 484, stomach, 84
487, 488, 489, 490, 492, 552 storage, 141, 181, 207, 208, 209, 212, 213, 338, 344,
South Africa, 539 345, 378, 511, 531
South America, 28, 31, 95, 138, 139, 149, 151, 154, storms, 202
157, 159, 255, 256, 257, 260, 261, 262, 263, 264, strategic position, 527
265, 310, 348, 438, 459, 494, 495, 496, 498, 499, strategy use, 489
501, 504, 506, 507, 508, 544, 545, 548 stratification, 473
South Korea, 524 stress, xi, 18, 51, 52, 53, 54, 58, 59, 60, 64, 70, 74,
Soxhlet extractor, 422 83, 106, 115, 116, 117, 133, 169, 228, 235, 259,
Spain, x, xii, xvi, 1, 133, 156, 198, 239, 285, 419, 267, 270, 328, 430, 438, 439, 449, 513, 533, 542,
420, 559 554
Spatial and Temporal Replication, 218, 219, 229 stress factors, 554
spatial distribution, 6, 7, 221, 239, 240, 251, 252, stress response, 74
268, 337, 546 stressors, 10, 19, 125, 493, 494, 495, 496, 498
speciation, 70, 200, 256, 310, 323, 331, 337, 338, structure, ix, 2, 52, 53, 120, 124, 149, 163, 165, 170,
561 175, 178, 224, 230, 232, 240, 251, 254, 257, 273,
species richness, 295, 303, 554 285, 289, 290, 295, 298, 301, 302, 418, 473, 513,
specific surface, 510, 519 530, 556
specific tax, 301 subgroups, 381
specifications, 88 sublethal effects, 2, 6, 24, 54, 95, 96, 267, 297, 334,
spectrophotometric method, 107, 117 340, 493, 500
spectroscopy, 241, 434 subsidy, 161
spending, 38 subsistence, 406
sperm, 83, 501, 506, 547 substitution, 59
spindle, 515 substrate, 15, 27, 28, 30, 34, 113, 127, 165, 298, 341,
spine, 500 421, 426, 443, 482, 513, 515
spleen, 513, 514 subsurface flow, 470
Spring, 42, 46, 533, 535 succession, 299
Sri Lanka, 410 sucrose, 467, 469
stability, 71, 88, 186, 197, 254, 271, 281, 299, 477, sugarcane, 17, 171, 473
492, 512, 518, 519, 534, 542 sulfate, 9, 11, 43, 87, 91, 92, 95, 171, 328, 471, 491
standard deviation, 45, 79, 80, 81, 82, 92, 93, 94, sulfur, 139, 256
123, 190, 312, 316, 382, 383, 411, 445, 553, 555, sulphur, 419, 430, 431, 439, 440, 449, 453, 455
556, 557 Sun, 260, 270, 338, 531, 534, 535
standard error, 109, 110, 111, 112, 244 Superfund, 361
standard length, 16 Superoxide dismutase (SOD), 29, 57, 58, 59, 61, 63,
standardization, 39, 95, 102 65, 67, 68, 105, 107, 109, 112, 113, 117, 260, 543
starvation, 115 supervision, 526
suppression, 514

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588 Index

surface area, 421, 473, 510, 517, 519 test species, 19, 87, 88, 94, 96, 97, 100, 173, 174,
surface layer, 258, 485, 486 253, 272, 321
surface mining, 156 testing, 10, 32, 95, 96, 98, 99, 100, 253, 269, 275,
surface properties, 511, 515 281, 283, 292, 303, 305, 306, 328, 336, 341, 346,
surfactants, 253, 257, 497 493, 495, 499, 524, 527, 529, 533
surveillance, xi, 355, 497 testosterone, 543
survival, 2, 3, 9, 11, 15, 28, 33, 50, 51, 54, 59, 90, tetrachlorodibenzo-p-dioxin, 405
101, 106, 165, 167, 253, 259, 290, 304, 311, 341, tetrad, 234
356, 402, 457, 494, 498, 500, 501, 505, 506 tetrahydrofuran, 514
survival rate, 90, 498 textiles, 257
susceptibility, 6, 10, 11, 25, 272, 433, 473, 496, 512 texture, 297, 327, 335, 373
suspensions, 514, 519 thin films, 331
sustainability, 260, 285, 322, 528, 530 Third World, 155
sustainable development, 285, 309, 348, 357, 505 threats, xi, 21, 138, 151, 178, 201, 203, 208, 236,
Sweden, 20, 22, 115, 197, 199, 420 269, 350, 356, 359, 390, 495, 496, 504, 506
swelling, 337 threats to biodiversity, 496
Switzerland, xii, 85, 304, 335, 414, 534 threonine, 120
SWNTs, 530 threshold level, 188, 372, 387
symbiosis, 418 Thunnus albacares, viii, 407, 408, 409, 411, 412,
symptoms, 360, 395, 401, 403, 404, 418 413, 414
synergistic effect, 469 tides, 473
synthesis, 74, 83, 84, 128, 133, 305, 419, 515 Tiered approach, 289, 291, 292, 502
time frame, 4, 539
time periods, 186, 397, 402, 488
T time series, 214
tin, 38
tanks, 208, 274, 281
TiO2, 512, 513, 531
target, x, 6, 24, 25, 33, 75, 84, 121, 128, 187, 270,
tissue, 6, 15, 27, 29, 30, 34, 37, 38, 39, 42, 46, 48,
377, 384, 504, 513, 539, 541, 542
49, 75, 76, 77, 78, 79, 84, 113, 116, 127, 128,
target identification, 377
145, 152, 159, 171, 172, 253, 281, 332, 353, 408,
target organs, 75, 513
409, 410, 412, 414, 460, 464, 494, 503, 511, 514,
target variables, 187
517
taxa, 171, 179, 427
titanium dioxide, 509, 531, 534, 535
taxonomy, 427
tolerance mechanisms, v, 57, 58, 60, 63, 66, 68, 70
teams, x, 418, 504
toluene, 461, 465
technical assistance, 214, 452
total costs, 310
techniques, 14, 19, 35, 140, 148, 166, 224, 232, 293,
total organic carbon, 187, 190, 195, 241, 243, 502
355, 417, 422, 423, 424, 425, 461, 498, 531
total product, 273
technology, 148, 166, 174, 208, 354, 361, 457, 459,
tourism, 114, 117, 143, 203, 209, 212, 497
473, 510, 511, 515, 519, 525, 526, 527, 528, 529,
toxic effect, 3, 4, 5, 6, 7, 24, 25, 31, 53, 154, 256,
533, 539
261, 326, 498, 507, 543
teeth, 364
toxic metals, 120, 124, 127, 128, 263, 321, 407, 489,
temperature, 2, 28, 39, 54, 71, 86, 121, 122, 124,
559
164, 170, 185, 228, 236, 237, 274, 275, 303, 312,
toxic substances, 166, 174, 364
372, 385, 393, 421, 422, 443, 471, 496, 497, 502,
toxicity tests, vi, xii, 10, 15, 32, 54, 87, 89, 90, 93,
505, 511, 519
95, 96, 97, 98, 99, 100, 167, 170, 176, 196, 234,
temporal variation, 400, 432, 540
314, 325, 326, 327, 328, 336, 346, 473, 492, 499,
tenure, 234
503, 555
terminals, 209
toxicology, 2, 9, 21, 27, 55, 74, 100, 101, 115, 137,
territory, 138
138, 166, 177, 284, 323, 405, 524, 526, 529, 538,
test procedure, 88, 346
546

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 Index 589

toxicology studies, 27
toxin, 154
U
trace elements, 132, 160, 184, 199, 234, 322, 323,
ultrasound, 199, 422
333, 335, 414, 438, 443, 456, 481, 482, 489, 555,
UNESCO, 160, 181, 262, 270, 504
558, 560
uniform, 510
trace metals, 69, 102, 134, 184, 187, 196, 199, 200,
United Kingdom, 266, 269, 287
255, 256, 260, 263, 265, 268, 269, 285, 286, 335,
United Nations, 161, 270, 356, 361, 368, 390, 414,
337, 432, 456, 482, 485, 486, 488, 491, 559
465, 496, 508, 524, 530, 534, 549
trade, 128, 203, 408
United Nations Industrial Development
trafficking, 545
Organization, 361
traits, 228, 234, 253, 300, 302
United States (USA), x, xii, 32, 49, 52, 54, 75, 77,
transcription, 84, 120, 131
88, 99, 102, 124, 134, 156, 161, 177, 200, 268,
transcripts, 75, 78, 79
286, 303, 306, 342, 346, 406, 409, 413, 419, 422,
transduction, 120
434, 460, 486, 524, 525, 527, 559, 560
transformation, 42, 515, 516, 517, 532, 544, 546
universities, 509, 527, 528, 538
transformations, 95, 298, 532
updating, 19
Transgenic Crops, 496
uranium, 101, 141, 155, 303, 451
transition metal, 120, 492
urban, ix, x, 50, 99, 113, 117, 140, 152, 157, 158,
translation, 120
167, 204, 206, 212, 218, 239, 241, 255, 256, 258,
translocation, 237, 514
260, 263, 269, 293, 385, 405, 419, 420, 426, 428,
transparency, 351, 393
429, 431, 432, 433, 434, 439, 449, 496, 505, 539,
transplant, 57, 60, 61, 63, 66, 67, 68, 70, 232, 421,
541, 545, 548
437, 439, 440, 441, 442, 443, 444, 445, 448, 449,
urea, 108, 467, 483
450, 451
uric acid (UA), xiii, 105, 107, 108, 109, 111, 113,
transport, 58, 59, 74, 121, 143, 168, 181, 196, 203,
114, 115, 116
212, 258, 299, 310, 321, 378, 452, 492, 515, 530,
uric acid levels, 111
531, 532
urine, 116, 353
treatment, xii, 53, 75, 77, 78, 90, 106, 125, 138, 141,
Uruguay, x, xii, xvii, xx, 141, 142, 145, 146, 147,
146, 151, 152, 158, 161, 211, 212, 214, 219, 224,
150, 156, 157, 159, 255, 263, 264, 266, 269, 430,
230, 231, 275, 310, 316, 338, 386, 417, 421, 422,
493, 496, 497, 498, 500, 501, 502, 504, 506, 507,
426, 457, 458, 459, 462, 467, 468, 469, 470, 471,
508, 525
473, 475, 477, 479, 482, 483, 484, 485, 486, 487,
US Department of Health and Human Services, 541,
489, 497,498, 500, 511, 515, 518, 523, 537, 539,
549
544, 545, 546, 548, 549, 556, 558
US Food and Drug Administration (US FDA), 541
triad, 234, 289, 290, 291, 292, 302, 303
UV-radiation, 484
trial, 283
tributyltin, 84, 254, 256, 266, 268, 269
triggers, 25 V
Trinidad, 88, 122
trophic state, 460, 463 valence, 518
tropical aquatic environments, 88 Valencia, 133, 419, 434
tropical forests, 138, 538 validation, 22, 73, 75, 91, 156, 243, 548
tropical species, 13, 88, 89, 95, 96 Valuation, 300
Tsunamis, 201, 205, 213, 214 vanadium, 461
Tukey Test, 62 vapor, 384, 386
turbulence, 49 variables, 122, 124, 228, 229, 233, 254, 262, 274,
Turkey, 125, 132, 454 275, 372, 393, 395, 396, 439, 461, 485, 554
turnover, 28 variations, 17, 19, 42, 83, 122, 124, 125, 127, 128,
turtle, 73, 76, 80, 85 131, 214, 281, 282, 320, 397, 401, 443, 452, 490,
495, 514, 548
varieties, 408

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590 Index

vegetables, 167, 334 Water Quality Index (WQI), 393, 395, 396, 401, 402,
vegetation, 165, 181, 289, 294, 295, 297, 298, 299, 404, 460
301, 302, 349, 351, 352, 358, 393, 395, 452, 469, water quality standards, 371
551, 552, 553, 559, 560 water resources, 138, 156, 175, 201, 202, 206, 356,
vegetative reproduction, 552 477, 504, 528
vehicles, 143, 420, 449 water supplies, 102, 150, 498
velocity, 3, 170 watershed, 21, 160, 167, 169, 175, 177, 263, 322,
Venezuela, vi, vii, viii, x, xii, xiv, xvi, xviii, xix, xx, 387, 392, 393, 459, 503, 540
xxi, xxiii, xxv, xxvii, xxviii, 119, 121, 122, 124, waterways, 170
125, 126, 127, 128, 130, 131, 132, 133, 134, 135, wavelengths, 342
139, 149, 152, 155, 266, 268, 347, 348, 351, 353, weakness, 401, 403
354, 356, 357, 358, 359, 360, 361, 417, 418, 419, wealth, 348, 357
420, 421, 426, 427, 428, 430, 431, 432, 433, 438, web, xv, 9, 253, 407, 414
457, 458, 459, 462, 463, 474, 475, 476, 477, 478, weight gain, 6
479, 506, 525 weight loss, 16, 165
Venus, 51 weight ratio, 41, 419
vertebrates, 120, 124, 260, 295, 297, 544 well-being, 305
vessels, 29, 256, 275, 281 wells, 148, 165, 207
viruses, 213 wetlands, 171, 210, 235, 392, 404, 469, 470, 471
vision, 2 wildland, 179
visual field, 240, 251 wildlife, 31, 74, 75, 78, 85, 86, 148, 161, 165, 268,
visualization, 428 326, 345, 348, 350, 360, 390, 495, 496, 503, 540
vitamin E, 113, 114 Wilson, Woodrow, 268, 512, 535
vitamins, 500 wind speed, 385
vitellogenin, 14, 15, 21, 73, 74, 75, 76, 77, 78, 79, wires, 512, 514
80, 81, 83, 84, 85, 86, 165, 257, 501, 502, 503, Wisconsin, 180
506, 507, 543 wood, 171, 336, 349
volatility, 274, 385 workers, 174, 357, 380, 385, 386, 389, 394, 513,
volatilization, 372 516, 525, 526, 529
vulnerability, xi, 2, 163, 202, 204, 253, 302, 384 working conditions, 385
workplace, 34, 403, 524
World Health Organization, 52, 85, 361, 364, 368,
W 372, 376, 388, 406, 408, 414
World Health Organization (WHO), 52, 160, 353,
Wales, 307, 433, 435
354, 361, 364, 368, 374, 385, 406, 408, 410, 412,
Washington, 10, 35, 100, 102, 156, 177, 200, 234,
413, 414, 465, 479, 510, 523, 524, 534
268, 284, 286, 287, 305, 346, 358, 361, 388, 406,
worldwide, xi, 23, 26, 60, 141, 184, 202, 378, 408,
413, 434, 474, 532, 560
410, 418, 420, 489, 513, 529, 558
waste, xi, 10, 106, 129, 139, 140, 141, 201, 203, 206,
worms, 519
208, 211, 212, 213, 311, 323, 328, 438, 472, 511,
515, 516, 523, 525, 529, 533, 537, 539, 545, 546
wastewater, viii, xii, 86, 138, 141, 144, 146, 151, X
154, 158, 161, 177, 201, 211, 212, 213, 214, 284,
457, 458, 459, 461, 462, 466, 467, 468, 469, 470, xenobiotic, v, 2, 37, 73, 75, 237
471, 472, 473, 474, 475, 477, 482, 493, 497, 498,
499, 503, 504, 506, 515, 523, 535, 537, 539, 541,
544, 545, 546,547, 548
Y
wastewater treatment plants (WWTPs), 141, 146,
yeast, 90
161, 214, 523, 537, 539, 544
yellowfin, viii, 407, 408, 409, 410, 412, 413, 414
water ecosystems, 171
yield, 330, 367, 370, 371, 373
water purification, 535

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 Index 591

zinc oxide, 515

Z ZnO, 512, 515, 517
zooplankton, 10, 52, 90, 95, 102, 145, 149, 150, 159,
zinc, 3, 10, 69, 119, 120, 132, 133, 135, 143, 154,
160, 497, 506
155, 184, 198, 215, 236, 265, 310, 311, 322, 323,
326, 334, 336, 337, 340, 346, 377, 413, 445, 474,
482, 483, 490, 491, 492, 515, 559

Complimentary Contributor Copy