INTERNATIONAL

STANDARD 10260
First edi tion
1992-07-15

Water quality - Measurement of biochemical

Parameters - Spectrometric
determination of
the chlorophyll-a concentration
iTeh STANDARD PREVIEW
(standards.iteh.ai)
Qualite de I’eau - Mesurage
spectrometrique
des paramefres
de la chlorophylle a
biochimiques - Dosage

ISO 10260:1992
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ca6047b90f0f/iso-10260-1992

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Reference number
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---. -.- ------ ISO 10260: 1992(E)
ISO 10260:1992(E)

Foreword

ISO (the International Organization for Standardization) is a worldwide

federation of national Standards bodies (ISO member bodies). The work
of preparing International Standards is normally carried out through ISO
technical committees. Esch member body interested in a subject for
which a technical committee has been established has the right to be
represented on that committee. International organizations, govern-
mental and non-governmental, in liaison with ISO, also take part in the
work. ISO collaborates closely with the International Electrotechnical
Commission (IEC) on all matters of electrotechnical standardization.

Draft International Standards adopted by the technical committees are

circulated to the member bodies for voting. Publication as an Inter-
national Standard requires approval by at least 75 % of the member
bodies casting a vote. iTeh STANDARD PREVIEW
International Standard (standards.iteh.ai)
ISO 10260 was prepared by Technical Committee
ISO/TC 147, Wafer quality, Sub-Committee SC 2, Physical, Chemical,
biochemical methods. ISO 10260:1992
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Annexes A and B of this International Standardca6047b90f0f/iso-10260-1992
are for information only.

0 ISO 1992
All righfs reserved. No part of this publication may be reproduced or utilized in any ferm
or by any means, electronie or mechanical, including photocopying and microfilm, without
permission in writing from the publisher.
International Organization for Standardization
Case Postale 56 l CH-1211 Geneve 20 l Seitzerland
Printed in Switzerland

ii
 ISO 10260:1992(E)

Introduction

Chlorophyll-a is the essential photosynthetic Pigment present in all

green plants. The chlorophyll content of a surface water is an indicator
of its trophic state. The determination of the chlorophyll-a concentration
provides information concerning the quantity and potential
photosynthetic activity of algae. The most important metabolites of
chlorophylls are phaeophytines and phaeophorbide. The ratio of
chlorophyll to phaeopigments is indicative of the physiological state of
the algae.

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(standards.iteh.ai)
ISO 10260:1992
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ca6047b90f0f/iso-10260-1992

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ISO 10260:1992
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ca6047b90f0f/iso-10260-1992
INTERNATIONAL STANDARD ISO 10260:1992(E)

Water quality - Measurement of biochemical Parameters -

Spectrometric determination of the chlorophyll-a
concentration

1 Scope 1.6 The Pigment of certain rarely occurring

phototrophic bacteria (e.g. Chlorobium) interferes
with the determination of chlorophyll-a concen-
1.1 This International Standard specifies a method tration [l]. The contribution of chlorophyll-b and
for the determination of the chlorophyll-a concen- chlorophyll-c to the absorbance at 665 nm is negli-
tration. The procedure tan be applied for gible [2].
phytoplankton in natura1 surface waters and for
iTeh STANDARD2 PREVIEW
testing algal growth in bio-assays. Using appropriate
sampling it tan also be applied to phytobenthic
Normative references
communities (see annex A). (standards.iteh.ai)
The following Standards contain provisions which,
through reference in this text, constitute provisions
1.2 Other algal Pigments such as chlorophyll-b ISO 10260:1992
and of this International Standard. At the time of publi-
chlorophyll-c cation, the editions indicated were valid. All stan-
and some https://standards.iteh.ai/catalog/standards/sist/d5952a45-7531-4427-ad7c-
chlorophyll metabolites do
not contribute to the determination. ca6047b90f0f/iso-10260-1992 are subject to revision, and Parties to
Phaeopigments dards
may be determined semiquantitatively, to correct for agreements based on this International Standard
interference with chlorophyll-a determination and to are encouraged to investigate the possibility of ap-
indicate the Portion of inactive algal biomass. plying the most recent editions of the Standards in-
dicated below. Members of IEC and ISO maintain
registers of currently valid International Standards.
1.3 Chlorophyll is sensitive to Iight and Oxygen,
especially when it is extracted. To avoid oxidative ISO 5667-1:1980, Water quality - Sampling -
and photochemical destruction, the samples shall Part 1: Guidance on the design of sampling pro-
not be exposed to bright light or air. Homogenization grammes.
of the Sample may in some cases increase the ex-
traction efficiency. ISO 5667-2:1991, Water quality - Sampling -
Part 2: Guidance oi7 sampling techniques.
1.4 The extraction procedure with ethanol involves
heating to 75 OC for 5 min to inactivate 3 Principle
chlorophyllase and accelerate the lysis of Pigments.
Storage of extracts (except filters containing sus- Collection of algae and other suspended matter from
pended matter) Prior to photometric measurement a water Sample by filtration. Extraction of algal pig-
should be kept to a minimum, but is possible up to ments from the filter residue into hot ethanol. Spec-
3 d under refrigeration at 4 OC. Storage of extracts trometric determination of the chlorophyll-a
at less than - 25 OC is possible for at least 30 d. concentration in the extract. Evaluation of the
chlorophyll-a and phaeopigment concentration from
the differente in absorbance at 665 nm Prior to and
1.5 Even though the procedure involves filtration after acidification of the extract [3] [4].
or centrifugation to clarify the final extract, a slight
turbidity may remain. The acidification step may
also Cause turbidity. Therefore, the absorbance 4 Reagents
measured at 665 nm has to be corrected for turbidity
by substracting the absorbance measured at Use only reagents of recognized analytical grade
750 nm. and only deionized water of equivalent purity.
ISO 10260:1992(E)

4.1 Hydrochlorit acid, c(HCI) = 3 mol/i. range 0,l litre to 2 litres, depending on the concen-
tration of algae) through a glass-fibre filter (5.3)
4.2 Ethanol. clamped in a suitable holder. Dry the filter in a vac-
(CJ-l~OH), aiueous Solution 90 % (V/V’). uum, as soon as it is dry remove it from the holder
and place it in the extraction vessel. If it does not fit
NOTE 1 Generally, a denaturant in ethanol does not in- into the extraction vessel, tear it into pieces.
terfere. Nevertheless, a comparative determination with
pure ethanol (90 Oh) is recommended with each unknown Avoid contact with fingers.
batch [4].

5 Apparatus 7.2 Extraction variant A

Ordinary Iaboratory apparatus and the following: Heat the required volume of ethanol (4.2) to 75 “C.

5.1 Spectrometer, for use in the visible range up to Pour a small volume (usually 30 ml to 40 ml) of the
750 nm, with a resolution of 1 nm, a bandwidth of hot ethanol into the vessel containing the filter or
2 nm or less, sensitivity less than or equal to 0,001 filter pieces. After cooling for a few minutes, grind
absorbance units and with Optical cells of path the filter to facilitate extraction, preferably with a
length between 1 cm and 5 cm. rod-shaped homogenizer. Wash the homogenizer
rod with a small volume of ethanol (4.2) to remove
particles of Sample adhering to it. Extract the sus-
5.2 Vacuum filtration device, filter holder with
Pension for at least 3 min.
clamp.
NOTE 2 Usually the extraction is carried out at room
5.3 Glas+fibre filters for fil-
free of organic binder, temperature for several hours or overnight. lf the ex-
tration of water samples, retaining more than 99 % traction is prolonged, or the extract is stored for several
days (e.g. over the weekend), the extraction vessels
range from 25 mm to 50 mm. iTeh STANDARD PREVIEW
of particles greater than 1 Pm. Suitable diameters
should be stored in a refrigerator.

5.4 Filters for filtration of extracts,

(standards.iteh.ai)
as described in Filter the slurry through a dense filter (5.4) into a
5.3 but of small diameter e.g. 25 mm. calibrated flask (of capacity 50 ml or 100 ml) with a
stopper. Wash the extraction vessel with ethanol
ISO 10260:1992
Alternative: Centrifuge, (4.2) to remove residual
withhttps://standards.iteh.ai/catalog/standards/sist/d5952a45-7531-4427-ad7c-
an acceleration of extract and transfer
6 000 g and a rotor suitable for appropriate ca6047b90f0f/iso-10260-1992
ex- quantitatively while rinsing the filter into the cali-
traction tubes. brated flask. Fill to the mark, stopper and mix thor-
oughly. This is the extract volume l/e.
5.5 Water bath, adjustable to 75 OC -+ 1 “C with a
Proceed with step 7.4.
rack for extraction vessels.

5.6 Extraction vessels, e.g. wide-necked amber

glass vials with polytetrafluoroethylene (PTFE)-lined 7.3 Extraction variant B
screw taps, of typical capacity 30 ml to 50 ml, suit-
able for centrifugation at 6 000 g. Dispense an exact volume VE (usually 20 ml or
25 ml) of the ethanol (4.2) into the extraction vessel
(5.6) and allow the filter pieces to submerge. Close
6 Sampling and storage the screw cap tightly to avoid losses from evap-
oration of extractant. Shake slightly to resuspend the
Sample according to ISO 5667-1 and ISO 5667-2. filter residue. Place the tube in the water bath (5.5)
Refrigerated storage of water samples in the dark so that the extractant level aligns with the level of
for less than 8 h is acceptable but should be the bath. Heat for 5 min, shaking slightly if necess-
avoided. If possible, perform Steps 7.1 to 7.3 im- ary. Take the extraction vessels from the water bath
mediately after sampling. lf necessary, store the raw and allow to cool to room temperature for 15 min.
extracts in air-tight brown glass extraction vessels
(5.6) below - 25 OC for up to 30 d. 00 not store fro- The time between extractio n and measurement
zen water samples or filters plus solids. shou ld be kept to a minimum.

NOTE 3 Extracts at this Stage may be stored in a

7 Procedure refrigerator overnight Prior to measurement (see 7.4).
Prolonged storage shall not exceed 3 d.
7.1 Filtration
Filter the supernatant extract through a filter (5.4)
Shake the samples in Order to mix thoroughly. Filter into a clean extraction vessel (5.6) but do not rinse
a measured volume of Sample l/s (normally in the with fresh solvent (see note 4).

2
 ISO 10260:1992(E)

Alternatively, centrifuge the extraction vessels for a is the volume, in litres, of fil-
period which is sufficient to obtain a clear tered Sample;
supernatant.
K, = 82 I/pgcm is the specific operational
Use a clear extract or supernatant for photometry. spectral a bsorption coef-
ficient for chlorophyll-a (the
NOTE 4 Using this procedure, it is sufficient to recover value is taken from [2]);
merely a part of the extract volume, because the initial
volume &, of extractant is known exactly and is not re- R= 1,7 is the ratio A/Aa for a sol-
duced due to evaporation during extraction because the ution of pure chlorophyll-a
taps are tightly closed. Furthermore, the residual water which is transferred to
content of the filters (see 7.1) is negligible, contributing to phaeophytin by acidification
far less than 5 % of the extract volume.
(see 7.4.2) (the value is taken
from [2]);

d is the path length, in centi-

metres, of the Optical cell;
7.4 Photometry
IO3 is the dimension to fit
7.4.1 Transfer patt of the clear extract into the Ves
spectrometer cuvette using a pipette, leaving a suf-
ficient volume for the acidification step (see 7.4.2). 8.2 The phaeopigment concentration pp, in micro-
grams per litre, is calculated according to the
Measure the absorbance at 665 nm (A& and equation
750 nm (A,,,) against a reference cell filled with
ethanol (4.2). 103v
pp = Aa X + X + - PC ... (2)
C
NOTE 5 iTeh STANDARD PREVIEW
The absorbance at 665 nm should fall between
0,Ol and 0,8 units. This may be achieved by suitably
S’

8.3 When the value 82 is taken for the specific

(standards.iteh.ai)
choosing the water volume filtered, extractant volume,
dilution, Optical pathlength, etc. To Start with, take 05 litre spectral absorption coefficient for chlorophyll-a in
of Sample, a 50 mm diameter filter, 20 ml of ethanol and 90 % ethanol, and 1,7 is taken for the maximum acid
a 5 cm cuvette. ISO 10260:1992
ratio (R) for pure chlorophyll-a, the chlorophyll-a
concentration pc in the water Sample simplifies to
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7.4.2 Acidify patt of the extract (usually 5 ml to
ca6047b90f0f/iso-10260-1992
10 ml) with 0,Ol ml of hydrochloric acid (4.1) per V
/IC= (A - Aa) X 2976X ti . . . . (3)
10 ml of extract volume, Shake and measure the c
absorbance again at 665 nm and 750 nm, after
5 min to 30 min. and for the concentration of phaeopigment, equation
2 simplifies Po equation (4)
I/e
,op = Aa x 20,8 x I/d - pc . . . (4)
8 Calculation and expression of results S’

NOTES
8.1 The chlorophyll-a concentration pc, in micro-
grams per litre, is calculated according to the 6 The calculated vaiues for phaeopigments are less re-
equation liable than for chlorophyll-a concentrations. lnterlabora-
tory trials have shown an interlaboratory Variation of
(A - Aa> - R 103ve 5 O/o to 11 % for chlorophyll-a determinations and 6 O/oto
Pc = P ...0 46 O/ofor phaeopigment determinations.
KC ’ R - 1 ’ V$;.d
where 7 The ratio A/A, is 1,7, if only undegraded chlorophyll-a
is present in the Sample. lt is 1, if only degradation prod-
ucts of chlorophyll-a are present in the Sample.
A = A665 - A,,, is the absorbance of the ex-
tract before acidification (see The operational absorption coefficient (82) for
7.4.1); chlorophyll-a in ethanol (4.2) is derived from the oper-
ational absorption coefficient, recommended in [5] at
A a= n 665 - A 750 is the absorbance of the ex- 665 nm. The value (84) given there makes allowance for
tract after acidification; the presence of chlorophyll-b and chlorophyll-c. The
absorbance of equal concentrations of chlorophyll-a in
Ve is the volume, in millilitres, acetone is 2 O/o to 3 O/o higher than in ethanol at 665 nm
of extract; Pl Pl*
ISO 10260:1992(E)

8.4 Note the results, in micrograms per litre (or 10 Test report
milligrams per cubic metre), with at most two sig-
nificant figures or one figure after the decimal Point, The test report shall include the following infor-
for example: mation:
Chlorophyll-a concentration 5s cis/1 a) a reference to this International Standard;
Phaeopigment concentration < O:l pg/l
b) identification of the water Sample;

c) expression of results, in accordance with

clause 8;

d) Sample pretreatment, if relevant;

9 Precision
e) any deviation from this method or any other cir-
An interlaboratory trial, carried out in 1983, pro- cumstances which may have influenced the re-
duced the results [6] given in table 1. sults.

Table 1 - Precision data

2 n 4 n % vc, CR VCR
Sample
Oh rd1 C~dI Oh dl Oh

A 18 71 0 126,5 5,46 493 6,36 570

B 18 70 278 20,3 3,.67 18,l 2,so 11,3
C 17
iTeh
67
STANDARD
576 24,6
PREVIEW
2,21 990 298 11,4

2 is the number of laboratories

(standards.iteh.ai)
a, is the repeatability Standard deviation
n is the number of values VC, is the repeatability Variation coefficient
ISO 10260:1992
n, is the percentage of outliers UR is the reproducibility Standard deviation
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2 is the total mean VC, is the
ca6047b90f0f/iso-10260-1992reproductibility Variation coefficient

4
 ISO 10260:1992(E)

Annex A
(informative)

Determination of phytobenthic concentration

A.l Determination of phytobenthos traction of algae. However, because it became

evident that the extraction efficiency of acetone was
The sampling of phytoplankton is carried out ac- rather poor in some cases, alcohols were used. lt
cording to ISO 5667-1 and ISO 5667-2. was shown that hot ethanol and methanol are
equally efficient, and considerably more efficient
Sampling of phytobenthos (attached algae) depends than cold acetone [4]. Special Problems may arise,
on the substratum colonized. A defined surface area however, from the use of methanol if the chlorophyll
on stones or other submerged material may be extracts are to be acidified in Order to correct for
scratched with a knife or a razor blade. Grave1 and interference from phaeopigments. A shifting of the
small stones are sampled directly into the solvent. absorption maximum to shorter wavelengths tan
The use of artificial Substrates exposed to water, lead to considerable Variation in the maximum acid
such as glass slides, facilitate quantitative sampling. ratio depending, for example, on the acid concen-
The colonized giass slides or the removed algae are tration, the water content of the solvent and filter,
transported from the sampling site with the water. the period between acidification and measurement.
The suspended phytobenthic algae or Sediments of These Problems tan only be overcome by including
known weight are afterwards treated similarly to additional Steps in the procedure, for example,
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phytoplankton according to the method described in
this International Standard.
neutralization with organic bases (dimethyl aniline
or diphenyl aniline). Furthermore, methanol is toxic.
(standards.iteh.ai) For these reasons, it is recommended to use ethanol
as a reference extractant for the determination of
A.2 Extracting agent chlorophyll-a in freshwater and marine samples.
ISO 10260:1992
Acetone has been widely used since it was rec-
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ommended by UNESCO-SCOR in 1966 ca6047b90f0f/iso-10260-1992
for the ex-