Iso 6341 2012

INTERNATIONAL ISO
STANDARD 6341
Fourth edition
2012-10-15
Water quality — Determination of the

inhibition of the mobility of Daphnia
magna Straus (Cladocera, Crustacea) —
Acute toxicity test
Qualité de l’eau — Détermination de l’inhibition de la mobilité de Daphnia
magna Straus (Cladocera, Crustacea) — Essai de toxicité aiguë
iTeh STANDARD PREVIEW

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ISO 6341:2012
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b45d51f40d93/iso-6341-2012
Reference number
ISO 6341:2012(E)
© ISO 2012
ISO 6341:2012(E)

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ISO 6341:2012
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ISO 6341:2012(E)
Contents Page
Foreword ............................................................................................................................................................................ iv
Introduction ........................................................................................................................................................................ v
1 Scope ...................................................................................................................................................................... 1
2 Normative references ......................................................................................................................................... 1
3 Terms and definitions ......................................................................................................................................... 1
4 Principle ................................................................................................................................................................. 2
5 Test environment ................................................................................................................................................. 2
6 Reagents, test organisms and media ............................................................................................................ 3
7 Apparatus and materials ................................................................................................................................... 4
8 Treatment and preparation of samples ......................................................................................................... 5
8.1 Special precautions for sampling, transportation, storage and treatment of water, effluent, or
aqueous extract samples to be tested........................................................................................................... 5
8.2 Preparation of solutions of substances to be tested ................................................................................ 6
9 Procedure .............................................................................................................................................................. 6
9.1 General ................................................................................................................................................................... 6
9.2 Preliminary test .................................................................................................................................................... 7
9.3 Definitive test ........................................................................................................................................................ 7
9.4
9.5
Check of the sensitivity of the Daphnia magna and conformity with the procedure ....................... 7
Limit test ................................................................................................................................................................ 8
10
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Interpretation and validity of the results ....................................................................................................... 8
10.1 Estimation of the EC50 ....................................................................................................................................... 8
10.2 ISO 6341:2012
Validity criteria ..................................................................................................................................................... 8
11 Expression of results .........................................................................................................................................
b45d51f40d93/iso-6341-2012 8
12 Test report ............................................................................................................................................................. 8
Annex A (informative) Preparation of the Elendt M4 medium............................................................................... 10
Annex B (informative) Example of graphical determination of the inhibition of mobility of Daphnia
magna by an effluent or stock solution of a substance at a concentration of 1 000 mg/l............. 13
Annex C (informative) General recommendations for stock culturing .............................................................. 16
Annex D (informative) Culturing of Daphnia magna for production of dormant eggs .................................. 17
Annex E (informative) Precision data .......................................................................................................................... 19
Annex F (informative) Dilution level D — Preparation of the dilution series for the determination of
the LID .................................................................................................................................................................. 20
Bibliography ..................................................................................................................................................................... 22
© ISO 2012 – All rights reserved iii

ISO 6341:2012(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. Each 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, governmental 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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 6341 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods.
This fourth edition cancels and replaces the third edition (ISO 6341:1996), which has been technically revised.
It also incorporates the Technical Corrigendum ISO 6341:1996/Cor. 1:1998.

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ISO 6341:2012
b45d51f40d93/iso-6341-2012
iv © ISO 2012 – All rights reserved

ISO 6341:2012(E)
Introduction
This International Standard specifies a procedure for the determination of the acute toxicity of chemicals,
waters and waste waters to the water flea Daphnia magna Straus.
The evaluation of harmful effects on water quality has for several years involved the performance of biological
tests. Crustaceans are of interest from the ecotoxicological point of view because they are primary consumers
and a major component of the zooplankton in aquatic ecosystems.
The test specified in this International Standard involves the determination of the immobilization of the water
flea Daphnia magna Straus after 24 h or 48 h exposure (depending on the requirement of users or national
authorities) to the test sample under the conditions specified in this International Standard.

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ISO 6341:2012
b45d51f40d93/iso-6341-2012
© ISO 2012 – All rights reserved v

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ISO 6341:2012
b45d51f40d93/iso-6341-2012
INTERNATIONAL STANDARD ISO 6341:2012(E)
Water quality — Determination of the inhibition of the mobility

of Daphnia magna Straus (Cladocera, Crustacea) — Acute
toxicity test
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This document does not purport to address all of the safety problems, if any, associated with
its use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this International
Standard be carried out by suitably qualified staff.
1 Scope
This International Standard specifies a method for the determination of the acute toxicity to Daphnia magna
Straus (Cladocera, Crustacea).
This method is applicable to:
— chemical substances which are soluble under the conditions of the test, or can be maintained as a stable
suspension or dispersion under the conditions of the test;
— industrial or sewage effluents;
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— treated or untreated waste water;
ISO 6341:2012
— aqueous extracts and leachates;
b45d51f40d93/iso-6341-2012
— fresh water (surface and ground water);
— eluates of fresh water sediment;
— pore water of fresh water sediment.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are indispensable
for its application. For dated references, only the edition cited applies. For undated references, the latest edition
of the referenced document (including any amendments) applies.
ISO 5667-16:1998, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method
ISO 10523, Water quality — Determination of pH
3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.
3.1
control batch
series of replicates containing control solution
[ISO 20665:2008,[3] 3.3]
© ISO 2012 – All rights reserved 1

ISO 6341:2012(E)
3.2
control solution
test medium without sample under test
3.3
immobilization
inability of the organisms to swim during the 15 s which follow gentle agitation of the test and control solutions,
even if they can still move their antennae
3.4
EC50
concentration at which there is an effect on 50 % of the organisms in line with the test criterion
[ISO 15088:2007,[1] 3.3]
3.5
neonate
newly born or newly hatched individual
NOTE In this International Standard, a neonate is a first-instar daphnid, <24 h old.
[ISO 20665:2008,[3] 3.6]
3.6
test batch
series of replicates filled with the same test solution
[ISO 20665:2008,[3] 3.8]

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4 Principle
ISO 6341:2012
Determination of the initial concentration (i.e. the concentration present at the beginning of the test) which,
in 24 h or 48 h, immobilizes 50 % of exposedb45d51f40d93/iso-6341-2012
D. magna, under the conditions specified in this International
Standard. This concentration, known as the effective initial inhibitory concentration, is designated 24 h EC50
or 48 h EC50.
An indication of the lowest concentration tested which immobilizes all the D. magna and the highest concentration
tested which does not immobilize any of the D. magna is desirable and provides useful information in cases
where the EC50 cannot be determined.
The test is carried out in one or two stages:
— a preliminary test which determines the range of concentrations to be tested in the definitive toxicity test
and gives an approximate value of the 24 h EC50 or 48 h EC50;
— a definitive test, conducted when the approximate value given by the preliminary test is not sufficient,
which permits calculation of the 24 h EC50 or 48 h EC50, and determines concentrations corresponding to
0 % and 100 % immobilization.
If the method specified in this International Standard is used for biotesting of chemical substances, a limit test
can be performed at 100 mg/l or at a lower concentration, at which the substance is soluble or is in stable
dispersion under the conditions of the test (see 9.5). If it provides useful information, a limit test may also be
performed at concentrations above 100 mg/l as long as the substance is soluble or in stable dispersion.
5 Test environment
The exposure of organisms as specified in this International Standard shall be carried out either in the dark or
under a 16 h + 8 h light + dark photoperiod, in a temperature-controlled room or incubator at (20 ± 2) °C in the
test containers.
2 © ISO 2012 – All rights reserved

ISO 6341:2012(E)
The testing atmosphere shall be free from vapours or dusts toxic to D. magna. Photodegradable chemicals shall be
tested in the dark, or using minimal lighting with the specified photoperiod, or minimal red lighting, as appropriate.
The use of controls (3.1) also allows checking that the test is performed in an atmosphere free from toxic
dusts and vapours.
6 Reagents, test organisms and media

Use only reagents of recognized analytical grade, unless otherwise specified.
6.1 Test organisms. The test organisms are neonates of D. magna Straus (Cladocera, Crustacea), obtained
by acyclical parthenogenesis under specified breeding conditions (see Annex C).
The animals used for the test shall be less than 24 h old and should not be first brood progeny. The D. magna
shall be from a healthy stock, showing no signs of stress such as mortality >20 % in 2 d, presence of males,
ephippia, or discoloured animals, and there shall be no delay in the production of the first brood. Isolate gravid
females and collect newly released neonates within 24 h.
If the culture conditions differ significantly from test conditions, it is recommended that one generation be
acclimated under the test conditions for about one week to avoid stressing the parent animals and the offspring.
The age of the stock culture and the source (including clone, if possible) of the D. magna culture shall be indicated
in the test report, since the sensitivity of D. magna to toxicants can be affected by the source of the culture.
The D. magna may also derive from the hatching of ephippia obtained from laboratory cultures of the crustacean as
iTeh STANDARD PREVIEW 1)
described in Annex D or can be purchased from a specialized company. The neonates hatched from the ephippia
may be used directly as test organisms if they comply with all validity criteria given in this International Standard.
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6.2 Pure water, conductivity below 10 µS/cm.
ISO 6341:2012
6.3 Dilution and culturing water. b45d51f40d93/iso-6341-2012
6.3.1 General. Natural water (surface or ground water), reconstituted water or dechlorinated tap water are
acceptable as culturing and dilution water if D. magna survives in it for the duration of the culturing, acclimation
and testing without showing signs of stress. These waters may be used if they comply with all criteria and
conditions specified in this International Standard. Waters in the range pH 6 to pH 9, with hardness between
140 mg/l and 275 mg/l (as CaCO3) are recommended.
For stock culture of D. magna in the laboratory, the M4 medium (see Annex A) may also be used.
M4 medium (Annex A) should not be used as dilution water for samples containing bivalent metal ions. The EDTA
in this medium can reduce the bioavailability of such ions, resulting in a decrease in apparent toxicity. In addition,
for the same reason, M4 medium should not be used as the dilution water for samples of unknown composition.
NOTE If the test is performed for purposes necessitating the use of a dilution water with characteristics differing from
those described in the preceding three paragraphs, state the main characteristics of the synthetic dilution water used in
the test report.
As an example, the preparation of dilution water meeting the requirements is described below.
Dissolve known quantities of reagents in pure water (6.2) The dilution water prepared shall have a pH of
7,8 ± 0,5, a hardness of (225 ± 50) mg/l (expressed as CaCO3), a molar Ca + Mg ratio close to 4 + 1 and a
dissolved oxygen concentration above 7 mg/l.
Prepare the solutions specified in 6.3.2 to 6.3.5.
1) MicroBioTests Inc., Mariakerke, Belgium, is an example of a suitable supplier. This information is given for the
convenience of the users of this document and does not constitute an endorsement by ISO of this supplier.

ISO 6341:2012(E)
6.3.2 Calcium chloride solution. Dissolve 11,76 g of calcium chloride dihydrate (CaCl2⋅2H2O) in pure water
(6.2) and make up to 1 l with pure water (6.2).
6.3.3 Magnesium sulfate solution. Dissolve 4,93 g of magnesium sulfate heptahydrate (MgSO4⋅7H2O) in
pure water (6.2) and make up to 1 l with pure water (6.2).
6.3.4 Sodium bicarbonate solution. Dissolve 2,59 g of sodium bicarbonate (NaHCO3) in pure water (6.2)
and make up to 1 l with pure water (6.2).
6.3.5 Potassium chloride solution. Dissolve 0,23 g of potassium chloride (KCI) in pure water (6.2) and
make up to 1 l with pure water (6.2).
6.3.6 Mixing. Mix 25 ml of each of the four solutions (6.3.2 to 6.3.5) and make up to 1 l with pure water (6.2).
The dilution water shall be aerated until the dissolved oxygen concentration has reached saturation and the
pH has stabilized. If necessary, adjust the pH to 7,8 ± 0,5 by adding sodium hydroxide (NaOH) solution or
hydrochloric acid (HCI). The dilution water prepared in this way shall not be further aerated before use.
6.4 Reference substance. Potassium dichromate (K2Cr2O7) is recommended.
Since K 2Cr 2O7 is a carcinogenic substance, toxic via inhalation, the use of a ready-made solution with a
defined concentration of K 2Cr 2O72) for the preparation of the stock solution of the reference substance can
reduce the risk of inhalation of the toxic dust in the laboratory.
6.5 iTeh STANDARD PREVIEW

Sodium hydroxide solution, e.g. [NaOH] = 1 mol/l.
6.6
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Hydrochloric acid, e.g. [HCl] = 1 mol/l.
ISO 6341:2012
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Apparatus and materials
b45d51f40d93/iso-6341-2012
Usual laboratory apparatus and in particular the following.
7.1 Temperature-controlled room or chamber.
7.2 Dissolved oxygen-measuring apparatus.
7.3 Culture vessels, of chemically inert material and of sufficient capacity, e.g. 2 l glass beakers.
7.4 Test containers, of chemically inert material and of sufficient capacity, e.g. glass test tubes or beakers.
7.5 Pipette for sampling the test organisms, with a sufficient diameter for capturing the animals while allowing
sampling of only a small volume of medium.
Micropipettes of inert plastic material with a bulb at the end are very suitable for the operations.
7.6 Sample collecting bottles, as specified in ISO 5667-16.
7.7 Sieves. Appropriate sieves (e.g. mesh 1,0 mm and 0,3 mm) to transfer the adult organisms to stock
culture and to separate the young from the adults.
2) Titrisol potassium dichromate solution is an example of a suitable product available commercially. This information
is given for the convenience of the users of this document and does not constitute an endorsement by ISO of this product.

ISO 6341:2012(E)
8 Treatment and preparation of samples
8.1 Special precautions for sampling, transportation, storage and treatment of water, efflu-
ent, or aqueous extract samples to be tested
Sampling, transportation and storage of the samples should be performed as specified in ISO 5667-16.
Carry out the toxicity test as soon as possible, preferably within 12 h of collection. If this time interval cannot
be met, cool the sample to 0 °C to 5 °C and test the sample within 24 h. If it is not possible to perform the test
within 72 h, the sample may be frozen as soon as possible after sampling and maintained deep-frozen (below
–18 °C) for testing within 2 months of collection (see ISO 5667-16:1998, Clause 5).
Immediately test the frozen samples after complete thawing, e.g. in a water bath at a maximum temperature of
30 °C. Do not use a microwave for thawing the samples.
At the time of testing, homogenize the sample to be analysed by shaking manually. High concentrations of
suspended inorganic or organic solids in a sample can be harmful to filter-feeding D. magna. Compensation for
this interference can be made by a sample treatment for turbidity. If necessary, allow to settle for a maximum
of 2 h in a container, and sample, e.g. by drawing off the required quantity of supernatant using a pipette,
maintaining the end of the pipette in the centre of the section of the test container and halfway between the
surface of the deposited substances and the surface of the liquid. If the raw sample of the decanted supernatant
is likely to interfere with the test (due to presence of residual suspended matter, protozoa, microorganisms,
etc.), centrifuge, for example, for 10 min at 5 000g or filter the raw or decanted sample. Test the residual toxicity
of the supernatant. The particular kind of filter to be used should be checked by a test with control medium run
through the filters.
NOTE Some filters and apparatus can add measurable toxicity, sometimes because of wetting agents added to the
(standards.iteh.ai)
filters. A filter paper can also absorb toxic substances and remove them from the sample filtrate.
The sample obtained by either of these methods is the sample submitted to testing.
ISO 6341:2012
Usually no aeration of https://standards.iteh.ai/catalog/standards/sist/2d02f429-4473-4403-bd6c-
sample or prepared test concentrations is necessary. If, and only if, the dissolved oxygen
is <40 % saturation, a pre-aerate of theb45d51f40d93/iso-6341-2012
sample or all test solutions for at most 20 min by appropriate methods,
e.g. aeration or stirring may be performed. Any supersaturation should be remedied.
Measure the pH (as specified in ISO 10523) and the dissolved oxygen concentration (as specified in ISO 5814)
and record these values in the test report.
Report any pre-aeration of test solutions or sample.
Tests shall be carried out without pH adjustment of the test sample.
The pH of test batches (3.6) is measured at the beginning and at the end of the test and reported.
However, in some cases, the final pH of a test solution may significantly differ from original pH of the test sample
due to the concentration range selected and as a result of the buffer capacity of the dilution water or test sample.
If toxic effects are observed at concentrations where the pH is not compatible with the survival of the organisms
(i.e. outside the pH 6,0 to pH 9,0 range), the test(s) can be repeated with pH adjustment of the test sample.
IMPORTANT — Adjustment of the pH can alter the nature of the sample.
If the pH is to be adjusted, the recommendation is to adjust to the pH of the dilution water (6.3) selected.
Choose the concentration of the hydrochloric acid (6.6) or the sodium hydroxide (6.5) solutions to restrict the
volume fraction added to not more than 5 %.
If, as a result of pH adjustment, there is an issue with suspended matter, separate the suspended matter from
the remaining sample as specified in ISO 5667-16. Any pH adjustment shall be included in the test report.
Adjust the temperature of the pretreated sample to the test temperature.

ISO 6341:2012(E)
8.2 Preparation of solutions of substances to be tested
8.2.1 Preparation of stock solutions
Prepare the stock solution of the substance to be tested by dissolving a known quantity of substance in a
specified volume of dilution water (6.3) at the time of use. However, if the stock solution of the substance is
stable under certain conditions, it can be prepared in advance and stored under these conditions.
For substances sparingly soluble in the test medium, refer to the specifications given in ISO 5667-16.
As far as possible, the use of solvents, emulsifiers or dispersants should be avoided. However, such compounds
may be required in some cases in order to produce a suitably concentrated stock solution. Guidance for
suitable solvents, emulsifiers and dispersants is given in Reference [4]. Special considerations concerning test
design and data evaluation are necessary (ISO/TS 20281[2]).
8.2.2 Preparation of test solutions
Prepare the test solution (see 9.1) by adding the stock solutions (8.2.1) to the dilution water (6.3) in specified quantities.
The test should comprise at least five concentrations of the sample to be tested. Select the dilutions within a
geometric series with a separation factor which depends on the nature of the sample to be analysed (chemical
substances, effluents, waters or extracts) and on the type of assay (range finding or definitive).
For the range finding test with chemical substances, the separation factor for the serial dilutions is usually 10
(one order of magnitude difference between two successive dilutions).
For treated or untreated waste water, fresh water, pore water or extracts, a separation factor of2 between
dilutions is usually performed (i.e. dilution of the previous dilution by half).
(standards.iteh.ai)
The preparation of the dilution series for lowest ineffective dilution (LID) determinations is described in Annex F.
Depending on the purpose of the test and the statistical ISOrequirements
6341:2012 concerning the test results, other dilution
schemes with concentrations in geometric or logarithmic series can also be appropriate.
b45d51f40d93/iso-6341-2012
Dilution series for the definitive test on chemical substances are prepared with a separation factor not exceeding 3,2.
If steep concentration–response curves are expected, it is recommended that a separation factor not
exceeding 2,2 be used.
Each dilution is preferably carried out in four replicates with a control (3.1) also in four replicates.
Substances which are poorly soluble in water may be solubilized or dispersed directly in pure water or dilution
water by suitable means using ultrasonic devices or solvents of low toxicity to D. magna. Solvents should
be used only when the EC50 is greater than the solubility of the test substance. If a solvent is used, the
concentration of the solvent in the final test solution shall not exceed 0,1 ml/l, and two control solutions, one
with no solvent, the other with the maximum concentration of solvent, shall be included in the test. Consider
special requirements concerning test design for chemicals with solvents, e.g. additional solvent-control and
statistical evaluations according to ISO/TS 20281.[2]
9 Procedure
9.1 General
Prepare a dilution series with the test solution (8.2.2) and the dilution water (6.3).
Combine increasing volumes of the test solution (8.2.2) with the dilution water (6.3), so as to obtain the desired
concentrations for the test and transfer to the test containers.
To obtain a test and solution temperature of (20 ± 2) °C, for example, place the containers in a temperature-
controlled room.

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INTERNATIONAL ISO

Water quality — Determination of the

iTeh STANDARD PREVIEW

iTeh STANDARD PREVIEW

COPYRIGHT PROTECTED DOCUMENT

ii © ISO 2012 – All rights reserved

© ISO 2012 – All rights reserved iii

iTeh STANDARD PREVIEW

iv © ISO 2012 – All rights reserved

iTeh STANDARD PREVIEW

© ISO 2012 – All rights reserved v

Water quality — Determination of the inhibition of the mobility

This method is applicable to:

— eluates of fresh water sediment;

— pore water of fresh water sediment.

ISO 10523, Water quality — Determination of pH

3 Terms and definitions

[ISO 20665:2008,[3] 3.3]

© ISO 2012 – All rights reserved 1

[ISO 15088:2007,[1] 3.3]

NOTE In this International Standard, a neonate is a first-instar daphnid, <24 h old.

[ISO 20665:2008,[3] 3.6]

[ISO 20665:2008,[3] 3.8]

The test is carried out in one or two stages:

2 © ISO 2012 – All rights reserved

6 Reagents, test organisms and media

Prepare the solutions specified in 6.3.2 to 6.3.5.

© ISO 2012 – All rights reserved 3

6.4 Reference substance. Potassium dichromate (K2Cr2O7) is recommended.

6.5 iTeh STANDARD PREVIEW

7.1 Temperature-controlled room or chamber.

7.2 Dissolved oxygen-measuring apparatus.

7.6 Sample collecting bottles, as specified in ISO 5667-16.

4 © ISO 2012 – All rights reserved

8 Treatment and preparation of samples

Report any pre-aeration of test solutions or sample.

Tests shall be carried out without pH adjustment of the test sample.

IMPORTANT — Adjustment of the pH can alter the nature of the sample.

Adjust the temperature of the pretreated sample to the test temperature.

© ISO 2012 – All rights reserved 5

8.2 Preparation of solutions of substances to be tested

8.2.1 Preparation of stock solutions

8.2.2 Preparation of test solutions

6 © ISO 2012 – All rights reserved

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