EN ISO 6341:2012

Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity test (ISO 6341:2012)

ISO 6341.2012 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; treated or untreated waste water; aqueous extracts and leachates; fresh water (surface and ground water); eluates of fresh water sediment; pore water of fresh water sediment.

Wasserbeschaffenheit - Bestimmung der Hemmung der Beweglichkeit von Daphnia magna Straus (Cladocera, Crustacea) - Akuter Toxizitäts-Test (ISO 6341:2012)

Diese Internationale Norm legt ein Verfahren zur Bestimmung der akuten Toxizität gegenüber Daphnia magna Straus (Cladocera, Crustacea) fest.
Das Verfahren ist anwendbar auf:
-   Chemikalien, die unter den Testbedingungen löslich sind, oder als stabile Suspension oder Dispersion unter den Testbedingungen erhalten bleiben können;
-   industrielle oder kommunale Abwässer;
-   behandelte oder unbehandelte Abwässer;
-   wässrige Extrakte und Sickerwasser;
-   Süßwasser (Oberflächen  und Grundwasser);
-   Eluate von Süßwassersedimenten;
-   Porenwasser von Süßwassersedimenten.

Qualité de l'eau - Détermination de l'inhibition de la mobilité de Daphnia magna Straus (Cladocera, Crustacea) - Essai de toxicité aiguë (ISO 6341:2012)

L'ISO 6341:2012 spécifie une méthode de détermination de la toxicité aiguë vis-à-vis de Daphnia magna Straus (Cladocera, Crustacea).
Cette méthode s'applique aux: a) substances chimiques solubles dans les conditions de l'essai ou pouvant être maintenues en suspension ou en dispersion stable dans les conditions de l'essai; b) effluents industriels ou domestiques; c) eaux usées traitées ou non; d) extraits aqueux et lixiviats; e) eaux douces (eaux de surface et eaux souterraines); f) éluats de sédiments d'eaux douces; g) eaux interstitielles de sédiments d'eau douce.

Kakovost vode - Določanje zaviranja gibanja Daphnia magna Straus (Cladocera, Crustacea) - Akutni preskus strupenosti (ISO 6341:2012)

Ta mednarodni standard opisuje metodo za določevanje akutne strupenosti za Daphnia magna Straus (Cladocera, Crustacea). Ta metoda se uporablja za: – kemične snovi, ki so topne pod pogoji preskusa ali se lahko pod pogoji preskusa ohranijo v stabilni suspenziji ali disperziji, – industrijske ali komunalne odplake, – obdelano ali neobdelano odpadno vodo, – vodne ekstrakte in izcedne vode, – sladko vodo (površinsko in podtalno), – izlužke sedimenta sladke vode, – porne vode svežega vodnega sedimenta.

General Information

• Status: Withdrawn
• Publication Date: 30-Jan-2013
• Withdrawal Date: 29-Apr-2013
• ICS: 07.100.20 - Microbiology of water
• ICS: 13.060 - Water quality
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230 - Water analysis
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 15-Oct-2012
• Completion Date: 15-Oct-2012
• Ref Project: SIST EN ISO 6341:2013 - Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity test (ISO 6341:2012)

Relations

• Replaces: EN ISO 6341:1996/AC:1998 - Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity test (ISO 6341:1996)
• Effective Date: 24-Oct-2012
• Replaces: EN ISO 6341:1996 - Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity test (ISO 6341:1996)
• Effective Date: 08-Jun-2022

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 6341:2013
01-januar-2013
1DGRPHãþD
SIST EN ISO 6341:1996
SIST EN ISO 6341:1996/AC:2004
.DNRYRVWYRGH'RORþDQMH]DYLUDQMDJLEDQMD'DSKQLDPDJQD6WUDXV&ODGRFHUD
&UXVWDFHD$NXWQLSUHVNXVVWUXSHQRVWL,62

Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus

(Cladocera, Crustacea) - Acute toxicity test (ISO 6341:2012)
Wasserbeschaffenheit - Bestimmung der Hemmung der Beweglichkeit von Daphnia
magna Straus (Cladocera, Crustacea) - Akuter Toxizitäts-Test (ISO 6341:2012)

Qualité de l'eau - Détermination de l'inhibition de la mobilité de Daphnia magna Straus

(Cladocera, Crustacea) - Essai de toxicité aiguë (ISO 6341:2012)
Ta slovenski standard je istoveten z: EN ISO 6341:2012
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 6341:2013 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 6341:2013
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SIST EN ISO 6341:2013
EUROPEAN STANDARD
EN ISO 6341
NORME EUROPÉENNE
EUROPÄISCHE NORM
October 2012
ICS 13.060.70 Supersedes EN ISO 6341:1996
English Version
Water quality - Determination of the inhibition of the mobility of
Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity
test (ISO 6341:2012)

Qualité de l'eau - Détermination de l'inhibition de la mobilité Wasserbeschaffenheit - Bestimmung der Hemmung der

de Daphnia magna Straus (Cladocera, Crustacea) - Essai Beweglichkeit von Daphnia magna Straus (Cladocera,

de toxicité aiguë (ISO 6341:2012) Crustacea) - Akuter Toxizitäts-Test (ISO 6341:2012)

This European Standard was approved by CEN on 14 October 2012.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European

Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national

standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation

under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same

status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United

Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 6341:2012: E

worldwide for CEN national Members.
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SIST EN ISO 6341:2013
EN ISO 6341:2012 (E)
Contents Page

Foreword ..............................................................................................................................................................3

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SIST EN ISO 6341:2013
EN ISO 6341:2012 (E)
Foreword

This document (EN ISO 6341:2012) has been prepared by Technical Committee ISO/TC 147 “Water quality”

in collaboration with Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by

DIN.

This European Standard shall be given the status of a national standard, either by publication of an identical

text or by endorsement, at the latest by April 2013, and conflicting national standards shall be withdrawn at the

latest by April 2013.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.

This document supersedes EN ISO 6341:1996.

According to the CEN/CENELEC Internal Regulations, the national standards organisations of the following

countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech

Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,

Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,

Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.

Endorsement notice

The text of ISO 6341:2012 has been approved by CEN as a EN ISO 6341:2012 without any modification.

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SIST EN ISO 6341:2013
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SIST EN ISO 6341:2013
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
Reference number
ISO 6341:2012(E)
ISO 2012
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SIST EN ISO 6341:2013
ISO 6341:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,

electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s

member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved
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SIST EN ISO 6341:2013
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 Check of the sensitivity of the Daphnia magna and conformity with the procedure ....................... 7

9.5 Limit test ................................................................................................................................................................ 8

10 Interpretation and validity of the results ....................................................................................................... 8

10.1 Estimation of the EC ....................................................................................................................................... 8

10.2 Validity criteria ..................................................................................................................................................... 8

11 Expression of results ......................................................................................................................................... 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
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SIST EN ISO 6341:2013
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.
iv © ISO 2012 – All rights reserved
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SIST EN ISO 6341:2013
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.

© ISO 2012 – All rights reserved v
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SIST EN ISO 6341:2013
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SIST EN ISO 6341:2013
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;
— treated or untreated waste water;
— aqueous extracts and leachates;
— 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
[3]
[ISO 20665:2008, 3.3]
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SIST EN ISO 6341:2013
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

concentration at which there is an effect on 50 % of the organisms in line with the test criterion

[1]
[ISO 15088:2007, 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.

[3]
[ISO 20665:2008, 3.6]
3.6
test batch
series of replicates filled with the same test solution
[3]
[ISO 20665:2008, 3.8]
4 Principle

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 exposed D. magna, under the conditions specified in this International

Standard. This concentration, known as the effective initial inhibitory concentration, is designated 24 h EC

or 48 h EC .

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 EC 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 EC or 48 h EC ;
50 50

— a definitive test, conducted when the approximate value given by the preliminary test is not sufficient,

which permits calculation of the 24 h EC or 48 h EC , and determines concentrations corresponding to

50 50
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.
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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

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.

6.2 Pure water, conductivity below 10 µS/cm.
6.3 Dilution and culturing water.

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 CaCO ) 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 CaCO ), 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.

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6.3.2 Calcium chloride solution. Dissolve 11,76 g of calcium chloride dihydrate (CaCl⋅2H O) in pure water

2 2
(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 (MgSO⋅7H O) in

4 2
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 (NaHCO ) 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 (K Cr O ) is recommended.
2 2 7

Since K Cr O is a carcinogenic substance, toxic via inhalation, the use of a ready-made solution with a

2 2 7

defined concentration of K Cr O for the preparation of the stock solution of the reference substance can

2 2 7
reduce the risk of inhalation of the toxic dust in the laboratory.
6.5 Sodium hydroxide solution, e.g. [NaOH] = 1 mol/l.
6.6 Hydrochloric acid, e.g. [HCl] = 1 mol/l.
7 Apparatus and materials
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.

4 © ISO 2012 – All rights reserved
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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

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.

Usually no aeration of sample or prepared test concentrations is necessary. If, and only if, the dissolved oxygen

is <40 % saturation, a pre-aerate of the 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.
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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 substa

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