SIST EN ISO 6341:2013
(Main)Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity test (ISO 6341:2012)
Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity test (ISO 6341:2012)
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.
Wasserbeschaffenheit - Bestimmung der Hemmung der Beweglichkeit von Daphnia magna Straus (Cladocera, Crustacea) - Akuter Toxizitäts-Test (ISO 6341:2012)
Diese Internationale Norm beschreibt ein Verfahren zur Bestimmung der akuten Toxizität gegenüber Daphnia magna Straus (Cladocera, Crustacea). 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.
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
Relations
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.
---------------------- Page: 1 ----------------------
SIST EN ISO 6341:2013
---------------------- Page: 2 ----------------------
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.
---------------------- Page: 3 ----------------------
SIST EN ISO 6341:2013
EN ISO 6341:2012 (E)
Contents Page
Foreword .3
2
---------------------- Page: 4 ----------------------
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.
3
---------------------- Page: 5 ----------------------
SIST EN ISO 6341:2013
---------------------- Page: 6 ----------------------
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
---------------------- Page: 7 ----------------------
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 copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved
---------------------- Page: 8 ----------------------
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
50
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
---------------------- Page: 9 ----------------------
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
---------------------- Page: 10 ----------------------
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
---------------------- Page: 11 ----------------------
SIST EN ISO 6341:2013
---------------------- Page: 12 ----------------------
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]
© ISO 2012 – All rights reserved 1
---------------------- Page: 13 ----------------------
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
EC
50
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
50
or 48 h EC .
50
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.
50
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.
2 © ISO 2012 – All rights reserved
---------------------- Page: 14 ----------------------
SIST EN ISO 6341:2013
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
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.
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.
3
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
3
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 2012 – All rights reserved 3
---------------------- Page: 15 ----------------------
SIST EN ISO 6341:2013
ISO 6341:2012(E)
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)
3
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
2)
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
---------------------- Page: 16 ----------------------
SIST EN ISO 6341:2013
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.
© ISO 2012 – All rights reserved 5
---------------------- Page: 17 ----------------------
SIST EN ISO 6341:2013
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
...
SLOVENSKI STANDARD
oSIST prEN ISO 6341:2011
01-marec-2011
.DNRYRVWYRGH'RORþDQMH]DYLUDQMDJLEDQMD'DSKQLDPDJQD6WUDXV &ODGRFHUD
&UXVWDFHD $NXWQLSUHVNXVVWUXSHQRVWL ,62',6
Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus
(Cladocera, Crustacea) - Acute toxicity test (ISO/DIS 6341:2010)
Wasserbeschaffenheit - Bestimmung der Hemmung der Beweglichkeit von Daphnia
magna Straus (Cladocera, Crustacea) - Akuter Toxizitäts-Test (ISO/DIS 6341:2010)
Qualité de l'eau - Détermination de l'inhibition de la mobilité de Daphnia magna Straus
(Cladocera, Crustacea) - Essai de toxicité aiguë (ISO/DIS 6341:2010)
Ta slovenski standard je istoveten z: prEN ISO 6341
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
oSIST prEN ISO 6341:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
oSIST prEN ISO 6341:2011
---------------------- Page: 2 ----------------------
oSIST prEN ISO 6341:2011
EUROPEAN STANDARD
DRAFT
prEN ISO 6341
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2010
ICS 13.060.70 Will supersede 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/DIS 6341:2010)
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/DIS 6341:2010) Crustacea) - Akuter Toxizitäts-Test (ISO/DIS 6341:2010)
This draft European Standard is submitted to CEN members for parallel enquiry. It has been drawn up by the Technical Committee
CEN/TC 230.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to
provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2010 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN ISO 6341:2010: E
worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
oSIST prEN ISO 6341:2011
prEN ISO 6341:2010 (E)
Contents Page
Foreword .3
2
---------------------- Page: 4 ----------------------
oSIST prEN ISO 6341:2011
prEN ISO 6341:2010 (E)
Foreword
This document (prEN ISO 6341:2010) 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 document is currently submitted to the parallel Enquiry.
This document will supersede EN ISO 6341:1996.
Endorsement notice
The text of ISO/DIS 6341:2010 has been approved by CEN as a prEN ISO 6341:2010 without any
modification.
3
---------------------- Page: 5 ----------------------
oSIST prEN ISO 6341:2011
---------------------- Page: 6 ----------------------
oSIST prEN ISO 6341:2011
DRAFT INTERNATIONAL STANDARD ISO/DIS 6341
ISO/TC 147/SC 5 Secretariat: DIN
Voting begins on: Voting terminates on:
2010-11-11 2011-04-11
INTERNATIONAL ORGANIZATION FOR STANDARDIZATION • МЕЖДУНАРОДНАЯ ОРГАНИЗАЦИЯ ПО СТАНДАРТИЗАЦИИ • ORGANISATION INTERNATIONALE DE NORMALISATION
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
[Revision of third edition (ISO 6341:1996) and ISO 6341:1996/Cor.1:1998]
ICS 13.060.70
ISO/CEN PARALLEL PROCESSING
This draft has been developed within the International Organization for Standardization (ISO), and
processed under the ISO-lead mode of collaboration as defined in the Vienna Agreement.
This draft is hereby submitted to the ISO member bodies and to the CEN member bodies for a parallel
five-month enquiry.
Should this draft be accepted, a final draft, established on the basis of comments received, will be
submitted to a parallel two-month approval vote in ISO and formal vote in CEN.
In accordance with the provisions of Council Resolution 15/1993 this document is circulated in
the English language only.
Conformément aux dispositions de la Résolution du Conseil 15/1993, ce document est distribué
en version anglaise seulement.
To expedite distribution, this document is circulated as received from the committee secretariat.
ISO Central Secretariat work of editing and text composition will be undertaken at publication
stage.
Pour accélérer la distribution, le présent document est distribué tel qu'il est parvenu du
secrétariat du comité. Le travail de rédaction et de composition de texte sera effectué au
Secrétariat central de l'ISO au stade de publication.
THIS DOCUMENT IS A DRAFT CIRCULATED FOR COMMENT AND APPROVAL. IT IS THEREFORE SUBJECT TO CHANGE AND MAY NOT BE
REFERRED TO AS AN INTERNATIONAL STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS BEING ACCEPTABLE FOR INDUSTRIAL, TECHNOLOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN NATIONAL REGULATIONS.
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT, WITH THEIR COMMENTS, NOTIFICATION OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPORTING DOCUMENTATION.
©
International Organization for Standardization, 2010
---------------------- Page: 7 ----------------------
oSIST prEN ISO 6341:2011
ISO/DIS 6341
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall
not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the
unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
Copyright notice
This ISO document is a Draft International Standard and is copyright-protected by ISO. Except as permitted
under the applicable laws of the user's country, neither this ISO draft nor any extract from it may be
reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, photocopying,
recording or otherwise, without prior written permission being secured.
Requests for permission to reproduce should be addressed to 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 copyright@iso.org
Web www.iso.org
Reproduction may be subject to royalty payments or a licensing agreement.
Violators may be prosecuted.
©
ii ISO 2010 – All rights reserved
---------------------- Page: 8 ----------------------
oSIST prEN ISO 6341:2011
ISO/DIS 6341
Contents Page
Foreword .iv
Introduction.iv
1 Scope.1
2 Normative references.1
3 Terms and definitions .2
4 Principle.2
5 Test environment.3
6 Reagents, test organisms and media.3
7 Apparatus and materials.5
8 Treatment and preparation of samples .5
9 Procedure.7
10 Interpretation and validity of the results.8
11 Expression of results.9
12 Test report.9
Annex A (informative) Preparation of the ELENDT M4 medium.11
Annex B (informative) Example of 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.14
Annex C (informative) References for selected Daphnia magna culture methods and general
recommendations for stock culturing.17
Annex D (informative) Culturing of Daphnia magna for production of dormant eggs .18
Annex E (informative) Precision data .20
Annex F (informative) Dilution level D Preparation of the dilution series for the determination of
the LID.21
Bibliography.23
© ISO 2010 – All rights reserved iii
---------------------- Page: 9 ----------------------
oSIST prEN ISO 6341:2011
ISO/DIS 6341
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.
iv © ISO 2010 – All rights reserved
---------------------- Page: 10 ----------------------
oSIST prEN ISO 6341:2011
ISO/DIS 6341
Introduction
This International Standard defines 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 immobilisation of the
water-flea Daphnia magna Straus after 24 h exposure to the toxicant under the conditions defined in this
International Standard.
© ISO 2010 – All rights reserved v
---------------------- Page: 11 ----------------------
oSIST prEN ISO 6341:2011
---------------------- Page: 12 ----------------------
oSIST prEN ISO 6341:2011
DRAFT INTERNATIONAL STANDARD ISO/DIS 6341
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 standard 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 lnternational Standard describes 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.
2 Normative references
The following referenced documents are indispensable for the application of this document. 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, 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
© ISO 2010 – All rights reserved 1
---------------------- Page: 13 ----------------------
oSIST prEN ISO 6341:2011
ISO/DIS 6341
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
control batch
series of replicates containing the solution employed as negative control
3.2
immobilisation
comparison of the number of mobile Daphnia magna at the end of the test between the control batch (3.1) and
the test batch (3.5)
NOTE Those which are not able to swim after gentle agitation of the liquid for 15 s shall be considered to be
immobilised, even if they can still move their antennae.
3.3
EC50
estimated concentration of a test sample giving rise to 50 % immobilisation with respect to the control batch
(3.1)
3.4
neonate
newly born or newly hatched individual
NOTE In this International Standard, a neonate is a first-instar daphnid, < 24 h old.
3.5
test batch
series of replicates filled with the same test solution
4 Principle
Determination of the initial concentration (that is the concentration present at the beginning of the test) which,
in 24 h, immobilises 50 % of exposed Daphnia magna, under the conditions defined in this International
Standard. This concentration, known as the effective initial inhibitory concentration, is designated 24 h - EC50.
If possible and if necessary, the initial concentration which immobilises 50 % of the exposed Daphnia magna
in 48 h can also be determined. This concentration is designated 48 h - EC50.
An indication of the lowest concentration tested which immobilises all the Daphnia magna and the highest
concentration tested which does not immobilise any of the Daphnia magna is desirable and provides useful
information in cases where the 24 h - EC50, and, where appropriate, the 48 h - 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 final toxicity test and
gives an approximate value of the 24 h - EC50, and, where appropriate, the 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, and, where appropriate, the 48 h - EC50, and determines
concentrations corresponding to 0 % and 100 % immobilisation.
If the method described in this International Standard is used for 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). A limit test may also be performed at concentrations above
100 mg/l, if this provides useful information, as long as the substance is soluble or in stable dispersion.
2 © ISO 2010 – All rights reserved
---------------------- Page: 14 ----------------------
oSIST prEN ISO 6341:2011
ISO/DIS 6341
Analysis of these substances should be conducted as soon as possible. The concentration of the test
substance is analysed at the beginning of the test and at the end of the test as a minimum at the lowest and
highest concentration. If these analyses show that, for each concentration tested, the deviation of the
concentrations measured during the test, randomly varying, is not greater than 20 % of the mean, the EC50
may be calculated from the means of the measurements rather than from the initial concentrations; they are
then designated 24 h - EC50 and, where appropriate, 48 h - EC50.
5 Test environment
The exposure of organisms as described 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.
The testing atmosphere shall be free from vapours or dusts toxic to Daphnia magna. Photo-degradable
chemicals shall be tested in the dark, or using the specified photoperiod, but with minimal red lighting, as
appropriate.
The use of negative controls is a double check that the test is performed in an atmosphere free from toxic
dusts and vapours.
6 Reagents, test organisms and media
Use only reagents of recognised analytical grade, unless otherwise specified.
6.1 Test organisms
The test organisms are neonates of Daphnia 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. Transfer
gravid females to vessels with fresh dilution water 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 so as to avoid stressing the parent animals.
The age 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 Daphnia magna may also derive from the hatching of ephippia obtained from laboratory cultures of the
1)
crustacean as described in Annex D or can be purchased from a specialised company . The neonates
hatched from the ephippia may be used directly as test organisms if they comply with all criteria and
conditions set by this International Standard for test organisms taken from laboratory cultures.
6.2 Pure water, having a conductivity below 10 µS/cm.
6.3 Dilution water
Dissolve known quantities of reagents of recognised analytical grade in pure water (6.2) The dilution water
thus prepared shall have a pH of 7,8 ± 0,5, a hardness of (225 ± 50) mg/l (expressed as CaCO ), a molar
3
Ca/Mg ratio close to 4:1 and a dissolved oxygen concentration above 7 mg/l.
1) MicroBioTests Inc., Mariakerke, Belgium, is an example of a supplier able to provide suitable Daphnia magna
ephippia. 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 2010 – All rights reserved 3
---------------------- Page: 15 ----------------------
oSIST prEN ISO 6341:2011
ISO/DIS 6341
Natural waters with pH and hardness characteristics similar to dilution water may be used for culturing.
Natural water (surface or ground water), reconstituted water or dechlorinated tap water are acceptable as
holding and dilution water if daphnids will survive 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 set
by this International Standard.
As an example, the preparation of water meeting these requirements is described below:
Prepare the following solutions:
6.3.1 Calcium chloride solution
Dissolve 11,76 g of calcium chloride dihydrate (CaCl · 2 H O) in pure water (6.2) and make up to 1 l with
2 2
pure water (6.2).
6.3.2 Magnesium sulfate solution
Dissolve 4,93 g of magnesium sulfate heptahydrate (MgSO · 7 H O) in pure water (6.2) and make up to 1 l
4 2
with pure water (6.2).
6.3.3 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).
3
6.3.4 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).
Mix 25 ml of each of the four solutions (6.3.1 to 6.3.4) 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 stabilised. 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.
For stock culture of Daphnia magna in the laboratory the M4-Medium (see Annex A) may also be used.
NOTE 1 M4-Medium (Annex A) is not suitable as dilution water for the acute tests, because on account of the high
hardness of the test medium and of the presence of EDTA within the M4-medium the bioavailability of the bivalent metal
ions can be reduced, thus resulting in a decrease in the apparent toxicity of these ions. Media containing chelating agents
such as Elendt M4 should not be used for test samples containing metals.
NOTE 2 If the test is performed for purposes necessitating the use of a dilution water with characteristics differing from
those described above, the main characteristics of the synthetic dilution water used should be stated in the test report.
6.4 Reference substance
Potassium dichromate (K Cr O ) of recognised analytical grade is the recommended reference chemical.
2 2 7
NOTE Since K Cr O is a carcinogenic substance, which might be toxic via inhalation, the use of a commercially
2 2 7
available solution with a defined concentration of K Cr O (Titrisol) for the preparation of the stock solution of the
2 2 7
reference substance might 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.
4 © ISO 2010 – All rights reserved
---------------------- Page: 16 ----------------------
oSIST prEN ISO 6341:2011
ISO/DIS 6341
7 Apparatus and materials
Usual laboratory apparatus and glassware 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 (for example 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) may be used to transfer the adult organisms to stock
culture and to separate the young from the adults.
8 Treatment and preparation of samples
8.1 Special precautions for sampling, transportation, storage and treatment of water,
effluent, 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, ideally 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 Clause 5 in ISO 5667-16).
Immediately measure 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, homogenise the sample to be analysed by shaking manually. High concentrations of
suspended inorganic or organic solids in a sample might be harmful to filter-feeding daphnids. This
interference can be compensated by a sample treatment for turbidity. If necessary, allow to settle for at most
2 h in a container, and sample e.g by drawing off (using a pipette) the required quantity of supernatant,
maintaining the end of the pipette in the centre of the section of the test tube and half way 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 5 000 g or filter the raw or decanted sample. Test
the residual toxicity in the liquid part of the sample. The particular kind of filters to be used should be checked
by a test with negative 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 might 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 2010 – All rights reserved 5
---------------------- Page: 17 ----------------------
oSIST prEN ISO 6341:2011
ISO/DIS 6341
Normally no aeration of sample or prepared test concentrations is necessary. If, and only if the dissolved
oxygen is < 40 % saturation, it is optional to pre-aerate the sample or all test solutions fo
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.