1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 6341:2012W a t e r q u a l i t y D e t e r m i n a t i o n of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) Acute toxicity testBS EN ISO
2、 6341:2012 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 6341:2012. It supersedes BS EN ISO 6341:1996, which is withdrawn.The UK participation in its preparation was entrusted to T e c h n i c a l C o m m i t t e e E H / 3 / 5 , B i o l o g i c a l M e t h
3、 o d s .A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2012. Published by B
4、SI Standards Limited 2012.ISBN 978 0 580 69874 3 ICS 13.060.70 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 October 2012.Amendments issued since publicatio
5、nDate T e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 6341 October 2012 ICS 13.060.70 Supersedes EN ISO 6341:1996English Version Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity test (ISO 6
6、341:2012) Qualit de leau - Dtermination de linhibition de la mobilit de Daphnia magna Straus (Cladocera, Crustacea) - Essai de toxicit aigu (ISO 6341:2012) Wasserbeschaffenheit - Bestimmung der Hemmung der Beweglichkeit von Daphnia magna Straus (Cladocera, Crustacea) - Akuter Toxizitts-Test (ISO 634
7、1:2012) 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 bibliograph
8、ical 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 responsibilit
9、y 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
10、 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 COMIT EUROPEN DE NORMALISATIO
11、N EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 6341:2012: EBS EN ISO 6341:2012EN ISO 6341:2012 (E) 3 Foreword This document (EN ISO 634
12、1:2012) 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 te
13、xt 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
14、 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, Cz
15、ech 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 Kingd
16、om. Endorsement notice The text of ISO 6341:2012 has been approved by CEN as a EN ISO 6341:2012 without any modification. BS EN ISO 6341:2012ISO 6341:2012(E) ISO 2012 All rights reserved iiiContents PageForeword ivIntroduction v1 Scope 12 Normative references . 13 Terms and definitions . 14 Principl
17、e . 25 Test environment . 26 Reagents, test organisms and media 37 Apparatus and materials . 48 Treatment and preparation of samples . 58.1 Special precautions for sampling, transportation, storage and treatment of water, effluent, or aqueous extract samples to be tested . 58.2 Preparation of soluti
18、ons of substances to be tested 69 Procedure 69.1 General . 69.2 Preliminary test 79.3 Definitive test 79.4 Check of the sensitivity of the Daphnia magna and conformity with the procedure . 79.5 Limit test 810 Interpretation and validity of the results . 810.1 Estimation of the EC50. 810.2 Validity c
19、riteria . 811 Expression of results . 812 Test report . 8Annex A (informative) Preparation of the Elendt M4 medium.10Annex 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/
20、l . 13Annex C (informative) General recommendations for stock culturing 16Annex D (informative) Culturing of Daphnia magna for production of dormant eggs 17Annex E (informative) Precision data 19Annex F (informative) Dilution level D Preparation of the dilution series for the determination of the LI
21、D 20Bibliography .22BS EN ISO 6341:2012ISO 6341:2012(E)ForewordISO (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.
22、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 Internat
23、ional 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 Standar
24、ds 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 o
25、f 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 te
26、chnically revised. It also incorporates the Technical Corrigendum ISO 6341:1996/Cor. 1:1998.iv ISO 2012 All rights reservedBS EN ISO 6341:2012ISO 6341:2012(E)IntroductionThis International Standard specifies a procedure for the determination of the acute toxicity of chemicals, waters and waste water
27、s 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 zooplank
28、ton 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 spec
29、ified in this International Standard. ISO 2012 All rights reserved vBS EN ISO 6341:2012BS EN ISO 6341:2012Water quality Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) Acute toxicity testWARNING Persons using this International Standard should be famili
30、ar 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.IMPORT
31、ANT It is absolutely essential that tests conducted in accordance with this International Standard be carried out by suitably qualified staff.1 ScopeThis International Standard specifies a method for the determination of the acute toxicity to Daphnia magna Straus (Cladocera, Crustacea).This method i
32、s 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
33、and ground water); eluates of fresh water sediment; pore water of fresh water sediment.2 Normative referencesThe 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
34、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 samplesISO 5814, Water quality Determination of dissolved oxygen Electrochemical probe methodISO 10523, Water quality Deter
35、mination of pH3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1control batchseries of replicates containing control solutionISO 20665:2008,33.3INTERNATIONAL STANDARD ISO 6341:2012(E) ISO 2012 All rights reserved 1BS EN ISO 6341:2012ISO 6341:2012(
36、E)3.2control solutiontest medium without sample under test3.3immobilizationinability 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 antennae3.4EC50concentration at which there is an effect on 50 % of the org
37、anisms in line with the test criterionISO 15088:2007,13.33.5neonatenewly born or newly hatched individualNOTE In this International Standard, a neonate is a first-instar daphnid, 20 % in 2 d, presence of males, ephippia, or discoloured animals, and there shall be no delay in the production of the fi
38、rst 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 offspr
39、ing.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 obtaine
40、d from laboratory cultures of the crustacean as described in Annex D or can be purchased from a specialized company.1)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, conductiv
41、ity below 10 S/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 showi
42、ng 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 medi
43、um (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
44、 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 w
45、ater 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 + M
46、g 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 consti
47、tute an endorsement by ISO of this supplier. ISO 2012 All rights reserved 3BS EN ISO 6341:2012ISO 6341:2012(E)6.3.2 Calcium chloride solution. Dissolve 11,76 g of calcium chloride dihydrate (CaCl22H2O) in pure water (6.2) and make up to 1 l with pure water (6.2).6.3.3 Magnesium sulfate solution. Dis
48、solve 4,93 g of magnesium sulfate heptahydrate (MgSO47H2O) 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. Di
49、ssolve 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 aera
