1、June 2009DEUTSCHE NORM English price group 11No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 13.060.70!$X8p“1532177www
2、.din.deDDIN EN ISO 15088Water quality Determination of the acute toxicity of waste water to zebrafish eggsEnglish version of DIN EN ISO 15088:2009-06Wasserbeschaffenheit (ISO 15088:2007)Englische Fassung DIN EN ISO 15088:2009-06SupersedesDIN 38415-6:2003-08www.beuth.de(Danio rerio) (ISO 15088:2007)B
3、estimmung der akuten Toxizitt von Abwasser auf Zebrafisch-Eier (Danio rerio)Document comprises 20 pagesDIN EN ISO 15088:2009-06 This standard is part of the series Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlamm-untersuchung Suborganismische Testverfahren (Gruppe T) (German standard me
4、thods for the examination of water, waste water and sludge Bio-assays with microorganisms (group T) and describes method T 6. National foreword This standard has been prepared by Technical Committee ISO/TC 147 “Water quality” in collaboration with Technical Committee CEN/TC 230 “Water analysis” (Sec
5、retariat: DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Wasserwesen (Water Practice Standards Committee), Technical Committees NA 119-01-03-05-01 AK Biotests and NA 119-01-03 AA Wasseruntersuchung. This standard has been published to implement the Wat
6、er Framework Directive (WFD), Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy1). Designation of method T 6 “Determination of the acute toxicity of waste water to zebrafish eggs (Danio rer
7、io)”: Method DIN EN ISO 15088 T 6 Expert assistance and specialized laboratories will be required to perform the analysis described in this standard. Existing safety regulations are to be taken into account. Depending on the objective of the analysis, a check shall be made on a case-by-case basis as
8、 to whether and to what extent additional conditions will have to be specified. Standard methods published as DIN Standards are obtainable from Beuth Verlag GmbH, either individually or grouped in volumes. The standard methods included in the loose-leaf publication entitled Deutsche Einheitsverfahre
9、n zur Wasser-, Abwasser- und Schlammuntersuchung will continue to be published jointly by Wiley-VCH Verlag and Beuth Verlag GmbH. All standard methods relevant to the Abwasserverordnung (Waste Water Regulation) (AbwV) included in the new Regulation on Section 7a of the Gesetz zur Ordnung des Wasserh
10、aushaltes (German Water Management Act) concerning Anforderungen an das Einleiten von Abwasser in Gewsser together with the Abwasserverordnung and the Gesetz zur Ordnung des Wasserhaushalts have been published by Beuth-Verlag GmbH as a loose-leaf collection Analysenverfahren in der Abwasserverordnun
11、g Rechtsvorschriften und Normen (Supplements). Standard methods or draft standards bearing the group title “German standard methods for the examination of water, waste water and sludge” are classified under the following categories (main titles): 1) Registered in the DITR database of DIN Software Gm
12、bH, obtainable from: Beuth Verlag GmbH, 10772 Berlin. 2 DIN EN ISO 15088:2009-06 General information (group A) (DIN 38402) Sensory analysis (group B) (DIN 38403) Physical and physicochemical parameters (group C) (DIN 38404) Anions (group D) (DIN 38405) Cations (group E) (DIN 38406) Substance group a
13、nalysis (group F) (DIN 38407) Gaseous constituents (group G) (DIN 38408) Parameters characterizing effects and substances (group H) (DIN 38409) Biological-ecological methods of analysis (group M) (DIN 38410) Microbiological methods (group K) (DIN 38411) Test methods using water organisms (group L) (
14、DIN 38412) Individual constituents (group P) (DIN 38413) Sludge and sediments (group S) (DIN 38414) Bio-assays with microorganisms (group T) (DIN 38415) In addition to the methods described in the DIN 38402 to DIN 38415 series of standards, there are a number of European and International Standards
15、available as DIN EN, DIN EN ISO and DIN ISO Standards, which also form part of the collection of German standard methods. Information on Parts of these series of standards that have already been published can be obtained from the offices of the Normenausschuss Wasserwesen, telephone +49 30 2601-2448
16、, or from Beuth Verlag GmbH, Burggrafenstr. 6, 10787 Berlin. The DIN Standards corresponding to the International Standards referred to in the EN are as follows: ISO 5667-16 DIN EN ISO 5667-16 ISO 5814 DIN EN 25814 ISO 7346-1 DIN EN ISO 7346-1 ISO 7346-2 DIN EN ISO 7346-2 Amendments This standard di
17、ffers from DIN 38415-6:2003-08 as follows: a) The title of the standard has been modified. b) The scope has been modified to cover the acute toxicity of substances. c) Table 1 has been extended to include the portion of sample in D. d) Figures 1 and 2 have been modified. e) Annex A “Scheme for layou
18、t and distribution of eggs in microplates“ has been added. f) The standard has been editorially revised. Previous editions DIN 38415-6: 2001-09, 2003-08 3 DIN EN ISO 15088:2009-06 National Annex NA (informative) Bibliography DIN EN 25814, Water quality Determination of dissolved oxygen by the electr
19、ochemical probe method DIN EN ISO 5667-16, Water quality Sampling Part 16: Guidance on biotesting of samples DIN EN ISO 7346-1, Water quality Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Part 1: Static metho
20、d DIN EN ISO 7346-2, Water quality Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Part 2: Semi-static method 4 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 15088 November 2008 ICS 13.060.70 English
21、 Version Water quality Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio) (ISO 15088:2007) Qualit de leau Dtermination de la toxicit aigu des eaux rsiduaires vis-vis des oeufs de poisson-zbre (Danio rerio) (ISO 15088:2007) Wasserbeschaffenheit Bestimmung der akuten To
22、xizitt von Abwasser auf Zebrafisch-Eier (Danio rerio) (ISO 15088:2007) This European Standard was approved by CEN on 29 October 2008. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national sta
23、ndard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any othe
24、r language made by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia,
25、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. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES
26、 KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 15088:2008: EContents Page Foreword 3 Introduction.4 1 Scope5 2 Normative references5 3 Terms and d
27、efinitions .5 4 Interferences 6 5 Principle7 6 Reagents.7 7 Apparatus.7 8 Procedure.8 9 Determination of toxicological endpoints.11 10 Validity criteria.12 11 Determination of LIDegg13 12 Expression of results13 13 Test report13 14 Reproducibility 14 Annex A (informative) Scheme for layout and distr
28、ibution of eggs in microplates.15 Bibliography16 EN ISO 15088:2008 (E) 2DIN EN ISO 15088:2009-06 Foreword The text of ISO 15088:2007 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 15088
29、:2008 by 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 May 2009, and conflicting national standards shall
30、 be withdrawn at the latest by May 2009. 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. According to the CEN/CENELEC Internal Regulat
31、ions, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherl
32、ands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 15088:2007 has been approved by CEN as a EN ISO 15088:2008 without any modification. EN ISO 15088:2008 (E) 3DIN EN ISO 15088:2009-06 Introduction Fish pl
33、ay a major role in the aquatic food web. They are high-order consumers (often final consumers) and have important functions of regulation in aquatic ecosystems. They are a confirmed part within test concepts regarding aquatic organisms from different trophic levels. The eggs of the zebrafish (Danio
34、rerio Hamilton-Buchanan) are used as test material. The zebrafish belongs to the family of Cyprinidae (carp-related fish) within the class of Osteichthyes (teleost fish). Zebrafish are easy to keep and produce transparent, nonadhesive eggs (diameter about 1 mm) throughout the whole year. Their embry
35、onic development is well described. The zebrafish is one of the most important model fish in research on the developmental biology of vertebrates and is recommended as a test fish, i.e. in the OECD Guidelines 203, 204 and 210. The development of fertilized fish eggs can be affected by water constitu
36、ents and effluents. Death of embryos and certain defined disturbances of embryonic development, which finally lead to death, are considered effects. EN ISO 15088:2008 (E) 4DIN EN ISO 15088:2009-06 WARNING Persons using this International Standard shall be familiar with normal laboratory practice. Th
37、is International 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 es
38、sential that tests conducted according to this International Standard be carried out by suitably qualified staff. When applying this International Standard, it is necessary in each case to determine if and to what extent additional conditions should be established. 1 Scope This International Standar
39、d specifies a method for the determination of degrees of dilution or of concentrations as a measure of the acute toxic effect of waste water to fish eggs within 48 h. This International Standard is also applicable to treated municipal waste water and industrial effluents. NOTE This International Sta
40、ndard has been elaborated as a substitute for the acute fish toxicity test. Applied to waste water, it gives the same or similar results as achieved from the acute fish toxicity test (e.g. ISO 7346-1 or ISO 7346-2). If used for single substances, different sensitivities from both test systems are po
41、ssible. 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
42、 quality Sampling Part 16: Guidance on biotesting of samples ISO 5814, Water quality Determination of dissolved oxygen Electrochemical probe method ISO 7346-1, Water quality Determination of the acute lethal toxicity of substances to a freshwater fish (Brachydanio rerio Hamilton-Buchanan (Teleostei,
43、 Cyprinidae) Part 1: Static method ISO 7346-2, Water quality Determination of the acute lethal toxicity of substances to a freshwater fish (Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Part 2: Semi-static method 3 Terms and definitions For the purposes of this document, the following
44、terms and definitions apply. 3.1 control dilution water with fertilized fish eggs EN ISO 15088:2008 (E) 5DIN EN ISO 15088:2009-06 3.2 dilution level D reciprocal value of the volume fraction of waste water in dilution water in which the test is conducted EXAMPLE 250 ml of waste water in a total volu
45、me of 1 000 ml (volume fraction of 25 %) represents dilution level D = 4. NOTE See ISO 5667-16. 3.3 EC50concentration at which there is an effect on 50 % of the organisms in line with the test criterion NOTE In this International Standard, LC50is the lethal concentration at which 50 % of the organis
46、ms are dead. 3.4 fish egg generally used term for all development stages of an egg cell (inside the chorion) within this International Standard NOTE If necessary, the following qualifiers for this term are used: “not fertilized”, immediately after spawning; “freshly fertilized”, between 4-cell stage
47、 and 128-cell stage; “embryo”, if the developing embryo is visible inside the egg before hatching. 3.5 lowest ineffective dilution dilution factor LID lowest ineffective dilution tested, expressed as dilution level D (3.2), at which no inhibition, or only effects not exceeding the test-specific vari
48、ability, are observed 3.6 LIDegglowest ineffective dilution within a test batch in which at least 90 % of the fish eggs do not show any effect according to this International Standard NOTE See Clauses 11 and 12 and Figure 2. 3.7 test batch defined dilutions with fertilized fish eggs 4 Interferences
49、The differentiation of eggs can be difficult in the case of heavily coloured and/or turbid waste water. In such cases, the following modified preparation order is used before starting the exposure: spawning; differentiation into fertilized and unfertilized eggs (8.2.2); transfer into dilution water (7.10, crystallization bowls); transfer into microplates without previous exposure. While transferring into microplates, the error in dilution caused by adherent water should be kept as small as possible
