1、BS EN ISO15088:2008ICS 13.060.70NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDWater quality Determination of theacute toxicity of wastewater to zebrafisheggs (Danio rerio) (ISO15088:2007)This British Standardwas published under theauthority of the StandardsPol
2、icy and StrategyCommittee on 31 December2008 BSI 2008ISBN 978 0 580 54630 3Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 15088:2008National forewordThis British Standard is the UK implementation of EN ISO 15088:2008.It is identical to ISO 15088:2007.The UK participation in its
3、 preparation was entrusted to TechnicalCommittee EH/3/5, Biological Methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct a
4、pplication.Compliance with a British Standard cannot confer immunityfrom legal obligations.EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 15088November 2008ICS 13.060.70English VersionWater quality - Determination of the acute toxicity of waste waterto zebrafish eggs (Danio rerio) (ISO 15088:
5、2007)Qualit de leau - Dtermination de la toxicit aigu deseaux rsiduaires vis-vis des oeufs de poisson-zbre(Danio rerio) (ISO 15088:2007)Wasserbeschaffenheit - Bestimmung der akuten Toxizittvon Abwasser auf Zebrafisch-Eier (kursiv(Danio rerio) (ISO15088:2007)This European Standard was approved by CEN
6、 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 standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards ma
7、y 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 other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN
8、 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, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands,
9、Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploitation in any form
10、 and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 15088:2008: EBS EN ISO 15088:2008EN ISO 15088:2008 (E) 3 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) an
11、d has been taken over as EN ISO 15088:2008 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
12、 conflicting national standards shall 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. Accordin
13、g to the CEN/CENELEC Internal Regulations, 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,
14、Lithuania, Luxembourg, Malta, Netherlands, 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. BS EN ISO 15088:2008ISO 15088:2
15、007(E) ISO 2007 All rights reserved iiiContents Page Foreword iv Introduction.v 1 Scope1 2 Normative references1 3 Terms and definitions .1 4 Interferences 2 5 Principle3 6 Reagents.3 7 Apparatus.3 8 Procedure.4 9 Determination of toxicological endpoints.7 10 Validity criteria.8 11 Determination of
16、LIDegg9 12 Expression of results9 13 Test report9 14 Reproducibility 9 Annex A (informative) Scheme for layout and distribution of eggs in microplates.11 Bibliography12 BS EN ISO 15088:2008ISO 15088:2007(E) iv ISO 2007 All rights reservedForeword ISO (the International Organization for Standardizati
17、on) 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 re
18、presented 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
19、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 Internati
20、onal 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 15088 was p
21、repared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. BS EN ISO 15088:2008ISO 15088:2007(E) ISO 2007 All rights reserved vIntroduction Fish play a major role in the aquatic food web. They are high-order consumers (often final consumers) and have important f
22、unctions 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 rerio Hamilton-Buchanan) are used as test material. The zebrafish belongs to the family of Cyprinidae (carp-related
23、 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 embryonic development is well described. The zebrafish is one of the most important model fish in research on the develo
24、pmental 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 constituents and effluents. Death of embryos and certain defined disturbances of embryonic development, which finally lead
25、to death, are considered effects. BS EN ISO 15088:2008BS EN ISO 15088:2008INTERNATIONAL STANDARD ISO 15088:2007(E) ISO 2007 All rights reserved 1Water quality Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio) WARNING Persons using this International Standard shall be
26、 familiar with normal laboratory practice. This 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 regulat
27、ory conditions. IMPORTANT It is absolutely essential 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 es
28、tablished. 1 Scope This International Standard 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 indu
29、strial effluents. NOTE This International Standard 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, differen
30、t sensitivities from both test systems are possible. 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 (includin
31、g 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 7346-1, Water quality Determination of the acute lethal toxicity of substances to a freshwater fish (Br
32、achydanio rerio Hamilton-Buchanan (Teleostei, 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
33、the purposes of this document, the following terms and definitions apply. 3.1 control dilution water with fertilized fish eggs BS EN ISO 15088:2008ISO 15088:2007(E) 2 ISO 2007 All rights reserved3.2 dilution level D reciprocal value of the volume fraction of waste water in dilution water in which th
34、e test is conducted EXAMPLE 250 ml of waste water in a total volume 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 Stand
35、ard, LC50is the lethal concentration at which 50 % of the organisms 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”, immed
36、iately after spawning; “freshly fertilized”, between 4-cell stage 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
37、 inhibition, or only effects not exceeding the test-specific variability, 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 b
38、atch defined dilutions with fertilized fish eggs 4 Interferences 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 fertil
39、ized 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. BS EN ISO 15088:2008ISO 15088:
40、2007(E) ISO 2007 All rights reserved 35 Principle In a dilution series, waste water graduated to integral volume ratio is mixed with dilution water giving defined dilution levels, D,. After exposure of fertilized fish eggs to the test batches for 48 h using microplates, the dilution limit in which n
41、o acute toxic effect occurs is determined (LID). At 26 C, the embryos hatch after 72 h to 96 h. The test duration is 48 h. As a positive control, a solution of 3,7 mg/l of the reference substance, 3,4-dichloroaniline, is tested with 10 fertilized eggs (8.3). This International Standard may also be u
42、sed to calculate dose-response-based EC50values as percentages of waste water without changing the test design. 6 Reagents As far as available, use only reagent grade chemicals. 6.1 Water, deionized or of equivalent purity (conductivity 10 %, see 8.3). As a positive control, a defined concentration
43、of reference substance (3,7 mg/l of 3,4-dichloroaniline) should be tested with 10 fertilized eggs (8.3) in parallel when the test is carried out. BS EN ISO 15088:2008ISO 15088:2007(E) ISO 2007 All rights reserved 911 Determination of LIDeggSummarize the number of dead eggs for each test batch after
44、48 h (9.2). If one of the criteria described in 9.4 is fulfilled, the egg is considered dead. The test result is the lowest tested value for D (LID) in which at least 90 % of the eggs have survived. This dilution level is specified as the result. If more than one egg dies in a microplate control, th
45、e respective microplate should be excluded from calculation. Mortality in microplate controls has not been considered within validity criteria. They give some indication of unsuitable microplates. Normally, mortality in microplate controls does not occur ( 0,1 %). If so, plausibility has to be check
46、ed. In case of doubt, the microplate is excluded from the evaluation of the test result. 12 Expression of results Values for LID are reported as integral numbers. EXAMPLE LIDegg= 2 It is generally possible to calculate EC50values from dilution series of different volume fractions of waste water. In
47、this case, ISO/TS 20281 should be taken into account. 13 Test report This clause specifies which information shall be included in the test report. It is required that information is given on at least the following aspects of the test: a) a reference to this International Standard (ISO 15088); b) ide
48、ntification of the method; c) identity of waste water; d) pH and oxygen concentration in milligrams per litre (or percentage saturation) of the original sample and the dilutions, if necessary, at the beginning of test; e) report of test results according to Clause 12; f) any deviation from the test
49、protocol and report of all marginal conditions of relevance according to the test result. 14 Reproducibility An interlaboratory trial based on the method described in this International Standard was carried out in 2000. Results with the reference substance 3,4-dichloroaniline and the percentage of mortality at a given concentration (3,7 mg/l of 3,4-dichloroaniline) are given in Table 2. Additional information referring to the test procedure and comparisons between the acute fish test and the fish egg test are
