1、BRITISH STANDARD BS EN ISO 7346-2:1998 BS 6068-5.3: 1998 Water quality Determination of the acute lethal toxicity of substances to a fresh water fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Semi-static method The European Standard EN ISO 7346-2:1997 has the status of a British St
2、andard ICS 13.060.01BSENISO7346-2:1998 This British Standard, having been prepared under the directionof the Health and Environment Sector Board, was published under the authority of the Standards Board and comes into effect on 15 January 1998 BSI 03-1999 ISBN 0 580 28489 1 Amendments issued since p
3、ublication Amd. No. Date CommentsBSENISO7346-2:1998 BSI 03-1999 i Contents Page National foreword ii Foreword 2 Foreword ii Text of EN ISO 7346-2 1BSENISO7346-2:1998 ii BSI 03-1999 National foreword This British Standard is the English language version of EN ISO7346-2:1997. It is identical with ISO7
4、346-2:1996. It supersedes BS6068-5.3:1985 which is withdrawn. The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/5, Biological methods, which has the responsibility to: aid enquirers to understand the text; present to the responsibl
5、e international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this subcommittee can be obtained on requ
6、est to its secretary. BS EN ISO 7346-2 is one of a series of standards on water quality, others of which have been, or will be, published as Sections of BS6068. This standard has therefore been given the secondary identifier BS6068-5.3. The various Sections of BS6068 are comprised within Parts 1 to
7、7, which, together with Part 0, are listed below. Part 0: Introduction; Part 1: Glossary; Part 2: Physical, chemical and biochemical methods; Part 3: Radiological methods; Part 4: Microbiological methods; Part 5: Biological methods; Part 6: Sampling; Part 7: Precision and accuracy. Cross-references
8、Attention is drawn to the fact that CEN and CENELEC Standards normally include an annex which lists normative references to international publications with their corresponding European publications. The British Standards which implement these international or European publications may be found in th
9、e BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are re
10、sponsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, the EN title page, page 2, the ISO title page, page ii, pages 1 to 8,
11、and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on theinside front cover.EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 7346-2 November 1997 ICS 13.060.01 Descriptors: See ISO docu
12、ment English version Water quality Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton (Teleostei, Cyprinidae) Part 2: Semi-static method (ISO 7346-2:1996) Qualit de leau Dtermination de la toxicit aigu ltale de substances vis-vis dun poisson deau
13、 douce Brachydanio rerio Hamilton-Buchanan (Tlostei, Cyprinidae) Partie 2: Mthode semi-statique (ISO 7346-2:1996) Wasserbeschaffenheit Bestimmung der akuten letalen Toxizitt von Substanzen gegenber einem Swasserfisch Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Teil 2: Semistatisches
14、Verfahren (ISO 7346-2:1996) This European Standard was approved by CEN on 30 October 1997. 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 l
15、ists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat 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 res
16、ponsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Iceland, Ireland, Italy, Luxembourg, Ne
17、therlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1997 CEN All rights of exploitation in any form and by any mea
18、ns reserved worldwide for CEN national Members. Ref. No. EN ISO 7346-2:1997 EENISO7346-2:1997 BSI 03-1999 2 Foreword The text of the International Standard from Technical Committee ISO/TC147 “Water quality” of the International Organization for Standardization (ISO) has been taken over as an Europea
19、n Standard 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 month of May 1998, and conflicting national
20、standards shall be withdrawn at the latest by May1998. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Icelan
21、d, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of the International Standard ISO 7346-2:1996 has been approved by CEN as a European Standard without any modification. Contents Page Foreword 2 Foreword ii In
22、troduction 1 1 Scope 1 2 Principle 1 3 Test organism and reagents 2 3.1 Test organism 2 3.2 Standard dilution water 2 3.3 Stock solutions of test substances 2 3.4 Test solutions 2 4 Apparatus 3 5 Test environment 3 6 Procedure 3 6.1 Condition of the fish 3 6.2 Limit test 3 6.3 Preliminary test 3 6.4
23、 Final test 4 7 Expression of results 4 7.1 Validity 4 7.2 Estimation of LC50 4 8 Test report 5 Annex A (informative) Environment parameters for maintenance and breeding of zebra fish (Brachydanio rerio Hamilton-Buchanan) 6 Annex B (informative) Suggested form for recording date 7 Annex C (informati
24、ve) Bibliography 8 Figure 1 Graphical interpolation of LC50 (liner scales) 5 Figure 2 Graphical interpolation of LC 50 (logarithmic and probability scales) 5ENISO7346-2:1997 ii Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (I
25、SO 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, gov
26、ernmental 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. Draft International Standards adopted by the technical committees are circulated to t
27、he member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard ISO 7346-2 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. This second edition ca
28、ncels and replaces the first edition (ISO 7346-2:1984), which has been technically revised. ISO 7346 consists of the following parts, under the general title Water quality Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cy
29、prinidae): Part 1: Static method; Part 2: Semi-static method; Part 3: Flow-through method. Annex A, Annex B and Annex C of this part of ISO 7346 are for information only. Descriptors: Water, quality, water pollution, tests, water tests, biological tests, determination, toxicity.ENISO7346-2:1997 BSI
30、03-1999 1 Introduction The three parts of ISO 7346 describe methods of determining the acute lethal toxicity of substances to the zebra fish (Brachydanio rerio Hamilton-Buchanan) but it must be emphasized that the recommended use of the zebra fish does not preclude the use of other species. The meth
31、odologies presented here may also be used for other species of freshwater, marine or brackish water fish, with appropriate modifications of, for example, dilution water quality and the temperature conditions of the test. Within the three parts of ISO 7346, a choice can be made between static, semi-s
32、tatic and flow-through methods. The static test, described in ISO 7346-1, in which the solution is not renewed, has the advantage of requiring simple apparatus, although the substances in the test vessel may become depleted during the course of the test and the general quality of the water may deter
33、iorate. The flow-through method, described in ISO7346-3, in which the test solution is replenished continuously, overcomes such problems but requires the use of more complex apparatus. In the semi-static procedure, described in this part of ISO 7346, the test solutions are renewed every 24h or 48h,
34、this method being a compromise between the other two. The flow-through method can be used for most types of substances, including those unstable in water, but the concentrations of the test substance are determined wherever possible. The static method is limited to the study of substances whose test
35、ed concentrations remain relatively constant during the test period. The semi-static method can be used for testing those substances whose concentrations can be maintained satisfactorily throughout the test by renewal of the solutions every 24h or 48h. Special arrangements may be necessary for subst
36、ances which are highly volatile. To assist in the preparation and maintenance of concentrations of substances which may be lethal at concentrations close to that of their aqueous solubility, a small volume of solvent may be used, as specified in the methods. 1 Scope This part of ISO 7346 specifies a
37、 semi-static method for the determination of the acute lethal toxicity of stable, non-volatile, single substances, soluble in water under specified conditions, to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) common name, zebra fish in water of a specified quality. Th
38、e method is applicable for assigning, for each test substance, broad categories of acute lethal toxicity to Brachydanio rerio under the test conditions. The results are insufficient by themselves to define water quality standards for environmental protection. The method is also applicable when using
39、 certain other species of freshwater fish as the test organism 1) . The method may be adapted for use with other freshwater fish and marine and brackish water fish with appropriate modification of the test conditions, particularly with respect to the quantity and quality of the dilution water and th
40、e temperature. 2 Principle Determination, under specified conditions, of the concentrations at which a substance is lethal to50% of a test population of Brachydanio rerio after exposure periods of24h, 48h, 72h and 96h tothat substance in the ambient water. These median lethal concentrations are desi
41、gnated the24h LC50, 48h LC50, 72h LC50 and 96h LC50. The test is carried out in two stages: a) a preliminary test which gives an approximate indication of the acute median lethal concentrations and serves to determine the range of concentrations for the final test; b) a final test, the results of wh
42、ich alone are reported. 1) The following species of freshwater fish can be used, in addition to Brachydanio rerio, without modification to this part of ISO7346. Lepomis macrochirus (Teleostei, Centrarchidae) Oryzias latipes (Teleostei, Poeciliidae) Pimephales promelas (Teleostei, Cyprinidae) Poecili
43、a reticulata (Teleostei, Poeciliidae)ENISO7346-2:1997 2 BSI 03-1999 Where evidence is available to show that test concentrations remain relatively constant (i.e. within about 20 % of the nominal values) throughout the test, then either measured or nominal concentrations are used in the estimation of
44、 the LC50. Where such analyses show that the concentrations present remain relatively constant but are less than about 80%, or greater than 120 %, of the nominal values, then the analytical values are used in estimating the LC50. Where evidence is not available to show that the test concentrations r
45、emained at an acceptable level throughout the test period, or where it is known (or suspected) that the concentrations of the test chemical have declined significantly at any stage during the test, then, irrespective of whether or not chemical analytical data are available, the LC50 cannot be define
46、d using this test method. In these cases, the test is not necessarily invalidated but it can only be stated that the LC50 of the substance is k x mg/l, the value, x, being estimated from the nominal concentrations used. 3 Test organism and reagents The reagents shall be of recognized analytical grad
47、e. The water used for the preparation of solutions shall be glass-distilled water or deionized water of at least equivalent purity. 3.1 Test organism The test species shall be Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae), commonly known as the zebra fish. Each test fish shall have a t
48、otal length of 30mm 5mm, which, in principle, corresponds to a mass of0,3g 0,1g. They shall be selected from a population of a single stock. This stock should have been acclimatized and, in any case, maintained for at least 7d prior to the test in dilution water, continuously aerated using bubbled a
49、ir (see3.2), under conditions of water quality and illumination similar to those used in the test. They shall be fed as normal up to the24h period immediately preceding the test. Test fish shall be free of overt disease or visible malformation. They shall not receive treatment for disease during the test or in the 2 weeks preceding the, test. Subsequent to the test, fish remaining alive should be suitably disposed of. Environmental conditions for the maintenance and breeding
