1、BRITISH STANDARD BS EN ISO 7346-3:1998 BS 6068-5.4: 1998 Water quality Determination of the acute lethal toxicity of substances to a fresh water fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Flow-through method The European Standard EN ISO 7346-3:1997 has the status of a British S
2、tandard ICS 13.060.01BSEN ISO 7346-3:1998 This British Standard, having been prepared under the directionof the Health and Environment Sector Board, waspublished under the authorityof the Standards Boardand comes into effect on 15January 1998 BSI 05-1999 ISBN 0 580 28490 5 Amendments issued since pu
3、blication Amd. No. Date CommentsBSEN ISO 7346-3:1998 BSI 05-1999 i Contents Page National foreword ii Foreword 2 Foreword ii Text of ISO 7346-3 1BSEN ISO 7346-3:1998 ii BSI 05-1999 National foreword This British Standard is the English language version of ENISO7346-3:1997. It is identical with ISO73
4、46-3:1996. It supersedes BS6068-5.4:1985 which is withdrawn.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 responsible
5、 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 reque
6、st to its secretary. BS EN ISO 7346-3 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 BS6068-5.4. The various Sections of BS6068 are comprised within Parts1 to7,
7、which, together with Part0, 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 Atte
8、ntion 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 the BS
9、I 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 respon
10、sible 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, theEN ISO title page, page 2, the ISO title page, page ii, pages 1 to 8 an
11、d abackcover. 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 7346-3 November 1997 ICS 13.060.01 Descriptors: See ISO document
12、 English version Water quality Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Part 3: Flow-through method (ISO 7346-3:1996) Qualit de leau Dtermination de la toxicit aigu ltale de subtances vis-vis dun poisson
13、 deau douce Brachydanio rerio Hamilton-Buchanan (Tlostei, Cyprinidae) Partie 3: Mthode avec renouvellement continu (ISO 7346-3:1996) Wasserbeschaffenheit Bestimmung der akuten letalen Toxizitt von Substanzen gegenber einem Swasserfisch Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Teil
14、 3: Durchfluverfahren (ISO 7346-3:1996) 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.
15、Up-to-date lists 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 u
16、nder the responsibility 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, CzechRepublic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Lu
17、xembourg, Netherlands, 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 an
18、d by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 7346-3:1997 EEN ISO 7346-3:1997 BSI 05-1999 2 Foreword The text of the International Standard from Technical Committee ISO/TC 147 “Water quality” of the International Organization for Standardization (ISO) has been taken ove
19、r as an European 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 May 1998, and conflicting nati
20、onal standards shall be withdrawn at the latest by May 1998. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, CzechRepublic, Denmark, Finland, France, Germany, Greece, I
21、celand, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and theUnitedKingdom. Endorsement notice The text of the International Standard ISO 7346-3:1996 has been approved by CEN as a European Standard without any modification. Contents Page Foreword 2 Introductio
22、n 1 1 Scope 1 2 Principle 1 3 Test organism and reagents 2 4 Apparatus 2 5 Test environment 3 6 Procedure 3 7 Expression of results 4 8 Test report 5 Annex A (informative) Environmental parameters for maintenance and breeding of zebra fish (Brachydanio rerio Hamilton-Buchanan) 6 A.1 General 6 A.2 En
23、vironmental parameters 6 A.3 Materials and methods 6 A.4 Conditioning 6 A.5 Breeding stage 6 A.6 Development of fry 6 Annex B (informative) Suggested form for recording data 7 Annex C (informative) Bibliography 8 Figure 1 Graphical interpolation of LC50 (linear scales) 5 Figure 2 Graphical interpola
24、tion of LC50 (logarithmic and probability scales) 5EN ISO 7346-3:1997 ii BSI 05-1999 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 thr
25、ough 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 collabo
26、rates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by a
27、t least 75% of the member bodies casting a vote. International Standard ISO 7346-3 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. This second edition cancels and replaces the first edition (ISO 7346-3:1984), which has been technically revised. I
28、SO 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, Cyprinidae): Part 1: Static method; Part 2: Semi-static method; Part 3: Flow-through method. An
29、nex A, Annex B and Annex C of this part of ISO7346 are for information only. Descriptors: Water, quality, water pollution, tests, water tests, biological tests, determination, toxicity.EN ISO 7346-3:1997 BSI 05-1999 1 Introduction The three parts of ISO7346 describe methods of determining the acute
30、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 methodologies presented here may also be used for other species of freshwater, marine or brackish
31、 water fish, with appropriate modifications of, for example, dilution water quality and the temperature conditions of the test. Within the three parts of ISO7346, a choice can be made between static, semi-static and flow-through methods. The static test, described in ISO7346-1, in which the solution
32、 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 deteriorate. The flow-through method, described in this part of ISO7346, in which the test solution
33、is replenished continuously, overcomes such problems but requires the use of more complex apparatus. In the semi-static procedure, described in ISO7346-2, the test solutions are renewed every 24h or 48h, this method being a compromise between the other two. The flow-through method can be used for mo
34、st 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 tested concentrations remain relatively constant during the test period. The semi-static method can
35、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 substances which are highly volatile. To assist in the preparation and maintenance of concentrations
36、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 ISO7346 specifies a flow-through method for the determination of the acute lethal toxicity of stable, non-volatile,
37、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. The method is applicable for assigning, for each test substance, broad categories of acute lethal
38、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 certain other species of freshwater fish as the test organism 1) . The method may be adapted fo
39、r 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 the temperature. 2 Principle Determination, under specified conditions, of the concentrations at w
40、hich a substance is lethal to50% of a test population of Brachydanio rerio after exposure periods of 24h, 48h, 72h and 96h to that substance in the ambient water. These median lethal concentrations are designated the 24h LC50, 48h LC50, 72h LC50 and 96h LC50. The test is carried out in two stages: a
41、) 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 which alone are reported. 1) The following species of freshwater fish can be used, in addition
42、to Brachydanio rerio, without modification to this part of ISO7346. Lepomis macrochirus (Teleostei, Centrarchidae) Oryzias latipes (Teleostei, Poeciliidae) Pimephales promelas (Teleostei, Cyprinidae) Poecilia reticulata (Teleostei, Poeciliidae)EN ISO 7346-3:1997 2 BSI 05-1999 Where evidence is avail
43、able 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 the LC50. Where such analyses show that the concentrations present remain relatively constan
44、t 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 remained at an acceptable level throughout the test period, or where it is known (or suspected)
45、 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 defined using this test method. In these cases, the test is not necessarily invalidated but it can o
46、nly 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 grade. The water used for the preparation of solutions shall be glass-distilled water or deionized
47、 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 total length of 30mm 5mm, which, in principle, corresponds to a mass of 0,3g 0,1g. They shall b
48、e 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 air (see 3.2), under conditions of water quality and illumination similar to those used in the
49、 test. They shall be fed as normal up to the 24h 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 of zebra fish are given in Annex A. 3.2 Standard dilution water The freshly prepared stan
