1、DEUTSCHE NORM March 1998 Determination of the acute lethal toxicity of substances in water to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Te leostei , Cyprin idae) Part 1: Static method (IS0 7346-1 : 1996) English version of DIN EN IS0 7346-1 DIN - EN IS0 7346-1 ICs 13.060.01 Descriptors:
2、 Water quality, toxicity, fish, bio-assays. Wasserbeschaffenheit - Bestimmung der akuten letalen Toxizitt von Substanzen gegenber einem Swasserfisch Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) - Teil 1 : Statisches Verfahren (IS0 7346-1 : 1996) European Standard EN IS0 7346-1 : 1997
3、has the status of a DIN Standard. A comma is used as the decimal marker. This standard is part of the series Deutsche Einheitsvetfahren zur Wasser-, Abwasser- und Schlamm- untersuchung - Testverfahren mit Wasserorganismen (Gruppe ) (German standard methods for the exami- nation of water, waste water
4、 and sludge - Bio-assays (group L). National foreword This standard has been published in accordance with a decision taken by CEN/TC 230 to adopt, without alteration, International Standard IS0 7346-1 as a European Standard. The responsible German body involved in its preparation was the Normenaussc
5、hu Wasserwesen (Water Practice Standards Committee). Expert assistance and specialized laboratories will be required to perform the analysis described in this standard. Depending on the objective of the analysis, a check shall be made in each individual case on whether and to what extent additional
6、boundary 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 Einheitsverfahren zur Wasser-, Abwasser- und Schl
7、ammuntersuchung will continue to be published by Wiley-VCH Verlag and Beuth Verlag GmbH. Continued overleaf. EN comprises 12 pages. No pari of this standard may be reproduced without the prior permission of Ref. No. DIN EN IS0 7346-1 : 1998-0 Y Deutsches Institut fr Normung e. V., Berlin. wth Verlag
8、 GmbH, D-10772 Berlin, has the exclusive right of sale for German Standards (DIN-Normen). English price group 08 Sales No. 1108 08.98 Page 2 DIN EN IS0 7346-1 : 1998-03 The standard methods relating to the Wasserhausha/fsgesefz (German Water Management Act), the Abwasser- abgabengesefz (Law on the c
9、harges leviable for the discharge of effluent into water), and all the regulations on waste water that have been issued to date are included in DIN-Taschenbuch (DIN Handbook) 230. Standard methods or draft standards bearing the group title German standard methods for the examination of water, waste
10、water and sludge are classified under the following categories (main titles): 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 anal
11、ysis (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 3841 O) Microbiological methods (group K) (DIN 3841 1) Bio-assays (group L) (DIN 38412) Individual con
12、stituents (group P) (DIN 3841 3) Sludge and sediments (group S) (DIN 3841 4) Bio-assays with microorganisms (group T) (DIN 3841 5) In addition to the methods described in the DIN 38402 to DIN 38415 series of standards, there are a number of European Standards available, which also form part of the c
13、ollection 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 Normenausschu Wasserwesen, telephone (O 30) 26 O1 -24 23, or from Beufh Verlag GmbH, Burggrafenstrae 6, D-10787 Berlin. EUROPEAN STANDARD N
14、ORME EUROPENNE EUROPISCHE NORM EN ISO7346-1 November 1997 ICs 13.060.01 Descriptors: Water quality, toxicity, fish, bio-assays. English version Water quality Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Ham i It on - Bu c han an (Teleost ei , Cyprin
15、 idae) Part 1 : Static method (IS0 7346-1 : 1996) Qualit de leau - Dtermination de la toxicit aigu ltale de substances vis- -vis dun poisson deau douce Brachydanio rerio Hamilton-Buchanan (Tlostei, Cyprinidae) - Partie 1 : Mthode statique (IS0 7346-1 : 1996) Wasserbeschaffenheit - Bestimmung der aku
16、ten letalen Toxizitt von Substanzen gegenber einem Swasserfisch Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprini- dae) -Teil 1 : Statisches Verfahren (IS0 7346-1 : 1996) This European Standard was approved by CEN on 1997-1 0-30. CEN members are bound to comply with the CENKENELEC Internal Reg
17、ulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national stand- ards may be obtained on application to the Central Secretariat or to any CEN member. The
18、 European Standards exist in three official versions (English, French, German). A version in any other language made by translation under 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
19、 national standards bodies of Austria, Belgium, the Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, and the United Kingdom. CEN European Committee for Standardization Comit Europen de Norma
20、lisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels O 1997. CEN - All rights of exploitation in any form and by any means reserved worldwide for CEN national members. Ref. No. EN IS0 7346-1 : 1997 E Page 2 EN IS0 7346-1 : 1997 Foreword International Stand
21、ard IS0 7346-1 : 1996 Water quality - Determination of the acute lethal toxicity of substances to afreshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) - Part 1 : Static method, which was prepared by ISO/TC 147 Water quality of the International Organization for Standardizatio
22、n, has been adopted by Technical Committee CEN/TC 230 Water analysis, the Secretariat of which is held by DIN, as a European Standard. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, and conflicting national stand
23、ards withdrawn, by May 1998 at the latest. In accordance with the CENKENELEC Internal Regulations, the national standards organizations of the follow- ing countries are bound to implement this European Standard: Austria, Belgium, the Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland
24、, Ireland, Italy, Luxembourg, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, and the United Kingdom. Endorsement notice The text of the International Standard IS0 7346-1 : 1996 was approved by CEN as a European Standard without any modification. Introduction The three parts of IS0 73
25、46 describe methods of determining the acute lethal toxicity of substances to the zebra fish (Brachydanio reno 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 oth
26、er 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 IS0 7346, a choice can be made between static, semi-static and flow-through methods. The static test,
27、described in this part of IS0 7346, 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 deteriorate. The flow-through method, descr
28、ibed in IS0 7346-3, in which the test solution is replenished continuously, over- comes such problems but requires the use of more complex apparatus. In the semi-static procedure, described in IS0 7346-2, the test solutions are renewed every 24 h or 48 h, this method being a compromise be- tween the
29、 other two. The flow-through method can be used for most types of substances, in- cluding those unstable in water, but the concentrations of the test sub- stance are determined wherever possible. The static method is limited to the study of substances whose tested concentrations remain relatively co
30、nstant during the test period. The semi-static method can be used for testing those substances whose concentrations can be maintained satis- factorily throughout the test by renewal of the solutions every 24 h or 48 h. Special arrangements may be necessary for substances which are highly volatile. T
31、o assist in the preparation and maintenance of concentrations of sub- stances 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. Page 3 EN IS0 7346-1 : 1997 1 Scope This part of IS0 7346 specifies a sta
32、tic 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 Brach ydanio rerio Ham i It o n-B uc ha na n (Teleost e, Cyprinidae) - common name, zebra fish in water of a specified quality.
33、The 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 usi
34、ng certain other species of freshwater fish as the test organism1. 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 the
35、 temperature. 2 Principle Determination, under specified conditions, of the concentrations at which a substance is lethal to 50 % of a test population of Brachydanio rerio after exposure periods of 24 h, 48 h, 72 h and 96 h to that substance in the ambient water. These median lethal concentrations a
36、re designated the 24 h - LC50, 48 h - LC50. 72 h - LC50 and 96 h - LC50. The test is carried out in two stages: a) a preliminary test which gives an approximate in- dication of the acute median lethal concentrations and serves to determine the range of concen- trations for the final test; b) a final
37、 test, the results of which alone are re- ported. 1) The following species of freshwater fish can be used, in addition to Brachydanio rerio, without modification to this part of IS0 7346. - Lepomis macrochirus (Teleostei, Centrarchidae) - Oryzias latipes (Teleostei, Poeciliidae) - Pimephales promela
38、s (Teleostei, Cyprinidae) - Poecilia reticulata (Teleostei, Poeciliidae) Page 4 EN IS0 7346-1 : 1997 Where evidence is available to show that test con- centrations remain relatively constant (.e. within about 20 % of the nominal values) throughout the test, then either measured or nominal concentrat
39、ions are used in the estimation of 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
40、show that the test concentrations remained at an acceptable level throughout the test period, or where it is known (or suspected) that the concentrations of the test chemi- cal have declined significantly at any stage during the test, then, irrespective of whether or not chemical analytical data are
41、 available, the LC50 cannot be de- fined 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 G x mg/l, the value, x, being estimated from the nominal concentrations used. 3 Test organism and reagents The reagents
42、 shall be of recognized analytical grade. 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 z
43、ebra fish. Each test fish shall have a total length of 30 mm f 5 mm, which, in principle, corre- sponds to a mass of 0,3 g rt 0.1 g. They shall be se- lected from a population of a single stock. This stock should have been acclimatized and, in any case, maintained for at least 7 d prior to the test
44、in dilution water, continuously aerated using bubbled air (see 3.2). under conditions of water quality and illumination similar to those used in the test. They shall be fed as normal up to the 24 h period immediately preceding the test. Test fish shall be free of overt disease or visible malformatio
45、n. 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
46、 The freshly prepared standard dilution water shall have a pH of 7,8 k 0.2. and a calcium hardness of approximately 250 mg/l, expressed as calcium carbonate, and shall contain the following concen- trations of salts dissolved in distilled or deionized water: 294,O mg/l CaC1,.2H,O 123,3 mg/i MgS0,.7H
47、20 63,O mg/l NaHCO, 5,5 mg/l KCI Aerate the dilution water until the concentration of dissolved oxygen reaches at least 90 % of its air saturation value (ASV) and the pH is constant at 7,8 f 0,2. If necessary, adjust the pH of the solution by adding sodium hydroxide solution or hydrochloric acid. Th
48、e dilution water thus prepared shall receive no further forced aeration before use in the tests. 3.3 Stock solutions of test substances A stock solution of the test substance should be pre- pared by dissolving a known amount of test sub- stance in a defined volume of dilution water, deionized water
49、or glass-distilled water. To enable stock solutions to be prepared and to assist in their transfer to the test vessels, substances of low aqueous solubility may be dissolved or dispersed by suitable means, including ultrasonic devices and or- ganic solvents of low toxicity to fish. If any such or- ganic solvent is used, its concentration in the test solution shall not exceed 0.1 ml/l, or the volume con- taining 0.1 g/i, whichever is the greater. Where a sol- vent is used, two sets of controls, one containing solvent at the maximum conc
