1、 STD.BS1 BS EN IS0 734b-1-ENGL L798 m Lb24bb9 Ob73491 2bb m I BRITISH STANDARD Water quality - Determination of the acute lethal toxicity of substances to a fresh water fish Brachydanio re% Hamilton-Buchanan (Teleostei, Cyprinidae) - Static method The European Standard EN IS0 73461 : 1997 has the st
2、atus of a British Standard ICs 13.060.01 BS EN IS0 BS 6068 : Section 5.2 : 1998 7346-1 : 1998 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGm LAW STD.BS1 BS EN IS0 73Llb-L-ENGL 1998 lb2Libb7 0b73i72 12 m been prepared under the diredion of the Health and Amd. No. Date Environment S
3、ector Board, was published under the authority of the Standards Board and comes into effect on 15 January 1998 O BSI 1998 ISBN O 080 28488 3 BS EN IS0 7346-1 : 1998 Text affected National foreword This British Standard is the English language version of EN IS0 73461 : 1997. It is identical with IS0
4、73461 : 1996. It supersedes BS 6068 : Section 5.2 : 1985 which is withdrawn. The UK participation in its preparation was entrusted by Technical Committee Ew3, Water quality, to Subcommittee EH/3/5, Biological methods, which has the responsibility to: - aid enquirers to understand the text; - present
5、 to the responsible intemationaVEuropean committee any enquiries on the interpretation, or proposais for change, and keep the UK interests informed; - monitor reiated intemational and European developments and promulgate them in the UK. A list of organizations represented on this subcommittee can be
6、 obtained on request to its secretary. BS EN IS0 73461 is one of a series of standards on water quality, others of which have been, or will be, published as Sections of ES 6068. This standard has therefore been given the secondary identifier BS 6068 : Section 5.2. The various Sections of BS 6068 are
7、 comprised within Parts 1 to 7, which, together with Part O, are listed below. Part O Introduction Part 1 Glossary Part 2 Phgsicai, chemical and biochemical methods Part 3 Rawlogica methods Part 4 Microbiological methods Part 5 Biological methods Part 6 Sampling Part 7 Precision and accuracy Cross-r
8、eferences 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 f
9、ound in the BSI Sandards Catalogue under the section entitled International Standards Correspondence Index, or by using the Find facility of the BSI Standards Electronic Catalogue. Compliance with a British Standard does not of itself confer immunity from legai obligations. Summary of pages This doc
10、ument comprises a front cover, an inside front cover, the EN Is0 title page, the EN IS0 foreword page, the IS0 titJe page, pages ii and iu, a blank page, pages 1 to 11, a biank page, an inside back cover and a back cover. STD-BSI BS EN IS0 73Vb-L-ENGL 1778 LbZLIbb7 b73473 037 EUROPEAN STANDARD NORME
11、 EUROPENNE EUROPISCHE NORM EN IS0 73464 November 1997 ICs 13.060.01 Descriptors: see IS0 document English version Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton- Buchanan (Teleostei, Cyprinidae) - Part 1 : Static method (IS0 7
12、346-1 : 1 996) Qualit de leau - Dlennination de la toxW aigu 18tale de substances vis-8-vis dun poisson deau douce Btad?nnk ah Hamiton-Buchanan (Thstei, Cyprinidae) - Partie 1: Mlhode statique (IC0 7346- 1:1996) Wasseibeschaienheit - Bestimmung der akuten letalen Toxizitt von Substanzen gegenber ein
13、em Swasseriisch BraiQsdank renb Haminon-Buchanan (Teleostei, Cyprinidae) -Teil 1: Statisches Verfahren (IS0 7346-1 :lQ) This European Standard was approved by CEN on 30 October 1997. CEN members are bound to comply wih the CEWCENELEC Internal Regulations which stipulate the conditions for giving thi
14、s European Standard the status of a national standard Without any alteration. Upto-date lists and biblographical references concerning such national standards may be obtained on applicatian lo the Central Secretariat or lo any CEN member. This European Standard exists in three official vernions (Eng
15、lish, French, German). A version in any other language made by translation under the responslbiiity of a CEN member into its own language and notied to the Central Secretariat has the same status as the b) a final test, the results of which alone are re- ported. 1) The following species of freshwate
16、r fish can be used, in addition to Brachydanio rerio, without modification to this part of IS0 7346. - Lepomis macrochirus (Teleostei, Centrarchidae) - Oryzias lafipes (Teleostei, Poeciliidae) - Prmephales promelas (Teleostei, Cyprinidae) - Poecilia rericulata (Teleostei, Poeciliidae) Previous page
17、is blank 1 STD.BSI BS EN IS0 73qb-L-ENGL 1998 m Lb2LibbS b73477 557 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 concentrations are use
18、d 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 show that th
19、e 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 available,
20、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 s x mg/l, the value, X, being estimated from the nominal concentrations used. 3 Test organism and reagents The reagents shall be of
21、 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 zebra fish. E
22、ach 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 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 in dilution wate
23、r, 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 malformation. They shall no
24、t 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 pre
25、pared standard dilution water shall have a pH of 7,8 f 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 CaC12.2H20 123,3 mg/l MgS0,.7H20 63,O mg/l NaH
26、CO, 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. The dilution water
27、 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 or glassdistille
28、d 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 u
29、sed, its concentration in the test solution shall not exceed 0.1 ml/l, or the volume con- taining 0,l g/i, whichever is the greater. Where a sol- vent is used, two sets of controls, one containing solvent at the maximum concentration used in any test vessel and one without solvent or test substance,
30、 shall be included. 3.4 Test solutions Test solutions are prepared by adding appropriate amounts of the stock solution of the test substance to the dilution water to give the required concen- trations. It is recommended that, when a stock sol- ution is prepared in distilled or deionized water, no mo
31、re than 100 ml of stock solution should be added per 10 litres of dilution water. 2 STD.BS1 BS EN IS0 734b-1-ENGL 1778 1624bb9 Ob73500 OT D EN IS0 7346-1 : 1997 4 Apparatus All materials which may come into contact with any liquid into which the fish are to be placed, or with which they may come int
32、o contact, shall be inert and should not absorb the test substance significantly. Maintain the temperature of the water in the stock tanks at 23 “C I 1 “C (4.2). 6.2 L,H test Using the procedures described in this part of Usual laboratory equipment and the following IS0 7346, a limit test may be per
33、formed at the limit of aqueous solubility under the conditions of the test with a securely fixed and close-fitting cover. The vol- ume of the test vessels should be sufficient that a loading rate of 1 g of fish per litre of water should not be exceeded at any time during the test. Before use, the te
34、st vessels shall be cleaned thoroughly, for example with a non-ionic detergent (followed by acid and solvent washes for substances expected to adsorb strongly to the vessel). 4.2 Temperature control equipment, to regulate the temperature of the test solutions and the water in the stock tanks to 23 “
35、C f 1 “C by a suitable method. 4.3 Dip-net, made of nylon or of another chemically inert material, for the control vessels and another for all the test vessels (4. 1). 5 Test environment The preparation and storage of solutions, the holding of fish, and all the manipulations and tests shall be carri
36、ed out in premises with an atmosphere free from harmful concentrations of airborne contaminants. Take care to avoid any unwanted disturbance that may change the behaviour of the fish. Carry out all tests under normal laboratory illumination with a daily photoperiod of 12 h to 16 h. 6 Procedure 6.1 C
37、ondition of the fish Whenever there is a change of stock population, carry out a toxicity test using the method specified in this part of IS0 7346 using a suitable reference chemical e.g. potassium dichromate (K2Cr20,). The results of such tests shall be in reasonable agreement with re- sults obtain
38、ed previously in the same laboratory. Test fish shall not have been used for any previous test i ng procedure. Perform the limit test using 10 fish, with the same number in the control(s1. NOTE 1 Binominal theory dictates that, when 10 fish are used, with zero mortality there is a 99.9 YO confidence
39、 that the 96 h - LC50 is greater than the limit-test concentration. If mortalities occur, a complete study (see 6.3 and 6.4) may need to be considered. If sublethal effects are observed, these should be recorded. 6.3 Preliminary test Add at least 2,5 litres, preferably 5 litres, of standard dilution
40、 water (3.2) to each of six test vesseis (4.1) and aerate if necesSan/ to restore the concentration of dissolved oxygen to at least 90 % of its air satu- ration value. Prepare test solutions by adding appropriate amounts of stock solution of the test substance (3.3) to five of the vessels in order t
41、o obtain an adequate geometric range of concentrations, for example 1 O00 mg/l; 100 mg/; 10 mgII; 1 mg/l and 0,l mgIl. Nothing is added to the sixth vessel, which serves as a control. The solutions shall be adjusted to and maintained at 23 “C I 1 “C (4.2) and shall not be forcibly aerated during the
42、 test. Place three fish in each vessel. At least twice a day, note the number of dead fish and the dissolved oxygen concentration in each vessel. Remove the dead fish. If there are insufficient data for establishing the range of concentrations required for the final test, repeat this preliminary tes
43、t with alternative ranges of con- ce nt ra t ions. 6.4 Final test Select at least five concentrations, forming an ap- proximately geometric series, for example 8 mg/l; 4 mg/l; 2 mg/l; 1 mg/l and 0,5 mg/i, between, but in- cluding, the lowest concentration killing all the fish in 3 STD-BSI BS EN IS0
44、7346-1-ENGL 1778 Lb24bb7 Ob73501 T35 EN IS0 7346-1 : 1997 the preliminaty test, and the highest non-lethal con- centration in 96 h. This selected series of concen- trations shall provide the possibility of obtaining mortalities of between O % and 100 YO in at least two consecutive concentrations of
45、the geometric series used, which is necessary for an estimation of the LC50 using the probit method. In some instances, a narrower range of concen- trations may be required to provide the necessary data and in others a wider range may be needed. Take at least six test vessels (4.1) and into each pou
46、r, for example, 10 litres of standard dilution water (3.2). Nothing is added to one of these (the control) but, to the remainder, add the different amounts of stock solution (3.3) required to give the particular range of concentrations of test substance which has been selected for testing. If an org
47、anic solvent has been used to dissolve a substance, prepare a second con- trol with the standard dilution water containing suf- ficient of the organic solvent to give the maximum concentration at which this solvent is present in any of the test solutions. When the test solution (3.4) has been adjust
48、ed to 23 “C f 1 “C (4.21, place at least seven fish in each of the vessels, as follows. Select the fish at random from the stock and place them at random in the test vessels, without delay, using a small mesh dip-net of soft inert material (4.3). Discard any fish dropped or otherwise mis- handled du
49、ring the transfer. In a given test, add all the fish within a period of 30 min. The solutions shall not be forcibly aerated. Record the number of dead fish in each vessel at least daily over the period of the test. Remove each dead fish from the vessel as soon as possible. Observations can be made more frequently, for example to enable median periods of survival to be calculated for each concen- tration. Note any abnormal behaviour of the fish. If the substance is shown to be stable over the period of exposure then, if possible, mea
