1、STD.BSI BS EN IS0 73qb-2-ENGL 1998 D lb24bb Ob735LO T48 E BRITISH STANDARD Water quality - Determination of the acute lethal toxicity of substances to a fresh water fish Brachydanio rerio Hamilt on-Buchanan (Teleostei, Cyprinidae) - Semi-static method I The European Standard EN IS0 73462 : 1997 has
2、the status of a British Standard ICs 13.060.01 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW BS EN IS0 BS 6068 : Section 5.3 : 1998 7346-2 : 1998 - _ STD.BS1 BS EN IS0 734b-2-ENGL 1778 = 1b24bb Ob73511 984 BS EN IS0 7346-2 : 1998 This British Standard, having been prepared u
3、nder the direction of the Health and Environment sector Board, was published under the authority of the Standards Board and comes into effect on 15 January 1998 O BSI i998 ISBN O 580 28489 1 National foreword This British Standard is the English language version of EN IS0 7346-2 : 1997. It is identi
4、cal with IS0 73462 : 1996. It supersedes BS 6068 : Section 5.3 : 1985 which is withdrawn. The UK participation in its preparation was entrusted by Technical Committee Em, Water quality, to Subcommittee EHM5, Biological methods, which has the responsibility to: - aid enquirers to understand the text;
5、 - present to the responsible internationallEuropean 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 subcom
6、mittee can be obtained on request to its secrem. BS EN IS0 73462 is one of a series of standards on water quality, others of which have been, or will be, published as Sections of BS 6068. This standard has therefore been given the secondary identifier Bs 6068 : Section 5.3. The various Sections of B
7、S 6068 are comprised within Paris 1 to 7, which, together with Part O, are listed below. Part O 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 Sam-pling Part 7 Pre, CyprinidaeJ
8、J - Partie 2: Mthode semi-statique Reference number IS0 73462:19961E3 STD.BSI BS EN IS0 734b-2-ENGL 1798 m 1b2qbb7 Ob73515 52T m EN IS0 7346-2 : 1997 Foreword IS0 (the International Organization for Standardization) is a worldwide federation of national standards bodies (IS0 member bodies). The work
9、 of preparing International Standards is normally carried out through IS0 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-governmen
10、tal, in liaison with ISO, also take part in the work. IS0 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 the member bodies for voting
11、. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard IS0 7346-2 was prepared by Technical Committee ISO/TC 147, Water qualify, Subcommittee SC 5, Biological methods. This second edition cancels and replaces the firs
12、t edition (IS0 7346-2:1984), which has been technically revised. IS0 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 fleleostei, C yprinidael : - Part 7: St
13、atic method - Part 2: Semi-static method - Part 3: Flow-through method Annexes A, B and C of this part of IS0 7346 are for information only. Ddptoir: water, quality, water pollution, tests, water tests, biological tests, determination, toxicity. II - 1TD.BSI BS EN IS0 734b-2-ENGL 1778 m 1b2Libb Ob73
14、5Lb 4bb W EN IS0 7346-2 : 1997 Introduction The three parts of IS0 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 ot
15、her species. The methodologies 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 IS0 7346, a choice can be made b
16、etween static, semi-static and flow-through methods. The static test, described in IS0 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 o
17、f the water may deteriorate. The flow-through method, described 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 this part of IS0 7346, the test solutions are re
18、newed every 24 h or 48 h, this method being a compro- mise between the 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 th
19、e study of substances whose tested concentrations remain relatively constant 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 arr
20、angements may be necessary for substances which are highly volatile. To 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. . 1
21、11 STD.BSI BS EN IS0 734b-2-ENGL 1998 = Lb24bb Ob73517 3T2 D EN IS0 7346-2 : 1997 Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) - Part 2: Sem -static met hod 1 Scope This part of IS0 7346 spec
22、ifies a 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 qu
23、ality. 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 w
24、hen using certain other species of freshwater fish as the test organism”. 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
25、and the 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 concentra
26、tions are 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)
27、a final 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 hripes (Teleostei, Poeciliidae) - fimephales p
28、romelas (Teleostei, Cyprinidae) - foecilia reticulata (Teleostei, Poeciliidae) Previous page is blank 1 EN IS0 7346-2 : 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 measur
29、ed or nominal concentrations 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 evide
30、nce 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) that the concentrations of the test chemi- cal have declined significantly at any stage during the test, then, irrespective of whether or not chem
31、ical analytical data are 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 Q x mg/l, the value, x, being estimated from the nominal concentrations used. 3.2 Standard dilu
32、tion water The freshly prepared 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/
33、l MgS0,.7H20 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 hydrochlori
34、c acid. The dilution water thus prepared shall receive no further forced aeration before use in the tests. 3 Test organism and reagents 3.3 Stock solutions of test substances The reagents shall be of recognized analytical grade. The water used for the preparation of solutions shall be glassdistilled
35、 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 total length of 30 mm f 5 mm, which, in principle, corre- sponds to a mass
36、of 0.3 g f 0,l g. 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 7 d prior to the test in dilution water, continuously aerated using bubbled air (see 3.2), under conditions of water quality and illuminati
37、on 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 not receive treatment for disease during the test or in the 2 weeks preceding the test. Subsequent to t
38、he test, fish remaining alive should be suitably disposed of. Environmental conditions for the maintenance and breeding of zebra fish are given in annex A. 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 wat
39、er, deionized water or glassdistilled water. The stock solution should be prepared at a frequency appropri- ate to the stability of the test substance. 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
40、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,l ml/l, or the volume con- taining 0,l g/i, whichever is the greater. Where a sol- vent is use
41、d, two sets of controls, one containing solvent at the maximum concentration used in any test vessel and one without solvent or test substance, 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
42、water to give the required concen- trations. It is recommended that, when a stock sol- ution is prepared in distilled or deionized water, no more than 100 ml of stock solution should be added per 10 litres of dilution water. 2 STD.BS1 BS EN IS0 734b-2-ENGL 1778 = Lb2Libb Ob73519 175 m 4 Apparatus Al
43、l materials which may come into contact with any liquid into which the fish are to be placed, or with which they may come into contact, shall be inert and should not absorb the test substance significantly. Usual laboratory equipment and the following. 4.1 Test vessels, of sufficient capacity (which
44、 may need to be greater than 10 litres), with a large area of interface between the air and the test medium (of about 800 cm2 for 10 litres of medium) and equipped 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
45、per litre of water should not be exceeded at any time during the test. Before use, the test 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, t
46、o regulate the temperature of the test solutions and the water in the stock tanks to 23 OC f 1 OC 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). * o * 5 Test environment The preparation and
47、storage of solutions, the holding of fish, and all the manipulations and tests shall be carried 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 und
48、er normal laboratory illumination with a daily photoperiod of 12 h to 16 h. 6 Procedure 6.1 Condition 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 dichromat
49、e (K2Cr20,). The results of such tests shall be in reasonable agreement with re- sults obtained previously in the same laboratory. EN IS0 7346-2 : 1997 Test fish shall not have been used for any previous testing procedure. Maintain the temperature of the water in the stock tanks at 23 “C f 1 “C (4.2). 6.2 Limit test Using the procedures described in this part of IS0 7346, a limit test may be performed at the limit of aqueous solubility under the conditions of the test or at 100 mg/l, whichever is the lower, in order to demonstrate that the 96 h - LC50 is gr
