ISO-7346-2-1996.pdf
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1、INTERNATIONAL STANDARD IS0 7346-2 Second edition 1996-06-l 5 Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) - Part 2: Semi-static method Qua/it de l eau - DBtermination de la toxicit b) a final
2、 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 macroc/irus (Teleostei, Centrarchidae) - Oyzias latipes (Teleostei, Poeciliidae) - Pimephales promelas
3、 (Teleostei, Cyprinidae) - foecilia reticulata (Teleostei, Poeciliidae) 1 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 09:37:58 MDTNo reproduction or networking permitted with
4、out license from IHS -,-,- IS0 7346-Z: 1996(E) 0 IS0 Where evidence is available to show that test con- centrations 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. Wh
5、ere 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 remained at a
6、n 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, the LC50 cannot be de- fined using
7、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. 3.2 Standard dilution water The freshly prepared standard dilution water shall have a pH
8、 of 7.8 + 0,2, and a calcium hardness of approximately 250 mgll, expressed as calcium carbonate, and shall contain the following concen- trations of salts dissolved in distilled or deionized water: 294,O mg/l CaClz.2HzO 123,3 mg/l MgSO,.7H,O 63,0 mg/l NaHCO, 5,5 mg/l KCI Aerate the dilution water un
9、til the concentration of dissolved oxygen reaches at least 90 % of its air saturation value (ASV) and the pH is constant at 7.8 + 0,2. If necessary, adjust the pH of the solution by adding sodium hydroxide solution or hydrochloric acid. The dilution water thus prepared shall receive no further force
10、d 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 glass-distilled water or deionized water of at least equivalent purity. 3.1 Test orga
11、nism 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 of 0,3 g f 0.1 g. They shall be selected from a population of a single
12、 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 illumination similar to those used in the test. They shall be fed as normal up t
13、o 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 the test, fish remaining alive should be suitably disposed of. Environm
14、ental 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 water, deionized water or glass-distilled water. The stock solution shoul
15、d 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 or dispersed by suitable means, including ultrasonic devices and or-
16、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/l, whichever is the greater. Where a sol- vent is used, two sets of controls, one containing solvent at the maximum concen
17、tration 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 water to give the required concen- trations. It is recommended that,
18、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 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001
19、 Not for Resale, 04/21/2007 09:37:58 MDTNo reproduction or networking permitted without license from IHS -,-,- Q1 IS0 IS0 7346-2:1996(E) 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 into contact, shall be iner
20、t and should not absorb the test substance significantly. Usual laboratory equipment and the following. 4.1 Test vessels, of sufficient capacity (which 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
21、) 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 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 n
22、on-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 “C + 1 “C by a suitable method. 4.3 Dip-net, made of nylon or
23、 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 carried out in premises with an atmosphere free from harmful conce
24、ntrations 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 Condition of the fish Whenever there is a change of stock popu
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