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1、INTERNATIONAL STANDARD IS0 7346-l 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 1: Static method Qua/it6 de I eau - DBtermination de la toxicit aiguB I b) a f
2、inal 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) - fimephales pru
3、melas (Teleostei, Cyprinidae) - Poecilia 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 09:37:57 MDTNo reproduction or networking permitted
4、 without license from IHS -,-,- IS0 7346-1:1996(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
5、. 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 the test concentrations remained
6、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, the LC50 cannot be de- fined us
7、ing 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 4 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. T
8、he 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. Each test fish shall have a tota
9、l length of 30 mm f 5 mm, which, in principle, corre- sponds to a mass of 0.3 g If: 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 water, continuously aerated us
10、ing 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 not receive treatment for di
11、sease 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 standard dilution wa
12、ter shall have a pH of 7,8 + 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: 294,0 mg/l CaCl,.2H,O 123,3 mg/l MgSO,.X-i,O 63,0 mg/l NaHCO, 5,5 mg/l KCI Aerate t
13、he 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 thus prepared shall rece
14、ive 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 glass-distilled water. To enable stock
15、 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 i
16、n 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 concentration used in any test vessel and one without solvent or test substance, shall be included. 3.4
17、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 more than 100 ml of stock
18、solution should be added per 10 litres of dilution water. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 09:37:57 MDTNo reproduction or networking permitted without license from
19、 IHS -,-,- Q IS0 IS0 7346-1:1996(E) 4 Apparatus Maintain the temperature of the water in the stock tanks at 23 “C 100 mg/l; 10 mg/l; 1 mg/l and 0,l mg/l. Nothing is added to the sixth vessel, which serves as a control. The solutions shall be adjusted to and maintained at 23 “C k 1 “C (4.2) and shall
20、 not be forcibly aerated during the 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. 6.1 Condition of the fish If there are insufficient data for establishing the range of concentrat
21、ions required for the final test, repeat Whenever there is a change of stock population, carry out a toxicity test using the method specified in this this preliminary test with alternative ranges of con- centrations. part of IS0 7346 using a suitable reference chemical e.g. potassium dichromate (K,C
22、r,O,). The results of such tests shall be in reasonable agreement with re- sults obtained previously in the same laboratory. 6.4 Final test Select at least five concentrations, forming an ap- proximately geometric series, for example 8 mg/l; Test fish shall not have been used for any previous testin
23、g procedure. 4 mg/l; 2 mgll; 1 mg/l and 0,5 mg/l, between, but in- eluding, the lowest concentration killing all the fish in 3 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:
24、37:57 MDTNo reproduction or networking permitted without license from IHS -,-,- the preliminary 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 0 % and 100 % in at least two consecuti
25、ve concentrations of the geometric series used, which is necessan/ 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
26、.1) and into each pour, 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 f
27、or testing. If an organic 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
28、(3.4) has been adjusted to 23 “C f 1 “C (4.2). 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 othe
29、rwise mis- handled during 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. Obser
30、vations 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, measure the concen- trations of the test sub
31、stance in the test vessels at least at the beginning and end of the test. Measure the dissolved oxygen concentration, the pH and temperature in each vessel at least once daily and at the beginning and end of the test. The temperature and pH of the test solutions should not vary by more than + 1 “C o
32、r 0,2 pH units, respectively. A suggested form which is suitable for recording the data is given in annex B. 7 Expression of results 7.1 Validity The results shall be considered valid if the following requirements are fulfilled: a) the dissolved oxygen concentration in the test solutions during the
33、test was at least 60 % ASV; b) the concentrations of the test substance were not known (or suspected) to have declined signifi- cantly throughout the test (but see clause 2); c) the mortality of the control fish did not exceed 10 % or one per tank; d) the proportion of control fish showing abnormal
34、behaviour did not exceed 10 % or one per tank; e) the 24 h - LC50 of the reference chemical e.g. potassium dichromate (K,Cr,O,) if available, for the stock of fish was in reasonable agreement with results obtained previously in the same lab- oratory. 7.2 Estimation of LC50 Where a simple graphical e
35、stimation of the LC50 is considered adequate, this can be obtained by plotting mortality (expressed as a percentage of test fish in each test vessel) against concentration of test sub- stance. Using axes with linear scales, this will produce a sigmoid relationship from which the LC50 can be derived
36、by interpolating the concentration expected to cause 50 % mortality (see figure I). It is more appropriate to plot the data on graph paper having axes with logarithmic and probability scales. Data plotted in this way should produce a linear re- lationship from which the LC50 can be interpolated as a
37、bove (see figure 2). Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 09:37:57 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 7346-1:1996(E) Figure
38、1 - Graphical interpolation of LC50 (linear scales) 1 I 1 I ,t I 1 0,s 1 2 4 8 Concentration (mg/l) Figure 2 - Graphical interpolation of LC50 (logarithmic and probability scales) Where estimation of slope and 95 % confidence limits of this and the LC50 are required, and it is rec- ommended that the
39、se statistics are frequently valu- able in expressing results, the data can be analysed graphically (2 in annex C). These concentrations will indicate the limits within which the LC50 probably lies. 8 Test report Where computing facilities are available, probit analy- sis can be applied (l in annex
40、C). If insufficient data are available to estimate the LC50 at 24 h and 48 h and, if available, at 72 h and 96 h, record the minimum concentration in which 100 % mortality occurred and the maximum concentration giving 0 % mortality at 24 h; 48 h; 72 h and 96 h. The test report shall include the foll
41、owing information: a) a reference to this part of IS0 7346; b) the chemical identity and any additional available information about the test substance (e.g. water solubility, volatility, octanol/water partition coef- ficient, degradation rate); c) the method of preparing the dilution water, stock so
42、lutions and test solutions; 5 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 09:37:57 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 7346-1:1996(E
43、) 63 IS0 d) all chemical, biological and physical data pertaining to the test and not otherwise specified in this part of IS0 7346, including details of the acclimatiz- ation conditions of the test fish, and the mass of fish, in grams per litre; e) the data taken into account when assessing the vali
44、dity of the test: I) concentration of dissolved oxygen; 2) mortality observed among control fish; 3) proportion of control fish showing abnormal behaviour; 4) LC50 of the reference substance; f) a tabulated list showing the nominal concen- trations tested (with chemical analytical values, where avai
45、lable), and the total percentage mor- talities in each 24 h, 48 h, 72 h and 96 h after the start of the test; g) the LC50 values and confidence limits, if available, at 24 h, 48 h, 72 h and 96 h, of the substance tested; reference should be made to the method of calculation, and the method of chemic
46、al analy- sis, where applicable; h) the slope of the concentration-response curve (and its 95 % confidence limit if available); i) a graphical illustration of the concentration- response relationship; j) any unusual reactions by the fish under the test conditions and any visible external effects pro
47、- duced by the test substance; k) any deviation from the procedure specified in this part of IS0 7346, and the reason for it. 6 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
48、:37:57 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 7346-1:1996(E) Annex A (informative) Environmental parameters for maintenance and breeding of zebra fish (Brachydanio rerio Hamilton-Buchanan) A.1 General The species originates from the Coromandel coast of India wh
49、ere it inhabits fast flowing streams. It is a common aquarium fish, so that information about procedures for its care and culture can be found in standard reference books on tropical fish culture. Its biology has recently been reviewed by Laalefsl. The fish rarely exceeds 45 mm in length. The body is cylindrical with 7 to