1、BRITISH STANDARD BS 6068-5.17: 1995 ISO 10229: 1994 Water quality Part 5: Biological methods Section 5.17 Determination of the prolonged toxicity of substances to freshwater fish Method for evaluating the effects of substances on the growth rate of rainbow trout Oncorhynchus mykiss Walbaum (Teleoste
2、i, Salmonidae)BS6068-5.17:1995 This British Standard, having been prepared under the directionof the Health and Environment Sector Board, waspublished under the authorityof the Standards Boardand comes into effect on 15April 1995 BSI 07-1999 The following BSI references relate to the work on this st
3、andard: Committee reference EPC/44/5 Draft for comment 93/500175 DC ISBN 0 580 24131 9 Committees responsible for this British Standard The preparation of this British Standard was entrusted by Technical Committee EPC/44, Water quality, to Subcommittee EPC/44/5, Biological methods, upon which the fo
4、llowing bodies were represented: British Laboratory Ware Association Ltd. Department of the Environment (Water Directorate) Department of the Environment for Northern Ireland Department of Health Freshwater Biological Association Institution of Water and Environmental Management Ministry of Agricult
5、ure, Fisheries and Food National Rivers Authority Royal Society of Chemistry Scottish Natural Heritage Soap and Detergent Industry Association Water Companies Association Water Research Centre Water Services Association of England and Wales Amendments issued since publication Amd. No. Date CommentsB
6、S6068-5.17:1995 BSI 07-1999 i Contents Page Committees responsible Inside front cover National foreword ii 1 Scope 1 2 Normative references 1 3 Principle 1 4 Test organism 2 5 Reagents 2 6 Apparatus 3 7 Test environment 3 8 Procedure 3 9 Precision of the test procedure 5 10 Expression of results 5 1
7、1 Test report 5 Annex A (informative) Two examples of report forms 8 Annex B (informative) Bibliography 11 Figure 1 Examples of marks used for freeze-branding fish 4 Figure 2 Responses of fish to 3, 4-dichloroaniline 7 List of references Inside back coverBS6068-5.17:1995 ii BSI 07-1999 National fore
8、word This Section of BS6068 has been prepared by Subcommittee EPC/44/5 and is identical with ISO10229:1994 Water quality Determination of the prolonged toxicity of substances to freshwater fish Method for evaluating the effects of substances on the growth rate of rainbow trout Oncorhynchus mykiss Wa
9、lbaum (Teleostei, Salmonidae). The international standard was prepared by Subcommittee 5, Biological methods, of Technical Committee 147, Water quality, of the International Organization for Standardization (ISO) with the active participation and approval of the UK. BS6068 is being published in a se
10、ries of Parts subdivided into Sections that will generally correspond to particular international standards. Sections are being, or will be, published in Parts1 to7, which, together with Part0, are listed below. Part 0: Introduction; Part 1: Glossary; Part 2: Physical, chemical and biochemical metho
11、ds; Part 3: Radiological methods; Part 4: Microbiological methods; Part 5: Biological methods; Part 6: Sampling; Part 7: Precision and accuracy. NOTEThe tests described in this Section of BS6068 should only be carried out by suitably qualified persons with an appropriate level of biological expertis
12、e. Usual biological procedures should be followed throughout. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity fro
13、m legal obligations. Cross-references International standard Corresponding British Standard ISO 7346-2:1984 a BS 6068-5.3:1985 Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae): Semi-static method (Identical) ISO
14、 7346-3:1984 a Section 5.4:1985 Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae): flow-through method (Identical) a Revision to be published shortly. Summary of pages This document comprises a front cover, an in
15、side front cover, pages i and ii, pages1to12, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on theinside front cover.ISO 10229:1994(E) BSI 07-1999 1 1 Scope This Inter
16、national Standard specifies a method for the determination of the long-term sublethal toxicity of substances soluble in water under specified conditions to a species of anadromous fish Oncorhynchus mykiss Walbaum (Teleostei, Salmonidae) common name, rainbow trout in fresh water. NOTE 1“Substances” i
17、s considered to include pure chemicals tested singly or in mixtures, waste waters, process waters or other waters whose sublethal toxicity to fish it is necessary to determine. A range of concentrations of waste and process waters should be tested (see 8.2). The response measured is the change in ra
18、te of growth of fish exposed to the test substance over periods of14d and28d (see 1 in Annex B). NOTE 2The mass of each fish at the start of the test, the period of exposure, the feeding regime and temperature range have been chosen to ensure that the control fish will be growing exponentially throu
19、ghout the test. The method is applicable for assigning, for each test substance, a category of prolonged sublethal toxicity to Oncorhynchus mykiss under the test conditions. The results are insufficient by themselves to define water quality limits for environmental protection. It is possible for the
20、 method to 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 temperature, the quantity and quality of the dilution water, the food and the fish-marking technique. 2 Normative referen
21、ces The following standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this International Stan
22、dard are encouraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 7346-2:1984, Water quality Determination of the acute lethal toxicity of substances to a
23、 freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Part 2: Semi-static method. ISO 7346-3:1984, Water quality Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Part 3: Flow-through metho
24、d. 3 Principle Determination, under specified conditions, of the concentrations at which a substance statistically and significantly reduces the rate of growth of a test population of Oncorhynchus mykiss after exposure periods of14 d and28 d to that substance in the ambient water. The test is carrie
25、d out in two stages: a) a preliminary test, carried out using rainbow trout as the test species but in accordance with ISO7346-2 or ISO7346-3 (although the rainbow trout is not specifically named in these parts of ISO7346), which determines the concentrations at which the substance is lethal to50% o
26、f a test population after exposure to the substance in the ambient water for 96h: this median lethal concentration is designated as96h LC50 and serves to determine the range of concentrations for the final test (seenote 11 to 8.1). b) a final test, using16 fish per concentration, in which the result
27、s are recorded as both the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC). They are prefixed by an indication of the exposure period concerned, for example “28 d LOEC”. The data may also be fitted to a regression model in order to calculate the IC10 (inhi
28、bition concentration for a10% effect), or a similar statistic. Where analytical data show that median test concentrations remain relatively constant (i.e.within20% of the nominal values) throughout the test, the method allows the use of either measured or nominal concentrations in the statements on
29、LOEC and NOEC. Where such analyses show that the concentrations present remain relatively constant (i.e. 20% of the median) but are less than80% or greater than120% of the nominal values, then the analytical values derived from this method are used in presenting the LOEC and NOEC. Where no evidence
30、is available to show that the test concentrations remained at an acceptable level throughout the test period or where it is known that the concentrations of the test chemical have changed significantly (i.e. by greater than20% of the median) at any stage during the test, then irrespective of whether
31、 or not chemical analytical data are available the LOEC and NOEC cannot be defined using this test method. In these cases, the test is not necessarily invalidated but it can only be stated that the LOEC of the substance is u x mg/l, and that the NOEC of the substance is u y mg/l, the values x and y
32、given being estimated from the nominal concentrations used.ISO 10229:1994(E) 2 BSI 07-1999 NOTE 3Wherever possible, it is very important to determine the concentrations of the test substance to which the fish are exposed. Fish can be individually recognized throughout the test if the freeze-branding
33、 technique or an equivalent method (e.g. tagging) is used for marking, but this is not mandatory. 4 Test organism The recommended test species is Oncorhynchus mykiss Walbaum (Teleostei, Salmonidae), commonly known as the rainbow trout. Test fish shall be selected from a population of a single stock.
34、 This stock shall have been acclimatized and, in any case, maintained for at least2 weeks prior to the test in dilution water, continuously aerated (using bubbled air), under conditions of water quality, feeding regime and illumination similar to those used in the test. Except where otherwise stated
35、 (see clause 8), the fish shall be fed at a minimum rate of1% of their wet body mass per day during any pre-acclimation and acclimation period and4% of their mass per day during the final test (see 8.2). NOTE 4This latitude in feeding rate during the pretest period enables the fish to be grown to, o
36、r held at, a suitable size (seebelow) for the start of the test. The food shall be a dry proprietary salmonid fry food and shall be divided into two equal portions and given to the fish in two feeds per day separated by at least5 h. After exposure of14 d to the test substance, when the fish are weig
37、hed again, the rations shall be recalculated for each test vessel. At the start of the test, each fish shall have a mass of not less than3g and not more than5g (but see8.2). For the whole batch of fish used in the test, the range in individual masses at the start of the test shall lie within 10% of
38、the arithmetic mean of the masses. Test fish shall be free of overt disease or visible malformation. They shall not receive treatment for disease during the test or in the2weeks preceding the test. Maintain the fish in environmental conditions suitable for rainbow trout. NOTE 5Fish cultivation manua
39、ls should be consulted, for example 2 in Annex B. 5 Reagents Use only reagents of recognized analytical grade. The water used for the preparation of stock solutions shall be glass-distilled or deionized water of at least equivalent purity. 5.1 Dilution water The dilution water shall be suitable for
40、the long-term survival and growth of the test fish. The average pH of the dilution water shall be within the range6,7to8,5, but during a given test the pH of the dilution water shall not vary by more than 0,2pH units from the mean value. NOTE 6Such a pH range can be expected to be associated with a
41、bicarbonate alkalinity of1mg/l to100mg/l CaCO 3and total hardness values of20mg/l to300mg/l CaCO 3 , but these ranges will depend on the precise constitution of the dilution water and the time available for equilibration of the water. A suitable artificial water can be made with distilled water, as
42、follows: 294,0mg/l CaCl 2 2H 2 O 123,3mg/l MgSO 4 .7H 2 O 63,0mg/l NaHCO 3 5,5mg/l KCl 5.2 Stock solutions of test substances Prepare a stock solution of the test substance by dissolving a known amount of test substance in a defined volume of dilution water, deionized water or glass-distilled water.
43、 NOTE 7In the case where the test “substance” is a waste water, process water or other water (see clause 1, note 1) and not one or more pure chemicals, these waters are to be taken as the stock solution. Prepare or obtain the stock solution as frequently as necessary to maintain stable solutions. Al
44、though the use of solvents is not desirable, to enable stock solutions to be prepared and to assist in their transfer to the test vessels, it shall be permissible to dissolve or disperse substances of low aqueous solubility by suitable means, for example ultrasonic devices or the use of organic solv
45、ents of low toxicity to fish. If any such organic solvent is used, its concentration in the test solution shall not exceed0,1ml/l (and preferably be below0,01ml/l), and two sets of controls, one containing solvent at the maximum concentration used in any test vessel and one without solvent or test s
46、ubstance, shall be included. NOTE 8It is preferable that the same concentration of solvent be used in all test vessels. 5.3 Test solutions Prepare test solutions by adding appropriate amounts of the stock solution of the test substance(5.2) to the dilution water to give the required concentrations (
47、8.2). It is recommended that when a stock solution is prepared in distilled or deionized water, no more than100ml of stock solution should be added per10litres of dilution water. Wherever possible, it is very important to determine the concentrations of the test substance in each test vessel, with t
48、he exception of waste waters where this may not be possible.ISO 10229:1994(E) BSI 07-1999 3 6 Apparatus 6.1 General 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 inert and shall not absorb the test s
49、ubstance significantly. Usual laboratory equipment including a dip-net, made of nylon or of another chemically inert material, for the control vessels and another for all the test vessels (6.2) shall be used. 6.2 Test vessels, of capacity at least45litres. Their sides shall be covered with an opaque material to minimize disturbance of the fish. Before use, carefully wash new test vessels and then rinse successively with water and the dilution water(5.1). At the end of the test, empty the vessels, clean them, for examp
