2002 ASTM水生生物急性毒性试验操作指南

Designation: E 729 – 96 (Reapproved 2002) Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians1 This standard is issued under the fixed designation E 729; the number immediately following the designation indicates the year of original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A superscript epsilon (e) indicates an editorial change since the last revision or reapproval. This standard has been approved for use by agencies of the Department of Defense. 1. Scope 1.1 This guide (1)2 describes procedures for obtaining laboratory data concerning the adverse effects (for example, lethality and immobility) of a test material added to dilution water, but not to food, on certain species of freshwater and saltwater fishes, macroinvertebrates, and amphibians during 2 to 8-day exposures, depending on the species. These proce- dures will probably be useful for conducting acute toxicity tests with many other aquatic species, although modifications might be necessary. 1.2 Other modifications of these procedures might be justi- fied by special needs or circumstances. Although using appro- priate procedures is more important than following prescribed procedures, results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting acute tests. 1.3 This guide describes tests using three basic exposure techniques: static, renewal, and flow-through. Selection of the technique to use in a specific situation will depend on the needs of the investigator and on available resources. Tests using the static technique provide the most easily obtained measure of acute toxicity, but conditions often change substantially during static tests; therefore, static tests should not last longer than 96 h, and test organisms should not be fed during such tests. Static tests should probably not be conducted on materials that have a high oxygen demand, are highly volatile, are rapidly trans- formed biologically or chemically in aqueous solution, or are removed from test solutions in substantial quantities by the test chambers or organisms during the test. Because the pH and concentrations of dissolved oxygen and test material are maintained at desired levels and degradation and metabolic products are removed, tests using renewal and flow-through methods are preferable and may last longer than 96 h; test organisms may be fed during renewal and flow-through tests. Although renewal tests might be more cost-effective, flow- through tests are generally preferable. 1.4 Acute tests may be performed to meet regulatory data requirements or to obtain time-independent estimates of toxic- ity. 1.4.1 If the objective is to obtain data to meet regulatory requirements, it may be necessary to limit the number of observation times based on stipulations of the regulatory agency and cost considerations. 1.4.2 If the objective of an acute toxicity test is to determine a time-independent (that is, incipient, threshold, or asymptotic) toxicity level, an appropriate number of observations must be taken over an exposure duration of sufficient length to establish the shape of the toxicity curve or allow the direct or math- ematically estimated determination of a time-independent tox- icity value (1), or both. 1.5 In the development of these procedures, an attempt was made to balance scientific and practical considerations and to ensure that the results will be sufficiently accurate and precise for the applications for which they are commonly used. A major consideration was that the common uses of the results of acute toxicity tests do not require or justify stricter require- ments than those set forth herein. Although the tests may be improved by using more organisms, longer acclimation times, and so forth, the requirements presented herein should usually be sufficient. 1.6 Results of acute toxicity tests should usually be reported in terms of an LC50 (median lethal concentration) or EC50 (median effective concentration) at the end of the test, but it is desirable to provide information concerning the dependence of adverse effects on both time and concentration. Thus, when feasible, flow-through and renewal tests should be conducted so that LC50s or EC50s can be reported from 6 h to an asymptotic (time-independent, threshold, incipient) value, if one exists. In some situations, it might only be necessary to determine whether a specific concentration is acutely toxic to the test species or whether the LC50 or EC50 is above or below a specific concentration. 1 This guide is under the jurisdiction of ASTM Committee E47 on Biological Effects and Environmental Fate and is the direct responsibility of Subcommittee E47.01 on Aquatic Assessment and Toxicology. Current edition approved Oct. 10, 2002. Published March 2003. Originally approved in 1980. Last previous edition approved in 1996 as E 729 – 96. 2 The boldface numbers in parentheses refer to the list of references at the end of this standard. 1 Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States. ??? www.zhunbiao.com ???? The standard is downloaded from www.zhunbiao.com Administrator 下划线 Administrator 高亮 Administrator 下划线 Administrator 下划线 1.7 This guide is arranged as follows: Section Referenced Documents 2 Terminology 3 Summary of Guide 4 Significance and Use 5 Apparatus 6 Facilities 6.1 Special Requirements 6.2 Construction Materials 6.3 Metering System 6.4 Test Chambers 6.5 Cleaning 6.6 Acceptability 6.7 Hazards 7 Dilution Water 8 Requirements 8.1 Source 8.2 Treatment 8.3 Characterization 8.4 Test Material 9 General 9.1 Stock Solution 9.2 Test Concentration(s) 9.3 Test Organisms 10 Species 10.1 Age 10.2 Source 10.3 Care and Handling 10.4 Feeding 10.5 Disease Treatment 10.6 Holding 10.7 Acclimation 10.8 Quality 10.9 Procedure 11 Experimental Design 11.1 Dissolved Oxygen 11.2 Temperature 11.3 Loading 11.4 Beginning the Test 11.5 Feeding 11.6 Duration of Test 11.7 Biological Data 11.8 Other Measurements 11.9 Analytical Methodology 12 Acceptability of Test 13 Calculation of Results 14 Report 15 1.8 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only. 1.9 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appro- priate safety and health practices and determine the applica- bility of regulatory limitations prior to use. Specific hazard statements are given in Section 7. 2. Referenced Documents 2.1 ASTM Standards: E 724 Guide for Conducting Static Acute Toxicity Tests Starting with Embryos of Four Species of Saltwater Bivalve Molluscs3 E 943 Terminology Relating to Biological Effects and En- vironmental Fate3 E 1023 Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses3 E 1191 Guide for Conducting Life-Cycle Toxicity Tests with Saltwater Mysids3 E 1192 Guide for Conducting Acute Toxicity Tests on Aqueous Effluents with Fishes, Macroinvertebrates, and Amphibians3 E 1203 Practice for Using Brine Shrimp Nauplii as Food for Test Animals in Aquatic Toxicology3 E 1563 Guide for Conducting Static Acute Toxicity Tests with Echinoid Embryos3 E 1604 Guide for Behavioral Testing in Aquatic Toxicol- ogy3 IEEE/ASTM SI 10 Standard for Use of the International System of Units (SI) (the Modernized Metric System)4 3. Terminology 3.1 Acute toxicity tests are generally used to determine the concentration of test material that produces a specific adverse effect on a specified percentage of test organisms during a short exposure. Because death is an obviously important adverse effect and is easily detected for many species, the most common acute toxicity test is the acute lethality test. Experi- mentally, effect on 50 % of a group of test organisms is the most reproducible and easily determined measure of toxicity, and 96 h is often a convenient, useful exposure duration. Therefore, the measure of acute toxicity most often used with fishes, macroinvertebrates, and amphibians is the 96-h LC50. However, because immobilization is a severe effect and is not easy to distinguish from death for some species, the measure of acute toxicity most often used with daphnids and midge larvae is the 48-h EC50 based on death plus immobilization. The terms LC50 and EC50 are consistent with the widely used toxicological terms LD50 (median lethal dose) and ED50 (median effective dose), respectively. The terms LC50 and EC50 should be used whenever results are calculated based on the concentration of test material in dilution water, whereas the terms LD50 and ED50 should be used whenever results are calculated based on the quantity of test material that enters or is applied directly to test organisms. For toxic agents or tests for which neither concentration nor dose is appropriate, such as tests on temperature or with poorly water-soluble materials, the terms LL50 (median lethal level) and EL50 (median effective level) should be used, if the effect is dichotomous. For tests in which the effect is expressed as a percent inhibition compared to the control, for example, a percent inhibition in growth, and not as the percentage of the individual organisms that were affected, the term IC50 should be used to denote the concen- tration that causes a 50 % inhibition compared to the control. 3.2 Acute toxicity tests in which test organisms are exposed to test solutions containing a test material can be conducted by at least four techniques: 3.2.1 In the static technique, test solutions and organisms are placed in chambers and kept there for the duration of the test. 3.2.2 The recirculation technique is like the static technique except that each test solution is continuously circulated through 3 Annual Book of ASTM Standards, Vol 11.05. 4 Annual Book of ASTM Standards, Vol 14.02. E 729 – 96 (2002) 2 ??? www.zhunbiao.com ???? The standard is downloaded from www.zhunbiao.com Administrator 高亮 Administrator 高亮 Administrator 高亮 Administrator 高亮 Administrator 高亮 Administrator 高亮 Administrator 下划线 Administrator 下划线 an apparatus designed to maintain water quality, and possibly remove degraded, but not undegraded, test material by such means as aeration, filtration, and sterilization and then returned to the test chamber. 3.2.3 The renewal technique is like the static technique except that test organisms are periodically exposed to fresh test solution of the same composition, usually once every 24 h, either by transferring the organisms from one test chamber to another or by replacing nearly all the test solution. 3.2.4 In the flow-through technique, test solution flows through the test chamber on a once-through basis throughout the test. 3.2.4.1 Two procedures may be used. In the first a large volume of each test solution is prepared before the beginning of the test, and these solutions flow through the respective chambers. In the second and more common procedure, fresh test solutions are prepared continuously or every few minutes just before they enter the respective test chambers. In both procedures a metering system controls the flow of test solution, and in the latter procedure the test solutions are prepared by the metering system. Both of the procedures may be used to conduct continuous-flow tests. Many tests conducted using the second procedure, however, are intermittent-flow tests because the metering system cycles and delivers test solution every few minutes. 3.2.5 With any of these techniques a pump or stirrer can be used to create a current in the test chamber to accommodate particular species, but the current will often increase both aeration and volatilization. 3.3 In flow-through tests a “volume addition” is the intro- duction into the test chamber of a volume of test solution equal to the volume of solution in the chamber. 3.4 For the purposes of 8.4.1, the term“ organophosphorus pesticides” refers to chlorpyrifos, demeton, diazinon, disulfo- ton, fenitrothion, malathion, methyl parathion, and parathion; the term “organochlorine pesticides” refers to aldrin, chlor- dane, DDD, DDE, DDT, dieldrin, endosulfan, endrin, hep- tachlor, heptachlor epoxide, lindane, methoxychlor, mirex, and toxaphene; and the term “chlorinated phenoxy herbicides” refers to the free acids, salts, and esters of 2,4-D, dicamba, silvex, and 2,4,5-T. The term “organic chlorine” refers to chlorine that would be detected if, when samples are prepared for gas chromatographic analysis for polychlorinated biphenyls (PCBs) and the organochlorine pesticides listed above, a chloride detector is used instead of an electron capture detector to measure compounds that elute from just before lindane to just after mirex on the gas chromatograph being used. Organic chlorine does not refer only to chlorine associated with organochlorine pesticides and PCBs; it refers to all chlorine that elutes within the specified period. 3.5 reconstituted water—a dilution water that is prepared by adding sea salt or appropriate amounts of selected chemicals to water, which is usually prepared using deionization, distilla- tion, or reverse osmosis, so that the concentrations and ratios of the major ions in the dilution water are similar to those in comparable natural surface waters. 3.6 The words “must,” “should,”“ may,” “can,” and “might” have very specific meanings in this guide. “Must” is used to express an absolute requirement, that is, to state that the test ought to be designed to satisfy the specified condition, unless the purpose of the test requires a different design. “Must” is only used in connection with factors that directly relate to the acceptability of the test (see 13.1). “Should” is used to state that the specified condition is recommended and ought to be met if possible. Although violation of one “should” is rarely a serious matter, violation of several will often render the results questionable. Terms such as “is desirable,” “is often desirable,” and “might be desirable” are used in connection with less important factors. “May” is used to mean “is (are) allowed to,”“ can” is used to mean “is (are) able to,” and “might” is used to mean “could possibly.” Thus the classic distinction between “may” and “can” is preserved, and “might” is never used as a synonym for either “may” or “can.” 3.7 IC50—a statistically or graphically estimated concen- tration of test material that is expected to cause a 50 % inhibition of one or more specified biological processes (such as growth or reproduction), for which the data are not dichoto- mous, under specified conditions. 3.8 For definitions of other terms used in this guide, refer to Terminology E 943 and Guide E 1023. For an explanation of units and symbols, refer to Standard IEE/ASTM SI 10. 4. Summary of Guide 4.1 In each of two or more treatments, test organisms of one species are maintained for 2 to 8 days in one or more test chambers. In each of the one or more control treatments, the organisms are maintained in dilution water to which no test material has been added in order to provide (1) a measure of the acceptability of the test by giving an indication of the quality of the test organisms and the suitability of the dilution water, test conditions, handling procedures, and so forth, and (2) the basis for interpreting data obtained from the other treatments. In each of the one or more other treatments, the organisms are maintained in dilution water to which a selected concentration of test material has been added. Data concerning effects on the organisms in each test chamber are usually obtained periodically during the test and analyzed to determine LC50s, EC50s, or IC50s for various lengths of exposure. 5. Significance and Use 5.1 An acute toxicity test is conducted to obtain information concerning the immediate effects on test organisms of a short-term exposure to a test material under specific experi- mental conditions. An acute toxicity test does not provide information about whether delayed effects will occur, although a post-exposure observation period, with appropriate feeding, if necessary, might provide such information. 5.2 Results of acute toxicity tests might be used to predict acute effects likely to occur on aquatic organisms in field situations as a result of exposure under comparable conditions, except that (1) motile organisms might avoid exposure when possible, and (2) toxicity to benthic organisms might be dependent on sorption or settling of the test material onto the substrate. 5.3 Results of acute tests might be used to compare the acute sensitivities of different species and the acute toxicities of E 729 – 96 (2002) 3 ??? www.zhunbiao.com ???? The standard is downloaded from www.zhunbiao.com Administrator 下划线 Administrator 下划线 Administrator 高亮 Administrator 高亮 Administrator 下划线 Administrator 下划线 different test materials, and to study the effects of various environmental factors on results of such tests. 5.4 Results of acute toxicity tests might be an important consideration when assessing the hazards of materials to aquatic organisms (see Guide E 1023) or when deriving water quality criteria for aquatic organisms (2). 5.5 Results of acute toxicity tests might be useful for studying the biological availability of, and structure-activity relationships between, test materials. 5.6 Results of acute toxicity tests will depend on the temperature, composition of the dilution water, condition of the test organisms, exposure technique, and other factors. 6. Apparatus 6.1 Facilities—Although some small organisms can be held and acclimated in static or renewal systems, most organisms are held, acclimated, and cultured in flow-through systems. Test chambers should be in a constant-temperature room, incubator, or recirculating water bath. For static and renewal tests a dilution-water tank, which may be used to prepare reconstituted water, is often elevated so that dilution water can be gravity fed into holding and acclimation tanks and test chambers. For flow-through tests an elevated headbox is often desirable so that dilution water can be gravity fed into holding and acclimation tanks and into the metering system (see 6.4), which prepares the test solutions and delivers them to the test chambers. Strainers and air traps should be included in the water-supply system. Headboxes and holding, acclimation, culture, and dilution-water tanks should be equipped for temperature control and aeration (see 8.3.1). Air used for aeration should be free of fumes, oil, and water; filters to remove oil and water are desirable. Filtration of air through a 0.22-µm bacterial filter might be desirable (3). The facility should be well-ventilated and free of fumes. To further reduce the possibility of contamination by test materials and other substances, especially volatile ones, holding, acclimation, and culture tanks should not be in a room in which toxicity tests are conducted, stock solutions or test solutions are prepared, or equipment is cleaned. Organisms should be shielded from disturbances with curtains or partitions to prevent unnecessary stress during holding, acclimation, culture, and testing. A timing device should be used to provide a 16-h light and 8-h dark photoperiod. A15 to 30-min transition period (4) when the lights go on might be desirable to reduce the possibility of organisms being stressed by large, sudden increases in light intensity. A transition period when the lights go off might also be desirable. 6.2 Special Requi