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ASTM E729-23e1

Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians

Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians E0729-23E01 ASTM|E0729-23E01|en-US Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians Standard new BOS Vol. 11.06 Committee E50
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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 experimental 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. Bioavailability of the test substance may also differ between real-world exposures and laboratory exposures due to site-specific water quality conditions (see Guides E1192, E1563, and E2455).

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 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 E1023) or when deriving water quality criteria for aquatic organisms (3).

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.


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, usually during 2 to 4-day exposures, depending on the species. These procedures 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 justified by special needs or circumstances such as meeting specific study goals, regulatory needs, or to accommodate specific test organism life stages. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual or novel 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 unless the test organisms are severely stressed without feeding over 48 h. Static tests should probably not be conducted on materials that have a high oxygen demand, are highly volatile, are rapidly transformed 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; 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 toxicity.

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 mathematically estimated determination of a time-independent toxicity 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 requirements 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.

1.7 This guide is arranged as follows:





Referenced Documents




Summary of Guide


Significance and Use






 Special Requirements


 Construction Materials


 Metering System


 Test Chambers








Dilution Water










Test Material




 Stock Solution


 Test Concentration(s)


Test Organisms








 Care and Handling




 Disease Treatment










 Experimental Design


 Dissolved Oxygen






 Beginning the Test




 Duration of Test


 Biological Data


 Other Measurements


Analytical Methodology


Acceptability of Test


Calculation of Results




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 appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 7.

1.10 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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Book of Standards Volume: 11.06
Developed by Subcommittee: E50.47
Pages: 23
DOI: 10.1520/E0729-23E01
ICS Code: 07.080