5.1 An important goal of aquatic toxicology is to determine the effects of toxic compounds on species that play a central role in aquatic communities. Rotifers have a major impact on several important ecological processes in freshwater and coastal marine environments. As filter-feeders on phytoplankton and bacteria, rotifers exert substantial grazing pressure that at times exceeds that of the larger crustacean zooplankton ( 1 , 2 ) . ³ Rotifer grazing on phytoplankton is highly selective ( 2- 4 ) and can influence phytoplankton composition, the coexistence of competitors, and overall water quality ( 5 ) . The contribution of rotifers to the secondary production of many aquatic communities is substantial ( 6- 9 ) . In fresh water, rotifers often account for the major fraction of zooplankton biomass at certain times of the year ( 10 , 11 ) . Rotifers and other zooplankton are a significant food source for many larval fish, planktivorous adult fish ( 12 , 13 ) , and several invertebrate predators ( 14- 16 ) . The high metabolic rates of rotifers contribute to their role in nutrient cycling, which might make rotifers more important than crustaceans in certain communities ( 17 , 18 ) .

5.2 In addition to their important ecological role in aquatic communities, rotifers are attractive organisms for toxicological studies because an extensive database exists on the basic biology of this group. Techniques have been published for the culture of many rotifer species ( 3 , 19 ) . The rotifer life cycle is well defined ( 20 , 21 ) , and the factors regulating it are reasonably well understood ( 22- 25 ) . Several aspects of rotifer behavior have been examined closely ( 26- 29 ) . The biogeography of many rotifer species has been characterized ( 30 , 31 ) , and the systematics of the group are well described ( 32 , 33 ) .

5.3 Toxicity tests with rotifers of the genus Brachionus are more easily performed than with many other aquatic animals because of their rapid reproduction, short generation times, sensitivity ( 34 ) , and the commercial availability of rotifer cysts. Brachionus spp. have a cosmopolitan distribution that spans six continents ( 31 ) , and they are ecologically important members of many aquatic communities impacted by pollution. The use of B. plicatilis in an acute toxicity test for estuarine and marine environments and B. rubens in fresh water has been described, as well as their sensitivity to several toxicants ( 35 , 36 ) .

5.4 The test described here is fast, easy to execute, sensitive, and cost-effective. Obtaining test animals from cysts greatly reduces some of the major problems in routine aquatic toxicological testing such as the limited availability of test animals and the inconsistency of sensitivity over time. Rotifers hatched from cysts are of similar age and are physiologically uniform, thus eliminating pre-test conditions as a source of variability in the toxicity test. Cysts can be shipped inexpensively world-wide, allowing all laboratories to use standard, genetically defined strains that have been calibrated with reference toxicants. The convenience of an off-the-shelf source of test animals that require no pre-conditioning is likely to permit new applications of aquatic toxicity tests.

5.5 Sensitivity to toxicants is compound and species specific, but the sensitivity of B. calyciflorus is generally comparable to that of Daphnia ( 37 ) .

5.6 Rotifer cysts are commercially available, but they can also be obtained from natural populations and from laboratory cultures. Techniques for rotifer cyst production in laboratory populations have been described ( 24 , 25 , 38 , 39 ) . However, using a well-characterized rotifer strain is best since strains are known to have differing toxicant sensitivities.

1.1 This guide describes procedures for obtaining laboratory data concerning the acute toxicity of chemicals and aqueous effluents released into fresh, estuarine, or marine waters. Acute toxicity is measured by exposing Brachionus newly hatched from cysts to a series of toxicant concentrations under controlled conditions. This guide describes a test for using B. calyciflorus , a fresh water rotifer, and the Appendix describes modifications of this test for estuarine and marine waters using B. plicatilis . These procedures lead to an estimation of acute toxicity, including the concentration expected to kill 50?% of the test rotifers (LC50) in 24 h. Procedures not specifically stated in this guide should be conducted in accordance with Guide E729 and Guide E1192 .

1.2 Modifications of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, the results of tests conducted using modified procedures might not be comparable to rotifer acute tests that follow the protocol described here. Comparison of the results using modified procedures might provide useful information concerning new concepts and procedures for conducting acute toxicity tests on chemicals and aqueous effluents.

1.3 This guide is organized as follows:

1.4 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or mixtures. This guide can also be used to conduct investigations of the effects on rotifer survival of pH, hardness, and salinity and on materials such as aqueous effluents, leachates, oils, particulate matter, sediments, and surface waters. This guide might not be appropriate for materials with high oxygen demand, with high volatility, subject to rapid biological or chemical transformation, or that readily sorb to test chambers.

1.5 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 and health practices and determine the applicability of regulatory limitations prior to use. For specific hazards statements, see Section 8 .