1.1 This standard covers procedures for obtaining laboratory data concerning the toxicity of test material (for example, sediment or hydric soil (that is, a soil that is saturated, flooded, or ponded long enough during the growing season to develop anaerobic (oxygen-lacking) conditions that favor the growth and regeneration of hydrophytic vegetation)) to amphibians. This test procedure uses larvae of the northern leopard frog (Lithobates pipiens). Other anuran species (for example, the green frog (Lithobates clamitans), the wood frog (Lithobates sylvatica), the American toad (Bufo americanus)) may be used if sufficient data on handling, feeding, and sensitivity are available. Test material may be sediments or hydric soil collected from the field or spiked with compounds in the laboratory.

1.2 The test procedure describes a 10-d whole sediment toxicity test with an assessment of mortality and selected sublethal endpoints (that is, body width, body length). The toxicity tests are conducted in 300 to 500-mL chambers containing 100 mL of sediment and 175 mL of overlying water. Overlying water is renewed daily and larval amphibians are fed during the toxicity test once they reach Gosner stage 25 (operculum closure over gills). The test procedure is designed to assess freshwater sediments, however, R. pipiens can tolerate mildly saline water (not exceeding about 2500 mg Cl^-/L, equivalent to a salinity of about 4.1 when Na⁺ is the cation) in 10-d tests, although such tests should always include a concurrent freshwater control. Alternative test durations and sublethal endpoints may be considered based on site-specific needs. Statistical evaluations are conducted to determine whether test materials are significantly more toxic than the laboratory control sediment or a field-collected reference sample(s).

1.3 Where appropriate, this standard has been designed to be consistent with previously developed methods for assessing sediment toxicity to invertebrates (for example, Hyalella azteca and Chironomus dilutus toxicity tests) described in the United States Environmental Protection Agency (USEPA, (1))2 freshwater sediment testing guidance, Test Methods E1367 and E1706, and Guides E1391, E1525, E1611, and E1688. Tests extending to 10 d or beyond, and including sublethal measurements such as growth, are considered more effective in identifying chronic toxicity and thus delineating areas of moderate contamination (1-3).

1.4 Many historical amphibian studies, both water and sediment exposure, have used tests of shorter duration (5 days or less) (for example, 4-7) and, although both survival and sublethal endpoints were often assessed, there is substantive evidence that tests of longer duration are likely to be more sensitive to some contaminants (8-10). Research performed to develop and validate this test protocol included long-term (through metamorphosis) investigations and other researchers have also conducted long-duration tests with anurans (7-20). Interestingly, some studies with anurans have shown significantly reduced growth (for example, whole body mass, snout-vent length) can be detected earlier in a longer-term test (for example, at 14-20 d), but cannot be statistically distinguished in older organisms later in the test (11, 14). In the development of these procedures, an attempt was made to balance the needs of a practical assessment with the importance of assessing longer-term effects so that the results will demonstrate the needed accuracy and precision. The most recent sediment toxicity testing protocols for invertebrates have encompassed longer duration studies which allow the measurement of reproductive endpoints (1, 21). Such tests, because of increased sensitivity of the sublethal endpoints, may also be helpful in evaluating toxicity. Full life-cycle studies with anurans (including reproduction) are usually not feasible from either a technical or monetary standpoint. However, if site-specific information indicates that the contaminants present are likely to affect other endpoints (including teratogenicity), then the duration of the toxicity test may be increased through metamorphosis or additional sublethal endpoints may be measured (for example, impaired behavior, deformities, time-to-metamorphosis). The possible inclusion of these endpoints and extension of test length should be considered during development of the project or study plan (see 8.1.1).

1.5 The methodology presented in this standard was developed under a Department of Defense (DoD) research program and presented in a guidance manual for risk assessment staff and state/federal regulators involved in the review and approval of risk assessment work plans and reports (22). To develop this method, a number of tests with spiked sediment tests were conducted (22, 23). Since development of the methodology it has been used operationally to evaluate field-collected sediments from several state and federal environmental sites (24, 25). For most of these studies the preferred test organisms, Lithobates pipiens, was used. At a lead-contaminated state-led site, operated by the Massachusetts Highway Department, Xenopus laevis(African clawed frog) was used in the sediment test system because of availability problems with Lithobates pipiens (26), The test method was also used to evaluate sediment toxicity at a cadmium-contaminated USEPA Region 4-led site in Tennessee (27). The methodology was used to help characterize potential effects of contaminants on amphibians and to help develop preliminary remedial goals, if warranted. All tests evaluated survival and growth effects after 10 d of exposure in accordance with the methods presented in this standard.

1.6 The use of larval amphibians to assess environmental toxicity is not novel. Researchers have used tadpoles to examine toxicity of metals and organic compounds. Most of these studies have been through water exposure, usually in a manner similar to fish or invertebrate exposure as described in Guide E729 (28-40). Fewer studies have focused on exposure of anuran larvae to sediments, and the methods employed vary widely, from in situ enclosures (15, 41) to laboratory tests using variable exposure conditions and organism ages (4, 8, 14, 39, 42-44). No studies were identified that used the same test conditions as described in this standard. However, several laboratory-based evaluations of sediment effects on amphibians are described in the following subsections.

1.7 Amphibian species may be key receptors of potential chemicals of concern at contaminated sites. Although historically not often included in risk assessments, the importance of amphibians as both sensitive and keystone species is increasingly recognized, particularly considering the decline in amphibian worldwide populations, which may be driven by multiple localized stress agents rather than a single, dominating cause (45). The lack of amphibian representation as surrogate species is likely due to multiple factors including scant knowledge of local amphibian populations and life histories, the paucity of applicable toxicity data, and inconsistency in standardized assessment protocols. A review of ecological risk assessment methods for amphibians and gaps in existing amphibian toxicity data and methods is provided by Johnson et al. (46). The importance of amphibians in the ecological risk assessment process is recognized by Environment and Climate Change Canada in the Ecological Risk Assessment Guidance under the Federal Contaminated Sites Action Plan (47). Sediment toxicity tests are an effective means for evaluating the impact of sediment contamination on amphibians in a multiple lines of evidence paradigm. The evaluation is most powerful when toxicity testing sampling stations are co-located with sediment analytical chemistry samples and ecological surveys, allowing for a detailed evaluation of the co-occurring data in the ecological risk assessment. The spatial and temporal co-location of toxicity testing and analytical samples is particularly important for establishing contaminant-specific effects and assessing contaminant bioavailability.

1.8 In order for a sediment toxicity test to be sensitive it must be of sufficient duration to measure potential toxicity and it must be conducted during the appropriate developmental stage of the test organism’s life cycle. Using recently hatched tadpoles and conducting the sediment exposure test for 10 d to allow the evaluation of growth endpoints meets both of these sensitivity requirements.

1.9 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.10 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.

1.11 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.