【国外标准】 Standard Guide for Conducting Laboratory Soil Toxicity Tests with the Nematode Caenorhabditis elegans

5.1 Soil toxicity tests provide information concerning the toxicity and bioavailability of chemicals associated with soils to terrestrial organisms. As important members of the soil fauna, nematodes have a number of characteristics that make them appropriate organisms for use in the assessment of potentially hazardous soils. Bacterial-feeding nematodes such as C. elegans feed on soil microbes and contribute to the breakdown of organic matter. They are also of extreme importance in the cycling and degradation of key nutrients in soil ecosystems (9). Soil nematodes also serve as a source of prey and nutrients for fauna and microflora such as soil nematophagous fungi (10). A major change in the abundance of soil invertebrates such as nematodes, either as a food source or as organisms functioning properly in trophic energy transfer and nutrient cycling, could have serious adverse ecological effects on the entire terrestrial system.5.2 Results from soil tests might be an important consideration when assessing the hazards of materials to terrestrial organisms.5.3 The soil test might be used to determine the temporal or spatial distribution of soil toxicity. Test methods can be used to detect horizontal and vertical gradients in toxicity.5.4 Results of soil tests could be used to compare the sensitivities of different species.5.5 An understanding of the effect of these parameters on toxicity may be gained by varying soil characteristics such as pH, clay content, and organic material.5.6 Results of soil tests may be useful in helping to predict the effects likely to occur with terrestrial organisms in field situations.5.6.1 Field surveys can be designed to provide either a qualitative or quantitative evaluation of biological effects within a site or among sites.5.6.2 Soil surveys evaluating biological effects are usually part of more comprehensive analyses of biological, chemical, geological, and hydrographic conditions. Statistical correlation can be improved and costs reduced if subsamples of soil for laboratory tests, geochemical analyses, and community structure are taken simultaneously from the same grab of the same site.5.7 Soil toxicity tests can be an important tool for making decisions regarding the extent of remedial action necessary for contaminated terrestrial sites.1.1 This guide covers procedures for obtaining laboratory data to evaluate the adverse effects of chemicals associated with soil to nematodes from soil toxicity tests. This standard is based on a modification to Guide E1676. The methods are designed to assess lethal or sublethal toxic effects on nematodes in short-term tests in terrestrial systems. Soils to be tested may be (1) references soils or potentially toxic soil sites; (2) artificial, reference, or site soils spiked with compounds; (3) site soils diluted with reference soils; or (4) site or reference soils diluted with artificial soil. Test procedures are described for the species Caenorhabditis elegans (see Annex A1). Methods described in this guide may also be useful for conducting soil toxicity tests with other terrestrial species, although modifications may be necessary.1.2 Summary of Previous Studies—Initial soil toxicity testing using the free-living, bacterivorous soil nematode Caenorhabditis elegans was developed by Donkin and Dusenbery (1).2 Following the development of an effective method of recovery of C. elegans from test soils, the organism was used to identify factors that affect the toxicity of zinc, cadmium, copper, and lead (2) . Freeman et al. further refined the nematode bioassay by decreasing the quantity of soil and spiking solution volumes, determining test acceptability criteria, and developing control charts to assess worm health using copper as a reference toxicant (3). More recently, the toxicological effects of nitrate and chloride metallic salts in two natural soils were compared (4) . LC50 values for C. elegans exposed for 24-h to nitrate salts of cadmium, copper, zinc, lead and nickel in an artificial soil (see Annex A2) were found to be similar to LC50 values for the earthworm, Eisenia fetida (5). Increasing the exposure time to 48-h resulted in much lower LC50 values (6). However, longer exposure times necessitate the addition of food and lead to lower recovery percentages in soils high in organic matter. A modification of the recovery method has also been used with a transgenic strain of C. elegans used as a soil biomonitoring tool to assess sub-lethal effects of metal exposures in soil (7) . A variety of sub-lethal endpoints have been developed using C. elegans in aquatic media and may prove useful for assessing soil exposures (8).1.3 Modification of these procedures might be justified by special needs. The results of tests conducted using typical procedures may not be comparable to results using this guide. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting soil toxicity tests with terrestrial worms.1.4 The results from field-collected soils used in toxicity tests to determine a spatial or temporal distribution of soil toxicity may be reported in terms of the biological effects on survival or sublethal endpoints. These procedures can be used with appropriate modifications to conduct soil toxicity tests when factors such as temperature, pH, and soil characteristics (for example, particle size, organic matter content, and clay content) are of interest or when there is a need to test such materials as sewage sludge. These methods might also be useful for conducting bioaccumulation tests.1.5 The results of toxicity tests with (1) materials (for example, chemicals or waste mixtures) added experimentally to artificial soil, reference soils, or site soils, (2) site soils diluted with reference soils, and (3) site or reference soils diluted with artificial soil, so as to create a series of concentrations, may be reported in terms of an LC50 (median lethal concentration) and sometimes an EC50 (median effect concentration).1.6 This guide is arranged as follows: 1Referenced Documents 2Terminology 3Summary of Guide 4 5Interferences 6Apparatus 7Safety Precautions 8Soil 9Test Organism 10Procedure 11Analytical Methodology 12Acceptability of Test 13Calculation of Results 14Report 15Annexes A1. Caenorhabditis elegans  A2. Artificial Soil CompositionReferences  1.7 The values stated in SI units are to be regarded as the standard.1.8 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. While some safety considerations are included in this guide, it is beyond the scope of this standard to encompass all safety requirements necessary to conduct soil toxicity tests. Specific precautionary statements are given in Section 8.1.9 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.