ASTM_E_2172_-_01_2014.pdf

Designation:E217201(Reapproved 2014)Standard Guide forConducting Laboratory Soil Toxicity Tests with theNematode Caenorhabditis elegans1This standard is issued under the fixed designation E2172;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This guide covers procedures for obtaining laboratorydata to evaluate the adverse effects of chemicals associatedwith soil to nematodes from soil toxicity tests.This standard isbased on a modification to Guide E1676.The methods aredesigned to assess lethal or sublethal toxic effects on nema-todes in short-term tests in terrestrial systems.Soils to be testedmay 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 referencesoils diluted with artificial soil.Test procedures are describedfor the species Caenorhabditis elegans(see Annex A1).Methods described in this guide may also be useful forconducting soil toxicity tests with other terrestrial species,although modifications may be necessary.1.2 Summary of Previous StudiesInitial soil toxicity test-ing using the free-living,bacterivorous soil nematode Cae-norhabditis elegans was developed by Donkin and Dusenbery(1).2Following the development of an effective method ofrecovery of C.elegans from test soils,the organism was usedto identify factors that affect the toxicity of zinc,cadmium,copper,and lead(2).Freeman et al.further refined thenematode bioassay by decreasing the quantity of soil andspiking solution volumes,determining test acceptabilitycriteria,and developing control charts to assess worm healthusing copper as a reference toxicant(3).More recently,thetoxicological effects of nitrate and chloride metallic salts in twonatural soils were compared(4).LC50 values for C.elegansexposed for 24-h to nitrate salts of cadmium,copper,zinc,leadand nickel in an artificial soil(see AnnexA2)were found to besimilar to LC50 values for the earthworm,Eisenia fetida(5).Increasing the exposure time to 48-h resulted in much lowerLC50 values(6).However,longer exposure times necessitatethe addition of food and lead to lower recovery percentages insoils high in organic matter.A modification of the recoverymethod has also been used with a transgenic strain of C.elegans used as a soil biomonitoring tool to assess sub-lethaleffects of metal exposures in soil(7).A variety of sub-lethalendpoints have been developed using C.elegans in aquaticmedia and may prove useful for assessing soil exposures(8).1.3 Modification of these procedures might be justified byspecial needs.The results of tests conducted using typicalprocedures may not be comparable to results using this guide.Comparison of results obtained using modified and unmodifiedversions of these procedures might provide useful informationconcerning new concepts and procedures for conducting soiltoxicity tests with terrestrial worms.1.4 The results from field-collected soils used in toxicitytests to determine a spatial or temporal distribution of soiltoxicity may be reported in terms of the biological effects onsurvival or sublethal endpoints.These procedures can be usedwith appropriate modifications to conduct soil toxicity testswhen factors such as temperature,pH,and soil characteristics(for example,particle size,organic matter content,and claycontent)are of interest or when there is a need to test suchmaterials as sewage sludge.These methods might also beuseful for conducting bioaccumulation tests.1.5 The results of toxicity tests with(1)materials(forexample,chemicals or waste mixtures)added experimentallyto artificial soil,reference soils,or site soils,(2)site soilsdiluted with reference soils,and(3)site or reference soilsdiluted with artificial soil,so as to create a series ofconcentrations,may be reported in terms of an LC50(medianlethal concentration)and sometimes an EC50(median effectconcentration).1.6 This guide is arranged as follows:Scope1Referenced Documents2Terminology3Summary of Guide4Significance and Use5Interferences6Apparatus7Safety Precautions8Soil9Test Organism10Procedure111This guide is under the jurisdiction ofASTM Committee E50 on EnvironmentalAssessment,Risk Management and Corrective Action and is the direct responsibil-ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.Current edition approved Oct.1,2014.Published December 2014.Originallyapproved in 2002.Last previous edition approved in 2008 as E217202(2008).DOI:10.1520/E2172-01R14.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 Analytical Methodology12Acceptability of Test13Ca