1、Designation: E 2172 01Standard Guide forConducting Laboratory Soil Toxicity Tests with theNematode Caenorhabditis elegans1This standard is issued under the fixed designation E 2172; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, t
2、he year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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 ass
3、ociatedwith soil to nematodes from soil toxicity tests. This standard isbased on a modification to Guide E 1676. 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
4、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). Meth-ods described in this guid
5、e may also be useful for conductingsoil toxicity tests with other terrestrial species, althoughmodifications 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
6、(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 s
7、olution volumes, determining test acceptability crite-ria, and developing control charts to assess worm health usingcopper as a reference toxicant (3). More recently, the toxico-logical effects of nitrate and chloride metallic salts in twonatural soils were compared (4). LC50 values for C. elegansex
8、posed for 24-h to nitrate salts of cadmium, copper, zinc, leadand nickel in an artificial soil (see Annex A2) 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 necess
9、itatethe 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 su
10、b-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 gui
11、de.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
12、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,
13、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
14、) 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 of concen-trations, may be reported in terms of an LC50 (median lethalconcentration) and sometim
15、es an EC50 (median effect concen-tration).1.6 This guide is arranged as follows:Scope 1Referenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Interferences 6Apparatus 7Safety Precautions 8Soil 9Test Organism 10Procedure 111This guide is under the jurisdiction of ASTM Committee E4
16、7 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.02 on Terrestrial Assessment and Toxicology.Current edition approved Nov. 10, 2001. Published January 2002.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard
17、.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Analytical Methodology 12Acceptability of Test 13Calculation of Results 14Report 15Annexes A1. Caenorhabditisel-egansA2. ArtificialSoilCompositionReferences1.7 The values stated in SI
18、units are to be regarded as thestandard.1.8 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory
19、requirements prior to use. While somesafety considerations are included in this guide, it is beyond thescope of this standard to encompass all safety requirementsnecessary to conduct soil toxicity tests. Specific precautionarystatements are given in Section 8.2. Referenced Documents2.1 ASTM Standard
20、s:D 4447 Guide for the Disposal of Laboratory Chemicalsand Samples3E 943 Terminology Relating to Biological Effects and En-vironmental Fate4E 1295 Guide for Conducting Three-Brood, Renewal Tox-icity Tests with Ceriodaphnia dubia4E 1676 Guide for Conducting a Laboratory Soil Toxicity orBioaccumulatio
21、n Tests with the Lumbricid EarthwormEisenia fetida4E 1706 Test Methods for Measuring the Toxicity ofSediment-Associated Contaminants with Fresh Water In-vertebrates43. Terminology3.1 Definitions:3.1.1 The words “must,” “should,” “may,” “can,” and“might” have very specific meanings in this guide. “Mu
22、st” isused to express an absolute requirement, that is, to state that thetest must be designed to satisfy the specified condition, unlessthe purpose of the test requires a different design. “Must” isused only in connection with the factors that relate directly tothe acceptability of the test (see Se
23、ction 13). “Should” is usedto state that the specified condition is recommended and oughtto be met if possible. Although a violation of one “should” israrely a serious matter, the violation of several will often renderthe results questionable. Terms such as “is desirable,” “is oftendesirable,” and “
24、might be desirable” are used in connectionwith less important factors. “May” is used to mean “is (are)allowed to,” “can” is used to mean “is (are) able to,” and“might” is used to mean “could possibly.” Thus, the classicdistinction between “may” and “can” is preserved, and “might”is never used as a s
25、ynonym for either “may” or “can.”3.1.2 For definitions of terms used in this guide, refer toTerminology E 943.3.2 Definitions of Terms Specific to This Standard:3.2.1 artificial soila synthetic soil, prepared with a spe-cific formulation, designed to simulate a natural soil (seeAnnex A2). Artificial
26、 soil may be used as a diluent medium toprepare concentrations of site or reference soil and may be usedas a negative control medium.3.2.2 batchthe total amount of test soil prepared for eachconcentration in a test. A batch is any hydrated test soil readyfor separation into replicates.3.2.3 concentr
27、ationthe ratio of the weight of test materi-als to the weight of soil (artificial, reference, or site), usuallyexpressed on a dry weight basis as percent or milligram/kilogram.3.2.4 diluent soilthe artificial or reference soil used todilute site soils.3.2.5 hydration waterwater used to hydrate test
28、soils tocreate an environment with a moisture level suitable for thespecies being tested. The water used for hydration is often testwater (see 3.2.18); however, depending on the nature of the testbeing implemented, site surface water or ground water mayalso be utilized for hydration.3.2.6 negative c
29、ontrol soilartificial or field collected soilto be used for evaluating the acceptability of a test.3.2.7 reference soila field-collected soil that has physico-chemical and biological properties as similar as possible to thesite soil but does not contain the potentially toxic compoundsof the site soi
30、l. It is used to describe matrix effects on the testin question. It may be used as a diluent medium to prepareconcentrations of site soil and may be used as a negativecontrol medium.3.2.8 sampling stationa specific location, within a site orsampling unit, depending on the field study design, at whic
31、hsoil is collected for chemical, physical, and biological evalua-tion.3.2.9 sampling unitan area of land within a site distin-guished by habitat and topography.3.2.10 sedimentparticulate materials that usually lie be-low water. Formulated particulate material that is intended tolie below water in a
32、test.3.2.11 sitea delineated tract of land that is being consid-ered as a study area, usually from the standpoint of its beingpotentially affected by contaminants.3.2.12 site soila soil collected from the field to beevaluated for potential toxicity. A site soil may be a naturallyoccurring soil or on
33、e that has been influenced by contaminants.3.2.13 soilsolid particles produced by the physical andchemical disintegration of rocks, which may or may notcontain organic material.3.2.14 spikingthe experimental addition of a test materialto an artificial, site, or reference soil, such that the toxicity
34、 ofthe material added can be determined. After the test material isadded, which may involve a solvent carder, the soil is mixedthoroughly to distribute the test material evenly throughout thesoil.3.2.15 test chamberan enclosed space or compartment inwhich environmental parameters such as temperature
35、 and3Annual Book of ASTM Standards, Vol 11.044Annual Book of ASTM Standards, Vol 11.05E2172012lighting are controlled (for example, incubator or modifiedroom). Test containers are placed in the test chamber forbiological evaluation.3.2.16 test containerthe experimental unit; the smallestphysical ent
36、ity to which treatments can be assigned indepen-dently.3.2.17 test soila soil prepared to receive a test organism.Site or reference soil mixed with artificial soil or reference soilmixed with site soil in known concentrations for evaluation aretest soils. Artificial, site, or reference soils spiked
37、with testmaterials such as chemicals, oils, or manufacturing productsare test soils. Once a site, reference, or artificial soil ishydrated, even though it is not mixed with artificial orreference soil or spiked with a material, it may be called a testsoil.3.2.18 test waterwater used to prepare stock
38、 solutions,rinse test organisms, rinse glassware, and apparatus or for anyother purpose associated with the test procedures or culture ofthe test organism. Test water should be deionized water orbetter, such as reagent-grade water produced by a system ofreverse osmosis, carbon, and ion-exchange cart
39、ridges.4. Summary of Guide4.1 Toxicity of Test Soils is Assessed During the ContinuousExposure of Terrestrial OrganismsSoils tested may be thefollowing: (1) soils collected from potentially contaminatedsites, (2) soils collected from reference sites, (3) artificial soil(see Annex A2) spiked with com
40、pounds, (4) site soil spikedwith compounds, (5) reference soil spiked with compounds, (6)site soil diluted with artificial soil, (7) site soil diluted withreference soil, or (8) reference soil diluted with artificial soil.Anegative control of artificial or reference soil is used for thefollowing: (1
41、) to yield a measure of the acceptability of the test;(2) to provide evidence of the health and relative quality of thetest organisms; (3) to determine the suitability of test condi-tions, food, and handling procedures; and (4) to provide a basisfor interpreting data obtained from the test soils. Sp
42、ecified dataare obtained to determine the toxic effects on survival for 24 hexposures to the terrestrial nematodes C. elegans.5. Significance and Use5.1 Soil toxicity tests provide information concerning thetoxicity and bioavailability of chemicals associated with soilsto terrestrial organisms. As i
43、mportant members of the soilfauna, nematodes have a number of characteristics that makethem appropriate organisms for use in the assessment ofpotentially hazardous soils. Bacterial-feeding nematodes suchas C. elegans feed on soil microbes and contribute to thebreakdown of organic matter. They are al
44、so of extremeimportance in the cycling and degradation of key nutrients insoil ecosystems (9). Soil nematodes also serve as a source ofprey and nutrients for fauna and microflora such as soilnematophagous fungi (10).Amajor change in the abundance ofsoil invertebrates such as nematodes, either as a f
45、ood source oras organisms functioning properly in trophic energy transferand nutrient cycling, could have serious adverse ecologicaleffects on the entire terrestrial system.5.2 Results from soil tests might be an important consider-ation when assessing the hazards of materials to terrestrialorganism
46、s.5.3 The soil test might be used to determine the temporal orspatial distribution of soil toxicity. Test methods can be used todetect horizontal and vertical gradients in toxicity.5.4 Results of soil tests could be used to compare thesensitivities of different species.5.5 An understanding of the ef
47、fect of these parameters ontoxicity may be gained by varying soil characteristics such aspH, clay content, and organic material.5.6 Results of soil tests may be useful in helping to predictthe effects likely to occur with terrestrial organisms in fieldsituations.5.6.1 Field surveys can be designed t
48、o provide either aqualitative or quantitative evaluation of biological effectswithin a site or among sites.5.6.2 Soil surveys evaluating biological effects are usuallypart of more comprehensive analyses of biological, chemical,geological, and hydrographic conditions. Statistical correlationcan be im
49、proved and costs reduced if subsamples of soil forlaboratory tests, geochemical analyses, and community struc-ture are taken simultaneously from the same grab of the samesite.5.7 Soil toxicity tests can be an important tool for makingdecisions regarding the extent of remedial action necessary forcontaminated terrestrial sites.6. Interferences6.1 Limitations to the methods described in this guide mightarise and thereby influence soil test results and complicate datainterpretation. The following factors should be consideredwhen testing soils:6.1.1 The alteration of field
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