ASTM E2172-22
(Guide)Standard Guide for Conducting Laboratory Soil Toxicity Tests with the Nematode Caenorhabditis elegans
Standard Guide for Conducting Laboratory Soil Toxicity Tests with the Nematode <emph type="ital"> Caenorhabditis elegans</emph>
SIGNIFICANCE AND USE
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 c...
SCOPE
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 additio...
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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.
Designation: E2172 − 22
Standard Guide for
Conducting Laboratory Soil Toxicity Tests with the
1
Nematode Caenorhabditis elegans
This standard is issued under the fixed designation E2172; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope soils high in organic matter. A modification of the recovery
method has also been used with a transgenic strain of C.
1.1 This guide covers procedures for obtaining laboratory
elegans used as a soil biomonitoring tool to assess sub-lethal
data to evaluate the adverse effects of chemicals associated
effects of metal exposures in soil (7). A variety of sub-lethal
withsoiltonematodesfromsoiltoxicitytests.Thisstandardis
endpoints have been developed using C. elegans in aquatic
based on a modification to Guide E1676. The methods are
media and may prove useful for assessing soil exposures (8).
designed to assess lethal or sublethal toxic effects on nema-
todesinshort-termtestsinterrestrialsystems.Soilstobetested
1.3 Modification of these procedures might be justified by
may be (1) references soils or potentially toxic soil sites; (2)
special needs. The results of tests conducted using typical
artificial, reference, or site soils spiked with compounds; (3)
procedures may not be comparable to results using this guide.
site soils diluted with reference soils; or (4) site or reference
Comparisonofresultsobtainedusingmodifiedandunmodified
soils diluted with artificial soil. Test procedures are described
versions of these procedures might provide useful information
for the species Caenorhabditis elegans (see Annex A1).
concerning new concepts and procedures for conducting soil
Methods described in this guide may also be useful for
toxicity tests with terrestrial worms.
conducting soil toxicity tests with other terrestrial species,
1.4 The results from field-collected soils used in toxicity
although modifications may be necessary.
tests to determine a spatial or temporal distribution of soil
1.2 Summary of Previous Studies—Initial soil toxicity test-
toxicity may be reported in terms of the biological effects on
ing using the free-living, bacterivorous soil nematode Cae-
survival or sublethal endpoints. These procedures can be used
norhabditis elegans was developed by Donkin and Dusenbery
with appropriate modifications to conduct soil toxicity tests
2
(1). Following the development of an effective method of
when factors such as temperature, pH, and soil characteristics
recovery of C. elegans from test soils, the organism was used
(for example, particle size, organic matter content, and clay
to identify factors that affect the toxicity of zinc, cadmium,
content) are of interest or when there is a need to test such
copper, and lead (2). Freeman et al. further refined the
materials as sewage sludge. These methods might also be
nematode bioassay by decreasing the quantity of soil and
useful for conducting bioaccumulation tests.
spiking solution volumes, determining test acceptability
1.5 The results of toxicity tests with (1) materials (for
criteria, and developing control charts to assess worm health
example, chemicals or waste mixtures) added experimentally
using copper as a reference toxicant (3). More recently, the
to artificial soil, reference soils, or site soils, (2) site soils
toxicologicaleffectsofnitrateandchloridemetallicsaltsintwo
diluted with reference soils, and (3) site or reference soils
natural soils were compared (4). LC50 values for C. elegans
diluted with artificial soil, so as to create a series of
exposedfor24-htonitratesaltsofcadmium,copper,zinc,lead
concentrations, may be reported in terms of an LC50 (median
andnickelinanartificialsoil(seeAnnexA2)werefoundtobe
lethal concentration) and sometimes an EC50 (median effect
similar to LC50 values for the earthworm, Eisenia fetida (5).
concentration).
Increasing the exposure time to 48-h resulted in much lower
LC50 values (6). However, longer exposure times necessitate
1.6 This guide is arranged as follows:
the addition of food and lead to lower recovery percentages in
Scope 1
Referenced Documents 2
Terminology 3
1
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental Summary of Guide 4
Assessment, Risk Management and CorrectiveAction and is the direct responsibil- Significance and Use 5
Interferences 6
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Apparatus 7
Current edition approved Aug. 1, 2022. Published September 2022. Originally
S
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E2172 − 01 (Reapproved 2014) E2172 − 22
Standard Guide for
Conducting Laboratory Soil Toxicity Tests with the
1
Nematode Caenorhabditis elegans
This standard is issued under the fixed designation E2172; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
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
2
elegans was developed by Donkin and Dusenbery (1). 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
1
This guide is under the jurisdiction of ASTM Committee E50 on Environmental Assessment, Risk Management and Corrective Action and is the direct responsibility
of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Oct. 1, 2014Aug. 1, 2022. Published December 2014September 2022. Originally approved in 2002. Last previous edition approved in 20082014
as E2172–02(2008).E2172–01(2014). DOI: 10.1520/E2172-01R14.10.1520/E2172-22.
2
The boldface numbers in parentheses refer to the list of references at the end of this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
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E2172 − 22
size, organic matter content, and clay content) are of interest or when there is a need to test such materials as sewage sludge.
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