ASTM E1676-12(2021)
(Guide)Standard Guide for Conducting Laboratory Soil Toxicity or Bioaccumulation Tests with the Lumbricid Earthworm Eisenia Fetida and the Enchytraeid Potworm Enchytraeus albidus
Standard Guide for Conducting Laboratory Soil Toxicity or Bioaccumulation Tests with the Lumbricid Earthworm <emph type="ital">Eisenia Fetida</emph > and the Enchytraeid Potworm <emph type="ital">Enchytraeus albidus</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, lumbricid earthworms and enchytraeid potworms have a number of characteristics that make them appropriate organisms for use in the assessment of potentially hazardous soils. Earthworms may ingest large quantities of soil, have a close relationship with other soil biomasses (for example, invertebrates, roots, humus, litter, and microorganisms), constitute up to 92 % of the invertebrate biomass of soil, and are important in recycling nutrients (1, 2).4 Enchytraeids contribute up to 5.2 % of soil respiration, constitute the second-highest biomass in many soils (the highest in acid soils in which earthworms are lacking) and effect considerably nutrient cycling and community metabolism (3-5). Earthworms and potworms accumulate and are affected by a variety of organic and inorganic compounds (2-10, 11-14). In addition, earthworms and potworms are important in terrestrial food webs, constituting a food source for a very wide variety of organisms, including birds, mammals, reptiles, amphibians, fish, insects, nematodes, and centipedes (15, 16, 3). A major change in the abundance of soil invertebrates such as lumbricids or enchytraeids, 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 A number of species of lumbricids and enchytraeid worms have been used in field and laboratory investigations in the United States and Europe. Although the sensitivity of various lumbricid species to specific chemicals may vary, from their study of four species of earthworms (including E. fetida) exposed to ten organic compounds representing six classes of chemicals, Neuhauser, et al (7) suggest that the selection of earthworm test species does not affect the a...
SCOPE
1.1 This guide covers procedures for obtaining laboratory data to evaluate the adverse effects of contaminants (for example, chemicals or biomolecules) associated with soil to earthworms (Family Lumbricidae) and potworms (Family Enchytraeidae) from soil toxicity or bioaccumulation tests. The methods are designed to assess lethal or sublethal toxic effects on earthworms or bioaccumulation of contaminants in short-term tests (7 to 28 days) or on potworms in short to long-term tests (14 to 42 days) in terrestrial systems. Soils to be tested may be (1) reference soils or potentially toxic site soils; (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 Eisenia fetida (see Annex A1) and for the species Enchytraeus albidus (see Annex A4). Methods described in this guide may also be useful for conducting soil toxicity tests with other lumbricid and enchytraeid terrestrial species, although modifications may be necessary.
1.2 Modification of these procedures might be justified by special needs. The results of tests conducted using atypical 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 and bioaccumulation tests with terrestrial worms.
1.3 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 (see Section 14). 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 c...
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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: E1676 − 12 (Reapproved 2021)
Standard Guide for
Conducting Laboratory Soil Toxicity or Bioaccumulation
Tests with the Lumbricid Earthworm Eisenia Fetida and the
Enchytraeid Potworm Enchytraeus albidus
This standard is issued under the fixed designation E1676; 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 soil characteristics (for example, particle size, organic matter
content, and clay content) are of interest or when there is a
1.1 This guide covers procedures for obtaining laboratory
need to test such materials as sewage sludge and oils. These
data to evaluate the adverse effects of contaminants (for
methods might also be useful for conducting bioaccumulation
example, chemicals or biomolecules) associated with soil to
tests.
earthworms (Family Lumbricidae) and potworms (Family
Enchytraeidae)fromsoiltoxicityorbioaccumulationtests.The
1.4 The results of toxicity tests with (1) materials (for
methods are designed to assess lethal or sublethal toxic effects
example, chemicals or waste mixtures) added experimentally
on earthworms or bioaccumulation of contaminants in short-
to artificial soil, reference soils, or site soils, (2) site soils
term tests (7 to 28 days) or on potworms in short to long-term
diluted with reference soils, and (3) site or reference soils
tests (14 to 42 days) in terrestrial systems. Soils to be tested
diluted with artificial soil, so as to create a series of
may be (1) reference soils or potentially toxic site soils; (2)
concentrations, may be reported in terms of an LC50 (median
artificial, reference, or site soils spiked with compounds; (3)
lethal concentration) and sometimes an EC50 (median effect
site soils diluted with reference soils; or (4) site or reference
concentration).Test results may be reported in terms of NOEC
soils diluted with artificial soil. Test procedures are described
(no observed effect concentration), LOEC (lowest observed
for the species Eisenia fetida (see Annex A1) and for the
effect concentration) or as an ECx (concentration where x%
species Enchytraeus albidus (see Annex A4). Methods de-
reduction of a biological effect occurs. Bioaccumulation test
scribed in this guide may also be useful for conducting soil
results are reported as the magnitude of contaminant concen-
toxicity tests with other lumbricid and enchytraeid terrestrial
tration above either the Day 0 tissue baseline analysis or the
species, although modifications may be necessary.
Day 28 tissues from the negative control or reference soil (that
is, 2x, 5x, 10x) (see A3.9).
1.2 Modification of these procedures might be justified by
special needs. The results of tests conducted using atypical
1.5 This guide is arranged as follows:
procedures may not be comparable to results using this guide.
Scope 1
Comparisonofresultsobtainedusingmodifiedandunmodified
Referenced Documents 2
versions of these procedures might provide useful information Terminology 3
Summary of Guide 4
concerning new concepts and procedures for conducting soil
Significance and Use 5
toxicity and bioaccumulation tests with terrestrial worms.
Interferences 6
Apparatus 7
1.3 The results from field-collected soils used in toxicity
Safety Precautions 8
tests to determine a spatial or temporal distribution of soil
Soil 9
Test Organism 10
toxicity may be reported in terms of the biological effects on
Procedure 11
survival or sublethal endpoints (see Section 14). These proce-
Analytical Methodology 12
dures can be used with appropriate modifications to conduct
Acceptability of Test 13
Calculation of Results 14
soil toxicity tests when factors such as temperature, pH, and
Report 15
Annexes
Annex A1. Eisenia fetida
Annex A2. Artificial Soil Composition
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Annex A3. Bioaccumulation Testing Utilizing Eisenia fetida
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
Annex A4. Enchytraeid Reporduction Test (ERT)
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
References
An ASTM guide is defined as a series of options or instructions that do not
recommend a specific course of action.
1.6 The values stated in SI units are to be regarded as
Current edition approved Jan. 1, 2021. Published February 2021. Originally
standard. No other units of measurement are included in this
approved in 1995. Last previous edition approved in 2012 as E1676–12. DOI:
10.1520/E1676-12R21. standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1676 − 12 (2021)
1.7 This standard does not purport to address all of the “might” is used to mean “could possibly.” Thus, the classic
safety concerns, if any, associated with its use. It is the distinctionbetween“may”and“can”ispreserved,and“might”
responsibility of the user of this standard to establish appro- is never used as a synonym for either “may” or “can.”
priate safety, health, and environmental practices and deter-
3.1.2 For definitions of terms used in this guide, refer to
mine the applicability of regulatory limitations prior to use.
Terminology E943 and Guide E1023. For an explanation of
While some safety considerations are included in this guide, it
units and symbols, refer to Practice E380.
is beyond the scope of this standard to encompass all safety
3.2 Definitions of Terms Specific to This Standard:
requirements necessary to conduct soil toxicity tests. Specific
3.2.1 artificial soil—a synthetic soil, prepared with a spe-
precautionary statements are given in Section 8.
cific formulation, designed to simulate a natural soil (see
1.8 This international standard was developed in accor-
AnnexA2).Artificial soil may be used as a diluent medium to
dance with internationally recognized principles on standard-
prepareconcentrationsofsiteorreferencesoilandmaybeused
ization established in the Decision on Principles for the
as a negative control medium.
Development of International Standards, Guides and Recom-
3.2.2 batch—the total amount of test soil prepared for each
mendations issued by the World Trade Organization Technical
concentration in a test.Abatch is any hydrated test soil ready
Barriers to Trade (TBT) Committee.
for separation into replicates.
2. Referenced Documents
3.2.3 bioaccumulation—thenetaccumulationofasubstance
by an organism as a result of uptake from all environmental
2.1 ASTM Standards:
sources. (See Guide E1688.)
D653Terminology Relating to Soil, Rock, and Contained
Fluids
3.2.4 bioaccumulation factor (BAF)—the ratio of tissue
D4447Guide for Disposal of Laboratory Chemicals and
residue to sediment or soil contaminant concentration at
Samples
steady-state. (See Guide E1688.)
E380Practice for Use of the International System of Units
3.2.5 bioaccumulation potential—a qualitative assessment
(SI) (the Modernized Metric System) (Withdrawn 1997)
of whether a contaminant in a particular sediment or soil is
E943Terminology Relating to Biological Effects and Envi-
bioavailable. (See Guide E1688.)
ronmental Fate
3.2.6 bioconcentration—the net assimilation of a substance
E1023Guide for Assessing the Hazard of a Material to
by an organism as a result of uptake directly from aqueous
Aquatic Organisms and Their Uses
solution. (See Guide E1688.)
E1383Guide for Conducting Sediment Toxicity Tests with
Freshwater Invertebrates (Withdrawn 1995)
3.2.7 bioconcentration factor (BCF)—the ratio of tissue
E1688Guide for Determination of the Bioaccumulation of
residue to water contaminant concentration as steady-state.
Sediment-Associated Contaminants by Benthic Inverte-
(See Guide E1688.)
brates
3.2.8 biota-sediment accumulation factor (BSAF)— the ra-
E1706TestMethodforMeasuringtheToxicityofSediment-
tio of lipid-normalized tissue residue to organic carbon-
Associated Contaminants with Freshwater Invertebrates
normalizedsedimentcontaminantconcentrationatsteadystate,
with units of g-carbon/g-lipid. (See Guide E1688.)
3. Terminology
3.2.9 clitellum—the fleshy “ring” or “saddle” of glandular
3.1 Definitions:
tissue found on certain mid-body segments of oligochaete
3.1.1 The words “must,” “should,” “may,”“ can,” and
(Lumbricidae and Enchytraeidae) worms. It is the most visible
“might” have very specific meanings in this guide. “Must” is
feature of an adult earthworm or potworm and secretes the
usedtoexpressanabsoluterequirement,thatis,tostatethatthe
cocoon into which eggs and sperm are deposited.
test must be designed to satisfy the specified condition, unless
the purpose of the test requires a different design. “Must” is
3.2.10 concentration—the ratio of the weight of test mate-
used only in connection with the factors that relate directly to
rials to the weight of soil (artificial, reference, or site), usually
the acceptability of the test (see Section 13). “Should” is used
expressed on a dry weight basis as percent or milligram/
to state that the specified condition is recommended and ought
kilogram.
to be met if possible. Although a violation of one “should” is
3.2.11 depuration—loss of a substance from an organism as
rarelyaseriousmatter,theviolationofseveralwilloftenrender
a result of any active (for example, metabolic breakdown) or
the results questionable.Terms such as “is desirable,” “is often
passive process.
desirable,” and “might be desirable” are used in connection
3.2.12 diluent soil—the artificial or reference soil used to
with less important factors. “May” is used to mean “is (are)
dilute site soils.
allowed to,” “can” is used to mean “is (are) able to,” and
3.2.13 enchytraeid—potworm members of the Family En-
chytraeidae of the Class Oligochaeta of the PhylumAnnelida.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
3.2.14 hydration water—water used to hydrate test soils to
Standards volume information, refer to the standard’s Document Summary page on
create an environment with a moisture level suitable for the
the ASTM website.
species being tested. The water used for hydration is often test
The last approved version of this historical standard is referenced on
www.astm.org. water(see3.2.27);however,dependingonthenatureofthetest
E1676 − 12 (2021)
beingimplemented,sitesurfacewaterorgroundwatermayalso the test organism. Test water must be deionized or distilled
be utilized for hydration. water or better, such as reagent-grade water produced by a
system of reverse osmosis, carbon, and ion-exchange car-
3.2.15 lumbricid—earthworm members of the Family Lu-
tridges.
bricidae of the Class Oligochaeta of the Phylum Annelida.
3.2.16 negativecontrolsoil—artificialorreferencesoiltobe
4. Summary of Guide
used for evaluating the acceptability of a test.
4.1 The toxicity of test soils or the bioavailability of
3.2.17 reference soil—a field-collected soil that has physi-
contaminants are assessed during the continuous exposure of
cochemical and biological properties as similar as possible to
terrestrial organisms. Soils tested may be the following: (1)
the site soil but does not contain the potentially toxic com-
soils collected from potentially contaminated sites, (2 ) soils
pounds of the site soil. It is used to describe matrix effects on
collectedfromreferencesites,(3)artificialsoil(seeAnnexA2)
the test in question. It may be used as a diluent medium to
spiked with compounds, (4) site soil spiked with compounds,
prepare concentrations of site soil and may be used as a
(5) reference soil spiked with compounds, (6) site soil diluted
negative control medium.
withartificialsoil,(7)sitesoildilutedwithreferencesoil,or(8)
reference soil diluted with artificial soil.Anegative control of
3.2.18 samplingstation—aspecificlocation,withinasiteor
artificial or reference soil is used for the following: (1) to yield
sampling unit, depending on the field study design, at which
a measure of the acceptability of the test; (2) to provide
soil is collected for chemical, physical, and biological evalua-
tion. evidence of the health and relative quality of the test organ-
isms; (3) to determine the suitability of test conditions, food,
3.2.19 sampling unit—an area of land within a site distin-
and handling procedures; and (4) to provide a basis for
guished by habitat and topography.
interpretingdataobtainedfromthetestsoils.Specifieddataare
3.2.20 site—a delineated tract of land that is being consid-
obtained to determine the toxic effects on survival or sublethal
ered as a study area, usually from the standpoint of its being
endpoints for 7 to 28-day exposures or containment bioaccu-
potentially affected by xenobiotics.
mulation for 28-day exposures to terrestrial lumbricids and the
3.2.21 site soil—a soil collected from the field to be evalu- toxic effects on survival or sublethal endpoints for 4 to 42-day
ated for potential toxicity. A site soil may be a naturally exposures to enchytraeids.
occurring soil or one that has been influenced by xenobiotics.
4.2 Summary of Changes—This current version of the
3.2.22 soil—sediments or other unconsolidated accumula-
standard is a revision of the E1676-97 version. Changes made
tions of solid particles produced by the physical and chemical
since 1997 involve toxicity testing procedures for the En-
disintegration of rocks, and that may or may not contain
chytraied potworm, Enchytraeus albidus. There has been an
organic material. (See Terminology D653.)
additionalannexadded(AnnexA4)andthemaindocumenthas
been modified to include this species.
3.2.23 spiking—the experimental addition of a test material
to an artificial, site, or reference soil, such that the toxicity of
5. Significance and Use
the material added can be determined.After the test material is
5.1 Soil toxicity tests provide information concerning the
added, which may involve a solvent carrier, the soil is mixed
toxicity and bioavailability of chemicals associated with soils
thoroughly to distribute the test material evenly throughout the
to terrestrial organisms. As important members of the soil
soil.
fauna,lumbricidearthwormsandenchytraeidpotwormshavea
3.2.24 test chamber—an enclosed space or compartment in
number of characteristics that make them appropriate organ-
which environmental parameters such as temperature and
isms for use in the assessment of potentially hazardous soils.
lighting are co
...
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