ASTM E1676-12(2021)

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 controlled (for example, incubator or modified
Earthworms may ingest large quantities of soil, have a close
room). Test containers are placed in the test chamber for
relationship with other soil biomasses (for example,
biological evaluation.
invertebrates, roots, humus, litter, and microorganisms), con-
3.2.25 test container—the experimental unit; the smallest
stitute up to 92% of the invertebrate biomass of soil, and are
physical entity to which treatments can be assigned indepen-
important in recycling nutrients (1, 2). Enchytraeids contrib-
dently.
ute up to 5.2% of soil respiration, constitute the second-
3.2.26 test soil—a soil prepared to receive a test organism. highest biomass in many soils (the highest in acid soils in
Siteorreferencesoilmixedwithartificialsoilorreferencesoil whichearthwormsarelacking)andeffectconsiderablynutrient
mixedwithsitesoilinknownconcentrationsforevaluationare cycling and community metabolism (3-5). Earthworms and
test soils. Artificial, site, or reference soils spiked with test potworms accumulate and are affected by a variety of organic
materials such as chemicals, oils, or manufacturing products and inorganic compounds (2-10, 11-14). In addition, earth-
are test soils. Once a site, reference, or artificial soil is worms and potworms are important in terrestrial food webs,
hydrated, even though it is not mixed with artificial or constitutingafoodsourceforaverywidevarietyoforganisms,
reference soil or spiked with a material, it may be called a test including birds, mammals, reptiles, amphibians, fish, insects,
soil. nematodes, and centipedes (15, 16, 3). A major change in the
3.2.27 test water—water used to prepare stock solutions,
rinse test organisms, rinse glassware, and apparatus or for any
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
other purpose associated with the test procedures or culture of this standard.
E1676 − 12 (2021)
abundance of soil invertebrates such as lumbricids or 5.4 Information might also be obtained on the bioaccumu-
enchytraeids, either as a food source or as organisms function- lation of chemicals associated with soil by analysis of animal
ing properly in trophic energy transfer and nutrient cycling, tissues for the chemicals being monitored. These results are
could have serious adverse ecological effects on the entire useful for studying the biological availability of chemicals.
terrestrial system.
5.5 The soil toxicity test might be used to determine the
temporal or spatial distribution of soil toxicity. Test methods
5.2 A number of species of lumbricids and enchytraeid
can be used to detect horizontal and vertical gradients in
worms have been used in field and laboratory investigations in
toxicity.
the United States and Europe. Although the sensitivity of
variouslumbricidspeciestospecificchemicalsmayvary,from
5.6 Results of soil toxicity tests could be used to compare
their study of four species of earthworms (including E. fetida)
the sensitivities of different species.
exposed to ten organic compounds representing six classes of
5.7 An understanding of the effect of these parameters on
chemicals, Neuhauser, et al (7) suggest that the selection of
toxicity and bioaccumulation may be gained by varying soil
earthworm test species does not affect the assessment of a
characteristics such as pH, clay content, and organic material.
chemical’s toxicity markedly. The sensitivity of various en-
chytraeid species has not been investigated in a comparable
5.8 Results of soil toxicity tests may be useful in helping to
way so far, but ecological importance and practicability rea-
predict the effects likely to occur with terrestrial organisms in
sons favor strongly the selection of a species belonging to the
field situations.
genus Enchytraeus.
5.8.1 Field surveys can be designed to provide either a
5.2.1 E. fetida is a species whose natural habitats are those
qualitative or quantitative evaluation of biological effects
ofveryhighorganicmattersuchascompostsandmanurepiles.
within a site or among sites.
It was selected as the test species because it (1) is bred in the
5.8.2 Soil surveys evaluating biological effects are usually
laboratory easily; (2) is the earthworm species used most
part of more comprehensive analyses of biological, chemical,
commonlyinlaboratoryexperiments (17);(3)hasbeenstudied
geological, and hydrographic conditions. Statistical correlation
extensively, producing a data pool on the toxicity and bioac-
can be improved and costs reduced if subsamples of soil for
cumulation of a variety of compounds (2, 7, 8, 18-23);(4) has
laboratorytoxicitytests,geochemicalanalyses,andcommunity
been approved for use in toxicity testing by the European
structure are taken simultaneously from the same grab of the
Union (EU) and the Organization for Economic Cooperation
same site.
and Development (OECD); and (5) has been used by the
5.9 Soil toxicity and bioaccumulation tests can be an im-
Environmental Protection Agency (EPA) for the toxicity
portant tool for making decisions regarding the extent of
screening of hazardous waste sites (24).
remedial action necessary for contaminated terrestrial sites.
5.2.2 The recommended enchytraeid test species is En-
chytraeus albidus Henle 1837 (white potworm). E. albidus is
6. Interferences
one of the biggest (up to 15 mm) species of the oligochaete
family Enchytraeidae and it is distributed world-wide (25, 26). 6.1 Limitationstothemethodsdescribedinthisguidemight
E. albidus is found in marine, limnic, and terrestrial habitats, arise and thereby influence soil toxicity test results and
mainly in decaying organic matter (seaweed, compost) and complicatedatainterpretation.Thefollowingfactorsshouldbe
rarely in meadows (4, 26).This broad ecological tolerance and considered when testing soils:
some morphological variations might indicate that there are
6.1.1 The alteration of field samples in preparation for
different races for this species. E. albidus is commercially
laboratory testing (for example, transport, screening, or mix-
available, sold as food for fish, can be bred easily in a wide
ing).
range of organic waste materials and has a short life cycle (33
6.1.1.1 Maintaining the integrity of soils during their
to 74 days; 27, 28). E. albidus was studied in various tests,
removal, transport, and testing in the laboratory is extremely
which covered a wide range of compounds (28-30).In
difficult. The soil environment is composed of a myriad of
addition, it is currently under investigation for use in toxicity
microenvironments, redox gradients, and other interacting
testing and soil quality assessment by the European Union
physicochemical and biological processes. Many of these
(EU), the Organization for Economic Cooperation and Devel-
characteristics influence soil toxicity and the availability of
opment (OECD), and the International Organization for Stan-
compounds to organisms, microbial degradation, and chemical
dardization (ISO). Other species of the genus Enchytraeus are
sorption. Any disruption of this environment complicates
also suitable, for example, E. buchholzi Vejdovsky 1879 or E.
interpretations of treatment effects, causative factors, and in
crypticus Westheide and Graefe 1992 (see Annex A4). Those
situ comparisons.
species are true soil inhabitants and are smaller in size. Other
6.1.1.2 Soils tested at temperatures other than those from
species of Enchytraeus may be used, but they should be
the field in which they are collected might affect chemical
identified clearly and the rationale for their selection should be
solubility, partitioning coefficients, and other physical and
reported.
chemical characteristics.
6.1.2 Interaction among chemicals present in the soil.
5.3 Results from soil toxicity tests might be an important
consideration when assessing the hazards of materials to 6.1.3 The use of laboratory-spiked soils that might not be
terrestrial organisms. representative of chemicals associated with soils in the field.
E1676 − 12 (2021)
6.1.4 The addition of food to test containers may affect the 7.3.2 Temperature-recording devices should be used to
resultsofatoxicitytest,butitmaybenecessarytofeedthetest monitorthetemperatureoftestandculturechambers.Bothtest
organisms in long-duration tests (see 11.7, A1.9.1.2, A1.9.5, and culture chambers should be at the same temperature
and A4.10.8). (except in the case of the Enchytraeid Reproduction Test).
6.1.5 The addition of solvents to the test containers might
7.4 Culture Containers—Containers used to culture test
obscuretheadverseinfluenceofchemicalsassociatedwithsoil
organismsshouldbemadeofmaterialsthatwillnotaffecttheir
and affect soil quality characteristics.
survival, growth, or reproduction adversely. Consideration
6.1.6 The natural geochemical properties of test soil col-
should be given to cleaning and organizational space.The size
lectedfromthefieldmightnotbewithinthetolerancelimitsof
ofculturecontainersmaydependonthespeciesbeingcultured.
the test species.
7.5 Test Containers—Test containers should be made of
6.1.7 Field-collected soils may contain indigenous organ-
isms including (1) the same or closely related species to that materials that minimize the sorption and leaching of test
beingtestedand(2)microorganisms(forexample,bacteriaand compounds and do not affect the survival, growth, and repro-
molds) and algae species that might grow in or on the soil and duction of the test organism adversely. Glass is an ideal
test container surfaces. material.
7.5.1 All test containers used in a soil toxicity test must be
6.2 Tests may not be applicable with materials that are
identical. The test containers should be covered with a lid to
highly volatile (that is, substances for which the Henry’s
prevent escape of the test organisms and help reduce drying of
constant or the air/water partition coefficient is greater than
the test soil.
one,orsubstancesforwhichthevaporpressureexceeds0.0133
Pa at 25ºC) or rapidly transformed biologically or chemically. 7.5.2 Species-specific information on test containers and
The dynamics of test material breakdown products should test conditions is given in Annex A1, Annex A3, and Annex
therefore be considered, especially in relation to assumptions A4.
of chemical equilibria.
7.6 Cleaning—Testcontainersandequipmentandapparatus
should be cleaned before use. Items may be cleaned in the
7. Apparatus
following manner: (1) scrub thoroughly with a scratch pad to
7.1 General Facilities—Thefacilityshouldincludeseparate
remove visible soil and residue; (2) detergent wash; (3) water
constanttemperatureareas(chambers)forculturingandtesting
rinse;(4)organicsolventwash(forexample,acetone);(5)acid
toreducethepossibilityofcontaminationbytestmaterialsand
wash (for example, 10% concentrated hydrochloric acid); (6)
other substances, especially volatile compounds. Culture con-
tapwaterrinse;(7)rinseatleasttwicewithdistilled,deionized,
tainers should not be in a room (chamber) in which toxicity
orreagentgradewater;and(8)driedatroomtemperatureorin
tests are conducted, stock solutions or test solutions are
a low-temperature (up to 90°C) air-drying oven. Care must be
prepared,orequipmentiscleaned.Thefacilitiesshouldbewell
taken to avoid the use of “plastics” that may breakdown in the
ventilated and free of fumes.
presence of the solvent used or at prolonged exposures near
90°C. For acceptable items, the following steps may be used
7.2 Equipment and Apparatus—Equipment and apparatus
alternatively for cleaning: (1) scrub thoroughly with a scratch
that contact stock solutions, test solutions, site soils, and test
pad to remove visible soil and residue; (2) detergent wash; (3)
soils, into which test organisms will be placed, should not
water rinse; (4) acid wash (for example, 10% concentrated
containsubstancesthatcanbeleachedordissolvedinamounts
hydrochloric acid); (5) tap water rinse; (6) rinse at least twice
that affect the test organisms adversely. In addition, equipment
with distilled, deionized, or reagent grade water; and (7) bake
and apparatus that contact soils or solutions should be chosen
in an oven at 350°C. Clean lids should be placed on test
to minimize the sorption of test materials. Glass, Type 316
containers after the containers have cooled.
stainless steel, nylon, high-density polyethylene,
7.6.1 A laboratory dish-washing machine may be used to
polycarbonate,andfluorocarbonplasticsshouldbeusedwhen-
accomplish the detergent wash/water rinse and tap water rinse
ever possible to minimize leaching, dissolution, and sorption.
stages. If a dish-washing machine is used, a neutralizing rinse
Copper, brass, lead, galvanized metal, and natural rubber
may be necessary after the acid wash to prevent acid damage
should not be used. Items made of neoprene rubber and other
to the machine’s metal parts.
materials not previously mentioned should not be used unless
it has been shown that their use will not affect the survival,
7.6.2 Many organic solvents leave a film that is insoluble in
growth, or reproduction of test organisms adversely.
water.Adichromate-sulfuric acid cleaning solution can gener-
ally be used in place of both the organic solvent and the acid,
7.3 Test and Culture Chambers—A test or culture chamber
but the solution might leave chromium residues on glass.
isanenclosedspaceorcompartmentinwhichtemperatureand
7.6.3 Upon completion of a test, all items to be reused
lighting are controlled (for example, incubator or modified
should immediately be (1) emptied of soil, (2) rinsed with
room). The ventilation of chambers, especially test chambers,
water, and (3) cleaned by the procedures previously outlined.
is desired.
Test organisms and soil should be disposed of using appropri-
7.3.1 Test and culture chambers usually require continuous
ate procedures (see Guide D4447).
lighting (except in the case of the Enchytraeid Reproduction
Test). A timing device should be used to provide a light:dark 7.6.4 Test containers should be stored with their lids on to
cycle if a photoperiod other than continuous light is used. keep them clean.
E1676 − 12 (2021)
7.7 Acceptability—Before a toxicity test is conducted in 8.7 The use of ground fault systems and leak detectors is
new test facilities, it is desirable to conduct a “non-toxicant” recommended strongly to help prevent electrical shocks.
test, in which all test containers contain a negative control of
9. Soil
artificial or reference soil. Survival, growth, or reproduction of
the test species will demonstrate whether the facilities, hydra-
9.1 General—Beforethepreparationorcollectionofsoil,an
tion water, artificial soil, and handling techniques are adequate
approved, written procedure should be prepared for the han-
to result in acceptable species-specific control numbers. The
dling of soils that might contain unknown quantities of toxic
magnitude of the within-chamber and between-chamber vari-
chemicals(seeSection8).Allsoilsshouldbecharacterizedand
ance should also be determined.
have at least the following determined: pH, percent organic
matter,cationexchangecapacity(CEC),totalnitrogen,particle
8. Safety Precautions
size distribution (percent sand, silt, and clay), and percent
water content. In addition, chemical analyses should be per-
8.1 Many substances pose health risks to humans if ad-
formed for compounds suspected of occurring in the particular
equate precautions are not taken. Information on the chemical
soil (for example, heavy metals and organics). Toxicological
and physical properties, toxicity to humans (31-34), and
results might provide information directing a more intensive
recommended handling procedures (35-39) of the test material
analysis.SoiltoxicitytestingproceduresaredetailedinSection
shouldbestudiedandmadeavailabletoallpersonnelinvolved
11.
beforeatestisbegun.Contactwiththetestmaterialsshouldbe
avoided. 9.2 Negative Control and Reference Soil—A negative con-
8.1.1 Many materials can affect humans adversely if pre- trol soil is used for the following: (1) to yield a measure of the
acceptability of the test, (2) to provide evidence of the health
cautions are inadequate. Field-collected soils might contain
toxic materials, and respiratory exposure and skin contact and relative quality of the test organisms, (3) to determine the
suitability of the test conditions and handling procedures, and
should be prevented or minimized. As much information as
(4) to provide a basis for interpreting data obtained from the
possible should be collected on the history of the site and the
test soils.Areference soil is used to describe the matrix effects
potentialproblemsfromhumanexposure.Exposuretoworkers
ofatest.Everytestmusthaveanegativecontrolofartificialor
mightbeminimizedbywearingrubberboots,disposablesafety
referencesoilandmayalsohaveareferencesoilifthenegative
gear, gloves, and a cartridge respirator. Information or direc-
control is an artificial soil.Areference soil should be collected
tives on necessary precautions should be available from a site
fromthefieldinacleanareaandrepresentthetestsoilasmuch
safety manager at some sites.
as possible in soil characteristics (for example, percent organic
8.1.2 When screening, mixing, or distributing hazardous
matter, particle size distribution, and pH). This provides a
soils in the laboratory, proper handling procedures might
site-specific basis for comparison of toxic and nontoxic con-
include working (1) under a ventilated hood, wearing protec-
ditions. The same conditions, procedures, and organisms must
tive gloves, laboratory coats, aprons, and safety glasses; or (2)
beusedwiththenegativecontrolandreferencesoilasareused
in a ventilated room, wearing rubber boots, disposable safety
in the other treatments, except that contaminated soil or test
gear, gloves, and a full-face bottled air respirator. When
materials are not added. In addition, a reference control
initiating toxicity tests in the laboratory, procedures might
(artificialorreferencesoilspikedwithacompoundwithknown
include wearing appropriate protective gloves, laboratory
toxicity at the concentrations(s) used) is desirable.
coats, aprons, and safety glasses and working in a ventilated
hood.
9.3 FieldSamplingDesign—Asiteisdefinedasadelineated
tract of land that is being considered as the overall study area,
8.2 Careful consideration should be given to those chemi-
usually from the standpoint of its being potentially affected by
cals that might biodegrade, transform to more toxic
xenobiotics. The field collection is often conducted in areas in
components, volatilize, oxidize, or photolyze during the test
which little is known concerning contamination or contamina-
period.
tion patterns. The object of a qualitative field sampling design
8.3 Healthandsafetyprecautionsandapplicableregulations
is to identify sites that contain potentially toxic conditions that
for the disposal of stock solutions, test organisms, and soils
may warrant further study. The collection design might divide
should be considered before beginning a test (see Guide
the site into sampling units based on habitat or topography to
D4447).
allow for maximum spatial coverage. Sampling stations may
be set up within each unit (see 3.2). One sample is collected
8.4 Cleaning of equipment with a volatile solvent such as
from each station. The lack of field replication at each station
acetone should be performed only in a well-ventilated area in
usually precludes statistical comparisons; however, the identi-
whichnosmokingisallowedandnoopenflamesuchasapilot
ficationofsamplesforfurtherstudyispossible,whensurvival,
light is present.
growth, or reproduction differ between sampling stations or
8.5 Anacidicsolutionshouldnotbemixedwithahypochlo-
sampling stations differ from a reference soil. Information on
rite solution because hazardous fumes might be produced.
field sampling design is presented byWarren-Hicks, et al (40),
8.6 Concentrated acid should be added to water, not vice Eberhardt and Thomas (41), Gilbert (42), and ISO (43).
versa, to prepare dilute acid solutions. Opening a bottle of 9.3.1 If the object of the field sampling design is to test for
concentratedacidandaddingconcentratedacidtowatershould statistically significant differences in the effects between nega-
be performed only in a fume hood. tive control or reference soils and test soils from several sites
E1676 − 12 (2021)
orbetweensamplingstationswithinasinglesite,aquantitative 9.4.3.1 There may be some instances when an intact core
methodisusedthatrequiresreplicatesampling.Thenumberof sample needs to be tested, and no processing is therefore
necessary.
field replicates (that is, separate soil samples at a single
9.4.4 Qualitative descriptions of the soil may include color,
sampling station) necessary per sampling station is a function
texture, or the presence of roots, leaves, and soil organisms.
of the need for sensitivity or power.Aminimum of three field
Monitoringtheodorofsoilsamplesshouldbeavoidedbecause
replicates from each station is recommended. These field
of potentially hazardous volatile chemicals (see Section 8).
replicates are each treated as a separate sample in the
9.4.5 The natural geochemical properties (for example, pH)
laboratory, that is, they are not mixed together. The field
of test soil collected from the field should be within the
replicates from a single sampling station might be used (1)to
tolerance limits of the test species, or controls for the variable
test for within-sampling station variability, (2) to compare
should be run (for example, a pH-adjusted soil). Limits for the
laboratorytestprocedures,or(3)tocomparesensitivityamong
test species should be determined in advance (see 10.1).
test species.
9.5 Laboratory-Spiked Test Soil—Test soil can also be
9.3.2 Sampling stations might be distributed along a known
prepared in the laboratory by adding materials such as chemi-
pollution gradient within a site or at random within sampling
cals or waste mixtures to artificial, reference, or site soils (see
units. Comparisons can be made between both space and time
1.4).
if the sampling and testing take place during different times of
9.5.1 Test chemicals should be reagent grade or better,
the year.
unless technical or other grade material is specifically needed.
9.4 Field-Collected Test Soil:
Before a test is started, the following should be known
concerning the test material: (1) identity and concentration of
9.4.1 Collection—A shovel or auger (preferably stainless
major ingredients and impurities; (2) water solubility in hydra-
steel) should be used to collect soil samples (see Section 8).
tion water, log P , and vapor pressure; (3) estimated toxicity
The surface of the location at which the sample is to be ow
to the test species and to humans; (4) precision and bias of the
collected should be cleared of debris such as leaves and twigs.
analytical method at the planned concentrations of the test
If the location is an area of grass or other plants, the plants
material, if the test concentrations are to be measured; and (5)
should be cut to ground level and removed before the sample
recommended handling and disposal procedures. Additional
iscollected.Thesampleshouldbeplacedinathickplasticbag
informationonthefateofthetestsubstanceinsoilisdesirable.
(for example, 4 mil) and taped closed.This bag should then be
9.5.2 Stock Solutions—Test materials to be tested in
placed in a second plastic bag, taped closed, and placed in a
artificial,reference,orsitesoilshouldbedissolvedinasolvent
cleansamplecontainerwithalid(forexample,plasticpailwith
(the preferred solvent is water) to form a stock solution. The
O-ring seal). Direct sunlight should be minimized during
stock solution itself, or dilutions of it, are then added to the
collection if the chemicals associated with soils include com-
soil. The concentration and stability of the chemical in the
pounds that photolyze readily. All soil samples should be
stock solution should be determined before beginning the test.
placed in an ice chest and kept cold in the field. Field
The stock solution should be shielded from light both before
observations concerning habitat and type of vegetation and
and during the process of mixing into the soil if the chemical
measurements such as soil temperature and moisture may be
is subject to photolysis. Concentrations of the chemical in the
taken in the field.
solvent and soil should be monitored before the test begins.
9.4.2 Storage—Soil samples should be utilized as soon as
9.5.3 Non-Water Solvents—If a solvent other than water is
possible in accordance withTest Methods E1706 stored at 4 6
necessary, it should be one that is water-miscible and can be
2°C for no longer than eight weeks before the start of the test.
driven off (for example, can be evaporated), leaving only the
Freezing and longer storage times might change the soil
test chemical on the soil. Both a solvent control and a negative
propertiesandshouldbeavoided.Thesoilmaybestoredinthe
control soil must be included in the test if a solvent other than
sample containers in which it was collected in the field. It is water is used. The solvent control must contain the highest
desirable to avoid contact with metals and plastics.
concentrationofsolventaddedtothesoilandmustusesolvent
fromthesamebatchusedtomakethestocksolution.Thesame
9.4.3 Processing—The following procedures should be fol-
concentration of solvent should be used in all treatments.
lowed if a homogenous sample is needed. The samples should
9.5.3.1 Acetone is an organic solvent used for preparing
be screened to remove oversize material such as rocks. A
stocksolutions (7, 21, 23, 44)becauseofitshighvolatilityand
6.30-mm mesh, stainless steel screen may be used. The soil
ability to dissolve many organic chemicals. Other water-
should be mixed after screening (for example, in a stainless
miscibleorganicsolvents,suchasmethanolorethanol (9),may
steel mixer) to ensure homogeneity (see Section 6). Sub-
be used. Organic solvents may affect total organic carbon
samples of the processed soil should be removed for pH and
levels,introducetoxicity,oralterthegeochemicalpropertiesof
moisture content determination. Moisture content is deter-
mined gravimetrically by drying a subsample for 24 h at
100°C. Information on moisture content is necessary to
Reagent Chemicals, American Chemical Society Specifications, American
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
determinetheamountofhydrationwatertoaddtothetestsoils
listed by the American Chemical Society, see Analar Standards for Laboratory
(see A1.9.3). Each replicate is screened, mixed, and treated
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
separately if a quantitative method of field sampling with
and National Formulary,U.S.PharmaceuticalConvention,Inc.(USPC),Rockville,
replicates was used. MD.
E1676 − 12 (2021)
the soil (see 6.1.5). A surfactant should not be used in the ismsareexposedtoacontrolandfiveconcentrationsofthetest
preparation of a stock solution because it might affect the material that differ by a factor of ten.
9.5.5 The addition of test materials to soil may be accom-
bioavailability, form, and toxicity of the test material.
plished using various methods such as hand mixing or using a
9.5.3.2 If the concentration of solvent is not the same in all
mechanical mixer (see 9.4.3).
testsolutionsthatcontaintestmaterial,asolventtestshouldbe
9.5.5.1 If tests are repeated, mixing conditions such as the
conductedtodeterminewhethersurvival,growth,orreproduc-
duration and temperature of mixing and time of mixing before
tion of the test organisms are related to the solvent concentra-
the test starts should be kept constant. Care should be taken to
tionovertherangeusedinthetoxicitytest.Ifsurvival,growth,
ensure that a test material added to a soil is distributed
or reproduction are found to be related to solvent
thoroughly and evenly within the soil. The homogeneity of
concentration, a soil toxicity test with that species in that
laboratory-dosedmaterialshouldalwaysbedeterminedpriorto
amount of solvent is unacceptable if any treatment contained a
testing.
concentration of solvent in that range.
9.5.3.3 For compounds insoluble in water and in organic
10. Test Organism
solvents, 10 g of finely ground quartz sand should be mixed
10.1 Species—Only one species is currently described in
with the quantity of test substance to obtain the desired test
this guide (see Annex A1 and Annex A4); however, descrip-
concentration. This mixture of quartz sand and test substance
tions of additional species may be included in revisions of this
shouldbeaddedtothepremoistenedsoilandthoroughlymixed
guide. The use of these species is encouraged to increase the
by adding an appropriate amount of deionized water to obtain
comparability of results. The source and type of soil being
the moisture required as-described by OECD (45).
tested or the type of test to be implemented might dictate the
9.5.3.4 The survival, growth, or reproduction of the organ-
selection of a particular species. The species used should be
isms tested in the two controls should be compared if the test
selected based on (1) availability; (2) sensitivity to test
containsbothanegativecontrolandasolventcontrol.Onlythe
materials; (3) tolerance to parameters such as temperature, pH,
solventcontrolmaybeusedformeetingtheacceptabilityofthe
and grain size; and (4) ease of handling in the laboratory. The
test and as the basis for the calculation of results if a
species used should be identified using an appropriate taxo-
statistically significant difference in either survival, growth, or
nomic key.
reproduction is detected between the two controls. The nega-
10.2 Ag
...