ASTM E2591-22

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: E2591 − 22
Standard Guide for
Conducting Whole Sediment Toxicity Tests with
Amphibians
This standard is issued under the fixed designation E2591; 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 sediment toxicity to invertebrates (for example, Hyalella az-
teca and Chironomus dilutus toxicity tests) described in the
1.1 This standard covers procedures for obtaining labora-
UnitedStatesEnvironmentalProtectionAgency(USEPA, (1))
tory data concerning the toxicity of test material (for example,
freshwatersedimenttestingguidance,TestMethodsE1367and
sediment or hydric soil (that is, a soil that is saturated, flooded,
E1706, and Guides E1391, E1525, E1611, and E1688. Tests
or ponded long enough during the growing season to develop
extending to 10 d or beyond, and including sublethal measure-
anaerobic (oxygen-lacking) conditions that favor the growth
ments such as growth, are considered more effective in
and regeneration of hydrophytic vegetation)) to amphibians.
identifying chronic toxicity and thus delineating areas of
This test procedure uses larvae of the northern leopard frog
moderate contamination (1-3).
(Lithobates pipiens). Other anuran species (for example, the
green frog (Lithobates clamitans), the wood frog (Lithobates
1.4 Many historical amphibian studies, both water and
sylvatica), theAmerican toad (Bufo americanus)) may be used
sediment exposure, have used tests of shorter duration (5 days
if sufficient data on handling, feeding, and sensitivity are
or less) (for example, 4-7) and, although both survival and
available. Test material may be sediments or hydric soil
sublethal endpoints were often assessed, there is substantive
collected from the field or spiked with compounds in the
evidence that tests of longer duration are likely to be more
laboratory.
sensitive to some contaminants (8-10). Research performed to
1.2 The test procedure describes a 10-d whole sediment develop and validate this test protocol included long-term
(through metamorphosis) investigations and other researchers
toxicity test with an assessment of mortality and selected
sublethal endpoints (that is, body width, body length). The have also conducted long-duration tests with anurans (7-20).
Interestingly, some studies with anurans have shown signifi-
toxicity tests are conducted in 300 to 500-mL chambers
containing100mLofsedimentand175mLofoverlyingwater. cantly reduced growth (for example, whole body mass, snout-
Overlyingwaterisreneweddailyandlarvalamphibiansarefed vent length) can be detected earlier in a longer-term test (for
during the toxicity test once they reach Gosner stage 25 example, at 14-20 d), but cannot be statistically distinguished
(operculum closure over gills). The test procedure is designed
in older organisms later in the test (11, 14). In the development
to assess freshwater sediments, however, R. pipiens can toler- of these procedures, an attempt was made to balance the needs
-
ate mildly saline water (not exceeding about 2500 mg Cl /L,
of a practical assessment with the importance of assessing
+
equivalent to a salinity of about 4.1 when Na is the cation) in longer-term effects so that the results will demonstrate the
10-d tests, although such tests should always include a con-
needed accuracy and precision. The most recent sediment
current freshwater control. Alternative test durations and sub- toxicity testing protocols for invertebrates have encompassed
lethal endpoints may be considered based on site-specific
longer duration studies which allow the measurement of
needs. Statistical evaluations are conducted to determine
reproductiveendpoints (1, 21).Suchtests,becauseofincreased
whether test materials are significantly more toxic than the
sensitivity of the sublethal endpoints, may also be helpful in
laboratory control sediment or a field-collected reference evaluatingtoxicity.Fulllife-cyclestudieswithanurans(includ-
sample(s).
ing reproduction) are usually not feasible from either a
technical or monetary standpoint. However, if site-specific
1.3 Where appropriate, this standard has been designed to
information indicates that the contaminants present are likely
be consistent with previously developed methods for assessing
to affect other endpoints (including teratogenicity), then the
duration of the toxicity test may be increased through meta-
morphosis or additional sublethal endpoints may be measured
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Jan. 1, 2022. Published April 2022. Originally
approvedin2007.Lastpreviouseditionapprovedin2013asE2591–07(2013).DOI: The boldface numbers in parentheses refer to the list of references at the end of
10.1520/E2591-22. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2591 − 22
(for example, impaired behavior, deformities, time-to- bins (8), or swimming pools (44). Endpoints evaluated at test
metamorphosis).The possible inclusion of these endpoints and termination included survival (4, 8, 14, 42-44), growth (8, 14,
extension of test length should be considered during develop- 42-44), bioaccumulation of metals (8), developmental rates (8,
ment of the project or study plan (see 8.1.1). 14, 43), deformities (14, 42, 43), swimming speed (44) and
foraging activity levels (43).
1.5 The methodology presented in this standard was devel-
1.6.2 To assess the effect of direct contact with the sedi-
oped under a Department of Defense (DoD) research program
ments containing PCBs, Savage et al. (43) exposed larval
and presented in a guidance manual for risk assessment staff
tadpoles (Gosner stage 23 to 25; wood frogs (R. sylvatica)) to
and state/federal regulators involved in the review and ap-
field-collected sediments under conditions that allowed both
proval of risk assessment work plans and reports (22).To
direct contact with the sediment and separation from the
develop this method, a number of tests with spiked sediment
sediment with a 500 µm mesh barrier. The study found that
tests were conducted (22, 23). Since development of the
lethal and sublethal effects on tadpoles observed through
methodology it has been used operationally to evaluate field-
metamorphosis were more pronounced when direct contact
collectedsedimentsfromseveralstateandfederalenvironmen-
with the sediment was allowed. Fuentes et al. (39) evaluated
tal sites (24, 25). For most of these studies the preferred test
the acute toxicity of two Roundup (a widely used herbicide
organisms, Lithobates pipiens, was used. At a lead-
with the active ingredient glyphosate) formulations to six
contaminated state-led site, operated by the Massachusetts
anuranspecies,includingLithobatespipiens,underbothwater-
Highway Department, Xenopus laevis(African clawed frog)
only and water-+sediment conditions. The study found that
was used in the sediment test system because of availability
toxicity of the glyphosate-based herbicides was reduced in the
problems with Lithobates pipiens (26), The test method was
presence of sediment, likely due to sorption to sediment
also used to evaluate sediment toxicity at a cadmium-
particles and associated organic matter. The test conditions
contaminated USEPARegion 4-led site in Tennessee (27). The
described in this standard allow tadpoles to maintain direct
methodology was used to help characterize potential effects of
contact with the sediment.
contaminants on amphibians and to help develop preliminary
1.6.3 Sediment toxicity testing with Xenopus laevis has
remedial goals, if warranted. All tests evaluated survival and
focused on evaluating the developmental effects of sediment
growth effects after 10 d of exposure in accordance with the
extracts, as opposed to whole sediments, on frog embryos.
methods presented in this standard.
Methods have been developed which expose blastula stage
1.6 The use of larval amphibians to assess environmental
embryos to sediment by enclosing the embryos in a Teflon
toxicity is not novel. Researchers have used tadpoles to
mesh insert that rests over the top of the sediment in the
examine toxicity of metals and organic compounds. Most of
sediment–water interface region ((42), Guide E1439-98 Ap-
these studies have been through water exposure, usually in a
pendix X2). These studies are conducted evaluate survival,
manner similar to fish or invertebrate exposure as described in
growth, and physical malformations of the embryos after a 4-d
Guide E729 (28-40). Fewer studies have focused on exposure
exposure period. The test conditions described in this standard
of anuran larvae to sediments, and the methods employed vary
allow more direct contact with the sediment, using older test
widely, from in situ enclosures (15, 41) to laboratory tests
organisms, and a longer exposure duration.
usingvariableexposureconditionsandorganismages (4, 8, 14,
1.7 Amphibian species may be key receptors of potential
39, 42-44). No studies were identified that used the same test
chemicals of concern at contaminated sites. Although histori-
conditions as described in this standard. However, several
cally not often included in risk assessments, the importance of
laboratory-based evaluations of sediment effects on amphib-
amphibians as both sensitive and keystone species is increas-
ians are described in the following subsections.
ingly recognized, particularly considering the decline in am-
1.6.1 Sediment toxicity tests conducted in the laboratory
phibian worldwide populations, which may be driven by
with amphibians were performed over a range of test durations
multiplelocalizedstressagentsratherthanasingle,dominating
from4d(4, 39, 42,GuideE1439-98AppendixX2)to12d (44)
cause (45). The lack of amphibian representation as surrogate
and through metamorphosis (8, 14, 43). Sediment toxicity tests
species is likely due to multiple factors including scant
with anurans native to North America were started with larval
knowledge of local amphibian populations and life histories,
tadpoles between Gosner stages 23 and 25 (8, 43, 44). Test
the paucity of applicable toxicity data, and inconsistency in
temperatureswerebetween21 °Cand23 °Candfeedingbegan
standardized assessment protocols.Areview of ecological risk
after tadpoles reached Gosner stage 25. Food sources were
assessment methods for amphibians and gaps in existing
TetraMin (8), boiled romaine lettuce (43), boiled romaine
amphibian toxicity data and methods is provided by Johnson et
lettuce and flaked fish food (14), or boiled romaine lettuce and
al. (46). The importance of amphibians in the ecological risk
dissipated rabbit food pellets (44). Tests were conducted in
assessment process is recognized by Environment and Climate
static renewal mode with water replacements conducted at
Change Canada in the Ecological Risk Assessment Guidance
varying rates (daily (42, 44), weekly (8), every 3 to 5 d (43)).
under the Federal Contaminated Sites Action Plan (47). Sedi-
Test design (number of replicates, test vessel size, number of
ment toxicity tests are an effective means for evaluating the
organisms per replicate) varied depending on the objective of
impact of sediment contamination on amphibians in a multiple
the study with several tests conducted in aquaria (14, 43), large
lines of evidence paradigm. The evaluation is most powerful
3 4
TetraMin is a trademark of TETRA GMBH. Roundup is a registered trademark of Monsanto Company.
E2591 − 22
when toxicity testing sampling stations are co-located with for Selection of Samplers Used to Collect Benthic Inver-
sediment analytical chemistry samples and ecological surveys, tebrates
allowing for a detailed evaluation of the co-occurring data in E1439 Guide for Conducting the Frog Embryo Teratogen-
the ecological risk assessment. The spatial and temporal esis Assay-Xenopus (FETAX)
co-location of toxicity testing and analytical samples is par- E1525 Guide for Designing BiologicalTests with Sediments
ticularlyimportantforestablishingcontaminant-specificeffects E1611 Guide for Conducting Sediment Toxicity Tests with
and assessing contaminant bioavailability. Polychaetous Annelids
E1688 Guide for Determination of the Bioaccumulation of
1.8 In order for a sediment toxicity test to be sensitive it
Sediment-Associated Contaminants by Benthic Inverte-
must be of sufficient duration to measure potential toxicity and
brates
it must be conducted during the appropriate developmental
E1706 Test Method for Measuring theToxicity of Sediment-
stage of the test organism’s life cycle. Using recently hatched
Associated Contaminants with Freshwater Invertebrates
tadpoles and conducting the sediment exposure test for 10 d to
E1733 Guide for Use of Lighting in Laboratory Testing
allow the evaluation of growth endpoints meets both of these
E1847 Practice for Statistical Analysis of Toxicity Tests
sensitivity requirements.
Conducted Under ASTM Guidelines (Withdrawn 2022)
1.9 The values stated in SI units are to be regarded as
SI10-02 IEEE/ASTM SI 10 American National Standard for
standard. No other units of measurement are included in this
UseoftheInternationalSystemofUnits(SI):TheModern
standard.
3. Terminology
1.10 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
3.1 The words “must”, “should”, “may”, “can” and “might”
responsibility of the user of this standard to establish appro-
have very specific meanings in this guide. “Must” is used to
priate safety, health, and environmental practices and deter-
express an absolute requirement, that is, to state that the design
mine the applicability of regulatory limitations prior to use.
of a test ought to be in a manner that satisfies the specified
1.11 This international standard was developed in accor-
conditions, unless project goals dictate needed alterations in
dance with internationally recognized principles on standard-
ordertoaddressthestudyhypotheses.“Should”isusedtostate
ization established in the Decision on Principles for the
that the specified condition is recommended and ought to be
Development of International Standards, Guides and Recom-
metifpossible.Althoughtheviolationofone“should”israrely
mendations issued by the World Trade Organization Technical
a serious matter, violation of several could render the results
Barriers to Trade (TBT) Committee.
questionable. Terms such as “is desirable”, “is often desirable”
and “might be desirable” are used in association with less
2. Referenced Documents
important factors, the alteration of which will probably not
have substantive effects on test outcome. “May” means “is
2.1 ASTM Standards:
(are) allowed to,” “can” means “is (are) able to” and “might”
D4447 Guide for Disposal of Laboratory Chemicals and
means “could possibly.” In this manner, the classic distinction
Samples
between “may” and “can” is preserved and “might” is never
E177 Practice for Use of the Terms Precision and Bias in
used as a synonym for either “may” or “can.”
ASTM Test Methods
E691 Practice for Conducting an Interlaboratory Study to
3.2 Definitions—For definitions of general terms related to
Determine the Precision of a Test Method
toxicity testing and used in this guide, refer to Guides E943,
E729 Guide for Conducting Acute Toxicity Tests on Test
E1023, E1192, E1367, and E1525. For an explanation of units
Materials with Fishes, Macroinvertebrates, and Amphib-
and symbols, refer to SI10-02 IEEE/ASTM SI 10.
ians
3.3 Definitions of Terms Specific to This Standard:
E943 Terminology Relating to Biological Effects and Envi-
3.3.1 IC25 (25 % inhibition concentration),
ronmental Fate
n—concentration at which there is a 25 % reduction in organ-
E1023 Guide for Assessing the Hazard of a Material to
ism performance, relative to the control. Performance may be
Aquatic Organisms and Their Uses
survival or a sublethal measurement such as growth.
E1192 Guide for ConductingAcute Toxicity Tests onAque-
3.3.2 overlying water, n—water that is placed over the
ous Ambient Samples and Effluents with Fishes,
sediment for the duration of the study. Overlying water may be
Macroinvertebrates, and Amphibians
surface water collected from the project site or from a clean
E1367 Test Method for Measuring theToxicity of Sediment-
lake or reservoir, or may be reconstituted water prepared in the
Associated Contaminants with Estuarine and Marine In-
laboratory (for example, moderately hard water; (48)).
vertebrates
E1391 Guide for Collection, Storage, Characterization, and
3.3.3 reference-toxicant test, n—a test conducted with a
Manipulation of Sediments for Toxicological Testing and
reagent-gradereferencechemicaltoassessthesensitivityofthe
test organisms. Deviations outside an established normal range
may indicate a change in the sensitivity of the test organism
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
Standards volume information, refer to the standard’s Document Summary page on The last approved version of this historical standard is referenced on
the ASTM website. www.astm.org.
E2591 − 22
population. Reference-toxicity tests are most often performed (IC25s), or 50 % inhibition concentrations (IC50s)) may be
in the absence of sediment. calculated. Field-collected sediments often contain more than
one potential chemical stressor and therefore calculating
3.3.4 test sediment or test material, n—sediment that may
chemical-specific point estimates should only be done with
contain contaminants, which is being evaluated using this test
caution. A reference-toxicant test should be run concurrently
procedure.
with a sediment test whenever a new batch or lot of organisms
4. Summary of Guide is used.
4.1 Each test consists of eight replicates of the test material
5. Significance and Use
(for example, field-collected sediment or spiked sediment) and
5.1 While federal criteria and state standards exist that
overlying water with five test organisms (recently-hatched
tadpoles) per replicate. A laboratory control sediment (some- define acute and chronic “safe” levels in the water column,
times called a negative control) is used to provide (1) a effects levels in the sediment are poorly defined and may be
measureoftheacceptabilityofthetestbyindicatingthequality dependent upon numerous modifying factors. Even where
of tadpoles, test conditions and handling procedures, and (2) a USEPArecommended Water Quality Criteria (WQC, (49)) are
basis for interpreting data from other treatments. The test not exceeded by water-borne concentrations, organisms that
duration is ten days with an assessment of mortality and liveinornearthesedimentmaystillbeadverselyaffected (50).
selected sublethal endpoints (that is, body width, body length) Therefore,simplymeasuringtheconcentrationofachemicalin
at the end of the test. Assessments of mortality can be made the sediment or in the water is often insufficient to evaluate its
daily during the test and dead organisms removed. However, actual environmental toxicity. Concentrations of contaminants
similar coloration of the tadpoles and sediment may make it in sediment may be much higher than concentrations in
difficult to see the organisms and sediment disturbance should overlying water; this is especially true of hydrophobic organic
be kept to a minimum.Alternative test durations and sublethal compounds as well as inorganic ions that have a strong affinity
endpoints may be considered based on site-specific needs. The for organic ligands and negatively-charged surfaces. Higher
objective of the test is to evaluate whether test materials chemical concentrations in sediment do not, however, always
(spiked or field-collected sediments) are significantly more translate to greater toxicity or bioaccumulation (51), although
toxic than the laboratory control or reference sediment(s). research also suggests that amending sediment with organic
Additional evaluations may be performed if an exposure matter actually increases the bioaccumulation of contaminant
gradient is tested. Statistical evaluations may be conducted to particles (52, 53). Other factors that can potentially influence
determine whether test materials are significantly more toxic sediment bioaccumulation and toxicity include pH mineralogi-
than the laboratory control sediment or field-collected refer- cal composition, acid-volatile sulfide (AVS) grain size, and
ence sample(s). If the test material is sediment spiked with a temperature (54-56). Laboratory toxicity tests provide a direct
knownconcentrationofachemicalstressororiffield-collected and effective way to evaluate the impacts of sediment contami-
sediment contains a measured gradient of a particular chemical nation on environmental receptors while providing empirical
of concern, then point estimates (for example, median lethal consideration of all of the physical, chemical and biological
concentrations (LC50s), 25 % inhibition concentrations parameters that may influence toxicity.
TABLE 1 Advantages and Disadvantages for Use of Sediment Tests (Modified from Test Method E1706)
Advantages
Measure bioavailable fraction of contaminant(s).
Provide a direct measure of effects on sediment-associated receptors (benthos, larval amphibians), assuming no field adaptation or amelioration of effects.
Limited special equipment is required.
Methods are rapid and inexpensive.
Legal and scientific precedence exist for use; USEPA and ASTM standard methods and guides are available.
Measure unique information relative to chemical analyses or community analyses.
Tests with spiked chemicals provide data on cause-effect relationships.
Sediment-toxicity tests can be applied to all chemicals of concern.
Tests applied to field samples reflect cumulative effects of contaminants and contaminant interactions.
Toxicity tests are amenable to confirmation with natural populations (invertebrate or amphibian surveys).
Disadvantages
Sediment collection, handling, and storage may alter bioavailability.
Spiked sediment may not be representative of field contaminated sediment.
Natural geochemical characteristics of sediment may affect the response of test organisms.
Indigenous animals may be present in field-collected sediments.
Route of exposure may be uncertain and data generated in sediment toxicity tests may be difficult to interpret if factors controlling the bioavailability of contaminants
in sediment are unknown.
Tests applied to field samples may not discriminate effects of individual chemicals.
Few comparisons have been made of methods or species.
Only a few chronic methods for measuring sublethal effects have been developed or extensively evaluated.
Laboratory tests have inherent limitations in predicting ecological effects.
Tests do not directly address human health effects.
Motile organisms may be able to avoid prolonged exposure to contaminated media so tests may overestimate actual exposure.
Species used in toxicity testing programs are typically chosen to be representative and protective of the organisms found on-site, but the use of surrogate species
cannot precisely predict the health of ecological communities on-site.
Toxicity to organisms in situ may be dependent upon physical characteristics and equilibrium partitioning that are not readily replicated under laboratory conditions.
E2591 − 22
5.2 Amphibians are often a major ecosystem component of these species to all potential sediment-associated contaminants
wetlands around the world, however limited data are available is unknown, use of test organisms for which more toxicity data
regarding the effects of sediment-bound contaminants to am- are available is recommended.
phibians (39, 41, 43, 55, 57, 58).Laboratorystudiessuchasthe
7. Facilities, Equipment, and Supplies
procedure described in this standard are one means of directly
assessing sediment toxicity to amphibians in order to evaluate
7.1 Facilities—While larval amphibians can be acclimated
potential ecological risks in wetlands.
and held for short periods of time in static or static-renewal
systems, continuous-renewal/flow-through conditions are pref-
5.3 Results from sediment testing with this procedure may
erable shortly after hatching. Tadpoles grow rapidly and, once
be useful in developing chemical-specific sediment screening
feeding begins at about Gosner Stage 25 (59), ammonia
values for amphibians.
concentrations are likely to increase and oxygen levels may be
5.4 Sediment toxicity test can be used to demonstrate the
depressed, making flow-through conditions desirable. Culture/
reaction of test organisms to the specific combination of
holding tanks and test chambers should be held at a constant
physical and chemical characteristics in an environmental
temperature, either in an environmental chamber or
medium. The bioavailability of chemicals is dependent on a
temperature-controlled water bath.Addition of overlying water
number of factors, which are both site-specific and medium-
in a flow-through system should be gravity-fed from a water
specific.Althoughmanyofthesefactorscanbeestimatedusing
source that may be replaced via pumps. Overlying water
equilibrium partitioning techniques, it is difficult to account for
should be near culture/test temperature although small tem-
all the physical and chemical properties which could poten-
perature deviations should have little impact upon test water
tially affect bioavailability. Sediment toxicity tests may be
temperature at the slow rate of water replacement. Low
particularly applicable to evaluating hydrophobic compounds
dissolved oxygen concentrations may be remedied by increas-
which may not readily partition into the water column. See
ing water replacement rates in small increments. If aeration is
Table 1 for a summary of advantages and disadvantages
necessary,airshouldbefreeofcontaminantsincludingoil,dust
associated with sediment toxicity tests.
and water; a filtration system may be desirable to remove
bacterial contaminants. Lighting should be maintained at a
6. Interferences
16-h light and 8-h dark cycle unless the test-specific protocol
calls for an alternative photoperiod.
6.1 General Interferences:
6.1.1 An interference is a characteristic of a sediment or a
7.2 Special Requirements—Amphibian eggs and tadpoles
test system that can potentially affect test organism response
can be highly sensitive to alterations in temperature, oxygen
aside from those related to sediment-associated contaminants.
deprivation and handling. If eggs are received from an out-of-
These interferences can potentially confound interpretation of
laboratory source, attention should be paid to how embryos are
test results in two ways: (1) toxicity is observed in the test
packed for shipment, shipment time and handling at the
sediment when contamination is low or there is more toxicity
laboratory. Shipping containers should be durable, insulated
than expected, and (2) no toxicity is observed when contami-
and water tight. Embryos may be contained in large plastic
nants are present at elevated concentrations or there is less
bags sealed with rubber bands. Double bagging is recom-
toxicity than expected.
mended for added security. Oxygenation of the water contain-
6.1.2 These general interferences may include: potential
ing the embryos is recommended before sealing the bags for
changes in contaminant bioavailability due to manipulation of
shipment. Coolers containing embryos should be firmly taped
field-collected sediments during collection, shipping, and stor-
shut before shipment. The use of ice packs or additional
age; the influence of natural physico-chemical characteristics
insulation in the shipping containers may be needed when
such as sediment texture, grain size, and organic carbon on the
outdoor temperatures are elevated or reduced. It is recom-
response of test organisms; tests conducted with field-collected
mended that temperatures be monitored during shipment, if
samples usually cannot discriminate between effects of mul-
possible, or upon receipt at the laboratory. Upon receipt at the
tiple contaminants. See Guide E1706 Section 6 for a detailed
laboratory, eggs should be allowed to hatch with minimal
discussion of several general interferences that pertain to
disturbance.
sediment toxicity testing.
7.3 Equipment and Supplies—All equipment used to pre-
6.1.3 Some interferences, such as the presence of indig-
pare test sediments or reagents, transfer sediments or organ-
enous organisms in field-collected sediments, may have less of
isms and conduct tests, should be decontaminated as outlined
an impact on toxicity tests conducted with larval amphibians
below. Table 2 provides a list of the general equipment needed
than on tests conducted with sediment invertebrates.
to conduct testing. Glass is the preferable material in which to
6.2 Species-Specific Interferences:
conduct tests, however, alternative materials such as stainless
6.2.1 Particular characteristics of individual species that steel, high-density polyethylene (HDPE), polycarbonate and
were tested during the development of this method will fluorocarbon plastics may be appropriate, depending upon the
probably not act as substantial interferences to completion of contaminants of concern that might be present in the sediment.
successfultests.ThosespeciesincludeLithobatespipiens,Bufo Used equipment should not be used if there is a possibility of
americanus, Lithobates clamitans, Lithobates palustris (pick- residualcontaminationthatcannotberemovedviathewashing
erel frog), Lithobates sylvatica, Hyla chrysoscelis (gray tree process. In some cases, test substances present in field-
frog) and Xenopus laevis. However, because the sensitivity of collected sediments or introduced into spiked sediments may
E2591 − 22
TABLE 2 General Equipment Required for Conducting a 10-d
8.1.5 In general, unless project specific conditions dictate
Sediment Toxicity Test with Lithobates pipiens
otherwise, sediment should be collected from the top 15 cm of
Stainless steel bowls and spoons or auger to homogenize sediment
the native horizon, which generally represents the maximum
Testing chambers (usually 300 to 500 mL beaker with a small-mesh
bioactive zone and area of most probable exposure.
(300 µm) screen covering a hole drilled in the side of the beaker
(secured with nontoxic silicone adhesive)) 8.1.6 The exact collection procedures will depend upon
Transfer pipettes
study design. In deeper water where a boat is used, a benthic
Small nets
grab, dredge or corer should be used (Guide E1391). At
Dissecting microscopes
Dissolved oxygen meter and probe locations where the water is very shallow, including saturated
Conductivity meter and probe
hydric soils, these devices can also be used or a clean trowel or
pH meter/selection ion meter and probe
shovel can be used.Whatever collection method is selected, all
Ammonia meter and probe
Reagents and equipment for hardness and alkalinity determinations
cleaning and decontamination protocols need to be followed to
Temperature-controlled water bath or environmental chamber
minimize sample contamination.
capable of controlling to 23 °C ± 1 °C
8.1.7 The testing procedure described in this standard re-
Flow-through water delivery system
Buffered 3-aminobenzoic acid ethyl ester, methanesulfonate salt
quires a minimum of about one liter of sediment. Since this
(MS-222 anesthetic) solution.
amount does not allow for accidental loss, spillage, analytical
Food source (dried fish food flakes)
chemistry, or test reruns, collection of a minimum of two liters
Appropriate data forms
Metric ruler
is recommended.
Forceps
8.1.8 The most convenient sample containers are wide-
Statistical software
mouth, high-density polyethylene (HDPE) bottles with a
screw-on cap. Glass jars may be desirable for some studies
where adsorption to plastic surfaces is of concern. However,
glass containers require greater care in handling and packing
for shipment and are generally more expensive than plastic
not be thoroughly washed from the test vessels. In these cases
jars.
the test vessels should not be re-used. All new and used
equipment needs to be washed in detergent and should be
8.2 Storage:
rinsed with dilute acid and deionized water. Rinsing with an
8.2.1 Light and heat can stimulate and accelerate chemical
organic solvent (for example, acetone) should also be consid-
and biological reactions that may alter chemical composition,
eredforthosematerialsthatwillnotbedamagedbythesolvent
promote degradation of potential toxicants, and affect bioavail-
(for example, some plastics) (see Test Method E1706 section
ability. Samples, therefore, should be kept out of sunlight and
9.3.6 for a step-by-step cleaning procedure). Materials that
stored in the dark under refrigeration. Samples should be
should not contact overlying water include copper, cast iron,
cooled before shipping, unless the ambient temperature is
brass, lead, galvanized metal (that may contain zinc) and
already <10ºC. Target cooling temperature for sediments is
natural rubber.
about 4°C (Test Method E1367). Ice or blue ice should be
included with the samples when they are shipped. Samples
8. Test Material Collection and Processing
should not be frozen as freezing can alter sediment character-
istics.
8.1 Collection:
8.2.2 For additional information on sediment collection and
8.1.1 Before field collection and preparation of sediments, a
shipment see Guide E1391.
sampling/processing procedure should be established that out-
8.2.3 It is desirable to initiate tests as soon as possible
lines the site- or project-specific steps to be followed. The
following field collection of sediments (Test Method E1706).
statistical analyses that will be applied to the data should be
Several studies have addressed the question of storage time for
considered during the development of the sampling/processing
procedure (see Practice E1847). See Guide E1391 for addi- sediments, and the conclusions reached in these studies vary
considerably.Wherethepotentialchemicalstressorsareknown
tional detail regarding methods for collecting, storing, and
characterizing sediment samples. to be recalcitrant, storage under the conditions described in
8.2.1 should allow the sample to remain stable for longer
8.1.2 Sediment should be collected with as little disturbance
as possible. It may be desirable to collect sediments from a periods. However, some labile chemicals (for example, ammo-
nia and volatile organics) can degrade or volatize during
boat (even if wading is possible) to minimize sediment
disruption. storage. For these labile materials, a maximum holding time of
twoweeks(fromthetimeofsamplecollectiontotestinitiation)
8.1.3 Since the distribution of contaminants in sediment
is recommended (61). However, more stable sediments can be
matrices can demonstrate a great deal of spatial variability
stored for much longer periods of time with little change in
(60), it is desirable to collect multiple replicates from within
toxicity.
the delineated study area. At a minimum, multiple samples
should be collected and thoroughly composited in the field so 8.2.4 During even short periods of storage, density differ-
the sample better represents environmental conditions. ences will results in settling in samples, resulting in a hetero-
8.1.4 Large pieces of plant material and other debris, such geneous mixture. Therefore, prior to test initiation, the sedi-
as large rocks and glass, should be removed and discarded in ment should be homogenized again, even if it was already
the field. Alternatively, these materials can be removed in the mixed in the field. In most situations, overlying water should
laboratory prior to test setup. not be drained off the sample, but should be remixed with solid
E2591 − 22
material.If,after24hoursofundisturbedsettling,>75 %ofthe in the sediment will remain open, (3) using a 10-mL or 5-mL
sample volume can still be considered standing water, it may pipet, punch at least five holes into the sediment to different
be desirable to remove some or all of that water so as to ensure
depths, (4) distribute equally to each hole the volume of the
that the test material will be a solid matrix.
stock solution needed to achieve the desired target concentra-
tion of test material. The stock solution may be an inorganic
8.3 Manipulation:
salt dissolved in water (for example, copper as CuCl ). If a
8.3.1 Homogenization: 2
hydrophobic chemical is to be tested, it may first be dissolved
8.3.1.1 Homogenization can be accomplished by using a
into a stock solution using a carrier solvent (for example,
tumbling or rolling mixer or other suitable apparatus. It can
acetone or methanol). A surfactant should not be used in the
also be done using a stainless steel auger and drill or simply by
preparation of a stock solution because it might affect the
hand with a stainless steel spoon.Aminimum interval (at least
threeminutes)shouldbeestablishedformixingeachsample.A bioavailability, form or toxicity of the test material. If a carrier
more heterogeneous sample would indicate the need for a solvent is used, a solvent control must also be prepared which
longer mixing time.Additional large debris should be removed
contains the solvent but not the contaminant to be tested. See
at this time. Sieving of samples is not recommended, however,
USEPA (1), Guide E1391, and Test Method E1706 for addi-
indigenous organisms can be removed by hand during the
tional details regarding sediment spiking techniques.
mixing process. Special attention should be paid to any
8.3.2.4 Once spiked, the sediments need to be thoroughly
predaceous organisms that might be present in the collected
mixed to incorporate the chemical into the sediment and create
sample.Augers,spoons,andanyotherequipmentthatcomesin
ahomogenizedmatrix.Homogenizationmethodsincluderoller
contact with the sediment during homogenization must be
mixers, end-over-end mixers stainless steel kitchen mixers,
washed and decontaminated between samples.
mixing manually with a spoon or a combination of these.
8.3.2 Sediment Spiking:
Mixing times, speeds and temperatures should be consistent
8.3.2.1 Test sediment can be prepared by manipulating the
among treatments, replicates and tests.
properties of a control sediment (Test Method E1706). Mixing
8.3.3 Test Concentration(s) for Laboratory-Spiked Sedi-
time (60) and aging (62) of spiked sediment can affect
ments:
bioavailability of chemicals. If tests are initiated within only a
few days of spiking a sediment, the spiked chemicals may not 8.3.3.1 If a test is intended to generate an LC50, IC50 or
be at equilibrium with the sediment. There are not, however, IC25 of a test chemical, a concentration series should be
specified equilibrium intervals for all chemicals that might be
created that will bracket that effect concentration. If mortality
spiked into sediment. Such specifications would not be reason-
is one of the desired endpoints, at least one test concentration
able since sediment characteristics will play a major role in
shouldproducegreaterthan50 %mortalityandthereshouldbe
time to equilibration as well as equilibration concentrations.
two or more concentrations with partial mortality. Determining
For a series of spiked sediment studies, where results will be
the concentration(s) that will result in desired lethal or sub-
compared, spiking methods should be consistent and the
lethaleffectscanbedifficultif(1)theenvironmentaltoxicityof
amount of time between spiking and test initiation should also
the test material is unknown and/or (2) the impact(s) of
be consistent.
sediment characteristics is/are unknown. The latter can be
8.3.2.2 The test material(s) should be at least reagent grade,
particularly important since there are many factors that can
unlessatestusingaformulatedcommercialproduct,technical-
significantly affect toxicity (39, 51-56). It may be desirable to
gradeoruse-gradematerialisspecificallyneeded.Beforeatest
conduct a range-finding test in which the organisms are
is initiated, the following should be known about the test
exposed to a control and three or more concentrations of the
material (not all of this information may be available): (1) the
test material that differ by a factor of ten. For example, test
identity and concentration of major ingredients and impurities,
concentrations in a range-finding test may include the control,
(2) solubility in test water and water used to prepare any stock
10, 100 and 1000 mg/kg.
solutions, (3) log K , BCF for aquatic vertebrates (preferably
ow
amphibians),persistenceinwaterandsediment,hydrolysisand
8.4 Sediment Characterization:
photolysis rates, (4) estimated toxicity to the test organism, (5)
8.4.1 It is recommended that a subsample of each field-
toxicity to humans and potential handling hazards, (6) if and
collected or spiked sediment be analyzed for at least the
when analytical samples will be collected, how much material
following parameters: pH, total organic carbon (TOC), particle
willbeneededtoobtaintheneededresolutionandpreservation
size distribution (percent sand, silt, clay). Similar analyses
methods, and (7) recommended handling and disposal meth-
should also be conducted on laboratory control sediment and
ods.
reference sediment(s).
8.3.2.3 Different sediment spiking methods are available.
8.4.2 Further characterization may be warranted depending
Sediment spiking techniques used during development and
on the objectives of the study. This may include chemical
validation of the amphibian sediment test method (22) were
analyses of inorganic and organic compounds of interest,
previously employed for incorporation of both inorganic con-
ammonia, pore water chemistry, chemical oxygen demand,
taminants and organic chemicals into sediment (57). The
sediment oxygen demand, oxidation-reduction potential (Eh),
procedure included: (1) place appropriate (considering testing
acid volatile sulfides (AVS), and simultaneously extracted
and analytical needs) amount of sediment in a mixing jar, (2)
if sediment is dry, wet it with deionized water to ensure holes metals (SEM), or other analyses depending on the program.
E2591 − 22
8.4.3 Chemical and physical data should be obtained using left intact, organisms should be transferred to an aquarium or
appropriate standard methods whenever possible. For those other holding container and slowly brought to test temperature.
measurements for which standard methods do not exist or are 9.3.1 Time to hatch will depend upon age at the time of
not sensitive enough, methods should be obtained from other shipping. Once the young embryos have developed into a
reliable sources. recognizable tadpole and are actively moving, the bag can be
8.4.4 Sediment characterization helps to evaluate sediment openedandtheeggs/earlystagetadpolesplacedinanaquarium
homogenization and accuracy of sediment-spiking, and identi- or other large chamber.
fies potential chemical or physical stressors for test organisms. 9.3.2 Once the eggs/tadpoles are released for the shipping
container to an aquarium or other chamber, shipping water
9. Test Organisms
should be slowly replaced with culture/overlying water. This
should be done by initially adding culture/overlying water at a
9.1 Species—Test organisms are recently hatched tadpoles
proportion of no more than 10 % for one hour. If organisms do
of small NorthAmerican anurans. The preferred species is the
not appear to be adversely affected, increase the amount of
Northern Leopard Frog, L. pipiens. Sediment toxicity testing
culture/overlying water by about 15 %⁄ hour to 25 %⁄ hour for
conducted with both L. pipiens and the American toad, B.
4 hours to 5 hours.
americanus, during the development of this standard indicated
9.3.3 Additionalacclimationoftestorganismsshouldnotbe
that L. pipiens was generally more sensitive to spiked sedi-
needed under most
...