ASTM E2122-22

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: E2122 − 22
Standard Guide for
Conducting In-situ Field Bioassays With Caged Bivalves
This standard is issued under the fixed designation E2122; 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 rangeandcompartmentalizedcagesformultiplemeasurements
on the same individuals.
1.1 This guide describes procedures for conducting con-
1.2 The test is referred to as a field bioassay because it is
trolled experiments with caged bivalves under field conditions.
conducted in the field and because it includes an element of
The purpose of this approach is to facilitate the simultaneous
relative chemical potency to satisfy the bioassay definition.
collection of field data to help characterize chemical exposure
Relative potency is established by comparing tissue concen-
and associated biological effects in the same organism under
trations with effects levels for various chemicals with toxicity
environmentally realistic conditions. This approach of charac-
and bioaccumulation end points (6, 7, 8, 9, 10) even though
terizing exposure and effects is consistent with the US EPA
there may be more uncertainty associated with effects mea-
ecological risk assessment paradigm. Bivalves are useful test
surements in field studies. Various pathways of exposure can
organisms for in-situ field bioassays because they (1) concen-
be evaluated because filter-feeding and deposit-feeding are the
trate and integrate chemicals in their tissues and have a more
primary feeding strategies for bivalves. Filter-feeding bivalves
limitedabilitytometabolizemostchemicalsthanotherspecies,
may be best suited to evaluate the bioavailability and associ-
(2) exhibit measurable sublethal effects associated with expo-
atedeffectsofchemicalsinthewatercolumn(thatis,dissolved
suretothosechemicals,(3)providepairedtissuechemistryand
and suspended particulates); deposit-feeding bivalves may be
response data which can be extrapolated to other species and
best suited to evaluate chemicals associated with sediments
trophic levels, (4) provide tissue chemistry data which can be
(11, 12, 13, 14). It may be difficult to demonstrate pathways of
used to estimate chemical exposure from water or sediment,
exposure under field conditions, particularly since filter-
and (5) facilitate controlled experimentation in the field with
feedingbivalvescaningestsuspendedsedimentandfacultative
large sample sizes because they are easy to collect, cage, and
deposit-feeding bivalves can switch between filter- and deposit
measure (1, 2) . The experimental control afforded by this
feeding over relatively small temporal scales. Filter-feeding
approach can be used to place a large number of animals of a
bivalves caged within 1 m of bottom sediment have also been
known size distribution in specific areas of concern to quantify
used effectively in sediment assessments from depths of 10 to
exposure and effects over space and time within a clearly
650 m (5, 15, 16). Caged bivalve studies have also been
defined exposure period. Chemical exposure can be estimated
conducted in the intertidal zone (17). The field testing proce-
by measuring the concentration of chemicals in water,
dures described here are useful for testing most bivalves
sediment, or bivalve tissues, and effects can be estimated with
although modifications may be necessary for a particular
survival, growth, and other sublethal end points (3). Although
species.
a number of assessments have been conducted using bivalves
to characterize exposure by measuring tissue chemistry or
1.3 These field testing procedures with caged bivalves are
associated biological effects, relatively few assessments have
applicable to the environmental evaluation of water and
been conducted to characterize both exposure and biological
sediment in marine, estuarine, and freshwater environments
effects simultaneously (2, 4, 5). This guide is specifically
with almost any combination of chemicals, and methods are
designed to help minimize the variability in tissue chemistry
being developed to help interpret the environmental signifi-
and response measurements by using a practical uniform size
cance of accumulated chemicals (6, 7, 9, 18, 19). These
procedures could be regarded as a guide to an exposure system
to assess chemical bioavailability and toxicity under natural,
1 site- specific conditions, where any clinical measurements are
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
possible.
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
1.4 Tissue chemistry results from exposures can be reported
Current edition approved April 1, 2022. Published May 2022. Originally
approved in 2001. Last previous edition approved in 2013 as E2122 – 02(2013)
in terms of concentrations of chemicals in bivalve tissues (for
which was withdrawn in January 2022 and reinstated in April 2022. DOI:
example, µg/g), amount (that is, weight or mass) of chemical
10.1520/E2122-22.
per animal (for example, µg/animal), rate of uptake, or bioac-
The boldface numbers in parentheses refer to references at the end of this
standard. cumulation factor (BAF, the ratio between the concentration of
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2122 − 22
a chemical in bivalve tissues and the concentration in the of this standard to establish appropriate safety, health, and
external environment, including water, sediment, and food). environmental practices and determine the applicability of
Tissue chemistry results can only be used to calculate a BAF regulatory limitations prior to use. Specific hazard statements
because caged bivalves in the field are exposed to multiple are given in Section 7.
sources of chemicals and can accumulate chemicals from 1.9 This international standard was developed in accor-
water, sediment, and food. Toxicity results can be reported in dance with internationally recognized principles on standard-
terms of survival (3, 20), growth rate (3, 20), or reproductive ization established in the Decision on Principles for the
effects (21, 22) after a defined exposure period. Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
1.5 Other modifications of these procedures might be justi-
Barriers to Trade (TBT) Committee.
fied by special needs or circumstances. Although using appro-
priate procedures is more important than following prescribed 2. Referenced Documents
procedures,resultsoftestsconductedusingunusualprocedures
2.1 ASTM Standards:
are not likely to be comparable to results of standardized tests.
D1129 Terminology Relating to Water
Comparisons of results obtained using modified and unmodi-
D1193 Specification for Reagent Water
fied versions of these procedures might provide useful infor-
D3976 Practice for Preparation of Sediment Samples for
mation concerning new concepts and procedures for conduct-
Chemical Analysis
ing field bioassays with bivalves.
D4447 Guide for Disposal of Laboratory Chemicals and
Samples
1.6 This guide is arranged as follows:
E724 Guide for Conducting Static Short-Term Chronic Tox-
Section
icity Tests Starting with Embryos of Four Species of
Referenced Documents 2
Saltwater Bivalve Molluscs
Terminology 3
E729 Guide for Conducting Acute Toxicity Tests on Test
Summary of Guide 4
Significance and Use 5 Materials with Fishes, Macroinvertebrates, and Amphib-
Interferences 6
ians
Hazards 7
E943 Terminology Relating to Biological Effects and Envi-
Experimental Design 8
ronmental Fate
Apparatus 9
Facilities
E1022 Guide for Conducting Bioconcentration Tests with
Construction Materials
Fishes and Saltwater Bivalve Mollusks (Withdrawn
Cages
Test Organisms 10 2022)
Species
E1023 Guide for Assessing the Hazard of a Material to
Commonly Used Taxa
Aquatic Organisms and Their Uses
Size and Age of Test Organisms
Source E1191 Guide for Conducting Life-Cycle Toxicity Tests with
Number of Specimens
Saltwater Mysids
Collection
E1367 Test Method for Measuring theToxicity of Sediment-
Handling
Holding
Associated Contaminants with Estuarine and Marine In-
Animal Quality
vertebrates
Field Procedures 11
E1391 Guide for Collection, Storage, Characterization, and
Test Initiation: Presort
Final Measurements and Distribution Manipulation of Sediments for Toxicological Testing and
Attachment of PVC Frames
for Selection of Samplers Used to Collect Benthic Inver-
Deployment
tebrates
Retrieval and End-of-Test Measurements
Analysis of Tissues for Background Contamination
E1525 Guide for Designing BiologicalTests with Sediments
Decontamination
E1688 Guide for Determination of the Bioaccumulation of
Collection and Preparation of Tissues for Analysis
Sediment-Associated Contaminants by Benthic Inverte-
Quality Assurance/Quality Control Procedures
Sample Containers, Handling, and Preservation
brates
Ancillary Methodology 12
E1706 Test Method for Measuring theToxicity of Sediment-
Temperature
Associated Contaminants with Freshwater Invertebrates
Food
Acceptability of Test 13
E1847 Practice for Statistical Analysis of Toxicity Tests
Report 14 4
Conducted Under ASTM Guidelines (Withdrawn 2022)
Keywords 15
E2455 Guide for Conducting Laboratory Toxicity Tests with
References
Freshwater Mussels (Withdrawn 2022)
1.7 The values stated in SI units are to be regarded as
SI10-16 IEEE/SI 10American National Standard for Metric
standard. No other units of measurement are included in this
Practice
standard.
1.8 This standard may involve hazardous materials,
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
operations, and equipment – particularly during field opera-
Standards volume information, refer to the standard’s Document Summary page on
tions in turbulent waters or extreme weather conditions. This
the ASTM website.
standard does not purport to address all of the safety concerns,
The last approved version of this historical standard is referenced on
if any, associated with its use. It is the responsibility of the user www.astm.org.
E2122 − 22
3. Terminology 3.2.10 growth dilution, n—a process whereby the rate of
accumulation is exceeded by the rate of tissue growth so that
3.1 Definitions:
when the concentration is expressed on mass of chemical per
3.1.1 The words “must,” “should,” “may,” “can,” and
mass of tissue over time, it appears as though depuration or
“might,” have very specific meanings in this guide. “Must” is
elimination is occurring because the concentration (µg/g) is
used to express an absolute requirement, that is, to state that a
decreasing.
test ought to be designed to satisfy the specified condition,
3.2.11 reference station, n—a station similar to the test
unless the purpose of the test requires a different design.
station(s) in physical and chemical characteristics and with
“Must” is only used in connection with factors that directly
relatively little to no contamination by the particular chemi-
relate to the acceptability of the test. “Should” is used to state
cal(s) under study. A reference station should ideally contain
that a specified condition is recommended and ought to be met
only background concentrations of chemicals characteristic of
if possible. Although violation of one “should” is rarely a
the region.
serious matter, violation of several will often render the results
questionable. Terms such as “is desirable” are used in connec- 3.2.12 scope for growth, n—an integrated physiological
tion with less important factors. “May” is used to mean “is
measure of the energy status of an organism at a particular
(are) allowed to,” “can” is used to mean “is (are) able to,” and time, based on the concept that energy in excess of that
“might” is used to mean “could possibly.” Thus the classic
required for normal maintenance will be available for the
distinction between “may” and “can” is preserved and “might” growth and reproduction of the organism.
is never used as a synonym for either “may” or “can.”
3.2.13 shell length, n—thedistancefromthetipoftheumbo
3.1.2 For definitions of other terms used in this guide, refer
to the distal valve edge.
to Terminology D1129, Guide E729, Terminology E943, and
3.2.14 site, n—a geographical area within a somewhat
Guide E1023. For an explanation of units and symbols, refer to
defined boundary that is being studied. The size of a site is
SI10-16.
dependentontheextentofsuspectedperturbation,generallyon
3.2 Definitions of Terms Specific to This Standard:
the order of 0.1 to 50–km . Part of the vagueness in size is due
3.2.1 bioaccumulation, n—the accumulation of a chemical
to variability in spatial scale and inadequate results from
in an organism. preliminary reconnaissance survey that clearly define the
boundary of suspected stressors.
3.2.2 bioaccumulation factor (BAF), n—the ratio of tissue
3.2.15 steady state, n—the state in which fluxes of material
chemical residue to chemical concentration in the external
environment. BAF is measured at steady state in situations moving bidirectionally across a membrane or boundary be-
tween compartments or phases have reached a balance. An
where organisms are exposed from multiple sources (that is,
water, sediment, food), unless noted otherwise. equilibrium between the phases is not necessarily achieved.
3.2.16 station, n—a specific sampling location or area
3.2.3 bioassay, n—an experiment that includes both an
within a site. The size of a station can vary from a single point
estimate of toxicity and an estimate of relative potency.
with one cage to an area of approximately 10 by 10 m
3.2.4 bioavailability, n—the fraction of the total chemical
including several cages. Vagueness in size is due to variability
concentration in water, on sediment particles, and on food that
in spatial scale and experimental design. Several stations in a
is available for bioaccumulation.
small geographic area could comprise a site.
3.2.5 biomonitoring, v—use of living organisms as “sen-
3.2.17 tissue loss magnification, n—theprocesswherebythe
sors”inwaterorsedimentqualitysurveillancetodetectcurrent
tissue mass is lost during the exposure period and the chemical
conditionsorchangesinaneffluentorwaterbodyortoidentify
mass remains constant over time, so that when the concentra-
exposure to chemicals and risks to aquatic life.
tion is expressed on mass of chemical per mass of tissue over
3.2.6 chemical concentration, n—the ratio of the weight or
time, it appears as though bioaccumulation is occurring be-
volume of chemicals to the weight or volume of a test sample.
cause the concentration (µg/g) is increasing.
3.2.7 chemical content, n—mass of chemical per whole 3.2.18 uptake, n—acquisition of a substance from the envi-
animal (for example, µg/animal) can be used to normalize the ronment by an organism as a result of any active or passive
expression of chemical uptake per unit time by eliminating the process.
effects of growth on changing tissues masses.
3.2.19 whole-animal wet-weight, n—the wet weight (g) of
the entire bivalve, including water trapped between the valves.
3.2.8 chemical fingerprinting, v—theuseofspecificpatterns
in the ratios of chemicals accumulated in bivalve tissues to
identify chemical sources; for example, the ratio of PAH 4. Summary of Guide
alkylated homologs to parent compounds.
4.1 This guide describes procedures for exposing marine,
3.2.9 compartmentalized cage, n—a rigid or flexible mesh estuarine, and freshwater bivalves to chemicals in water,
cage with individual compartments for holding bivalves in a sediment, and food in the field under natural in-situ field
controlled position so that multiple measurements can be made conditions. The purpose of this guide is to provide a standard
on the same individual organism. The compartmentalized cage approach for in-situ testing with bivalves. Because of its
helps maximize water flow around individual test organisms application to a wide variety of species, many of which have a
and provides even exposure to the test environment. range of tolerance limits for water quality conditions, it is
E2122 − 22
outside the scope of this guide to provide the tolerance limits and sediment particles make them appropriate species for
for all water quality conditions for all species that can be used conducting field bioassays to assess bioaccumulation potential
for in-situ testing. Tolerance limits are provided for selected
and associated biological effects. The test procedures in this
species as examples and points of reference (6.4).
guide are intended to provide guidance for conducting con-
trolled experiments with caged bivalves under “natural,” site-
4.2 The approach can be used to characterize exposure and
specific conditions. It is important to acknowledge that a
effects over space and time. The primary measurement end
points are bioaccumulation of chemicals in bivalve tissues to number of “natural” factors can affect bivalve growth and the
assess biological availability or bioaccumulation potential, and accumulation of chemicals in their tissues (Section 6, Interfer-
sublethal effects, like growth, to assess adverse biological
ences). This field bioassay can also be conducted in conjunc-
effects. The bioavailability of chemical(s) in water, sediment,
tion with laboratory bioassays to help answer questions raised
and food and associated biological effects are determined by
in the field exposures. The field exposures can also be used to
the relative differences in these exposure and effects end points
validate the results of laboratory bioassays.
among stations over time.
5.2 The ultimate resources of concern are communities.
4.3 In practice, the two most commonly measured effects
However, it is often difficult or impossible to adequately assess
end points are survival and growth. Survival is the easiest
theecologicalfitnessorconditionofthecommunityoridentify
effects end point to measure and provides an estimate of
and test the most sensitive species. Bivalves are recommended
toxicity in exposures of any duration that is determined to be
as a surrogate test species for other species and communities
appropriate to meet the study needs (see 8.10). The survival
for the following reasons: (1) They readily accumulate many
end point may be insensitive for some chemicals but can
chemicals and show sublethal effects associated with exposure
provide important corroborative effects information. Sublethal
to those chemicals (2);(2) they accumulate many chemicals
endpointslikegrowtharegenerallymoresensitive.Growthcan
through multiple pathways of exposure, including water,
be estimated from changes in whole-animal wet-weight, shell
sediment, and food (24, 25, 26, 27, 28, 29), and (3) caged
length, tissue weight, or shell weight, with baseline tissue and
bivalves have been shown to represent effects on the benthos
shell weights for the entire test population estimated from a
more accurately than traditional laboratory tests (30, 31).
subsample of that population. Reproduction is another sensi-
Although bivalve species might be considered insensitive
tive end point, but is more difficult to measure in bivalves.
because of their wide use as indicators of chemical
4.4 Bioaccumulation and growth are compared among test
bioavailability, it has been suggested that sensitivity is related
stations for ranking purposes, among reference and treatment
to the type of test, end points being measured, and duration of
stations, or among stations for temporal and spatial variability
exposure (2). In relatively short-term toxicity assessments in
as well as short- and long-term trends. It is also possible to use
which survival is typically determined as the measurement end
the data to construct dose-response relationships (6, 7) and to
point, bivalves may appear to be more tolerant to and less
identify sources of point and non-point discharges by compar-
affected by chemicals because of their ability to close their
ing bioaccumulation and biological effects at various distances
valves for short periods and avoid exposure (32, 33, 34, 35).
away from suspected sources of contamination in a gradient
approach (23). However,studiescomparingthemortalityendpointinbivalves
and other test species have found bivalves to be equally (36,
5. Significance and Use
37) or more sensitive (38, 39) than the other species (Table 1).
When the bivalve growth end point was compared to the
5.1 The ecological importance of bivalves, their wide geo-
graphic distribution, ease of handling in the laboratory and the mortalityendpointinothertestspecies,thebivalvegrowthend
field,andtheirabilitytofilterandingestlargevolumesofwater point was more sensitive (20, 30, 31, 40, 41).
TABLE 1 Relative Sensitivity of Bivalves Compared to Other Test Species
Bivalve Species Species Compared Exposure End Point Sensitivity
Anodonta grandis (37) daphnia, fathead minnow, municipal effluent LC-50 equal
(giant floater; currently Pyganodon grandis) rainbow trout
Anodonata imbecilis (38) daphnia pulp and paper 10-d vs 7-d mortality more
(paper pondshell; currently Utterbackia mill effluent
imbecilis)
Anodonata imbecilis (36) daphnia, midge, metals 7-d mortality equal
(paper pondshell; currently Utterbackia fathead minnow
imbecilis)
Musculium transversum (39) 17 different species ammonia 20-d mortality more sensitive than
(fingernail clam) 16 species
Mercenaria mercenaria (30, 31) 2 amphipods, microtox sediment 7-d growth, 10-d mortality more
(hard clam)
Caged Mercenaria more sensitive than lab Mercenaria (30, 31)
Mullinia lateralis (40) amphipod sediment 7-d growth, 10-d mortality more
(dwarf surf clam)
Mytilus galloprovincialis (20) amphipod in-situ water column 84-d growth, 10-d more, [tissue TBT]
(Mediterranean mussel) mortality
E2122 − 22
5.2.1 Chronic tests designed to monitor sublethal end in sediment may not exclusively accumulate or be affected by
points, such as growth, are recommended because bivalves chemicals in a particular medium. That is, bivalves in or on
generally show increasing sensitivity with increasing exposure sediment may still filter and accumulate chemicals from
period. Sublethal end points measured in bivalves that have overlying water. Conversely, bivalves transplanted in the water
demonstrated high levels of sensitivity include growth (3, 20), column may filter suspended sediment and accumulate chemi-
reproduction (21),DNAdamage (42, 43),metallothioneinsand calsfromthatsediment.Bivalvescanalsoassimilatechemicals
other biochemical markers (44, 45, 46). as they ventilate overlying water.
5.2.2 There are many field monitoring programs in the US
5.6 Field bioassays are conducted to obtain information
which use bivalves, including the NOAA Status and Trends
concerning the bioavailability of chemicals in the water col-
Program (47), the California Mussel Watch (48), and the
umn or bedded sediments and subsequent biological effects on
California Toxics Monitoring Program, a freshwater monitor-
bivalves after short- and long-term exposure to water and
ing program (49). Similar field-monitoring programs exist in
sedimentundersite-specificconditions.Thesebioassaysdonot
other countries. Numerous laboratory studies throughout the
necessarily provide information about whether delayed effects
worldhaveexaminedbioaccumulationandbiologicaleffectsin
will occur, although a post-exposure observation period could
bivalves. The existing databases which have compiled bioac-
provide such information. Sublethal post-exposure observa-
cumulation and effects in bivalves and other species (8, 9)
tions may include gonad development, spawning success,
makeitpossibletousetissueresiduesassociatedwitheffectsin
gamete survival, and development. The decision to conduct
bivalves as surrogates to estimate effects in both water column
post-exposure studies in the field or in the laboratory depends
and benthic organisms in many freshwater, estuarine, and
on the observations being made, test conditions required, and
marine environments.
experimental logistics.
5.3 Bivalves are an abundant component of many soft
5.7 The in-situ exposures described in this guide could be
bottom marine, estuarine, and freshwater environments. Inter-
followed by laboratory measurements, such as scope for
tidal marine bivalves make up a significant portion of many
growth (2), filtration rate (51), byssal thread production (52,
habitats and provide habitats for many additional species. It is
53, 54), and biomarkers (55, 56).
important to monitor freshwater bivalves for the following
reasons:theyareamongthefirsttaxatodisappearfrombenthic
5.8 The bivalve field bioassay can be used to determine the
communities impacted by chemicals; they have been shown to
spatial or temporal trends of chemical bioavailability in water
be more sensitive than several other major taxa in laboratory
and sediment and effects due to exposure to those chemicals.
tests.(50) The threatened and endangered status of many
Spatial comparisons of parameters of concern can be made by
freshwater bivalve species also make them an important group
distributing the caged bivalves along physical and chemical
to monitor.
gradients at scales commensurate with the desired level of
discrimination. For example, station locations might be distrib-
5.4 If practical, the species to be used in a field bioassay
uted along a known physical or chemical gradient in relation to
should be one that is endemic to the area under investigation.
the boundary of a disposal site (57, 58, 59, 60, 61), sewage
In many cases, the specific area under investigation may not
outfall (62), or effluent pipe or at stations identified as
support bivalves due to a variety of factors including high
containing elevated concentrations of chemicals in water or
concentrations of chemicals, competition or predation, or lack
sediment as identified in a reconnaissance survey (3, 63, 64).
of suitable habitat or substrate. Under these conditions, it may
This can be accomplished by placing caged bivalves along
be desirable to use a species that would normally be found in
horizontal transects or at different depths in the water column.
the environment if all conditions were favorable; however, it
Temporal comparisons can be made by conducting before-and-
may be necessary to use a surrogate species, that is, a species
after studies. For example, the effectiveness of dredge
that can tolerate the environmental conditions but is not
activities, effluent diffuser construction, effluent reduction, or
normally found in the area, if native species are unavailable in
remedial action can be determined by conducting field bioas-
the test area.
says before the action, during the action, and after the action.
5.5 Bivalves generally utilize one of two primary modes of
feeding: filter-feeding or deposit feeding. However, all known 5.9 The relative bioavailability of chemicals from the vari-
deposit-feeding bivalves are facultative in that they can either ous pathways of exposure (that is, aqueous phase, suspended
deposit- or filter-feed. Filter-feeders assimilate dissolved or- particulate matter, sediment) and subsequent effects can be
ganics as well as suspended particulate matter, including determined by simultaneously deploying bivalves with differ-
plankton and suspended sediments, from the water column and ent feeding strategies and making supplementary measure-
have the potential for exposure to chemicals associated with ments.Acombinationoffiltrationandtheuseofsedimenttraps
this ingested material. Facultative deposit-feeding bivalves can followed by chemical analysis of the various environmental
be exposed to chemicals associated with sediments as they compartments can be used to identify the relative contribution
ingestsediments.Theyalsoingestparticulatematerialfromthe of the aqueous phase, suspended particulate matter, and sedi-
water column as they filter feed.As such, bivalves are capable ment. Lipid bags or semi-permeable membrane devices
of integrating exposure to chemicals dissolved in water and (SPMDs), which predominantly collect the dissolved fraction
sorbed on sediment particles on the bottom or in suspension. It of chemicals, could also be used ( 65, 66, 67, 68, 69, 70). The
should be acknowledged that bivalves transplanted in the bioaccumulation of chemicals and effects among different
overlying water above sediment or transplanted directly on or bivalve species deployed either side-by-side, at exposure and
E2122 − 22
reference locations, or before and after exposures can be temperature tolerance for the most commonly used species are
compared and used to help explain the spatial variability of provided in Table 2. Temperature conditions during the expo-
chemicalcontamination,particularlyifthedifferentspeciesare sure period can be quantified using in-situ monitoring devices.
placed in different locations (that is, in the water column, on These devices can be attached to the deployment cages and set
top of the sediments, within the sediments) as determined to collect temperature data at specified time intervals for the
appropriate for the study design. This field assessment ap- duration of the test.
proachcouldbesupplementedwithlaboratorystudiesdesigned
6.5 Lack of acclimation to deployment water quality con-
to answer specific questions regarding dissolved versus par-
ditions could be an interference. If water quality conditions
ticulate pathways of exposure.
differatcollectionanddeploymentsites,itmaybenecessaryto
5.10 Results of bivalve field bioassays might be an impor-
acclimate the test organisms gradually to the deployment
tant consideration when assessing the hazards of materials to
conditions. This transition is particularly important near the
aquatic organisms (see Guide E1023) or when deriving water
bivalve’s tolerance limits and may be accomplished using
or sediment quality guidelines for aquatic organisms (17, 71).
serial water dilutions until the proper water quality conditions
Bivalve field bioassays can be useful in making decisions
(for example, temperature, salinity, and pH) are reached.
regarding the extent of remedial action needed for contami-
Acclimation for temperature should proceed no faster than 3°C
nated sites. They also provide a convenient method for
in 72 h (Guides E1022 and E1688). Once acclimated, bivalves
manipulative field experiments, hypothesis testing, and moni-
should be maintained under these conditions for a minimum
toringspecificsitesbefore,during,andaftercleanupoperations
period of time. Holding bivalves for extended periods under
(63, 64).
laboratory conditions can induce stress because bivalves are
particularly sensitive to temperature, nutrition, and water flow.
6. Interferences
If test specimens are held for an extended period of time in the
laboratory, the effect of this holding can be assessed by
6.1 As with all bioassay procedures, there are limitations to
comparing soft tissue weights, or other indicators of bivalve
themethodsdescribedinthisguide.However,theselimitations
health, to that of bivalves of the same size group freshly
should not be considered as a reason for not using the methods
collected from the field. Alternatively, bivalves could be
described in this guide.
acclimated in the field under conditions similar to the proposed
6.2 Results of bivalve field bioassays will depend, in part,
transplant sites.
on natural factors, including temperature, food supply, other
6.6 Food supply is important because it affects both biologi-
physical and chemical properties of the test environment,
cal availability and associated biological effects. Food avail-
selection of adequate reference areas, species selected, condi-
ability may be more difficult to quantify during the test than
tion of the test organisms, exposure technique, and handling of
temperature or other physical factors. Until in-situ monitoring
the bivalves prior to deployment. Taking bivalves out of their
devices for chlorophyll and other nutrient sources are
habitat and weighing and measuring them may be stressful to
developed, it is suggested that food availability be estimated at
the bivalves. The degree of handling, holding time, and
least three times during the study (that is, beginning, middle,
differences between water and sediment conditions at the
and end of test). The measurements made (that is,
collection site versus the transplant site may also be stressful.
chlorophyll-a, particulate or total organic carbon, and sus-
Careful handling and appropriate acclimation can minimize
pended solids) will depend on the feeding strategy of the test
these stresses.
species.
6.3 Condition of the test organisms is critical to the success
6.7 Current speed is important for filter-feeding bivalves
of the field bioassay. The most important consideration is
becausecurrentsregulatethefoodsupplytothetestorganisms.
spawning cycle because of possible interferences on bioaccu-
Currents are also important to facultative deposit-feeding and
mulation and growth and with subsequent data interpretation.
filter-feeding bivalves in the benthos because flushing may
Generally, chemicals are lost during spawning, resulting in
reduce the potential effects of chemicals by dilution with clean
potential underestimation of chemical bioavailability (72).
water from outside the assessment area. Currents can be
Conversely, the energy used for gonad development and
quantified during the exposure period with a continuously
spawning can make bivalves more sensitive to chemicals,
recording, in-situ current meter or quantified intermittently
reduce their growth rates, and overestimate potential toxicity.
during the suggested sampling intervals used to measure food
Tests should be conducted with populations that will most
availability.
likely not spawn during the exposure period. The spawning
cycle of candidate test species should be evaluated prior to
6.8 Salinity is particularly important in estuarine areas,
developing the study design, and species that do not spawn
where salinity can range from 0 ppt at the head of a river to 33
during the proposed exposure period should be selected.
ppt at the mouth. Salinity should be evaluated prior to species
6.4 Temperature, conductivity, hardness, pH, and dissolved selection. If there is a wide salinity range, it may be necessary
oxygen concentrations of the test environment could affect to identify two or more bivalve species for the assessment: one
both bioaccumulation and biological effects. These water species for the lower end of the salinity range and another for
quality parameters should be monitored over the course of the theupperendofthesalinityrange.Itisrecommendedthatboth
study to quantify the exposure conditions and the potential species be deployed in the area where salinity is in the middle,
effects of temperature. As a general guide, examples of as this provides a means to compare results between species.
E2122 − 22
TABLE 2 Temperature (°C) and Salinity (Parts per Thousand (ppt)) Tolerance Limits for Selected Bivalve Species
(Months when spawning may occur and species distribution are also shown)
Temperature Salinity
Species and Reference Spawning Distribution
Range Range
Corbicula fluminea (Asian clam) (73) 2–25 0–5 may be continuous, usually twice/year All west, gulf, and east coastal United
spring/early summer; later summer States to DE River; NM; OH & MS River
systems
Dreissena polymorpha (Zebra mussel) (74) <0–35 0–6 May to September Canada and Northeastern United States;
Great Lakes, St. Lawrence River; MS, OH,
IL & TN River drainages; NY Canals,
Hudson River, Finger Lakes
Elliptio complanata (Eastern Elliptio) (75, 76, 77) 0–30 0–3 most June to July; some May to Gulf St. Lawrence to GA; Great Lakes,
September except Lake Michigan & Lake Erie
Pyganodon (Anodonta) grandis (floater mussel) (75) 0–30 0–3 most April to May; some to late August Canada Interior & St. Lawrence River
drainage; Hudson Bay, MI and MO Rivers
drainages; NM, CO, TX, Mex
Rangia cuneata (Atlantic Rangia) (78, 79) 8–32 0<19 VA: early April to summer; FL: July- Gulf of Mexico coast from northwest FL to
November; LA: Mar-May and late Campeche, Mexico; along Atlantic coast
summer to November; Mexico: to NJ
February-June and
September to November
Argopecten irradians (Bay scallop) (79) >7 >30 >14–28 mid-Atlantic: mid-April through early Atlantic coast; Cape Cod to Gulf of Mexico
September; NY: June and July; NC
and FL: August and December
Crassostrea gigas (Pacific oyster) (79) 4–24 25–35 July to August Pacific coast; Pacific Northwest
Crassostrea virginica (Eastern oyster) (80) -2–36 5–32 Gulf of Mexico: April-October; Malpeque Gulf of Mexico to Cape Cod
Bay, PEI: July-August; Bideford River
Estuary, PEI: July
Macoma balthica (Baltic clam) (81, 82, 83) -2–23 5–30 June-August (Europe); July-September Greenland to France; Baltic and Wadden
(United States) Seas; UK; N. Canada to Chesapeake; AK
to San Francisco Bay
Mercenaria mercenaria (Hard clam) (84) <0–35 12–35 March-November depending on latitude Atlantic and Gulf coasts; abundant MA to
and temperature. Peaks in July VA
Mya arenaria (Soft-shell clam) (85) -1.7–32 10–32 June-September; once/year north of Atlantic coast from Labrador to SC; less in
Cape Cod, twice/year south of Cape Cod FL; AK and CA
Mytilus californianus (California mussel) (79) 7–28 25–33 Continuous throughout year; peaks in AK to southern CA
July and December
Mytilus edulis (Blue mussel) (86) 0–27 5–33 differs between populations; some low- Atlantic coast, from Labrador to Cape
level throughout year; first in early Hatteras, NC
summer, second in the fall
Mytilus galloprovincialis (Mediterranean mussel) (87) 8–25 10–33 Similar to M. edulis, but several weeks Mediterranean, Europe, Atlantic France
later when temperature is maximum and British Isles, Japan, East China to
Korea, Australia, South Africa; southern
CA to OR
Mytilus trossulus (Pacific blue mussel) (88) 0–29 4–33 July to September Baltic Sea; west Coast, Central CA to AK;
east Coast, Canadian Maritimes
Ostrea lurida (Olympia oyster) (89) 6–20 NA–25 Spring to fall: peaks in spring in south, Southeast AK to Baja California
mid summer in mid-range and north
Protothaca staminea (Littleneck clam) (90) 0–25 20-32 BC, Canada, January to March; AK, Aleutian Islands, AK to Cape San Lucas,
mid-July; southern CA, June Baja California
Venerupis japonica (Manila clam) (79) 13–21 24–31 Washington: once/year May-September; British Columbia to CA
peaks in June/July
6.9 Possibleinterferencesinfluencingretrievaloftestorgan- surfaces of the cages by hand or with a stiff brush. If the cages
isms from the field include caged bivalves being washed away are heavily fouled and it is difficult to remove the attached
during storm events, buried by underwater sediment shifts,
biomass with brushing or scraping, the bivalves should be
theft, vandalism, fouling, disease, and consumption by preda-
transferred to clean, unfouled cages for the remainder of the
tors.
exposure period.
6.10 Depending on the environment under assessment, it is
6.11 Possible interferences associated with interpretation of
possible for the bivalve cages, including the external predator
tissuechemistrydataincludetheuseofinappropriateanalytical
mesh(see11.3)andthemeshbags,tobecomefouledwithboth
procedures. It is critical to use the most appropriate method for
epiphytic plant and animal growth. Fouling occurs most
eachchemicalanalysis.Forexample,whenmeasuringthesuite
frequently in highly productive embayments or areas with
of PAH-alkylated homologs, it is essential to use sufficient
restricted flow, such as marinas. Excessive fouling can reduce
silica gel to clean up excess lipids in the sample. A more
or eliminate flow of water through the cage material, resulting
specific approach for these analyses developed as part of the
in highly stressful conditions to the test bivalves. If such
Exxon Valdez oil spill assessment program included advanced
conditions are anticipated, the deployed cages should be
methods specific to that group of researchers. These methods
examined for fouling at regular intervals during the exposure
period. Fouling organisms can be removed from the exterior
E2122 − 22
are recommended for bivalve tissues when source identifica- 8. Experimental Design
tion through chemical fingerprinting is necessary (91, 92, 93,
8.1 Field bioassays can be designed to provide either a
94).
qualitative reconnaissance or a quantitative assessment involv-
ing statistical comparisons of measured end points (that is,
6.12 Natural variability in the concentrations of chemicals
chemical concentration in tissues and effects end points)
of concern in water or sediment coupled with intermittent
among stations. The object of a qualitative reconnaissance
chemical discharges may increase the difficulty in interpreting
survey is to identify sites with the potential for bioaccumula-
exposure concentrations in these pathways. However, weekly
tion and associated biological effects. Qualitative surveys are
measurements of chemicals in the water column coupled with
often conducted in areas where little is known about contami-
measurements of bioaccumulation and growth have proven
nation patterns. Quantitative assessments are conducted to test
effective in explaining the environmental significance of these
for statistically significant differences among stations.
variables (3, 20). In practice, it is usually difficult to sample
with that frequency, and water samples are generally taken 8.2 Experimental design considerations, such as station
location, number of stations per site, number of cages per
only at the beginning and end of the test. Since the variability
station, and number of bivalves per cage, should be based on
in sediment chemistry is generally less extreme than in water,
the purpose of the test and the procedure(s) used to analyze the
collecting sediment samples for chemical analysis at the
results. Various experimental designs can be applied, with the
beginning and end of test may be sufficient to characterize
most common used to:
exposure conditions (Practice D3976). However, sediments
(1) Compare bivalve tissue chemistry and growth at one or
may also be highly variable on a small spatial scale (95).
more stations to reference, background, or pre-test conditions.
6.13 In assessing effects of effluents with high organic
(2) Compare bivalve tissue chemistry and growth among
loads, it is possible that the organic enrichment from the
multiple stations to characterize patterns, trends, or gradients.
effluent will increase bivalve growth rates and make it more
8.3 Experimental control of all test variables can be difficult
difficult to assess the adverse effects of associated chemicals.
to achieve in field tests that assess or monitor resident
Differentiating between the positive effects of nutrient enrich-
populations. The use of in-situ field bioassays allows the
ment and the adverse effects of toxic chemicals is best
investigator to control the following: species; number; and size
accomplished by maximizing the number of stations in the
range of test animals, specific location(s) to be assessed, and
assessment area, deploying caged bivalves at various depths,
exposureduration.Generally,theconcentrationofchemicalsof
and maximizing the number of effects end points. The pro-
concern and natural factors, such as temperature, salinity,
cesses involved could be better characterized and understood
dissolved oxygen, pH, current speed, and food supply, are not
byusingvariousbiomarkersinadditiontothebioaccumulation
manipulated or controlled as they are in laboratory testing.
and effects end points (96).
However, temperature could be increased by heating dissolved
oxygen by aeration, current speed by pumping, and food
7. Hazards
supply by adding nutrients. The intent of field bioassays is to
determine chemical bioavailability and subsequent effects
7.1 Water and sediment might be contaminated with un-
undernatural,site-specificconditions,
...