ASTM E1022-22

Standard Guide for Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Mollusks

SIGNIFICANCE AND USE
5.1 A bioconcentration test is conducted to obtain information concerning the ability of an aquatic species to accumulate a test material directly from water. This guide provides guidance for designing bioconcentration tests on the properties of the test material so that each material is tested in a cost-effective manner.  
5.2 Because steady-state is usually approached from the low side and the definition of apparent steady-state is based on a statistical hypothesis test, the apparent steady-state BCF will usually be lower than the steady-state BCF. With the variation and sample sizes commonly used in bioconcentration tests, the actual steady-state BCF will usually be no more than twice the apparent BCF.  
5.3 When both are determined in the same test, the projected steady-state BCF will usually be higher than the apparent steady-state BCF because the models used to calculate the projected BCF assume that the BCF steadily increases until infinite time.  
5.4 The BCFs and rates and extents of uptake and depuration will depend on temperature, water quality, the species and its size, physiological condition, age, and other factors (1).4 Although organisms are fed during tests, uptake by means of sorption onto food is probably negligible during tests.  
5.5 Results of bioconcentration tests are used to predict concentrations likely to occur in aquatic organisms in field situations as a result of exposure under comparable conditions, except that mobile organisms might avoid exposure when possible. Under the experimental conditions, particulate matter is deliberately minimized compared to natural water systems. Exposure conditions for the tests may therefore not be comparable for an organic chemical that has a high octanol-water partition coefficient or for an inorganic chemical that sorbs substantially onto particulate matter. The amount of the test substance in solution is thereby reduced in both cases, and therefore the material is less available to many organisms...
SCOPE
1.1 This guide describes procedures for obtaining laboratory data concerning bioconcentration of a test material added to dilution water—but not to food—by freshwater and saltwater fishes and saltwater bivalve mollusks using the flow-through technique. These procedures also should be useful for conducting bioconcentration tests with other aquatic species, although modifications might be necessary.  
1.2 Other modifications of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, the results of tests conducted using unusual procedures are not likely to be comparable to those of many other tests. The comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting bioconcentration tests.  
1.3 These procedures are applicable to all chemicals that can be measured accurately at the necessary concentrations in water and in appropriate tissues. Bioconcentration tests are usually conducted on individual chemicals but can be conducted on mixtures if appropriate measurements can be made. Some techniques described in this guide were developed for tests on non-ionizable organic chemicals (see 11.1.2.1) and might not apply to ionizable or inorganic chemicals.  
1.4 Results of bioconcentration tests should usually be reported in terms of apparent steady-state and projected steady-state bioconcentration factors (BCFs) and uptake and depuration rate constants. Results should be reported in terms of whole body for fishes and in terms of total soft tissue for bivalve mollusks. For fishes and scallops consumed by humans, some results should also be reported in terms of the edible portion, especially if ingestion of the test material by humans is a major concern. For tests on organic and organometallic chemicals, the ...

General Information

• Status: Published
• Publication Date: 31-Mar-2022
• ICS: 07.080 - Biology. Botany. Zoology
• Technical Committee: E50 - Environmental Assessment, Risk Management and Corrective Action
• Drafting Committee: E50.47 - Biological Effects and Environmental Fate

Relations

• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM E1688-19 - Standard Guide for Determination of the Bioaccumulation of Sediment-Associated Contaminants by Benthic Invertebrates
• Effective Date: 01-Dec-2019
• Refers: ASTM E1706-19 - Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates
• Effective Date: 01-Apr-2019
• Refers: ASTM E1688-10(2016) - Standard Guide for Determination of the Bioaccumulation of Sediment-Associated Contaminants by Benthic Invertebrates
• Effective Date: 01-Feb-2016
• Refers: ASTM E1706-05(2010) - Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates (Withdrawn 2019)
• Effective Date: 01-Sep-2010
• Refers: ASTM E1688-10 - Standard Guide for Determination of the Bioaccumulation of Sediment-Associated Contaminants by Benthic Invertebrates
• Effective Date: 01-Apr-2010
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• Refers: ASTM E943-08 - Standard Terminology Relating to Biological Effects and Environmental Fate
• Effective Date: 01-Mar-2008
• Refers: ASTM E1733-95(2008) - Standard Guide for Use of Lighting in Laboratory Testing
• Effective Date: 01-Feb-2008
• Refers: ASTM E1192-97(2008) - Standard Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents with Fishes, Macroinvertebrates, and Amphibians
• Effective Date: 01-Feb-2008
• Refers: ASTM E1191-03a(2008) - Standard Guide for Conducting Life-Cycle Toxicity Tests with Saltwater Mysids
• Effective Date: 01-Feb-2008
• Refers: ASTM E1023-84(2007) - Standard Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses
• Effective Date: 01-Oct-2007
• Refers: ASTM E729-96(2007) - Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
• Effective Date: 01-Oct-2007
• Refers: ASTM D4447-06 - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 15-Nov-2006
• Refers: ASTM D4447-10 - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 15-Nov-2006

Overview

ASTM E1022-22, Standard Guide for Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Mollusks, provides comprehensive procedures for laboratory bioconcentration tests. Developed by ASTM International, this guide outlines best practices for evaluating the ability of aquatic species such as freshwater and saltwater fishes and saltwater bivalve mollusks to accumulate chemicals directly from water.

This standard is vital for environmental scientists and regulatory professionals seeking accurate data on the bioconcentration of various substances in aquatic organisms. It promotes scientifically sound, cost-effective methodologies for testing both individual chemicals and, when appropriate, chemical mixtures using the flow-through technique. The guide also applies, with minor adjustments, to other aquatic species.

Key Topics

• Bioconcentration Test Design: Guidance on structuring tests to reflect the properties of each test material, ensuring results are meaningful and cost-effective.
• Steady-State and Apparent Steady-State BCF: Clarification of steady-state versus apparent steady-state bioconcentration factors (BCF), uptake and depuration phases, and the statistical evaluation of results.
• Environmental Variables: Consideration of factors influencing bioconcentration such as temperature, water quality, organism species, size, physiological condition, and age.
• Reporting Results: Best practices for reporting BCFs and rates of uptake and depuration, including distinctions between whole-organism, tissue-specific, and edible portions (relevant to human consumption).
• Water Quality and Test Material Handling: Emphasis on water source selection, quality control, chemical measurement accuracy, and safety precautions in laboratory practice.
• Flexibility and Modifications: Recommendations for adapting procedures to meet special research needs, noting the impact of modifications on comparability of results.
• Safety and Environmental Considerations: Details on laboratory safety, waste management, and regulatory compliance.

Applications

The ASTM E1022-22 standard is employed in several practical scenarios:

• Regulatory Compliance: By providing reliable data on chemical accumulation, this standard supports regulatory decision-making concerning permissible levels of substances in aquatic environments and food sources.
• Hazard Assessment: Results from bioconcentration tests are crucial for assessing the risks of chemical exposure to wildlife and humans, feeding into water quality criteria and environmental risk assessments.
• Comparative Analysis: Enables comparison among chemicals or species regarding their propensity to bioconcentrate, supporting product stewardship and safer chemical selection.
• Environmental Fate Modeling: Uptake and depuration rates can be used to model the fate of chemicals in aquatic ecosystems, aiding in prediction and mitigation planning.
• Research and Development: Supports studies on the relationship between chemical properties and bioconcentration, helping improve predictive models and understanding of environmental behavior.

Related Standards

ASTM E1022-22 references and complements several related ASTM and international standards:

• ASTM E729: Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians.
• ASTM E1023: Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses.
• ASTM E1191/E1192/E1193/E1241/E1295/E1688/E1706/E1733/E1847/E2122/E2455: Various guides for conducting toxicity and bioaccumulation tests in aquatic environments.
• ASTM D1193: Specification for Reagent Water.
• IEEE/ASTM SI10: Standard for use of the International System of Units (SI).
• Guide D4447: Guide for Disposal of Laboratory Chemicals and Samples.

Following ASTM E1022-22 ensures adherence to internationally accepted methodologies, facilitating credible bioconcentration data for environmental management and regulatory processes. For additional guidance on related assessment and toxicity test methods, consult the above-mentioned ASTM standards.

Standards Content (Sample)

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: E1022 − 22
Standard Guide for
Conducting Bioconcentration Tests with Fishes and
Saltwater Bivalve Mollusks
This standard is issued under the fixed designation E1022; 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 humans is a major concern. For tests on organic and organo-
metallic chemicals, the percent lipids of the tissue should be
1.1 Thisguidedescribesproceduresforobtaininglaboratory
reported.
data concerning bioconcentration of a test material added to
1.5 This guide is arranged as follows:
dilution water—but not to food—by freshwater and saltwater
fishes and saltwater bivalve mollusks using the flow-through
Section
Referenced Documents 2
technique.Theseproceduresalsoshouldbeusefulforconduct-
Terminology 3
ing bioconcentration tests with other aquatic species, although
Summary of Guide 4
modifications might be necessary.
Significance and Use 5
Safety Precautions 7
1.2 Other modifications of these procedures might be justi-
Apparatus 6
fied by special needs or circumstances.Although using appro- Facilities 6.1
Construction Materials 6.2
priate procedures is more important than following prescribed
Metering System 6.3
procedures, the results of tests conducted using unusual pro-
Test Chambers 6.4
Cleaning 6.4.4
cedures are not likely to be comparable to those of many other
Acceptability 6.5
tests. The comparison of results obtained using modified and
Dilution Water 8
unmodified versions of these procedures might provide useful
Requirements 8.1
Source 8.2
information concerning new concepts and procedures for
Treatment 8.3
conducting bioconcentration tests.
Characterization 8.4
Test Material 9
1.3 These procedures are applicable to all chemicals that
General 9.1
can be measured accurately at the necessary concentrations in
Radiolabeled Material 9.2
water and in appropriate tissues. Bioconcentration tests are
Stock Solution 9.3
Test Concentration(s) 9.4
usually conducted on individual chemicals but can be con-
Test Organisms 10
ducted on mixtures if appropriate measurements can be made.
Species 10.1
Some techniques described in this guide were developed for
Size 10.2
Source 10.3
tests on non-ionizable organic chemicals (see 11.1.2.1) and
Care and Handling 10.4
might not apply to ionizable or inorganic chemicals.
Feeding 10.5
Disease Treatment 10.6
1.4 Results of bioconcentration tests should usually be
Holding 10.7
reportedintermsofapparentsteady-stateandprojectedsteady-
Acclimation 10.8
Quality 10.9
state bioconcentration factors (BCFs) and uptake and depura-
Procedure 11
tion rate constants. Results should be reported in terms of
Experimental Design 11.1
whole body for fishes and in terms of total soft tissue for
Dissolved Oxygen 11.2
Temperature 11.3
bivalve mollusks. For fishes and scallops consumed by
Loading 11.4
humans, some results should also be reported in terms of the
Beginning the Test 11.5
edible portion, especially if ingestion of the test material by
Care of Organisms 11.6
Feeding 11.7
Cleaning 11.8
Biological Data 11.9
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Measurements on Test Solutions 11.10
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
Analytical Methodology 12
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Acceptability of Test 13
Current edition approved April 1, 2022. Published June 2022. Originally
Calculation of Results 14
approved in 1984. Last previous edition approved in 2013 as E1022–94(2013)
Documentation 15
which was withdrawn January 2022 and reinstated in April 2022. DOI: 10.1520/
Keywords 16
E1022-22.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1022 − 22
1.6 The values stated in SI units are to be regarded as Conducted Under ASTM Guidelines (Withdrawn 2022)
standard. No other units of measurement are included in this E2122Guide for Conducting In-situ Field Bioassays With
standard. Caged Bivalves
E2455GuideforConductingLaboratoryToxicityTestswith
1.7 This standard does not purport to address all of the
Freshwater Mussels
safety concerns, if any, associated with its use. It is the
SI10IEEE/ASTM SI10 Standard for Use of the Interna-
responsibility of the user of this standard to establish appro-
tional System of Units (SI) (the Modernized Metric
priate safety, health, and environmental practices and deter-
System)
mine the applicability of regulatory limitations prior to us-
e.Specific precautionary statements are given in Section 7.This
3. Terminology
standard does not purport to address all of the safety concerns,
3.1 Definitions:
if any, associated with its use. It is the responsibility of the user
3.1.1 The words “must,” “should,” “may,” “can,” and
of this standard to establish appropriate safety and health
“might” have very specific meanings in this guide. “Must” is
practices and determine the applicability of regulatory limita-
usedtoexpressanabsoluterequirement,thatis,tostatethatthe
tions prior to use.
test ought to be designed to satisfy the specified condition,
1.8 This international standard was developed in accor-
unless the purpose of the test requires a different design.
dance with internationally recognized principles on standard-
“Must” is used only in connection with factors that relate
ization established in the Decision on Principles for the
directly to the acceptability of the test (see 13.1). “Should” is
Development of International Standards, Guides and Recom-
used to state that the specified condition is recommended and
mendations issued by the World Trade Organization Technical
oughttobemetifpossible.Althoughviolationofone“should”
Barriers to Trade (TBT) Committee.
is rarely a serious matter, violation of several will often render
the results questionable. Terms such as “is desirable” are used
2. Referenced Documents
in connection with less important factors.“ May” is used to
2.1 ASTM Standards:
mean“is(are)allowedto,”“can”isusedtomean“is(are)able
D1129Terminology Relating to Water
to,” and “might” is used to mean “could possibly.” Thus the
D1193Specification for Reagent Water
classic distinction between“ may” and “can” is preserved, and
D4447Guide for Disposal of Laboratory Chemicals and
“might” is never used as a synonym for either “may” or “can.”
Samples
3.1.2 For definitions of other terms used in this guide, refer
E729Guide for Conducting Acute Toxicity Tests on Test
to Terminologies D1129 and E943 and Guide E729. For an
Materials with Fishes, Macroinvertebrates, and Amphib-
explanation of units and symbols, refer to Standard SI10.
ians
3.2 Definitions of Terms Specific to This Standard:
E943Terminology Relating to Biological Effects and Envi-
3.2.1 apparent steady-state bioconcentration factor, n—a
ronmental Fate
BCF that does not change significantly over a period of two to
E1023Guide for Assessing the Hazard of a Material to
four days at a uniform concentration (as defined in 11.10.3.2)
Aquatic Organisms and Their Uses
ofthetestmaterialinthesolutioncontainingtheorganism,that
E1191Guide for Conducting Life-Cycle Toxicity Tests with
is, the BCF that exists when uptake and depuration are equal
Saltwater Mysids
andbioconcentration(netaccumulation)iszerofortwotofour
E1192Guide for ConductingAcute Toxicity Tests onAque-
days.
ous Ambient Samples and Effluents with Fishes,
3.2.2 bioaccumulation, n—the net accumulation of a sub-
Macroinvertebrates, and Amphibians
stance by an organism as a result of uptake from all environ-
E1193Guide for Conducting Daphnia magna Life-Cycle
mental sources.
Toxicity Tests
3.2.3 bioconcentration, n—the net accumulation of a sub-
E1241GuideforConductingEarlyLife-StageToxicityTests
stance by an aquatic organism as a result of uptake directly
with Fishes
from aqueous solution.
E1295Guide for Conducting Three-Brood, Renewal Toxic-
3.2.4 bioconcentration factor (BCF), n—thequotient,atany
ity Tests with Ceriodaphnia dubia
E1688Guide for Determination of the Bioaccumulation of time during the uptake phase of a bioconcentration test, of the
concentrationofamaterialinoneormoretissuesofanaquatic
Sediment-Associated Contaminants by Benthic Inverte-
brates organism at that time, divided by the effective average expo-
sure concentration at that time of the same material in the
E1706TestMethodforMeasuringtheToxicityofSediment-
Associated Contaminants with Freshwater Invertebrates solution which contains the organism, in units of volume of
solutionpermassoforganism.(BCFsareusuallycalculatedso
E1733Guide for Use of Lighting in Laboratory Testing
E1847Practice for Statistical Analysis of Toxicity Tests that the volume of solution, for example, 1 L, is about
comparable to the mass of tissue, for example, 1 kg, and the
BCF is reported without units.)
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. 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.
E1022 − 22
3.2.5 depuration, n—loss of a substance from an organism suitability of the dilution water, food, test conditions, handling
as a result of any active or passive process. procedures, etc. In the other treatment the organisms are (a)
exposed during the uptake phase to dilution water, to which a
3.2.6 depuration curve, n—the line obtained by plotting the
selected concentration of test material has been intentionally
measured concentration of a test material in aquatic organisms
added, at least until either apparent steady-state or 28 days is
versus time during the depuration phase of a bioconcentration
reached and (b) exposed during the depuration phase to
test.
dilutionwatertowhichnotestmaterialhasbeenadded.During
3.2.7 depuration phase, n—the portion of a bioconcentra-
both phases of the test, representative organisms and water
tiontestaftertheuptakephaseandduringwhichtheorganisms
samples are removed periodically from each test chamber and
are in dilution water to which no test material has been added.
analyzed for test material.Apparent steady-state and projected
3.2.8 depuration rate constant, n—the mathematically de-
steady-stateBCFsanduptakeanddepurationrateconstantsare
rived value(s) that expresses how rapidly test material is
usually calculated from the measured concentrations of test
eliminated from previously exposed aquatic organisms when
material in tissue and water samples. If it is desired to
placed in dilution water to which no test material has been
determine whether BCFs and rate constants are dependent on
added, usually expressed in units of reciprocal time.
the concentration of test material in water, additional
treatments, utilizing different concentrations of test material
3.2.9 effective average exposure concentration, n—the av-
during the uptake phase, must be used.
erage concentration, at any time during the uptake phase of a
bioconcentrationtest,oftestmaterialinthetestsolutionduring
5. Significance and Use
the preceding period of time equal to the shorter of (a) the
length of the uptake phase to that point and (b) one half the
5.1 Abioconcentration test is conducted to obtain informa-
time to apparent steady-state. Effective exposure concentra- tion concerning the ability of an aquatic species to accumulate
tions cannot be calculated until after the time to apparent
a test material directly from water. This guide provides
steady-state has been determined, unless the concentration of guidancefordesigningbioconcentrationtestsontheproperties
test material is constant.
of the test material so that each material is tested in a
cost-effective manner.
3.2.10 projected steady-state bioconcentration factor, n—a
BCFcalculatedforinfinitetime(a)fromuptakeanddepuration
5.2 Becausesteady-stateisusuallyapproachedfromthelow
rate constants derived using an appropriate compartmental
side and the definition of apparent steady-state is based on a
model or (b) by fitting an appropriate equation to data
statistical hypothesis test, the apparent steady-state BCF will
concerning BCF versus time.
usually be lower than the steady-state BCF. With the variation
and sample sizes commonly used in bioconcentration tests, the
3.2.11 uptake, n—acquisition of a substance from the envi-
actual steady-state BCF will usually be no more than twice the
ronment by an organism as a result of any active or passive
apparent BCF.
process.
5.3 Whenbotharedeterminedinthesametest,theprojected
3.2.12 uptake curve, n—the line obtained by plotting the
steady-state BCF will usually be higher than the apparent
measured concentration of test material in aquatic organisms
steady-state BCF because the models used to calculate the
versus time during the uptake phase of a bioconcentration test.
projected BCF assume that the BCF steadily increases until
3.2.13 uptake phase, n—the portion of a bioconcentration
infinite time.
test during which organisms are exposed to test material
intentionally added to dilution water. (Although uptake and 5.4 The BCFs and rates and extents of uptake and depura-
depuration both occur during the uptake phase, uptake always tion will depend on temperature, water quality, the species and
predominates at the beginning, but depuration often becomes its size, physiological condition, age, and other factors (1).
nearly equal to uptake at the end of the uptake phase. Although organisms are fed during tests, uptake by means of
Occasionallydepurationexceedsuptakeduringaportionofthe sorption onto food is probably negligible during tests.
uptake phase.)
5.5 Results of bioconcentration tests are used to predict
3.2.14 uptake rate constant, n—the mathematically derived
concentrations likely to occur in aquatic organisms in field
value(s) that express how rapidly test material is accumulated situationsasaresultofexposureundercomparableconditions,
by aquatic organisms during the uptake phase of a bioconcen-
except that mobile organisms might avoid exposure when
trationtest,inunitsofvolumeofsolutionpermassoforganism possible.Undertheexperimentalconditions,particulatematter
per time.
is deliberately minimized compared to natural water systems.
Exposure conditions for the tests may therefore not be compa-
4. Summary of Guide rable for an organic chemical that has a high octanol-water
partition coefficient or for an inorganic chemical that sorbs
4.1 Each of two groups of test organisms of one species is
substantially onto particulate matter. The amount of the test
administered a treatment, consisting of an uptake phase and a
substance in solution is thereby reduced in both cases, and
depuration phase, using the flow-through technique. The con-
therefore the material is less available to many organisms.
trol treatment, in which organisms are exposed during both
phases to dilution water to which no test material has been
added, provides a measure of the acceptability of the test by
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
givinganindicationofthequalityofthetestorganismsandthe this standard.
E1022 − 22
However, sorption might increase bioaccumulation by aquatic control and aeration (see 8.3).Air used for aeration should be
species that ingest particulate matter (2), or food may be a freeoffumes,oil,andwater;filterstoremoveoilandwaterare
more important source of residues in fish than water per se for desirable. Filtration of air through a 0.22-µm bacterial filter
stable neutral organic chemicals that have a Log K between might be desirable. During holding, acclimation, and testing,
ow
4 and 6 (3). organisms should be shielded from disturbances with curtains
or partitions to prevent unnecessary stress. The test facility
5.6 Resultsofbioconcentrationtestscanbeusedtocompare
should be well ventilated and free of fumes. To further reduce
the propensity of different materials to be accumulated. Non-
the possibility of the contamination of test organisms by test
ionizable organic chemicals can also be ranked for bioconcen-
materials and other substances, especially volatile ones,
tration using correlations that have been reported between
culture,holding,andacclimationtanksshouldnotbeinaroom
steady-state BCFs and physical–chemical properties, such as
inwhichbioconcentrationortoxicitytestsareconducted,stock
the octanol–water partition coefficient and solubility in water
solutions or test solutions are prepared, or equipment is
(4). However, when such predictions are impossible, exceed
cleaned.
the demonstrated limits of the correlation, or might be other-
6.1.1 A 16-h light and 8-h dark photoperiod should be
wise questionable (1, 5), a bioconcentration test may be
provided to reflect natural cycles as is done for other inverte-
necessary.
brate toxicity tests (see Guide E1192). This can be controlled
5.7 Results of bioconcentration tests can also be used to
by a timing device. Alternatively, a 12–12 or 14–10 photope-
compare the abilities of different species to accumulate mate-
riod might be desirable to delay maturation of some species.A
rials. At steady-state the concentration of a nonionizable
15 to 30-min transition period (8) when the lights go on might
organic chemical in individual organisms, and in various
be desirable to reduce the possibility of organisms being
tissues within an organism, will probably be related to the
stressed by instantaneous illumination; a transition period
concentration of lipids in the organisms and tissues (6).
when the lights go off may also be desirable (see Guide
E1733).
5.8 Results of bioconcentration tests might be an important
consideration when assessing hazard (see Guide E1023)or
6.2 Construction Materials—Equipment and facilities that
deriving water-quality criteria because consumer animals
might contact stock solutions, test solutions, or any water into
mightbeadverselyaffectedbyingestingaquaticorganismsthat
which test organisms will be placed should not contain
contain toxic materials. However, assessment of hazard to
substances that can be leached or dissolved by aqueous
consumer organisms must take into account not only the
solutions in amounts that can adversely affect aquatic organ-
quantity of material accumulated in tissues of aquatic
isms. In addition, equipment and facilities that contact stock
organisms,butalsothetoxicityofthematerialtotheconsumer.
solutions or test solutions should be chosen to minimize the
Further, humans eat only certain portions of most aquatic
sorptionoftestmaterialsfromwater.Glass,Type316stainless
organisms, whereas other predators often consume additional
steel, nylon, and fluorocarbon plastics should be used when-
tissues.
ever possible to minimize leaching, dilution, and sorption,
exceptthatstainlesssteelshouldnotbeusedfortestsonmetals
5.9 Bioconcentration tests might be useful for studying
insaltwater.Concreteandrigid(unplasticized)plasticsmaybe
structure–activityrelationshipsbetweentestmaterials,biologi-
used for culture, holding, and acclimation tanks and in the
cal availability, metabolism of materials in aquatic organisms,
water-supply system, but they should be soaked, preferably in
and effects of various environmental factors on results of such
flowing dilution water, for several days before use (9). Cast-
tests.
iron pipe should not be used with salt water and probably
5.10 Uptake and depuration rate constants might be useful
should not be used in freshwater supply systems, because
for predicting environmental fate using compartmental models
colloidal iron will be added to the dilution water, and strainers
(7).
will be needed to remove rust particles. Specially designed
5.11 Tissues collected after organism exposures based on systems are usually necessary to obtain salt water from a
othertestingmethodsmightalsobeconsideredfordetermining
natural water source (see Guide E729). Copper, brass, lead,
bioconcentration according to this guide (see Guides E1241, galvanized metal, and natural rubber should not contact dilu-
E1688, E2122, E2455, and Test Method E1706). tion water, stock solutions, or test solutions before or during
thetest.Itemsmadeofneoprenerubberandothermaterialsnot
mentioned above should not be used unless a sensitive aquatic
6. Apparatus
species(see8.2.3)cansurvivefor48or96h(seeGuideE729)
6.1 Facilities—Flow-through tanks should be available for
in static water in which the items are soaking.
culturing,holding,andacclimatingtestorganisms.Anelevated
6.3 Metering System:
dilution-water tank or headbox, or both, might be desirable so
dilution water can be gravity-fed into holding and acclimation 6.3.1 The metering system must be designed to accommo-
tanksandthemeteringsystem(see6.3),whichpreparesthetest date the type and concentration(s) of test material and the
solutions and delivers them to the test chambers. Strainers and necessary flow rates of test solutions. The system must
air traps should be included in the water supply system. The reproducibly (see 11.10.3.2) supply the selected concentra-
test chambers should be in a constant temperature area or tion(s)oftestmaterial(see9.4).Avarietyofmeteringsystems,
recirculating water bath. Headboxes and holding, acclimation, using various combinations of syringes, “dipping birds,”
and dilution-water tanks should be equipped for temperature siphons, pumps, solenoids, valves, etc. (see Guide E729), has
E1022 − 22
beenusedsuccessfully.Becauseabioconcentrationtestusually adhesiveareneededforstrength,theyshouldbeontheoutside
consists of a control treatment and one concentration of test of chambers rather than on the inside.
material,themeteringsystemusuallyconsistsofonedevicefor
6.4.3 The minimum dimensions of test chambers and the
metering a solution of the test material, two devices for
minimum depth of test solution depend on the size of the
metering dilution water, and two small chambers for mixing
individual test organisms (see 10.2) and the loading (see 11.4).
(and splitting, if replicate test chambers are used) the indi-
The smallest horizontal dimension of test chambers should be
vidual test solutions before they enter test chambers.
atleast1.5timesthelargesthorizontaldimensionofthelargest
6.3.2 The metering system should be calibrated before each
test organism. For fish the depth of test solution should be at
testbydeterminingtheflowratethrougheachtestchamberand
least3timestheheightofthelargesttestorganism;inaddition,
measuring either the concentration of test material in each test
the test solution should be at least 150 mm deep for fish over
chamber or the volume of solution used in each portion of the
0.5 g (wet weight) each, and at least 50 mm deep for smaller
meteringsystem.Thegeneraloperationofthemeteringsystem
fish. Chambers filled to within 150 mm of the top sometimes
should be visually checked each morning and afternoon
need to be covered to prevent fish from jumping out. With
throughout the test. The metering system should be adjusted
bivalvemollusks,thetestsolutionshouldcompletelysubmerge
during the test if necessary.
the organisms throughout the test. Tests with bivalve mollusks
6.3.3 The flow rate through each test chamber should be at
for which the distance from the tip of the umbo to the distal
least five volume additions per 24 h, but might need to be
valve edge is less than 60 mm and tests with small fish, for
greater depending on the loading (see 11.4). In tests with
example,lessthan10g,areoftenconductedin300by600mm
bivalve mollusks, the minimum necessary flow rate might also
by 300mm deep all-glass test chambers containing 30 L of
depend on the amount of food available in the dilution water
solution. Use of excessively large volumes of solution in test
(see 11.4.3). It is usually desirable to use a flow rate of at least
chambers will unnecessarily increase the amount of dilution
ten volume additions per 24 h, especially at the beginning of
water and test material used or the average retention time, or
the test when uptake is greatest, but a higher flow rate will
both.
increase the amount of dilution water and test material used.A
6.4.4 Cleaning—The metering system, test chambers, and
higher flow rate is also desirable if there is rapid loss of test
equipment used to prepare and store dilution water, stock
material due to microbial degradation, hydrolysis, oxidation,
solutions,andtestsolutionsshouldbecleanedbeforeuse.New
photolysis, reduction, sorption, or volatilization. At any par-
items should be washed with detergent and rinsed with water,
ticular time during a test, the flow rates through any two test
a water-miscible organic solvent, water, acid (such as 10%
chambers should not differ by more than 10%. If comparable
concentrated hydrochloric acid), and at least twice with
numbers of test organisms are removed from all chambers, the
distilled, deionized, or dilution waterr, orASTMType II water
depth of solution or the flow rate, or both, in all test chambers
may be equally reduced, as long as the flow rate remains at (Specification D1193). (Some lots of some organic solvents
least five volume additions per 24 h and the loading (see 11.4) may leave a film that is insoluble in water.) A dichromate-
and temperature (see 11.3) remain acceptable.
sulfuricacidcleaningsolutionmaybeusedinplaceofboththe
organic solvent and the acid, but it might attack silicone
6.4 Test Chambers:
adhesiveandrequiresspecialdisposaltechniques.Attheendof
6.4.1 In a toxicity test with aquatic organisms, a test
every test, all items that are to be used again should be
chamberisdefinedasthesmallestphysicalunitbetweenwhich
immediately (a) emptied, (b) rinsed with water, (c) cleaned by
there are no water connections. However, screens and cups
a procedure appropriate for removing the test material (for
may be used to create two or more compartments within each
example, acid to remove metals and bases; detergent, organic
chamber. Test solution can therefore flow from one compart-
solvent, or activated carbon to remove organic chemicals), and
menttoanotherwithinatestchamberbut,bydefinition,cannot
(d) rinsed at least twice with distilled, deionized, dilution
flow from one chamber to another. Because solution can flow
water, or ASTM Type II water. Acid is useful for removing
fromonecompartmenttoanotherinthesametestchamber,the
mineral deposits, and 200 mg of hypochlorite/Lis often useful
temperature, concentration of test materials, and levels of
for removing organic matter and for disinfection. A solution
pathogens and extraneous contaminants are likely to be more
−
containing about 200 mg of OCl /L is conveniently prepared
similar between compartments in the same test chamber than
by adding 6 mL of liquid household chlorine bleach to 1 L of
between compartments in different test chambers in the same
water. Hypochlorite is quite toxic to most aquatic animals (10)
treatment.Chambersshouldbecoveredtokeepoutextraneous
and is difficult to rinse from some construction materials. It is
contaminantsandtoreducetheevaporationoftestsolutionand
often removed by reaction with sodium thiosulfate, sodium
test material. All of the chambers and compartments in a test
sulfite, or sodium bisulfite or by autoclaving in distilled water
must be identical.
for20minorbydryingtheitemandlettingitsitforatleast24
6.4.2 Testchambersareusuallyconstructedbywelding(not
h before use.An item cleaned or disinfected with hypochlorite
soldering) stainless steel or by gluing double-strength or
should not be used unless it has been demonstrated at least
stronger window glass with clear silicone adhesive. Stoppers
once that unfed test organisms held for at least 48 h in static
and silicone adhesive sorbs some organochlorine and organo-
phosphorus pesticides that are then difficult to remove. dilutionwaterinwhichthecleaneditemissoakingdonotshow
moresignsofstress,suchasdiscoloration,unusualbehavior,or
Therefore, as few stoppers and as little adhesive as possible
should be in contact with the test solution. If extra beads of death,thandounfedtestorganismsheldinstaticdilutionwater
E1022 − 22
containing a similar item that was not treated with hypochlo- potentially hazardous materials, including those used for ana-
rite. Alternatively, peracetic acid is a less noxious anti- lytical measurements (for example, acid used during alkalinity
microbial than hypochlorite that avoids these issues. The titrations),usersneedtowearappropriateprotectiveequipment
metering system and test chambers should be rinsed with (for example, safety glasses and gloves). Common laboratory
dilution water just before use. protective wear should also be used to reduce exposure to
potential biological hazards (for example Salmonella, Vibrio
6.5 Acceptability—New holding, acclimation, and testing
ssp.). All laboratory-specific health and safety considerations
facilities should be tested for toxicity before use (see 6.2 and
should be followed.
8.1.1).
7. Safety Precautions 8. Dilution Water
7.1 Many materials can affect humans adversely if precau-
8.1 Requirements—The dilution water should (a)bein
tions are inadequate. Therefore, skin contact with all test
adequatesupply;(b)beacceptabletothetestorganisms;(c)be
materials and solutions of them should be minimized by
of uniform quality; and (d) except as stated in 8.1.4, not affect
wearing appropriate protective gloves (especially when wash-
the results of the test unnecessarily.
ing equipment or putting hands in test solutions), laboratory
8.1.1 The dilution water should not adversely affect the test
coats, aprons, and glasses, and by using dip nets or forceps to
organisms.Forbioconcentrationtests,theminimalcriterionfor
remove organisms from test solutions.
acceptability of dilution water to test organisms is that healthy
7.1.1 Special precautions, such as covering test chambers,
test organisms will survive in it during acclimation and testing
ventilatingtheareasurroundingthechambers,anduseoffume
without showing signs of stress, such as discoloration or
hoods, should be taken when conducting tests on volatile
unusual behavior. In addition, the water should not affect the
materials. Information concerning toxicity to humans (11),
ability of organisms to sorb and depurate test material.
recommended handling procedures (12), and chemical and
Therefore, a better criterion for acceptability of dilution water
physicalpropertiesofthetestmaterialshouldbestudiedbefore
to test organisms is that at least one aquatic animal species can
a test is begun. Special procedures may be necessary with
survive,grow,andreproducesatisfactorilyinit.Unlessaccept-
radiolabeled test materials (13) and with materials that are, or
ability of the dilution water has been demonstrated during the
are suspected of being, carcinogenic (14).
previous year, it should be demonstrated during the test by
7.2 Although disposal of stock solutions, test solutions, and showing that either (a) at least one species will survive, grow,
andreproduceacceptablyinalaboratorycultureoralife-cycle
test organisms poses no special problems in most cases, health
and safety precautions and applicable regulations should be toxicity test (see Guides E1191, E1193, and E1295)or(b)at
least one species of fish will perform acceptably in a partial
considered before beginning a test. Removal or degradation of
test material might be desirable before disposal of stock and life-cycle or early life-stage toxicity test (see Guide E1241).
test solutions.
8.1.2 The dilution water should not unnecessarily affect the
results of a bioconcentration test because of such things as
7.3 Cleaning equipment with a volatile solvent such as
sorption or complexation of test material. Therefore, except as
acetone should be performed only in a well-ventilated area in
stated in 8.1.4, concentrations of both total organic carbon
whichnosmokingisallowedandnoopenflame,suchasapilot
(TOC) and particulate matter should be less than 5 mg/L for
light, is present.
tests with fish and less than 20 mg/L for tests with saltwater
7.4 Anacidicsolutionshouldnotbemixedwithahypochlo-
bivalve mollusks (see 10.5.3).
rite solution because hazardous fumes might be produced.
8.1.3 The quality of the dilution water should be uniform
7.5 To protect hands from being cut by sharp edges of
during the test. During a test in freshwater the range of
shells, cotton work gloves should be worn (over appropriate
hardness should be less than 5 mg/L or 10% of the average,
protectivegloves(see7.1)ifnecessary)whenbivalvemollusks
whichever is higher. During a test in salt water the range of
are handled.
salinity should be less than 2 g/kg or 20% of the average,
whichever is higher.
7.6 Because dilution water and test solutions are usually
good conductors of electricity, use of ground fault systems and
8.1.4 If it is desired to study the effect of an environmental
leak detectors should be considered to help avoid electrical factor such asTOC, particulate matter, or dissolved oxygen on
shocks. Salt water is such a good conductor that protective
theresultsofabioconcentrationtest,itwillbenecessarytouse
devices are strongly recommended. a water that is naturally or artificially high in TOC or
particulate matter or low in dissolved oxygen. If such a water
7.7 To prepare dilute acid solutions, concentrated acid
is used, it is important that adequate analyses be performed to
should be added to water, not vice versa. Opening a bottle of
characterize the water and that a comparable test be available
concentratedacidandaddingconcentratedacidtowatershould
or conducted in a more usual dilution water to facilitate
be performed only in a fume hood.
interpretation of the results in the special water.
7.8 Some test materials, as well as some materials used to
8.2 Source:
preserve test organisms, may be inherently hazardous. Caution
needstobeusedwhenhandlingthesematerials.Guidelinesfor 8.2.1 Although reconstituted water (see Guide E729) may
the handling and disposal of hazardous materials should be be used, its use generally is not practical for bioconcentration
strictly followed (Guide D4447). When working with any tests because large volumes are necessary. In addition, it may
E1022 − 22
bedifficulttoprovidesaltwaterbivalvemolluskswithadequate 8.3.4 Except possibly for tests with bivalve mollusks (see
amounts of acceptable food (see 10.5.3) when reconstituted 8.3.2), it might be desirable to pass salt water from a surface
water is used. water source through a filter effective to 15 µm or less to
remove parasites.
8.2.2 If a natural water is used, it should be obtained from
8.3.5 Except possibly for tests with bivalve mollusks (see
an uncontaminated, uniform quality source. For fresh water, a
8.3.2), dilution water that might be contaminated with unde-
well or spring is usually preferable to a surface water. If a
sirable microorganisms may be passed through a properly
surface water is used for fresh or salt waters, the intake should
be positioned to minimize fluctuations in quality and the maintainedultravioletsterilizer (22)equippedwithanintensity
meter and flow controls or passed through a filter with a pore
possibility of contamination.
size of 0.22 µm or less.
8.2.2.1 For bioconcentration tests with saltwater bivalve
8.3.6 Hardness, salinity, and pH may be adjusted by the
mollusks, it might also be desirable to position the intake to
additionofappropriatereagentgradechemicals, seasalt,acid,
maximize the amount of plankton that will support growth and
base, and deionized or distilled water if it has been shown that
survival (see 8.3.2).
the addition will not adversely affect the test organisms.
8.2.3 Chlorinated water should not be used as, or in the
preparation of, dilution water because total residual chlorine
8.4 Characterization—The following items should be mea-
and chlorine-produced oxidants are quite toxic to aquatic
sured at least twice each year and more often if such measure-
animals (10).Dechlorinatedwatershouldbeusedonlyasalast
ments have not been made semiannually for at least two years
resort because dechlorination is often incomplete. Sodium
or if a surface water is used:
bisulfite should be better for dechlorinating water than sodium
8.4.1 All Waters—Alkalinity, pH, conductivity, particulate
sulfiteandbotharemorereliablethancarbonfilters,especially
matter, TOC, total organophosphorus pesticides, organic chlo-
for removing chloramines (15). Some organic chloramines,
rine(ortotalorganochlorinepesticidesplusPCBs),chlorinated
however,reactslowlywithsodiumbisulfite (16).Inadditionto
phenoxy herbicides, ammonia, cyanide, sulfide, bromide,
residual chlorine, municipal drinking water often contains
fluoride, iodide, nitrate, phosphate, sulfate, calcium,
unacceptably high concentrations of copper, lead, zinc, and
magnesium, potassium, aluminum, arsenic, beryllium, boron,
fluoride, and quality is often rather variable. When necessary,
cadmium, chromium, cobalt, copper, iron, lead, manganese,
excessive concentrations of most metals can usually be re-
mercury, molybdenum, nickel, selenium, silver, and zinc.
moved with a chelating resin (17). If dechlorinated water is
8.4.2 Freshwater—Hardness, chloride, and sodium.
used as dilution water or in its preparation, either (1) the
8.4.3 Salt Water—Salinity.
acceptability of the dilution water must be demonstrated (see
8.4.4 For each method used (see 12.3), the detection limit
8.1.1) during the test or (2) it must be shown three times each
should be below either (a) the concentration in the dilution
week on nonconsecutive days that in fresh samples of dilution
water or (b) the lowest concentration that has been shown to
watereither(a) Acartia tonsa,mysids(notmorethan30-hpost
adversely affect the test organisms (23).
releaseofaspeciesthatcanlivefor48hwithoutfood),bivalve
9. Test Material
mollusk larvae, or first-instar daphnids can survive for 48 h
withoutfood,or(b)theconcentrationoftotalresidualchlorine
9.1 General—The test material should be reagent-grade or
infreshwaterorchlorine-producedoxidantsinsaltwaterisless
better, unless a test on a formulation, commercial product, or
than 8 µg/L (10).
technical-grade or use-grade material is specifically needed.
Before a test is begun, the following should be known about
8.3 Treatment:
the test material:
8.3.1 Dilution water should be aerated intensively by using
9.1.1 Identities and concentrations of major ingredients and
air stones, surface aerators, or column aerators (18, 19) before
major impurities, for example, impurities constituting more
addition of test material. Adequate aeration will bring the pH
than approximately 1% of the material.
and concentrations of dissolved oxygen and other gases into
9.1.2 Solubility and stability in dilutions with water.
equilibrium with air and minimize oxygen demand and con-
9.1.3 Expected steady-state BCF. This might be obtained
centrationsofvolatiles.Theconcentrationofdissolvedoxygen
from the results of tests on the same or a similar material with
in dilution water should be between 90 and 100% saturation
the same or a different species. For organic chemicals, this
(20) to help ensure that dissolved oxygen concentrations in the
might be obtained from correlations that have been reported
test chambers are acceptable. Supersaturation by dissolved
between steady-state BCFs and such physical–chemical prop-
gases, which can occur when dilution water is heated, should
ertiesastheoctanol-waterpartitioncoefficientandsolubilityin
be avoided to prevent gas bubble disease (18, 21).
water (4).
8.3.2 For tests with bivalve mollusks, unfiltered, unsteril-
9.1.4 Estimated time to apparent steady-state (see 11.1.2.1).
ized natural salt water is often used to provide as much natural
9.1.5 Acutetoxicitytothetestorganisms(ameasurementor
planktonic food as possible (see 10.5.3).
estimate of chronic toxicity is desirable).
8.3.3 Except possibly for tests with bivalve mollusks (see
8.3.2), filtration through sand, sock, bag, or depth-type car-
Reagent Chemicals, American Chemical Society Specifications , American
tridge filters may be used to keep the concentration of
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
particulate matter acceptably low (see 8.1.2) and as a pretreat-
listedbytheAmericanChemicalSociety,see Reagent Chemicals and Standards,by
ment before ultraviolet sterilization or filtration through a finer
Joseph Rosen, D. Van Nostrand Co., Inc., New York, NY, and the United States
filter, or both. Pharmacopeia.
E1022 − 22
9.1.6 Precision and bias of the analytical methods at might stimulate undesirable growths of microorganisms, and
planned water concentration(s), the expected steady-state tis- acetone is also quite volatile. If an organic solvent is used, it
sue concentration, and one-tenth the expected steady-state shouldbereagent-grade orbetter,anditsconcentrationinany
tissue concentration. test solution should not exceed 0.1 mL/L. Surfactants should
not be used in the preparation of a stock solution because they
9.2 Radiolabeled Test Materials—Radiolabeled test materi-
might affect the form of the test material in test solutions.
als have been used occasionally in an effort to simplify the
(These limitations do not apply to any ingredient of a mixture,
analyses of test solutions and test organisms. Their usefulness
formulation, or commercial product unless an extra amount of
is limited greatly by two serious complications: (a) Many
solvent is used in preparation of the stock solution.)
radiolabeled materials contain more than 1% radiolabeled
9.3.3 If a solvent other than water is used, it might be
impurities;asmallamountofanimpuritywithahighBCFcan
desirable to conduct simultaneous tests using two chemically
greatly affect the apparent BCF of a chemical with a much
unrelated solvents or two different concentrations of the same
lowerBCF;and(b)iftheradiolabeledchemicalismetabolized
solventtoobtaininformationconcerningpossibleeffectsofthe
substantially in the test organisms and one or more of the
solvent on results of the test.
metabolitesareradiolabeled,theapparentBCFofthechemical
willbetoohigh.Theonlywaytoovercomethesetwoproblems 9.4 Test Concentration(s)—Several factors might influence
is to verify that the radioactivity in the tissue and water is selection of the test concentration(s):
associated with the parent chemical. Such techniques as thin 9.4.1 The concentration of test material in a test solution
layer chromatography are more useful for demonstrating that must not stress, irritate, or otherwise adversely affect organ-
theradioactivityisnotassociatedwiththeparentchemicalthan isms during the test. The highest acceptable test concentration
verifying that it is associated with the parent chemical. Verifi- can often be estimated by dividing, for fish, the 96-h LC50, or,
cation usually requires gas chromatography, which means that for bivalve mollusks, the 48-h EC50 based on survival and
the use of radiolabeled test material does not save resources in development of embryos and larvae, by an appropriate acute-
the determination of the BCF in the long run. Because of these chronicratio.Appropriateratiosforsomematerialsareabout3,
complications, radiolabeled test materials are more useful for but a few are above 100.
demonstrating the presence of metabolites than for measuring 9.4.2 Thetestconcentrationmustbehighenoughsothatthe
BCFs. test material can be accurately measured in the test solution
during the uptake phase and in the test organisms after 90%
9.3 Stock Solution—The test material can be added directly
depuration.Therefore,thetestconcentrationshouldbeequalto
to dilution water in the metering system in some cases, but
or higher than the highest of (a) three times the background in
usually it is dissolved in a solvent to form a stock solution that
dilution water; (b) three times the detection limit of the
is then added to dilution water in the metering system. If a
analytical method in dilution water; (c) thirty times the
stocksolutionisused,theconcentrationandstabilityofthetest
background in the test organisms divided by the expected
material in the solvent should be de
...