ASTM E724 - 21

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Designation: E724 −21
Standard Guide for
Conducting Static Short-Term Chronic Toxicity Tests
Starting with Embryos of Four Species of Saltwater Bivalve
Molluscs
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This standard is issued under the fixed designation E724; 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
1.1 This guide describes procedures for obtaining laboratory
data concerning the acute effects of a test material on embryos
and the resulting larvae of four species of saltwater bivalve
molluscs (Pacific oyster, Crassostrea gigas Thunberg; eastern
oyster, Crassostrea virginica Gmelin; quahog or hard clam,
Mercenaria mercenaria Linnaeus; and the mussel species
complex (Mytilus spp.) including the blue mussel, Mytilus
edulis Linnaeus; the Mediterranean mussel, Mytilus gallopro-
vincialis Lamark; and the Northern Bay Mussel, Mytilus
trossulus Gould) during static 48-h exposures. These proce-
dures will probably be useful for conducting static short-term
chronic toxicity tests starting with embryos of other bivalve
species (1)
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although modifications might be necessary.
1.2 Other modifications of these procedures might be justi-
fied by special needs or circumstances. Although using proce-
dures appropriate to a particular species or special needs and
circumstances is more important than following prescribed
procedures, results of tests conducted by using unusual proce-
dures are not likely to be comparable to results of many other
tests. Comparison of results obtained by using modified and
unmodified versions of these procedures might provide useful
information concerning new concepts and procedures for
conducting 48-h acute tests starting with embryos of bivalve
molluscs.
1.3 These procedures are applicable to most chemicals,
either individually or in formulations, commercial products, or
known mixtures. With appropriate modifications these proce-
dures can be used to conduct acute tests on temperature,
dissolved oxygen, and pH and on such materials as aqueous
effluents (see also Guide E1192), leachates, oils, particulate
matter, sediments, and surface waters. Renewal tests might be
preferable to static tests for materials that have a high oxygen
demand, are highly volatile, are rapidly biologically or chemi-
cally transformed in aqueous solution, or are removed from test
solutions in substantial quantities by the test chambers or
organisms during the test.
1.4 Results of toxicity tests with embryos of bivalve mol-
luscs should usually be reported as the EC50 based on the total
incompletely developed and dead organisms. It might also be
desirable to report the LC50 based only on death. In some
situations, it might only be necessary to determine whether a
specific concentration is toxic to embryos or whether the EC50
is above or below a specific concentration.
1.5 This guide is arranged as follows:
Section
Referenced Documents 2
Terminology 3
Summary of Guide 4
Significance and Use 5
Hazards 6
Apparatus 7
Facilities 7.1
Construction Materials 7.2
Test Chambers 7.3
Cleaning 7.4
Acceptability 7.5
Dilution Water 8
Requirements 8.1
Source 8.2
Treatments 8.3
Characterization 8.4
Test Material 9
General 9.1
Stock Solution 9.2
Test Concentration(s) 9.3
Test Organisms 10
Species 10.1
Age 10.2
Handling 10.3
Brood Stock Source and Condition 10.4
Spawning and Fertilization 10.5
Quality 10.6
Procedure 11
Experimental Design 11.1
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This guide is under the jurisdiction of ASTM Committee E50 on Environmental
Assessment, Risk Management and Corrective Action and is the direct responsibil-
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Jan. 15, 2021. Published March 2021. Originally
approved in 1980. Last previous edition approved in 2012 as E74 – 98 (2012). DOI:
10.1520/E0724-21.
2
The boldface numbers in parentheses refer to the list of references at the end of
this guide.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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.
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Dissolved Oxygen 11.2
Temperature 11.3
Beginning the Test 11.4
Feeding 11.5
Duration of Test 11.6
Biological Data 11.7
Other Measurements 11.8
Analytical Methods 12
Acceptability of Test 13
Calculation of Results 14
Report 15
Annex Annex A1
1.6 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. Specific hazard
statements are given in Section 6.
1.7 This international standard was developed in accor-
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
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E380 Practice for Use of the International System of Units
(SI) (the Modernized Metric System)
4
D4447 Guide for Disposal of Laboratory Chemicals and
Samples
E729 Guide for Conducting Acute Toxicity Tests on Test
Materials with Fishes, Macroinvertebrates, and Amphib-
ians
E943 Terminology Relating to Biological Effects and Envi-
ronmental Fate
E1023 Guide for Assessing the Hazard of a Material to
Aquatic Organisms and Their Uses
E1192 Guide for Conducting Acute Toxicity Tests on Aque-
ous Ambient Samples and Effluents with Fishes,
Macroinvertebrates, and Amphibians
E1367 Test Method for Measuring the Toxicity of Sediment-
Associated Contaminants with Estuarine and Marine In-
vertebrates
E1391 Guide for Collection, Storage, Characterization, and
Manipulation of Sediments for Toxicological Testing and
for Selection of Samplers Used to Collect Benthic Inver-
tebrates
E1706 Test Method for Measuring the Toxicity of Sediment-
Associated Contaminants with Freshwater Invertebrates
E1847 Practice for Statistical Analysis of Toxicity Tests
Conducted Under ASTM Guidelines
3. Terminology
3.1 Definitions:
3.1.1 The words “must,” “should,” “may,” “can,” and
“might” have very specific meanings in this guide. “Must” is
used to express an absolute requirement, that is, to state that the
test ought to be designed to satisfy the specified condition,
unless the purpose of the test requires a different design.
“Must” is only used in connection with factors that directly
relate to the acceptability of the test (see 13.1). “Should” is
used to state that the specified condition is recommended and
ought to be met if possible. Although violation of one “should”
statement is rarely a serious matter, violation of several will
often render the results questionable. Terms such as “is
desirable,” “is often desirable,” and “might be desirable” are
used in connection with less important factors. “May” is used
to mean “is (are) allowed to,” “can” is used to mean “is (are)
able to,” and “might” is used to mean “could possibly.” Thus
the classic distinction between “may” and “can” is preserved,
and “might” is never used as a synonym for either “may” or
“can.”
3.1.2 For definitions of other terms used in this guide, refer
to Guide E729, Terminology E943, and Guide E1023. For an
explanation of units and symbols, refer to Practice E380.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 embryo—used herein to denote the stages between the
fertilization of the egg and the trochophore (2)(Figs. 1 and 2).
3.2.2 larva—used herein to include the trochophore and the
straight hinge stage (2)(Figs. 1 and 2).
4. Summary of Guide
4.1 Adult saltwater bivalve molluscs are brought into the
laboratory, cleaned of detritus and fouling organisms, and
identified to species. If the gonads are not ripe (see 10.4.5), the
bivalves are conditioned to bring them into a suitable repro-
ductive state. Bivalves with ripe gonads are maintained under
conditions that keep the gonads ripe without inducing unde-
sired spontaneous spawning or resorption of gametes. Mytilus
spp. can be held for several months in a gravid state by keeping
them in clean cold seawater without feeding. In order to start a
test, spawning is induced by using one or more stimuli which
may be physical (for example, temperature), biological (for
example, heat-killed bivalve sperm), or chemical (for example,
serotonin).
4.2 In each of two or more treatments, embryos and the
resulting larvae of one species are maintained for 48 h. In each
of one or more control treatments, the embryos and resulting
larvae are maintained in dilution water to which no test
material has been added in order to provide (a) a measure of
the acceptability of the test by giving an indication of the
quality of the organisms and the suitability of the dilution
water, test conditions, handling procedures, and so forth, and
(b) the basis for interpreting data obtained from the other
treatments. In each of one or more other treatments, the
embryos and resulting larvae are maintained in dilution water
to which a selected concentration of test material has been
added. The 48-h EC50 is calculated based on the proportion of
live larvae with completely developed shells in chambers
3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
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Withdrawn. The last approved version of this historical standard is referenced
on www.astm.org.
E724 − 21
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containing the test material to live larvae with completely
developed shells in the controls at the termination of the 48-h
test.
5. Significance and Use
5.1 An acute toxicity test is conducted to assess the effects
of a short term exposure of organisms to a test material under
specific experimental conditions. An acute toxicity test does
not provide information concerning whether delayed effects
will occur and typically evaluates effects on survival. A chronic
test is typically longer in duration and includes a sublethal
endpoint to assess effects on a population that might occur
beyond the exposure period. Because the bivalve embryo
development test includes a sublethal endpoint, but is also
short in duration, these tests are considered to be short-term
chronic tests.
FIG. 1 Drawings Exemplifying Five Key Developmental Stages of Bivalve Larvae Occurring During the First 48 h of Development
E724 − 21
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