ASTM E1563 - 21a

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Designation: E1563 −21a
Standard Guide for
Conducting Short-Term Chronic Toxicity Tests with Echinoid
Embryos
1,2
This standard is issued under the fixed designation E1563; 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 covers procedures for obtaining laboratory
data concerning the short-term chronic effects of a test material
on echinoderm embryos and the resulting larvae (sea urchins
and sand dollars) during static 48- to 96-h exposures. These
procedures have generally been used with U.S. East Coast
(Arbacia punctulata and Strongylocentrotus droebachiensis )
(1)
3
and West Coast species (Strongylocentrotus purpuratus, S.
droebachiensis, and Dendraster excentricus)(2).The basic
procedures described in this guide first originated in Japan and
Scandanavia (3),and parallel procedures have been used with
foreign species, especially in Japan and the Mediterranean (4).
These procedures will probably be useful for conducting static
toxicity tests with embryos of other echinoid species, 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, the results of tests conducted by using unusual
procedures are not likely to be comparable with those of many
other tests. The comparison of results obtained by using
modified and unmodified versions of these procedures might
provide useful information concerning new concepts and
procedures for conducting tests starting with embryos of
echinoids.
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 tests on temperature, dissolved
oxygen, and pH and on such materials as aqueous effluents (see
also Guide E1192), leachates, oils, particulate matter, surface
waters, effluents, and sediments (Annex A1). Renewal tests
might be preferable to static tests for materials that have a high
oxygen demand, are highly volatile, are rapidly transformed
biologically or chemically in aqueous solution, or are removed
from test solutions in substantial quantities by the test cham-
bers or organisms during the test.
1.4 Results of short-term chronic toxicity tests with echi-
noid embryos should usually be reported as the 50 % effect
concentration (EC50) based on the total abnormally developed
embryos and larvae. 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
Scope 1
Referenced Documents 2
Terminology 3
Summary of Guide 4
Significance and Use 5
Apparatus 6
Facilities 6.1
Construction Materials 6.2
Test Chambers 6.3
Cleaning 6.4
Acceptability 6.5
Safety Precautions 7
Dilution Water 8
Requirements 8.1
Source 8.2
Treatment 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
Source of Embryos 10.3
Handling 10.4
Test Animal Source and Condition 10.5
Spawning and Fertilization 10.6
Quality 10.7
Procedure 11
Experimental Design 11.1
Dissolved Oxygen 11.2
1
A Standard Guide is a document, developed using the consensus mechanisms
of ASTM that provides guidance for the selection of procedures to accomplish a
specific test, but which does not stipulate specific procedures.
2
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 Nov. 1, 2021. Published January 2022. Originally
approved in 1995. Last previous edition approved in 2021 as E1563 – 21. DOI:
10.1520/E1563-21A.
3
The boldface numbers in parentheses refer to the list of references at the end of
this standard.
*A Summary of Changes section appears at the end of this standard
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.
1
Temperature 11.3
Beginning the Test 11.4
Feeding 11.5
Duration of Test 11.6
Biological Data 11.7
Control Performance 11.8
Other Measurements 11.9
Analytical Methods 12
Acceptability of Test 13
Calculation of Results 14
Report 15
Keywords 16
Annex
Sediment Tests Annex A1
1.6 The values stated in SI units are to be regarded as the
standard.
1.7 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, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
Specific precautionary statements are given in Section 7.
1.8 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:
4
E380 Practice for Use of the International System of Units
(SI) (the Modernized Metric System) (Withdrawn 1997)
5
E724 Guide for Conducting Static Short-Term Chronic Tox-
icity Tests Starting with Embryos of Four Species of
Saltwater Bivalve Molluscs
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
E1525 Guide for Designing Biological Tests with Sediments
E1706 Test Method for Measuring the Toxicity of Sediment-
Associated Contaminants with Freshwater Invertebrates
E1733 Guide for Use of Lighting in Laboratory Testing
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 and Terminology E943. For sediment tests
(Annex A1), refer to Guides E1391 and E1525. For an
explanation of units and symbols, refer to Practice E380.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 embryo, n—the stages of a multicellular organism’s
development that occur between the fertilization of the egg and
the pluteus larva.
3.2.2 pluteus larva, n—a juvenile lifecycle stage character-
istic of all echinoids The term “embryo” is used herein to
denote the stages between the fertilization of the egg and the
pluteus larva. The term “larva” is used herein to refer to the
pluteus larva characteristic of all echinoids (5)(Fig. 1 and Fig.
2).
4. Summary of Guide
4.1 Adult sea urchins and sand dollars are brought into the
laboratory and identified to species. If the gonads are not ripe,
the sea urchins or sand dollars should be held and fed until the
gonads are brought into a suitable reproductive state. Echinoids
with ripe gonads are maintained under conditions that keep the
gonads ripe without inducing undesired spontaneous spawning
or resorption of gametes. In order to start a test, spawning is
induced by using one or more stimuli, which may be physical
(for example, heat or electrical current) or chemical (for
example, potassium chloride).
4.2 In each of two or more treatments, embryos and the
resulting larvae of one species are maintained for 48 to 96 h,
depending on the species and test temperature. 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 (1) a measure of the accept-
ability of the test by giving an indication of the quality of the
4
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.
5
The last approved version of this historical standard is referenced on
www.astm.org.
E1563 − 21a
2
organisms and the suitability of the dilution water, test
conditions, handling procedures, etc.; and (2) 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 concen-
tration of test material has been added. The EC50 is calculated
based on the proportion of larvae that develop into normal
pluteus larvae in chambers containing the test material relative
to normal larvae in the controls at the termination of the test.
5. Significance and Use
5.1 An acute toxicity test is conducted to assess 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 echinoderm embryo
development test includes a sublethal endpoint, but is also
short in duration, these tests are considered to be short-term
chronic tests, consistent with EPA guidance.
5.2 Because embryos and larvae are usually assumed to be
the most sensitive life stages of these echinoid species, and
because some of these species are commercially and recre-
ationally important, the results of these tests are often consid-
ered to be a good indication of the acceptability of pollutant
concentrations to saltwater species in general. The results of
these toxicity tests are often assumed to be an important
consideration when assessing the hazard of materials to other
saltwater organisms (see Guides E724 and E1023) or when
deriving water quality criteria for saltwater organisms (7).
5.3 The results of short-term chronic toxicity tests might be
used to predict effects likely to occur to aquatic organisms in
field situations as a result of exposure under comparable
conditions, except that toxicity to benthic species might depend
on sorption or settling of the test material onto the substrate.
FIG. 1 Drawings Exemplifying Key Developmental Stages of Normal Echinoid Larvae Occurring During the First 48 to 96 h of Develop-
ment and Examples of Abnormal or Arrested Development (6)
E1563 − 21a
3
5.4 The results of short-term chronic tests might be used to
compare the sensitivities of different species and the acute
toxicities of different test materials, and to determine the effects
of various environmental factors on the results of such tests.
5.5 The results of short-term chronic toxicity tests might be
useful for studying the biological availability of, and structure-
activity relationships between, test materials.
5.6 The results of any toxicity tests will depend on
temperature, composition of the dilution water, condition of the
test organisms, and other factors.
5.7 Results of short-term chronic toxicity tests might be
used to predict effects likely to occur to aquatic organisms
exposed to suspended particulates of dredged sediments dis-
posed through the water column.
5.8 Results of short-term chronic toxicity tests might be
used to predict effects likely to occur to aquatic organisms
exposed to bedded whole sediments.
6. Apparatus
6.1 Facilities:
6.1.1 Flow-through troughs with appropriate trays should be
available for holding and conditioning test animals (8). The
water-supply system should be equipped for temperature con-
trol and aeration (see 8.3) and should contain strainers and air
traps. Air used for aeration should be free of fumes, oil, and
water; filters to remove oil and water are desirable. Test
chambers should be in a constant-temperature room, incubator,
or recirculating water bath. A dilution-water tank or headbox,
which may be used to prepare reconstituted water, is often
elevated so that dilution water can be gravity-fed into holding
and conditioning troughs and test chambers. The facility
should be well ventilated and free of fumes. To further reduce
the possibility of contamination of the test organisms by test
materials and other substances, especially volatile ones, hold-
ing and conditioning troughs should not be in a room in which
the toxicity tests are conducted, stock solutions or test solutions
are prepared, or equipment is cleaned. Organisms should be
shielded from disturbances, with curtains or partitions, to
prevent unnecessary stress during holding, conditioning, and
testing.
FIG. 2 (a) Examples of Normal and Abnormal Development of
Purple Sea Urchin (Strongylocentrotus purpuratus) Embryos at
the Conclusion of a 72 to 96-h Toxicity Test. Figs. 2a and 2b
Show Examples of Normal Echinopluteus’ with Four Distinct
Arms and Good Symmetrical Development. Fig. 2c Shows a Bor-
derline Abnormal Pluteus with Poor Symmetrical Form and One
Missing Arm. Figs. 2d through 2j Show Examples of Increasing
Abnormal and/or Retarded (Considered Abnormal) Development.
Other Species of Sea Urchins and Sand Dollars Will Have the
Same General Larval Form, But Will Vary in Size, Conformation,
and Number of Larval Arms. Each Investigator Using a Different
Species Should Carefully Compare Well-Developed Embryos
from Controls with Gradations of Abnormal Development in a
Toxicant to Identify Distinctions between Normal and Abnormal
for Their Given Species
FIG. 2 (b) (continued)
E1563 − 21a
4
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