ASTM E729-23e1

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
Designation: E729 − 23
Standard Guide for
Conducting Acute Toxicity Tests on Test Materials with
Fishes, Macroinvertebrates, and Amphibians
This standard is issued under the fixed designation E729; 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.
This standard has been approved for use by agencies of the U.S. Department of Defense.
ε NOTE—Sections 3 and 4 were editorially reorganized, and 7.8 and 8.2.1.1 were editorially corrected in February 2023.
1. Scope materials that have a high oxygen demand, are highly volatile,
2 are rapidly transformed biologically or chemically in aqueous
1.1 This guide (1) describes procedures for obtaining
solution, or are removed from test solutions in substantial
laboratory data concerning the adverse effects (for example,
quantities by the test chambers or organisms during the test.
lethality and immobility) of a test material added to dilution
Because the pH and concentrations of dissolved oxygen and
water, but not to food, on certain species of freshwater and
test material are maintained at desired levels and degradation
saltwater fishes, macroinvertebrates, and amphibians, usually
and metabolic products are removed, tests using renewal and
during 2 to 4-day exposures, depending on the species. These
flow-through methods are preferable; test organisms may be
procedures will probably be useful for conducting acute
fed during renewal and flow-through tests. Although renewal
toxicity tests with many other aquatic species, although modi-
tests might be more cost-effective, flow-through tests are
fications might be necessary.
generally preferable.
1.2 Other modifications of these procedures might be justi-
1.4 Acute tests may be performed to meet regulatory data
fied by special needs or circumstances such as meeting specific
requirements or to obtain time-independent estimates of toxic-
study goals, regulatory needs, or to accommodate specific test
ity.
organism life stages. Although using appropriate procedures is
1.4.1 If the objective is to obtain data to meet regulatory
more important than following prescribed procedures, results
of tests conducted using unusual or novel procedures are not requirements, it may be necessary to limit the number of
observation times based on stipulations of the regulatory
likely to be comparable to results of many other tests. Com-
parison of results obtained using modified and unmodified agency and cost considerations.
versions of these procedures might provide useful information
1.4.2 If the objective of an acute toxicity test is to determine
concerning new concepts and procedures for conducting acute
a time-independent (that is, incipient, threshold, or asymptotic)
tests.
toxicity level, an appropriate number of observations must be
taken over an exposure duration of sufficient length to establish
1.3 This guide describes tests using three basic exposure
the shape of the toxicity curve or allow the direct or math-
techniques: static, renewal, and flow-through. Selection of the
ematically estimated determination of a time-independent tox-
technique to use in a specific situation will depend on the needs
icity value (1), or both.
of the investigator and on available resources. Tests using the
static technique provide the most easily obtained measure of
1.5 In the development of these procedures, an attempt was
acute toxicity, but conditions often change substantially during
made to balance scientific and practical considerations and to
static tests; therefore, static tests should not last longer than 96
ensure that the results will be sufficiently accurate and precise
h, and test organisms should not be fed during such tests unless
for the applications for which they are commonly used. A
the test organisms are severely stressed without feeding over
major consideration was that the common uses of the results of
48 h. Static tests should probably not be conducted on
acute toxicity tests do not require or justify stricter require-
ments than those set forth herein. Although the tests may be
This guide is under the jurisdiction of ASTM Committee E50 on Environmental improved by using more organisms, longer acclimation times,
Assessment, Risk Management and Corrective Actionand is the direct responsibility
and so forth, the requirements presented herein should usually
of Subcommittee E50.47 on Biological Effects and Environmental Fate.
be sufficient.
Current edition approved Jan. 1, 2023. Published February 2023. Originally
approved in 1980. Last previous edition approved in 2014 as E729 – 96(2014). DOI:
1.6 Results of acute toxicity tests should usually be reported
10.1520/E0729-23E01.
in terms of an LC50 (median lethal concentration) or EC50
The boldface numbers in parentheses refer to the list of references at the end of
this standard. (median effective concentration) at the end of the test, but it is
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
´1
E729 − 23
desirable to provide information concerning the dependence of Development of International Standards, Guides and Recom-
adverse effects on both time and concentration. Thus, when mendations issued by the World Trade Organization Technical
feasible, flow-through and renewal tests should be conducted Barriers to Trade (TBT) Committee.
so that LC50s or EC50s can be reported from 6 h to an
2. Referenced Documents
asymptotic (time-independent, threshold, incipient) value, if
one exists. In some situations, it might only be necessary to
2.1 ASTM Standards:
determine whether a specific concentration is acutely toxic to
D4447 Guide for Disposal of Laboratory Chemicals and
the test species or whether the LC50 or EC50 is above or below Samples
a specific concentration.
E724 Guide for Conducting Static Short-Term Chronic Tox-
icity Tests Starting with Embryos of Four Species of
1.7 This guide is arranged as follows:
Saltwater Bivalve Molluscs
Section
E943 Terminology Relating to Biological Effects and Envi-
Referenced Documents 2
ronmental Fate (Withdrawn 2023)
Terminology 3
E1023 Guide for Assessing the Hazard of a Material to
Summary of Guide 4
Aquatic Organisms and Their Uses
Significance and Use 5
Apparatus 6
E1191 Guide for Conducting Life-Cycle Toxicity Tests with
Facilities 6.1
Saltwater Mysids
Special Requirements 6.2
E1192 Guide for Conducting Acute Toxicity Tests on Aque-
Construction Materials 6.3
Metering System 6.4
ous Ambient Samples and Effluents with Fishes,
Test Chambers 6.5
Macroinvertebrates, and Amphibians
Cleaning 6.6
E1203 Practice for Using Brine Shrimp Nauplii as Food for
Acceptability 6.7
Hazards 7
Test Animals in Aquatic Toxicology (Withdrawn 2013)
Dilution Water 8
E1563 Guide for Conducting Short-Term Chronic Toxicity
Requirements 8.1
Tests with Echinoid Embryos
Source 8.2
Treatment 8.3
E1604 Guide for Behavioral Testing in Aquatic Toxicology
Characterization 8.4
E1706 Test Method for Measuring the Toxicity of Sediment-
Test Material 9
Associated Contaminants with Freshwater Invertebrates
General 9.1
Stock Solution 9.2
E1733 Guide for Use of Lighting in Laboratory Testing
Test Concentration(s) 9.3
E2455 Guide for Conducting Laboratory Toxicity Tests with
Test Organisms 10
Species 10.1 Freshwater Mussels
Age 10.2
IEEE/ASTM SI 10 Standard for Use of the International
Source 10.3
System of Units (SI) (the Modernized Metric System)
Care and Handling 10.4
Feeding 10.5
Disease Treatment 10.6 3. Terminology
Holding 10.7
3.1 The words “must,” “should,”“ may,” “can,” and “might”
Acclimation 10.8
Quality 10.9
have very specific meanings in this guide. “Must” is used to
Procedure 11
express an absolute requirement, that is, to state that the test
Experimental Design 11.1
ought to be designed to satisfy the specified condition, unless
Dissolved Oxygen 11.2
Temperature 11.3
the purpose of the test requires a different design. “Must” is
Loading 11.4
only used in connection with factors that directly relate to the
Beginning the Test 11.5
acceptability of the test (see 13.1). “Should” is used to state
Feeding 11.6
Duration of Test 11.7
that the specified condition is recommended and ought to be
Biological Data 11.8
met if possible. Although violation of one “should” is rarely a
Other Measurements 11.9
serious matter, violation of several will often render the results
Analytical Methodology 12
Acceptability of Test 13
questionable. Terms such as “is desirable,” “is often desirable,”
Calculation of Results 14
and “might be desirable” are used in connection with less
Report 15
important factors. “May” is used to mean “is (are) allowed
1.8 The values stated in SI units are to be regarded as the
to,”“ can” is used to mean “is (are) able to,” and “might” is
standard. The values given in parentheses are for information
used to mean “could possibly.” Thus the classic distinction
only.
between “may” and “can” is preserved, and “might” is never
1.9 This standard does not purport to address all of the
used as a synonym for either “may” or “can.”
safety concerns, if any, associated with its use. It is the
3.2 Definitions:
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.
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
Specific hazard statements are given in Section 7.
Standards volume information, refer to the standard’s Document Summary page on
1.10 This international standard was developed in accor-
the ASTM website.
dance with internationally recognized principles on standard-
The last approved version of this historical standard is referenced on www.ast-
ization established in the Decision on Principles for the m.org.
´1
E729 − 23
3.2.1 acute test, n—a comparative study in which toxicological terms LD50 (median lethal dose) and ED50
organisms, that are subjected to different treatments, are (median effective dose), respectively. The terms LC50 and
observed for a relatively short period usually not constituting a EC50 should be used whenever results are calculated based on
substantial portion of their life span. the concentration of test material in dilution water, whereas the
terms LD50 and ED50 should be used whenever results are
3.2.2 dilution water, n—non-toxic aqueous exposure media
calculated based on the quantity of test material that enters or
(that is, water) used to reduce the concentration of a test
is applied directly to test organisms. For toxic agents or tests
substance in aquatic toxicity tests and is used as the control
for which neither concentration nor dose is appropriate, such as
water.
tests on temperature or with poorly water-soluble materials, the
3.2.3 reconstituted water, n—a dilution water that is pre-
terms LL50 (median lethal level) and EL50 (median effective
pared by adding sea salt or appropriate amounts of reagent-
level) should be used, if the effect is dichotomous. For tests in
grade salts to water, which is usually prepared using
which the effect is expressed as a percent inhibition compared
deionization, distillation, or reverse osmosis, so that the con-
to the control, for example, a percent inhibition in shell growth
centrations and ratios of the major ions in the dilution water are
in acute 96-h shell deposition tests with saltwater bivalve
similar to those in comparable natural surface waters.
molluscs (2), and not as the percentage of the individual
3.2.4 IC50, n—a statistically or graphically estimated con-
organisms that were affected, the term IC50 should be used to
centration of test material that is expected to cause a 50 %
denote the concentration that causes a 50 % inhibition com-
inhibition of one or more specified biological processes (such
pared to the control.
as shell growth of saltwater bivalve molluscs in acute shell
4.3 Acute toxicity tests in which test organisms are exposed
deposition tests), for which the data are not dichotomous, under
to test solutions containing a test material can be conducted by
specified conditions.
at least four techniques:
3.3 For definitions of other terms used in this guide, refer to
4.3.1 In the static technique, test solutions and organisms
Terminology E943 and Guide E1203. For an explanation of
are placed in chambers and kept there for the duration of the
units and symbols, refer to IEEE/ASTM SI 10.
test.
4.3.2 The recirculation technique is like the static technique
4. Summary of Guide
except that each test solution is continuously circulated through
an apparatus designed to maintain water quality, and possibly
4.1 In each of two or more treatments, test organisms of one
remove degraded, but not undegraded, test material by such
species are maintained for 2 to 8 days in one or more test
means as aeration, filtration, and sterilization and then returned
chambers. In each of the one or more control treatments, the
to the test chamber.
organisms are maintained in dilution water to which no test
4.3.3 The renewal technique is like the static technique
material has been added in order to provide (1) a measure of
except that test organisms are periodically exposed to fresh test
the acceptability of the test by giving an indication of the
solution of the same composition, usually once every 24 h or
quality of the test organisms and the suitability of the dilution
48 h, either by transferring the organisms from one test
water, test conditions, handling procedures, and so forth, and
chamber to another or by replacing nearly all the test solution.
(2) the basis for interpreting data obtained from the other
4.3.4 In the flow-through technique, test solution flows
treatments. In each of the one or more other treatments, the
through the test chamber on a once-through basis throughout
organisms are maintained in dilution water to which a selected
the test.
concentration of test material has been added. Data concerning
effects on the organisms in each test chamber are usually 4.3.4.1 Two procedures may be used. In the first a large
volume of each test solution is prepared before the beginning
obtained periodically during the test and analyzed to determine
LC50s, EC50s, or IC50s for various lengths of exposure. of the test, and these solutions flow through the respective
chambers. In the second and more common procedure, fresh
4.2 Acute toxicity tests are generally used to determine the
test solutions are prepared every few minutes or hours just
concentration of test material that produces a specific adverse
before they enter the respective test chambers. In both proce-
effect on a specified percentage of test organisms during a short
dures a metering system controls the flow of test solution, and
exposure, relative to the life-cycle of the test organism.
in the latter procedure the test solutions are prepared by the
Because death is an obviously important adverse effect and is
metering system. Both of the procedures may be used to
easily detected for many species, the most common acute
conduct continuous-flow tests. Many tests conducted using the
toxicity test is the acute lethality test. Experimentally, effect on
second procedure, however, are intermittent-flow tests because
50 % of a group of test organisms is the most reproducible and
the metering system cycles and delivers test solution every few
easily determined measure of toxicity, and 96 h is often a
minutes or hours.
convenient, useful exposure duration. Therefore, the measure
4.3.5 With any of these techniques a pump or stirrer can be
of acute toxicity most often used with fishes,
used to create a current in the test chamber to accommodate
macroinvertebrates, and amphibians is the 96-h LC50.
particular species, but the current will often increase both
However, because immobilization is a severe effect and is not
aeration and volatilization.
easy to distinguish from death for some species, the measure of
acute toxicity most often used with daphnids and midge larvae 4.4 In flow-through tests a “volume addition” is the intro-
is the 48-h EC50 based on death plus immobilization. The duction into the test chamber of a volume of test solution equal
terms LC50 and EC50 are consistent with the widely used to the volume of solution in the chamber.
´1
E729 − 23
4.5 For the purposes of 8.4.1, the term“ organophosphorus incubator, or recirculating water bath. For static and renewal
pesticides” refers to chlorpyrifos, demeton, diazinon, tests a dilution-water tank, which may be used to prepare
disulfoton, fenitrothion, malathion, methyl parathion, and para- reconstituted water, is often elevated so that dilution water can
thion; the term “organochlorine pesticides” refers to aldrin, be gravity fed into holding and acclimation tanks and test
chlordane, DDD, DDE, DDT, dieldrin, endosulfan, endrin, chambers. For flow-through tests an elevated headbox is often
heptachlor, heptachlor epoxide, lindane, methoxychlor, mirex, desirable so that dilution water can be gravity fed into holding
and toxaphene; and the term “chlorinated phenoxy herbicides” and acclimation tanks and into the metering system (see 6.4),
refers to the free acids, salts, and esters of 2,4-D, dicamba, which prepares the test solutions and delivers them to the test
silvex, and 2,4,5-T. The term “organic chlorine” refers to chambers. Strainers and air traps should be included in the
chlorine that would be detected if, when samples are prepared water-supply system. Headboxes and holding, acclimation,
for gas chromatographic analysis for polychlorinated biphenyls culture, and dilution-water tanks should be equipped for
(PCBs) and the organochlorine pesticides listed above, a temperature control and aeration (see 8.3.1). Air used for
chloride detector is used instead of an electron capture detector aeration should be free of fumes, oil, and water; filters to
to measure compounds that elute from just before lindane to remove oil and water are desirable. Filtration of air through a
just after mirex on the gas chromatograph being used. Organic 0.22 μm bacterial filter might be desirable (4). The facility
chlorine does not refer only to chlorine associated with should be well-ventilated and free of fumes. To further reduce
organochlorine pesticides and PCBs; it refers to all chlorine the possibility of contamination by test materials and other
that elutes within the specified period. substances, especially volatile ones, holding, acclimation, and
culture tanks should not be in a room in which toxicity tests are
5. Significance and Use
conducted, stock solutions or test solutions are prepared, or
5.1 An acute toxicity test is conducted to obtain information equipment is cleaned. Organisms should be shielded from
disturbances with curtains or partitions to prevent unnecessary
concerning the immediate effects on test organisms of a
short-term exposure to a test material under specific experi- stress during holding, acclimation, culture, and testing. A
timing device should be used to provide a 16 h light and 8-h
mental conditions. An acute toxicity test does not provide
information about whether delayed effects will occur, although dark photoperiod. A 15 min to 30 min transition period (5)
when the lights go on might be desirable to reduce the
a post-exposure observation period, with appropriate feeding,
if necessary, might provide such information. Bioavailability of possibility of organisms being stressed by large, sudden
increases in light intensity. A transition period when the lights
the test substance may also differ between real-world expo-
sures and laboratory exposures due to site-specific water go off might also be desirable (see Guide E1733).
quality conditions (see Guides E1192, E1563, and E2455).
6.2 Special Requirements—Some organisms require special
5.2 Results of acute toxicity tests might be used to predict conditions during holding, acclimation, and testing. For
acute effects likely to occur on aquatic organisms in field example, burrowing mayfly nymphs should be provided a
situations as a result of exposure under comparable conditions, substrate suitable for burrowing (6) or artificial burrows (7, 8);
except that (1) motile organisms might avoid exposure when immature stream insects should be provided with a current (7)
possible, and (2) toxicity to benthic organisms might be or mild aeration, or both (8); and amphipods, midge larvae
dependent on sorption or settling of the test material onto the crabs, shrimp, and bottom-dwelling fish should be provided a
substrate. silica sand substrate. Nylon or stainless steel mesh can also be
used to provide a substrate to which amphipods can cling.
5.3 Results of acute tests might be used to compare the
Because cannibalism might occur among many species of
acute sensitivities of different species and the acute toxicities of
decapod crustaceans or midges, the claws of crabs and crayfish
different test materials, and to study the effects of various
should be banded, or the individuals should be physically
environmental factors on results of such tests.
isolated by such means as screened compartments or held
5.4 Results of acute toxicity tests might be an important
individually in test chambers during testing.
consideration when assessing the hazards of materials to
6.3 Construction Materials—Equipment and facilities that
aquatic organisms (see Guide E1023) or when deriving water
contact stock solutions, test solutions, or any water into which
quality criteria for aquatic organisms (3).
test organisms will be placed should not contain substances
5.5 Results of acute toxicity tests might be useful for
that can be leached or dissolved by aqueous solutions in
studying the biological availability of, and structure-activity
amounts that adversely affect test organisms. In addition,
relationships between, test materials.
equipment and facilities that contact stock solutions or test
5.6 Results of acute toxicity tests will depend on the solutions should be chosen to minimize sorption of test
temperature, composition of the dilution water, condition of the
materials from water. Glass, Type 316 stainless steel, nylon,
test organisms, exposure technique, and other factors. and non-fluorocarbon plastics should be used whenever pos-
sible to minimize dissolution, leaching, and sorption, except
6. Apparatus
that stainless steel should not be used in tests on metals in salt
6.1 Facilities—Although some small organisms can be held water. Concrete and rigid plastics may be used for holding,
and acclimated in static or renewal systems, most organisms acclimation, and culture tanks in the water-supply system, but
are held, acclimated, and cultured in flow-through systems. these materials should be soaked, preferably in flowing dilution
Test chambers should be in a constant-temperature room, water, for a week or more before use (8). Cast iron pipe should
´1
E729 − 23
not be used with salt water and probably should not be used in 6.5.1 In a toxicity test with aquatic organisms, test chambers
a freshwater-supply system because colloidal iron will prob- are defined as the smallest physical units between which no
ably be added to the dilution water, and strainers will be needed water connections exist. However, screens, cups, and so forth,
to remove rust particles. A specially designed system is usually may be used to create two or more compartments within each
necessary to obtain salt water from a natural water source (9). chamber. Therefore, the test solution can flow from one
Brass, copper, lead, galvanized metal, and natural rubber compartment to another within a test chamber, but, by
should not contact dilution water, stock solutions, or test definition, cannot flow from one chamber to another. Because
solutions before or during the test. Items made of neoprene the solution can flow from one compartment to another in the
rubber or other materials not previously mentioned should not same test chamber, the temperature, concentration of test
be used unless it has been shown that either (1) unfed material, and levels of pathogens and extraneous contaminants
individuals of a sensitive aquatic species (see 8.1.1.1 and are likely to be more similar between compartments in the
8.1.1.2) do not show more signs of stress, such as same test chamber than between compartments in different test
discoloration, unusual behavior, or death, when held for at least chambers in the same treatment. Chambers should be covered
48 h in static dilution water in which the item is soaking than to keep out extraneous contaminants and, especially in static
when held in static dilution water that does not contain the and renewal tests, to reduce evaporation of test solution and
item, or (2) their use will not adversely affect survival, growth, test material. Also, chambers may need to be covered to
or reproduction of a sensitive species. prevent organisms from jumping out. All chambers (and
compartments) in a test must be identical.
6.4 Metering System:
6.5.2 Test chambers may be constructed by welding, but not
6.4.1 For flow-through tests, the metering system should be
soldering, stainless steel or by gluing double-strength or
designed to accommodate the type and concentration(s) of the
stronger window glass with clear silicone adhesive. Stoppers
test material and the necessary flow rates of test solutions. The
and silicone adhesive sorb some organochlorine and organo-
system should permit the mixing of test material with dilution
phosphorus pesticides, which are then difficult to remove.
water immediately before entrance to the test chambers and
Therefore, as few stoppers and as little adhesive as possible
permit the supply of the selected concentration(s) of test
should be in contact with test solution. If extra beads of
material (see 9.3 and 11.9.3.4) in a reproducible fashion.
adhesive are needed for strength, they should be on the outside
Various metering systems, using different combinations of
of chambers rather than on the inside. Especially in static and
syringes, “dipping birds”, siphons, pumps, saturators,
renewal tests, the size and shape of the test chamber might
solenoids, valves, and so forth, have been used successfully to
affect the results of tests on materials that volatilize or sorb
control the concentrations of test material in, and the flow rates
onto the chambers in substantial quantities.
of, test solutions (10). Proportional diluters (11) use an
6.5.3 The minimum acceptable dimensions of test chambers
intermittent flow design and various devices for metering the
and the minimum depth of test solution depend on the size of
test material (12). Continuous-flow metering systems are also
the individual test organisms and the loading (see 11.4). The
available, as are systems that prepare the different test solutions
smallest horizontal dimension of the test chambers should be at
independently of each other (10).
least three times the largest horizontal dimension of the largest
6.4.2 The metering system should be calibrated before use,
test organism. The depth of the test solution should be at least
and verified after the test to confirm that the targeted flow rates
three times the height of the largest test organism. In addition,
were met, by determining the flow rate through each test
the test solution should be at least 150 mm deep for organisms
chamber and by measuring either the concentration of test
over 0.5 g (wet weight). Use of excessively large volumes of
material in each test chamber or the volume of solution used in
solution in test chambers will probably unnecessarily increase
each portion of the metering system. The general operation of
the amount of dilution water and test material used, and, in
the metering system should be visually checked daily in the
flow-through tests, increase the average retention time.
morning and afternoon throughout the test. The metering
6.5.4 For static and renewal tests, organisms weighing more
system should be adjusted during the test if necessary.
than 0.5 g (wet weight) each are often exposed in 19 L (5 gal)
6.4.3 The flow rate through each test chamber should be at
wide-mouth soft-glass bottles (13) containing 15 L of solution
least five-volume additions per 24 h depending on test species.
or in 30 L of solution in 300 mm by 600 mm by 300 mm deep
It is usually desirable to construct the metering system so that
all-glass test chambers. Smaller organisms are often exposed in
it can provide at least ten-volume additions per 24 h, if desired,
500 mL to 1 L glass beakers containing 200 mL to 800 mL of
in case (1) the loading is high (see 11.4) or (2) there is rapid
solution. Daphnids, amphipods, midge larvae, juvenile fresh-
loss of test material due to microbial degradation, hydrolysis,
water mussels, and mayflies are often exposed in 100 mL
oxidation, photolysis, reduction, sorption, or volatilization (see
beakers containing 50 mL of solution or 50 mL beakers
11.9.3.4). In shell deposition tests with saltwater bivalve
containing 30 mL of solution.
molluscs (2), the minimum necessary flow rate might also
6.5.5 For flow-through tests, chambers may be constructed
depend on the amount of food available in the dilution water
by modifying glass bottles, glass canning jars, or beakers to
(see 10.5.3). At any particular time during the test, the flow
provide screened overflow holes, standpipes, or V-shaped
rates through any two test chambers should not differ by more
notches. Organisms weighing more than 0.5 g (wet weight)
than 10 %.
each are often exposed in 30 L of solution in 300 by 600 by
6.5 Test Chambers: 300-mm deep all-glass test chambers. Smaller organisms are
´1
E729 − 23
often exposed in 2 L to 4 L of solution. In tests with daphnids means as wearing appropriate protective gloves (especially
and other small species, the test chambers or metering system, when washing equipment or putting hands in test solutions),
or both, should be constructed so that the organisms are not laboratory coats, aprons, and glasses, and by using dip nets,
stressed by turbulence (14). forceps, or tubes to remove organisms from test solutions.
6.5.6 Embryos are often exposed in glass cups with stainless Special precautions, such as covering test chambers and
steel or nylon screen bottoms or cups constructed by welding ventilating the area surrounding the chambers, should be taken
stainless steel screen or gluing nylon screen with clear silicone when conducting tests on volatile materials. Information on
adhesive. The cups should be suspended in the test chambers in toxicity to humans (16), recommended handling procedures
such a way as to ensure that the embryos are always submerged (17), and biological, chemical, and physical properties of the
and that test solution regularly flows into and out of the cups test material should be studied before a test is begun. (See
without creating too much turbulence. Appendixes X2, X3, and X4 of Guide E1023.) Special proce-
dures might be necessary with radiolabeled test materials (18)
6.6 Cleaning—The metering system, test chambers, and
and with test materials that are, or are suspected of being,
equipment used to prepare and store dilution water, stock
carcinogenic (19).
solutions, and test solutions must be cleaned after use and may
need to be cleaned before use. New items should be washed
7.2 Although disposal of stock solutions, test solutions, and
with detergent and rinsed with water, a water-miscible organic
test organisms poses no special problems in most cases, health
solvent, water, acid (such as 10 % concentrated hydrochloric
and safety precautions and applicable regulations should be
acid (HCl)), and rinsed at least twice with deionized, distilled,
considered before beginning a test. Removal or degradation of
or dilution water. Reagent grade solvents are recommended. If
test material might be desirable before disposal of stock and
lesser grades are used, possible contaminants should be con-
test solutions.
sidered with respect to the purpose of the test. (Some lots of
7.3 Cleaning of equipment with a volatile solvent such as
some organic solvents might leave a film that is insoluble in
acetone should be performed only in a well-ventilated area in
water.) A dichromate-sulfuric acid cleaning solution may be
which no smoking is allowed and no open flame, such as a pilot
used in place of both the organic solvent and the acid, but it
light, is present.
might attack silicone adhesive. At the end of the test, all items
7.4 An acidic solution should not be mixed with a hypo-
that are to be used again should be immediately (1) emptied,
chlorite solution because hazardous fumes might be produced.
(2) rinsed with water, (3) cleaned by a procedure appropriate
for removing the test material (for example, acid to remove
7.5 To prepare dilute acid solutions, concentrated acid
metals and bases, detergent, organic solvent, or activated
should be added to water, not vice versa. Opening a bottle of
carbon to remove organic chemicals), and (4) rinsed at least
concentrated acid and adding concentrated acid to water should
twice with deionized, distilled, or dilution water. Acid is often
be performed only in a fume hood.
used to remove mineral deposits, and 200 mg of hypochlorite
−
7.6 Because dilution water and test solutions are usually
(ClO )/L is often used to remove organic matter and for
−
good conductors of electricity, use of ground fault systems and
disinfection. (A solution containing about 200 mg of ClO /L
leak detectors should be considered to help prevent electrical
may be prepared by adding 6 mL of liquid household chlorine
−
shocks. Salt water is such a good conductor that protective
bleach to 1 L of water. However, ClO is quite toxic to many
devices are strongly recommended.
aquatic animals (15) and is difficult to remove from some
construction materials. It is often removed by soaking in a
7.7 To protect hands from being cut by sharp edges of
sodium thiosulfate, sodium sulfite, or sodium bisulfite solution,
shells, cotton work gloves should be worn (over appropriate
by autoclaving in distilled water for 20 min, or by drying the
protective gloves (see 7.1) if necessary) when juvenile and
item and letting it sit for at least 24 h before use. An item
adult bivalve molluscs are handled.
cleaned or disinfected with hypochlorite should not be used
7.8 Special handling and precautionary guidance in Safety
unless it has been demonstrated at least once that unfed
Data Sheets (SDS) should be followed for reagents and other
individuals of a sensitive aquatic species (see 8.1.1.1 and
chemicals purchased from supply houses.
8.1.1.2) do not show more signs of stress, such as
discoloration, unusual behavior, or death, when held for at least 7.9 For further guidance on safe practices when handling
48 h in static dilution water in which the item is soaking than
field-collected samples and conducting toxicity tests, check
when held in static dilution water containing a similar item that with the permittee and consult general industrial safety manu-
−
was not treated with ClO .) The metering system and test
als (Test Method E1706).
chambers should be rinsed with dilution water just before use.
7.10 Work with some field-collected samples may require
6.7 Acceptability—The acceptability of new holding,
compliance with rules pertaining to the handling of hazardous
acclimation, and testing facilities should be demonstrated with
materials. Guidelines for the handling and disposal of hazard-
a sensitive species (see 8.1.1.1 and 8.1.1.2) before use.
ous materials should be strictly followed (Guide D4447). Some
regulatory authorities have developed requirements for the
7. Hazards
management of hazardous waste. As a handler of hazardous
7.1 Many materials can affect humans adversely if precau- materials, it is your responsibility to know and comply with
tions are inadequate. Therefore, skin contact with all test applicable regulations where you are operating (Test Method
materials and solutions of them should be minimized by such E1706).
´1
E729 − 23
7.11 Appropriate measures and practices should be imple- which they will survive for 48 h without showing signs of
mented to prevent the spread of non-target species when stress is probably acceptable for acute tests with most saltwater
acquiring or transferring the target test species. Additionally, animal species. Water in which Acartia tonsa or mysids will
bio-secure practices should be utilized when working with survive, grow, and reproduce satisfactorily in a life-cycle test is
either nonlocalized or non-native species to prevent escape- probably an acceptable dilution water for tests with most
ment into local watersheds and potentially altering or nega- saltwater animal species.
tively influencing existing ecosystems.
8.1.2 The quality of the dilution water should be uniform so
that the test organisms are cultured or acclimated and the test
8. Dilution Water
conducted in water of the same quality. In fresh water, the
8.1 Requirements—The dilution water should (1) be avail- range of hardness should be less than 10 % of the average. In
able in adequate supply, (2) be acceptable to the test organisms, salt water, the range of salinity should be less than 20 % of the
(3) be of uniform quality, and (4) except as stated in 8.1.4, not average.
unnecessarily affect results of the test.
8.1.3 The dilution water should not unnecessarily affect the
8.1.1 The minimal requirement for an acceptable dilution
results of an acute test because of such things as sorption or
water for acute toxicity tests is that healthy test organisms
complexation of test material. Except as in accordance with
survive in it through acclimation and testing without showing
8.1.4, it is desirable for the purpose of reducing interlaboratory
signs of stress, such as discoloration, unusual behavior, or
variability that the concentrations of both total organic carbon
death. A better criterion for an acceptable dilution water is that
(TOC) and particulate matter should be less than 20 mg/L for
at least one species of aquatic animal (preferably of the one
shell deposition tests with saltwater bivalve molluscs (see
being tested or one taxonomically similar) will survive, grow,
10.5.2) and less than 5 mg/L for all other tests.
and reproduce satisfactorily in it.
8.1.4 If it is desired to study the effect of an environmental
8.1.1.1 Fresh Water—Because daphnids are more acutely
factor such as TOC, particulate matter, or dissolved oxygen on
sensitive to some test materials than many other aquatic animal
the results of an acute test, it will be necessary to use a water
species, water in which daphnids (less than 24-h old) will
that is naturally or artificially high in TOC or particulate matter
survive for 48 h without showing signs of stress is probably
or low in dissolved oxygen. If such a water is used, it is
acceptable for acute tests with most freshwater animal species.
important that adequate analyses be performed to characterize
Water in which daphnids will survive, grow, and reproduce
the water and that a comparable test be available or be
satisfactorily in a life-cycle test is probably an acceptable
conducted in a more usual dilution water to facilitate interpre-
dilution water for tests with most freshwater animal species-
tation of the results in the special water.
.The “US Lab” strain of amphipod (Hyalella azteca) has an
8.2 Source:
additional requirement for chloride concentrations higher than
8.2.1 Reconstituted Water:
those present in many natural or reconstituted waters (20).
8.2.1.1 Reconstituted water used for tests with freshwater
Growth and reproduction of H. azteca also declines when
species is generally prepared by adding specified amounts of
chloride concentrations are below 15 mg/L (21). Importantly,
reagent-grade salts to deionized water (Tables 1 and 2).
the “US Lab” strain of H. azteca also showed greater sensitiv-
However, the buffers used in Table 2 might react chemically
ity to the acute toxic effects of sodium sulfate and sodium
with some test materials. Because these waters might be
nitrate at lower chloride concentrations of <10 mg/L. Special
deficient in some trace nutrients, addition of about 2 μg of
attention to this constituent is required when selecting culture
selenium(IV) and 1 μg of crystalline vitamin B /L might be
and test water for H. azteca (Test Method E1706). Selenium is
desirable (23), especially if daphnids are cultured in these
also recommended as a micronutrient for C. dubia culture and
waters. Examples of other reconstituted fresh waters that have
testing by adding sufficient sodium selenate (Na SeO ) to
2 4
been used successfully for toxicity testing in some laboratories
provide 2 μg selenium per liter of final dilution water (22).
are also available ((24), (25), Test Method E1706 Section
8.1.1.2 Salt Water—Because Acartia tonsa (copepod), my-
7.1.3.5).
sids (less than 24 h post-release from the brood sac), and
8.2.1.2 If a reconstituted water is used for tests with
bivalve mollusc larvae are more acutely sensitive to many test
materials than many other saltwater animal species, water in saltwater species, the reconstituted water described in Table 3
TABLE 1 Quantities of Reagent Grade Chemicals Added to Deionized Water to Prepare Reconstituted Fresh Waters (23) and the
Resulting Water Qualities
Reagent grade salts Required, mg/L
A B B
pH Hardness Alkalinity
NaHCO CaSO ·2H O MgSO KCl
3 4 2 4
Very soft 12 7.5 7.5 0.5 6.7–6.8 10–13 10–13
Soft 48 30 30 2.0 7.3–7.5 40–48 30–35
Moderately 96 60 60 4.0 7.4–7.8 80–100 57–64
hard
Hard 192 120.0 120.0 8.0 7.8–8.0 160–180 110–120
Very hard 384 240.0 240.0 16.0 8.0–8.2 280–320 225–245
A
Approximate equilibrium pH after aeration.
B
Expressed as mg CaCO /L.
´1
E729 − 23
TABLE 2 Quantities of Reagent Grade Chemicals to Be Added to
8.2.1.3 Reconstituted water is prepared by adding a sea salt
Aerated Soft Reconstituted Fresh Water 5
or specified amounts of reagent grade chemicals to highqual-
to Buffer pH (26)
ity water with (1) conductivity less than 1 μS/cm and (2) either
NOTE 1—The solutions should not be aerated after addition of these
total organic carbon (TOC) less than 2 mg/L or chemical
chemicals.
oxygen demand (COD) less than 5 mg/L. Acceptable water can
Milliliters of Solution to Add to 15 L of Soft Water
usually be prepared using properly operated deionization,
A
pH
1.0 N NaOH 1.0 M KH PO 0.5 M H BO
2 4 3 3 distillation, or reverse osmosis units. Conductivity should be
B B
Solution Solution Solution
measured on each batch and TOC or COD should be measured
6.0 1.3 80.0 .
at least twice a year and whenever substantial changes might be
6.5 5.0 30.0 .
7.0 19.0 30.0 . expected. If the water is prepared from a surface water, TOC or
7.5 . . .
COD should be measured on each batch. The reconstituted
8.0 19.0 20.0 .
water should be intensively aerated before use, except that the
8.5 6.5 . 40.0
buffered soft fresh waters (Table 2) should be aerated before,
9.0 8.8 . 30.0
9.5 11.0 . 20.0
but not after, addition of buffers. Problems have been encoun-
10.0 16.0 . 18.0
tered with some species in some fresh and salt reconstituted
A
Approximate equilibrium pH with fish in water.
waters, but sometimes these problems have been overcome by
B
Buffers containing ions such as phosphate and borate should not be used when
aging the reconstituted water for one or more weeks.
conducting tests on metals unless it has been shown that the buffers do not affect
the toxicity of the metal to the test species.
8.2.2 Natural Dilution Water:
8.2.2.1 If a natural dilution water is used, it should be
obtained from an uncontaminated, uniform quality source. The
TABLE 3 Reconstituted Salt Water (27)
quality of water from a well or spring is usually more uniform
Add the following reagent grade chemicals in the amounts and order listed
than that of water from a surface water. If a surface water is
to 890 mL of water. Each chemical must be dissolved before the next is
A
used as a sourc
...