ASTM E1192-23

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: E1192 − 23
Standard Guide for
Conducting Acute Toxicity Tests on Aqueous Ambient
Samples and Effluents with Fishes, Macroinvertebrates, and
Amphibians
This standard is issued under the fixed designation E1192; 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 obtained measure of acute toxicity but should not last longer
than 48 h. Renewal and flow-through tests may last longer than
1.1 This guide covers procedures for obtaining laboratory
48 h because the pH and concentrations of dissolved oxygen
data concerning the adverse effects of an aqueous effluent on
and effluent are maintained at desired levels and degradation
certain species of freshwater and saltwater fishes,
and metabolic products are removed. Static tests might not be
macroinvertebrates, and amphibians, usually during 2 day to
applicable to effluents that have a high oxygen demand or
4 day exposures, depending on the species, using the static,
contain materials that (1) are highly volatile, (2) are rapidly
renewal, and flow-through techniques. These procedures will
biologically or chemically transformed in aqueous solutions, or
probably be useful for conducting acute toxicity tests on
(3) are removed from test solutions in substantial quantities by
aqueous effluents with many other aquatic species, although
the test chambers or organisms during the test. Flow-through
modifications might be necessary.
tests are generally preferable to renewal tests, although in some
1.2 Other modifications of these procedures might be justi-
situations a renewal test might be more cost-effective than a
fied by special needs or circumstances. Although using appro-
flow-through test.
priate procedures is more important than following prescribed
1.5 In the development of these procedures, an attempt was
procedures, results of tests conducted using unusual procedures
made to balance scientific and practical considerations and to
are not likely to be comparable to results of many other tests.
ensure that the results will be sufficiently accurate and precise
Comparison of results obtained using modified and unmodified
for the applications for which they are commonly used. A
versions of these procedures might provide useful information
major consideration was that the common uses of the results of
concerning new concepts and procedures for conducting acute
acute tests on effluents do not require or justify stricter
toxicity tests on aqueous effluents.
requirements than those set forth in this guide. Although the
1.3 This guide is based in large part on Guide E729 where
tests may be improved by using more organisms, longer
addition details are provided for test elements that may be
acclimation times, and so forth, the requirements presented in
applicable to the ambient and effluent toxicity testing described
this guide should usually be sufficient.
in this method. The major differences between the two guides
1.6 Results of acute toxicity tests should usually be reported
are (1) the maximum test concentration is 100 % effluent or
in terms of a median lethal concentration (LC50) or median
ambient sample, (2) testing is not chemical-specific, and (3) the
effective concentration (EC50). In some situations, it might be
holding time of effluent and ambient samples is often consid-
necessary only to determine whether a specific concentration is
erably less than that for chemicals and other test materials.
acutely toxic to the test species or whether the LC50 or EC50
Because the sample is often a complex mixture of chemicals,
is above or below a specific concentration.
analytical tests cannot generally be used to confirm exposure
concentrations.
1.7 This guide is arranged as follows:
Section
1.4 Selection of the technique to be used in a specific
situation will depend upon the needs of the investigator and
Referenced Documents 2
upon available resources. Static tests provide the most easily
Terminology 3
Summary of Guide 4
Significance and Use 5
Hazards 7
Apparatus 6
This guide is under the jurisdiction of ASTM Committee E50 on Environmental
Facilities 6.1
Assessment, Risk Management and Corrective Action and is the direct responsibil-
Special Requirements 6.2
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Construction Materials 6.3
Current edition approved Jan. 1, 2023. Published February 2023. Originally
Metering System 6.4
approved in 1988. Last previous edition approved in 2014 as E1192 – 97(2014).
Test Chambers 6.5
DOI: 10.1520/E1192-23.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1192 − 23
E943 Terminology Relating to Biological Effects and Envi-
Cleaning 6.6
Acceptability 6.7
ronmental Fate (Withdrawn 2023)
Dilution Water 8
E1203 Practice for Using Brine Shrimp Nauplii as Food for
Requirements 8.1
Test Animals in Aquatic Toxicology (Withdrawn 2013)
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-
Effluent 9
Associated Contaminants with Freshwater Invertebrates
Sampling Point 9.1
Collection 9.2
E1733 Guide for Use of Lighting in Laboratory Testing
Preservation 9.3
IEEE/ASTM SI 10 American National Standard for Use of
Treatment 9.4
Test Concentration(s) 9.5 the International System of Units (SI): The Modern Metric
Test Organisms 10
System
Species 10.1
Age 10.2
3. Terminology
Source 10.3
Care and Handling 10.4
3.1 Definitions:
Feeding 10.5
3.1.1 acute test, n—a comparative study in which
Disease Treatment 10.6
organisms, that are subjected to different treatments, are
Holding 10.7
Acclimation 10.8
observed for a relatively short period usually not constituting a
Quality 10.9
substantial portion of their life span.
Procedure 11
Experimental Design 11.1
3.1.2 dilution water, n—non-toxic aqueous exposure media
Dissolved Oxygen 11.2
(that is, water) used to reduce the concentration of a test
Temperature 11.3
Loading 11.4 substance in aquatic toxicity tests and is used as the control
Beginning the Test 11.5
water.
Feeding 11.6
3.1.3 reconstituted water, n—a dilution water that is pre-
Duration of Test 11.7
Biological Data 11.8
pared by adding sea salt or appropriate amounts of reagent-
Other Measurements 11.9
grade salts to water, which is usually prepared using
Analytical Methodology 12
Acceptability of Test 13 deionization, distillation, or reverse osmosis, so that the con-
Calculation or Results 14
centrations and ratios of the major ions in the dilution water are
Report 15
similar to those in comparable natural surface waters.
1.8 This standard does not purport to address all of the
3.2 The words “must,” “should,” “may,” “can,” and “might”
safety concerns, if any, associated with its use. It is the
have very specific meanings in this guide. “Must” is used to
responsibility of the user of this standard to establish appro-
express an absolute requirement, that is, to state that the test
priate safety, health, and environmental practices and deter-
ought to be designed to satisfy the specified condition, unless
mine the applicability of regulatory limitations prior to use.
the purpose of the test requires a different design. “Must” is
Specific hazard statements are given in Section 7.
only used in connection with factors that directly relate to the
1.9 This international standard was developed in accor-
acceptability of the test (see 13.1). “Should” is used to state
dance with internationally recognized principles on standard-
that the specified condition is recommended and ought to be
ization established in the Decision on Principles for the
met if possible. Although violation of one “should” is rarely a
Development of International Standards, Guides and Recom-
serious matter, violation of several will often render the results
mendations issued by the World Trade Organization Technical
questionable. Terms such as “is desirable,” “is often desirable,”
Barriers to Trade (TBT) Committee.
and “might be desirable” are used in connection with less
important factors. “May” is used to mean “is (are) allowed to,”
2. Referenced Documents
“can” is used to mean “is (are) able to,” and “might” is used to
2.1 ASTM Standards:
mean “could possibly.” Thus the classic distinction between
D4447 Guide for Disposal of Laboratory Chemicals and
“may” and “can” is preserved, and “might” is never used as a
Samples
synonym for either “may” or “can.”
E724 Guide for Conducting Static Short-Term Chronic Tox-
3.3 The term “effluents” refers to aqueous discharges regu-
icity Tests Starting with Embryos of Four Species of
lated under the National Pollutant Discharge Elimination
Saltwater Bivalve Molluscs
System (NPDES) collected at the sampling point specified in
E729 Guide for Conducting Acute Toxicity Tests on Test
the NPDES permit.
Materials with Fishes, Macroinvertebrates, and Amphib-
ians
3.4 The term “ambient samples” refers to water samples
collected from the environment. Examples include surface
waters, storm waters, leachates, and ground water.
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 last approved version of this historical standard is referenced on www.ast-
the ASTM website.
m.org.
E1192 − 23
3.5 For definitions of other terms used in this guide, refer to incubator, or recirculating water bath. A dilution-water tank,
Guide E729 and Terminology E943. For an explanation of which may be used to store receiving water, or a headbox is
units and symbols, refer to IEEE/ASTM SI 10. often elevated so dilution water can be gravity-fed into holding
and acclimation tanks and test chambers. Pumps are often used
4. Summary of Guide
to deliver dilution water and effluent to headboxes and tanks.
Strainers and air traps should be included in the water supply.
4.1 In each of two or more treatments, test organisms of one
Headboxes and holding, acclimation, culture, and dilution-
species are maintained for 2 days to 8 days in one or more test
water tanks should be equipped for temperature control and
chambers. In each of the one or more control treatments, the
aeration (see 8.3). Air used for aeration should be free of
organisms are maintained in dilution water to which no effluent
fumes, oil, and water; filters to remove oil and water are
has been added in order to provide (1) a measure of the
desirable. Filtration of air through a 0.22 μm bacterial filter
acceptability of the test by giving an indication of the quality
might be desirable (1) . The facility should be well ventilated
of the test organisms and the suitability of the dilution water,
and free of fumes. To further reduce the possibility of contami-
test conditions, handling procedures, and so forth, and (2) the
nation by components of the effluent and other substances,
basis for interpreting data obtained from the other treatments.
especially volatile ones, holding, acclimation, and culture tanks
In each of the one or more other treatments, the organisms are
should not be in a room in which toxicity tests are conducted,
maintained in dilution water to which a selected concentration
effluent is stored, test solutions are prepared, or equipment is
of effluent has been added. Data on effects on the organisms in
cleaned. During holding, acclimation, culture, and testing,
each test chamber are usually obtained periodically during the
organisms should be shielded from disturbances with curtains
test and analyzed to determine LC50s or EC50s for various
or partitions to prevent unnecessary stress. A timing device
lengths of exposure.
should be used to provide a 16 h light and 8 h dark photope-
5. Significance and Use riod. A 15 to 30-min transition period (2) when the lights go on
might be desirable to reduce the possibility of organisms being
5.1 An acute effluent toxicity test is conducted to obtain
stressed by large, sudden increases in light intensity. A transi-
information concerning the immediate effects on test organ-
tion period when the lights go off might also be desirable (see
isms of a short-term exposure to an effluent under specific
Guide E1733).
experimental conditions. One can directly examine acute
effects of complex mixtures of chemicals as occurs in effluents
6.2 Special Requirements—Some organisms require special
and some ambient waters. Acute effluent toxicity tests can be conditions during holding, acclimation, and testing. For
used to evaluate the potential for designated-use or aquatic life
example, burrowing mayfly nymphs should be provided a
impairment in the receiving stream, lake, or estuary. An acute
substrate suitable for burrowing (3); immature stream insects
toxicity test does not provide information about whether
should be in a current (4); and amphipods, midge larvae, crabs,
delayed effects will occur, although a post-exposure observa-
shrimp, and bottom-dwelling fish should be provided a silica-
tion period, with appropriate feeding if necessary, might
sand substrate. Nylon or stainless steel mesh can also be used
provide such information.
to provide a substrate to which amphipods can cling. Because
cannibalism might occur among many species of decapod
5.2 Results of acute effluent tests might be used to predict
crustaceans, the claws of crabs and crayfish should be banded,
acute effects likely to occur on aquatic organisms in field
or the individuals should be physically isolated by means of
situations as a result of exposure under comparable conditions,
screened compartments or held individually in test chamber
except that (1) motile organisms might avoid exposure when
during testing.
possible, (2) toxicity to benthic species might be dependent on
sorption or settling of components of the effluent onto the
6.3 Construction Materials—Equipment and facilities that
substrate, and (3) the effluent might physically or chemically contact effluent samples, test solutions, or any water into which
interact with the receiving water.
test organisms will be placed should not contain substances
that can be leached or dissolved by aqueous solutions in
5.3 Results of acute effluent tests might be used to compare
amounts that adversely affect aquatic organisms. In addition,
the acute sensitivities of different species and the acute
equipment and facilities that contact effluent samples or test
toxicities of different effluents, and to study the effects of
solutions should be chosen to minimize sorption of effluent
various environmental factors on results of such tests.
components from water. Glass, Type 316 stainless steel, nylon,
5.4 Acute tests are usually the first step in evaluating the
and non-fluorocarbon plastics should be used whenever pos-
effects of an effluent on aquatic organisms.
sible to minimize dissolution, leaching, and sorption, except
5.5 Results of acute effluent tests will depend on the
that stainless steel should not be used in tests on metals in salt
temperature, composition of the dilution water, condition of the water. Concrete and rigid plastics may be used for holding,
test organisms, exposure technique, and other factors.
acclimation, and culture tanks and in the water supply, but they
should be soaked, preferably in flowing dilution water, for a
6. Apparatus
week or more before use (5). Cast iron pipe should not be used
with salt water and probably should not be used in a
6.1 Facilities—Although some small organisms can be held
and acclimated in static or renewal systems, most organisms
are held, acclimated, and cultured in flow-through systems.
The boldface numbers in parentheses refer to a list of references at the end of
Test chambers should be in a constant-temperature room, this standard.
E1192 − 23
freshwater-supply system because colloidal iron will be added 6.5.1 In a toxicity test with aquatic organisms, test chambers
to the dilution water and strainers will be needed to remove rust are defined as the smallest physical units between which there
particles. A specially designed system is usually necessary to are no water connections. However, screens, cups, and so forth,
obtain salt water from a natural water source (see Guide E729). may be used to create two or more compartments within each
Brass, copper, lead, galvanized metal, and natural rubber chamber. Therefore, the test solution can flow from one
should not contact dilution water, effluent, or test solutions compartment to another within a test chamber, but, by
before or during the test. Items made of neoprene rubber or definition, cannot flow from one chamber to another. Because
other materials not mentioned above should not be used unless solution can flow from one compartment to another in the same
it has been shown that either (1) unfed individuals of a test chamber, the temperature, concentration of test material,
sensitive aquatic species (see 8.2.3) do not show more signs of and levels of pathogens and extraneous contaminants are likely
stress, such as discoloration, unusual behavior, or death, when to be more similar between compartments in the same test
held for at least 48 h in static dilution water in which the item chamber than between compartments in different test chambers
is soaking than when held in static dilution water that does not in the same treatment. Chambers should be covered to keep out
contain the item, or (2) their use will not adversely affect extraneous contaminants and, especially in static and renewal
survival, growth, or reproduction of a sensitive species. tests, to reduce evaporation of test solution and components of
the effluent. Also, chambers may need to be covered to prevent
6.4 Metering System:
organisms from jumping out. All chambers and compartments
6.4.1 For flow-through tests, the metering system should be
in a test must be identical.
designed to accommodate the type and concentration(s) of the
6.5.2 Test chambers may be constructed by welding, but not
effluent and the necessary flow rates of test solutions. The
soldering, stainless steel or by gluing double-strength or
system should mix the effluent with the dilution water imme-
stronger window glass with clear silicone adhesive. Stoppers
diately before they enter the test chambers and reproducibly
and silicone adhesive sorb some organochlorine and organo-
(see 6.4.4) supply the selected concentration(s) of effluent (see
phosphorus pesticides, which are then difficult to remove.
9.5). Various metering systems, using different combinations of
Therefore, as few stoppers and as little adhesive as possible
syringes, “dipping birds”, siphons, pumps, saturators,
should be in contact with test solution. If extra beads of
solenoids, valves, and so forth, have been used successfully to
adhesive are needed for strength, they should be on the outside
control the concentrations of effluent in, and the flow rates of,
of chambers rather than on the inside. Especially in static and
test solutions (see Guide E729).
renewal tests, the size and shape of the test chambers might
6.4.2 The following factors should be considered when
affect the results of tests on effluents that contain components
selecting a metering system: (1) the installation and use of the
that volatilize or sorb onto the chambers in substantial quan-
apparatus in a fixed or mobile laboratory; (2) availability of
tities.
adequate space and structural requirements for the system, test
6.5.3 The minimum dimensions of test chambers and the
chambers, and effluent and dilution water storage; (3) the
minimum depth of test solution depend on the size of the
applicability of the metering system to specific effluent char-
individual test organisms and the loading (see 11.4). The
acteristics (for example, high suspended solids, volatiles, and
smallest horizontal dimension of the test chambers should be at
so forth.); (4) the system’s dependability, durability, flexibility,
least three times the largest horizontal dimension of the largest
and ease of maintenance and replacement; (5) the ability to
test organism. The depth of the test solution should be at least
achieve the necessary precision for both flow rate and concen-
three times the height of the largest test organism. In addition,
tration; and (6) cost.
the test solution should be at least 150 mm deep for organisms
6.4.3 The metering system should be calibrated before use,
over 0.5 g (wet weight). Use of excessively large volumes of
and verified after the test to confirm that the targeted flow rates
solution in test chambers will probably unnecessarily increase
were met, by determining the flow rate through each test
the amount of dilution water and effluent used, and, in
chamber and measuring either the concentration of effluent in
flow-through tests, increase the average retention time.
each test chamber or the volume of solution used in each
6.5.4 For static and renewal tests, organisms weighing more
portion of the metering system. The general operation of the
than 0.5 g each (wet weight) are often exposed in 19 L (5 gal)
metering system should be visually checked daily in the
wide-mouth soft-glass bottles containing 15 L of solution or in
morning and afternoon throughout the test. The metering
300 mm by 600 mm by 300 mm deep all-glass test chambers.
system should be adjusted during the test if necessary.
Smaller organisms are often exposed in 3.8 L (1 gal) wide-
6.4.4 The flow rate through each test chamber should be at
mouth soft-glass bottles or battery jars containing 2 L to 3 L of
least five volume additions per 24 h. It is usually desirable to
solution. Daphnids, amphipods, juvenile freshwater mussels,
construct the metering system to provide at least ten volume
mayflies, and midge larvae are often exposed in 250 mL
additions per 24 h, in case (1) the loading is high (see 11.4) or
beakers containing 150 mL to 200 mL of solutionor 50-mL
(2) there might be rapid loss of components of the effluent due
beakers containing 30 mL of solution .
to microbial degradation, hydrolysis, oxidation, photolysis,
6.5.5 For flow-through tests, chambers may be constructed
reduction, sorption, or volatilization. At any particular time
by modifying glass bottles, glass canning jars, or beakers to
during the test, the flow rates through any two test chambers
provide screened overflow holes, standpipes, or V-shaped
should not differ by more than 10 %.
notches. Organisms weighing more than 0.5 g each (wet
6.5 Test Chambers: weight) are often exposed in 30 L of solution in 300 mm by
E1192 − 23
600 mm by 300 mm deep all-glass test chambers. Smaller putting hands in test solutions), laboratory coats, aprons, and
organisms are often exposed in 2 L to 4 L of solution. In tests glasses, and by using dip nets, forceps, or tubes to remove
with daphnids and other small species, the test chambers or organisms from test solutions. Special precautions, such as
metering system, or both, should be constructed so that the covering test chambers and ventilating the area surrounding the
organisms are not stressed by turbulence (6). chambers, should be taken when conducting tests on effluents
6.5.6 Embryos are often exposed in glass cups with stainless containing volatile materials. Information on toxicity to hu-
steel or nylon-screen bottoms or cups constructed by welding mans (8), recommended handling procedures (9), and chemical
stainless steel screen or gluing nylon screen with clear silicone and physical properties of components of the effluent should be
adhesive. The cups should be suspended in the test chambers so studied before a test is begun. Special procedures might be
as to ensure that the embryos are always submerged and that necessary with effluents that contain materials that are radio-
test solution regularly flows into and out of the cups without active (10), or are, or might be, carcinogenic (11).
creating too much turbulence.
7.2 Although disposal of effluent, test solutions, and test
6.6 Cleaning—The metering system, test chambers, and organisms poses no special problems in most cases, health and
equipment used to prepare and store dilution water, effluent, safety precautions and applicable regulations should be con-
and test solutions must be cleaned after use and may need to be sidered before beginning a test. Treatment of effluent and test
cleaned before use. New items should be washed with deter-
solutions might be desirable before disposal.
gent and rinsed with water, a water-miscible organic solvent,
7.3 Cleaning of equipment with a volatile solvent, such as
water, acid (such as 10 % concentrated hydrochloric acid
acetone, should be performed only in a well-ventilated area in
(HCl)), and at least twice with deionized, distilled, or dilution
which no smoking is allowed and no open flame, such as a pilot
water. (Some lots of organic solvents might leave a film that is
light, is present.
insoluble in water.) A dichromate-sulfuric acid cleaning solu-
7.4 An acidic solution should not be mixed with a hypo-
tion may be used in place of both the organic solvent and the
chlorite solution because hazardous fumes might be produced.
acid, but it might attack silicone adhesive. At the end of the
test, all items that will be used again should be immediately (1)
7.5 To prepare dilute acid solutions, concentrated acid
emptied, (2) rinsed with water, (3) cleaned by a procedure
should be added to water, not vice versa. Opening a bottle of
appropriate for removing known components of the effluent
concentrated acid and adding concentrated acid to water should
(for example, acid to remove metals and bases; detergent,
be performed only in a fume hood.
organic solvent, or activated carbon to remove organic
7.6 Because dilution water and test solutions are usually
chemicals), and (4) rinsed at least twice with deionized,
good conductors of electricity, use of ground fault systems and
distilled, or dilution water. Acid is often used to remove
− leak detectors should be considered to help prevent electrical
mineral deposits, and 200 mg of hypochlorite (ClO )/L is often
shocks. Salt water is such a good conductor that protective
used to remove organic matter and for disinfection. (A solution
− devices are strongly recommended.
containing about 200 mg ClO /L may be prepared by adding 6
7.7 To protect hands from being cut by sharp edges of
mL of liquid household chlorine bleach to 1 L of water.
However, hypochlorite is quite toxic to many aquatic animals shells, cotton work gloves should be worn (over appropriate
protective gloves (see 7.1) if necessary) when juvenile and
(7) and is difficult to remove from some construction materials.
It is often removed by soaking in a sodium thiosulfate, sodium adult bivalve molluscs are handled.
sulfite, or sodium bisulfite solution, by autoclaving in distilled
7.8 Personnel who will be handling an effluent or solutions
water for 20 min, or by drying the item and letting it sit for at
of it should discuss the advisability of immunization shots with
least 24 h before use. An item cleaned or disinfected with
medical personnel and should wash immediately after coming
hypochlorite should not be used unless it has been demon-
in contact with effluent or test solutions.
strated at least once that unfed individuals of a sensitive aquatic
7.9 Special handling and precautionary guidance in Material
species (see 8.2.3) do not show more signs of stress, such as
Safety Data Sheets (MSDS) should be followed for reagents
discoloration, unusual behavior, or death, when held for at least
and other chemicals purchased from supply houses.
48 h in static dilution water in which the item is soaking than
when held in static dilution water containing a similar item that
7.10 For further guidance on safe practices when handling
was not treated with hypochlorite.) The metering system and
field-collected samples and conducting toxicity tests, check
test chambers should be rinsed with dilution water just before
with the permittee and consult general industrial safety manu-
use.
als (Test Method E1706).
6.7 Acceptability—The acceptability of new holding,
7.11 Work with some field-collected samples may require
acclimation, and testing facilities should be demonstrated with
compliance with rules pertaining to the handling of hazardous
a sensitive species (see 8.2.3) before use.
materials. Guidelines for the handling and disposal of hazard-
ous materials should be strictly followed (Guide D4447). The
7. Hazards
Federal Government has published regulations for the manage-
7.1 Many materials can adversely affect humans if precau- ment of hazardous waste and has given the States the option of
tions are inadequate. Therefore, skin contact with all effluents either adopting those regulations or developing their own. If
and solutions should be minimized by wearing appropriate States develop their own regulations, these regulations are
protective gloves (especially when washing equipment or required to be at least as stringent as the Federal regulations. As
E1192 − 23
a handler of hazardous materials, it is your responsibility to and pH within 0.2 units of those of the receiving water at the
know and comply with the pertinent regulations applicable in time of the test may be used. It is also desirable that the
the State in which you are operating (Test Method E1706). alkalinity and conductivity be within 25 % of those of the
receiving water at the time of the test. If a reconstituted water
7.12 Appropriate measures and practices should be imple-
is used for the dilution water, procedures for preparing the
mented to prevent the spread of non-target species when
water should be carefully followed (see Guide E729).
acquiring or transferring the target test species. Additionally,
8.2.3 Chlorinated water should not be used as, or in the
bio-secure practices should be utilized when working with
preparation of, dilution water because residual chlorine and
either nonlocalized or non-native species to prevent escape-
chlorine-produced oxidants are quite toxic to many aquatic
ment into local watersheds and potentially altering or nega-
animals (7). Dechlorinated water should be used only as a last
tively influencing existing ecosystems.
resort because dechlorination is often incomplete. Sodium
8. Dilution Water bisulfite is probably better for dechlorinating water than
sodium sulfite and both are more reliable than carbon filters,
8.1 Requirements—Besides being available in adequate
especially for removing chloramines (14). Some organic
supply, the dilution water should be acceptable to the test
chloramines, however, react slowly with sodium bisulfite (15).
organisms and the purpose of the test. The minimal require-
In addition to residual chlorine, municipal drinking water often
ment for an acceptable dilution water for acute toxicity tests is
contains unacceptably high concentrations of copper, lead,
that healthy organisms survive in it through acclimation and
zinc, and fluoride, and quality is often rather variable. Exces-
testing without showing signs of stress, such as discoloration,
sive concentrations of most metals can usually be removed
unusual behavior, or death. A better criterion for an acceptable
with a chelating resin (16), but use of a different dilution water
dilution water is that at least one species of aquatic animal
might be preferable. If dechlorinated water is used as dilution
(preferably the one being tested or one taxonomically similar)
water or in its preparation, during the test either it must be
can survive, grow, and reproduce satisfactorily in it.
shown at least three times each week on nonconsecutive days
8.2 Source:
that in fresh samples of dilution water either (1) copepods
8.2.1 The dilution water for effluent toxicity tests should be
(Acartia tonsa), mysids (less than 24-h post-release from the
a representative sample of the receiving water obtained as close
brood sac), bivalve mollusc larvae, or daphnids (less than 24-h
to the point of discharge as possible but upstream of or outside
old) do not show more signs of stress, such as discoloration,
the zone of influence of the effluent. Other factors, such as
unusual behavior, or death, when held in the water for at least
possible toxicity, eutrophication, and indigenous food should
48 h without food than when similarly held in a water that was
be considered in selecting a collecting site. The dilution water
not chlorinated and dechlorinated; or (2) the concentration of
should be obtained from the receiving water as close to the start
residual chlorine in fresh water is less than 11 μg/L or the
of the test as practical but never more than 96 h prior to the
concentration of chlorine-produced oxidants in salt water is
beginning of the test. If the receiving water contains effluent
less than 6.5 μg/L (7).
from one or more other dischargers, it might be desirable to
8.2.4 When dilution water is to be transported to the test
collect dilution water further upstream or further away from the
facility, one or more tanks of adequate capacity may be filled
point of discharge either in addition to or as an alternative to
daily. With highly toxic effluents requiring very large volumes
the receiving water. When a test is conducted on effluent being
of dilution water to produce the desired test concentrations, it
discharged into an estuary, it might be more practical to
might be convenient to conduct the test near the source of
transport the dilution water to the test facility. Dilution water is
dilution water and transport the effluent.
often collected from an estuary at slack high tide, but this
8.2.5 In some situations the selected dilution water might
might contain effluent that was backwashed upstream during
adversely affect the test organisms. Therefore it is sometimes
the incoming tide. Therefore, it might be preferable to collect
desirable to include a performance control in the test, that is, to
the dilution water on the outgoing tide close to, but upstream
maintain organisms during the test in the water from which
of, the mixing zone. Also note that some receiving waters
they were obtained in order to determine whether any effects
contain ion concentrations outside or near the tolerance limits
seen in the dilution-water control were due to the quality of the
of typical toxicity testing organisms. For example, The “US
water or the quality of the organisms.
Lab” strain of amphipod (Hyalella azteca) has an additional
8.3 Treatment:
requirement for chloride concentrations higher than those
present in many natural or reconstituted waters (12). Growth 8.3.1 Dilution water may be aerated by such means as air
and reproduction of H. azteca also declines when chloride stones, surface aerators, or column aerators (17, 18), prior to
concentrations are below 15 mg/L (13). Importantly, the “US addition of the effluent. Adequate aeration will bring the pH
Lab” strain of H. azteca also showed greater sensitivity to the and concentrations of dissolved oxygen and other gases into
acute toxic effects of sodium sulfate and sodium nitrate at equilibrium with air and minimize oxygen demand and con-
lower chloride concentrations of <10 mg/L. Special attention to centrations of volatiles. The concentration of dissolved oxygen
this constituent is required when selecting culture and test in dilution water should be between 90 and 100 % of saturation
water for H. azteca (Test Method E1706). (19) to help ensure that dissolved oxygen concentrations are
8.2.2 If an acceptable dilution water cannot be obtained acceptable in test chambers. Supersaturation by dissolved
from the receiving water, an uncontaminated, well-aerated gases, which might be caused by heating the dilution water,
surface or ground water with hardness or salinity within 10 % should be avoided to prevent gas bubble disease (18, 20).
E1192 − 23
8.3.2 Dilution water may be filtered through a noncontami- 9.2.2 Ambient samples may be collected using a variety of
nating (for example, nylon) sieve with 2 mm or larger holes to methods, depending on the nature of the source. For example,
remove debris and break up large floating or suspended solids. flow proportional sampling is often appropriate for collection
If necessary, dilution water may be filtered through a sieve with of storm water run-off; grab samples might be adequate for
smaller holes (for example, 35 μm is sufficiently small) to pond samples; tidal estuaries might be sampled using a
remove parasites and predatory organisms if the test organisms composite sample.
are small. 9.2.2.1 Regardless of the sampling technique employed,
effluent samples should be used for testing within 36 h after the
8.3.3 When toxicity tests are conducted with saltwater
end of the collection period, unless it has been shown that
species, the freshwater component of an effluent might cause
toxicity does not change with time.
an additional stress just as would an extreme pH. Similarly, an
9.2.3 Flow-through toxicity tests should generally be con-
effluent with a high salt content might cause an additional
ducted on effluent.
stress in tests with freshwater species. In order to measure the
9.2.4 In most cases, continuous, composite, or grab sam-
whole impact of the effluent, the salinity of the effluent should
pling . will be suitable for testing. In some cases (such as storm
not be adjusted and the salinity of dilution water should be
water run-off events or in ambient sample collection) flow-
equal to that of the receiving water outside the zone of
proportional sampling might be most appropriate. It is recom-
influence of the effluent. This same dilution water without the
mended that provision be made for cooling samples to 4 °C
addition of effluent should be used in the dilution-water control
during the collection of composite samples. In some cases,
treatment. If it is desired to determine the toxicity of the
flow-proportional sampling might be desirable. Such situations
effluent in the absence of any stress due to high or low salinity,
will be governed by the effect of significant flow variation on
the salinity of the effluent or the dilution water, or both, may be
the retention time of the effluent, and in turn, the effect of
adjusted. Adjustment of the salinity of the effluent might affect
altered retention time on loss of components of the effluent.
the toxicity of the effluent. As an alternative to adjusting
Generally, losses will occur either (1) in a treatment basin, or
salinity, it might be desirable to conduct a test with a species
(2) due to hydrolysis or other naturally occurring phenomenon.
that can tolerate both fresh and salt water.
Flow-proportional sampling, therefore, is recommended only
8.4 Characterization—The following items should be mea-
when the variation in flow has a substantial effect relative to
sured on each batch of dilution water (or daily if dilution water
these factors. Other sampling techniques are described in detail
is pumped continuously from a surface water source):
by Shelley (21).
8.4.1 Fresh Water—Hardness, alkalinity, conductivity, pH,
9.3 Preservation—If samples are not used within approxi-
particulate matter, and total organic carbon.
mately 2 h of collection, they should be preserved by storing
8.4.2 Salt Water—Salinity or chlorinity, pH, particulate
them in the dark at about 4°C.
matter, and total organic carbon.
9.4 Treatment—Except as per 8.3.3, the sample of effluent
8.4.3 For each analytical method used (see
12.2) the detec-
must not be altered except that it may be filtered through a
tion limit should be below the concentration in the dilution
nylon (or comparable) sieve or screen with 2 mm or larger
water.
holes. Undissolved materials should be uniformly dispersed by
gentle agitation immediately before any sample of effluent is
9. Effluent
distributed to test chambers.
9.1 Sampling Point—The effluent sampling point should be
9.5 Test Concentration(s):
the same as that specified in the National Pollutant Discharge
9.5.1 If the test is intended to allow calculation of an LC50
Elimination System (NPDES) permit if the test is conducted
or EC50, the test concentrations (see 11.1.1.1) should bracket
for NPDES monitoring purposes (21). In some cases, a
the predicted LC50 or EC50. A prediction might be based on
sampling point between first treatment and the discharge point
the results of a test on the same or a similar effluent with the
might provide much better access. If the treated waste is
same or a similar species. If a useful prediction is not available,
chlorinated, it might be desirable to have sampling points both
it is usually desirable to include additional lower effluent
upstream and downstream of the chlorine contact point to
concentrations in the design to ensure bracketing of the LC50.
determine the toxicity of both chlorinated and unchlorinated
9.5.2 In some situations (usually regulatory), it is only
effluent. The schedule of effluent sampling should be based on
necessary to determine (1) whether a specific concentration is
an understanding of the short- and long-term operations and
acutely toxic to the test species or (2) whether the LC50 or
schedules of the discharger. Although it is usually desirable to
EC50 is above or below a specific concentration. For example,
evaluate an effluent sample that most clearly represents the
the specific concentration might be a concentration specified by
normal or typical discharge, conducting tests on atypical
a regulatory agency. When there is interest only in a specific
samples might also be informative.
concentration, it is often necessary only to test that concentra-
9.2 Collection:
tion (see 11.1.1.2), and it is not necessary to actually determine
9.2.1 Several different methods may be used to collect
the LC50 or EC50.
effluent samples for toxicity tests. However, a specific sam-
10. Test Organisms
pling method is frequently specified in the NPDES permit.
Selection of a method should be based on the type of test that 10.1 Species—For many effluent and ambient water tests the
is to be conducted and the characteristics of the effluent. test species is recommended by a regulatory agency. Whenever
E1192 − 23
possible, effluent tests should be conducted with a sensitive, luted areas, although some amphibian species are also avail-
important species indigenous to or regularly stocked into the able from commercial suppliers. Importing and collecting
receiving water. However, species sensitivity will depend on permits might be required by local and state agencies. Organ-
the receiving water, the composition of the effluent, and so isms captured by electroshocking, chemical treatment, and gill
forth, and is, therefore, generally difficult to determine without nets should not be used.
conducting tests with a variety of species. If the objective of
10.4 Care and Handling—Organisms should be cared for
the test is to determine the site-specific toxicity of an effluent
and handled properly (22) so they are not unnecessarily
or ambient sample, tests are usually conducted with a readily
stressed.
available, commercially, or recreationally important indig-
10.4.1 Whenever aquatic animals are brought into a facility,
enous species (see Guide E729). The species used should be
they should be quarantined (1) until used or (2) for 14 days or
identified using an appropriate taxonomic key.
until they appear to be disease free, whichever is longer. Dip
10.2 Age—All organisms in a test should be uniform in age
nets, brushes, other equipment, organisms, or water should not
and size.
be transferred from a quarantined tank to any other tank
10.2.1 Fish—Use of fish weighing between 0.1 g and 5.0 g
without being autoclaved in distilled water or sterilized.
each is usually desirable. Unless data on another life stage are
10.4.2 To maintain aquatic animals in good condition and
specifically desired, tests should be conducted with juvenile
avoid unnecessary stress, they should not be crowded or
fish, that is, post-larval or older and actively feeding, but not
subjected to rapid changes in temperatu
...