ASTM E1604-20

Standard Guide for Behavioral Testing in Aquatic Toxicology

SIGNIFICANCE AND USE
5.1 Protection of a species requires the prevention of detrimental effects of chemicals on the survival, growth, reproduction, and health of that species. Behavioral toxicity provides information concerning sublethal effects of chemicals and signals the presence of toxic test substances.  
5.1.1 The behavioral responses of all organisms are adaptive and essential to survival. Major changes in the behavioral responses of fish, amphibians, and macroinvertebrates may result in a diminished ability to survive, grow, or reproduce and cause significant changes in the natural population (8).  
5.2 The results from behavioral toxicity tests may be useful for measuring injury in the assessment of damages resulting from the release of hazardous materials (9) .  
5.3 Behavioral toxicity test methods may be useful for long-term monitoring of effluents (10) .  
5.4 The results from behavioral toxicity data can be used to predict the effects of exposure on fish, amphibians, and aquatic invertebrates likely to occur in field situations as a result of exposure under similar conditions, including the avoidance of exposure by motile organisms (11).  
5.5 The results from behavioral toxicity tests might be an important consideration for assessing the hazard of materials to aquatic organisms. Such results might also be used when deriving water quality criteria for fish and aquatic invertebrate organisms.  
5.6 The results from behavioral toxicity tests can be used to compare the sensitivities of different species, relative toxicity of different chemical substances on the same organism, or effect of various environmental variables on the toxicity of a chemical substance.  
5.7 The results from behavioral toxicity tests can be used to predict the effects of long-term exposure.  
5.8 The results of behavioral toxicity tests can be useful for guiding decisions regarding the extent of remedial action needed for contaminated aquatic and terrestrial sites.  
5.9 The behavioral charac...
SCOPE
1.1 This guide covers some general information on the selection and application of behavioral methods useful for determining the sublethal effects of chemicals to fish, amphibians, and macroinvertebrates.  
1.2 Behavioral toxicity occurs when chemical or other stressful conditions, such as changes in water quality or temperature, induce a behavioral change that exceeds the normal range of variability  (1).2 Behavior includes all observable, recordable, or measurable activities of a living organism and reflects genetic, neurobiological, physiological, and environmental determinants  (2).  
1.3 Behavioral methods can be used in biomonitoring, the determination of no-observed-effect and lowest-observed-effect concentrations, and the prediction of hazardous chemical impacts on natural populations (3).  
1.4 Behavioral methods can be applied to fish, amphibians, and macroinvertebrates in standard laboratory toxicity tests, tests of effluents, and sediment toxicity tests.  
1.5 The various behavioral methods included in this guide are categorized with respect to seven interdependent, functional responses that fish, amphibians, and macroinvertebrates must perform in order to survive. These functional responses include respiration, locomotion, habitat selection, feeding, predator avoidance, competition, and reproduction (4). These responses can be documented visually or through video or acoustic imagery. Electronically recorded information can be derived through manual techniques or through the use of digital image analysis software (5, 6, 7).  
1.5.1 The functional responses are not necessarily mutually exclusive categories. For instance, locomotion, of some form of movement, is important to all behavioral functions.  
1.6 Additional behavioral methods for any category may be added when new tests are developed as well as when methods are adapted to different species or different life stages of an organism.  
1.7 This gui...

General Information

• Status: Published
• Publication Date: 30-Nov-2020
• ICS: 07.060 - Geology. Meteorology. Hydrology
• Technical Committee: E50 - Environmental Assessment, Risk Management and Corrective Action
• Drafting Committee: E50.47 - Biological Effects and Environmental Fate

Relations

• Refers: ASTM E943-08 - Standard Terminology Relating to Biological Effects and Environmental Fate
• Effective Date: 01-Mar-2008
• Refers: ASTM E1733-95(2008) - Standard Guide for Use of Lighting in Laboratory Testing
• Effective Date: 01-Feb-2008
• Refers: ASTM E1192-97(2008) - Standard Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents with Fishes, Macroinvertebrates, and Amphibians
• Effective Date: 01-Feb-2008
• Refers: ASTM E729-96(2007) - Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
• Effective Date: 01-Oct-2007
• Refers: ASTM E1023-84(2007) - Standard Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses
• Effective Date: 01-Oct-2007

Overview

ASTM E1604-20: Standard Guide for Behavioral Testing in Aquatic Toxicology provides comprehensive guidance on selecting and applying behavioral test methods to evaluate the sublethal effects of chemicals on aquatic organisms, including fish, amphibians, and macroinvertebrates. Recognizing behavior as an adaptive function essential for survival, this standard supports toxicological assessment by focusing on behavioral endpoints that directly relate to the ecological fitness and health of aquatic populations. Behavioral toxicity tests are vital tools for detecting the presence of hazardous substances before more severe effects occur, making them crucial for environmental risk assessments and long-term ecosystem monitoring.

Key Topics

• Behavioral Toxicity: This guide emphasizes the importance of observable, measurable activities in aquatic organisms as indicators of sublethal stress. Behavioral changes can reveal impacts not detected through lethality tests.
• Functional Behavioral Responses: Key behavioral categories include:
  • Respiration
  • Locomotion
  • Habitat selection
  • Feeding
  • Predator avoidance
  • Competition
  • Reproduction
• Test Organisms: Methods apply to a range of aquatic species and life stages, supporting broad ecological relevance.
• Testing Applications: Behavioral methods can be integrated into standard laboratory toxicity tests, effluent assessments, and sediment toxicity studies.
• Experimental Design: Robust test facility requirements, water quality controls, and randomized assignment of organisms and treatments are critical for data reliability.
• Data Utilization: Results from behavioral tests can be used to:
  • Compare species sensitivities
  • Assess chemical hazards
  • Guide water quality criteria development
  • Inform remedial action for contaminated sites

Applications

Behavioral toxicity assessments under ASTM E1604-20 offer several practical uses in environmental science and regulatory frameworks:

• Biomonitoring: Behavioral endpoints help track the ongoing impact of effluents and chemical discharges in aquatic environments.
• Early Warning & Risk Assessment: Sublethal effects often precede population declines. Timely detection through behavioral observations enables proactive environmental management.
• Water Quality Criteria: Data supports the derivation of science-based criteria for aquatic life protection, often required in regulatory decision-making.
• Injury Assessment: Behavioral responses aid in evaluating damages from accidental releases of hazardous substances.
• Comparative Toxicology: Sensitivities of different organisms to specific chemicals are compared, informing both site-specific and broader ecological risk assessments.
• Remediation Guidance: Behavioral tests inform the extent of cleanup necessary at contaminated aquatic or terrestrial sites, ensuring recovery of ecosystem functions.

Related Standards

ASTM E1604-20 is closely linked to several other key ASTM standards that provide additional guidance for aquatic toxicity testing and terminology:

• ASTM E729 - Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
• ASTM E943 - Terminology Relating to Biological Effects and Environmental Fate
• ASTM E1023 - Assessing the Hazard of a Material to Aquatic Organisms
• ASTM E1192 - Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents
• ASTM E1241 - Conducting Early Life-Stage Toxicity Tests with Fishes
• ASTM E1711 - Measurement of Behavior During Fish Toxicity Tests
• ASTM E1847 - Statistical Analysis of Toxicity Tests Conducted Under ASTM Guidelines

These supporting standards ensure harmonized terminology, robust experimental design, and data quality across aquatic toxicology assessments.

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Keywords: behavioral toxicity, aquatic toxicology, sublethal effects, ASTM E1604, environmental monitoring, chemical hazard assessment, water quality, ecological risk assessment, fish, amphibians, macroinvertebrates, biomonitoring.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E1604 − 20
Standard Guide for
Behavioral Testing in Aquatic Toxicology
This standard is issued under the fixed designation E1604; 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 1.6 Additional behavioral methods for any category may be
added when new tests are developed as well as when methods
1.1 This guide covers some general information on the
are adapted to different species or different life stages of an
selection and application of behavioral methods useful for
organism.
determining the sublethal effects of chemicals to fish,
1.7 This guide is arranged as follows:
amphibians, and macroinvertebrates.
Section Number
1.2 Behavioral toxicity occurs when chemical or other
Scope 1
Referenced Documents 2
stressful conditions, such as changes in water quality or
Terminology 3
temperature, induce a behavioral change that exceeds the
Summary of Guide 4
normal range of variability (1). Behavior includes all Significance and Use 5
Interferences 6
observable, recordable, or measurable activities of a living
Test Facility 7
organism and reflects genetic, neurobiological, physiological,
Water Supply 8
Safety Precautions 9
and environmental determinants (2).
Test Material 10
Test Organisms 11
1.3 Behavioral methods can be used in biomonitoring, the
Responses Measured 12
determination of no-observed-effect and lowest-observed-
Behavioral Test Method Selection Criteria 13
effectconcentrations,andthepredictionofhazardouschemical Experimental Design 14
Acceptability of Test 15
impacts on natural populations (3).
Calculation of Test Results 16
Report 17
1.4 Behavioral methods can be applied to fish, amphibians,
1.8 The values stated in SI units are to be regarded as the
and macroinvertebrates in standard laboratory toxicity tests,
standard. For an explanation of units and symbols, refer to
tests of effluents, and sediment toxicity tests.
IEEE/ASTM SI 10.
1.5 The various behavioral methods included in this guide
1.9 This standard does not purport to address all of the
are categorized with respect to seven interdependent, func-
safety concerns, if any, associated with its use. It is the
tional responses that fish, amphibians, and macroinvertebrates
responsibility of the user of this standard to establish appro-
must perform in order to survive. These functional responses
priate safety, health, and environmental practices and deter-
include respiration, locomotion, habitat selection, feeding,
mine the applicability of regulatory limitations prior to use.
predator avoidance, competition, and reproduction (4). These
Specific precautionary statements are given in Section 9.
responses can be documented visually or through video or
1.10 This international standard was developed in accor-
acoustic imagery. Electronically recorded information can be
dance with internationally recognized principles on standard-
derivedthroughmanualtechniquesorthroughtheuseofdigital
ization established in the Decision on Principles for the
image analysis software (5, 6, 7).
Development of International Standards, Guides and Recom-
1.5.1 The functional responses are not necessarily mutually
mendations issued by the World Trade Organization Technical
exclusive categories. For instance, locomotion, of some form
Barriers to Trade (TBT) Committee.
of movement, is important to all behavioral functions.
2. Referenced Documents
2.1 ASTM Standards:
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
E729 Guide for Conducting Acute Toxicity Tests on Test
Assessment, Risk Management and CorrectiveActionand is the direct responsibility
of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Dec. 1, 2020. Published January 2021. Originally
approved in 1994. Last previous edition approved in 2012 as E1604-12. DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/E1604-20. contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
The boldface numbers in parentheses refer to the list of references at the end of Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1604 − 20
Materials with Fishes, Macroinvertebrates, and Amphib- stancesinwater,effluents,food,orsedimentsiscomparedwith
ians the behavioral responses of control organisms. The behavioral
E943 Terminology Relating to Biological Effects and Envi- responses measured during toxicity tests are highly sensitive to
ronmental Fate sublethal exposure. The behavioral measures are relevant to
E1023 Guide for Assessing the Hazard of a Material to essential life functions that fish, amphibians, and macroinver-
Aquatic Organisms and Their Uses tebrates often must perform in order to survive and include
E1192 Guide for ConductingAcute Toxicity Tests onAque- respiration, locomotion, habitat selection, feeding, predator
ous Ambient Samples and Effluents with Fishes, avoidance, competition, and reproduction. Data are obtained to
Macroinvertebrates, and Amphibians determine the effects of toxic substances on behavior from
E1241 GuideforConductingEarlyLife-StageToxicityTests short (for example, 1 h) or long-term (partial to full life cycle)
with Fishes exposures.
E1383 Guide for Conducting Sediment Toxicity Tests with
Freshwater Invertebrates (Withdrawn 1995)
5. Significance and Use
E1711 Guide for Measurement of Behavior During Fish
5.1 Protection of a species requires the prevention of detri-
Toxicity Tests
mental effects of chemicals on the survival, growth,
E1733 Guide for Use of Lighting in Laboratory Testing
reproduction, and health of that species. Behavioral toxicity
E1847 Practice for Statistical Analysis of Toxicity Tests
provides information concerning sublethal effects of chemicals
Conducted Under ASTM Guidelines
and signals the presence of toxic test substances.
IEEE/ASTM SI 10 American National Standard for Metric
5.1.1 Thebehavioralresponsesofallorganismsareadaptive
Practice
and essential to survival. Major changes in the behavioral
responses of fish, amphibians, and macroinvertebrates may
3. Terminology
resultinadiminishedabilitytosurvive,grow,orreproduceand
3.1 Definitions—The words “must,” “should, “may,” “can,”
cause significant changes in the natural population (8).
and “might” have very specific meanings. “Must” is used to
5.2 The results from behavioral toxicity tests may be useful
express an absolute requirement, that is, to state that the test
for measuring injury in the assessment of damages resulting
ought to be designed to satisfy the specified condition, unless
from the release of hazardous materials (9).
the purpose of the test requires a different design. “Must” is
used only in connection with the factors that directly relate to
5.3 Behavioral toxicity test methods may be useful for
the acceptability of the test. “Should” is used to state that the
long-term monitoring of effluents (10).
specified condition is recommended and ought to be met if
5.4 The results from behavioral toxicity data can be used to
possible. Although the violation of one “should” is rarely a
predict the effects of exposure on fish, amphibians, and aquatic
serious matter, violation of several will often render the results
invertebrates likely to occur in field situations as a result of
questionable.Termssuchas“isdesirable,”“isoftendesirable,”
exposure under similar conditions, including the avoidance of
and “might be desirable” are used in connection with less
exposure by motile organisms (11).
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
5.5 The results from behavioral toxicity tests might be an
mean “could possibly.” Thus the classic distinction between
importantconsiderationforassessingthehazardofmaterialsto
“may” and “can” is preserved, and “might” is never used as a
aquatic organisms. Such results might also be used when
synonym for either “may” or “can.”
deriving water quality criteria for fish and aquatic invertebrate
3.2 Definitions of Terms Specific to This Standard:
organisms.
3.2.1 behavior—the complex of observable, recordable, or
5.6 The results from behavioral toxicity tests can be used to
measurable activities of a living organism.
compare the sensitivities of different species, relative toxicity
3.2.2 behavioral toxicity—the phenomenon observed when
of different chemical substances on the same organism, or
a behavioral response varies beyond the range of normal as a
effect of various environmental variables on the toxicity of a
result of exposure to chemical or other stressors.
chemical substance.
3.3 For definitions of other terms used in this guide, refer to
5.7 The results from behavioral toxicity tests can be used to
Guides E729, E1192, E1241, E1383, E1711, E1847, and
predict the effects of long-term exposure.
Terminology definitions in Guide E943.
5.8 The results of behavioral toxicity tests can be useful for
4. Summary of Guide
guiding decisions regarding the extent of remedial action
needed for contaminated aquatic and terrestrial sites.
4.1 The potential toxicity of chemical substances in water,
food, or sediments is assessed by measuring the behavior of
5.9 The behavioral characteristics of a particular organism
fish, amphibians, and macroinvertebrates during exposure,
must be understood and defined before a response can be used
using static, flow-through, or food exposure systems. The
as a measure of toxicity. The range of variability of any
behavioral response of organisms exposed to chemical sub-
behavioral response of unexposed organisms is influenced by
genetic, experiential, physiological, and environmental factors.
Thus it is important to avoid selecting test organisms from
The last approved version of this historical standard is referenced on
www.astm.org. populations that may vary significantly in these factors.
E1604 − 20
5.10 The results of behavioral toxicity tests will depend on 7.1.2 Atiming device should be used to provide a light:dark
the behavioral response measured, testing conditions, water cycle.A15 to 30-min transition period, allowing for a gradual
quality, species, genetic strain, life stage, health, and general
change in light intensity when the lights are turned on or off,
condition of test organisms.Therefore, the behavioral response may be desirable for reducing stress caused by instantaneous
may be affected by the test environment.
illumination or darkness (See Guide E1733).
6. Interferences
7.2 Construction Materials—Consistent with specifications
delineated, for example, in Guide E1241, equipment and
6.1 A number of factors can suppress, elicit, or alter
facilities that come into contact with stock solutions, test
behavioral responses and thus influence behavioral test results
solutions, food, sediment, air, or water, into which the test
and complicate data interpretation. The following factors
organismsareplaced,shouldnotcontainsubstancesthatcanbe
should be considered in the experimental protocol or in the
leached or dissolved in amounts that affect the test organisms
discussion of results when measuring behavioral responses
adversely.Thematerialsshouldbechosentominimizesorption
during toxicity tests:
of test materials.
6.1.1 The pretest handling of test organisms resulting from
collection, transfer, and maintenance of the culture environ-
7.3 Water and Air Delivery Systems—The water delivery
ment can affect the response observed during exposure to toxic
system used in flow-through testing can be one of several
substances.
designs. The system should be capable of delivering equal
6.1.2 The health, nutritional state, and physical condition of
volumes of water at an equal rate of flow to each replicate
the organism can influence the test.
treatment container. Various metering systems, using different
6.1.3 Behavioral responsiveness may vary by species, ge-
combinations of siphons, pumps, solenoids, valves, etc., have
neticstrain,population,gender,anddevelopmentalstageofthe
been used successfully to control the flow rates of water and
organism.
toxic substances (see Guides E729, E1023, E1241, E1383, and
6.1.4 Prior exposure to hazardous materials, environmental
E1847).
stresses, and pathogens can affect the behavioral responses.
7.3.1 The metering system should be calibrated before the
6.1.5 Social status, such as dominance or sex of the indi-
test by determining the flow rate of water and air through each
viduals tested, and experiential factors, such as prior experi-
test chamber. The general operation of the metering system
encewithpredatororpreyspecies,caninfluencethebehavioral
shouldbevisuallycheckeddailythroughoutthetest.Thewater
response. Individuals tested in isolation may respond differ-
delivery system should be adjusted during the test if necessary.
ently than when tested in groups.
At any particular time during the test, flow rates through any
6.1.6 Cyclical changes (circadian, seasonal, annual, and
two test chambers should not differ by more than 10 %.
reproductive) in behavioral responses can occur.
6.1.7 The behavioral response can be affected by apparatus
7.4 Test Chambers—In a behavioral toxicity test with fish,
design and by the procedural sequence of the measurement
amphibians, and macroinvertebrates, the measurement of be-
method.
havioral response may take place directly in the exposure
6.1.8 Behavioral responses will vary according to the extent
vessel, or the organisms may be transferred to a specific
to which test organisms acclimate to the physical variables of
apparatusorobservationchamberforthepurposeofmeasuring
the testing environment, including water quality, temperature,
a behavioral response (see Section 12). The independent
water flow, light, cover, and substrate, as well as their recovery
experimental unit for such tests is based on the smallest
from handling, acceptance of diet, and adjustment to novel
physical exposure unit between which there are no water, air
testing chambers.
connections, or common access to sediment or food. All test
6.1.9 Behavioral responses to toxic substances may change
chambers must be identical, and the test compartments within
over time.
each chamber must be identical and placed in analogous
locations within each test chamber.
7. Test Facility
7.4.1 Testchambersmaybeconstructedinseveralwaysand
7.1 Facilities—The facility should include a constant tem-
ofvariousmaterials,dependingontheexperimentaldesignand
perature area for culturing and testing. Test and culture
contaminants of interest. Clear silicone adhesives, suitable for
chambers may be placed in a temperature-controlled recircu-
aquaria, should be used sparingly since they sorb some organic
latingwaterbathorinaconstant-temperaturearea.Airusedfor
compounds that may be difficult to remove. New test chambers
aerationshouldbefreeoffumes,oil,andwaterandcanrequire
sealed with silicone adhesives should be weathered for at least
filters to remove oil, water, and bacteria. The test facility
48hinwaterofthesamequalityasthatusedinthetoxicitytest
shouldbewell-ventilatedandfreeoffumes.Enclosuresmaybe
to leach potentially toxic compounds from the adhesive.
necessary to ventilate test chambers. Descriptions of toxicity
testing equipment appear Guides E729, E1023, E1192, E1241, 7.4.2 Apparatus will vary according to the response being
measured and species and life stage being tested. Organisms
E1383, and E1711. These provide guidance on construction
materials, water and air delivery systems, test chambers and maybeobserveddirectlyintheexposurechamber,ortheymay
be transferred to specialized apparatus for measurement of the
cleaning, and water supply
7.1.1 Culture and animal care facilities should not be in a response. Recording of response may require (1) direct visual
observation, (2) video-recorded observation, or (3) electroni-
room in which toxicity tests are conducted, stock solutions or
test solutions are prepared, or equipment is cleaned. cally recorded observation.
E1604 − 20
7.5 Cleaning—Test chambers, water delivery systems, surface water is used, the intake should be positioned to
equipment used for preparing and storing exposure water, and minimize fluctuations in quality and the possibility of contami-
stock solutions should be cleaned before use. Consistent with nation;tomaximizetheconcentrationofdissolvedoxygen;and
Guide E729, new items should be washed in the following to help ensure low concentrations of sulfide and iron. Munici-
manner: (1) detergent wash, (2) tap water rinse, (3) water- pal water supplies can contain unacceptably high concentra-
miscible organic solvent wash, (4) tap water rinse, (5) acid tions of copper, lead, zinc, fluoride, chlorine, or chloramines,
wash (such as 10 % concentrated hydrochloric acid), and (6) and quality can be variable. Chlorinated water should not be
rinse at least twice with distilled, deionized, or test water. Test used for, or in the preparation of, exposure water because
chambers should be rinsed with test water just before use. residual chlorine and chlorine-produced oxidants are toxic to
7.5.1 Many organic solvents leave a film that is insoluble in many aquatic animals (12). Dechlorinated water should be
water. A 10 % nitric acid solution, for example, may cause used only as a last resort because dechlorination can be
deterioration of silicone adhesive. A rinse with 10 % concen- incomplete.
trated hydrochloric acid may be preferable. A dichromate-
8.2.2 For certain applications, the experimental design
sulfuricacidcleaningsolutioncangenerallybeusedinplaceof
mightrequiretheuseofwaterfromthetesteffluentorsediment
both the organic solvent and the acid (see Guide E729), but the
collection site.
solution might attack silicone adhesive and leave potentially
8.2.3 Reconstituted water is prepared by adding specified
mutagenic residues of chromium on glass. Non-chromium
amounts of reagent grade chemicals to high-quality distilled or
cleaning solutions are also available.
deionized water (see Guide E729).
7.5.2 Upon completion of a test, all items that are to be used
8.3 Characterization—The following items should be mea-
againshouldbeimmediately(1)emptiedofwater,sediment,or
sured at least twice each year, and more often if (1) such
effluent (which should be disposed of properly; (2) rinsed with
measurements have not been determined semiannually for at
water; (3) cleaned by a procedure appropriate for removing the
least two years or (2) if surface water is used: pH, particulate
testmaterial(forexample,acidtoremovemetalsandbasesand
matter, TOC, organophosphorus pesticides, organic halides,
detergent, organic solvent, or aqueous slurry of activated
organochlorine pesticides, polychlorinated biphenols (PCBs),
carbon to remove organic chemicals); and (4) rinsed at least
chlorinated phenoxy herbicides, ammonia, cyanide, sulfide,
twice with distilled, deionized, or overlying water.
bromide, chloride, fluoride, iodide, nitrate, phosphate, sulfate,
7.6 Acceptability—Before a toxicity test is conducted in
calcium, magnesium, sodium, potassium, aluminum, arsenic,
new test facilities, it is desirable to conduct a non-toxicant test,
beryllium, boron, cadmium, chromium, cobalt, copper, iron,
in which all test chambers contain uncontaminated water or
lead, manganese, mercury, molybdenum, nickel, selenium,
sediment. The behavior of the test species will demonstrate
silver, and zinc, hardness, alkalinity, and conductivity (see
whether facilities, water, control sediment, and handling tech-
Guide E729).
niques are adequate to result in acceptable species-specific
8.3.1 For each method used, the detection limit should be
control numbers. The magnitude of the within-chamber and
below (1) the concentration in the dilution water or (2) the
between-chamber variance should also be determined.
lowest concentration that has been shown to affect the test
species adversely (13).
8. Water Supply
8.3.2 Water that might be contaminated with facultative
8.1 Requirements—In addition to being available in ad-
pathogens may be passed through a properly maintained
equate supply, dilution water used in behavioral toxicity tests,
ultraviolet sterilizer (14) equipped with an intensity meter and
and water used to hold organisms before testing, should be
flow controls or passed through a filter with a pore size of 0.45
acceptable to test species and uniform in quality. To be
µm or less. Carbon filtration may be required to remove the
acceptable to the test species, the water must permit satisfac-
pathogenic toxins.
tory survival and growth, without inducing signs of disease or
8.3.3 Water may require aeration using air stones, surface
apparent stress, such as discoloration, or unusual behavior.
aerators, or column aerators (15-17). Adequate aeration will
8.2 Source—Natural overlying water should be uncontami-
stabilize the pH, bring the concentrations of dissolved oxygen
nated and of constant quality and should meet the following
and other gases into equilibrium with air, and minimize the
specifications as established in Guide E729. The values stated
oxygen demand and concentrations of volatiles. The concen-
help to ensure that the test organisms are not apparently
tration of dissolved oxygen in water should be between 90 and
stressed during holding, acclimation, and testing and that the
100 % saturation (17) to help ensure that the dissolved oxygen
test results are not affected unnecessarily by water character-
concentrations are acceptable in the test chambers. Precautions
istics: particulate matter, <5 mg/L; total organic carbon (TOC),
shouldbetaken,however,toensurethatglassairstonesarenot
<5 mg/L; chemical oxygen demand (COD), <5 mg/L; and
breaking down with use and that plastic air stones are not
residual chlorine, <11 µg/L.
absorbing organic chemicals.
8.2.1 A natural water source is considered to be of uniform
quality if the monthly ranges of the hardness, alkalinity, and
9. Safety Precautions
specific conductance are less than 10 % of their respective
averages and if the monthly range of pH is less than 0.4 unit. 9.1 Many substances may pose health risks to humans if
Natural waters should be obtained from an uncontaminated adequate precautions are not taken. Information on toxicity to
well or spring, if possible, or from a surface water source. If humans, recommended handling procedures, and chemical and
E1604 − 20
physicalpropertiesofthetestmaterialshouldbestudiedandall 11.5 All organisms should be as uniform as possible in age
personnel informed before an exposure is initiated. and size class.
11.6 All organisms in a test must be from the same source.
NOTE 1—Warning: Special procedures might be necessary with
radiolabeledtestmaterialsandwithtestmaterialsthatare,oraresuspected Organisms may be obtained from (1) laboratory cultures; (2)
of being, carcinogenic.
commercial, state, or federal institutions; or (3) natural popu-
lationsfromcleanareas.Laboratoryculturesoftestspeciescan
9.2 Many materials can affect humans adversely. Contact
provide organisms whose history, age, and quality are known.
with test material, sediments, and water should be minimized.
Local and state agencies may require collecting permits.
Where appropriate, protective gloves, laboratory coats, aprons,
protective clothing, and safety glasses should be worn, and dip
11.7 To maintain organisms in good condition and avoid
nets, sieves, or tubes should be used to remove test organisms.
unnecessary stress, they should not be crowded and should not
When handling potentially hazardous materials, proper han-
be subjected to rapid changes in temperature or water quality
dling procedures may include (1) manipulating test materials
characteristics.
under a ventilated hood or in an enclosed glovebox; (2)
11.8 The addition of shelter or refuge may be required for
enclosingandventilatingtheexposurechambers;and(3)using
certain species.
respirators, aprons, safety glasses, and gloves.
12. Responses Measured
10. Test Material
12.1 Respiration—Respiratory tissue is frequently in imme-
10.1 Test materials may include pure compounds or com-
diate contact with injurious substances. Disruptions in respira-
mercial formulations of compounds that are added to water or
tory behavior arise when the substance reduces respiratory
sediment.Testmaterialscollectedfromfieldlocationsmayalso
efficiency, affects neurological control of respiration, or irri-
include complex mixtures of chemical compounds in effluents
tates respiratory membranes (10). Respiratory variables com-
and sediments.
monly measured include respiratory frequency, respiratory
10.2 Considerations for technical test materials for use in
volume, and the analog waveform characteristics of the respi-
aqueous tests, preparations of stock solutions, use of solvents,
ratory cycle.
andselectionoftestconcentrationsofaqueoussolutionsshould
12.2 Locomotion—Locomotory responses are essential to
follow those outlined in Guide E1241.
survival in most organisms and are often very sensitive to
10.3 Tests using sediments as the exposure media should
hazardous substances (18). Disruption of locomotory behavior
follow Guide E1383 for the characterization, collection,
can impair the ability of fish, amphibians, and macroinverte-
storage, preparation of spiked sediment samples, and test
brates to perform essential life functions that might rely on
concentrations of spiked sediment samples.
agile, efficient, and vigorous swimming. Variables of locomo-
tory behavior commonly measured include the frequency and
11. Test Organisms duration of activity, form and posture of locomotion, larval
development of locomotion, physical capacity for swimming,
11.1 Species and life stages selected for study will depend
and bioenergetics. Locomotion may also include the respira-
on the focus of the study and may include standard bioassay
tory and feeding movements of sessile organisms.
organisms when the relative toxicity of a compound is to be
determine
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E1604 − 12 E1604 − 20
Standard Guide for
Behavioral Testing in Aquatic Toxicology
This standard is issued under the fixed designation E1604; 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 some general information on the selection and application of behavioral methods useful for determining the
sublethal effects of chemicals to fish, amphibians, and macroinvertebrates.
1.2 Behavioral toxicity occurs when chemical or other stressful conditions, such as changes in water quality or temperature, induce
a behavioral change that exceeds the normal range of variability (1). Behavior includes all observable, recordable, or measurable
activities of a living organism and reflects genetic, neurobiological, physiological, and environmental determinants (2).
1.3 Behavioral methods can be used in biomonitoring, the determination of no-observed-effect and lowest-observed-effect
concentrations, and the prediction of hazardous chemical impacts on natural populations (3).
1.4 Behavioral methods can be applied to fish, amphibians, and macroinvertebrates in standard laboratory toxicity tests, tests of
effluents, and sediment toxicity tests.
1.5 The various behavioral methods included in this guide are categorized with respect to seven interdependent, functional
responses that fish, amphibians, and macroinvertebrates must perform in order to survive. These functional responses include
respiration, locomotion, habitat selection, feeding, predator avoidance, competition, and reproduction (4). These responses can be
documented visually or through video or acoustic imagery. Electronically recorded information can be derived through manual
techniques or through the use of digital image analysis software.software (5, 6, 7)).
1.5.1 The functional responses are not necessarily mutually exclusive categories. For instance, locomotion, of some form of
movement, is important to all behavioral functions.
1.6 Additional behavioral methods for any category may be added when new tests are developed as well as when methods are
adapted to different species or different life stages of an organism.
1.7 This guide is arranged as follows:
Section Number
Scope 1
Referenced Documents 2
Terminology 3
This guide is under the jurisdiction of ASTM Committee E50 on Environmental Assessment, Risk Management and Corrective Actionand is the direct responsibility of
Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Dec. 1, 2012Dec. 1, 2020. Published February 2013January 2021. Originally approved in 1994. Last previous edition approved in 20072012 as
E1604-04(2007).E1604-12. DOI: 10.1520/E1604-12.10.1520/E1604-20.
The boldface numbers in parentheses refer to the list of references at the end of this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1604 − 20
Summary of Guide 4
Significance and Use 5
Interferences 6
Test Facility 7
Water Supply 8
Safety Precautions 9
Test Material 10
Test Organisms 11
Responses Measured 12
Behavioral Test Method Selection Criteria 13
Experimental Design 14
Acceptability of Test 15
Calculation of Test Results 16
Report 17
1.8 The values stated in SI units are to be regarded as the standard. For an explanation of units and symbols, refer to IEEE/ASTM
SI 10.
1.9 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 appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use. Specific precautionary statements are given in Section 9.
1.10 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.
2. Referenced Documents
2.1 ASTM Standards:
E729 Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
E943 Terminology Relating to Biological Effects and Environmental Fate
E1023 Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses
E1192 Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents with Fishes, Macroinvertebrates,
and Amphibians
E1241 Guide for Conducting Early Life-Stage Toxicity Tests with Fishes
E1383 Guide for Conducting Sediment Toxicity Tests with Freshwater Invertebrates (Withdrawn 1995)
E1711 Guide for Measurement of Behavior During Fish Toxicity Tests
E1733 Guide for Use of Lighting in Laboratory Testing
E1847 Practice for Statistical Analysis of Toxicity Tests Conducted Under ASTM Guidelines
IEEE/ASTM SI 10 American National Standard for Metric Practice
3. Terminology
3.1 Definitions—The words “must,” “should, “may,” “can,” and “might” have very specific meanings. “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 used only in connection with the factors that directly relate to the acceptability of
the test. “Should” is used to state that the specified condition is recommended and ought to be met if possible. Although the
violation of one “should” 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.”
3.2 Definitions of Terms Specific to This Standard:
3.2.1 behavior—the complex of observable, recordable, or measurable activities of a living organism.
3.2.2 behavioral toxicity—the phenomenon observed when a behavioral response varies beyond the range of normal as a result
of exposure to chemical or other stressors.
3.3 For definitions of other terms used in this guide, refer to Guides E729, E1192, E1241, E1383, E1711, E1847, and Terminology
definitions in Guide E943.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
The last approved version of this historical standard is referenced on www.astm.org.
E1604 − 20
4. Summary of Guide
4.1 The potential toxicity of chemical substances in water, food, or sediments is assessed by measuring the behavior of fish,
amphibians, and macroinvertebrates during exposure, using static, flow-through, or food exposure systems. The behavioral
response of organisms exposed to chemical substances in water, effluents, food, or sediments is compared with the behavioral
responses of control organisms. The behavioral responses measured during toxicity tests are highly sensitive to sublethal exposure.
The behavioral measures are relevant to essential life functions that fish, amphibians, and macroinvertebrates often must perform
in order to survive and include respiration, locomotion, habitat selection, feeding, predator avoidance, competition, and
reproduction. Data are obtained to determine the effects of toxic substances on behavior from short (for example, 1 h) or long-term
(partial to full life cycle) exposures.
5. Significance and Use
5.1 Protection of a species requires the prevention of detrimental effects of chemicals on the survival, growth, reproduction, health,
and useshealth of individuals of that species. Behavioral toxicity provides information concerning sublethal effects of chemicals
and signals the presence of toxic test substances.
5.1.1 The behavioral responses of all organisms are adaptive and essential to survival. Major changes in the behavioral responses
of fish, amphibians, and macroinvertebrates may result in a diminished ability to survive, grow, or reproduce and cause significant
changes in the natural population (8).
5.2 The results from behavioral toxicity tests may be useful for measuring injury in the assessment of damages resulting from the
release of hazardous materials (9).
5.3 Behavioral toxicity test methods may be useful for long-term monitoring of effluents (10).
5.4 The results from behavioral toxicity data can be used to predict the effects of exposure on fish, amphibians, and aquatic
invertebrates likely to occur in field situations as a result of exposure under similar conditions, including the avoidance of exposure
by motile organisms (11).
5.5 The results from behavioral toxicity tests might be an important consideration for assessing the hazard of materials to aquatic
organisms. Such results might also be used when deriving water quality criteria for fish and aquatic invertebratesinvertebrate
organisms.
5.6 The results from behavioral toxicity tests can be used to compare the sensitivities of different species, relative toxicity of
different chemical substances on the same organism, or effect of various environmental variables on the toxicity of a chemical
substance.
5.7 The results from behavioral toxicity tests can be used to predict the effects of long-term exposure.
5.8 The results of behavioral toxicity tests can be useful for guiding decisions regarding the extent of remedial action needed for
contaminated aquatic and terrestrial sites.
5.9 The behavioral characteristics of a particular organism must be understood and defined before a response can be used as a
measure of toxicity. The range of variability of any behavioral response of unexposed organisms is influenced by genetic,
experiential, physiological, and environmental factors. Thus it is important to avoid selecting test organisms from populations that
may vary significantly in these factors.
5.10 The results of behavioral toxicity tests will depend on the behavioral response measured, testing conditions, water quality,
species, genetic strain, life stage, health, and general condition of test organisms. Therefore, the behavioral response may be
affected by the test environment.
6. Interferences
6.1 A number of factors can suppress, elicit, or alter behavioral responses and thus influence behavioral test results and complicate
E1604 − 20
data interpretation. The following factors should be considered in the experimental protocol or in the discussion of results when
measuring behavioral responses during toxicity tests:
6.1.1 The pretest handling of test organisms resulting from collection, transfer, and maintenance of the culture environment can
affect the response observed during exposure to toxic substances.
6.1.2 The health, nutritional state, and physical condition of the organism can influence the test.
6.1.3 Behavioral responsiveness may vary by species, genetic strain, population, gender, and developmental stage of the organism.
6.1.4 Prior exposure to hazardous materials, environmental stresses, and pathogens can affect the behavioral responses.
6.1.5 Social status, such as dominance or sex of the individuals tested, and experiential factors, such as prior experience with
predator or prey species, can influence the behavioral response. Individuals tested in isolation may respond differently than when
tested in groups.
6.1.6 Cyclical changes (circadian, seasonal, annual, and reproductive) in behavioral responses can occur.
6.1.7 The behavioral response can be affected by apparatus design and by the procedural sequence of the measurement method.
6.1.8 Behavioral responses will vary according to the extent to which test organisms acclimate to the physical variables of the
testing environment, including water quality, temperature, water flow, light, cover, and substrate, as well as their recovery from
handling, acceptance of diet, and adjustment to novel testing chambers.
6.1.9 Behavioral responses to toxic substances may subsidechange over time.
7. Test Facility
7.1 Facilities—The facility should include a constant temperature area for culturing and testing. Test and culture chambers may
be placed in a temperature-controlled recirculating water bath or in a constant-temperature area. Air used for aeration should be
free of fumes, oil, and water and can require filters to remove oil, water, and bacteria. The test facility should be well-ventilated
and free of fumes. Enclosures may be necessary to ventilate test chambers. Descriptions of toxicity testing equipment appear
Guides E729, E1023, E1192, E1241, E1383, and E1711. These provide guidance on construction materials, water and air delivery
systems, test chambers and cleaning, and water supply
7.1.1 Culture and animal care facilities should not be in a room in which toxicity tests are conducted, stock solutions or test
solutions are prepared, or equipment is cleaned.
7.1.2 A timing device should be used to provide a light:darknesslight:dark cycle. A15 A 15 to 30-min transition period, allowing
for a gradual change in light intensity when the lights are turned on or off, may be desirable for reducing stress caused by
instantaneous illumination or darkness.darkness (See Guide E1733).
7.2 Construction Materials—Consistent with specifications delineated, for example, in Guide E1241, equipment and facilities that
come into contact with stock solutions, test solutions, food, sediment, air, or water, into which the test organisms are placed, should
not contain substances that can be leached or dissolved in amounts that affect the test organisms adversely. The materials should
be chosen to minimize sorption of test materials.
7.3 Water and Air Delivery Systems—The water delivery system used in flow-through testing can be one of several designs. The
system should be capable of delivering equal volumes of water at an equal rate of flow to each replicate treatment container.
Various metering systems, using different combinations of siphons, pumps, solenoids, valves, etc., have been used successfully to
control the flow rates of water and toxic substances (see GuideGuides E729, E1023, E1241, E1383, and E1847).
7.3.1 The metering system should be calibrated before the test by determining the flow rate of water and air through each test
chamber. The general operation of the metering system should be visually checked daily throughout the test. The water delivery
system should be adjusted during the test if necessary. At any particular time during the test, flow rates through any two test
chambers should not differ by more than 10 %.
E1604 − 20
7.4 Test Chambers—In a behavioral toxicity test with fish, amphibians, and macroinvertebrates, the measurement of behavioral
response may take place directly in the exposure vessel, or the organisms may be transferred to a specific apparatus or observation
chamber for the purpose of measuring a behavioral response (see sectionSection 128.1.8).). The independent experimental unit for
such tests is based on the smallest physical exposure unit between which there are no water, air connections, or common access
to sediment or food. All test chambers must be identical, and the test compartments within each chamber must be identical and
placed in analogous locations within each test chamber.
7.4.1 Test chambers may be constructed in several ways and of various materials, depending on the experimental design and
contaminants of interest. Clear silicone adhesives, suitable for aquaria, should be used sparingly since they sorb some organic
compounds that may be difficult to remove. New test chambers sealed with silicone adhesives should be weathered for at least 48
h in water of the same quality as that used in the toxicity test to leach potentially toxic compounds from the adhesive.
7.4.2 Apparatus will vary according to the response being measured and species and life stage being tested. Organisms may be
observed directly in the exposure chamber, or they may be transferred to specialized apparatus for measurement of the response.
Recording of response may require (1) direct visual observation, (2) video-recorded observation, or (3) electronically recorded
observation.
7.5 Cleaning—Test chambers, water delivery systems, equipment used for preparing and storing exposure water, and stock
solutions should be cleaned before use. Consistent with Guide E729, new items should be washed in the following manner: (1)
detergent wash, (2) tap water rinse, (3) water-miscible organic solvent wash, (4) tap water rinse, (5) acid wash (such as 10 %
concentrated hydrochloric acid), and (6) rinse at least twice with distilled, deionized, or test water. Test chambers should be rinsed
with test water just before use.
7.5.1 Many organic solvents leave a film that is insoluble in water. A10 % A 10 % nitric acid solution, for example, may cause
deterioration of silicone adhesive. A rinse with 10 % concentrated hydrochloric acid may be preferable. A dichromate-sulfuric acid
cleaning solution can generally be used in place of both the organic solvent and the acid (see Guide E729), but the solution might
attack silicone adhesive and leave potentially mutagenic residues of chromium on glass. Non-chromium cleaning solutions are also
available.
7.5.2 Upon completion of a test, all items that are to be used again should be immediately (1) emptied of water, sediment, or
effluent (which should be disposed of properly; (2) rinsed with water; (3) cleaned by a procedure appropriate for removing the test
material (for example, acid to remove metals and bases and detergent, organic solvent, or aqueous slurry of activated carbon to
remove organic chemicals); and (4) rinsed at least twice with distilled, deionized, or overlying water.
7.6 Acceptability—Before a toxicity test is conducted in new test facilities, it is desirable to conduct a non-toxicant test, in which
all test chambers contain uncontaminated water or sediment. The behavior of the test species will demonstrate whether facilities,
water, control sediment, and handling techniques are adequate to result in acceptable species-specific control numbers. The
magnitude of the within-chamber and between-chamber variance should also be determined.
8. Water Supply
8.1 Requirements—In addition to being available in adequate supply, dilution water used in behavioral toxicity tests, and water
used to hold organisms before testing, should be acceptable to test species and uniform in quality. To be acceptable to the test
species, the water must permit satisfactory survival and growth, without inducing signs of disease or apparent stress, such as
discoloration, or unusual behavior.
8.2 Source—Natural overlying water should be uncontaminated and of constant quality and should meet the following
specifications as established in Guide E729. The values stated help to ensure that the test organisms are not apparently stressed
during holding, acclimation, and testing and that the test results are not affected unnecessarily by water characteristics: particulate
matter, <5 mg/L; total organic carbon (TOC), <5 mg/L; chemical oxygen demand (COD), <5 mg/L; and residual chlorine, <11
μg/L.
8.2.1 A natural water source is considered to be of uniform quality if the monthly ranges of the hardness, alkalinity, and specific
conductance are less than 10 % of their respective averages and if the monthly range of pH is less than 0.4 unit. Natural waters
should be obtained from an uncontaminated well or spring, if possible, or from a surface water source. If surface water is used,
the intake should be positioned to minimize fluctuations in quality and the possibility of contamination; to maximize the
E1604 − 20
concentration of dissolved oxygen; and to help ensure low concentrations of sulfide and iron. Municipal water supplies oftencan
contain unacceptably high concentrations of copper, lead, zinc, fluoride, chlorine, or chloramines, and quality is oftencan be
variable. Chlorinated water should not be used for, or in the preparation of, exposure water because residual chlorine and
chlorine-produced oxidants are toxic to many aquatic animals (12). Dechlorinated water should be used only as a last resort
because dechlorination is oftencan be incomplete.
8.2.2 For certain applications, the experimental design might require the use of water from the test effluent or sediment collection
site.
8.2.3 Reconstituted water is prepared by adding specified amounts of reagent grade chemicals to high-quality distilled or deionized
water (see Guide E729).
8.3 Characterization—The following items should be measured at least twice each year, and more often if (1) such measurements
have not been determined semiannually for at least two years or (2) if surface water is used: pH, particulate matter, TOC,
organophosphorus pesticides, organic halides, organochlorine pesticides, polychlorinated biphenols (PCBs), chlorinated phenoxy
herbicides, ammonia, cyanide, sulfide, bromide, chloride, fluoride, iodide, nitrate, phosphate, sulfate, calcium, magnesium, sodium,
potassium, aluminum, arsenic, beryllium, boron, cadmium, chromium, cobalt, copper, iron, lead, manganese, mercury,
molybdenum, nickel, selenium, silver, and zinc, hardness, alkalinity, and conductivity (see Guide E729).
8.3.1 For each method used, the detection limit should be below (1) the concentration in the dilution water or (2) the lowest
concentration that has been shown to affect the test species adversely (13).
8.3.2 Water that might be contaminated with facultative pathogens may be passed through a properly maintained ultraviolet
sterilizer (14) equipped with an intensity meter and flow controls or passed through a filter with a pore size of 0.45 μm or less.
Carbon filtration may be required to remove the pathogenic toxins.
8.3.3 Water may require aeration using air stones, surface aerators, or column aerators (15-17). Adequate aeration will stabilize
the pH, bring the concentrations of dissolved oxygen and other gases into equilibrium with air, and minimize the oxygen demand
and concentrations of volatiles. The concentration of dissolved oxygen in water should be between 90 and 100 % saturation (17)
to help ensure that the dissolved oxygen concentrations are acceptable in the test chambers. Precautions should be taken, however,
to ensure that glass air stones are not breaking down with use and that plastic air stones are not absorbing organic chemicals.
9. Safety Precautions
9.1 Many substances may pose health risks to humans if adequate precautions are not taken. Information on toxicity to humans,
recommended handling procedures, and chemical and physical properties of the test material should be studied and all personnel
informed before an exposure is initiated.
NOTE 1—Warning: Special procedures might be necessary with radiolabeled test materials and with test materials that are, or are suspected of being,
carcinogenic.
9.2 Many materials can affect humans adversely if precautions are inadequate. adversely. Contact with test material, sediments,
and water should be minimized. Where appropriate, protective gloves, laboratory coats, aprons, protective clothing, and safety
glasses should be worn, and dip nets, sieves, or tubes should be used to remove test organisms. When handling potentially
hazardous materials, proper handling procedures may include (1) manipulating test materials under a ventilated hood or in an
enclosed glovebox; (2) enclosing and ventilating the exposure chambers; and (3) using respirators, aprons, safety glasses, and
gloves.
10. Test Material
10.1 Test materials may include pure compounds or commercial formulations of compounds that are added to water or sediment.
Test materials collected from field locations may also include complex mixtures of chemical compounds in effluents and sediments.
10.2 Considerations for technical test materials for use in aqueous tests, preparations of stock solutions, use of solvents, and
selection of test concentrations of aqueous solutions should follow those outlined in Guide E1241.
E1604 − 20
10.3 Tests using sediments as the exposure media should follow Guide E1383 for the characterization, collection, storage,
preparation of spiked sediment samples, and test concentrations of spiked sediment samples.
11. Test Organisms
11.1 Species and life stages selected for study will depend on the focus of the study and may include standard bioassay organisms
when the relative toxicity of a compound is to be determined.
11.2 The species and life stage selected for study should be appropriate for the experimental setting, tolerant of handling and
confinement within a reasonable acclimation time, and be willing to accept food in the setting in which the behavioral responses
will be observed. The species used should be selected based on (1) availability; (2) sensitivity to a test material(s); (3) ecological
relevance to the habitat under study (for example, saltwater or freshwater); and (4) tolerance to ecological conditions such as
temperature, grain size, and ease of handling in the laboratory. The species of test organism used should be determined using an
appropriate taxonomic key.
11.3 Test organisms must not be diseased or injured and must be obtained from relatively uncontaminated field sites or
contaminant-free cultures. The organisms must be acclimated to the water quality and testing conditions following the procedures
outlined in Guide E729.
11.4 The relative health and quality of test organisms can be verified through an assessment of their behavioral repertoire and
bioassays in response to reference toxicants.
11.5 All organisms should be as uniform as possible in age and size class.
11.6 All organisms in a test must be from the same source. Organisms may be obtained from (1) laboratory cultures; (2)
commercial, state, or federal institutions; or (3) natural populations from clean areas. Laboratory cultures of test species can
provide organisms whose history, age, and quality are known. Local and state agencies may require collecting permits.
11.7 To maintain organisms in good con
...