ASTM E1241-22

Standard Guide for<brk type="line"/> Conducting Early Life-Stage Toxicity Tests with Fishes

SIGNIFICANCE AND USE
5.1 Protection of a species requires prevention of unacceptable effects on the number, weight, health, and uses of the individuals of that species. An early life-stage toxicity test provides information about the chronic toxicity of a test material to a species of fish. The primary adverse effects studied are reduced survival and growth.  
5.2 Results of early life-stage toxicity tests are generally useful estimates of the results of comparable life-cycle tests with the same species (1).4 However, results of early life-stage tests are sometimes under estimative of those obtained with the same species in the longer life-cycle tests (2).  
5.3 Results of early life-stage toxicity tests might be used to predict long-term effects likely to occur on fish in field situations as a result of an exposure under comparable conditions, except that motile organisms might avoid exposure when possible.  
5.4 Results of early life-stage toxicity tests might be used to compare the chronic sensitivities of different fish species and the chronic toxicities of different materials, and to study the effects of various environmental factors on results of such tests.  
5.5 Results of early life-stage toxicity tests might be an important consideration when assessing the hazards of materials to aquatic organisms (see Guide E1023) or when deriving water quality criteria for aquatic organisms (3).  
5.6 Results of an early life-stage test might be useful for predicting the results of chronic tests on the same test material with the same species in another water or with another species in the same or a different water. Most such predictions take into account the results of acute toxicity tests, and so the usefulness of the results of an early life-stage test is greatly increased by reporting also the results of an acute toxicity test (see Guide E729) conducted with juveniles of the same species under the same conditions.  
5.7 Results of early life-stage toxicity tests might be useful for...
SCOPE
1.1 This guide describes procedures for obtaining laboratory data concerning the adverse effects of a test material added to dilution water—but not to food—on certain species of freshwater and saltwater fishes during 28 day to 120 day (depending on species) continuous exposure, beginning before hatch and ending after hatch, using flow-through exposures. This guide will probably be useful for conducting early life-stage toxicity tests with some other species of fish, although modifications might be necessary.  
1.2 Other modifications of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting early life-stage toxicity tests with fishes.  
1.3 These procedures are applicable to all chemicals, either individually or in formulations, commercial products, or known mixtures, that can be measured accurately at the necessary concentrations in water. With appropriate modifications these procedures can be used to conduct tests on temperature, dissolved oxygen, and pH and on such materials as aqueous effluents (see Guide E1192), leachates, oils, particulate matter, sediments, and surface waters.  
1.4 This guide is arranged as follows:    
Section  
Referenced Documents  
2  
Terminology  
3  
Summary of Standard  
4  
Significance and Use  
5.1  
Hazards  
6  
Apparatus  
7  
Facilities  
7.1  
Construction Materials  
7.2  
Metering System  
7.3  
Test Chambers and Incubation Cups  
7.4  
Cleaning  
7.5  
Acceptability  
7.6  
Dilution Water  
8  
Requirements  
8.1  
S...

General Information

• Status: Published
• Publication Date: 31-Mar-2022
• ICS: 65.150 - Fishing and fish breeding
• Technical Committee: E50 - Environmental Assessment, Risk Management and Corrective Action
• Drafting Committee: E50.47 - Biological Effects and Environmental Fate

Relations

• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• 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
• Refers: ASTM D4447-10 - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 15-Nov-2006
• Refers: ASTM D4447-06 - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 15-Nov-2006
• Refers: ASTM D1129-06ae1 - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1129-06a - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM D1129-06 - Standard Terminology Relating to Water
• Effective Date: 15-Feb-2006
• Refers: ASTM D1129-04e1 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2004
• Refers: ASTM D1129-04 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2004

Overview

ASTM E1241-22 is the Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes, developed by ASTM International. This guideline details standardized laboratory procedures for evaluating the chronic toxicity of various chemicals and materials on fish during the sensitive early life stages. The guide focuses on both freshwater and saltwater species and involves continuous exposure lasting between 28 and 120 days, depending on the test fish species.

The primary aim is to provide reliable data on the adverse effects of test materials-such as chemicals, effluents, leachates, and surface waters-added to dilution water. Results inform assessments of chronic toxicity, with the most significant adverse effects being reduced survival and growth among fish embryos and larvae. These toxicity tests play a crucial role in environmental risk assessment, regulatory compliance, and the development of water quality criteria.

Key Topics

• Chronic Toxicity Assessment: Detailed procedures are provided to assess long-term adverse effects of chemicals on early fish developmental stages, focusing on survival and growth.
• Comparative Sensitivity Studies: Results can be used to compare the chronic sensitivities among different fish species as well as the toxicity profiles of various substances.
• Laboratory Procedures: Covers specifications for dilution water, test chambers, metering systems, and handling of test organisms to ensure reproducibility and reliability of toxicity data.
• Test Modifications: The guide allows for justified procedural modifications to accommodate special needs, while stressing the importance of procedural consistency for result comparability.
• Test Material Scope: Applicable to a wide range of test materials, including individual chemicals, commercial formulations, and environmental samples, provided concentrations can be accurately measured in water.
• Environmental Factor Studies: Procedures can be adapted to study effects of variables such as temperature, dissolved oxygen, and pH, enhancing the relevance of results to real-world conditions.
• Predicting Environmental Impact: Data from early life-stage toxicity tests may be used to predict potential long-term impacts on fish populations and assist in the derivation of water quality standards.

Applications

• Hazard Assessment for Aquatic Organisms: These standardized toxicity tests are indispensable in evaluating the ecological risks posed by new and existing chemicals, effluents, and environmental samples to aquatic life.
• Regulatory Compliance: Early life-stage toxicity data support regulatory submissions concerning the environmental safety of chemicals, industrial discharges, and water management practices.
• Water Quality Criteria Development: The results are often integral to the establishment or revision of water quality criteria, helping ensure the protection of aquatic species over their entire lifecycle.
• Comparative Toxicology: Researchers and environmental managers use this guide to compare the relative toxicity of different substances across fish species, contributing to informed decision-making and prioritization of environmental interventions.
• Environmental Monitoring: The guide underpins monitoring programs, helping to identify sources of chronic toxicity in natural waters and track the effectiveness of pollution control strategies.

Related Standards

• ASTM E1023: Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses, essential for broader hazard assessments.
• ASTM E729: Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians, often used in conjunction with chronic toxicity tests.
• ASTM E1192: Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents with Fishes, Macroinvertebrates, and Amphibians, for evaluating complex samples.
• ASTM D1129, D1193: Terminology and specifications for water used in toxicity testing.
• ASTM E943, E1022: Terminology and guidance for bioconcentration and related biological effects testing.
• Other References within E1241-22: Includes guides for test organism handling, water quality characterization, and apparatus specifications, ensuring comprehensive support for test execution and data integrity.

By offering a clear and consistent framework, ASTM E1241-22 enhances the reliability and regulatory acceptance of early life-stage toxicity testing with fishes, ultimately contributing to the protection of aquatic environments.

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: E1241 − 22
Standard Guide for
Conducting Early Life-Stage Toxicity Tests with Fishes
This standard is issued under the fixed designation E1241; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
Section
1.1 Thisguidedescribesproceduresforobtaininglaboratory
Referenced Documents 2
data concerning the adverse effects of a test material added to Terminology 3
Summary of Standard 4
dilution water—but not to food—on certain species of fresh-
Significance and Use 5.1
waterandsaltwaterfishesduring28dayto120day(depending
Hazards 6
on species) continuous exposure, beginning before hatch and Apparatus 7
Facilities 7.1
ending after hatch, using flow-through exposures. This guide
Construction Materials 7.2
will probably be useful for conducting early life-stage toxicity
Metering System 7.3
Test Chambers and Incubation Cups 7.4
tests with some other species of fish, although modifications
Cleaning 7.5
might be necessary.
Acceptability 7.6
Dilution Water 8
1.2 Other modifications of these procedures might be justi-
Requirements 8.1
fied by special needs or circumstances.Although using appro-
Source 8.2
priate procedures is more important than following prescribed Treatment 8.3
Characterization 8.4
procedures,resultsoftestsconductedusingunusualprocedures
Test Material 9
are not likely to be comparable to results of many other tests.
General 9.1
Comparisonofresultsobtainedusingmodifiedandunmodified
Stock Solution 9.2
Test Concentration(s) 9.3
versions of these procedures might provide useful information
Test Organisms 10
concerning new concepts and procedures for conducting early
Species 10.1
life-stage toxicity tests with fishes.
Age 10.2
Source 10.3
1.3 These procedures are applicable to all chemicals, either
Brood Stock 10.4
individually or in formulations, commercial products, or Handling 10.5
Procedure 11
known mixtures, that can be measured accurately at the
Experimental Design 11.1
necessary concentrations in water. With appropriate modifica-
Dissolved Oxygen 11.2
Temperature 11.3
tions these procedures can be used to conduct tests on
Beginning the Test 11.4
temperature, dissolved oxygen, and pH and on such materials
Thinning 11.5
as aqueous effluents (see Guide E1192), leachates, oils, par-
Feeding 11.6
Duration of Test 11.7
ticulate matter, sediments, and surface waters.
Biological Data 11.8
1.4 This guide is arranged as follows: Other Measurements 11.9
Analytical Methodology 12
Acceptability of Test 13
Calculation of Results 14
Documentation 15
Appendixes
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental Appendix X1 Salmon, Trout, and Char
Appendix X2 Northern pike
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
Appendix X3 Fathead minnow
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Appendix X4 White sucker
Current edition approved April 1, 2022. Published May 2022. Originally
Appendix X5 Channel catfish
approved in 1988. Last previous edition approved in 2013 as E1241–05(2013)
Appendix X6 Bluegill
which was withdrawn January 2022 and reinstated in April 2022. DOI: 10.1520/
Appendix X7 Gulf toadfish
E1241-22.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1241 − 22
acceptability of the test (see 13.1). “Should” is used to state
Appendix X8 Sheepshead minnow
Appendix X9 Silversides
that the specified condition is recommended and ought to be
Appendix X10 Statistical Guidance
met if possible.Although violation of one “should” is rarely a
Appendix X11. Striped Bass
serious matter, violation of several will often render the results
questionable.Termssuchas“isdesirable,”“isoftendesirable,”
and “might be desirable” are used in connection with less
1.5 This standard does not purport to address all of the
importantfactors.“May”isusedtomean“is(are)allowedto,”
safety concerns, if any, associated with its use. It is the
“can”isusedtomean“is(are)ableto,”and“might”isusedto
responsibility of the user of this standard to establish appro-
mean “could possibly.” Thus the classic distinction between
priate safety, health, and environmental practices and deter-
“may” and “can” is preserved, and “might” is never used as a
mine the applicability of regulatory limitations prior to use.
synonym for either “may” or “can.”
Specific hazard statements are given in Section 6 and 9.
3.2 Definitions:
1.6 This international standard was developed in accor-
dance with internationally recognized principles on standard-
3.2.1 For definitions of other terms used in this guide, refer
ization established in the Decision on Principles for the toTerminologyD1129,GuideE729,TerminologyE943,Guide
Development of International Standards, Guides and Recom- E1022 and Guide E1023. For an explanation of units and
mendations issued by the World Trade Organization Technical symbols, refer to SI10.
Barriers to Trade (TBT) Committee.
3.2.2 antagonism, n—a situation which an effect of an
exposuretomultiplesubstancesislessthanwouldbeexpected
2. Referenced Documents
if the known effects of the individual substances were added
2.1 ASTM Standards:
together.
D1129Terminology Relating to Water
3.2.3 confounding, n—a situation in which one or more
D1193Specification for Reagent Water
othervariablescovarywiththeindependentvariable,makingit
D4447Guide for Disposal of Laboratory Chemicals and
impossible to determine the influence of the independent
Samples
variable on the dependent variable.
E729Guide for Conducting Acute Toxicity Tests on Test
3.2.4 exposure, n—whenatestorganismisincontactwitha
Materials with Fishes, Macroinvertebrates, and Amphib-
test material for a duration of time.
ians
E943Terminology Relating to Biological Effects and Envi-
3.2.5 synergism, n—a situation in which an effect of an
ronmental Fate
exposure to multiple substances is more than would be
E1022Guide for Conducting Bioconcentration Tests with
expectediftheknowneffectsoftheindividualsubstanceswere
Fishes and Saltwater Bivalve Mollusks
added together.
E1023Guide for Assessing the Hazard of a Material to
Aquatic Organisms and Their Uses
4. Summary of Guide
E1192Guide for ConductingAcute Toxicity Tests onAque-
4.1 In each of two or more treatments, embryos and the
ous Ambient Samples and Effluents with Fishes,
subsequent larvae of one species of fish are maintained in two
Macroinvertebrates, and Amphibians
or more test chambers in a flow-through system for 28 to 120
E1203Practice for Using Brine Shrimp Nauplii as Food for
days, depending upon species. In each of the one or more
Test Animals in Aquatic Toxicology (Withdrawn 2013)
control treatments, the embryos and larvae are maintained in
E1733Guide for Use of Lighting in Laboratory Testing
dilutionwatertowhichnotestmaterialhasbeenaddedinorder
E1847Practice for Statistical Analysis of Toxicity Tests
to provide (a) a measure of the acceptability of the test by
Conducted Under ASTM Guidelines (Withdrawn 2022)
giving an indication of the quality of the embryos and larvae
SI10 IEEE/ASTM SI 10 American National Standard for
and the suitability of the dilution water, food, test conditions,
UseoftheInternationalSystemofUnits(SI):TheModern
handling procedures, and so forth, and (b) the basis for
Metric System
interpretingdataobtainedfromtheothertreatments.Ineachof
3. Terminology
the one or more other treatments, the embryos and larvae are
maintained in dilution water to which a selected concentration
3.1 Thewords“must,”“should,”“may,”“can,”and“might”
of test material has been added. Specified data on the concen-
haveveryspecificmeaningsinthisstandard.“Must”isusedto
tration of test material and the survival and growth of the
express an absolute requirement, that is, to state that the test
embryos and larvae in each test chamber are obtained and
ought to be designed to satisfy the specified condition, unless
analyzed to determine the effect(s) of the test material on the
the purpose of the test requires a different design. “Must” is
survival and growth of the test organisms.
only used in connection with factors that directly relate to the
5. Significance and Use
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
5.1 Protection of a species requires prevention of unaccept-
Standards volume information, refer to the standard’s Document Summary page on
able effects on the number, weight, health, and uses of the
the ASTM website.
individuals of that species. An early life-stage toxicity test
The last approved version of this historical standard is referenced on
www.astm.org. provides information about the chronic toxicity of a test
E1241 − 22
material to a species of fish. The primary adverse effects 6.2 Although disposal of stock solutions, test solutions, and
studied are reduced survival and growth. test organisms poses no special problems in most cases, health
and safety precautions and applicable regulations should be
5.2 Results of early life-stage toxicity tests are generally
considered before beginning a test. Removal or degradation of
useful estimates of the results of comparable life-cycle tests
4 test material might be desirable before disposal of stock and
with the same species (1). However, results of early life-stage
test solutions.
testsaresometimesunderestimativeofthoseobtainedwiththe
same species in the longer life-cycle tests (2). 6.3 Cleaning of equipment with a volatile solvent such as
acetone should be performed only in a well-ventilated area in
5.3 Resultsofearlylife-stagetoxicitytestsmightbeusedto
whichnosmokingisallowedandnoopenflame,suchasapilot
predict long-term effects likely to occur on fish in field
light, is present. (Warning—An acidic solution should not be
situations as a result of an exposure under comparable
mixed with a hypochlorite solution because hazardous fumes
conditions, except that motile organisms might avoid exposure
might be produced.
when possible.
6.4 To prepare dilute acid solutions, concentrated acid
5.4 Resultsofearlylife-stagetoxicitytestsmightbeusedto
should be added to water, not vice versa. Opening a bottle of
compare the chronic sensitivities of different fish species and
concentratedacidandaddingconcentratedacidtowatershould
the chronic toxicities of different materials, and to study the
be performed only in a fume hood.
effectsofvariousenvironmentalfactorsonresultsofsuchtests.
6.5 Sampled media potentially containing hazardous com-
5.5 Results of early life-stage toxicity tests might be an
pounds should be disposed of properly (Guide D4447).
important consideration when assessing the hazards of materi-
als to aquatic organisms (see Guide E1023) or when deriving 6.6 Because dilution water and test solutions are usually
water quality criteria for aquatic organisms (3). goodconductorsofelectricity,useofgroundfaultsystemsand
leak detectors should be considered to help avoid electrical
5.6 Results of an early life-stage test might be useful for
shocks. Salt water is such a good conductor that protective
predicting the results of chronic tests on the same test material
devices are strongly recommended.
with the same species in another water or with another species
inthesameoradifferentwater.Mostsuchpredictionstakeinto
7. Apparatus
accounttheresultsofacutetoxicitytests,andsotheusefulness
7.1 Facilities—Flow-through tanks should be available for
of the results of an early life-stage test is greatly increased by
culturing brood stock, and for holding and acclimating test
reporting also the results of an acute toxicity test (see Guide
organisms. The test chambers should be in a constant-
E729) conducted with juveniles of the same species under the
temperature area or recirculating water bath. An elevated
same conditions.
headbox might be desirable so dilution water can be gravity-
5.7 Results of early life-stage toxicity tests might be useful
fed into holding, acclimation, and culture tanks, and the
for studying the biological availability of, and structure-
metering system (see 7.3). Strainers and air traps should be
activity relationships between, test materials.
included in the water-supply system. Headboxes and holding,
5.8 Results of early life-stage toxicity tests will depend on
acclimation, culture, and dilution-water tanks should be
temperature,compositionofthedilutionwater,conditionofthe
equipped for temperature control and aeration (see 8.3). Air
test organisms, and other factors.
usedforaerationshouldbefreeoffumes,oil,andwater;filters
to remove oil and water are desirable. Filtration of air through
6. Hazards
a0.22µmbacterialfiltermightbedesirable.Thefacilityshould
6.1 Many materials can affect humans adversely if precau- be well ventilated and free of fumes. To further reduce the
tions are inadequate. Therefore, skin contact with all test possibility of contamination by test materials and other sub-
stances (especially volatile ones) holding, acclimation, and
materials and solutions of them should be minimized by such
means as wearing appropriate protective gloves (especially culturetanksshouldnotbeinaroominwhichtoxicitytestsare
conducted, stock solutions or test solutions are prepared, or
when washing equipment or putting hands in test solutions),
laboratory coats, aprons, and glasses, and by using dip nets, equipment is cleaned. During holding, acclimation, culture,
and testing, organisms should be shielded from disturbances
forceps, or tubes, to remove organisms from test solutions.
Special precautions, such as covering test chambers and with curtains or partitions to prevent unnecessary stress. A
timing device should be used to control the photoperiod (see
ventilating the area surrounding the chambers, should be taken
when conducting tests on volatile materials. Information on Appendix X1 – Appendix X9). A 15- to 30-min transition
period (8) might be desirable whenever the lights go on to
toxicity to humans (4), recommended handling procedures (5),
andchemicalandphysicalpropertiesofthetestmaterialshould reduce the possibility of organisms being stressed by large,
suddenincreasesinlightintensity.Atransitionperiodwhenthe
be studied before a test is begun. (Warning—Special proce-
dures might be necessary with radiolabeled test materials (6) lights go off might also be desirable.
and with test materials that are, or are suspected of being,
7.2 Construction Materials—Equipment and facilities that
carcinogenic (7).
come in contact with stock solutions, test solutions, or any
water into which test organisms will be placed should not
containsubstancesthatcanbeleachedordissolvedbyaqueous
Boldfacenumbersinparenthesesrefertothelistofreferencesattheendofthis
guide. solutions in amounts that adversely affect fish. In addition,
E1241 − 22
equipment and facilities that contact stock solutions or test ously during the test as long as the test temperature (see 11.3)
solutions should be chosen to minimize sorption of test and concentrations of test material (see 11.9.3) remain accept-
able.
materials from water.
7.2.1 Glass, Type 316 Stainless Steel, Nylon, and Fluoro-
7.4 Test Chambers and Incubation Cups:
carbon Plastics—Use whenever possible to minimize
7.4.1 Inatoxicitytestwithaquaticorganisms,testchambers
dissolution, leaching, and sorption, except that stainless steel
are defined as the smallest physical units between which there
should not be used for tests on metals in salt water.
arenowaterconnections.However,screens,cups,andsoforth,
7.2.2 Concrete and Rigid Plastics—May be used for may be used to create two or more compartments within each
holding,acclimation,andculturetanksandinthewater-supply chamber.Thus,testsolutioncanflowfromonecompartmentto
another within a test chamber but, by definition, cannot flow
system, but these materials should be soaked, preferably in
flowing dilution water, for a week or more before use (9). Cast from one chamber to another. Because solution can flow from
one compartment to another in the same test chamber, the
iron pipe should not be used with salt water and probably
temperature, concentration of test material, and levels of
should not be used in a freshwater-supply system because
pathogens and extraneous contaminants, are likely to be more
colloidal iron will be added to the dilution water and strainers
similar between compartments in the same test chamber than
will be needed to remove rust particles. A specially designed
between compartments in different test chambers in the same
system is usually necessary to obtain salt water from a natural
treatment. Chambers should be covered or in an enclosure to
watersource(seeGuideE729).Dilutionwater,stocksolutions,
keepoutextraneouscontaminantsandtoreduceevaporationof
or test solutions should not contact brass, copper, lead, galva-
test solution and test material. Also, chambers filled to within
nized metal, and natural rubber before or during the test. Items
150 mm of the top sometimes need to be covered to prevent
made of neoprene rubber or other materials not mentioned
organisms from jumping out. All chambers (and compart-
above should not be used unless it has been shown that their
ments) in a test must be identical.
use will not adversely affect either survival or growth of
7.4.2 Testchambersmaybeconstructedbywelding(butnot
embryos and larvae of the test species.
soldering) stainless steel, or gluing double-strength or stronger
7.3 Metering System:
window glass with clear silicone adhesive. Stoppers and
7.3.1 The metering system should be designed to accom- siliconeadhesivemaysorbsomenon-polarorganiccompounds
modate the type and concentration(s) of the test material and (for example, organochlorine and organophosphorus
pesticides), which are then difficult to remove. Therefore, as
the necessary flow rates of the test solutions. The system
few stoppers and as little adhesive as possible should be in
should mix the test material with the dilution water immedi-
contactwithtestsolution.Ifextrabeadsofadhesiveareneeded
ately before the water and the test material enter the test
for strength, they should be on the outside of chambers rather
chambers and this system should permit the supply of the
than on the inside.
selected concentration(s) of test material in a reproducible
7.4.3 Embryos and young fish can be exposed in glass
fashion (see 9.3, 11.1.1, and 11.9.3.4). Various metering
systems, using different combinations of syringes, “dipping incubation cups constructed by gluing (a) stainless steel or
nylon screen bottoms to lengths of glass tubing or bottles with
birds,” siphons, pumps, saturators, solenoids, valves, and so
the bottoms cut off, or (b) nylon or stainless steel screen tubes
forth,havebeenusedsuccessfullytocontroltheconcentrations
(collars) to petri dishes. To ensure that test solution regularly
of test material, and the flow rates of test solutions (see Guide
flows into and out of each cup, either (a) test solution should
E729).
flow directly into the cups, or (b) the cups should be oscillated
7.3.2 The metering system should be calibrated before the
in the test solution by means of a rocker arm apparatus driven
testbydeterminingtheflowratethrougheachtestchamberand
bya1to6r/minelectricmotor,or(c)thewaterlevelinthetest
measuring either the concentration of test material in each test
chamber should be varied by means of a self-starting siphon.
chamber or the volume of solution used in each portion of the
(Clogging of the screens on cups in some treatments might be
meteringsystem.Thegeneraloperationofthemeteringsystem
greater than in others because of bacterial or algal growth
should be visually checked daily in the morning and afternoon
caused by differences in the concentration of solvent, intensity
throughout the test. The metering system should be adjusted
of light, etc. If some cups must be changed, all cups in the test
during the test if necessary.
should be changed to minimize differences in growth not
7.3.3 The flow rate through each test chamber should be at
related to the test material.) Embryo exposures can also be
least5volumeadditionsper24hdependingonthetestspecies
conductedin50mlcentrifugetubesthataregentlyaeratedand
(see Appendix X1 – Appendix X9). It is usually desirable to
renewed daily by hand to minimize fungus growth which can
construct the metering system so that it can provide at least 10
occur in the glass incubation cups during warm-water fish
volume additions per 24 h if desired, in case (a) the loading is
testing(forexample,fatheadminnows).Afterhatching,fishare
high (see 11.5.4)or(b) there is rapid loss of test material due
transferred from the centrifuge tubes to a flow through system.
to microbial degradation, hydrolysis, oxidation, photolysis,
The metering system, test chambers, and incubation cups
reduction, sorption, or volatilization (see 11.4.2). At any
shouldbeconstructedsothattestorganismsremainsubmerged
particular time during the test, the flow rates through any two and are not unacceptably stressed by crowding or turbulence.
test chambers should not differ by more than 10%. Flow rates
Sufficient flow of the test water to ensure oxygenation and
through all test chambers may be equally changed simultane- exposure while discouraging fungus growth is an important
E1241 − 22
step in developing an appropriate exposure system which may 8. Dilution Water
need to vary for different tests, as appropriate for the species,
8.1 Requirements:
toxicant, and test temperature.
8.1.1 Besides being available in adequate supply, the dilu-
7.4.4 Species-specific information on test chambers and
tionwatershould(a)beacceptabletothetestorganisms,(b)be
incubation cups is given in Appendix X1 – Appendix X9. Use
of uniform quality, and (c) except as per 8.1.4, not unneces-
of excessively large volumes of solution in test chambers will
sarily affect results of the test.
probably unnecessarily increase the amount of dilution water
8.1.2 To be acceptable to the test organisms, the dilution
and test material used, and the average retention time.
water must allow satisfactory survival and growth of embryos
and larvae of the test species (see 13.1.8).
7.5 Cleaning—The metering system, test chambers, incuba-
8.1.3 The quality of the dilution water should be uniform
tion cups, and equipment used to prepare and store dilution
during the test. During a test in fresh water, the range of
water, stock solutions, and test solutions should be cleaned
hardness should be less than 5 mg/L or 10% of the average,
before use. New items should be washed with detergent and
whichever is higher. During a test in salt water, the range of
rinsedwithwater,awater-miscibleorganicsolvent,water,acid
salinity should be less than 2 g/kg or 20% of the average,
(such as 10% concentrated hydrochloric acid), and rinsed at
whichever is higher.
least twice with deionized, distilled, dilution water, or ASTM
8.1.4 The dilution water should not unnecessarily affect
Type II water (Specification D1193). (Some lots of some
results of an early life-stage test because of such things as
organic solvents might leave a film that is insoluble in water.)
sorption or complexation of test material. Therefore, except as
A dichromate-sulfuric acid cleaning solution may be used in
per 8.1.5, concentrations of both total organic carbon (TOC)
place of both the organic solvent and the acid, but it might
and particulate matter should be less than 5 mg/L.
attacksiliconeadhesive.Attheendofthetest,allitemsthatare
8.1.5 If it is desired to study the effect of an environmental
tobeusedagainshouldbeimmediately(a)emptied,(b)rinsed
factor such asTOC, particulate matter, or dissolved oxygen on
with water, (c) cleaned by a procedure appropriate for remov-
the results of an early life-stage test, it will be necessary to use
ing the test material (for example, acid to remove metals and
a water that is naturally or artificially high in TOC or
bases; detergent, organic solvent, or activated carbon to re-
particulate matter or low in dissolved oxygen. If such a water
move organic chemicals), and (d) rinsed at least twice with
is used, it is important that adequate analyses be performed to
deionized, distilled, dilution water, or ASTM Type II water.
characterize the water and that a comparable test be available
Acid is often used to remove mineral deposits, and 200 mg of
or conducted in a more usual dilution water to facilitate
hypochlorite/L is often used to remove organic matter and for
interpretation of the results in the special water.
−
disinfection.Asolution containing about 200 mg ClO /L may
8.2 Source:
be prepared by adding 6 mL of liquid household chlorine
8.2.1 Although reconstituted water (see Guide E729) may
bleach to 1 Lof water. However, hypochlorite is quite toxic to
beusedinearlylife-stagetoxicitytests,itsusegenerallyisnot
mostfishes (10)andisdifficulttoremovefromsomeconstruc-
practical because of the large volume that is necessary for use.
tion materials. It is often removed by soaking in a sodium
thiosulfate, sodium sulfite, or sodium bisulfite solution, or by 8.2.2 If a natural dilution water is used, it should be
obtainedfromanuncontaminated,uniformqualitysource.The
autoclavingindistilledwaterfor20min,orbydryingtheitem
quality of water from a well or spring is usually more uniform
andlettingitsitforatleast24hbeforeuse.Anitemcleanedor
than that of water from surface water. If surface water is used
disinfected with hypochlorite should not be used unless it has
as a source of fresh or salt water, the intake should be
been demonstrated at least once that unfed larvae held for at
positioned to minimize fluctuations in quality and the possi-
least 48 h in static dilution water in which the cleaned item is
bility of contamination, and to maximize the concentration of
soaking, do not show more signs of stress, such as
dissolved oxygen to help ensure low concentrations of sulfide
discoloration, unusual behavior, or death, than do unfed larvae
and iron.
held in static dilution water containing a similar item that was
8.2.3 Chlorinated water should not be used as, or in the
not treated with hypochlorite.Alternatively, peracetic acid is a
preparation of, dilution water because residual chlorine and
less noxious anti-microbial than hypochlorite that avoids these
chlorine-producedoxidantsarequitetoxictomanyfishes (10).
issues. The metering system, test chambers, and incubation
Dechlorinated water should be used only as a last resort
cups should be rinsed with dilution water just before use.
becausedechlorinationisoftenincomplete.Sodiumbisulfiteis
7.6 Acceptability—Before an early life-stage test is con-
probably better for dechlorinating water than sodium sulfite
ducted in new test facilities, a “non-toxicant” test should be
and both are more reliable than carbon filters, especially for
conducted, in which all test chambers contain dilution water
removing chloramines (11). Some organic chloramines,
without added test material. Determine before the first test, (a)
however,reactslowlywithsodiumbisulfite (12).Inadditionto
whether embryos and larvae will survive and grow acceptably
residual chlorine, municipal drinking water often contains
(see 13.1.8) in the new facilities, (b) whether the food, water,
unacceptably high concentrations of copper, lead, zinc, and
handling procedures, etc., are acceptable, (c) whether there are
fluoride, and quality is often rather variable. Excessive con-
any location effects on either survival or growth, and (d) the
centrations of most metals can usually be removed with a
magnitudes of the within-chamber and between-chamber vari-
chelating resin (13), but use of a different dilution water might
ances. be preferable.
E1241 − 22
8.3 Treatment: 9.1.1 The test material should be reagent-grade or better,
unless a test on a formulation, commercial product, or
8.3.1 Dilution water should be aerated intensively by such
technical-grade or use-grade material is specifically needed.
means as air stones, surface aerators, or column aerators (14,
Before a test is begun, the following should be known about
15) prior to addition of test material. Adequate aeration will
the test material:
stabilize pH, bring concentrations of dissolved oxygen and
9.1.1.1 Identities and concentrations of major ingredients
other gases into equilibrium with air, and minimize oxygen
and major impurities, for example, impurities constituting
demand and concentrations of volatiles. The concentration of
more than about 1% of the material,
dissolved oxygen in dilution water should be between 90 and
9.1.1.2 Solubility and stability in the dilution water,
100% of saturation (16) to help ensure that dissolved oxygen
9.1.1.3 Acute toxicity to the test species,
concentrationsareacceptableintestchambers.Supersaturation
9.1.1.4 Measured or estimated chronic toxicity to the test
by dissolved gases, which might be caused by heating the
species,
dilutionwater,shouldbeavoidedtopreventgas-bubbledisease
9.1.1.5 Precision and bias of the analytical method at the
(15, 17).
planned concentration(s) of test material,
8.3.2 Filtration through bag, sand, sock, or depth-type
9.1.1.6 Estimate of toxicity to humans, and
cartridge filters may be used to keep the concentration of
9.1.1.7 Recommended handling procedures (see 6.1).
particulate matter acceptably low (see 8.1.3) and as a pretreat-
9.2 Stock Solution:
ment before ultraviolet sterilization or filtration through a finer
9.2.1 Insomecasesthetestmaterialcanbeaddeddirectlyto
filter.
the dilution water in the metering system, but some chemicals
8.3.3 Dilution water that might be contaminated with fac-
mayneedtobedissolvedinasolventotherthandilutionwater
ultative pathogens may be passed through a properly main-
toformastocksolutionthatisthenaddedtothedilutionwater
tained ultraviolet sterilizer (18) equipped with an intensity
in the metering system. If a stock solution is used, the
meter and flow controls or passed through a filter with a pore
concentration and stability of the test material in it should be
size of 0.45 µm or less.
determined before the beginning of the test. If the test material
8.3.4 Salt water from a surface water source should be
is subject to photolysis, the stock solution should be shielded
passed through a filter effective to 15 µm or less to remove
from light.
parasites and larval stages of predators.
9.2.2 Except possibly for tests on hydrolyzable, oxidizable,
and reducible materials, it is preferred to dissolve the test
8.3.5 When necessary, sea salt may be added to prevent
materialindilutionwater,althoughfiltration,orsterilization,or
excessive decreases in salinity, (see 8.1.2), if the salt has been
both, of the water might be necessary. If the hardness (or
shown to cause no adverse effects on either survival or growth
salinity)ofthedilutionwaterwillnotbeaffected,deionizedor
of embryos and larvae of the test species at the concentration
distilled water may be used. Several techniques have been
used.
specificallydevelopedforpreparingaqueousstocksolutionsof
8.4 Characterization:
slightly soluble materials (20). The minimum necessary
8.4.1 Thefollowingitemsshouldbemeasuredatleasttwice
amountofastrongacidorbasemaybeusedinthepreparation
each year and more often if such measurements have not been
ofanaqueousstocksolution,butsuchacidorbasemightaffect
made semiannually for at least two years or if a surface water
the pH of test solutions appreciably. Use of a more soluble
is used:
form of the test material, such as chloride or sulfate salts or
organic amines, sodium or potassium salts of phenols or
8.4.1.1 All Waters: pH, particulate matter, Total Organic
organic acids, and chloride or nitrate salts of metals, might
Carbon (TOC), organophosphorus pesticides, organic chlorine
affectthepHmorethantheuseofminimumnecessaryamounts
(or organochlorine pesticides plus Polychlorinated Biphenyls,
of strong acids and bases.
(PCBs)), chlorinated phenoxy herbicides, ammonia, cyanide,
9.2.3 If the test material cannot be dissolved in the dilution
sulfide, bromide, fluoride, iodide, nitrate, phosphate, sulfate,
water, the concentration of the solvent in test solutions should
calcium, magnesium, potassium, aluminum, arsenic,
be kept to a minimum and should be low enough that it does
beryllium, boron, cadmium, chromium, cobalt, copper, iron,
not affect either survival or growth of the test organisms.
lead, manganese, mercury, molybdenum, nickel, selenium,
Because of its low toxicity to aquatic animals (21), low
silver, and zinc.
volatility, and high ability to dissolve many organic chemicals,
8.4.1.2 Fresh Water: hardness, alkalinity, conductivity,
triethylene glycol is often a good organic solvent for preparing
sodium, and chloride.
stock solutions. Other water-miscible organic solvents such as
8.4.1.3 Salt Water: salinity or chlorinity.
methanol, ethanol, and acetone may also be used, but they
8.4.1.4 The methods used (see 12.3) should either (a)be
might stimulate undesirable growths of microorganisms. If an
accurate and precise enough to adequately characterize the
organic solvent is used, it should be reagent grade or better
dilutionwateror(b)havedetectionlimitsbelowconcentrations
that have been shown to adversely affect fish (19).
“Reagent Chemicals, American Chemical Society Specifications,” American
9. Test Material Chemical Society, Washington, DC. For suggestions on the testing of reagents not
listedintheAmericanChemicalSociety,see“AnalarStandardsforLaboratoryU.K.
9.1 General: Chemicals, BDH Ltd., Poole, Dorset, and the United States Pharmacopeia.”
E1241 − 22
anditsconcentrationinanytestsolutionshouldnotexceed0.1 be no higher than 50 ug/L. Testing at least double the
mL/L. A surfactant should not be used in the preparation of a concentrationofsolventusedinthetoxicitytestwouldprovide
stock solution because it might affect the form and toxicity of some margin of safety in extrapolating results of toxicity tests
the test material in the test solutions. (These limitations do not between species in the same family. For example, Dwyer et al.
(2005a,b) (22,23) and Besser et al. (2005) (24) reported the
apply to any ingredient of a mixture, formulation, or commer-
cial product unless an extra amount of solvent is used in the sensitivity of endangered species of fish was within a factor of
about2ofcommonlytestedsurrogatefishspeciesforavariety
preparation of the stock solution.) (Warning—Acetone is also
quite volatile.) of organic and inorganic chemicals in acute or chronic toxicity
tests.Similarly,USEPA(2003) (25)reportedsimilarsensitivity
9.2.4 If the test material cannot be dissolved in the dilution
of aquatic species to a variety of organic or inorganic chemi-
water, at least one solvent control using solvent from the same
cals in toxicity tests conducted within a family.
batch used to make the stock solution, must be included in the
9.2.4.4 If the test contains both a dilution-water control and
test. If no other solvent other than water is used, a dilution-
a solvent control, the survival and growth, or both, of the
water control must be included in the test and the survival and
organismsinthetwocontrolsshouldbecompared(seeX10.6).
growth, or both, of test organisms in the dilution water control
Ifastatisticallysignificantdifferenceinsurvivalandgrowthor
must meet test acceptability requirements in order for the test
both is detected between the two controls, only the solvent
tobeconsideredacceptable(13.1).Usingnosolventotherthan
control should be used for meeting the requirements of 13.1.8
dilution water is the most desirable option (9.2.2) because
and as the basis for calculation of results. If no statistically
usinganyothersolventmeansthatantagonism,synergism,and
significant difference is detected, the data from both controls
confounding are possible. Using different concentrations of a
shouldbepooledformeetingtherequirementsof13.1.8andas
solvent at the different concentrations of the test material
the basis for calculation of results.
should be avoided because both the concentration of the
9.2.5 If a solvent other than water is used to prepare a stock
solvent and the concentration of the test material vary across
solution,itmightbedesirabletoconductsimultaneoustestson
thetreatments,potentiallyresultinginconfounding.Therefore,
the test material using two chemically unrelated solvents or
itisdesirabletotestthesameconcentrationofsolventinallof
two different concentrations of the same solvent to obtain
the test solutions.
information concerning possible effects of solvent on the
9.2.4.1 If the concentration of solvent is the same in all test
toxicityofthetestmaterialorthesensitivityofthetestspecies.
solutions that contain test material, the solvent control must
9.3 Test Concentration(s):
contain the same concentration of solvent.
9.3.1 Ifthetestisintendedtoprovideagoodestimateofthe
9.2.4.2 If the concentration of solvent is not the same in all
highestconcentrationoftestmaterialthatwillnotunacceptably
test solutions that contain test material, either (a) an early
affect survival or growth of the early life stages of the test
life-stagetestmustbeconductedtodeterminewhethersurvival
species (see Section 14), the test concentrations (see 11.1.1.1)
and growth, or both of the test organisms is related to the
shouldbracketthebestpredictionofthatconcentration.Sucha
concentration of the solvent over the range used in the toxicity
prediction is usually based on the results of a flow-through
test, or (b) such an early life-stage test must have been
acute toxicity test (see Guide E729) using the same dilution
conductedonthesolventusingthesamedilutionwaterandtest
water, test material, and species. If an acute-chronic ratio has
species. If survival and growth, or both, is found to be related
been determined for the test material with a species of
totheconcentrationofsolvent,anearlylife-stagetestwiththat
comparable sensitivity, the result of the acute test can be
speciesinthatwaterisunacceptableifanytreatmentcontained
divided by the acute-chronic ratio. Except for a few materials,
a concentration of solvent in that range. If neither survival nor
acute-chronic ratios with sensitive species are often less than
growthisfoundtoberelatedtotheconcentrationofsolvent,an
five. Thus, if no other useful information is available, the
earlylife-stagetoxicitytestwiththatsamespeciesinthatsame
highest concentration of test material in an early life-stage test
water may contain solvent concentrations within the tested
is often selected to be equal to the lowest concentration that
range,butthesolventcontrolmustcontainthehighestconcen-
caused adverse effects in a comparable acute test.
tration of solvent present in any of the other treatments.
9.3.2 In some (usually regulatory) situations, it is only
9.2.4.3 There may be instances when a toxicity test is to be
necessary to determine whether one specific concentration of
conducted with a species that is not routinely available for
the test material reduces survival or growth. For example, the
testing (for example, such as with an endangered species
specific concentration might be the concentration occurring in
(Dwyer et al. 2005a,b ; Besser et al. 2005 (22,23,24)). In these
a surface water, the concentration resulting from the direct
instances, the toxicity test used to evaluate potential effects of
application of the material to a body of water, or the solubility
a solvent outlined in 9.2.4.2 may be conducted with a species
limit of the material in water. When there is only interest in a
in the same family (preferably the same genus) as long as the
specific concentration, it is often only necessary to test that
concentrations of solvent are at least double the concentration
concentration (see 11.1.1.2).
of solvent used in the toxicity test on the test material. For
example if data were available for a commonly tested species
10. Test Organisms
such as rainbow trout (Oncorhynchus mykiss) demonstrating
no effect of a solvent of interest at 100 ug/L, then the solvent 10.1 Species—Whenever possible and appropriate, tests
concentration for toxicity test conducted with a species in the should be conducted with species listed in Appendix X1 –
same family (for example, the Oncorhynchus of interest) can Appendix X9 mainly because these species have been used
E1241 − 22
successfullyinearlylife-stagetests.Useofthespecieslistedin 10.4.3 Thebroodstockshouldbecaredforproperly (26)so
theappendicesisencouragedtoincreasetheavailabilityofdata it is not unnecessarily stressed. To maintain fish in good
for comparison among these few species. However, other condition and avoid unnecessary stress, they should not be
speciescanalsobeusedwithappropriatemodificationofthese crowded and should not be subjected to rapid changes in
procedures and when necessary to meet the needs of a specific
temperature or water quality. Fish should not be subjected to
study. Use of a specific strain should be specified only when it more than a 3°C change in water temperature in any 12h
is of special concern. The species used should be determined
period and preferably not more than 3°C in 72 h. The
using an appropriate taxonomic key. concentration of dissolved oxygen should be maintained be-
tween 60% and 100% saturation (16) and continuous gentle
10.2 Age—Except possibly with salmonids (see Appendix
aeration is usually desirable. Supersaturation by dissolved
X1) and with Gulf toadfish (see Appendix X7), tests should be
gasesshouldbeavoidedtopreventgas-bubbledisease (15, 17).
begun with newly fertilized (uneyed) embryos (≤24 h after
Water that might be contaminated with facultative pathogens
fertilization) and must be begun with embryos less than 48 h
may be passed through a properly maintained ultraviolet
after fertilization so that the exposure encompasses the early
sterilizer (18) equipped with an intensity meter and flow
stages of organogenesis.
controls or passed through a filter with a pore size of 0.22 µm
10.3 Source—Allorganismsinatestmustbefromthesame
or less. The un-ionized ammonia concentration in holding and
source. Gametes or embryos may be obtained from: (a) brood
acclimation tanks should be less than 35 µg/L (27).
fish cultured in the laboratory: (b) commercial, state or federal
NOTE 1—The value given is for 15°C at pH=8.0 to 9.0. Other values
hatcheries; or (c) wild populations from relatively unpolluted
mightbemoreappropriatedependingonthespecies,temperature,andpH.
areas. Laboratory cultures of species such as fathead and
sheepshead minnows and Atlantic and tidewater silversides 10.4.4 At least once a day, brood fish should be fed a food
usually can provide at any time of the year gametes whose
that will support survival, growth, and reproduction (see
history, age, and quality are known.Whenever salmon or trout AppendixX1–AppendixX9).Analysisofthefoodforthetest
aretobeused,gametesshouldbeobtainedfromahatcherythat
material is desirable if the material might be present in the
has been certified disease-free, for example, free of infectious environment.
pancreatic necrosis, furunculosis, kidney disease, enteric
10.4.5 Tanks should be scraped or brushed as needed.
redmouth, and whirling disease. Requirements for certification
Between use with different groups of fish, tanks should be
varyfromstatetostateandfromspeciestospecies.Gametesof
sterilizedbyautoclavingorbytreatmentwithaniodophor (28)
the other suggested species are usually obtained directly from
or with 200 mg of hypochlorite/L for 1 h, brushed well once
wildpopulations(seeAppendixX1–AppendixX9forspecific
during the hour, and then rinsed well.Although iodophors are
methodsregardingcareofbroodfishandobtaininggametesfor
not very acutely toxic to fish, hypochloride is (see 7.5
testing).Importingandcollectingpermitsmightberequiredby
concerning preparation and removal of hypochlorite).
local and state agencies.
10.4.6 Brood fish should be carefully observed daily during
10.4 Brood Stock: quarantine,holding,acclimation,andcultureforsignsofstress,
physical damage, mortality, disease, and external parasites.
10.4.1 Brood fish can be obtained from either another
Abnormal, dead, and injured individua
...