ASTM E1193-20

Standard Guide for Conducting <emph type="ital">Daphnia magna</emph> Life-Cycle Toxicity Tests

SIGNIFICANCE AND USE
5.1 Protection of an aquatic species requires prevention of unacceptable effects on populations in natural habitats. Toxicity tests are conducted to provide data that may be used to predict what changes in numbers and weights of individuals might result from similar exposure to the test material in the natural aquatic environment. Information might also be obtained on the effects of the material on the health of the species.  
5.2 Results of life-cycle tests with D. magna are used to predict chronic effects likely to occur on daphnids in field situations as a result of exposure under comparable conditions.  
5.2.1 Life-cycle tests with D. magna are used to compare the chronic sensitivities of different species, the chronic toxicities of different materials, and study the effects of various environmental factors on the results of such tests.  
5.2.2 Life-cycle tests with D. magna are used to assess the risk of materials to aquatic organisms (see Guide E1023) or derive water quality criteria for aquatic organisms (1).3  
5.2.3 Life-cycle tests with D. magna are used to extrapolate the results of chronic toxicity 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 a life-cycle test with D. magna may be increased by reporting the results of an acute toxicity test (see Guide E729) conducted under the same conditions. In addition to conducting an acute toxicity test with unfed D. magna, it may be relevant to conduct an acute test in which the daphnids are fed the same as in the life-cycle test to see if the presence of that concentration of that food affects the results of the acute test and the acute-chronic ratio (ACR) (see 10.3.1).  
5.2.4 Life-cycle tests are used to evaluate the biological availability of, and structure-activity relationships between, test materials and t...
SCOPE
1.1 This guide covers procedures for obtaining laboratory data concerning the adverse effects of a test material (added to dilution water, but not to food) on Daphnia magna Straus, 1820, during continuous exposure throughout a life-cycle using the renewal or flow-through techniques. These procedures also should be useful for conducting life-cycle toxicity tests with other invertebrate species and cladocerans from the same genus (for example, Daphnia pulex), although modifications might be necessary.  
1.2 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or known mixtures. With appropriate modifications, these procedures can be used to conduct tests on temperature, dissolved oxygen, pH, and on such materials as aqueous effluents (also see Guide E1192), leachates, oils, particulate matter, sediments, and surface waters. The technique, (renewal or flow-through), will be selected based on the chemical characteristics of the test material such as high oxygen demand, volatility, susceptibility to transformation (biologically or chemically), or sorption to glass.  
1.3 Modification 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 standard test procedures. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information on new concepts and procedures for conducting life-cycle toxicity tests with D. magna. Appendix X3 provides modifications for conducting the chronic toxicity test method with D. pulex Leydig, 1860.  
1.4 This guide is arranged as follows:    
Section  
Referenced Documents  
2  
Terminology  
3  
Summary of Guide  
4  
Si...

General Information

• Status: Published
• Publication Date: 30-Nov-2020
• ICS: 07.100.20 - Microbiology of water
• ICS: 13.020.30 - Environmental impact assessment
• 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 E1193-20: Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests provides a comprehensive framework for evaluating the effects of chemicals and other test materials on the freshwater invertebrate Daphnia magna. This standard, developed by ASTM International, outlines laboratory procedures for life-cycle toxicity testing using renewal or flow-through exposure methods. The guide addresses testing requirements to assess the chronic toxicity, survival, growth, and reproduction of D. magna under controlled laboratory conditions, supporting regulatory assessments and environmental risk evaluations.

Key Topics

• Scope and Flexibility
  ASTM E1193-20 specifies techniques for assessing adverse effects of test materials-such as chemicals, effluents, leachates, and sediments-on Daphnia magna throughout their life cycle. Procedures may be adapted for use with other cladoceran species, enhancing the guide's applicability to broader ecological studies.

• Test Design and Exposure

  • Describes renewal and flow-through techniques to ensure continuous, controlled exposure.
  • Specifies conditions for culturing and handling test organisms, including water quality, temperature, and food requirements.
  • Recommends a structured experimental design with multiple concentrations and control groups to evaluate dose-response relationships.

• Significance of Life-Cycle Tests

  • Provides predictive data for changes in populations due to environmental exposures.
  • Supports comparison of chronic sensitivities among species and the analysis of environmental factors’ effects.
  • Enables extrapolation of chronic toxicity results across different water sources or species.

• Data Analysis and Reporting

  • Details endpoints such as survival, reproduction, and growth to evaluate chronic effects.
  • Emphasizes the importance of reporting test conditions and analytical results for regulatory compliance and reproducibility.

Applications

ASTM E1193-20 is widely used in ecotoxicology, environmental protection, and regulatory settings where accurate assessment of long-term aquatic toxicity is crucial. Key applications include:

• Regulatory Compliance
  Enables industries and laboratories to generate data required for chemical registration, pesticide approvals, and meeting environmental protection regulations.

• Water Quality Assessment
  Supports the derivation of water quality criteria to protect aquatic organisms in freshwater environments.

• Risk Assessment
  Supplies data for ecological risk assessments, helping to predict chronic impacts and potential population-level effects of test substances on aquatic habitats.

• Product Development and Environmental Monitoring
  Informs the development of environmentally safer chemicals and products by identifying adverse effects early in their life cycle.

• Comparative Toxicity Studies
  Facilitates comparison of toxicological profiles across different materials, species, and environmental conditions, guiding environmental management decisions.

Related Standards

To ensure comprehensive aquatic toxicity testing, ASTM E1193-20 frequently references additional ASTM guides and standards, including:

• ASTM E729: Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians
• ASTM E1023: Guide for Assessing the Hazard of a Material to Aquatic Organisms
• ASTM E1192: Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents
• ASTM E943: Terminology Relating to Biological Effects and Environmental Fate
• ASTM E1733: Guide for Use of Lighting in Laboratory Testing

These related standards provide further procedures and terminology necessary for consistent, reliable aquatic toxicity assessments. Together, they form a robust toolkit for laboratories, regulatory bodies, and industries dedicated to safeguarding aquatic environments and ensuring responsible chemical management.

---

Keywords: Daphnia magna life-cycle toxicity test, chronic aquatic toxicity, ASTM E1193-20, environmental risk assessment, water quality criteria, ecotoxicology, aquatic organism protection, chronic toxicity guideline, flow-through toxicity test, renewal toxicity test, regulatory ecotoxicology testing.

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: E1193 − 20
Standard Guide for
Conducting Daphnia magna Life-Cycle Toxicity Tests
This standard is issued under the fixed designation E1193; 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 This guide covers procedures for obtaining laboratory Referenced Documents 2
Terminology 3
data concerning the adverse effects of a test material (added to
Summary of Guide 4
dilution water, but not to food) on Daphnia magna Straus,
Significance and Use 5
Apparatus 6
1820,duringcontinuousexposurethroughoutalife-cycleusing
Facilities 6.1
the renewal or flow-through techniques.These procedures also
Construction Materials 6.2
should be useful for conducting life-cycle toxicity tests with
Test Chambers 6.3
otherinvertebratespeciesandcladoceransfromthesamegenus Cleaning 6.4
Acceptability 6.5
(forexample, Daphnia pulex),althoughmodificationsmightbe
Reagents 7
necessary.
Purity of Reagents 7.1
Hazards 8
1.2 These procedures are applicable to most chemicals,
Dilution Water 9
Requirements 9.1
either individually or in formulations, commercial products, or
Source 9.2
known mixtures. With appropriate modifications, these proce-
Treatment 9.3
dures can be used to conduct tests on temperature, dissolved
Characterization 9.4
Test Material 10
oxygen, pH, and on such materials as aqueous effluents (also
General 10.1
see Guide E1192), leachates, oils, particulate matter,
Stock Solutions 10.2
sediments, and surface waters. The technique, (renewal or Test Concentrations(s) 10.3
Test Organisms 11
flow-through), will be selected based on the chemical charac-
Species 11.1
teristics of the test material such as high oxygen demand,
Age 11.2
volatility, susceptibility to transformation (biologically or Source 11.3
Brood Stock 11.4
chemically), or sorption to glass.
Food 11.5
Handling 11.6
1.3 Modification of these procedures might be justified by
Harvesting Young 11.7
special needs or circumstances. Although using appropriate
Quality 11.8
Procedure 12
procedures is more important than following prescribed
Experimental Design 12.1
procedures,resultsoftestsconductedusingunusualprocedures
Dissolved Oxygen 12.2
are not likely to be comparable to results of standard test
Temperature 12.3
Loading 12.4
procedures.Comparisonofresultsobtainedusingmodifiedand
Selection of Test System 12.5
unmodified versions of these procedures might provide useful
Beginning the Test 12.6
information on new concepts and procedures for conducting
Care and Maintenance 12.7
Feeding 12.8
life-cycle toxicity tests with D. magna. Appendix X3 provides
Duration 12.9
modifications for conducting the chronic toxicity test method
Biological Data 12.10
with D. pulex Leydig, 1860.
Other Measurements 12.11
Analytical Methodology 13
1.4 This guide is arranged as follows: Acceptability of Test 14
Calculation of Results 15
Report 16
Keywords 17
Appendixes
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Appendix X1 Statistical Guidance
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
Appendix X2 Food
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Appendix X3 Modifications for Conducting Chronic Life
Current edition approved Dec. 1, 2020. Published January 2021. Originally
Cycle Analysis Tests with Daphnia Pulex
approved in 1987. Last previous edition approved in 2012 as E1193–97 (2012).
DOI: 10.1520/E1193-20.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1193 − 20
1.5 The values stated in SI units are to be regarded as “can”ispreserved,and“might”isneverusedasasynonymfor
standard. No other units of measurement are included in this either“ may” or “can.”
standard.
3.5 Fordefinitionsofothertermsusedinthisguide,referto
1.6 This standard does not purport to address all of the
Guide E729 and Terminology E943. For an explanation of
safety concerns, if any, associated with its use. It is the
units and symbols, refer to IEEE/ASTM SI 10.
responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter-
4. Summary of Guide
mine the applicability of regulatory limitations prior to use.
Specific hazard statements are given in Section 8. 4.1 A 21-day life-cycle toxicity test for Daphnia magna is
1.7 This international standard was developed in accor- described. The test design allows for the test organisms to be
dance with internationally recognized principles on standard-
exposed to a test material using either the renewal technique
ization established in the Decision on Principles for the
(withexchangeofthetotalvolumeoftestwaterandtoxicantat
Development of International Standards, Guides and Recom-
least three times a week) or the flow-through technique (with
mendations issued by the World Trade Organization Technical
continual water and toxicant addition, usually at least four
Barriers to Trade (TBT) Committee.
volumeadditionsperday).Atleastfiveconcentrationsofatest
material, a control, and a solvent control (if applicable)
2. Referenced Documents
replicated at least four times are recommended. Each test
2.1 ASTM Standards:
concentrationhasatleastten Daphniapertreatment.Thereare
E729Guide for Conducting Acute Toxicity Tests on Test
two applicable exposure approaches (both may be renewal or
Materials with Fishes, Macroinvertebrates, and Amphib-
flow-through): (1) use of a minimum of ten daphnids per
ians
treatment and one daphnid per replicate (renewal or flow-
E943Terminology Relating to Biological Effects and Envi-
through); or (2) use of four replicates with at least five
ronmental Fate
daphnids per replicate (≥20 daphnids per treatment).Acontrol
E1023Guide for Assessing the Hazard of a Material to
consists of maintaining daphnids in dilution water to which no
Aquatic Organisms and Their Uses
test material has been added to provide (1) a measure of the
E1192Guide for ConductingAcute Toxicity Tests onAque-
acceptability of the test by giving an indication of the quality
ous Ambient Samples and Effluents with Fishes,
of the test organisms and the suitability of the dilution water,
Macroinvertebrates, and Amphibians
food,testconditions,handlingprocedures,andsoforth,and(2)
E1733Guide for Use of Lighting in Laboratory Testing
the basis for interpreting data obtained from the other treat-
E1847Practice for Statistical Analysis of Toxicity Tests
ments. In each of the other treatments, the daphnids are
Conducted Under ASTM Guidelines
maintained in dilution water, to which a selected concentration
IEEE/ASTM SI 10American National Standard for Use of
oftestmaterialhasbeenintentionallyadded.Measurementend
theInternationalSystemofUnits(SI):TheModernMetric
points obtained during the test include the concentration of the
System
test material and final number alive, final weight, final length,
and number of progeny per daphnid. Data are analyzed to
3. Terminology
determine the effect of the test material on survival, growth,
3.1 Thewords“must,”“should,”“may,”“can,”and“might”
and reproduction of D. magna. These methods are also
have very specific meanings in this guide.
applicable for chronic toxicity testing using the conspecific
3.2 must—used to express an absolute requirement, that is,
Daphnia pulex,withslightmodificationsprovidedinAppendix
tostatethatthetestoughttobedesignedtosatisfythespecified
X3.
condition, unless the purpose of the test requires a different
design. “Must” is used only in connection with factors that
5. Significance and Use
directly relate to the acceptability of the test (see 14.1).
5.1 Protection of an aquatic species requires prevention of
3.3 should—used to state that the specified condition is
unacceptable effects on populations in natural habitats. Toxic-
recommended and ought to be met if possible. Although
ity tests are conducted to provide data that may be used to
violationofone“should”israrelyaseriousmatter,violationof
predict what changes in numbers and weights of individuals
several will often render the results questionable. Terms such
might result from similar exposure to the test material in the
as“isdesirable,”“isoftendesirable,”and“mightbedesirable”
natural aquatic environment. Information might also be ob-
are used in connection with less important factors.
tainedontheeffectsofthematerialonthehealthofthespecies.
3.4 may—used to mean “is (are) allowed to,” “can” is used
5.2 Results of life-cycle tests with D. magna are used to
to mean “is (are) able to,” and “might” is used to mean “could
possibly.”Thereforetheclassicdistinctionbetween“may”and predict chronic effects likely to occur on daphnids in field
situationsasaresultofexposureundercomparableconditions.
5.2.1 Life-cycle tests with D. magna are used to compare
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
the chronic sensitivities of different species, the chronic tox-
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
icities of different materials, and study the effects of various
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. environmental factors on the results of such tests.
E1193 − 20
5.2.2 Life-cycle tests with D. magna are used to assess the 6.2 Construction Materials—Equipment and facilities that
risk of materials to aquatic organisms (see Guide E1023)or contact stock solutions, test solutions, or any water into which
derive water quality criteria for aquatic organisms (1). daphnids will be placed should not contain substances that can
5.2.3 Life-cycle tests with D. magna are used to extrapolate be leached or dissolved by aqueous solutions in amounts that
the results of chronic toxicity tests on the same test material can adversely affect daphnids. In addition, equipment and
with the same species in another water or with another species facilitiesthatcontactstocksolutionsortestsolutionsshouldbe
inthesameoradifferentwater.Mostsuchpredictionstakeinto chosen to minimize sorption of test materials from water.
accounttheresultsofacutetoxicitytests,andsotheusefulness Glass, Type 316 stainless steel, nylon, fiberglass, silicon, and
of the results of a life-cycle test with D. magna may be fluorocarbon plastics should be used whenever possible to
increased by reporting the results of an acute toxicity test (see minimize leaching, dissolution, and sorption. Concrete and
Guide E729) conducted under the same conditions. In addition rigid (unplasticized) plastics may be used for culture tanks and
to conducting an acute toxicity test with unfed D. magna,it in the water-supply system, but they should be soaked, prefer-
mayberelevanttoconductanacutetestinwhichthedaphnids ably in flowing dilution water, for several days before use (6).
are fed the same as in the life-cycle test to see if the presence Cast-iron pipe may be used in supply systems, but colloidal
ofthatconcentrationofthatfoodaffectstheresultsoftheacute iron probably will be added to the dilution water and strainers
test and the acute-chronic ratio (ACR) (see 10.3.1). will be needed to remove rust particles. Copper, brass, lead,
5.2.4 Life-cycle tests are used to evaluate the biological galvanized metal, and natural rubber should not contact dilu-
availability of, and structure-activity relationships between, tion water, stock solutions, or test solutions before or during
test materials and test organisms. thetest.Itemsmadeofneoprenerubberandothermaterialsnot
previously mentioned should not be used unless it has been
5.3 Results of life-cycle tests with D. magna might be
shown that their use will not adversely affect survival, growth,
influenced by temperature (2), quality of food, composition of
and reproduction of D. magna (see Section 14).
dilution water, condition of test organisms, test media (for
example, water hardness), and other factors. 6.3 Test Chambers:
6.3.1 Flow-through tests, 500-mL to 2-L glass beakers (or
6. Apparatus equivalent)withanotch(approximately4by13cm)cutinthe
lipmaybeusedtoexposethe Daphniatothetestmaterial.The
6.1 Facilities—Cultureandtestchambersareoftenkeptina
notchshouldbecoveredwith0.33-mmopening(U.S.standard
separate room maintained at about 20°C. Alternatively, cul-
sieve size No. 50) stainless steel or polyethylene screening
tures and test chambers may be placed in a temperature-
small enough to retain first instar Daphnia. The screen can be
controlled water bath, environmental chamber, or incubator.
attached to the beaker with silicone adhesive. The chambers
Thewater-supplysystemshouldprovideanadequatesupplyof
shouldprovideatleast30mLofsolutionforeachoftheinitial
dilution water to the culture tanks and test chambers. The
test daphnid(s).
water-supply system should be equipped for temperature con-
6.3.2 Renewal tests,beakerranginginsizefrom100to1000
trolandaeration,andstrainersandairtrapsshouldbeincluded
mL.Anotchedchamberisnotrequiredforarenewaltest.Each
inthewater-supplysystem.Airusedforaerationshouldbefree
chamber should provide at least 40 mLof solution for each of
of fumes, oil, and water; filters to remove oil and water are
the initial test daphnid(s).
desirable. Filtration of air through a 0.22-µm bacterial filter
6.3.3 Any container made of glass,Type 316 stainless steel,
might be desirable (3). During culturing and testing, daphnids
or a fluorocarbon plastic may be used if (1) each chamber is
should be shielded from disturbances to prevent unnecessary
separatewithnointerconnections,(2)eachchambercontainsat
stress. The test facility should be well-ventilated and free of
least 30 mL of test solution (see 12.4) per first-generation
fumes.Atiming device should be used to provide a 16-h light
daphnid for flow-through tests and at least 40 mL for renewal
and 8-h dark photoperiod (4).A15 to 30-min transition period
tests,(3)thereisatleast1000mm ofairtowaterinterfaceper
when lights go on might be desirable to reduce the possibility
daphnid,and(4)thetestsolutionisatleast30mmdeep.Static
of daphnids being stressed by instantaneous illumination; a
test chambers should be covered with glass, stainless steel,
transition period when lights go off may also be desirable (see
nylon, or fluorocarbon plastic covers to keep out extraneous
Guide E1733).
contaminants and to reduce evaporation of test solution. All
6.1.1 When D. magna are fed live algae, a high-light
chambersandcoversinatestmustbeidentical.Coversarenot
intensity might cause sufficient photosynthesis to result in an
required for flow-through studies.
increaseofpHhighenoughtokilldaphnids (5).Therefore,the
maximum acceptable intensity is dependent on the buffer
6.4 Cleaning—Test chambers and equipment used to pre-
capacity of the dilution water, species, and density of algae,
pareandstoredilutionwater,stocksolutions,andtestsolutions
and the kind of test chamber and cover (see Guide E1733).
should be cleaned before use. New equipment should be
Light intensities up to 600 lx or a fluence rate of 1 w/m will
washed with detergent and rinsed with water, a water-miscible
usually be acceptable, but higher intensities might result in an
organic solvent, water, acid (such as 5% concentrated nitric
unacceptably high pH in the culture water.
acid), and washed at least twice with distilled, deionized, or
dilutionwater.Somelotsofsomeorganicsolventsmightleave
a film that is insoluble in water. Also, stronger nitric acid, for
example, 10%, might cause deterioration of silicone adhesive;
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this guide. aninitialrinsewith10%concentratedhydrochloricacidmight
E1193 − 20
preventsuchdeterioration.Adichromate-sulfuricacidcleaning degradation of test material might be desirable before disposal
solution can generally be used in place of both the organic of stock and test solutions.
solvent and the acid, but it might attack silicone adhesives.At
8.3 Cleaning equipment with a volatile solvent such as
the end of every test, all items that are to be used again should
acetone should be performed only in a well-ventilated area
be immediately (1) emptied, (2) rinsed with water, (3) cleaned
withnosmokingallowedandnoopenflame,forexample,pilot
by a procedure appropriate for removing the test material (for
light, is present.
example, acid to remove metals and bases; detergent, organic
8.4 Acidic solutions and hypochlorite solutions should not
solvent, or activated carbon to remove organic chemicals), and
be mixed together because hazardous fumes might be pro-
(4) rinsed at least twice with distilled, deionized, or dilution
duced.
water. Acid is useful for removing mineral deposits. Test
chambers should be rinsed with dilution water just before use.
8.5 Because dilution water and test solutions are usually
good conductors of electricity, use of ground fault systems and
6.5 Acceptability—Before a toxicity test is conducted in
leak detectors should be considered to help prevent electrical
new test facilities, it is desirable to conduct a control water-
shocks.
only (that is, no test material) test, in which all test chambers
contain dilution water. This test will reveal (1) whether D. 8.6 To prepare dilute acid solutions, concentrated acid
magna will survive, grow, and reproduce acceptably (see
should be added to water, not vice versa. Opening a bottle of
Section 14) in the new facilities, (2) whether there are any concentrated acid and mixing concentrated acid with water
location effects on survival, growth, or reproduction, and (3)
should be performed only in a well-ventilated area.
the magnitude of the within-chamber and between-chamber
9. Dilution Water
variance.
9.1 Requirements—The dilution water should (1) be accept-
7. Reagents
able to D. magna,(2) be of uniform quality, and (3), except as
stated in 9.1.4, not unnecessarily affect results of the test.
7.1 Purity of Reagents—Reagent grade chemicals shall be
9.1.1 The dilution water must allow satisfactory survival,
used in all tests. Unless otherwise indicated, it is intended that
growth, and reproduction of D. magna (see Section 14).
all reagents shall conform to the specifications of the Commit-
tee onAnalytical Reagents of theAmerican Chemical Society 9.1.2 The quality of the dilution water should be uniform,
allowing the brood stock to be cultured and the test conducted
where such specifications are available. Other grades may be
used, provided it is first ascertained that the reagent is of in water of the same quality. In particular, during culture or
testing, or both, the range of hardness should be 610% of the
sufficiently high purity to permit its use without lessening the
accuracy of the test. average.
9.1.3 The dilution water should not unnecessarily affect
8. Hazards
results of a life-cycle test with D. magna because of such
things as sorption or complexation of test material. Therefore,
8.1 Many materials can affect humans adversely if precau-
except as stated in 9.1.4, concentrations of both total organic
tions are inadequate. Therefore, skin contact with all test
carbon (TOC) and particulate matter should be less than 5
materials and solutions should be minimized by wearing
mg/L.
appropriate protective gloves (especially when washing equip-
9.1.4 If it is desired to study the effect of an environmental
ment or putting hands in test solutions), laboratory coats,
factor such asTOC, particulate matter, or dissolved oxygen on
aprons, and glasses, and by using dip nets or tubes to remove
the results of a life-cycle test with D. magna, it will be
daphnids from test solutions. Special precautions, such as
necessary to use a water that is naturally or artificially high in
coveringtestchambersandventilatingtheareasurroundingthe
TOC or particulate matter or low in dissolved oxygen. If such
chambers, should be taken when conducting tests on volatile
a water is used, it is important that adequate analyses be
materials. Information on toxicity to humans (7), recom-
performedtocharacterizethewater,andthatacomparabletest
mended handling procedures (8), and chemical and physical
be available or conducted in the laboratory’s usual culture
properties of the test material should be studied before a test is
dilution water to facilitate interpretation of the results in the
begun. Special procedures will be necessary with radiolabeled
special water.
test materials (9) and with materials that are, or are suspected
of being, carcinogenic (10).
9.2 Source:
9.2.1 The use of reconstituted water might increase compa-
8.2 Disposal of stock solutions, test solutions, and test
rability of test results between laboratories. The hard reconsti-
organisms might pose special problems in some cases;
tuted fresh water (160 to 180 mg/L as CaCO ) described in
therefore, health and safety precautions and applicable regula- 3
Guide E729 has been used successfully. Addition of 2 µg of
tionsshouldbeconsideredbeforebeginningatest.Removalor
selenium(IV) and 1 µg of crystalline vitamin B /L might be
desirable (11). Other water sources (natural or reconstituted)
may be used if they have been demonstrated to provide
Reagent Chemicals, American Chemical Society Specifications, American
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
adequate daphnid survival, growth, and reproduction.
listed by the American Chemical Society, see Analar Standards for Laboratory
9.2.2 Natural fresh waters have been used successfully.
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
Natural waters should be obtained from an uncontaminated
and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
MD. source of consistent quality. A well or spring is usually
E1193 − 20
preferable to a surface water. If a surface water is used, the 10. Test Material
intake should be positioned to minimize fluctuations in quality 4
10.1 General—Thetestmaterialshouldbereagentgrade or
and the possibility of contamination and should maximize the
better, unless a test on a formulation, commercial product, or
concentration of dissolved oxygen to help ensure low concen-
technical-grade or use-grade material is specifically needed.
trations of sulfide and iron.
Before a test is begun, the following should be known about
9.2.3 Dechlorinated water is not recommended as a dilution
the test material:
waterfor Daphnia magna.Dechlorinatedwatershouldbeused
10.1.1 Identities and concentrations of major ingredients
only as a last resort because dechlorination is often incomplete
and major impurities. For example, impurities constituting
and residual chlorine is quite toxic to D. magna (12). Sodium more than about 1% of the material.
bisulfite is probably better for dechlorinating water than
10.1.2 Solubility and stability in the dilution water and
sodium sulfite, and both are more reliable than carbon solvents.
filtration, especially for removing chloramines (13). Some 10.1.3 Measured acute toxicity to D. magna.
organic chloramines, however, react slowly with sodium
10.1.4 Measured or estimated chronic toxicity to D. magna.
bisulfite (14). In addition to residual chlorine, municipal 10.1.5 Precision and bias of the analytical method at the
drinkingwateroftencontainsunacceptablyhighconcentrations planned concentration(s) of test material.
of copper, lead, zinc, and fluoride, and quality is often rather 10.1.6 Estimate of toxicity to humans.
variable. When necessary, excessive concentrations of most 10.1.7 Recommended handling procedures (see 8.1).
metals can usually be removed with a chelating resin (15).
10.2 Stock Solutions:
10.2.1 Stock solutions are usually prepared prior to dosing
9.3 Treatment:
the dilution water to obtain the desired test concentrations.
9.3.1 Dilution water should be aerated intensively by such
Water-soluble test materials can often be added directly to
means as air stones, surface aerators, or column aerators
dilution water to prepare a stock solution (or in some cases the
(16,17)priortotheadditionoftestmaterial.Adequateaeration
test solution). Test materials that are moderately soluble or
will bring the pH and concentrations of dissolved oxygen and
insoluble in water are often dissolved in a solvent to form a
othergasesintoequilibriumwiththeair,andminimizeoxygen
stock solution that is then added to dilution water. If a stock
demand and concentrations of volatiles. The concentration of
solution is used, the concentration and stability of the test
dissolved oxygen in dilution water should be between 90 and
material in the stock solution should be determined before
100% saturation to help ensure that dissolved oxygen concen-
beginning the test. If the test material is subject to photolysis,
trations are acceptable in test chambers. Supersaturation of
the stock solution should be shielded from light. If the test
dissolved gases, which might be caused by heating dilution
material hydrolyzes or biodegrades rapidly, it might be neces-
water,shouldbeavoidedtopreventgasbubbledisease (18,19).
sary to prepare new stock solutions daily.
9.3.2 Filtration through sand, sock, bag, or depth-type
10.2.2 The preferred carrier for stock solutions is dilution
cartridge filters may be used to keep the concentration of
waterexceptpossiblyfortestsonhydrolyzable,oxidizable,and
particulate matter acceptably low (see 9.1.3).
reducible materials. Filtration or sterilization, or both, of the
9.3.3 Dilution water that might be contaminated with unde-
water might be necessary. If the hardness of the dilution water
sirable microorganisms may be passed through a properly in the test system will not be affected, distilled and deionized
maintainedultravioletsterilizer (20)equippedwithanintensity water are also acceptable for stock solution preparation.
meter and flow controls or passed through a filter with a pore Several techniques have been specifically developed for pre-
size of 0.45 µm. Water that might be contaminated with paring aqueous stock solutions of slightly soluble materials
Aphanomyces daphniae should be autoclaved (3). (22). Minimum necessary amounts of strong acids and bases
may be used to prepare aqueous stock solutions, but such
9.4 Characterization:
reagents might affect the pH of test solutions appreciably. Use
9.4.1 Thefollowingitemsshouldbemeasuredatleasttwice
of a more soluble form of the test material, such as chloride or
each year, and more often if, (1) such measurements have not
sulfate salts of organic amines, sodium or potassium salts of
been made semiannually for at least two years, or (2) surface
phenols and organic acids, and chloride or nitrate salts of
waterisused:hardness,alkalinity,conductivity,pH,particulate
metals, might affect the pH even more than the use of the
matter, TOC, organophosphorus pesticides, polychlorinated
minimum necessary amount of strong acid or base.
biphenyls (PCBs), chlorinated phenoxy herbicides, ammonia,
10.2.3 If a solvent other than dilution water is used, its
cyanide, sulfide, chloride, bromide, fluoride, iodide, nitrate,
concentration in test solutions should be kept to a minimum
phosphate, sulfate, calcium, magnesium, sodium, potassium,
and should not affect survival, growth, or reproduction of D.
aluminum, arsenic, beryllium, boron, cadmium, chromium,
magna. Because of their low toxicities to aquatic animals (23),
cobalt, copper, iron, lead, manganese, mercury, molybdenum,
low volatilities, and high abilities to dissolve many organic
nickel, selenium, silver, and zinc.
chemicals,dimethylformamideandtriethyleneglycolareoften
9.4.2 Foreachanalyticalmethodused(see13.3)tomeasure good organic solvents for preparing stock solutions. Other
theparameterslistedin9.4.1,quantificationofthelimitshould
water-miscible organic solvents, such as methanol, ethanol,
be below either (1) the concentration in the dilution water or and acetone, may also be used as carriers, but they might
(2) the lowest concentration that has been shown to adversely
stimulate undesirable growths of microorganisms, and acetone
affect the survival, growth, or reproduction of D. magna (21). is quite volatile. If an organic solvent is used, its concentration
E1193 − 20
in any test solution should not exceed 0.1 mL/L. Surfactants specific concentration, it is often only necessary to test that
shouldnotbeusedinthepreparationofstocksolutionsbecause specific concentration (see 12.1.2).
they might affect the form and toxicity of the test material in
11. Test Organisms
test solutions. (These limitations do not apply to any ingredi-
11.1 Species—D. magnahasbeenextensivelyusedforacute
ents of a mixture, formulation, or commercial product, unless
and life-cycle toxicity tests because it is one of the largest
an extra amount of solvent is used in the preparation of the
cladoceran species, is easy to identify, and is available from
stock solution.)
many laboratories and commercial sources. These procedures
10.2.4 If a solvent other than water is used as a carrier, at
might also be suitable for other daphnid species, (that is, D.
least one solvent control, using solvent from the same batch
pulex) although modifications might be necessary. The identi-
used to make the stock solution, in addition to the dilution-
ties of daphnids obtained from laboratories and commercial
water control, must be included in the test.
sources should be verified, regardless of any information that
10.2.4.1 If the test contains both a dilution-water control
comes with the organisms. Daphnia magna should be verified
andasolventcontrol,thesurvival,growth,andreproductionof
using the scheme of Brooks (27). The identification of other
D. magna in the two controls should be compared (see X1.4).
daphnids may vary with the taxonomic reference used (28,29).
Ifastatisticallysignificantdifferenceineithersurvival,growth,
or reproduction is detected between the two controls, the
11.2 Age—Life-cycletestswith D. magnashouldbeginwith
solvent control is normally used for meeting the requirements organisms less than 24-h old.Neonates shipped overnight from
specified in Section 14 and as the basis for the calculation of
avendormaybegreaterthan24-holdandthustheirageshould
results. Judgment might be required in the choice of which be specifically documented. It is preferable that neonates used
controldatatousetocomparewithtreatments,especiallywhen
in testing be obtained from in-house cultures (see 11.4)to
thesolventconcentrationisnotconstantinthetreatments.Ifno ensure organisms are the right age and to avoid unnecessary
statistically significant difference is detected, the data from
handling stresses (see 11.6).
both controls should be used for meeting the requirements
11.3 Source—Alldaphnidsusedinatestshouldbefromthe
specified in Section 14 and as the basis for calculating the
same brood stock. This brood stock must have been cultured
results (see Appendix X1).
for at least two generations using the same food, water, and
10.2.5 If a solvent other than water is used as a carrier, it
temperature as will be used in the life-cycle test. This will not
might be desirable to conduct simultaneous tests using two
only acclimate the daphnids, but will also demonstrate the
chemically unrelated solvents or two different concentrations
acceptability of the food, water, and so forth, before the test.
of the same solvent to obtain information concerning possible
11.4 Brood Stock:
effects of solvent on results of the test.
11.4.1 Brood stock can be obtained from in-house cultures,
10.3 Test Concentration(s):
anotherlaboratoryoracommercialsource.Whendaphnidsare
10.3.1 If the test is intended to provide a good estimate of
brought into the laboratory, they should be acclimated to the
the highest concentration that will not unacceptably affect the
dilution water by gradually changing the water in the culture
survival, growth, or reproduction of D. magna, the test
chamber from the water in which they were transported to
concentrations(see12.1.1.1)shouldbracketthebestprediction
100% dilution water over a period of two or more days.
of that concentration. Such a prediction is usually based on the
Daphnids should be acclimated to the test temperature by
results of an acute toxicity test (see Guide E729) with the test
changing the water temperature at a rate not to exceed 3°C
material using the same dilution water and D. magna neonates
within12huntilthedesiredtemperatureisreached.Generally,
(forexample,individualslessthan24-hold).Becausethefood
acclimation to pH should not exceed more than 1.5 pH units
used in the life-cycle test sometimes affects the results of the
per day.
acute test (24,25), acute tests should be conducted with and
11.4.2 D. magna has been cultured in a variety of systems,
withoutthefoodaddedtothedilutionwaterpriortoconducting
such as in large groups in aquaria, in groups of one to five in
thechronicstudy.Ifanacute-chronicratiohasbeendetermined
100 to 250-mL beakers, or in specially designed chambers
for the test material with a species of comparable sensitivity,
(30).
theresultoftheacutetestwith D. magnacanbedividedbythe
11.4.3 To maintain D. magna in good condition, the brood
acute-chronic ratio. Except for a few materials (26), acute-
stock should be cultured so as to avoid unnecessary stress due
chronic ratios determined with daphnids are typically less than
to crowding, rapid changes in temperature, and water quality.
ten. Thus, the highest concentration of test material in a
Daphnids should not be subjected to more than a 3°C change
life-cycle test with D. magna is typically selected to be equal
in water temperature in any 12-h period and preferably not
to the lowest concentration that caused adverse effects in a
morethana3°Cchangeinany72-hperiod.Culturesshouldbe
comparable acute test.
regularly fed enough food to support adequate reproduction.
10.3.2 In some situations (usually regulatory), it is only Culture chambers should be cleaned periodically to remove
necessary to determine whether one specific concentration of feces, debris, and uneaten food. If culture chambers are
test material unacceptably affects survival, growth, or repro- properly cleaned and the density of daphnids is kept low, for
duction. These situations usually arise when the concentration example, no more than 1 daphnid/30 mL, the surface water/air
resulting from the direct application of a material to a body of interface should provide adequate dissolved oxygen. Organ-
water is known, or when the material is thought to be nontoxic isms used for testing must produce at least 60 young per adult
atitssolubilitylimitinwater.Whenthereisonlyinterestinone during a 21-day test.
E1193 − 20
TABLE 1 Experimental Design
11.5 Food—Various combinations (see Appendix X2)of
trout chow, yeast, alfalfa, and algae, such as Ankistrodesmus Design Parameter Renewal Test Flow-Through Test
convolutus, Ankistrodesmus falcatus, Chlorella vulgaris, Chla- Number of test $5 $5
concentrations
mydomonas reinhardtii, and Raphidocelis subcapitata (for-
Control Yes Yes
merly Selenastrum capricornutumand Pseudokirchneriella
Solvent control If appropriate If appropriate
subcapitata ) (31), have been successfully used for culturing Number of chambers At least 4 At least 4
Minimum number of 10 (individual daphnid/ 10 (individual daphnid/
and testing D. magna. The concentration of test material
daphnids/treatment chamber) chamber)
(number of cells for algae) in the batch of food used should be
20 20
determined. The experience gained over the past decade has (multiple daphnids/ (multiple daphnids/
chamber) chamber)
shown that it is critical to incorporate algae into the diet to
Number of daphnids/test At least 1 At least 5
maintain consistently healthy daphnids (32-34).
chamber
Feeding Once daily 2 to 3 times daily (or
11.6 Handling—D. magna should be handled as little as
continuous)
possible.Whenhandlingisnecessary,itshouldbedonegently, Renewal of test solution At least 3 times/week At least 1 volume
replacement/day
carefully, and quickly so that the daphnids are not unnecessar-
Temperature 20°C 20°C
ily stressed. Daphnids should be introduced into solutions
Water quality parameters New solutions at each Initially and at least
beneath the air-water interface. Daphnids that touch dry renewal, old solutions weekly thereafter
after longest time
surfaces or are dropped or injured during handling should be
hour interval
discarded. Smooth glass tubes with an inside diameter of at
Analytical confirmation Initially and at least Initially and at least
of test material weekly thereafter, old weekly thereafter
least5mmshouldbeusedfortransferringadult D. magna,and
solutions at least
the amount of solution carryover should be minimized. Equip-
once during the study
ment used to handle daphnids should be sterilized between use
by autoclaving or by treatment with an iodophor (35) or with
200 mg of hypochlorite/L for at least 1 h (see 6.4 ).
of test material. In the controls, daphnids are exposed to
11.7 Harvesting Young—Young less than 24-h old can be dilution water to which neither test material nor solvent has
obtained using specially designed chambers (27) or by trans- been added. One or more solvent controls might also be
ferring to chambers containing dilution water and food, allow- necessary (see 10.2.3). Except for the control(s) and the high
ing an overnight period for brood release. concentration, each concentration should be at least 50% of
the next higher one, unless information concerning the
11.8 Quality—To decrease the chances of a test being
concentration-effect curve indicates that a different dilution
unacceptable (see 14.1), the test should not begin with young
factor is more appropriate. At a dilution factor of 0.5, five
thatwereinthefirstbrood (32),norwithyoungfromadaphnid
concentrations are a reasonable compromise between cost and
that (1) is sick (3,36) or incompletely developed (11),(2)is
the risk of all concentrations being either too high or too low.
more than 50 days old, (3) did not produce young before Day
If the estimate of chronic toxicity is particularly uncertain (see
10, (4) did not produce at least nine young in the previous
10.3.1), six or seven concentrations might be desirable. Range
brood,or(5)isfromacultureinwhichephippiawereproduced
finder tests (for example, low replication tests with a 90%
or in which substantial mortality (>10%) occurred during the
dilution series) may also be informative in planning robust
week prior to the test. These factors are most easily monitored
chronic toxicity tests.
if an appropriate number of daphnids from brood stock are
12.1.1.2 If the purpose of the test is to determine whether a
individually isolated for the seven days prior to the test, and
specified concentration causes adverse effects (see 10.3.2),
young produced by these daphnids are used to start the test.
only that concentration and appropriate control(s) are neces-
sary. Two additional concentrations at about one-half and two
12. Procedure
timesthespecifiedconcentrationmightbedesirabletoincrease
12.1 Experimental Design—It is recommended that at least
confidence in the results.
four replicate chambers per treatment containing at least ten
12.1.2 The primary focus of the physical and experimental
daphnids per treatment (for example, one individual in each of
test design and the statistical analysis of the data is the
10 replicates; or 5 individuals in each of four replicates) be
experimental unit, which is defined as the smallest physical
used for renewal and flow-through tests. A comparison of the
entity to which treatments can be independently assigned (37).
experimental design for renewal and flow-through tests is
Therefore, the test chamber is the experimental unit. All
presented in Table 1.
chambers in the test should be treated as similarly as possible.
12.1.1 Decisions concerning the various aspects of experi- For example, the temperature in all test chambers should be as
mental design, such as the number of treatments, dilution
similar as possible unless the purpose of the test is to study the
factor, and numbers of test chambers and daphnids per
effect of temperature.
treatment, should be based on the purpose of the test and the
12.1.3 A renewal test system should consist of at least five
type of procedure that is to be used to calculate results (see
test concentrations plus a control and solvent control (if
Section 15).
necessary). At least four chambers should be used for each
12.1.1.1 Alife-cycletestintendedtoallowcalculationofan treatment and control, with at least five daphnids per chamber.
end point (see X1.2) usually consists of one or more control A common design is five treatment levels with ten chambers
treatmentsandageometricseriesofatleastfiveconcentrations each with one daphnid per chamber.
E1193 − 20
12.1.4 The flow-through test can be any of several designs more than one test chamber, the highest and lowest tempera-
and should be capable of (1) delivering at least five test tures must not differ by more than 2°C.
concentrationsplusacontrolandsolventcontrol;(2)delivering
12.4 Loading—There should be at least 30 mL of test
test material concentrations that vary less than 630% of the
solutionpereachfirst-generationdaphnidinflow-throughtests
mean measured amount over a 21-day period, and (3) supply-
and 40 mL per each daphnid in renewal tests.
ing four to six volume exchanges of each test solution per day.
12.5 Selection of Test System:
At least four chambers must be used for each treatment and
12.5.1 Arenewal test can be used for test materials that are
control, with at least ten daphnids per test concentration. A
stable in the dilution water and testing conditions.Also, when
common design is five treatments plus controls with four
testing at or near the test material’s water solubility, the
chambers per treatment and with ten daphnids per chamber.
renewalallowsformoretimetoadequatelystirtestsolutionsto
12.1.5 Test Material Measurement—Ageneral guide is that
approach expected water solubility.
the highest values for a given treatment level divided by the
12.5.2 Aflow-throughtestsystemcanbeusedformanytest
lowest measured value for the same treatment level should not
materials, but should be selected for test materials that have a
varybymorethanafactorof1.5.Thisvariesforchemicalsfor
tendency to dissipate rapidly by hydrolysis, oxidation,
which the method of analysis is not precise or for chemicals
photolysis, reduction, sorption, and volatilization. Several
which are measured at extremely low levels. In these cases,
dilutersystemsarecurrentlyinuse.MountandBrungsdiluters
every effort should be made to make the measurements as
(38) have been successfully modified for Daphnia testing and
accurate and precise as possible.
other diluter systems have also been useful (39-45).
12.1.6 Assignment of Daphnia to the chambers within the
treatments as well as assignment of treatment chambers within
12.6 Beginning the Test:
the test system must be randomized. The following format is
12.6.1 Selecting the Test System and Preparing Test Solu-
suggested:(1)randomassignmentoftreatmentchamberstothe
tions:
test system, (2) random selection of the sequence of chambers
12.6.1.1 For a renewal test, fresh test solutions containing
to be followed when placing the Daphnia into the system, and
appropriate amounts of test material and food should be
(3)randomassignmentofthe Daphniatothebeakersinagiven
prepared less than 4 h before each renewal. The fresh test
sequence.
solutionsshouldbeplacedineachchamber.Thetestorganisms
should be added after the food has been added. Analytical
12.2 Dissolved Oxygen—The dissolved oxygen (DO) con-
confirmation of the test material concentrations prior to the
centration in each test chamber should be at least 3.0 mg/Lfor
initiation of the test is recommended. Test solutions should be
both the renewal and flow-through tests. Because results are
renewedatleastthreetimesaweek(atleasttwodaysapart;for
basedonmeasuredratherthancalculatedconcentrationsoftest
example, Monday,Wednesday, Friday).The test concentrations
material, some loss of test material by aeration is not neces-
should vary less than 630% of the mean measured amounts
sarily detrimental and test solutions m
...

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: E1193 − 97 (Reapproved 2012) E1193 − 20
Standard Guide for
Conducting Daphnia magna Life-Cycle Toxicity Tests
This standard is issued under the fixed designation E1193; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This guide covers procedures for obtaining laboratory data concerning the adverse effects of a test material (added to dilution
water, but not to food) on Daphnia magna Straus, 1820, during continuous exposure throughout a life-cycle using the renewal or
flow-through techniques. These procedures also should be useful for conducting life-cycle toxicity tests with other invertebrate
species, species and cladocerans from the same genus (for example, Daphnia pulex), although modifications might be necessary.
1.2 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or known
mixtures. With appropriate modifications, these procedures can be used to conduct tests on temperature, dissolved oxygen, pH, and
on such materials as aqueous effluents (also see Guide E1192), leachates, oils, particulate matter, sediments, and surface waters.
The technique, (renewal or flow-through), will be selected based on the chemical characteristics of the test material such as high
oxygen demand, volatility, susceptibility to transformation (biologically or chemically), or sorption to glass.
1.3 Modification 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 standard test procedures. Comparison of results obtained using modified and unmodified versions of these
procedures might provide useful information on new concepts and procedures for conducting life-cycle toxicity tests with D.
magna.Appendix X3 provides modifications for conducting the chronic toxicity test method with D. pulex Leydig, 1860.
1.4 This guide is arranged as follows:
Section
Referenced Documents 2
Terminology 3
Summary of Guide 4
Significance and Use 5
Apparatus 6
Facilities 6.1
Construction Materials 6.2
Test Chambers 6.3
Cleaning 6.4
Acceptability 6.5
Reagents 7
Purity of Reagents 7.1
Hazards 8
Dilution Water 9
Requirements 9.1
This guide is under the jurisdiction of ASTM Committee E50 on Environmental Assessment, Risk Management and Corrective Action and is the direct responsibility
of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Dec. 1, 2012Dec. 1, 2020. Published December 2012January 2021. Originally approved in 1987. Last previous edition approved in 20042012
as E1193 – 97 (2004).(2012). DOI: 10.1520/E1193-97R12.10.1520/E1193-20.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1193 − 20
Section
Source 9.2
Treatment 9.3
Characterization 9.4
Test Material 10
General 10.1
Stock Solutions 10.2
Test Concentrations(s) 10.3
Test Organisms 11
Species 11.1
Age 11.2
Source 11.3
Brood Stock 11.4
Food 11.5
Handling 11.6
Harvesting Young 11.7
Quality 11.8
Procedure 12
Experimental Design 12.1
Dissolved Oxygen 12.2
Temperature 12.3
Loading 12.4
Selection of Test System 12.5
Beginning the Test 12.6
Care and Maintenance 12.7
Feeding 12.8
Duration 12.9
Biological Data 12.10
Other Measurements 12.11
Analytical Methodology 13
Acceptability of Test 14
Calculation of Results 15
Report 16
Keywords 17
Appendixes
Appendix X1 Statistical Guidance
Appendix X2 Food
Appendix X3 Modifications for Conducting Chronic Life
Cycle Analysis Tests with Daphnia Pulex
E1193 − 20
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 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 hazard statements are given in Section 8.
1.7 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
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 Use of the International System of Units (SI): The Modern Metric System
3. Terminology
3.1 The words “must,” “should,”“ may,” “can,” and “might” have very specific meanings in this guide.
3.2 must—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 factors that directly
relate to the acceptability of the test (see 14.1).
3.3 should—used to state that the specified condition is recommended and ought to be met if possible. Although 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.
3.4 may—used to mean “is (are) allowed to,” “can” is used to mean “is (are) able to,” and “might” is used to mean “could
possibly.” Therefore the classic distinction between “may” and “can” is preserved, and “might” is never used as a synonym for
either“ may” or “can.”
3.5 For definitions of other terms used in this guide, refer to Guide E729 and Terminology E943. For an explanation of units and
symbols, refer to IEEE/ASTM SI 10.
4. Summary of Guide
4.1 A 21-day life-cycle toxicity test for Daphnia magna is described. The test design allows for the test organisms to be exposed
to a toxicant test material using either the renewal technique (with exchange of the total volume of test water and toxicant at least
three times a week) or the flow-through technique (with continual water and toxicant addition, usually at least four volume
additions per day). At least five concentrations of a test material, a control, and a solvent control (if applicable) replicated at least
four times are recommended. Each test concentration has at least ten Daphnia per treatment. The technique (renewal or
flow-through)There are two applicable exposure approaches (both may be renewal or flow-through): which(1) uses use of a
minimum of ten daphnids per treatment has only and one daphnid per replicate, whereas the typical technique replicate (renewal
or flow-through) utilizesflow-through); or (2) use of four replicates with at least five daphnids per replicate (≥20 daphnids per
treatment). A control consists of maintaining daphnids in dilution water to which no test material has been added to provide (1)
a measure of the acceptability of the test by giving an indication of the quality of the test organisms and the suitability of the
dilution water, food, test conditions, handling procedures, and so forth, and (2) the basis for interpreting data obtained from the
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.
E1193 − 20
other treatments. In each of the other treatments, the daphnids are maintained in dilution water, to which a selected concentration
of test material has been intentionally added. Measurement end points obtained during the test include the concentration of the test
material and final number alive, final weight, final length, and number of progeny per daphnid. Then data Data are analyzed to
determine the effect of the test material on survival, growth, and reproduction of D. magna. These methods are also applicable for
chronic toxicity testing using the conspecific Daphnia pulex, with slight modifications provided in Appendix X3.
5. Significance and Use
5.1 Protection of an aquatic species requires prevention of unacceptable effects on populations in natural habitats. Toxicity tests
are conducted to provide data that may be used to predict what changes in numbers and weights of individuals might result from
similar exposure to the test material in the natural aquatic environment. Information might also be obtained on the effects of the
material on the health of the species.
5.2 Results of life-cycle tests with D. magna are used to predict chronic effects likely to occur on daphnids in field situations as
a result of exposure under comparable conditions.
5.2.1 Life-cycle tests with D. magna are used to compare the chronic sensitivities of different species, the chronic toxicities of
different materials, and study the effects of various environmental factors on the results of such tests.
5.2.2 Life-cycle tests with D. magna are used to assess the risk of materials to aquatic organisms (see Guide E1023) or derive
water quality criteria for aquatic organisms (1).
5.2.3 Life-cycle tests with D. magna are used to predictextrapolate the results of chronic toxicity 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 a life-cycle test with D. magna is greatlymay be
increased by also reporting the results of an acute toxicity test (see Guide E729) conducted under the same conditions. In addition
to conducting an acute toxicity test with unfed D. magna, it may be desirablerelevant to conduct an acute test in which the daphnids
are fed the same as in the life-cycle test to see if the presence of that concentration of that food affects the results of the acute test
and the acute-chronic ratio (ACR) (see 10.3.1).
5.2.4 Life-cycle tests are used to evaluate the biological availability of, and structure-activity relationships between, test materials
and test organisms.
5.3 Results of life-cycle tests with D. magna might be influenced by temperature (2), quality of food, composition of dilution
water, condition of test organisms, test media (for example, water hardness), and other factors.
6. Apparatus
6.1 Facilities—Culture and test chambers are often kept in a separate room maintained at about 20°C but at separate locations.
Alternatively, culture20 °C. Alternatively, cultures and test chambers may be placed in a temperature-controlled water bath or bath,
environmental chamber, or incubator. The water-supply system should provide an adequate supply of dilution water to the culture
tanks and test chambers. The water-supply system should be equipped for temperature control and aeration, and strainers and air
traps should be included in the water-supply system. Air used for aeration should be free of fumes, oil, and water; filters to remove
oil and water are desirable. Filtration of air through a 0.22-μm bacterial filter might be desirable (3). During culturing and testing,
daphnids should be shielded from disturbances to prevent unnecessary stress. The test facility should be well-ventilated and free
of fumes. A timing device should be used to provide a 16-h light and 8-h dark photoperiod (4). A 15 to 30-min transition period
when lights go on might be desirable to reduce the possibility of daphnids being stressed by instantaneous illumination; a transition
period when lights go off may also be desirable.desirable (see Guide E1733).
6.1.1 When D. magna are fed live algae, a high-light intensity might cause sufficient photosynthesis to result in an increase of pH
high enough to kill daphnids (5). Therefore, the maximum acceptable intensity is dependent on the buffer capacity of the dilution
water, species, and density of algae, and the kind of test chamber and cover. cover (see Guide E1733). Light intensities up to 600
lx or a fluence rate of 1 w/m will usually be acceptable, but higher intensities might result in an unacceptably high pH in the
culture water.
The boldface numbers in parentheses refer to the list of references at the end of this guide.
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6.2 Construction Materials—Equipment and facilities that contact stock solutions, test solutions, or any water into which daphnids
will be placed should not contain substances that can be leached or dissolved by aqueous solutions in amounts that can adversely
affect daphnids. In addition, equipment and facilities that contact stock solutions or test solutions should be chosen to minimize
sorption of test materials from water. Glass, Type 316 stainless steel, nylon, fiberglass, silicon, and fluorocarbon plastics should
be used whenever possible to minimize leaching, dissolution, and sorption. Concrete and rigid (unplasticized) plastics may be used
for culture tanks and in the water-supply system, but they should be soaked, preferably in flowing dilution water, for several days
before use (6). Cast-iron pipe may be used in supply systems, but colloidal iron probably will be added to the dilution water and
strainers will be needed to remove rust particles. Copper, brass, lead, galvanized metal, and natural rubber should not contact
dilution water, stock solutions, or test solutions before or during the test. Items made of neoprene rubber and other materials not
previously mentioned should not be used unless it has been shown that their use will not adversely affect survival, growth, and
reproduction of D. magna (see Section 14).
6.3 Test Chambers:
6.3.1 Flow-through tests, 500-mL to 2-L glass beakers (or equivalent) with a notch (approximately 4 by 13 cm) cut in the lip may
be used to expose the Daphnia to the test material. The notch should be covered with 0.33-mm opening (U.S. standard sieve size
No. 50) stainless steel or polyethylene screening small enough to retain first instar Daphnia. The screen can be attached to the
beaker with silicone adhesive. The chambers should provide at least 30 mL of solution for each of the initial test daphnid(s).
6.3.2 Renewal tests, beaker ranging in size from 100 to 1000 mL. A notched chamber is not required for a renewal test. Each
chamber should provide at least 40 mL of solution for each of the initial test daphnid(s).
6.3.3 Any container made of glass, Type 316 stainless steel, or a fluorocarbon plastic may be used if (1) each chamber is separate
with no interconnections, (2) each chamber contains at least 30 mL of test solution (see 12.4) per first-generation daphnid for
flow-through tests and at least 40 mL for renewal tests, (3) there is at least 1000 mm of air to water interface per daphnid, and
(4) the test solution is at least 30 mm deep. Static test chambers should be covered with glass, stainless steel, nylon, or fluorocarbon
plastic covers to keep out extraneous contaminants and to reduce evaporation of test solution. All chambers and covers in a test
must be identical. Covers are not required for flow-through studies.
6.4 Cleaning—Test chambers and equipment used to prepare and store dilution water, stock solutions, and test solutions should
be cleaned before use. New equipment should be washed with detergent and rinsed with water, a water-miscible organic solvent,
water, acid (such as 5 % concentrated nitric acid), and washed at least twice with distilled, deionized, or dilution water. Some lots
of some organic solvents might leave a film that is insoluble in water. Also, stronger nitric acid, for example, 10 %, might cause
deterioration of silicone adhesive; an initial rinse with 10 % concentrated hydrochloric acid might prevent such deterioration. A
dichromate-sulfuric acid cleaning solution can generally be used in place of both the organic solvent and the acid, but it might
attack silicone adhesives. At the end of every test, all items that are to be used again should be immediately (1) emptied, (2) rinsed
with water, (3) cleaned by a procedure appropriate for removing the test material (for example, acid to remove metals and bases;
detergent, organic solvent, or activated carbon to remove organic chemicals), and (4) rinsed at least twice with distilled, deionized,
or dilution water. Acid is useful for removing mineral deposits. Test chambers should be rinsed with dilution water just before use.
6.5 Acceptability—Before a toxicity test is conducted in new test facilities, it is desirable to conduct a “non-toxicant” control
water-only (that is, no test material) test, in which all test chambers contain dilution water with no added test material. water. This
test will reveal (1) whether D. magna will survive, grow, and reproduce acceptably (see Section 14) in the new facilities, (2)
whether there are any location effects on survival, growth, or reproduction, and (3) the magnitude of the within-chamber and
between-chamber variance.
7. Reagents
7.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where
such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high
purity to permit its use without lessening the accuracy of the test.
Reagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC. For suggestions on the testing of reagents not listed by
the American Chemical Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National
Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
E1193 − 20
8. Hazards
8.1 Many materials can affect humans adversely if precautions are inadequate. Therefore, skin contact with all test materials and
solutions should be minimized by wearing appropriate protective gloves (especially when washing equipment or putting hands in
test solutions), laboratory coats, aprons, and glasses, and by using dip nets or tubes to remove daphnids from test solutions. Special
precautions, such as covering test chambers and ventilating the area surrounding the chambers, should be taken when conducting
tests on volatile materials. Information on toxicity to humans (7), recommended handling procedures (8), and chemical and
physical properties of the test material should be studied before a test is begun. Special procedures will be necessary with
radiolabeled test materials (9) and with materials that are, or are suspected of being, carcinogenic (10).
8.2 Disposal of stock solutions, test solutions, and test organisms might pose special problems in some cases; therefore, health and
safety precautions and applicable regulations should be considered before beginning a test. Removal or degradation of test material
might be desirable before disposal of stock and test solutions.
8.3 Cleaning equipment with a volatile solvent such as acetone should be performed only in a well-ventilated area with no
smoking allowed and no open flame, for example, pilot light, is present.
8.4 Acidic solutions and hypochlorite solutions should not be mixed together because hazardous fumes might be produced.
8.5 Because dilution water and test solutions are usually good conductors of electricity, use of ground fault systems and leak
detectors should be considered to help prevent electrical shocks.
8.6 To prepare dilute acid solutions, concentrated acid should be added to water, not vice versa. Opening a bottle of concentrated
acid and mixing concentrated acid with water should be performed only in a well-ventilated area.
9. Dilution Water
9.1 Requirements—The dilution water should (1) be acceptable to D. magna, (2) be of uniform quality, and (3), except as stated
in 9.1.4, not unnecessarily affect results of the test.
9.1.1 The dilution water must allow satisfactory survival, growth, and reproduction of D. magna (see Section 14).
9.1.2 The quality of the dilution water should be uniform, allowing the brood stock to be cultured and the test conducted in water
of the same quality. In particular, during culture or testing, or both, the range of hardness should be 610 % of the average.
9.1.3 The dilution water should not unnecessarily affect results of a life-cycle test with D. magna because of such things as
sorption or complexation of test material. Therefore, except as stated in 9.1.4, concentrations of both total organic carbon (TOC)
and particulate matter should be less than 5 mg/L.
9.1.4 If it is desired to study the effect of an environmental factor such as TOC, particulate matter, or dissolved oxygen on the
results of a life-cycle test with D. magna, it will be necessary to use a water that is naturally or artificially high in TOC or
particulate matter or low in dissolved oxygen. If such a water is used, it is important that adequate analyses be performed to
characterize the water, and that a comparable test be available or conducted in the laboratory’s usual culture dilution water to
facilitate interpretation of the results in the special water.
9.2 Source:
9.2.1 The use of reconstituted water might increase comparability of test results between laboratories. The hard reconstituted fresh
water (160 to 180 mg/L as CaCO ) described in Guide E729 has been used successfully. Addition of 2 μg of selenium(IV) and
1 μg of crystalline vitamin B /L might be desirable (11). Other water sources (natural or reconstituted) may be used if they have
been demonstrated to provide adequate daphnid survival, growth, and reproduction.
9.2.2 Natural fresh waters have been used successfully. Natural waters should be obtained from an uncontaminated source of
consistent quality. A well or spring is usually preferable to a surface water. If a surface water is used, the intake should be
positioned to minimize fluctuations in quality and the possibility of contamination and should maximize the concentration of
dissolved oxygen to help ensure low concentrations of sulfide and iron.
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9.2.3 Dechlorinated water is not recommended as a dilution water for Daphnia magna. Dechlorinated water should be used only
as a last resort because dechlorination is often incomplete and residual chlorine is quite toxic to D. magna(12). Sodium bisulfite
is probably better for dechlorinating water than sodium sulfite, and both are more reliable than carbon filtration, especially for
removing chloramines (13). Some organic chloramines, however, react slowly with sodium bisulfite (14). In addition to residual
chlorine, municipal drinking water often contains unacceptably high concentrations of copper, lead, zinc, and fluoride, and quality
is often rather variable. When necessary, excessive concentrations of most metals can usually be removed with a chelating resin
(15).
9.3 Treatment:
9.3.1 Dilution water should be aerated intensively by such means as air stones, surface aerators, or column aerators (16,17) prior
to the addition of test material. Adequate aeration will bring the pH and concentrations of dissolved oxygen and other gases into
equilibrium with the air, and minimize oxygen demand and concentrations of volatiles. The concentration of dissolved oxygen in
dilution water should be between 90 and 100 % saturation to help ensure that dissolved oxygen concentrations are acceptable in
test chambers. Supersaturation of dissolved gases, which might be caused by heating dilution water, should be avoided to prevent
gas bubble disease (18,19).
9.3.2 Filtration through sand, sock, bag, or depth-type cartridge filters may be used to keep the concentration of particulate matter
acceptably low (see 9.1.3).
9.3.3 Dilution water that might be contaminated with undesirable microorganisms may be passed through a properly maintained
ultraviolet sterilizer (20) equipped with an intensity meter and flow controls or passed through a filter with a pore size of 0.45 μm.
Water that might be contaminated with Aphanomyces daphniae should be autoclaved (3).
9.4 Characterization:
9.4.1 The following items should be measured at least twice each year, and more often if, (1) such measurements have not been
made semiannually for at least two years, or (2) surface water is used: hardness, alkalinity, conductivity, pH, particulate matter,
TOC, organophosphorus pesticides, polychlorinated biphenyls (PCBs), chlorinated phenoxy herbicides, ammonia, cyanide, sulfide,
chloride, bromide, 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.
9.4.2 For each analytical method used (see 13.3) to measure the parameters listed in 9.4.1, quantification of the limit should be
below either (1) the concentration in the dilution water or (2) the lowest concentration that has been shown to adversely affect the
survival, growth, or reproduction of D. magna(21).
10. Test Material
10.1 General—The test material should be reagent grade or better, unless a test on a formulation, commercial product, or
technical-grade or use-grade material is specifically needed. Before a test is begun, the following should be known about the test
material:
10.1.1 Identities and concentrations of major ingredients and major impurities. For example, impurities constituting more than
about 1 % of the material.
10.1.2 Solubility and stability in the dilution water and solvents.
10.1.3 Measured acute toxicity to D. magna.
10.1.4 Measured or estimated chronic toxicity to D. magna.
10.1.5 Precision and bias of the analytical method at the planned concentration(s) of test material.
10.1.6 Estimate of toxicity to humans.
10.1.7 Recommended handling procedures (see 8.1).
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10.2 Stock Solutions:
10.2.1 Stock solutions are usually prepared prior to dosing the dilution water to obtain the desired test concentrations.
Water-soluble test materials can often be added directly to dilution water to prepare a stock solution (or in some cases the test
solution). Test materials that are moderately soluble or insoluble in water are often dissolved in a solvent to form a stock solution
that is then added to dilution water. If a stock solution is used, the concentration and stability of the test material in the stock
solution should be determined before beginning the test. If the test material is subject to photolysis, the stock solution should be
shielded from light. If the test material hydrolyzes or biodegrades rapidly, it might be necessary to prepare new stock solutions
daily.
10.2.2 The preferred carrier for stock solutions is dilution water except possibly for tests on hydrolyzable, oxidizable, and
reducible materials. Filtration or sterilization, or both, of the water might be necessary. If the hardness of the dilution water in the
test system will not be affected, distilled and deionized water are also acceptable for stock solution preparation. Several techniques
have been specifically developed for preparing aqueous stock solutions of slightly soluble materials (22). Minimum necessary
amounts of strong acids and bases may be used to prepare aqueous stock solutions, but such reagents might affect the pH of test
solutions appreciably. Use of a more soluble form of the test material, such as chloride or sulfate salts of organic amines, sodium
or potassium salts of phenols and organic acids, and chloride or nitrate salts of metals, might affect the pH even more than the use
of the minimum necessary amount of strong acid or base.
10.2.3 If a solvent other than dilution water is used, its concentration in test solutions should be kept to a minimum and should
not affect survival, growth, or reproduction of D. magna. Because of their low toxicities to aquatic animals (23), low volatilities,
and high abilities to dissolve many organic chemicals, dimethylformamide and triethylene glycol are often good organic solvents
for preparing stock solutions. Other water-miscible organic solvents, such as methanol, ethanol, and acetone, may also be used as
carriers, but they might stimulate undesirable growths of microorganisms, and acetone is quite volatile. If an organic solvent is
used, its concentration in any test solution should not exceed 0.1 mL/L. Surfactants should not be used in the preparation of stock
solutions because they might affect the form and toxicity of the test material in test solutions. (These limitations do not apply to
any ingredients of a mixture, formulation, or commercial product, unless an extra amount of solvent is used in the preparation of
the stock solution.)
10.2.4 If a solvent other than water is used as a carrier, at least one solvent control, using solvent from the same batch used to
make the stock solution, in addition to the dilution-water control, must be included in the test.
10.2.4.1 If the test contains both a dilution-water control and a solvent control, the survival, growth, and reproduction of D. magna
in the two controls should be compared (see X1.4). If a statistically significant difference in either survival, growth, or reproduction
is detected between the two controls, the solvent control is normally used for meeting the requirements specified in Section 14 and
as the basis for the calculation of results. Judgment might be required in the choice of which control data to use to compare with
treatments, especially when the solvent concentration is not constant in the treatments. If no statistically significant difference is
detected, the data from both controls should be used for meeting the requirements specified in Section 14 and as the basis for
calculating the results.results (see Appendix X1).
10.2.5 If a solvent other than water is used as a carrier, it might be desirable to conduct simultaneous tests using two chemically
unrelated solvents or two different concentrations of the same solvent to obtain information concerning possible effects of solvent
on results of the test.
10.3 Test Concentration(s):
10.3.1 If the test is intended to provide a good estimate of the highest concentration that will not unacceptably affect the survival,
growth, or reproduction of D. magna, the test concentrations (see 12.1.1.1) should bracket the best prediction of that concentration.
Such a prediction is usually based on the results of an acute toxicity test (see Guide E729) with the test material using the same
dilution water and D. magna neonates (for example, individuals less than 24-h old). Because the food used in the life-cycle test
sometimes affects the results of the acute test (24,25), acute tests should be conducted with and without the food added to the
dilution water prior to conducting the chronic study. If an acute-chronic ratio has been determined for the test material with a
species of comparable sensitivity, the result of the acute test with D. magna can be divided by the acute-chronic ratio. Except for
a few materials (26), acute-chronic ratios determined with daphnids are typically less than ten. Thus, the highest concentration of
test material in a life-cycle test with D. magna is typically selected to be equal to the lowest concentration that caused adverse
effects in a comparable acute test.
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10.3.2 In some situations (usually regulatory), it is only necessary to determine whether one specific concentration of test material
unacceptably affects survival, growth, or reproduction. These situations usually arise when the concentration resulting from the
direct application of a material to a body of water is known, or when the material is thought to be nontoxic at its solubility limit
in water. When there is only interest in one specific concentration, it is often only necessary to test that specific concentration (see
12.1.2).
11. Test Organisms
11.1 Species—D. magna has been extensively used for acute and life-cycle toxicity tests because it is one of the largest cladoceran
species, is easy to identify, and is available from many laboratories and commercial sources. These procedures might also be
suitable for other daphnid species, (that is, D. pulex) although modifications might be necessary. The identities of daphnids
obtained from laboratories and commercial sources should be verified, regardless of any information that comes with the
organisms. D.Daphnia magna should be verified using the scheme of Brooks (27). The identification of other daphnids may vary
with the taxonomic reference used (28,29).
11.2 Age—Life-cycle tests with D. magna should begin with organisms less than 24-h old.old.Neonates shipped overnight from
a vendor may be greater than 24-h old and thus their age should be specifically documented. It is preferable that neonates used
in testing be obtained from in-house cultures (see 11.4) to ensure organisms are the right age and to avoid unnecessary handling
stresses (see 11.6).
11.3 Source—All daphnids used in a test should be from the same brood stock. This brood stock must have been cultured for at
least two generations using the same food, water, and temperature as will be used in the life-cycle test. This will not only acclimate
the daphnids, but will also demonstrate the acceptability of the food, water, and so forth, before the test.
11.4 Brood Stock:
11.4.1 Brood stock can be obtained from in-house cultures, another laboratory or a commercial source. When daphnids are brought
into the laboratory, they should be acclimated to the dilution water by gradually changing the water in the culture chamber from
the water in which they were transported to 100 % dilution water over a period of two or more days. Daphnids should be
acclimated to the test temperature by changing the water temperature at a rate not to exceed 3°C3 °C within 12 h until the desired
temperature is reached. Generally, acclimation to pH should not exceed more than 1.5 pH units per day.
11.4.2 D. magna has been cultured in a variety of systems, such as in large groups in aquaria, in groups of one to five in 100 to
250-mL beakers, or in specially designed chambers (30).
11.4.3 To maintain D. magna in good condition, the brood stock should be cultured so as to avoid unnecessary stress due to
crowding, rapid changes in temperature, and water quality. Daphnids should not be subjected to more than a 3°C3 °C change in
water temperature in any 12-h period and preferably not more than a 3°C3 °C change in any 72-h period. Cultures should be
regularly fed enough food to support adequate reproduction. Culture chambers should be cleaned periodically to remove feces,
debris, and uneaten food. If culture chambers are properly cleaned and the density of daphnids is kept low, for example, no more
than 1 daphnid/30 mL, the surface water/air interface should provide adequate dissolved oxygen. Organisms used for testing must
produce at least 60 young per adult during a 21-day test.
11.5 Food—Various combinations (see Appendix X2) of trout chow, yeast, alfalfa, and algae, such as Ankistrodesmus
convolutus,Ankistrodesmus falcatus,Chlorella vulgaris,Chlamydomonas reinhardtii, and Raphidocelis subcapitata (formerly
Selenastrum capricornutumand Pseudokirchneriella subcapitata ) (31), have been successfully used for culturing and testing D.
magna. The concentration of test material (number of cells for algae) in the batch of food used should be determined. The
experience gained over the past decade has shown that it is very important critical to incorporate algae into the diet to maintain
consistently healthy daphnids (32-34).
11.6 Handling—D. magna should be handled as little as possible. When handling is necessary, it should be done gently, carefully,
and quickly so that the daphnids are not unnecessarily stressed. Daphnids should be introduced into solutions beneath the air-water
interface. Daphnids that touch dry surfaces or are dropped or injured during handling should be discarded. Smooth glass tubes with
an inside diameter of at least 5 mm should be used for transferring adult D. magna, and the amount of solution carryover should
be minimized. Equipment used to handle daphnids should be sterilized between use by autoclaving or by treatment with an
iodophor (35) or with 200 mg of hypochlorite/L for at least 1 h (see 6.4 ).
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11.7 Harvesting Young—Young less than 24-h old can be obtained using specially designed chambers (27) or by transferring to
chambers containing dilution water and food, allowing an overnight period for brood release.
11.8 Quality—To decrease the chances of a test being unacceptable (see 14.1), the test should not begin with young that were in
the first brood (32), nor with young from a daphnid that (1) is sick (3,36) or incompletely developed (11), (2) is more than 50 days
old, (3) did not produce young before Day 10, (4) did not produce at least nine young in the previous brood, or (5) is from a culture
in which ephippia were produced or in which substantial mortality (>10 %) occurred during the week prior to the test. These factors
are most easily monitored if an appropriate number of daphnids from brood stock are individually isolated for the seven days prior
to the test, and young produced by these daphnids are used to start the test.
12. Procedure
12.1 Experimental Design—It is recommended that at least four replicate chambers per treatment containing at least ten daphnids
per treatment be used for renewal and flow-through tests. As a minimum for flow-through and renewal tests, ten daphnids per
treatment could be used when each chamber contains only one daphnid and ten chambers per treatment are used. (for example,
one individual in each of 10 replicates; or 5 individuals in each of four replicates) be used for renewal and flow-through tests. A
comparison of the experimental design for renewal and flow-through tests is presented in Table 1.
12.1.1 Decisions concerning the various aspects of experimental design, such as the number of treatments, dilution factor, and
numbers of test chambers and daphnids per treatment, should be based on the purpose of the test and the type of procedure that
is to be used to calculate results (see Section 15).
12.1.1.1 A life-cycle test intended to allow calculation of an end point (see X1.2) usually consists of one or more control
treatments and a geometric series of at least five concentrations of test material. In the controls, daphnids are exposed to dilution
water to which neither test material nor solvent has been added. One or more solvent controls might also be necessary (see 10.2.3).
Except for the control(s) and the high concentration, each concentration should be at least 50 % of the next higher one, unless
information concerning the concentration-effect curve indicates that a different dilution factor is more appropriate. At a dilution
factor of 0.5, five concentrations are a reasonable compromise between cost and the risk of all concentrations being either too high
or too low. If the estimate of chronic toxicity is particularly uncertain (see 10.3.1), six or seven concentrations might be desirable.
Range finder tests (for example, low replication tests with a 90 % dilution series) may also be informative in planning robust
chronic toxicity tests.
TABLE 1 Experimental Design
Design Parameter Renewal Test Flow-Through Test
Number of test $5 $5
concentrations
Control Yes Yes
Solvent control If appropriate If appropriate
Number of chambers At least 4 At least 4
Minimum number of 10 (individual daphnid/ 10 (individual daphnid/
daphnids/treatment chamber) chamber)
20 20
(multiple daphnids/ (multiple daphnids/
chamber) chamber)
Number of daphnids/test At least 1 At least 5
chamber
Feeding Once daily 2 to 3 times daily (or
continuous)
Renewal of test solution At least 3 times/week At least 1 volume
replacement/day
Temperature 20°C 20°C
Water chemistry New solutions at each Initially and at least
renewal, old solutions weekly thereafter
after longest time
hour interval
Water quality parameters New solutions at each Initially and at least
renewal, old solutions weekly thereafter
after longest time
hour interval
Analytical confirmation Initially and at least Initially and at least
of test material weekly thereafter, old weekly thereafter
solutions at least
once during the study
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12.1.1.2 If the purpose of the test is to determine whether a specified concentration causes adverse effects (see 10.3.2), only that
concentration and appropriate control(s) are necessary. Two additional concentrations at about one-half and two times the specified
concentration might be desirable to increase confidence in the results.
12.1.2 The primary focus of the physical and experimental test design and the statistical analysis of the data is the experimental
unit, which is defined as the smallest physical entity to which treatments can be independently assigned (37). Therefore, the test
chamber is the experimental unit. All chambers in the test should be treated as similarly as possible. For example, the temperature
in all test chambers should be as similar as possible unless the purpose of the test is to study the effect of temperature.
12.1.3 A renewal test system should consist of at least five test concentrations plus a control and solvent control (if necessary).
At least four chambers should be used for each treatment and control, with at least five daphnids per chamber. A common design
that is frequently used is is five treatment levels with ten chambers each with one daphnid per chamber.
12.1.4 The flow-through test can be any of several designs and should be capable of (1) delivering at least five test concentrations
plus a control and solvent control; (2) delivering test material concentrations that vary less than 630 % of the mean measured
amount over a 21-day period, and (3) supplying four to six volume exchanges of each test solution per day. At least four chambers
must be used for each treatment and control, with at least ten daphnids per test concentration. A common design that is frequently
used is is five treatments plus controls with four chambers per treatment and with ten daphnids per chamber.
12.1.5 Test Material Measurement—A general guide is that the highest values for a given treatment level divided by the lowest
measured value for the same treatment level should not vary by more than a factor of 1.5. This varies for chemicals for which the
method of analysis is not precise or for chemicals which are measured at extremely low levels. In these cases, every effort should
be made to make the measurements as accurate and precise as possible.
12.1.6 Assignment of Daphnia to the chambers within the treatments as well as assignment of treatment chambers within the test
system must be randomized. The following format is suggested: (1) random assignment of treatment chambers to the test system,
(2) random selection of the sequence of chambers to be followed when placing the Daphnia into the system, and (3) random
assignment of the Daphnia to the beakers i
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