ASTM E1295-22

Standard Guide for Conducting Three-Brood, Renewal Toxicity Tests with <emph type="ital">Ceriodaphnia dubia</emph>

SIGNIFICANCE AND USE
5.1 Ceriodaphnia  was first used as a toxicity test organism by Mount and Norberg (2). Introduced for use in effluent and ambient water evaluations, Ceriodaphnia have also been a valuable addition to single chemical test procedures.  
5.2 Protection of a population requires prevention of unacceptable effects on the number, weight, health, and uses of the individuals of that species, or species for which the test species serves as a surrogate. A three-brood toxicity test is conducted to help determine changes in survival and the number of neonates produced that result from exposure to the test material.  
5.3 Results of three-brood toxicity tests with C. dubia might be used to predict chronic or partial chronic effects on species in field situations as a result of exposure under comparable conditions.  
5.4 Results of three-brood toxicity tests with C. dubia might be compared with the chronic sensitivities of different species and the chronic toxicities of different materials, and to study the effects of various environmental factors on results of such tests.  
5.5 Results of three-brood toxicity tests with C. dubia might be useful for predicting the results of chronic tests on the same test material with the same species in another water or with another species in the same or a different water. Most such predictions are based on the results of acute toxicity tests, and so the usefulness of the results of a three-brood toxicity test with C. dubia might be greatly increased by also reporting the results of an acute toxicity test (see Guides E729 and E1192) conducted under the same conditions. In addition to conducting an acute test with unfed C. dubia, it might also be desirable to conduct an acute test in which the organisms are fed the same as in the three-brood test, to see if the presence of that concentration of that food affects the results of the acute test and the acute chronic ratio (see 10.4.1).  
5.5.1 A 48 or 96-h EC50 or LC50 can sometimes be obtaine...
SCOPE
1.1 This guide describes procedures for obtaining data concerning the adverse effects of an effluent or a test material (added to dilution water, but not to food) on Ceriodaphnia dubia Richard 1894, during continuous exposure throughout a portion of the organism's life. These procedures should also be useful for conducting life cycle toxicity tests with other Cladocera (Guide E1193), although modifications will be necessary.  
1.2 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or known mixtures, that can be measured accurately at the necessary concentrations in water. With appropriate modifications these procedures can be used to conduct tests on temperature, dissolved oxygen, pH, dissolved ions, and on such materials as aqueous effluents (see also Guide E1192), leachates, oils, particulate matter, sediments (see also Guide E1706), and surface waters. Renewal tests might not be applicable to materials that have high oxygen demand, are highly volatile, are rapidly biologically or chemically transformed, or sorb to test chambers. If the concentration of dissolved oxygen falls below 4 mg/L or the concentration of test material decreases by more than 20 % in test solution(s) at any concentration between renewals, more frequent renewals might be necessary.  
1.3 Other modifications of these procedures might be justified by special needs or circumstances. Results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparisons of results obtained using modified and unmodified versions of these procedures might provide useful information on new concepts and procedures for conducting three-brood toxicity tests with C. dubia.  
1.4 This guide is arranged as follows:    
Section  
Referenced Documents  
2  
Terminology  
3  
Summary of Guide  
4  
Significance and Use  
5  
Apparatus ...

General Information

• Status: Published
• Publication Date: 31-Dec-2021
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: E50 - Environmental Assessment, Risk Management and Corrective Action
• Drafting Committee: E50.47 - Biological Effects and Environmental Fate

Relations

• Refers: ASTM E1706-19 - Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates
• Effective Date: 01-Apr-2019
• Refers: ASTM E1706-05(2010) - Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates (Withdrawn 2019)
• Effective Date: 01-Sep-2010
• Refers: ASTM E943-08 - Standard Terminology Relating to Biological Effects and Environmental Fate
• Effective Date: 01-Mar-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 E1733-95(2008) - Standard Guide for Use of Lighting in Laboratory Testing
• Effective Date: 01-Feb-2008
• Refers: ASTM E1023-84(2007) - Standard Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses
• Effective Date: 01-Oct-2007
• Refers: ASTM 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 D4447-10 - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 15-Nov-2006
• Refers: ASTM D4447-06 - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 15-Nov-2006
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM D4447-84(2003) - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 10-Mar-2003
• Refers: ASTM D1193-99e1 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999
• Refers: ASTM D1193-99 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999
• Refers: ASTM D4447-84(1997) - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 10-Oct-1997

Overview

ASTM E1295-22 is the internationally recognized standard guide for conducting three-brood, renewal toxicity tests with Ceriodaphnia dubia. Published by ASTM International, this standard provides detailed procedures for evaluating the chronic toxicity of chemicals, effluents, and other materials on the freshwater organism Ceriodaphnia dubia. The purpose is to generate scientifically valid data on the adverse effects of test materials through continuous exposure during a significant portion of the test organisms’ life cycle. Results are critical for assessing aquatic toxicity, supporting water quality criteria, and ensuring environmental protection.

Key Topics

• Test Objective: The three-brood toxicity test with Ceriodaphnia dubia is designed to assess both survival and reproduction over multiple broods. This is crucial for predicting chronic or partially chronic effects in aquatic environments.

• Test Organism:Ceriodaphnia dubia is widely recognized as a sensitive test organism for freshwater toxicity due to its rapid life cycle and sensitivity to contaminants.

• Applicability: The procedures are suitable for chemicals (individual or mixtures), effluents, leachates, oils, sediments, and surface waters, provided accurate measure at relevant concentrations is possible.

• Test Conditions:

  • Neonates (less than 24 hours old) from the same broodstock are individually exposed in separate chambers.
  • Assays are typically conducted for 6-8 days, using static-renewal of solutions every 24 hours.
  • Consistent light, temperature, and feeding conditions are maintained to replicate natural environments while ensuring reproducible results.

• Quality Assurance: Control treatments are required to assess organism health and water suitability, ensuring data reliability. Performance criteria include control survival rates of 80% or greater and average reproduction of at least 15 young per surviving female.

• Significance and Use: Data from these tests inform on the risk posed by substances to aquatic populations and can guide comparisons among different species, materials, and testing environments.

Applications

• Environmental Risk Assessment: Results help predict how chemicals or effluents will impact aquatic life under real-world conditions, aiding in the derivation of water quality criteria or regulatory limits.
• Effluent and Water Quality Monitoring: Regulatory agencies and industries use these tests to evaluate the chronic toxicity of wastewater discharges, surface waters, leachates, and sediments.
• Comparative Toxicity Studies: By comparing chronic sensitivities across different organisms and materials, stakeholders can prioritize risks and design safer products or interventions.
• Product Development and Registration: Manufacturers of chemicals and commercial products use chronic toxicity data from C. dubia tests to demonstrate regulatory compliance and support environmental safety claims.

Related Standards

Several ASTM and related standards are commonly referenced or used alongside ASTM E1295-22 to support comprehensive aquatic toxicity testing and quality assurance:

• ASTM E729: Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians
• ASTM E1192: Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents
• ASTM E1193: Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests
• ASTM E1023: Guide for Assessing the Hazard of a Material to Aquatic Organisms
• ASTM D1193: Specification for Reagent Water

By following ASTM E1295-22, laboratories, regulatory agencies, and environmental professionals can obtain high-quality, reproducible data on the chronic toxicity of chemicals and effluents using Ceriodaphnia dubia. This supports reliable aquatic risk assessment, effective regulatory compliance, and environmental stewardship.

Keywords: ASTM E1295-22, three-brood toxicity test, Ceriodaphnia dubia, chronic toxicity, effluent testing, aquatic toxicity standard, water quality criteria, environmental risk assessment, ASTM International, reproductive toxicity test, aquatic toxicology.

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: E1295 − 22
Standard Guide for
Conducting Three-Brood, Renewal Toxicity Tests with
Ceriodaphnia dubia
This standard is issued under the fixed designation E1295; 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 describes procedures for obtaining data
Referenced Documents 2
concerning the adverse effects of an effluent or a test material Terminology 3
Summary of Guide 4
(added to dilution water, but not to food) on Ceriodaphnia
Significance and Use 5
dubia Richard 1894, during continuous exposure throughout a
Apparatus 6
portionoftheorganism’slife.Theseproceduresshouldalsobe Facilities 6.1
Construction Materials 6.2
useful for conducting life cycle toxicity tests with other
Test Chambers 6.3
Cladocera (Guide E1193), although modifications will be
Cleaning 6.4
Reagents and Materials 7
necessary.
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, that can be measured accurately at the
Treatment 9.3
necessary concentrations in water. With appropriate modifica- Characterization 9.4
Test Material 10
tions these procedures can be used to conduct tests on
General 10.1
temperature,dissolvedoxygen,pH,dissolvedions,andonsuch
Stock Solution 10.2
materials as aqueous effluents (see also Guide E1192),
Effluent 10.3
Test Concentration(s) 10.4
leachates, oils, particulate matter, sediments (see also Guide
Collection 10.5
E1706), and surface waters. Renewal tests might not be
Sample Containers 10.6
applicable to materials that have high oxygen demand, are
Preservation 10.7
Treatment 10.8
highly volatile, are rapidly biologically or chemically
Test Organisms 11
transformed, or sorb to test chambers. If the concentration of
Species 11.1
dissolved oxygen falls below 4 mg/L or the concentration of Age 11.2
Source 11.3
test material decreases by more than 20% in test solution(s) at
Brood Stock 11.4
any concentration between renewals, more frequent renewals
Food 11.5
might be necessary. Handling 11.6
Quality 11.7
1.3 Other modifications of these procedures might be justi-
Procedure 12
Demonstration of Feasibility 12.1
fied by special needs or circumstances. Results of tests con-
Experimental Design 12.2
ducted using unusual procedures are not likely to be compa-
Dissolved Oxygen 12.3
rable to results of many other tests. Comparisons of results
Temperature 12.4
Preparing Test Solutions 12.5
obtained using modified and unmodified versions of these
Conditioning Test Chambers 12.6
procedures might provide useful information on new concepts
Beginning a Test 12.7
and procedures for conducting three-brood toxicity tests with
Renewing Test Solutions 12.8
Duration of Test 12.9
C. dubia.
Biological Data 12.10
1.4 This guide is arranged as follows: Other Measurements 12.11
Test Material 12.12
Analytical Methodology 13
Acceptability of Test 14
Calculation 15
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Report 16
Assessment,RiskManagementandCorrectiveActionandisthedirectresponsibility
Appendixes
of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Food Appendix X1
Current edition approved Jan. 1, 2022. Published April 2022. Originally
Culture Techniques Appendix X2
approved in 1989. Last previous edition approved in 2013 as E1295–01(2013).
Test Chambers Appendix X3
DOI: 10.1520/E1295-22.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1295 − 22
rarelyaseriousmatter,violationofseveralwilloftenrenderthe
Statistical Guidance Appendix X4
results questionable. Terms such as “is desirable,” “is often
1.5 This standard does not purport to address all of the
desirable,” and “might be desirable” are used in connection
safety concerns, if any, associated with its use. It is the
with less important factors. “May” is used to mean “is (are)
responsibility of the user of this standard to establish appro-
allowed to,” “can” is used to mean “is (are) able to,” and
priate safety, health, and environmental practices and deter-
“might” is used to mean “could possibly.” Thus the classic
mine the applicability of regulatory limitations prior to use.
distinctionbetween“may”and“can”ispreserved,and“might”
Specific hazard statements are given in Section 8.
is never used as a synonym for either “may” or “can.”
1.6 This international standard was developed in accor-
dance with internationally recognized principles on standard-
3.2 Fordefinitionsofothertermsusedinthisstandard,refer
ization established in the Decision on Principles for the
to Guide E729, Terminology E943, and Guide E1023. For an
Development of International Standards, Guides and Recom-
explanation of units and symbols, refer to SI10-02SI10–02
mendations issued by the World Trade Organization Technical
IEEE/ASTM SI 10.
Barriers to Trade (TBT) Committee.
3.3 Definitions of Terms Specific to This Standard:
3.3.1 brood, n—the young neonates released at the time of
2. Referenced Documents
adult molt by the young/adult animal originally exposed to the
2.1 ASTM Standards:
control and test solutions.
D1193Specification for Reagent Water
3.3.1.1 Discussion—Thenumberofyoungineachbroodare
D3978Practice for Algal Growth Potential Testing with
typically counted over each 24h period of the test and should
Pseudokirchneriella subcapitata
increase over the duration of the test. Animals may be
D4447Guide for Disposal of Laboratory Chemicals and
transferred to fresh control or test solution before completing
Samples
the release of a brood, resulting in split broods. Care is needed
E729Guide for Conducting Acute Toxicity Tests on Test
when interpreting the results to determine the number of
Materials with Fishes, Macroinvertebrates, and Amphib-
broods released during a test.
ians
E943Terminology Relating to Biological Effects and Envi-
4. Summary of Guide
ronmental Fate
E1023Guide for Assessing the Hazard of a Material to
4.1 At the beginning of the test, at least ten C. dubia less
Aquatic Organisms and Their Uses
than 24h old, and within 8h of age, are introduced individu-
E1192Guide for ConductingAcute Toxicity Tests onAque-
ally in separate test chambers, and exposed to control water
ous Ambient Samples and Effluents with Fishes,
and a least one (preferably 2 or more) toxicant concentrations.
Macroinvertebrates, and Amphibians
One control treatment must be used but more may be used.
E1193Guide for Conducting Daphnia magna Life-Cycle
Controltreatmentsmayincludestandardlaboratorywateronly,
Toxicity Tests
or some combination of standard water(s) and uncontaminated
E1706TestMethodforMeasuringtheToxicityofSediment-
site water, to provide a measure of organism survival and
Associated Contaminants with Freshwater Invertebrates
reproduction based on specific test water conditions, such as
E1733Guide for Use of Lighting in Laboratory Testing
hardness, alkalinity, and so forth. Specified data on the con-
E1847Practice for Statistical Analysis of Toxicity Tests
centration of test material and the survival and reproduction of
Conducted Under ASTM Guidelines (Withdrawn 2022)
C. dubia are collected and analyzed to determine the effect of
SI10–02IEEE/ASTMSI10AmericanNationalStandardfor
the tested concentration (% effluent or ambient water) on C.
UseoftheInternationalSystemofUnits(SI):TheModern
dubia.
Metric System
4.2 Table 1(1) contains a summary of the conditions used
when conducting a three-brood test with C. dubia. Table 2 and
3. Terminology
Section 14 list the requirements that need to be met for a test
3.1 Thewords“must,”“should,”“may,”“can,”and“might”
to be deemed acceptable.
haveveryspecificmeaningsinthisstandard.“Must”isusedto
express an absolute requirement, that is, to state that the test
5. Significance and Use
has to be designed to satisfy the specified condition, unless the
5.1 Ceriodaphnia was first used as a toxicity test organism
purpose of the test requires a different design. “Must” is only
by Mount and Norberg (2). Introduced for use in effluent and
used in connection with factors that directly relate to the
ambient water evaluations, Ceriodaphnia have also been a
acceptability of the test (see Section 14). “Should” is used to
valuable addition to single chemical test procedures.
statethatthespecifiedconditionisrecommendedandhastobe
5.2 Protection of a population requires prevention of unac-
met in most tests. Although a violation of one “should” is
ceptable effects on the number, weight, health, and uses of the
individualsofthatspecies,orspeciesforwhichthetestspecies
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
serves as a surrogate. A three-brood toxicity test is conducted
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.
3 4
The last approved version of this historical standard is referenced on Boldfacenumbersinparenthesesrefertothelistofreferencesattheendofthis
www.astm.org. guide.
E1295 − 22
TABLE 1 Test Conditions for Conducting Three-Brood Toxicity TABLE 2 Test Acceptability Requirements for Three-Brood
Tests with Ceriodaphnia dubia Toxicity Test with Ceriodaphnia dubia
Test Criteria Specification A. The following performance criteria must be met when
1) Test Type Whole effluent, receiving water or conducting a three-brood test withCeriodaphnia dubia.
reference toxicity test, or both, with Additional criteria listed in Section 14.
static-renewal of test solution. 1. All C. dubia used in the test must be less than 24 h old
4) Test Duration 6–8 days, when 60% of control (hatched within 8 h window) and from the same brood stock.
animals produce 3 broods 2. The average survival of the C. dubia exposed in the control
3) Temperature 25°C (± 1°C) sample must be$ 80 %. Control treatments may include
4) Photoperiod 16 h light: 8 h dark, ambient standard laboratory water only, or some combination of
laboratory light levels standard water(s) and uncontaminated site water, to provide a
5) Test Chamber Size 30 mL measure of organism survival and reproduction based on
specific test water conditions, such as hardness, alkalinity, and
6) Test Solution Volume 15 mL
7) Renewal of Test Solution every 24 h so forth. A control treatment consists of maintaining organisms
in water to which no test material has been added in order to
8) Age of Test Organisms < 24 h old neonates, within 8 h of
same age provide (a) a measure of the acceptability of the test by giving
an indication of the quality of the test organisms and the
9) No. Organisms/Test Chamber 1
10) No. Replicate Test Chambers/ 10 suitability of the dilution water, food, test conditions, handling
procedures, and so forth, and (b) the basis for interpreting data
treatment
11) Feeding Regime Feed 0.1 mL each YCT and obtained from the other treatments. In each of the other
treatments the ten organisms are maintained in water to which
Raphidocelis subcapitata, (formerly
known as Selenastrum a selected concentration of test material (percentage of effluent
or river, or lake water) has been added.
capricornutum)
12) Test Solution Aeration None 3. At least 60 % of the control animals must produce 3 broods
in 8 days (6–8 days preferred), with the 3 brood average in the
13) Dilution Water Any appropriate water as determined
by purpose of test. See Section10 for control treatment of$ 15 young/surviving female.
4. All measured dissolved oxygen measurements be between
additional guidance.
14) Test Chamber Cleaning Replace or brush and rinse cups 4.0 and 8.4 mg/L.
5. All test containers must be the same and must be randomly
between uses.
15) Test Concentrations Test Dependent assigned to the control or test treatment.
6. Test animals should be stocked in test chambers via random
16) Biological Variables Survival and reproduction
block loading design, for example, all A replicates come from
17) Test Acceptability 80 % or greater control survival$ 15
the same brood cup, all B replicates come from the same
young/female in controls
brood cup and so on and so forth.
7. If required, a solvent control treatment must be included with
each test.
to help determine changes in survival and the number of B. The performance based criteria for culturing C. dubia include:
1. Seven day, three brood reference toxicity testing (12.1)
neonates produced that result from exposure to the test
should be performed on a monthly basis for labs routinely
material.
conducting three brood tests. For laboratories conducting tests
only occasionally, reference toxicant tests should be conducted
5.3 Resultsofthree-broodtoxicitytestswith C. dubiamight
at least once in association with each period of testing. For
be used to predict chronic or partial chronic effects on species
laboratories testing only occasionally, it may be desirable for a
reference toxicant test to be conducted prior to initiating other
in field situations as a result of exposure under comparable
testing, as a means to ensure that the testing procedures,
conditions.
facilities, and staff can successfully support conduct of the
procedure. If not performed on a monthly, it might be desirable
5.4 Resultsofthree-broodtoxicitytestswith C. dubiamight
to perform a reference toxicant test concurrently with any 6 day
be compared with the chronic sensitivities of different species
to 8 day 3 brood test with chemicals or environmental samples.
If tested concurrently, both tests must use the same lot of test
and the chronic toxicities of different materials, and to study
animals and same control/dilution water.
the effects of various environmental factors on results of such
2. Survival and reproduction of the culture animals and food
tests.
stocks used in culture should be tracked and recorded. Culture
restarts should be tracked in this same logbook.
5.5 Resultsofthree-broodtoxicitytestswith C. dubiamight
3. Characteristics such as pH, hardness, alkalinity, conductivity,
beusefulforpredictingtheresultsofchronictestsonthesame dissolved oxygen and temperature should be recorded for each
batch of culture water.
test material with the same species in another water or with
4. Water and food should be routinely analyzed for background
another species in the same or a different water. Most such
contamination. This can include chemical analysis, as well as
side-by-side testing of new and old lots of food and water to
predictions are based on the results of acute toxicity tests, and
determine the suitability of the new food and water for use in
so the usefulness of the results of a three-brood toxicity test
culture or testing, or both.
with C. dubia might be greatly increased by also reporting the
results of an acute toxicity test (see Guides E729 and E1192)
conductedunderthesameconditions.Inadditiontoconducting
an acute test with unfed C. dubia, it might also be desirable to
knowtheEC50orLC50beforebeginningthethree-broodtest,
conduct an acute test in which the organisms are fed the same
as a means to determine the concentrations for use in the
as in the three-brood test, to see if the presence of that
chronictest(see10.4.1).Itshouldbenotedthatresultsfroman
concentration of that food affects the results of the acute test
acute test may not necessarily correspond to those of a chronic
and the acute chronic ratio (see 10.4.1).
test, due to the addition of food to the chronic test.
5.5.1 A 48 or 96-h EC50 or LC50 can sometimes be
obtained from a three-brood toxicity test with a known test 5.6 Three-broodtoxicitytestswith C. dubiamightbeuseful
material, but often all the concentrations in the test will be for studying biological availability of, and structure activity
below the EC50 or LC50. In addition, it is usually desirable to relationships between, test materials.
E1295 − 22
5.7 Results of three-brood toxicity tests with C. dubia can particles. Copper, brass, lead, galvanized metal, and natural
vary with temperature, quality and quantity of food, dissolved rubber should not contact dilution water, stock solutions,
ion concentrations, quality of the dilution water, condition of effluents,ortestsolutionsbeforeorduringthetest.Itemsmade
the test organisms, and other factors. of neoprene rubber and other materials not mentioned above
should not be used unless it has been shown that their use will
5.8 Resultsofthree-broodtoxicitytestswith C. dubiamight
not adversely affect either survival, reproduction, or when
be an important consideration when assessing the hazards of
measured length or weight, or both, of C. dubia (see 14.1).
materials to aquatic organisms (see Guide E1023), or when
deriving water quality criteria for aquatic organisms. 6.3 Test Chambers:
6.3.1 Inatoxicitytestwithaquaticorganisms,testchambers
6. Apparatus
are defined as the smallest physical units between which there
are no water connections. However, screens, tubes, cups, and
6.1 Facilities—Culture and test chambers should be main-
so forth, may be used to create two or more compartments
tained in a constant temperature room, incubator, or recircu-
within each chamber if (a) first instar C. dubia cannot move
latingwaterbath.Ifdilutionwaterisnotpreparedbatchwise,it
from one compartment to another, and (b) it has been shown
is usually piped directly from the source of an elevated
thatsurvivalandreproductionarethesamewhenonlysomeof
headbox so it can be gravity-fed into culture tanks and
the compartments in a chamber contain first-generation organ-
containersusedtopreparetestsolutions.Strainersandairtraps
isms (organisms used to initiate a test) as when all the
should be included in the water supply system. The head-box
compartmentsinachambercontainfirst-generationorganisms.
should be equipped for temperature control and aeration. Air
Thus, test solution can flow (chambers are not considered
usedforaerationshouldbefreeoffumes,oil,andwater;filters
replicates in static tests), from one compartment to another
to remove oil and water are desirable. Filtration of air through
within a test chamber, but, by definition, cannot flow from one
a 0.22 µm bacterial filter might be desirable (3). The facility
chamber to another. Because solution can flow from one
should be well ventilated and free of fumes. To further reduce
the possibility of contamination by test materials and other compartment to another in the same test chamber, the
temperature, concentration of test material, and levels of
substances, especially volatile ones, the culture tanks should
notbeinaroominwhichtoxicitytestsareconducted,stockor pathogens and extraneous contaminants, will be more similar
between compartments in the same test chamber than between
testsolutionsareprepared,effluentortestmaterialisstored,or
compartmentsindifferenttestchambersinthesametreatment.
equipment is cleaned. During culture and testing, organisms
6.3.2 Manyseven-daytoxicitytestswithC.dubiahavebeen
shouldbeshieldedfromdisturbanceswithcurtainsorpartitions
to prevent unnecessary stress.Atiming device should be used conducted with each test organism in a separate 30 mLbeaker
containing 15 mL of test solution or disposable plastic food
to provide a 16-h light and 8-h dark photoperiod. A 15- to
30-min transition period (4) when lights go on might be quality cups.Any container made of glass, Type 316 stainless
steel, or a polystyrene may be used if as long as the composi-
desirable to reduce the possibility of organisms being stressed
by instantaneous illumination; a transition period when lights tion of the cup does not interact unacceptably with the test
material (a) each first generation C. dubia is in a separate
go off might also be desirable.
6.1.1 When C. dubia are fed algae, a high light intensity chamber or compartment, and (b) each chamber contains
sufficient test solution to provide adequate surface area to
might cause sufficient photosynthesis to result in a pH high
enough to kill Cladocera (5). Thus the maximum acceptable maintaindissolvedoxygenconcentrationsacceptabletothetest
organisms (12.2). All chambers (and compartments) in a test
intensity is dependent on the buffer capacity of the dilution
water, species and density of algae, and the kind of test must be identical. Chambers should be covered with clean
glass, stainless steel, nylon, or fluorocarbon plastic covers or
chamber and cover. Ambient laboratory light levels will
usually be acceptable, but higher intensities might be better or Shimatsu closures, to keep out extraneous contaminants and to
worse, depending on other conditions. limit evaporation of test solution.
6.2 Construction Materials—Equipment and facilities that 6.4 Cleaning—Test chambers and equipment used to pre-
contact stock solutions, effluents, test solutions, or any water pare and store dilution water, stock solutions, effluent, and test
into which organisms will be placed should not contain solution, should be cleaned before use. The methods used to
substances that can be leached or dissolved by aqueous clean the test containers might depend in part on the material
solutions in amounts that can adversely affect organisms. In from which they are made. New glass and stainless steel items
addition, equipment and facilities that contact stock solutions, should be washed with detergent and rinsed with water, a
test solutions, or effluents should be chosen to minimize water-miscible organic solvent, water, acid (such as 10%
sorption of test materials and components of effluents from concentrated hydrochloric acid), and at least twice with water
water. Glass, Type 316 stainless steel, nylon, and fluorocarbon that meets the specifications of ASTM Type II (see Specifica-
plastics should be used whenever possible to minimize tionD1193).Somelotsofsomeorganicsolventsmightleavea
leaching, dissolution, and sorption. Concrete and rigid plastics filmthatisinsolubleinwater.Attheendofatest,allitemsthat
may be used for culture tanks and in the water supply system, are to be used again should be immediately (a) emptied, (b)
buttheyshouldbesoaked,preferablyinflowingdilutionwater, rinsed with water, (c) cleaned by a procedure appropriate for
for several days before use (6). Cast iron pipe may be used in removingthetestmaterial(forexample,acidtoremovemetals
supply systems, but colloidal iron might be added to the and bases; detergent, organic solvent, or activated carbon to
dilution water and strainers will be needed to remove rust remove organic chemicals), and (d) rinsed at least twice with
E1295 − 22
ASTM Type II water. Test chambers should be rinsed with 8.6 Because dilution water and test solutions are usually
dilution water just before use. (Warning—Cleaning proce- goodconductorsofelectricity,useofgroundfaultsystemsand
dures which use dichromate-sulfuric acid or hypochlorite are leak detectors should be considered to help prevent electrical
discouraged because they are hazardous and might leave shocks.
residues which might contaminate test solutions.)
9. Dilution Water
7. Reagents and Materials
9.1 Requirements—Besides being available in adequate
7.1 General—The test material should be reagent grade or supply, dilutionwater should(a) beacceptableto C. dubia,(b)
better, unless a test on an effluent, a formulation, commercial not affect results of the test, and (c) be of uniform character-
product,ortechnical-gradeoruse-gradematerialisspecifically istics. In effluent testing, upstream dilution water might be
needed. toxic. If the objective of the effluent test is to determine the
toxicity of the effluent independent of the upstream water, a
7.2 Purity of Water—Unless otherwise indicated, references
reconstituted water of similar hardness, alkalinity and pH may
to water shall be understood to mean reagent water as defined
be used as the dilution water. However, use of a reconstituted
by Type II of Specification D1193.
waterwillnotonlyremovetheconfoundingresultsofupstream
toxicity, but also other factors (suspended solids, humic acids,
8. Hazards
and so forth) that might otherwise act to reduce or increase the
8.1 Many materials can affect humans adversely if precau-
toxicity of the effluent.
tions are inadequate. Therefore, guidelines for the safe han-
9.1.1 The dilution water must allow satisfactory survival
dling and disposal of hazardous materials should be strictly
(80% or greater in the cultured animals) and reproduction (at
followed (Guide D4447). Skin contact with all test materials,
least 15 live young/surviving female animals) of C. dubia (see
effluents, and solutions of them should be minimized, by
14.1 d, e, and f). However, acceptable dilution water typically
wearing appropriate protective gloves (especially when wash-
will produce >90% survival and >30 live young/surviving
ing equipment or putting hands in test solutions), laboratory
female during a 6day-8 day test using healthy cultures.
coats,aprons,glasses,andbyusingpipetstoremoveorganisms
9.1.2 The characteristics of the dilution water should be
from test solutions. Special precautions, such as covering test
consistentovertimesothatbroodstockiscultured,andthetest
chambers and ventilating the area surrounding the chambers,
conducted, in water of the same characteristics. In tests to
should be taken when conducting tests on volatile materials.
evaluate the toxicity of ambient waters, additional controls
Information on toxicity to humans (7), recommended handling
should be considered using acceptable quality dilution water
procedures (8),andchemicalandphysicalpropertiesofthetest
(see 9.1.1) with similar chemical characteristics (for example,
material or effluent should be studied before a test is begun.
pH, hardness, and alkalinity).
Special procedures may be necessary with radiolabeled test
9.1.3 The characteristics of the dilution water should be
materials (9) and with materials that are, or are suspected of
consistentduringthetest.Therangeofhardnessduringthetest
being, carcinogenic (10).
should be less than 5 mg/Lor 10% of the average, whichever
8.2 Although disposal of stock solutions, test solutions, is higher. In effluent testing where upstream water is used as
effluents,andtestorganismsposesnospecialproblemsinmost dilution water the variance associated with hardness might
naturally exceed these ranges.
cases, health and safety precautions and applicable regulations
should be considered before beginning a test. Removal or 9.1.4 If it is desired to study the effect of an environmental
degradation of test materials or effluents might be desirable factor such as total organic carbon (TOC), particulate matter,
before disposal of solutions. dissolved ion concentrations, or dissolved oxygen on the
results of a three-brood test with C. dubia, it will be necessary
8.3 Cleaning of equipment with a volatile solvent such as
touseawaterthatisnaturallyorartificiallyhighorlowinthat
acetone should be performed only in a well-ventilated area in
environmental factor. If such a water is used, it is important
whichnosmokingisallowedandnoopenflame,suchasapilot
that adequate analyses be performed to characterize the water
light, is present.
and that a comparable test be available or conducted in a more
8.4 Acidic solutions and hypochlorite solutions should not
usual dilution water to facilitate interpretation of the results in
be mixed because hazardous fumes might be produced.
the special water.
8.5 To prepare dilute acid solutions, concentrated acid
9.2 Source:
should be added to water, not vice versa. Opening a bottle of
9.2.1 If a natural fresh water is used it should be obtained
concentrated acid and mixing concentrated acid with water
from an uncontaminated source of consistent characteristics.A
should be performed only in a fume hood.
well or spring that has been shown to be of acceptable
characteristics is usually preferable to a surface water. If a
surface water is used, the intake should be positioned to
minimize fluctuations in characteristics and the possibility of
Reagent Chemicals, American Chemical Society Specifications, American.
Chemical Society., Washington, DC. For suggestions on the testing of reagents not
contamination,andtomaximizetheconcentrationofdissolved
listed by the American Chemical Society, see Analar Standards for Laboratory
oxygen to help ensure low concentrations of sulfide and iron.
Chemicals,BDHLtd.,Poole,Dorset,U.K.andthe United States Pharmacopeia and
Surface waters should be filtered (60-µm mesh) to remove
National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC0, Rockville,
M.D. potential predators and competitors of C. dubia.
E1295 − 22
9.2.2 Widespread use of one reconstituted water will in- beryllium, boron, cadmium, chromium, cobalt, copper, iron,
crease comparability of test results. The reconstituted fresh lead, manganese, mercury, molybdenum, nickel, selenium,
water described in Guide E729 has been used successfully by silver,andzinc.Foreachmethodused(see13.3),thedetection
several people.Addition of 5 µg of selenium (11)and1µgof limit should be below either (a) the concentration in the
crystalline vitamin B /L (12) might be desirable, (but see dilution water, or (b) the lowest concentration that has been
X1.1). C. dubia has also been cultured and tested in reconsti- shown to affect adversely the survival, and reproduction of C.
tuted soft water. Acclimation in one reconstituted water and dubia.
testing in another of different hardness or alkalinity should be
9.5 Control water—Dilution water can be analogous to the
avoided to minimize stress due to routine water quality
laboratorycontrolwatertreatmenttowhichnotestmaterialhas
changes.
been added. The control provides (a) a measure of the
9.2.3 Chlorinated water should not be used as, or in the
acceptability of the test by giving an indication of the quality
preparationof,dilutionwaterbecauseresidualchlorineisquite
of the test organisms and the suitability of the dilution water,
toxic to Cladocera (13). Dechlorinated water should be used
food,testconditions,handlingprocedures,andsoforth,and(b)
onlyasalastresortbecausedechlorinationisoftenincomplete.
the basis for interpreting data obtained from the other treat-
Sodium bisulfite is probably better for dechlorinating water
ments.
than sodium sulfite, and both are more reliable than carbon
9.5.1 Additional controls, also referred to as secondary
filters,especiallyforremovingchloramines (14).Someorganic
controls, can be useful for distinguishing effects due to a test
chloramines, however, react slowly with sodium bisulfite (15).
material from the water in which samples are tested. Surface
Inadditiontoresidualchlorine,municipaldrinkingwateroften
water samples or tested aqueous samples can contain ion
contains unacceptably high concentrations of copper, lead,
concentrationsorwaterqualitycharacteristicsthatareaboveor
zinc, and fluoride, and quality is often rather variable. Exces-
below the tolerance range of C. dubia (22, 23, 24). Including a
siveconcentrationsofmostmetalscanusuallybereducedwith
secondarycontrolthatreflectsthesamplewaterqualityandion
a chelating resin (16), but use of an alternative dilution water
concentrationscanidentifyifeffectstoa C. dubiaendpointare
might be preferable.
due to the test material or toxicant alone or if effects are also
9.3 Treatment: associated with the background water quality of the sample.
9.3.1 Dilutionwatershouldbeaeratedgentlybysuchmeans
10. Test Material
as air stones, surface aerators, or column aerators, (17, 18)
prior to addition of test material.Adequate aeration will bring
10.1 Before a test is begun with material other than
thepHandconcentrationsofdissolvedoxygenandothergases
effluents, the following should be known about the test
into equilibrium with air and minimize oxygen demand and
material:
concentrations of volatiles. The concentration of dissolved
10.1.1 Identities and concentrations of major ingredients
oxygenindilutionwatershouldbebetween90%and110%of
and major impurities, for example, impurities constituting
saturation (19) to help ensure that dissolved oxygen concen-
more than 1% of the material.
trations are acceptable in test chambers. Supersaturation by
10.1.2 Solubility and stability in the dilution water.
dissolvedgasesthatcanbecausedbyheatingthedilutionwater
10.1.3 An estimate of the lowest concentration of test
should be avoided (20).
material that is acutely toxic to C. dubia.
9.3.2 Filtration through sand, rock, bag, or depth type 10.1.4 Accuracy and precision of the analytical method at
cartridge filters may be used to keep the concentration of
planned test concentration(s).
particulatematteracceptablylow(see9.2.1),andasapretreat-
10.1.5 Estimate of toxicity to humans and recommended
ment before ultraviolet sterilization or filtration through a finer
handling procedures (see 8.1).
filter.
10.2 Stock Solution:
9.3.3 Dilution water that might be contaminated with fac-
10.2.1 In some cases the test material can be added directly
ultative pathogens may be passed through a properly main-
todilutionwater,butusuallyitisdissolvedinasolventtoform
tained ultraviolet sterilizer (21) equipped with an intensity
a stock solution that is then added to the dilution water. If a
meterandflowcontrols,passedthroughamembranefilterwith
stocksolutionisused,theconcentrationandstabilityofthetest
a pore size of 0.20 µm, or autoclaved. Water that might be
material in it should be determined before the beginning of the
contaminated with Aphanomyces daphniae should be auto-
test. If the test material is subject to photolysis, the stock
claved (3).
solution should be shielded from light.
9.4 Characterization—The following items should be mea- 10.2.2 Except possibly for tests on hydrolyzable,
sured in the dilution water at least twice each year and more oxidizable, and reducible materials, the preferred solvent is
often if such measurements have not been made semiannually dilutionwater,althoughfiltrationorsterilization,orboth,ofthe
for at least two years, or if a surface water is used: hardness, water might be necessary. If the hardness of the dilution water
alkalinity, conductivity, pH, particulate matter, total dissolved willnotbeaffected,distilledanddeionizedwatermaybeused.
solids, total suspended solids, TOC, selected pesticides (such Several techniques have been specifically developed for pre-
as those found in USGS Schedules 2001/2010), organic paring aqueous stock solutions of slightly soluble materials
chlorine, PCBs, phthalate esters, ammonia, cyanide, sulfide, (25). Minimum amounts of strong acids or bases may be used
chloride, bromide, fluoride, iodide, nitrate, phosphate, sulfate, inthepreparationofaqueousstocksolutions,butsuchreagents
calcium, magnesium, sodium, potassium, aluminum, arsenic, mightaffectthepHoftestsolutionsappreciably.Useofamore
E1295 − 22
soluble form of the test material, such as chloride or sulfate (for example, high or low TOC or dissolved ions) may be
salts of organic amines, sodium or potassium salts of phenols appropriate and would function as does the solvent control
described here.
andorganicacids,andchlorideornitratesaltsofmetals,might
affectthepHmoreorlessthanuseoftheminimumamountsof 10.2.5 Ifasolventotherthanwaterisusedtoprepareastock
solution, it might be desirable to conduct simultaneous tests
strong acids and bases.
using two chemically unrelated solvents or two different
10.2.3 If a solvent other than dilution water is used, its
concentrations of the same solvent to obtain information
concentration in test solutions should be kept to a minimum
concerning possible effects of solvent on results of the test.
and should be low enough that it does not affect survival or
reproductionofC.dubia(andlengthorweight,orboth,ifthese 10.3 Effluent:
characteristics are to be measured). Because of its low toxicity
10.3.1 Sampling Point—The effluent sampling point should
to aquatic animals (26), low volatility, and high ability to bebasedonthepurposeofthetest.Thecollectionpointforthe
dissolve many organic chemicals, triethylene glycol is often a National Pollutant Discharge Elimination System (NPDES)
permit testing purposes is often strictly defined. In some cases,
good organic solvent for preparing stock solutions. Other
asamplingpointbetweenlasttreatmentandthedischargepoint
water-miscibleorganicsolventssuchasmethanol,ethanol,and
might provide much better access. If the waste is chlorinated,
acetonemayalsobeused,buttheymightstimulateundesirable
it might be desirable to have sampling points both upstream
growth of microorganisms and besides, acetone is quite vola-
and downstream of the chlorine contact point to determine the
tile. If an organic solvent is used, it should be reagent grade or
toxicity of both chlorinated and unchlorinated effluent. The
better. Asurfactant should not be used in the preparation of a
schedule of effluent sampling should be based on an under-
stock solution because it might affect the form and toxicity of
standing of the short- and long-term operations and schedules
the test material in test solutions.
ofthedischarger.Althoughitisusuallydesirabletoevaluatean
10.2.4 If a solvent other than water is used, (a) at least one
effluent sample that most closely represents the normal or
solvent control, using solvent from the same batch used to
typical discharge, conducting tests on atypical samples might
make the stock solution, must be included in the test and (b)a
also be informative.
dilution water control, must be included in the test. If no
10.4 Test Concentration(s):
solvent other than water is used, a dilution water control must
10.4.1 If the test is intended to provide a good estimate of
be included in the test.
the highest concentration of test material or effluent that will
10.2.4.1 Iftheconcentrationofsolventisthesameinalltest
not unacceptably affect the survival, and reproduction of C.
solutions that contain test material, the solvent control must
dubia, the test concentrations (see 12.12.2) should bracket the
contain the same concentration of solvent.
best prediction of that concentration. Such a prediction is
10.2.4.2 Iftheconcentrationofsolventisnotthesameinall
usually based on the results of a 48-h static-acute toxicity test
test solutions that contain test material, either (a) a solvent test
(see Guide E729) on the test material using the same dilution
must be conducted to determine whether the survival, or
water and C. dubia less than 24-h old. Because the food used
reproduction of C. dubia is related to the concentration of the
in a three-brood toxicity test sometimes affects the results of
solvent over the range used in the toxicity test, or (b) such a
the acute test (27), the acute test should be conducted with and
solvent test must have already been conducted using the same
withoutthefoodaddedtothedilutionwater.Ifanacutechronic
typeofdilutionwaterandthesamesourceof C. dubia.Ifeither
ratiohasbeendeterminedforthetestmaterialwithaspeciesof
survival or reproduction is found to be related to the concen-
comparable sensitivity, the results of the acute test with C.
tration of solvent, a three-brood toxicity test with C. dubia in
dubia can be divided by the acute-chronic ratio to predict an
that water is unacceptable if any treatment contained a con-
appropriate range of concentrations for the chronic test.
centration of solvent in that range. If neither survival or
10.4.2 In some (usually regulatory) situations, it is only
reproduction is found to be related to the concentration of
necessary to determine whether one specific concentration of
solvent, a three-brood toxicity test with C. dubia in that water
test material or effluent unacceptably affects survival or repro-
maycontainsolventconcentrationswithinthetestedrange,but
duction. For example, the specific concentration might be the
the solvent control must contain the highest concentration of concentration occurring in a receiving water, the concentration
solvent present in any of the other treatments. resulting from the direct application of a material to a body of
water, or the solubility limit of a material in water.When there
10.2.4.3 Ifthetestcontainsbothadilutionwatercontroland
is interest only in a specific concentration, it is often necessary
a solvent control, the survival, and reproduction of C. dubia in
totestonlythatspecificconcentration(see12.2.1.3).However,
the controls should be compared (see X4.7 and Guide E1847).
use of multiple concentrations will provide data useful for
If a statistically significant difference in either survival or
determining toxicity thresholds and evaluating the concentra-
reproduction, is detected between the two controls, only the
tion response relationship (28).
solvent control may be used for meeting the requirements of
14.1 c, d, and e as the basis for calculation of results. If no 10.5 Collection:
statistically significant difference is detected, the data from
10.5.1 Several different methods may be used to collect
both controls should be used for meeting the requirements of
effluentsamplesfortoxicitytests.Selectionofamethodshould
14.1 c, d,and easthebasisforcalculationofresults.Likewise,
be based on the type of test that is to be conducted, the
a secondary control to account for background water quality characteristics of the effluent, any treatment technologies
E1295 − 22
employed, the rate and manner by which the effluent is 11. Test Organisms
discharged into the receiving water, and the average wastewa-
11.1 Species—The genus Ceriodaphnia went through a
ter retention time. Industrial or municipal facilities occasion-
revision. Berner (31) investigated the taxonomy of Ceri-
ally discharge directly, with no provision for effluent retention.
odaphnia in U.S. EPA cultures and based on this study the
In the more typical situation, however, holding and treatment
early published reference in toxicological literature to C.
ponds provide some duration of effluent retention. The reten-
dubia/affınis was most likely C. dubia. Identification of the
tion time should be measured because channeling sometimes
species employed in testing is the responsibility of the report-
causes the average retention time to be substantially less than
ing investigator.
the calculated or design retention time.
10.5.2 It is recommended that renewal toxicity tests be 11.2 Age—Three-brood toxicity tests with C. dubia should
conducted on effluent obtained by the following methods: be started with organisms less than 24h old. Using neonates
bornwithinanarrowagerange,forexample,lessthan24hold
10.5.2.1 If the average retention time of the effluent is less
than 24 h, a 24-h composite sample should be collected daily, and born within 8h of each other is required.
diluted appropriately, and used for daily renewals (see
11.3 Source—All organisms used in a test must be from the
10.5.2.3).
same brood stock. The two (and preferably five) prior genera-
10.5.2.2 If the average retention time is greater than 14
tions must have been raised from birth using the same food,
days, a grab sample should be collected daily, diluted
type of water, and temperature as will be used in the three-
appropriately, and used for daily renewals. If the average
broodtest.Thiswillnotonlyacclimatetheorganisms,butwill
retention time is greater than 24h and less than 14 days, either
also help demonstrate the acceptability of the food, water, and
composite or grab sampling can be used to collect effluent
so forth, before the test.Acclimation of organisms for effluent
samples.
testsinwhichnaturaldilutionwatersareusedmightbedifficult
10.5.2.3 If an effluent is known, or suspected, of being
to achieve. In some cases available (upstream) dilution water
highly variable in terms of constituents and retention time is
mightbetoxicandanalternativedilutionwaterwillhavetobe
less than 24 h, grab samples might be more representative of
employedinthosecasesinwhicheffluenttoxicityindependent
toxicity potential. In addition, more frequent renewal intervals
of ambient water toxicity is the testing objective. When a lab
might be desirable.
cannot raise organism
...

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: E1295 − 01 (Reapproved 2013) E1295 − 22
Standard Guide for
Conducting Three-Brood, Renewal Toxicity Tests with
Ceriodaphnia dubia
This standard is issued under the fixed designation E1295; 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*Scope
1.1 This guide describes procedures for obtaining data concerning the adverse effects of an effluent or a test material (added to
dilution water, but not to food) on Ceriodaphnia dubia Richard 1894, during continuous exposure throughout a portion of the
organism’s life. These procedures should also be useful for conducting life cycle toxicity tests with other Cladocera (Guide E1193),
although modifications will be necessary.
1.2 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or known
mixtures, that can be measured accurately at the necessary concentrations in water. With appropriate modifications these
procedures can be used to conduct tests on temperature, dissolved oxygen, pH, dissolved ions, and on such materials as aqueous
effluents (see also Guide E1192), leachates, oils, particulate matter, sediments (see also Guide E1383E1706), and surface waters.
Renewal tests might not be applicable to materials that have high oxygen demand, are highly volatile, are rapidly biologically or
chemically transformed, or sorb to test chambers. If the concentration of dissolved oxygen falls below 4 mg/L or the concentration
of test material decreases by more than 20 % in test solution(s) at any concentration between renewals, more frequent renewals
might be necessary.
1.3 Other modifications of these procedures might be justified by special needs or circumstances. Results of tests conducted using
unusual procedures are not likely to be comparable to results of many other tests. Comparisons of results obtained using modified
and unmodified versions of these procedures might provide useful information on new concepts and procedures for conducting
three-brood toxicity tests with C. dubia.
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
Reagents and Materials 7
This guide is under the jurisdiction of ASTM Committee E50 on Environmental Assessment, Risk Management and Corrective Actionand is the direct responsibility of
Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved March 1, 2013Jan. 1, 2022. Published March 2013April 2022. Originally approved in 1989. Last previous edition approved in 20062013 as
E1295 – 01(2006).(2013). DOI: 10.1520/E1295-01R13.10.1520/E1295-22.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1295 − 22
Hazards 8
Dilution Water 9
Requirements 9.1
Source 9.2
Treatment 9.3
Characterization 9.4
Test Material 10
General 10.1
Stock Solution 10.2
Effluent 10.3
Test Concentration(s) 10.4
Collection 10.5
Sample Containers 10.6
Preservation 10.7
Treatment 10.8
Test Organisms 11
Species 11.1
Age 11.2
Source 11.3
Brood Stock 11.4
Food 11.5
Handling 11.6
Quality 11.7
Procedure 12
Demonstration of Feasibility 12.1
Experimental Design 12.2
Dissolved Oxygen 12.3
Temperature 12.4
Preparing Test Solutions 12.5
Conditioning Test Chambers 12.6
Beginning a Test 12.7
Renewing Test Solutions 12.8
Duration of Test 12.9
Biological Data 12.10
Other Measurements 12.11
Test Material 12.12
Analytical Methodology 13
Acceptability of Test 14
Calculation 15
Report 16
Appendixes
Food Appendix X1
Culture Techniques Appendix X2
Test Chambers Appendix X3
Statistical Guidance Appendix X4
1.5 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.
1.5 This guide is arranged as follows:
1.6 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.
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
Reagents and Materials 7
Hazards 8
Dilution Water 9
Requirements 9.1
Source 9.2
Treatment 9.3
Characterization 9.4
Test Material 10
General 10.1
E1295 − 22
Stock Solution 10.2
Effluent 10.3
Test Concentration(s) 10.4
Collection 10.5
Sample Containers 10.6
Preservation 10.7
Treatment 10.8
Test Organisms 11
Species 11.1
Age 11.2
Source 11.3
Brood Stock 11.4
Food 11.5
Handling 11.6
Quality 11.7
Procedure 12
Demonstration of Feasibility 12.1
Experimental Design 12.2
Dissolved Oxygen 12.3
Temperature 12.4
Preparing Test Solutions 12.5
Conditioning Test Chambers 12.6
Beginning a Test 12.7
Renewing Test Solutions 12.8
Duration of Test 12.9
Biological Data 12.10
Other Measurements 12.11
Analytical Methodology 13
Acceptability of Test 14
Calculation 15
Report 16
Appendixes
Food Appendix X1
Culture Techniques Appendix X2
Test Chambers Appendix X3
Statistical Guidance Appendix X4
2. Referenced Documents
2.1 ASTM Standards:
D1193 Specification for Reagent Water
D3978 Practice for Algal Growth Potential Testing with Pseudokirchneriella subcapitata
D4447 Guide for Disposal of Laboratory Chemicals and Samples
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
E1193 Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests
E1383 Guide for Conducting Sediment Toxicity Tests with Freshwater Invertebrates (Withdrawn 1995)
E1706 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates
E1733 Guide for Use of Lighting in Laboratory Testing
E1847 Practice for Statistical Analysis of Toxicity Tests Conducted Under ASTM Guidelines (Withdrawn 2022)
IEEE/ASTM SI 10SI10–02 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 standard. “Must” is used to
express an absolute requirement, that is, to state that the test has to be designed to satisfy the specified condition, unless the purpose
of the test requires a different design. “Must” is only used in connection with factors that directly relate to the acceptability of the
test (see Section 14). “Should” is used to state that the specified condition is recommended and has to be met in most tests.
Although a violation of one “should” is rarely a serious matter, violation of several will often render the results questionable. Terms
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
The last approved version of this historical standard is referenced on www.astm.org.
E1295 − 22
such as “is desirable,” “is often desirable,” and “might be desirable” are used in connection with less important factors. “May” is
used to mean “is (are) allowed to,” “can” is used to mean “is (are) able to,” and “might” is used to mean “could possibly.” Thus
the classic distinction between “may” and “can” is preserved, and “might” is never used as a synonym for either “may” or “can.”
3.2 A brood refers, collectively, to the young neonates released at the time of adult molt by the young/adult animal originally
exposed to the control and test solutions. The number of young in each brood should increase over the period of the test. Animals
may be transferred to fresh control or test solution before completing the release of a brood, resulting in split broods. Care is needed
when interpreting the results to determine the number of broods released during a test.
3.2 For definitions of other terms used in this standard, refer to Guide E729, Terminology E943, and Guide E1023. For an
explanation of units and symbols, refer to SI10-02IEEE/ASTM SI 10SI10–02. IEEE/ASTM SI 10.
3.3 Definitions of Terms Specific to This Standard:
3.3.1 brood, n—the young neonates released at the time of adult molt by the young/adult animal originally exposed to the control
and test solutions.
3.3.1.1 Discussion—
The number of young in each brood are typically counted over each 24 h period of the test and should increase over the duration
of the test. Animals may be transferred to fresh control or test solution before completing the release of a brood, resulting in split
broods. Care is needed when interpreting the results to determine the number of broods released during a test.
4. Summary of Guide
4.1 At the beginning of the test, at least ten C. dubia less than 24-h old are maintained24 h old, and within 8 h of age, are
introduced individually in separate test chambers (or in separate compartments in two or more test chambers), chambers, and
exposed to control water and a least one (preferably 2 or more) toxicant concentrations. One or more control treatments control
treatment must be used but more may be used. Control treatments may include standard laboratory water only, or some
combination of standard water(s) and uncontaminated site water, to provide a measure of organism survival and reproduction based
on specific test water conditions, such as hardness, alkalinity, and so forth. A control treatment consists of maintaining organisms
in water to which no test material has been added in order to provide (a) 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 (b) the basis for interpreting data obtained from the other treatments. In each of the other treatments the ten
organisms are maintained in water to which a selected concentration of test material (percentage of effluent or river, or lake water)
has been added. Specified data on the concentration of test material and the survival and reproduction of C. dubia are collected
and analyzed to determine the effect of the tested concentration (% effluent or ambient water) on C. dubia.
4.2 Table 1(1) contains a summary of the conditions used when conducting a three-brood test with C. dubia.Table 2 and Section
14 list the requirements that need to be met for a test to be deemed acceptable.
5. Significance and Use
5.1 Ceriodaphnia was first used as a toxicity test organism by Mount and Norberg (2). Introduced for use in effluent and ambient
water evaluations, Ceriodaphnia have also been a valuable addition to single chemical test procedures.
5.2 Protection of a population requires prevention of unacceptable effects on the number, weight, health, and uses of the
individuals of that species, or species for which the test species serves as a surrogate. A three-brood toxicity test is conducted to
help determine changes in survival and the number of neonates produced that result from exposure to the test material.
5.3 Results of three-brood toxicity tests with C. dubia might be used to predict chronic or partial chronic effects on species in field
situations as a result of exposure under comparable conditions.
5.4 Results of three-brood toxicity tests with C. dubia might be compared with the chronic sensitivities of different species and
the chronic toxicities of different materials, and to study the effects of various environmental factors on results of such tests.
Boldface numbers in parentheses refer to the list of references at the end of this guide.
E1295 − 22
TABLE 1 Test Conditions for Conducting Three-Brood Toxicity TABLE 2 Test Acceptability Requirements for Three-Brood
Tests with Ceriodaphnia dubia Toxicity Test with Ceriodaphnia dubia
Test Criteria Specification A. The following performance criteria must be met when
1) Test Type Whole effluent, receiving water or conducting a three-brood test withCeriodaphnia dubia.
reference toxicity test, or both, with Additional criteria listed in Section 14.
static-renewal of test solution. 1. All C. dubia used in the test must be less than 24–h old and
from the same broodstock.
4) Test Duration 6–8 days, when 60% of control
animals produce 3 broods 1. All C. dubia used in the test must be less than 24 h old
(hatched within 8 h window) and from the same brood stock.
3) Temperature 25°C (± 1°C)
4) Photoperiod 16 h light: 8 h dark, ambient 2. The average survival of the C. dubia exposed in the control
laboratory light levels sample must be $ 90%.
5) Test Chamber Size 30 mL 2. The average survival of the C. dubia exposed in the control
6) Test Solution Volume 15 mL sample must be $ 80 %. Control treatments may include
7) Renewal of Test Solution every 24 or 48 hours standard laboratory water only, or some combination of
7) Renewal of Test Solution every 24 h standard water(s) and uncontaminated site water, to provide a
8) Age of Test Organisms < 24h old neonates, within 8–12h of measure of organism survival and reproduction based on
specific test water conditions, such as hardness, alkalinity, and
same age
8) Age of Test Organisms < 24 h old neonates, within 8 h of so forth. A control treatment consists of maintaining organisms
in water to which no test material has been added in order to
same age
9) No. Organisms/Test Chamber 1 provide (a) a measure of the acceptability of the test by giving
an indication of the quality of the test organisms and the
10) No. Replicate Test Chambers/ 10
suitability of the dilution water, food, test conditions, handling
treatment
procedures, and so forth, and (b) the basis for interpreting data
11) Feeding Regime Feed 0.1 mL each natural food and
obtained from the other treatments. In each of the other
synthetic food
treatments the ten organisms are maintained in water to which
11) Feeding Regime Feed 0.1 mL each YCT and
a selected concentration of test material (percentage of effluent
Raphidocelis subcapitata, (formerly
or river, or lake water) has been added.
known as Selenastrum
3. At least 60% of the control animals must produce 3 broods
capricornutum)
in 8 days ( 7 days preferred), with the 3 brood average $ 15
12) Test Solution Aeration None
young/female.
13) Dilution Water Any appropriate water as determined
3. At least 60 % of the control animals must produce 3 broods
by purpose of test. See Section10 for
in 8 days (6–8 days preferred), with the 3 brood average in the
additional guidance.
control treatment of $ 15 young/surviving female.
14) Test Chamber Cleaning Brush and rinse cups between uses.
4. All measured dissolved oxygen reading must be between
14) Test Chamber Cleaning Replace or brush and rinse cups
4.0 and 8.4 mg/L.
between uses.
4. All measured dissolved oxygen measurements be between
15) Test Concentrates Test Dependent
4.0 and 8.4 mg/L.
15) Test Concentrations Test Dependent
5. All test containers must be the same and must be randomly
16) Biological Variables Survival and reproduction
assigned to the control or test treatment.
17) Test Acceptability 90% or greater control survival $ 15
6. Test animals must be randomly assigned to a control or test
young/female in controls
treatment replicate test chamber.
17) Test Acceptability 80 % or greater control survival $ 15
6. Test animals should be stocked in test chambers via random
young/female in controls
block loading design, for example, all A replicates come from
the same brood cup, all B replicates come from the same
brood cup and so on and so forth.
7. If required, a solvent control treatment must be included with
each test.
B. The performance based criteria for culturing C. dubia include:
1. Seven day, three brood reference toxicity testing should be
performed on a monthly basis. If not performed on a monthly, it
might be desirable to perform a reference toxicant test
concurrently with any 7 day 3 brood test with chemicals or
environmental samples. If tested concurrently, both tests must
use the same lot of test animals and same control/dilution
water.
1. Seven day, three brood reference toxicity testing (12.1)
should be performed on a monthly basis for labs routinely
conducting three brood tests. For laboratories conducting tests
only occasionally, reference toxicant tests should be conducted
at least once in association with each period of testing. For
laboratories testing only occasionally, it may be desirable for a
reference toxicant test to be conducted prior to initiating other
testing, as a means to ensure that the testing procedures,
facilities, and staff can successfully support conduct of the
procedure. If not performed on a monthly, it might be desirable
to perform a reference toxicant test concurrently with any 6 day
to 8 day 3 brood test with chemicals or environmental samples.
If tested concurrently, both tests must use the same lot of test
animals and same control/dilution water.
2. Survival and reproduction of the culture animals and food
stocks used in culture should be tracked and recorded. Culture
restarts should be tracked in this same logbook.
3. Characteristics such as pH, hardness, alkalinity, conductivity,
dissolved oxygen and temperature should be recorded for each
batch of culture water.
E1295 − 22
4. Water and food should be routinely analyzed for background
contamination. This can include chemical analysis, as well as
side-by-side testing of new and old lots of food and water to
determine the suitability of the new food and water for use in
culture or testing, or both.
5.5 Results of three-brood toxicity tests with C. dubia might be useful for predicting the results of chronic tests on the same test
material with the same species in another water or with another species in the same or a different water. Most such predictions are
based on the results of acute toxicity tests, and so the usefulness of the results of a three-brood toxicity test with C. dubia might
be greatly increased by also reporting the results of an acute toxicity test (see Guides E729 and E1192) conducted under the same
conditions. In addition to conducting an acute test with unfed C. dubia, it might also be desirable to conduct an acute test in which
the organisms are fed the same as in the three-brood test, to see if the presence of that concentration of that food affects the results
of the acute test and the acute chronic ratio (see 10.4.1).
5.5.1 A 48 or 96-h EC50 or LC50 can sometimes be obtained from a three-brood toxicity test with a known test material, but often
all the concentrations in the test will be below the EC50 or LC50. In addition, it is usually desirable to know the EC50 or LC50
before beginning the three-brood test, as a means to determine the concentrations for use in the chronic test (see 10.4.1). It should
be noted that results from an acute test may not necessarily correspond to those of a chronic test, due to the addition of food to
the chronic test.
5.6 Three-brood toxicity tests with C. dubia might be useful for studying biological availability of, and structure activity
relationships between, test materials.
5.7 Results of three-brood toxicity tests with C. dubia can vary with temperature, quality and quantity of food, dissolved ion
concentrations, quality of the dilution water, condition of the test organisms, and other factors.
5.8 Results of three-brood toxicity tests with C. dubia might be an important consideration when assessing the hazards of materials
to aquatic organisms (see Guide E1023), or when deriving water quality criteria for aquatic organisms.
6. Apparatus
6.1 Facilities—Culture and test chambers should be maintained in a constant temperature room, incubator, or recirculating water
bath. If dilution water is not prepared batchwise, it is usually piped directly from the source of an elevated headbox so it can be
gravity-fed into culture tanks and containers used to prepare test solutions. Strainers and air traps should be included in the water
supply system. The head-box should be equipped for temperature control and aeration. 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). The facility should be well ventilated and free of fumes. To further reduce the possibility of contamination by test
materials and other substances, especially volatile ones, the culture tanks should not be in a room in which toxicity tests are
conducted, stock or test solutions are prepared, effluent or test material is stored, or equipment is cleaned. During culture and
testing, organisms should be shielded from disturbances with curtains or partitions to prevent unnecessary stress. A timing device
should be used to provide a 16-h light and 8-h dark photoperiod. A 15- to 30-min transition period (4) when lights go on might
be desirable to reduce the possibility of organisms being stressed by instantaneous illumination; a transition period when lights go
off might also be desirable.
6.1.1 When C. dubia are fed algae, a high light intensity might cause sufficient photosynthesis to result in a pH high enough to
kill Cladocera (5). Thus 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. Ambient laboratory light levels will usually be acceptable, but higher
intensities might be better or worse, depending on other conditions.
6.2 Construction Materials—Equipment and facilities that contact stock solutions, effluents, test solutions, or any water into which
organisms will be placed should not contain substances that can be leached or dissolved by aqueous solutions in amounts that can
adversely affect organisms. In addition, equipment and facilities that contact stock solutions, test solutions, or effluents should be
chosen to minimize sorption of test materials and components of effluents from water. Glass, Type 316 stainless steel, nylon, and
fluorocarbon plastics should be used whenever possible to minimize leaching, dissolution, and sorption. Concrete and rigid 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 might be added to the dilution water
E1295 − 22
and strainers will be needed to remove rust particles. Copper, brass, lead, galvanized metal, and natural rubber should not contact
dilution water, stock solutions, effluents, or test solutions before or during the test. Items made of neoprene rubber and other
materials not mentioned above should not be used unless it has been shown that their use will not adversely affect either survival,
reproduction, or when measured length or weight, or both, of C. dubia (see 14.1).
6.3 Test Chambers:
6.3.1 In a toxicity test with aquatic organisms, test chambers are defined as the smallest physical units between which there are
no water connections. However, screens, tubes, cups, and so forth, may be used to create two or more compartments within each
chamber if (a) first instar C. dubia cannot move from one compartment to another, and (b) it has been shown that survival and
reproduction are the same when only some of the compartments in a chamber contain first-generation organisms (organisms used
to initiate a test) as when all the compartments in a chamber contain first-generation organisms. Thus, test solution can flow
(chambers are not considered replicates in static tests), from one compartment to another within a test chamber, but, by definition,
cannot flow from one chamber to another. Because solution can flow from one compartment to another in the same test chamber,
the temperature, concentration of test material, and levels of pathogens and extraneous contaminants, will be more similar between
compartments in the same test chamber than between compartments in different test chambers in the same treatment.
6.3.2 Many seven-day toxicity tests with C. dubia have been conducted with each test organism in a separate 30 mL beaker
containing 15 mL of test solution or disposable plastic food quality cups. Any container made of glass, Type 316 stainless steel,
or a fluorocarbon plastic polystyrene may be used if as long as the composition of the cup does not interact unacceptably with the
test material (a) each first generation C. dubia is in a separate chamber or compartment, and (b) each chamber contains sufficient
test solution to provide adequate surface area to maintain dissolved oxygen concentrations acceptable to the test organisms (12.2).
All chambers (and compartments) in a test must be identical. Chambers should be covered with clean glass, stainless steel, nylon,
or fluorocarbon plastic covers or Shimatsu closures, to keep out extraneous contaminants and to reducelimit evaporation of test
solution.
6.4 Cleaning—Test chambers and equipment used to prepare and store dilution water, stock solutions, effluent, and test solution,
should be cleaned before use. The methods used to clean the test containers might depend in part on the material from which they
are made. New glass and stainless steel items should be washed with detergent and rinsed with water, a water-miscible organic
solvent, water, acid (such as 10 % concentrated hydrochloric acid), and at least twice with water that meets the specifications of
ASTM Type II (see Specification D1193). Some lots of some organic solvents might leave a film that is insoluble in water. At the
end of a test, all items that are to be used again should be immediately (a) emptied, (b) rinsed with water, (c) 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 (d) rinsed at least twice with ASTM Type II water. Test chambers should be rinsed with
dilution water just before use. (Warning—Cleaning procedures which use dichromate-sulfuric acid or hypochlorite are
discouraged because they are hazardous and might leave residues which might contaminate test solutions.)
7. Reagents and Materials
7.1 General—The test material should be reagent grade or better, unless a test on an effluent, a formulation, commercial product,
or technical-grade or use-grade material is specifically needed.
7.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water as defined by Type
II of Specification D1193.
8. Hazards
8.1 Many materials can affect humans adversely if precautions are inadequate. Therefore, skin guidelines for the safe handling and
disposal of hazardous materials should be strictly followed (Guide D4447). Skin contact with all test materials, effluents, and
solutions of them should be minimized, by wearing appropriate protective gloves (especially when washing equipment or putting
hands in test solutions), laboratory coats, aprons, glasses, and by using pipets to remove organisms 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
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. (USPC0, Rockville, M.D.
E1295 − 22
physical properties of the test material or effluent should be studied before a test is begun. Special procedures may be necessary
with radiolabeled test materials (9) and with materials that are, or are suspected of being, carcinogenic (10).
8.2 Although disposal of stock solutions, test solutions, effluents, and test organisms poses no special problems in most cases,
health and safety precautions and applicable regulations should be considered before beginning a test. Removal or degradation of
test materials or effluents might be desirable before disposal of solutions.
8.3 Cleaning of equipment with a volatile solvent such as acetone should be performed only in a well-ventilated area in which
no smoking is allowed and no open flame, such as a pilot light, is present.
8.4 Acidic solutions and hypochlorite solutions should not be mixed because hazardous fumes might be produced.
8.5 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 fume hood.
8.6 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.
9. Dilution Water
9.1 Requirements—Besides being available in adequate supply, the dilution water should (a) be acceptable to C. dubia, (b) not
unnecessarily affect results of the test, and (c) be of uniform characteristics. In effluent testing, upstream dilution water might be
toxic. If the objective of the effluent test is to determine the toxicity of the effluent independent of the upstream water, a
reconstituted water of similar hardness, alkalinity and pH may be used as the dilution water. However, use of a reconstituted water
will not only remove the confounding results of upstream toxicity, but also other factors (suspended solids, humic acids, and so
forth) that might otherwise act to reduce or increase the toxicity of the effluent.
9.1.1 The dilution water must allow satisfactory survival (90 %(80 % or greater in the cultured animals) and reproduction (at least
15 live young/surviving female animals) of C. dubia (see 14.1 d, e, and f). However, acceptable dilution water typically will
produce >90 % survival and >30 live young/surviving female during a 6 day-8 day test using healthy cultures.
9.1.2 The characteristics of the dilution water should be uniform consistent over time so that brood stock is cultured, and the test
conducted, in water of the same characteristics. In tests to evaluate the toxicity of ambient waters, additional controls should be
considered using acceptable quality dilution water (see 9.1.1) with similar chemical characteristics (for example, pH, hardness, and
alkalinity).
9.1.3 The characteristics of the dilution water should be uniformconsistent during the test. The range of hardness during the test
should be less than 5 mg/L or 10 % of the average, whichever is higher. In effluent testing where upstream water is used as dilution
water the variance associated with hardness might naturally exceed these values.ranges.
9.1.4 If it is desired to study the effect of an environmental factor such as total organic carbon,carbon (TOC), particulate matter,
dissolved ion concentrations, or dissolved oxygen on the results of a three-brood test with C. dubia, it will be necessary to use a
water that is naturally or artificially high in TOC or particulate matter or low in dissolved oxygen. or low in that environmental
factor. 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 a more usual dilution water to facilitate interpretation of the results in the special water.
9.2 Source:
9.2.1 If a natural fresh water is used it should be obtained from an uncontaminated source of uniformconsistent characteristics.
A well or spring that has been shown to be of acceptable characteristics is usually preferable to a surface water. If a surface water
is used, the intake should be positioned to minimize fluctuations in characteristics and the possibility of contamination, and to
maximize the concentration of dissolved oxygen to help ensure low concentrations of sulfide and iron. Surface waters should be
filtered (60-μm mesh) to remove potential predators and competitors of C. dubia.
9.2.2 Widespread use of one reconstituted water will increase comparability of test results. The reconstituted fresh water described
in Guide E729 has been used successfully by several people . people. Addition of 5 μg of selenium (11) and 1 μg of crystalline
E1295 − 22
vitamin B /L (12) might be desirable, (but see X1.1). C. dubia has also been cultured and tested in reconstituted soft water.
Acclimation in one reconstituted water and testing in another of different hardness or alkalinity should be avoided to minimize
stress due to routine water quality changes.
9.2.3 Chlorinated water should not be used as, or in the preparation of, dilution water because residual chlorine is quite toxic to
Cladocera (13). Dechlorinated water should be used only as a last resort because dechlorination is often incomplete. Sodium
bisulfite is probably better for dechlorinating water than sodium sulfite, and both are more reliable than carbon filters, especially
for removing chloramines (14). Some organic chloramines, however, react slowly with sodium bisulfite (15). 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. Excessive concentrations of most metals can usually be reduced with a chelating resin (16), but use of an
alternative dilution water might be preferable.
9.3 Treatment:
9.3.1 Dilution water should be aerated intensivelygently by such means as air stones, surface aerators, or column aerators, (17,
18) prior to addition of test material. Adequate aeration will bring the pH and concentrations of dissolved oxygen and other gases
into equilibrium with air and minimize oxygen demand and concentrations of volatiles. The concentration of dissolved oxygen in
dilution water should be between 9090 % and 100 %110 % of saturation (19) to help ensure that dissolved oxygen concentrations
are acceptable in test chambers. Supersaturation by dissolved gases that can be caused by heating the dilution water should be
avoided (20).
9.3.2 Filtration through sand, rock, bag, or depth type cartridge filters may be used to keep the concentration of particulate matter
acceptably low (see 9.2.1), and as a pretreatment before ultraviolet sterilization or filtration through a finer filter.
9.3.3 Dilution water that might be contaminated with facultative pathogens may be passed through a properly maintained
ultraviolet sterilizer (21) equipped with an intensity meter and flow controls, passed through a membrane filter with a pore size
of 0.20 μm, or autoclaved. Water that might be contaminated with Aphanomyces daphniae should be autoclaved (3).
9.4 Characterization—The following items should be measured in the dilution water at least twice each year and more often if
such measurements have not been made semiannually for at least two years, or if a surface water is used: hardness, alkalinity,
conductivity, pH, particulate matter, total dissolved solids, total suspended solids, TOC, selected pesticides (such as those found
in USGS Schedules 2001/2010), organic chlorine, PCBs, phthalate esters, 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. For each method used
(see 13.3), the detection limit should be below either (a) the concentration in the dilution water, or (b) the lowest concentration
that has been shown to affect adversely the survival, and reproduction of C. dubia.
9.5 Control water—Dilution water can be analogous to the laboratory control water treatment to which no test material has been
added. The control provides (a) 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 (b) the basis for interpreting
data obtained from the other treatments.
9.5.1 Additional controls, also referred to as secondary controls, can be useful for distinguishing effects due to a test material from
the water in which samples are tested. Surface water samples or tested aqueous samples can contain ion concentrations or water
quality characteristics that are above or below the tolerance range of C. dubia (22, 23, 24). Including a secondary control that
reflects the sample water quality and ion concentrations can identify if effects to a C. dubia endpoint are due to the test material
or toxicant alone or if effects are also associated with the background water quality of the sample.
10. Test Material
10.1 Before a test is begun with material other than effluents, 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 1 %
of the material.
10.1.2 Solubility and stability in the dilution water.
E1295 − 22
10.1.3 An estimate of the lowest concentration of test material that is acutely toxic to C. dubia.
10.1.4 Accuracy and precision of the analytical method at planned test concentration(s).
10.1.5 Estimate of toxicity to humans and recommended handling procedures (see 8.1).
10.2 Stock Solution:
10.2.1 In some cases the test material can be added directly to dilution water, but usually it is dissolved in a solvent to form a stock
solution that is then added to the dilution water. If a stock solution is used, the concentration and stability of the test material in
it should be determined before the beginning of the test. If the test material is subject to photolysis, the stock solution should be
shielded from light.
10.2.2 Except possibly for tests on hydrolyzable, oxidizable, and reducible materials, the preferred solvent is dilution water,
although filtration or sterilization, or both, of the water might be necessary. If the hardness of the dilution water will not be affected,
distilled and deionized water may be used. Several techniques have been specifically developed for preparing aqueous stock
solutions of slightly soluble materials (2225). Minimum amounts of strong acids or bases may be used in the preparation of
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 more or less than use of the minimum amounts of strong acids and bases.
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
be low enough that it does not affect survival,survival or reproduction of C. dubia (and length or weight, or both, if these
characteristics are to be measured). Because of its low toxicity to aquatic animals (2326), low volatility, and high ability to dissolve
many organic chemicals, triethylene glycol is often a good organic solvent for preparing stock solutions. Other water-miscible
organic solvents such as methanol, ethanol, and acetone may also be used, but they might stimulate undesirable growth of
microorganisms and besides, acetone is quite volatile. If an organic solvent is used, it should be reagent grade or better. A
surfactant should not be used in the preparation of a stock solution because it might affect the form and toxicity of the test material
in test solutions.
10.2.4 If a solvent other than water is used, (a) at least one solvent control, using solvent from the same batch used to make the
stock solution, must be included in the test and (b) a dilution water control, must be included in the test. If no solvent other than
water is used, a dilution water control must be included in the test.
10.2.4.1 If the concentration of solvent is the same in all test solutions that contain test material, the solvent control must contain
the same concentration of solvent.
10.2.4.2 If the concentration of solvent is not the same in all test solutions that contain test material, either (a) a solvent test must
be conducted to determine whether the survival, or reproduction of C. dubia is related to the concentration of the solvent over the
range used in the toxicity test, or (b) such a solvent test must have already been conducted using the same type of dilution water
and the same source of C. dubia. If either survival or reproduction is found to be related to the concentration of solvent, a
three-brood toxicity test with C. dubia in that water is unacceptable if any treatment contained a concentration of solvent in that
range. If neither survival or reproduction is found to be related to the concentration of solvent, a three-brood toxicity test with C.
dubia in that water may contain solvent concentrations within the tested range, but the solvent control must contain the highest
concentration of solvent present in any of the other treatments.
10.2.4.3 If the test contains both a dilution water control and a solvent control, the survival, and reproduction of C. dubia in the
two controls should be compared (see X4.6X4.7 and Guide E1847). If a statistically significant difference in either survival or
reproduction, is detected between the two controls, only the solvent control may be used for meeting the requirements of 14.1 c,
d, and e as the basis for calculation of results. If no statistically significant difference is detected, the data from both controls should
be used for meeting the requirements of 14.1 c, d, and e as the basis for calculation of results. Likewise, a secondary control to
account for background water quality (for example, high or low TOC or dissolved ions) may be appropriate and would function
as does the solvent control described here.
10.2.5 If a solvent other than water is used to prepare a stock solution, 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.
E1295 − 22
10.3 Effluent:
10.3.1 Sampling Point—The effluent sampling point should be based on the purpose of the test. The collection point for the
National Pollutant Discharge Elimination System (NPDES) permit testing purposes is often strictly defined. In some cases, a
sampling point between last treatment and the discharge point might provide much better access. If the waste is chlorinated, it
might be desirable to have sampling points both upstream and downstream of the chlo
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