P R O T O C O L 

 A 48-HOUR STATIC ACUTE TOXICITY TEST 

WITH THE CLADOCERAN (Daphnia magna) 

OECD Guideline 202 

U.S. EPA OPPTS Number 850.1010 
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SPONSOR: 

 

SPONSOR’S REPRESENTATIVE:

TESTING FACILITY: 

STUDY DIRECTOR: 

 

LABORATORY MANAGEMENT:

FOR LABORATORY USE ONLY 

Proposed Dates: 

Experimental

Start Date: ________________________ 

Experimental

Termination Date: _________________ 

Project No.: ________________________ 

Test Concentrations: _________________________________________________________ 

Test Substance No.: ___________ Reference Substance No. (if applicable): ____________ 

PROTOCOL APPROVAL

____________________________________ ___________________________ 

STUDY DIRECTOR DATE 

____________________________________ ___________________________ 

LABORATORY MANAGEMENT DATE 

____________________________________ ___________________________ 

SPONSOR’S REPRESENTATIVE DATE 
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INTRODUCTION 

 will conduct a static acute toxicity test with the cladoceran, Daphnia 

magna, for the Sponsor at the aquatic toxicology facility in Easton, Maryland.

The study will be performed based on procedures in the OECD Guideline for Testing of Chemicals, 202:

Daphnia sp. Acute Immobilization Test and Reproduction Test (1); U.S. EPA Series 850 - Ecological

Effects Test Guidelines OPPTS Number 850.1010: Aquatic Invertebrate Acute Toxicity Test (2); and

ASTM Standard E-729-96, Standard Guide for Conducting Acute Toxicity Tests on Test Materials with

Fishes, Macroinvertebrates and Amphibians(3). Raw data for all work performed at 

and a copy of the final report will be filed by project number in archives located on the 

 site, or at an alternative location to be specified in the final report.

OBJECTIVE 

The objective of this study is to determine the acute effects of a test substance on the cladoceran,

Daphnia magna, under static test conditions for a period of 48 hours.

EXPERIMENTAL DESIGN 

Daphnia magna will be exposed to a geometric series of at least five water accommodated

fractions (WAF) of the test substance and a negative (dilution water) control for 48 hours. Two replicate 

test chambers will be maintained for each treatment and the control group, with 10 Daphnia magna in

each test chamber for a total of 20 Daphnia magna per treatment level. 

 will be selected in consultation with the Sponsor, and will be based upon

information such as the results of exploratory range-finding toxicity data, known toxicity data,

physical/chemical properties of the test substance or other relevant information. For this test, the term

loading rate means the total amount of test substance added to the dilution water volume (mg/L) to achieve

the respective WAF solution. Generally, each test substance loading rate in the definitive test will be at 

least 60% of the next higher loading rate unless information concerning the concentration-effect curve 

indicates that a different dilution factor would be more appropriate. Water samples from each test 

concentration will be collected for analysis of Total Organic Carbon (TOC).
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To control bias, neonate daphnids will be impartially assigned to exposure chambers at test

initiation. No other potential sources of bias are expected to affect the results of the study. Observations

of mortality/immobility and other clinical signs will be made throughout the 48-hour test period.

Cumulative percent mortality/immobilty observed in the treatment groups will be used to calculate, when

possible, EL50 values at 24 and 48 hours. The no-observed-effect level (NOEL) will be determined by

visually interpreting the mortality, immobility and clinical observation data.

MATERIALS AND METHODS 

Test Substance

Information on the characterization of test, control or reference substances is required by Good

Laboratory Practice (GLP) Standards and Principles. The Sponsor is responsible for providing

verification that the test substance has been characterized according to GLPs prior to its 

use in the study. If verification of GLP test substance characterization is not provided to 

 it will be noted in the compliance statement of the final report.

The Sponsor is responsible for all information related to the test substance and agrees to accept 

any unused test substance and/or test substance containers remaining at the end of the study. 

Preparation of Test Loading rates

The test substance will be mixed directly with dilution water on a weight:volume basis. Each

WAF will be prepared individually in a 13.2 L Pyrex® aspirator bottle with tubulation by mixing an

amount of the test substance in approximately 12 L of dilution water using a Teflon®-coated stir bar on a 

magnetic stir plate. Care will be taken to maintain a vortex depth of approximately 30% of the test 

solution height. Following the mixing period, the WAF solutions will then be allowed to settle for 

approximately 1 hour and the test solution will be decanted off the top (or bottom, as appropriate).

Test Organism

The cladoceran, Daphnia magna, has been selected as the test species for this study. Daphnids are

representative of an important group of aquatic invertebrates, and have been selected for use in the test 

based upon past use history and ease of culturing in the laboratory. Daphnid neonatesto be used in the test

will be less than 24 hours old and will be obtained from cultures maintained at
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Easton, Maryland. The identity of the species will be verified by the supplier of the original culture or by

 personnel using appropriate taxonomic keys such as Pennak (4).

Daphnids will be cultured in water from the same source and at approximately the same

temperature as will be used during the test. Daphnids in the cultures producing neonates for the test will 

be held for at least 10 days prior to collection of the neonates for testing. Adult daphnids in the culture

will produce an average of at least 3 young per adult per day over the 7 day period prior to the test.

Neonates from daphnids that show signs of disease or stress will not be used as test organisms. Daphnids

in holding that produce ephippia also will not be used to supply neonates for testing.

Daphnids in the cultures will be fed once daily. The diet will be a mixture of yeast, Cerophyll®, 

and trout chow (YCT), supplemented with a suspension of the freshwater green alga Selenastrum 

capricornutum. Adults are fed during the 24-hour period prior to test initiation, but neonates will not be

fed during the test. Specifications for acceptable levels of contaminants in daphnid diets have not been 

established. However, there are no known levels of contaminants reasonably expected to be present in the

diet that are considered to interfere with the purpose or conduct of the test. 

Neonates will be obtained for testing from at least three individual adults that have already

produced at least one previous brood. Prior to test initiation, the neonates will be collected from cultures 

and transferred to small containers. The daphnids will be released into the test compartments below the

water surface using a wide-bore pipette.

Dilution Water 

The water used for culturing and testing will be obtained from a well approximately 40 meters 

deep located on the The water will be passed through a sand filter and

pumped into a 37,800-L storage tank where the water will be aerated with spray nozzles. Prior to use the

water will be filtered to 0.45 μm in order to remove fine particles, and may be UV-sterilized. Water used

for culturing and testing is characterized as moderately hard. Typical values for hardness, alkalinity, pH

and specific conductance are approximately:

Hardness, mg/L as CaCO3 145 

Alkalinity, mg/L as CaCO3 190 

pH 8.1 
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Specific Conductance, μmhos/cm 330 

Hardness, alkalinity, pH and specific conductance will be measured weekly to monitor the

consistency of the well water. Means and ranges of the measured parameters for the four-week period

preceding the test will be provided in the final report. Analyses will be performed at least once annually to

determine the concentrations of selected organic and inorganic constituents in the water and results of the

most recent analyses will be summarized in the final report.

Test Apparatus 

Test chambers will consist of glass beakers or glass aquaria. Alternatively, test chambers will 

consist of test compartments immersed in glass beakers or glass aquaria. When test compartments are 

used, they will be 300 mL glass beakers with nylon-covered holes on opposite sides of the beaker. The

size of the test chamber and the volume of test solution in each chamber will be sufficient to meet the 

requirements of sampling for analytical verification of test substance concentrations and routine water 

chemistry analyses. Test chambers will be indiscriminately positioned in a temperature-controlled water 

bath or an environmental chamber. The water bath will be enclosed in a plexigass ventilation hood. Test 

chambers will be labelled with the project number, test concentration and replicate.

Environmental Conditions

Lighting used to illuminate the cultures and test chambers during culturing and testing will be

provided by fluorescent tubes that emit wavelengths similar to natural sunlight (e.g., Colortone® 50). A

photoperiod of 16 hours of light and 8 hours of darkness will be controlled with an automatic timer. A 30-

minute transition period of low light intensity will be provided when lights go on and off to avoid sudden 

changes in light intensity. Light intensity will be measured at test initiation with a SPER Scientific Ltd.

light meter or equivalent. 

The target test temperature will be 20 ± 1°C. Temperature will be measured in each test chamber

at the beginning and end of the test, and at 24-hour intervals during the test, using a hand-held

thermometer. Temperature also will be measured with a continuous recorder in a container of water placed

adjacent to the test chambers. Recorder measurements will be verified with a hand-held thermometer prior

to test initiation.
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Dissolved oxygen and pH will be measured in each treatment and control replicate at test initiation

and at approximately 24-hour intervals during the test. In the event that dissolved oxygen levels approach

or fall below 60% saturation, the Sponsor will be notified, and appropriate corrective actions will be taken,

if necessary, to maintain dissolved oxygen levels above 60% saturation. Dissolved oxygen concentrations

will be measured with a Thermo Orion Model 850Aplus dissolved oxygen meter, or equivalent and pH 

will be measured with a Thermo Orion Model 720Aplus pH meter, or equivalent. If a treatment replicate

reaches 100% mortality, dissolved oxygen, pH and temperature measurements will be taken in that 

replicate at that time, and then discontinued.

Hardness, alkalinity, specific conductance and total organic carbon (TOC) will be measured in the 

dilution water at the beginning of the test. Hardness and alkalinity measurements will be made by titration

using procedures based on methods in Standard Methods for the Examination of Water and Wastewater

(5). Specific conductance will be measured using a Yellow Springs Instrument Model 33 SalinityConductivity-Temperature meter, or equivalent. Total organic carbon will be measured on a Shimadzu

Model 5000 TOC analyzer, or equivalent. Additional water quality measurements may be taken as

deemed necessary by study personnel. The reason for the additional measurements will be documented in

the raw data and summarized in the final report.

Biological Observations

Observations of mortality, immobilization and clinical signs of toxicity will be made between 0-24

hours, and at 24 and 48 hours ±1 hour. Immobilization is defined as a lack of movement by the test

organism except for minor activity of the appendages.

Sampling for Analytical Measurements

Water samples will be collected from the WAF preparation vessels for the control and each test 

concentration at the beginning of the test and from pooled (old) solutions from each test concentrations at 

the end of the test. Total organic carbon (TOC) will be measured in each sample using a Shimadzu Model

5000 TOC analyzer, or equivalent. 

Conditions for the Validity of the Test

The following criteria will be used to judge the validity of the test: 
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1) immobility of the daphnids in the negative control group will not exceed 10% by the end of

the test, and 

2) the dissolved oxygen concentration will be at least 60% the air-saturation value throughout

the test.

Data Analysis

When the dose-response pattern allows calculation of an EC50 value, the mortality/immobility

pattern will be used to calculate the 24 and 48-hour EL50 concentrations. The EL50 values will be based

on nominal loading rates and calculated using the computer software of C.E. Stephan (6). The program

was designed to calculate the EL50 value and the 95% confidence interval by probit analysis, the moving

average method, and the binomial probability method (7,8,9). The method used will depend upon the 

immobility pattern and its suitability for each method. Additional analysis of data may be conducted if 

deemed appropriate by the Study Director. The results of the analysis will be documented in the raw data 

and summarized in the final report. A no-observed-effect level (NOEL) will be reported if it is defined by

the concentration-response pattern. The maximum WAF level causing 0% immobility and the minimum

WAF level causing 100% immobility will be presented, if possible.

RECORDS TO BE MAINTAINED 

Recordsto be maintained for data generated by Wildlife International, Ltd. will include, but not be

limited to: 

 1. A copy of the signed protocol. 

 2. Identification and characterization of the test substance, if provided by the Sponsor. 

 3. Dates of initiation and termination of the test. 

 4. History of the test organism, culture and holding records.

 5. Stock solution calculation and preparation.

 6. Daily observations. 

 7. Water chemistry results (e.g., hardness and alkalinity). 

8. If applicable, the methods used to analyze test substance concentrations and the results of 

analytical measurements.

 9. Statistical calculations.

10. Test conditions (light intensity, photoperiod, etc.).
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11. Calculation and preparation of test concentrations. 

12. Copy of final report. 

FINAL REPORT 

A final report of the results of the study will be prepared by The report

will include, but not be limited to the following, when applicable: 

 1. Name and address of the facility performing the study.

2. Dates upon which the study was initiated and completed, and the definitive experimental start and

termination dates. 

3. A statement of compliance signed by the Study Director addressing any exceptions to Good

Laboratory Practice Standards.

4. Objectives and procedures, as stated in the approved protocol, including all changes to the 

protocol. 

5. The test substance identification including name, chemical abstract number or code number,

strength, purity, composition, and other information provided by the Sponsor.

6. Stability and solubility of the test substance under the conditions of administration, if provided by

the Sponsor.

 7. A description of the methods used to conduct the test. 

8. A description of the test organisms, including the source, scientific name, age or life stage and 

feed types.

 9. A description of the preparation of the test solutions. 

10. The methods used to allocate organisms to test chambers and begin the test, the number of

organisms and chambers per treatment, and the duration of the test. 

11. A description of circumstances that may have affected the quality or integrity of the data.

12. The name of the Study Director and the names of other scientists, professionals, and supervisory

personnel involved in the study. 

13. A description of the transformations, calculations, and operations performed on the data, a 

summary and analysis of the biological data and analytical chemistry data, and a statement of the 

conclusions drawn from the analyses. A graph plotting the concentration response curve at 48

hours, when sufficient data exists. If the data is conducive to evaluation by probit analysis, the

slope of the concentration-response curve will be reported. 
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14. Statistical methods used to evaluate the data.

15. The signed and dated reports of each of the individual scientists or other professionalsinvolved in

the study. 

16. The location where raw data and final report are to be stored. 

17. A statement prepared by the Quality Assurance Unit listing the dates that study inspections and

audits were made and the dates of any findings reported to the Study Director and Management. 

18. If it is necessary to make corrections or additions to a final report after it has been accepted, such

changes will be made in the form of an amendment issued by the Study Director. The amendment 

will clearly identify the part of the final report that is being amended and the reasons for the 

amendment, and will be signed by the Study Director.

CHANGES TO PROTOCOL 

Planned changes to the protocol will be in the form of written amendments signed by the Study

Director and approved by the Sponsor’s Representative. Amendments will be considered as part of the 

protocol and will be attached to the final protocol. Any other changes will be in the form of written

deviations signed by the Study Director and filed with the raw data. All changes to the protocol will be

indicated in the final report.

GOOD LABORATORY PRACTICES 

This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40 

CFR Part 160 and/or Part 792); OECD Principles of Good Laboratory Practices (ENV/MC/CHEM (98)

17); and Japan MAFF (11 NohSan, Notification No. 6283, Agricultural Production Bureau, 1 October

1999). Each study conducted by is routinely examined by the

Quality Assurance Unit for compliance with Good Laboratory Practices, Standard

Operating Procedures and the specified protocol. A statement of compliance with Good Laboratory

Practices will be prepared for all portions of the study conducted by The

Sponsor will be responsible for compliance with Good Laboratory Practices for procedures performed by

other laboratories (e.g., residue analyses or pathology). Raw data for all work performed at 

and a copy of the final report will be filed by project number in archives located on the 

 site or at an alternative location to be specified in the final report.
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REFERENCES 

1 Organization for Economic Cooperation and Development. 1984. Guideline 202: Daphnia sp. 

Acute Immobilization Test and Reproduction Test. OECD Guideline for Testing of Chemicals.

Updated Guideline, adopted April, 1984. 

2 U.S. Environmental Protection Agency. 1996. Aquatic Invertebrate Acute Toxicity Test,

Freshwater Daphnids. Series 850- Ecological Effects Test Guidelines (draft), OPPTS Number

850.1010. 

3 ASTM Standard E729-96. 1996. Standard Guide for Conducting Acute Toxicity Tests on Test

Materials with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and

Materials. 

4 Pennak, R.W. 1978. Freshwater Invertebrates of the United States. 2nd Ed. 365 p. 

5 APHA, AWWA, WPCF. 1998. Standard Methods for the Examination of Water and 

Wastewater. 20th Edition, American Public Health Association. American Water Works 

Association. Water Pollution Control Federation, New York.

6 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. 

Personal communication.

7 Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press,

London. 

8 Thompson, W.R. 1947. Bacteriological Reviews. Vol. II, No. 2. Pp. 115-145. 

9 Stephan, C.E. 1977. Methods for Calculating an LC50, Aquatic Toxicology and Hazard

Evaluations. American Society for Testing and Materials. Publication Number STP 634. Pp 65-

84.