P R O T O C O L 

 A 96-HOUR STATIC-RENEWAL ACUTE 

TOXICITY TEST WITH THE FATHEAD MINNOW (Pimephales promelas) 

OECD Guidelines, Section 203 

U.S. EPA OPPTS Number 850.1075 

Annex to Commission Directive 92/69/EEC, Method C.1. 
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 A 96-HOUR STATIC-RENEWAL ACUTE 

TOXICITY TEST WITH THE FATHEAD MINNOW (Pimephales promelas) 

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-renewal acute toxicity test with the fathead

minnow, Pimephales promelas, for the Sponsor at the aquatic toxicology

facility in Easton, Maryland. The study will be performed based on procedures in OECD Guideline for 

Testing of Chemicals 203: Fish, Acute Toxicity Test (1); U.S. EPA Series 850 - Ecological Effects Test

Guidelines OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine (2); U.S.

Environmental Protection Agency, Standard Evaluation Procedure, Acute Toxicity Test for Freshwater 

Fish (3); ASTM Standard E729-96 Standard Guide for Conducting Acute Toxicity Tests on Test Materials

with Fishes, Macroinvertebrates and Amphibians (4) and Official Journal of the European Communities 

No. L383 C.1. Acute Toxicity for Fish (5). Raw data for all work performed at 

and the original 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 fathead

minnow, Pimephales promelas, under static-renewal test conditions. 

EXPERIMENTAL DESIGN 

Fathead minnows 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 96 hours. Two replicate 

test chambers will be maintained in each treatment and control group, with 10 minnows in each chamber 

for a total of 20 minnows per test concentration. WAF solutions and control water will be renewed on Day

2 of the test. At each renewal period, fresh WAF solutions will be prepared and the fish will be transferred

from the old solutions to the new solutions.

WAF loading rates 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 
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indicates that a different dilution factor would be more appropriate. Water samples will be collected at 

specified intervals for analysis of Total Organic Carbon (TOC).

To control bias, fathead minnows will be indiscriminately 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 and other clinical signs will be made throughout the 96-hour test period. Cumulative percent

mortality observed in the treatment groups will be used to calculate, when possible, LL50 values at 24, 48,

72, and 96 hours. The no-mortality level and the no-observed-effect level (NOEL) will be determined by

visually interpreting the mortality 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).
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Test Organism

The fathead minnow (Pimephales promelas) has been selected as the test species for this study.

Fathead minnows are representative of an important group of aquatic vertebrates, and have been selected

for use in the test based upon past use history in the laboratory. Fish will be from the same source and year

class, and the standard length of the longest fish measured will be no more than twice that of the shortest.

Fish will weigh between 0.1 g and 3.0 g each and the total weight in each test chamber will not exceed 0.8

grams fish/L of solution. Total lengths and wet weights of the individual fish in one negative control 

replicate will be measured at the end of the test and will be considered representative of the length and

weight of all fish used in the study. Fish will be obtained from a commercial supplier or hatchery, and the

identity of the species will be verified by the supplier or by personnel using

appropriate taxonomic keys, such as Eddy (6).

Fathead minnows will be held for at least 14 days prior to the test in water from the same source 

and at approximately the same temperature as used during the test. Any changes in water temperature will 

not exceed 3°C per day. Fathead minnows will be held at the test temperature for a minimum of 7 days 

prior to test initiation. Fathead minnows will not be used in the test if they show signs of disease or stress 

or if more than 5% die during the 48 hours prior to the test. At test initiation, the fathead minnows will be

collected from holding or acclimation tanks and transferred to the test chambers.

During the holding period the test fish will be fed at least once daily. The diet will consist of live 

or frozen brine shrimp nauplii (Artemia sp.), and/or commercial food. Fish will not be fed for at least two

days prior to the test or during the test. Specifications for acceptable levels of contaminants in fish 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. 

Dilution Water 

Water used for the holding and testing of fathead minnows will be obtained from a well 

approximately 40 meters deep located on the t 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. Water used for
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holding 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 

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 ofselected organic and inorganic constituents of the well water and results of 

the most recent GLP-compliant analyses will be summarized in the final report.

Test Apparatus 

Test chambers will be Teflon®-lined, 25-L, polyethylene aquaria filled with approximately 15 L 

of water. Alternatively, test chambers may be all glass aquaria filled to appropriate volumes to meet 

loading guidelines. Test chambers will be positioned in an environmental chamber or temperaturecontrolled water bath to maintain a temperature of 22 ±1°C. 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 holding, acclimation, 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 dark 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 22 ± 1°C. Temperature will be measured in all replicates at the

beginning of the test and at approximately 24-hour intervals thereafter (including prior to and after

renewal) using a liquid-in-glass thermometer. Temperature also will be measured with a continuous
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recorder in one negative control replicate. Recorder measurements will be verified with a liquid-in-glass 

thermometer prior to test initiation.

Dissolved oxygen will be measured in all replicates of the treatment and control groups at test 

initiation and at approximately 24-hour intervals thereafter (including prior to and after renewal) using a 

Thermo Orion Model 850Aplus dissolved oxygen meter, or equivalent. 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.

Measurements of pH will be made in all replicates of each treatment and control group at test initiation and

at approximately 24-hour intervals thereafter (including prior to and after renewal) using a Thermo Orion

Model 525Aplus 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, and specific conductance will be measured in the dilution water at test

initiation. Hardness and alkalinity measurements will be made by titration using procedures based on

methods in Standard Methods for the Examination of Water and Wastewater (7). Specific conductance

will be measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter,

or equivalent. Additional water quality measurements may be taken as deemed necessary by study

personnel in consultation with the Study Director. The reason for the additional measurements will be

documented in the raw data and summarized in the final report. 

Biological Measurements

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

24, 48, 72 and 96 hours ± 1 hour. Lethality is defined as the lack of visible movement (e.g. lack of fin or 

operculum movement) in the fish. All clinical observations including abnormal behavior will be noted. 

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 at the beginning of each renewal, and from pooled (old) 
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solutions from each test concentrations at the end of each renewal period. 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: 

1) mortality of the fish 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

The mortality pattern in the definitive multiple concentration test will be used to calculate, if 

possible, the 24, 48, 72 and 96-hour LL50 concentrations. The LL50 values will be based on nominal 

loading rates and calculated using the computer software of C.E. Stephan (8). The program was designed

to calculate the LL50 value and the 95% confidence interval by probit analysis, the moving average 

method, and the binomial probability method (9,10,11). The method used will depend upon the mortality

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 

dose-response pattern. The maximum WAF level causing 0% mortality and the minimum WAF level 

causing 100% mortality will be presented, if possible.

RECORDS TO BE MAINTAINED 

Recordsto be maintained for data generated by 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. Test organism history, holding and acclimation records. 

 5. Results of rangefinding tests, if performed. 

 6. Stock solution calculation and preparation, if applicable.
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 7. Observations. 

 8. Water chemistry results (e.g., alkalinity and conductance). 

9. The methods used to analyze test substance concentrations and the results of analytical 

measurements.

10. Statistical calculations, if applicable.

11. Test conditions (light intensity, photoperiod, etc.).

12. Calculation and preparation of test concentrations. 

13. Copy of final report. 

FINAL REPORT 

A final report of the results of the study will be prepared byWildlife International, Ltd. 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, lengths

and weights of a representative group of test organisms, feed types, light intensity and photoperiod.

 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 
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conclusions drawn from the analyses. A graph of the concentration-mortality curve at the end of 

the test will be provided.

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. Organisation for Economic Cooperation and Development. 1993. OECD Guideline for

Testing of Chemicals. Guideline 203: Fish, Acute Toxicity Test. Adopted by the Council on 12 

July 1992. 

2. U.S. Environmental Protection Agency. 1996. Fish Acute Toxicity Test, Freshwater and 

Marine. Series 850- Ecological Effects Test Guidelines (draft), OPPTS Number 850.1075. 

3. U.S. Environmental Protection Agency. 1985. Standard Evaluation Procedure, Acute Toxicity

Test for Freshwater Fish. Hazard Evaluation Division. Office of Pesticide Programs. EPA540/9-85-006. Washington, DC. 

4. ASTM Standard E729-96. 19964. Standard Guide for Conducting Acute Toxicity Tests on Test

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

Materials. 

5. Official Journal of the European Communities. 1992. No. L383. Method C.1. Acute

Toxicity for Fish. 

6. Eddy, S. 1974. The Freshwater Fishes. Wm. C. Brown Company Publishers, Dubuque, Iowa. 

7. 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.

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

Personal communication.

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

London. 

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

11. 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.