A 96-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST 

WITH THE FATHEAD MINNOW (Pimephales promelas) 

FINAL REPORT 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 
Wildlife International, Ltd. 

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STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS 

No claim of confidentiality is made for any information contained in this study on the basis of its 

falling within the scope of FIFRA section 10(d)(1)(A), (B) or (C). 

 Company: _________________________ (Typed Name) 

 Company Agent: _________________________ (Typed Name) 

 Title: _________________________ 

 Signature: ________________________________ Date: ____________ 
Wildlife International, Ltd. Project Number 523~-104 

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

SPONSOR: Infineurn 

STUDY: A 96-Hour Static-Renewal Acute Toxicity Test with the Fathead Minnow (Pimephales 

promelas) 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 523A- 104 

STUDY COMPLETION: August 25,2005 

This study was conducted in compliance with Good Laboratory Practice Standards as 

published by the U.S. Environmental Protection Agency (40 CFR Parts 160 and 792, 17 August 

1989); OECD Principles of Good Laboratory Practice (ENVIMCKHEM (98) 17); and Japan MAFF 

(1 1 NohSan, Notification No. 6283, Agricultural Production Bureau, 1 October 1999), with the 

following exceptions: 

The characterization of the test substance, and the stability of the test substance under the 

conditions of storage at the test site, were not determined in accordance with Good Laboratory 

Practice Standards. 

Periodic analyses of well water for potential contaminants were performed using a certified 

laboratory and standard U.S. EPA analytical methods. 

STUDY DIRECTOR: 

Senior Biologist 

SPONSOR APPROVAL: 

Sponsor Date 

Submitter Date 
Wildlife International, Ltd. Project Number 523A- 104 

QUALITY ASSURANCE STATEMENT 

This study was examined for compliance with Good Laboratory Practice Standards as 

published by the U.S. Environmental Protection Agency (40 CFR Parts 160 and 792, 17 August 

1989); OECD Principles of Good Laboratory Practice (ENVIMCICHEM (98) 17); and Japan MAFF 

(1 1 NohSan, Notification No. 6283, Agricultural Production Bureau, 1 October 1999). The dates of 

all inspections and audits and the dates that any findings were reported to the Study Director and 

Laboratory Management were as follows: 

DATE REPORTED TO: 

ACTIVITY: DATE CONDUCTED: STUDY DIRECTOR: 

Protocol June 17,2005 June 17,2005 

Test Substance Preparation August 5,2005 August 5,2005 

Observations August 12,2005 August 12,2005 

Data and Draft Report August 18 - 19,2005 August 19,2005 

Final Report August 25,2005 August 25,2005 

All inspections were study-based unless otherwise noted. 

~inda R. Mitchell 

Manager of Regulatory and Ecotox Operations 

MANAGEMENT: 

June 20,2005 

August 9,2005 

August 16,2005 

August 24,2005 

August 25,2005 

Date 
Wildve International, L td. Project Number 523A- 104 

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REPORT APPROVAL 

SPONSOR: Infineurn 

STUDY: A 96-Hour Static-Renewal Acute Toxicity Test with the Fathead Minnow (Pimephales 

promelas) 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 523A- 104 

STUDY DIRECTOR: 

, 

4. 

 usa an J. Palmer, B.S/ 

Senior Biologist ' 

WILDLIFE 

Director 

INTERNATIONAL, 

of Aquatic Toxicology/Terrestrial 

LTD. MANAGEMENT: 

Plants and Insects 

* 
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TABLE OF CONTENTS

Title Page................................................................................................................................................ 1 

Statement of No Data Confidentiality Claims........................................................................................ 2 

Good Laboratory Practice Compliance Statement ................................................................................. 3

Quality Assurance Statement ................................................................................................................. 4 

Report Approval ..................................................................................................................................... 5 

Table of Contents ................................................................................................................................... 6 

Summary ................................................................................................................................................ 8 

Introduction ............................................................................................................................................ 9 

Objective ................................................................................................................................................ 9 

Experimental Design .............................................................................................................................. 9 

Materials and Methods ......................................................................................................................... 10 

 Test Substance......................................................................................................................... 10 

 Test Organism ......................................................................................................................... 10 

 Dilution Water......................................................................................................................... 11 

 Test Apparatus......................................................................................................................... 11 

 Preparation of Test Concentrations ......................................................................................... 12 

 Analytical Sampling................................................................................................................ 12 

 Environmental Conditions....................................................................................................... 12 

 Observations............................................................................................................................ 13 

 Conditions for the Validity of the Test.................................................................................... 13 

 Statistical Analyses.................................................................................................................. 14 

Results and Discussion......................................................................................................................... 14 

 Measurement of TOC in Test Solutions.................................................................................. 14 

 Observations and Measurements............................................................................................. 14 

Conclusions .......................................................................................................................................... 15 

References ............................................................................................................................................ 16 
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TABLE OF CONTENTS (Continued) 

TABLES AND FIGURES 

Table 1. Measurements of Total Organic Carbon in Test Solution Samples.................................. 17 

Table 2. Temperature, Dissolved Oxygen and pH of Water in the Test Chambers........................ 18 

Table 3. Specific Conductance, Hardness and Alkalinity Measured in Dilution Water 

 at Test Initiation................................................................................................................ 20 

Table 4. Cumulative Mortality and Observations........................................................................... 21 

Table 5. LL50 Values ..................................................................................................................... 22 

Figure 1. Concentration-Response Curve (96-Hour Mortality Data)............................................... 23 

APPENDICES

Appendix 1. Specific Conductance, Hardness, Alkalinity and pH of Well Water 

 Measured During the 4-Week Period Immediately Preceding the Test.................. 24 

Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. 

 Well Water .............................................................................................................. 25 

Appendix 3. Changes to Protocol ................................................................................................ 27 

Appendix 4. Personnel Involved in the Study ............................................................................. 28 
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SUMMARY 

SPONSOR: 

 

 

 

 

 

 

 

 

 

 

 
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MATERIALS AND METHODS 

The study was conducted according to the procedures outlined in the protocol, A 96-Hour 

Static-Renewal Acute Toxicity Test with the Fathead Minnow (Pimephales promelas). The protocol 

was based on procedures outlined 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. EPA 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). 

Test Substance 

The test substance was received from UK Ltd. on April 19, 2005. It was assigned 

Wildlife International, Ltd. identification number 7161 upon receipt and was stored under ambient 

conditions. The test substance, a liquid, was identified as Tall Oil Fatty Acid, Lot number 8488/035. 

Test Organism 

The fathead minnow, Pimephales promelas, was selected as the test species for this study. 

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

for use in the test based upon past history of use in the laboratory. Fathead minnows used in the test 

were juveniles obtained from cultures at Wildlife International, Ltd., Easton, Maryland. Identification 

of the species was verified by the supplier of the original culture. 

All fish used in the test were from the same source and year class, and the length of the 

longest fish measured was no more than twice the length of the shortest. The average total length of 

10 negative control fish measured at the end of the test was 3.1 cm, with a range of 2.6 to 3.6 cm. 

The average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 

0.19 grams, with a range of 0.12 to 0.26 grams. Loading was defined as the total wet weight of fish 

per liter of test water and was 0.16 g fish/L. 
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The fathead minnows were held for at least 14 days prior to the test in water from the same 

source as used during the test. The test fish were held at the test temperature for at least seven days 

prior to test initiation, with water temperatures ranging from 21.5 to 21.7°C, measured with a handheld thermometer. During the 14-day holding period prior to the test, the pH of the culture water 

ranged from 8.1 to 8.5, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved 

oxygen ranged from 7.2 to 8.4 mg/L (≥85% of saturation), measured with a Yellow Springs 

Instruments Model 51B dissolved oxygen meter. During the holding period, the fathead minnow 

were fed daily a commercially-prepared diet supplied by Zeigler Brothers, Inc., Gardners, 

Pennsylvania. The fish were not fed for at least two days prior to the test or during the test. 

During the 14-day period prior to the test, the fish used in the test showed no signs of disease 

or stress and no mortalities occurred during the 48-hour period immediately preceding the test. At 

test initiation, fathead minnows were collected from the culture tank and impartially distributed one 

and two at a time to the test chambers until each contained 10 fish. 

Dilution Water 

The water used for culturing and testing was freshwater obtained from a well approximately 

40 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as 

moderately-hard water. The specific conductance, hardness, alkalinity and pH of the well water 

during the four-week period immediately preceding the test are presented in Appendix 1. 

The well water was passed through a sand filter to remove particles greater than 

approximately 25 μm, and pumped into a 37,800-L storage tank where the water was aerated with 

spray nozzles. Prior to use, the water was filtered to 0.45 μm to remove fine particles. The results of 

periodic analyses performed to measure the concentrations of selected organic and inorganic 

constituents in the well water used by Wildlife International, Ltd. are presented in Appendix 2. 

Test Apparatus 

Test chambers were 25-L stainless steel aquaria filled with approximately 11.5 L of test 

water. The depth of the test water in a representative chamber was 14 cm. Test chambers were 

labeled with the project number, test concentration and replicate, and were indiscriminately 

positioned in a temperature-controlled environmental chamber set to maintain the desired test 

temperature. 
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Preparation of Test Concentrations 

Test solutions were prepared prior to test initiation on Day 0. New test solutions were 

prepared in separate test chambers on Day 2, and all surviving test fish were transferred from old to 

new solutions. Individual WAFs were prepared for each test concentration. Nominal WAF 

concentrations were 6.3, 13, 25, 50 and 100 mg/L. The test substance was mixed directly with 

dilution water on a weight:volume basis. Each 12-L WAF was stirred for approximately 24 hours in 

a 13.2 L pyrex aspirator bottle using a Teflon®-lined magnetic stir bar to create a vortex depth of 

approximately 30% of the test solution height. After mixing, the WAFs were allowed to settle for 

approximately one hour. The test solutions were then drawn from spigots placed near the bottom of 

the mixing vessels and added to the appropriate test chamber. 

The test solutions ranged in appearance from slightly cloudy to cloudy white in the test 

chambers, increasing in intensity with increasing test substance concentration. There was visible 

particulate matter suspended in the solutions at the start of each renewal period and on the surface of 

the solutions at the end of each renewal period. 

Analytical Sampling 

Samples of the test solutions were collected at approximately 0, 48 and 96 hours to measure 

concentrations of total organic carbon (TOC). Samples were collected from the WAF preparation 

vessels at test initiation and at 48 hours, and from pooled solution from each test chamber prior to 

renewal at 48 hours and at test termination. The samples were collected from mid-depth into glass 

vials, acidified with 2N HCl, and stored refrigerated, as needed, prior to analysis. TOC was measured 

using a Shimadzu Model TOC-5000 total organic carbon analyzer. 

Environmental Conditions 

Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone® 50) were 

used for illumination of the culture and test chambers. A photoperiod of 16 hours of light and 8 hours 

of darkness was controlled with an automatic timer. A 30-minute transition period of low light 

intensity was provided when lights went on and off to avoid sudden changes in lighting. Light 

intensity at test initiation was 211 lux at the surface of the water of one representative test chamber. 
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The target test temperature during the study was 22 ± 1oC. Temperature was measured in 

each test chamber at the beginning and end of the test and at 24-hour intervals during the test, 

including before and after renewal, using a liquid-in-glass thermometer. Temperature also was 

measured continuously during the test in one negative control test chamber using a Fulscope ER/C 

Recorder, which was verified prior to test initiation with a liquid-in-glass thermometer. Dissolved 

oxygen and pH were measured in each test chamber at the beginning and end of the test and at 24-

hour intervals during the test, including before and after renewal. Gentle aeration was applied to each 

test chamber throughout the exposure period to maintain dissolved oxygen concentrations above 60% 

of saturation. Hardness, alkalinity and specific conductance were measured in the dilution water at 

the beginning of the test. 

Light intensity was measured using a SPER Scientific Model 840006C light meter. 

Measurements of pH were made using a Thermo Orion Model 720Aplus meter, and dissolved oxygen 

was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter. Specific conductance 

was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature 

meter. Hardness and alkalinity measurements were made by titration based on procedures in 

Standard Methods for the Examination of Water and Wastewater (6). 

Observations 

Observations were made periodically to determine the number of mortalities. The numbers 

of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations 

were made approximately 16, 24, 48, 72 and 96 hours after test initiation. 

Conditions for the Validity of the Test 

The following criteria used to judge the validity of the test were met: 

1. mortality of the fish in the negative control group did not exceed 10% by the end of the 

test, and 

2. the dissolved oxygen concentration was at least 60% of the air saturation value throughout 

the test. 
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Statistical Analyses 

The results of the test are based on the nominal WAF loading rates. The mortality data were 

analyzed using the computer program of C. E. Stephan (7). The program was designed to calculate 

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

binomial probability with nonlinear interpolation (8, 9, 10). In this study, the binomial method was 

used to calculate the 24, 48, 72 and 96-hour LL50 values. The no-mortality level and the NOEL were 

determined by visual interpretation of the mortality and observation data. 

RESULTS AND DISCUSSION 

Measurement of TOC in Test Solutions 

Results of analyses for TOC in the test solutions are presented in Table 1. Measurements of 

TOC in the newly prepared WAF solution on Day 0 and Day 2 ranged from <1 to 39.7 mg C/L. 

Measurements of TOC in the 48-hour old solutions collected from the test chambers on Day 2 and 

Day 4 ranged from <1 to 34.1 mg C/L. TOC content increased with increasing test substance 

concentration, and was generally consistent over the test period. 

Observations and Measurements 

Measurements of temperature, dissolved oxygen and pH of the water in the test chambers are 

presented in Table 2. Water temperatures were within the 22 ± 1°C range established for the test. 

Dissolved oxygen concentrations remained ≥6.6 mg/L (≥76% of saturation) throughout the test. 

Measurements of pH ranged from 8.1 to 8.6. The measurements of hardness, alkalinity and specific 

conductance in the dilution water at test initiation were typical of Wildlife International, Ltd. well 

water (Table 3). 

Daily observations of mortality and signs of toxicity observed during the test are presented in 

Table 4. All fathead minnows in the negative control group and in the 6.3, 13 and 25 mg/L treatment 

groups appeared normal throughout the exposure period. Mortality in the 50 and 100 mg/L treatment 

groups at test termination was 20 and 100%, respectively. The no-mortality level and the NOEL were 

25 mg/L. LL50 values at 24, 48, 72 and 96 hours were calculated from the mortality data based on 

the nominal WAF loading rates, and are presented in Table 5. A graph of the concentration-response 

curve is presented in Figure 1. 
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CONCLUSIONS 

Fathead minnows (Pimephales promelas) were exposed for 96 hours under static-renewal 

conditions to five nominal water accommodated fractions (WAF) of ranging 

from 6.3 to 100 mg/L. The 96-hour LL50 value was 62 mg/L, with a 95% confidence interval of 50 

to 100 mg/L. The no-mortality level and the NOEL were both 25 mg/L. 
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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. 

EPA-540/9-85-006. Washington, DC. 

4. 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. 

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

Toxicity for Fish. 

6 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. 

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

Personal communication. 

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

London. 

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

10. 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. 
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Table 1 

Measurements of Total Organic Carbon in Test Solution Samples 

TOC (mg C/L) 1 Nominal WAF 

Loading Rate 

(mg/L) 

0 Hour 

New Solution 

48 Hours 

Old Solution 

48 Hours 

New Solution 

96 Hours 

Old Solution 

Negative Control <1 <1 1.6 1.2 

6.3 2.8 2.7 3.7 3.0 

13 5.6 4.2 5.8 5.1 

25 11.2 7.9 12.2 8.7 

50 20.5 16.1 20.4 14.7 

100 39.7 34.1 -- 2 -- 

1

 The limit of quantitation was 1 mg C/L. 

2

 Measurements were discontinued due to 100% mortality within 24 hours. 
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Table 2 

Temperature, Dissolved Oxygen and pH of Water in the Test Chambers 

0 Hour 1 Nominal WAF 24 Hours 48 

Loading Rate 

(mg/L) Replicate Temp. 2 (oC) DO 3 (mg/L) pH Temp. 2 (oC) DO 3 (mg/L) pH Temp

(oC) 

Negative Control A 21.2 8.4 8.3 22.1 8.4 8.5 22.0 

 B 21.1 8.6 8.3 22.1 8.3 8.5 22.2 

6.3 A 21.2 8.5 8.3 22.2 8.3 8.4 22.2 

 B 21.6 8.5 8.3 22.2 8.2 8.4 22.2 

13 A 21.7 8.2 8.3 22.1 7.9 8.4 22.0 

 B 21.7 8.2 8.3 22.1 7.8 8.4 22.1 

25 A 22.1 8.2 8.3 22.1 8.0 8.4 22.0 

 B 22.6 7.9 8.3 22.1 8.1 8.4 22.1 

50 A 22.4 7.9 8.3 22.1 8.1 8.4 22.1 

 B 22.4 7.9 8.3 22.1 8.0 8.4 22.2 

100 A 22.5 8.0 8.3 22.1 6.6 8.1 -- 4 

 B 22.1 8.1 8.3 22.1 7.4 8.3 -- 4 

1 Gentle aeration was maintained in each test chamber throughout the 96-hour exposure period. 2 Manual temperature measurements. Temperature measured continuously during the test ranged from approximately 21.0 to 22.5°C, mea3 A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22°C in freshwater. 4 Measurements were discontinued after 24 hours due to 100% mortality. 
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Table 2 (Continued) 

Temperature, Dissolved Oxygen and pH of Water in the Test Chambers 

48 Hours (Following Renewal) 1 Nominal WAF 72 Hours 

Loading Rate 

(mg/L) Replicate Temp. 2 (oC) DO 3 (mg/L) pH Temp. 2 (oC) DO 3 (mg/L) pH Temp

(oC) 

Negative Control A 21.3 8.6 8.3 22.1 8.5 8.6 22.1 

 B 21.3 8.6 8.3 22.2 8.3 8.5 22.2 

6.3 A 21.4 8.2 8.3 22.2 8.3 8.5 22.2 

 B 21.9 8.1 8.3 22.2 8.2 8.5 22.2 

13 A 22.1 8.0 8.3 22.1 7.8 8.4 22.1 

 B 22.0 8.1 8.3 22.1 7.7 8.4 22.1 

25 A 22.3 8.0 8.3 22.0 7.6 8.4 22.1 

 B 22.2 8.1 8.3 22.1 7.8 8.4 22.2 

50 A 22.1 8.1 8.3 22.1 7.9 8.5 22.2 

 B 21.9 8.4 8.4 22.1 6.7 8.4 22.2 

100 A -- 4 -- 4 -- 4 -- -- -- -- 

 B -- 4 -- 4 -- 4 -- -- -- -- 

1 Gentle aeration was maintained in each test chamber throughout the 96-hour exposure period. 2 Manual temperature measurements. Temperature measured continuously during the test ranged from approximately 21.0 to 22.5°C, mea3 A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22°C in freshwater. 4 Measurements were discontinued after 24 hours due to 100% mortality. 
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Table 3 

Specific Conductance, Hardness and Alkalinity 

Measured in Dilution Water at Test Initiation 

Parameter Day 0 

 

Specific Conductance 315 

 (μmhos/cm) 

 

Hardness 132 

 (mg/L as CaCO3) 

 

Alkalinity 178 

 (mg/L as CaCO3) 
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Table 5 

LL50 Values 

Time 

LL50 1

(mg/L) 

95% Confidence Interval 

(mg/L) 

Statistical 

Method 

24 Hours 64 50 – 100 Binomial Probability 

48 Hours 64 50 – 100 Binomial Probability 

72 Hours 62 50 – 100 Binomial Probability 

96 Hours 62 50 – 100 Binomial Probability 

1

 Based on nominal WAF loading rates. 
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Figure 1 

Concentration-Response Curve (96-Hour Mortality Data) 

Concentration (mg/L)

10 100

Mortality (Probits)

1

2

3

4

5

6

7

8

9
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Appendix 1 

Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured 

During the 4-Week Period Immediately Preceding the Test 

Parameter Mean Range 

 

Specific Conductance 313 300 – 320 

 (μmhos/cm) (N = 4) 

Hardness 136 132 – 140 

 (mg/L as CaCO3) (N = 4) 

Alkalinity 181 178 – 184 

 (mg/L as CaCO3) (N = 4) 

pH 8.3 8.2 – 8.4 

 (N = 4) 
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Appendix 2 

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 

Pesticides and Organics 

Component 

Measured Concentration 

(μg/L) Component 

Measured Concentration 

(μg/L) 

 

Aldrin < 0.0099 Heptachlor Epoxide < 0.0099 

Alpha BHC < 0.0099 Malathion < 2.0 

Beta BHC < 0.040 Merphos < 2.0 

Bolstar < 2.0 Methoxychlor < 0.099 

Chlordane < 0.50 Methyl Parathion < 2.0 

Coumaphos < 3.0 Mevinphos < 2.0 

Delta BHC < 0.0099 Mirex < 0.050 

Demeton-O < 2.0 Naled < 3.0 

Demeton-S < 2.0 o,p-DDD < 0.020 

Diazinon < 2.0 o,p-DDE < 0.020 

Dichlorvos < 2.0 o,p-DDT < 0.020 

Dieldrin < 0.020 p,p-DDD < 0.020 

Disulfoton < 2.0 p,p-DDE < 0.020 

Dursban (Chlorpyrifos) < 2.0 p,p-DDT < 0.025 

Endosulfan I < 0.0099 PCB-1016 < 0.50 

Endosulfan II < 0.042 PCB-1221 < 1.2 

Endosulfan Sulfate < 0.020 PCB-1232 < 0.89 

Endrin < 0.020 PCB-1242 < 0.50 

EPN < 4.0 PCB-1248 < 0.50 

Ethion < 2.0 PCB-1254 < 0.50 

Ethoprop < 2.0 PCB-1260 < 0.50 

Ethyl Parathion < 2.0 Phorate < 2.0 

Famphur < 2.0 Ronnel < 2.0 

Fensulfothion < 4.0 Stirophos < 2.0 

Fenthion < 2.0 Telodrin < 0.0099 

Gamma BHC – Lindane < 0.0099 Tokuthion < 2.0 

Guthion (Azinphos-methyl) < 4.0 Toxaphene < 0.99 

HCB < 0.099 Trichloronate < 2.0 

Heptachlor < 0.0099 Trithion < 2.0 

 

1

Analyses performed by Lancaster Laboratories on samples collected on December 22, 2004. 
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Appendix 2 (Continued) 

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 

Metals 

Component 

Measured Concentration 

(mg/L) Component 

Measured Concentration 

(mg/L) 

 

Aluminum < 0.200 Magnesium 12.7 

Antimony < 0.0200 Manganese < 0.0050 

Arsenic < 0.0100 Mercury < 0.00020 

Barium < 0.0050 Nickel < 0.0100 

Beryllium < 0.0050 Nitrate Nitrogen < 0.50 

Bromide < 2.5 Nitrite Nitrogen < 0.50 

Cadmium < 0.0050 Potassium 6.64 

Calcium 31.1 Selenium < 0.0100 

Chloride 6.9 Silver < 0.0050 

Chromium < 0.0050 Sodium 19.7 

Cobalt < 0.0050 Sulfate 5.5 

Copper < 0.0100 Thallium < 0.0200 

Fluoride < 0.50 Vanadium < 0.0050 

Iron < 0.200 Zinc < 0.0200 

Lead < 0.0200 

1

Analyses performed by Lancaster Laboratories on samples collected on December 22, 2004. 
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Appendix 3 

Changes to Protocol 

This study was conducted in accordance with the approved protocol with the following changes: 

1. The protocol was amended to add gentle aeration to the test chambers throughout the 96-hour 

exposure period. 

2. Test chambers used in the test were 25-L stainless steel aquaria containing 11.5 L of test 

solution, rather than the Teflon-lined polyethylene or glass aquaria specified in the protocol. 

Since stainless steel aquaria are interchangeable with polyethylene or glass for this type of test, 

this had no adverse impact on the study. 

3. Fathead minnows used in the test were obtained from Wildlife International, Ltd. cultures, rather 

than from a commercial supplier. Twelve days prior to the test, the change in culture water 

temperature slightly exceeded 3oC in one day (25.4 to 21.8oC) when the fish were transferred to 

a new culture unit. Since the fish were gradually adjusted to the new temperature, and no signs 

of stress were observed after the transfer, this had no adverse impact on the study. 
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