A 96-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST WITH THE FATHEAD MINNOW ( Pimephales promelas )

FINAL REPORT

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER:

# W ildlife International, Ltd.

- 2 - Project Number

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any inform ation 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

-5-



### 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 INTERNATIONAL, LTD. MANAGEMENT: 

# *

Director of Aquatic Toxicology/Terrestrial Plants and Insects

# W ildlife International, Ltd.

- 6 - Project Number

TABLE OF CONTENTS

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

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

Good Laboratory Practice Co mpliance 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

Refere nces ..................................................................................................................... ....................... 16

# W ildlife International, Ltd.

- 7 - Project Number

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

# W ildlife International, Ltd.

- 8 - Project Number

SUMMARY

SPONSOR:

# W ildlife International, Ltd.

- 9 - Project Number

# W ildlife International, Ltd.

- 10 - Project Number

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 num ber 7161 upon receipt and was stored under ambient

conditions. The test substance, a liquid, was id entified 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 Inte rnational, 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 sam e source and year class, and the length of the

longest fish measured was no more than twice the le ngth 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. Lo ading was defined as the total wet weight of fish

per liter of test water and was 0.16 g fish/L.

# W ildlife International, Ltd.

- 11 - Project Number

The fathead minnows were held for at least 14 da ys prior to the test in water from the same
source as used during the test. The test fish were he ld 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 Sc ientific 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 s upplied by Zeigler Brot hers, 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 fi sh 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 μ m, 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 con centrations of selected organic and inorganic
constituents in the well water used by Wildlife In ternational, 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 repr esentative chamber was 14 cm. Test chambers were
labeled with the project number, test concentr ation and replicate, and were indiscriminately
positioned in a temperature-controlled environmenta l chamber set to maintain the desired test

temperature.

# W ildlife International, Ltd.

- 12 - Project Number

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 a ll 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. Af ter 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 s lightly 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 th e 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 refrigera ted, 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 cham bers. 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 o ff 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.

# W ildlife International, Ltd.

- 13 - Project Number

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 liqui d-in-glass thermometer. Temperature also was

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

Recorder, which was verified prior to test initiati on 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 af ter renewal. Gentle aeration was applied to each

test chamber throughout the exposure period to main tain 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 Instrume nt Model 33 Salinity-Conductivity-Temperature

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

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

Observations

Observations were made periodically to determ ine 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.

# W ildlife International, Ltd.

- 14 - Project Number 4

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 va lues. 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 soluti ons 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 c ontent increased with increasing test substance

concentration, and was generally consistent over the test period.

Observations and Measurements

Measurements of temperature, dissolved oxygen a nd 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 we re typical of Wildlife International, Ltd. well

water (Table 3).

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

Table 4. All fathead minnows in the negative cont rol 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-mo rtality level and the NOEL were

25 mg/L. LL50 values at 24, 48, 72 and 96 hours we re 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.

# W ildlife International, Ltd.

- 15 - Project Number

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.

# W ildlife International, Ltd.

- 16 - Project Number

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.

# W ildlife International, Ltd.

- 17 - Project Number

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.Wildlife International, Ltd.Project Number- 18 -Table 2Temperature, Dissolved Oxygen and pH of Water in the Test Chambers0 Hour124 Hours 48 Hours (Prior to Renewal)Nominal WAFLoading Rate(mg/L)ReplicateTemp.2(oC)DO3(mg/L)pHTemp.2(oC)DO3(mg/L)pHTemp.2(oC)DO3(mg/L)pHNegative Control A 21.2 8.4 8.3 22.1 8.4 8.5 22.0 8.4 8.6B 21.1 8.6 8.3 22.1 8.3 8.5 22.2 8.1 8.56.3 A 21.2 8.5 8.3 22.2 8.3 8.4 22.2 8.2 8.5B 21.6 8.5 8.3 22.2 8.2 8.4 22.2 8.1 8.513 A 21.7 8.2 8.3 22.1 7.9 8.4 22.0 7.9 8.5B 21.7 8.2 8.3 22.1 7.8 8.4 22.1 8.0 8.525 A 22.1 8.2 8.3 22.1 8.0 8.4 22.0 7.6 8.4B 22.6 7.9 8.3 22.1 8.1 8.4 22.1 7.8 8.550 A 22.4 7.9 8.3 22.1 8.1 8.4 22.1 7.7 8.5B 22.4 7.9 8.3 22.1 8.0 8.4 22.2 7.8 8.5100 A 22.5 8.0 8.3 22.1 6.6 8.1 --4--4--4B 22.1 8.1 8.3 22.1 7.4 8.3 --4--4--41Gentle aeration was maintained in each testchamber throughout the 96-hour exposure period.2Manual temperature measurements. Temperature measured continuously during the test ranged from approximately 21.0 to 22.5°C,measured to the nearest 0.5°C.3A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22°C in freshwater.4Measurements were discontinuedafter 24 hours due to 100% mortality.Wildlife International, Ltd.Project Number- 19 -Table 2 (Continued)Temperature, Dissolved Oxygen and pH of Water in the Test Chambers48 Hours (Following Renewal)172 Hours 96 HoursNominal WAFLoading Rate(mg/L)ReplicateTemp.2(oC)DO3(mg/L)pHTemp.2(oC)DO3(mg/L)pHTemp.2(oC)DO3(mg/L)pHNegative Control A 21.3 8.6 8.3 22.1 8.5 8.6 22.1 8.6 8.6B 21.3 8.6 8.3 22.2 8.3 8.5 22.2 8.4 8.56.3 A 21.4 8.2 8.3 22.2 8.3 8.5 22.2 8.3 8.5B 21.9 8.1 8.3 22.2 8.2 8.5 22.2 8.2 8.513 A 22.1 8.0 8.3 22.1 7.8 8.4 22.1 7.9 8.4B 22.0 8.1 8.3 22.1 7.7 8.4 22.1 7.9 8.525 A 22.3 8.0 8.3 22.0 7.6 8.4 22.1 7.5 8.4B 22.2 8.1 8.3 22.1 7.8 8.4 22.2 7.7 8.450 A 22.1 8.1 8.3 22.1 7.9 8.5 22.2 7.6 8.4B 21.9 8.4 8.4 22.1 6.7 8.4 22.2 7.7 8.5100 A --4--4--4-- -- -- -- -- --B --4--4--4-- -- -- -- -- --1Gentle aeration was maintained in each testchamber throughout the 96-hour exposure period.2Manual temperature measurements. Temperature measured continuously during the test ranged from approximately 21.0 to 22.5°C,measured to the nearest 0.5°C.3A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22°C in freshwater.4Measurements were discontinuedafter 24 hours due to 100% mortality.

# W ildlife International, Ltd.

- 20 - Project Number

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 CaCO 3 )

Alkalinity 178 (mg/L as CaCO 3 )Wildlife International, Ltd.Project Number

# W ildlife International, Ltd.

- 22 - Project Number

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.

# W ildlife International, Ltd.

- 23 - Project Number

Figure 1

Concentration-Response Curve (96-Hour Mortality Data)

Concentration (mg/L) 10 100Mortality (Probits)

1 2 3 4 5 6 7 8 9

# W ildlife International, Ltd.

- 24 - Project Number

Appendix 1

Specific Conductance, Hardness, Alkalin ity 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 CaCO 3 ) (N = 4)

Alkalinity 181 178 – 184 (mg/L as CaCO 3 ) (N = 4)

pH 8.3 8.2 – 8.4 (N = 4)

# W ildlife International, Ltd.

- 25 - Project Number

Appendix 2

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water 1

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 Laborator ies on samples collected on December 22, 2004.

# W ildlife International, Ltd.

- 26 - Project Number

Appendix 2 (Continued)

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water 1

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 Laborator ies on samples collected on December 22, 2004.

# W ildlife International, Ltd.

- 27 - Project Number

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 aer ation 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 polyethylen e or glass aquaria specified in the protocol. Since stainless steel aquaria are interchangeable w ith 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 pr ior 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.

# W ildlife International, Ltd.

- 28 - Project Number