Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): A 48-HOUR STATIC ACUTE TOXICITY TEST

WITH THE CLADOCERAN ( Daphnia magna )

FINAL REPORT

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527A-102

OECD GUIDELINE 202 U.S. EPA OPPTS NUMBER 850.1010

AUTHORS:

Susan J. Palmer, B.S. Mary Beth Claude, B.S. Henry O. Krueger, Ph.D.

STUDY INITIATION DATE: October 30, 2009

STUDY COMPLETION DATE: January 12, 2010

SUBMITTED TO:

Sasol Germany GmbH Building 1033/PB 02 Paul-Baumann-Str. 1 D-45764 Marl Germany 

# Wildlife International, Ltd.

8598 Commerce Drive Easton, Maryland 21601 USA 1-410-822-8600

Page 1 of 24

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STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any informa tion 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: ____________

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GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

SPONSOR: Sasol Germany GmbH

TITLE: Alcohols, CI8-22, distn. residues (CAS No. 1160164-88-4): A 48-Hour Static Acute Toxicity Test with the Cladoceran (Daphnia magna)

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527A-102

STUDY COMPLETION: January 12, 2010

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

### V.S. Environmental Protection Agency (40 CFR Parts 160 and 792, 17 August 1989); OECD Principles of Good Laboratory Practice (ENVIMC/CHEM (98) 17); and Japan MAFF (11 NohSan, Notification No. 6283, Agricultural Production Bureau, I October 1999), with the following exceptions:

Periodic analyses of water for potential contaminants were not performed according to Good Laboratory Practice Standards, but were performed using a certified laboratory and standard 

### VS EPA analytical methods.

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.

STUDY DIRECTOR:

SPONSOR APPROVAL:

Sponsor Representative Date

ApplicantlSubmitter Date

# Wildlife International, Ltd. Project Number 

### 527A-I02

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QUALITY ASSURANCE STATEMENT

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

### V.S. Environmental Protection Agency (40 CFR Parts 160 and 792, 17 August 1989); OECD Principles of Good Laboratory Practice 

### (ENVIMC/CHEM (98) 17); and Japan MAFF (11 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: MANAGEMENT: Protocol October 12, 2009 October 12, 2009 October 14, 2009

Test Substance Preparation November 2, 2009 November 2, 2009 November 4, 2009

Obsevations/Temperature Measurements November 5, 2009 November 5, 2009 November 9, 2009

Biological Data and Draft Report December 22, 2009 December 22, 2009 Januaty 4, 2010

Final Report Januaty 12,2010 January 12, 2010 Januaty 12,2010

All inspections were study-based unless otherwise noted.

Linda R. Mitchell Date Director of Regulatory and Ecotox Operations

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### REPORT APPROVAL

SPONSOR: Sasol Gennany GmbH

TITLE: Alcohols, CIS-22, distil. residues (CAS No. II60164-SS-4): A 4S-Hour Static Acute Toxicity Test with the Cladoceran (Daphnia magna)

WILDLIFE INTERNATIONAL, LID. PROJECT NUMBER: 527A-102

Senior Biologist

PRINCIPAL BIOLOGIST



### Mary Beth Claude, B.S. Biologist

O. Krueger, Ph.D. Director of Aquatic, Plant and Insect Toxicology 

# I /;)/10

Date 

### 1 }

Date / 

### J

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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 Statistical Analyses.......................................................................................................... ........ 13

Results and Discussion......................................................................................................... ................ 13 Measurement of TOC in Test Solutions.................................................................................. 13 Observations and Measurements............................................................................................. 14

Conclusions .................................................................................................................... ...................... 14

Refere nces ..................................................................................................................... ....................... 15

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TABLE OF CONTENTS (Continued)

TABLES

Table 1 . Measurements of Total Organic Carbon in Test Solution Samples.................................. 16

Table 2. Temperature, Dissolved Oxygen and pH of Water in the Test Chambers........................ 17

Table 3. Specific Conductance, Hardness and Alkalinity Measured in Dilution Water at Test Initiation .................................................................. 18

Table 4 . Cumulative Immobility and Observations ........................................................................ 19

Table 5 . EL50 Values .................................................................................................................. ... 20

APPENDICES

Appendix 1. Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test.................. 21

Appendix 2. Analyses of Pesticides, Organics and Me tals in Wildlife International, Ltd. Well Water .............................................................................................................. 22

Appendix 3. Personnel Involved in the Study ............................................................................. 24

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SUMMARY

SPONSOR: Sasol Germany GmbH

TITLE: Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): A 48-Hour Static Acute Toxicity Test with the Cladoceran ( Daphnia magna )

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527A-102

TEST DATES: Experimental Start (OECD): November 2, 2009 Experimental Start (EPA): November 4, 2009 Biological Termination: November 6, 2009 Experimental Termination: November 11, 2009

LENGTH OF EXPOSURE: 48 Hours

TEST ORGANISMS: Cladoceran ( Daphnia magna )

SOURCE OF TEST ORGANISMS: Wildlife International, Ltd. Cultures Easton, Maryland 21601

AGE OF TEST ORGANISMS: Neonates <24 hours at test start

WAF LOADING RATE: Nominal Negative Control 100 mg/L

RESULTS: Based on nominal WAF loading rates: 48-Hour EL50: >100 mg/L No-Immobility Level: 100 mg/L 48-Hour NOEL: 100 mg/L

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INTRODUCTION This study was conducted by Wildlife Internati onal, Ltd. for Sasol Germany GmbH at the Wildlife International, Ltd. aquatic toxicology fac ility in Easton, Maryland. The in-life phase of the definitive toxicity test was conducted from Novemb er 4, 2009 to November 6, 2009. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number

527A-102 in archives located on the Wi ldlife International, Ltd. site.

OBJECTIVE The objective of this study was to determine the acute effects of the test substance (Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4)) on the cladoceran, Daphnia magna , during a 48- hour exposure period under static test conditions.

EXPERIMENTAL DESIGN Daphnids were exposed to a single limit concentr ation of test solution prepared using water accommodated fractions (WAF) of the test substan ce and a negative control (dilution water) for 48 hours under static conditions. Three replicate test chambers were maintained in each treatment and control group, with 10 daphnids in each test cham ber for a total of 30 daphnids per concentration. The nominal WAF loading rate was selected in c onsultation with the Sponsor based on information provided by the sponsor. For this test, the term lo ading rate means the total amount of test substance added to the dilution water volume (mg/L) to ach ieve the respective WAF solution. The nominal WAF loading rate selected was 100 mg test substance/L. Samples of test water were collected at test initiation and termination for analys is of total organic carbon (TOC).

Daphnids were impartially assigned to test cham bers at test initiation. Observations of immobility and other signs of toxicity were made approximately 4, 24 and 48 hours after test initiation. Cumulative percent immobility observed in the treatment groups was used to determine EL50 values (i.e., the theoretical WAF loading rate that produces 50% immobility) at 24 and 48 hours. The no-immobility level and the no-obser ved-effect level (NOEL) were determined by visual interpretation of the immobility and observation data.

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MATERIALS AND METHODS The study was conducted according to the pro cedures outlined in the protocol, “Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): A 48-Hour Static Acute Toxicity Test with the Cladoceran ( Daphnia magna )”. The protocol was based on procedures outlined in the OECD Guidelines for Testing of Chemicals, Guideline 202: Daphnia sp., Acute Immobilization Test (1); U.S. EPA Series 850 – Ecological Effects Test Guidelines (draft), OPPTS Number 850.1010: Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids (2); and ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates and Amphibians (3).

Test Substance

The test substance was received from Sasol Germany GmbH on September 11, 2009. It was assigned Wildlife International, Ltd. identificati on number 9187 upon receipt and was stored under ambient conditions. The test substance, a solid, w as identified as: Alcohols, C18-22, distn. Residues; Lot number 03585/MA, Spec: 59B1RN2; CAS numbe r 1160164-88-4. The test substance had a reported purity of 100% and an e xpiration date of August 2013.

Test Organism The cladoceran, Daphnia magna , was selected as the test species for this study. Daphnids are representative of an important group of aquatic inve rtebrates and were selected for use in the test based upon past history of use and ease of culturing in the laboratory. Daphnid neonates used in the

test were less than 24 hours old and were obtained from cultures maintained by Wildlife International, Ltd., Easton, Maryland.

Adult daphnids were cultured in water from th e same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 19.5 to 20. 6oC, measured with a hand-held liquid-in-glass thermometer. The pH of the water ranged from 8.3 to 8.6, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved ox ygen ranged from 7.6 to 9.2 mg/L ( ≥ 84% of saturation), measured with a Yellow Springs Instruments Model 85 dissolved oxygen meter. Daphnids in the cultures were fed daily a mixture of yeast, cereal grass media and trout chow (YCT), as well as a suspension of the

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freshwater green alga, Pseudokirchneriella subcapitata . The adults were fed prior to test initiation, but neonates were not fed during the test.

The three adult daphnids used to supply neonates for the test were held for at least 14 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. At test initiation, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 10 daphnids. E ach group of daphnids then was transferred to an indiscriminately assigned test chamber. All transfers were made below the water surface using wide-bore pipettes.

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 wa ter 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 μ m to remove fine particles and was passed through an ultraviolet (UV) sterilizer.

The well water is characterized as moderate ly-hard water. The specific conductance, hardness, alkalinity and pH of the well water dur ing the four-week period immediately preceding the test as well as the monthly total organic carbon of the well water are presented in Appendix 1. The results of periodic analyses performed to measur e the concentrations of selected organic and inorganic constituents in the well water are presented in Appendix 2.

Test Apparatus Test chambers were 250-mL glass beakers contai ning approximately 200 mL of test water. The depth of the test water in a representative ch amber was 5.5 cm. Each test chamber was labeled with the project number, test concentration and repl icate. Test chambers were impartially positioned

in an environmental chamber set to ma intain the desired test temperature.

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Preparation of Test Concentrations The test solution was prepared in the form of water accommodated fractions (WAF) at a nominal loading rate of 100 mg/L. Each WAF was pr epared by weighing the test substance in a tared beaker and rinsing it with dilution water ( UV sterilized well water) into a 13.2-L Pyrex ® aspirator

bottle to a final volume of 12 L. Each solution was stirred with a Teflon ® -coated stir bar on a magnetic stir plate for approximately 48 hours. Care was taken to maintain a vortex depth of approximately 30% of the test solution height. Following the mixing period, each WAF solution was allowed to settle for approximately one hour befo re the solution was decanted from mid-depth into the test chambers through a spigot and tubing placed approximately 2-3 cm from the bottom of the aspirator bottle. The negative control solution w as prepared using the same procedures, without

addition of test substance.

Analytical Sampling Duplicate samples were collected from each test concentration and control at the beginning of

the test and at 48 hours to measure concentrations of total organic carbon (TOC). The samples were collected from mid-depth into glass vials, acidified with 2N HCl, and stored under refrigeration prior to analysis. TOC was measured using a Shimadzu Model TOC-VCSH total organic carbon analyzer.

Environmental Conditions Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of dark ness. 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 was measured at the water surface of one representative test chamber at test initiation using

a SPER Scientific Model 840006C light meter.

The target test temperature during the study was 20 ± 1oC. Temperature was measured in each test chamber at the beginning and end of the test and at approximately 24 hours during the test using a liquid-in-glass thermometer. Temperature also was monitored continuously during the test in a container of water placed adjacent to the test chambers, using a Fulscope ER/C Recorder. The recorder was verified prior to test in itiation with a liquid-in-glass thermometer.

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Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at approximately 24 hours during th e test. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter, and measurements of pH were made using a Thermo Orion Model 525Aplus meter.

Hardness, alkalinity and specific conductance in th e dilution water at the beginning of the test

were measured. Hardness and alka linity measurements were made by titration based on procedures in Standard Methods for the Examination of Water and Wastewater (4). Specific conductance was measured using an Acorn Series Model C ON6 Conductivity-Temperature meter.

Observations Observations were made periodically to determ ine the numbers of immobile organisms in each treatment group. Immobility was defined as a lack of movement by the organism except for minor activity of the appendages. The numbers of individuals exhibiting signs of toxicity or

abnormal behavior also were evaluated. Observati ons were made approximate ly 4, 24 and 48 hours

after test initiation.

Statistical Analyses Because this is a limit test, the EL50 value cannot be statistically defined. However, based upon the biological response obtained in the exposur e group, the EL50 value was estimated to be greater than the limit loading rate. The no-immob ility level and the no-observed-effect level (NOEL) were determined by visual interpretati on of the immobility and observation data.

RESULTS AND DISCUSSION

Measurement of TOC in Test Solutions The nominal WAF loading rate selected for use in this study was 100 mg/L. Test solutions in the test chambers at this nominal WAF loading rate appeared clear and colorless during the test with white particles suspended throughout the solutions. Results of analy ses for TOC in the test solutions are presented in Table 1. Measurements of TOC in th e newly prepared test solutions on Day 0 and in the 48-hour old solutions on Day 2 were all be low the limit of quantitation (LOQ = 1 mg C/L).

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Observations and Measurements Measurements of temperature, dissolved oxygen a nd pH of the water in each test chamber are presented in Table 2. Water temperatures were within the 20 ± 1 ° C range established for the test. Dissolved oxygen concentrations remained ≥ 7.9 mg/L ( ≥ 88% of saturation) throughout the test. Measurements of pH ranged from 8.2 to 8.5. Th e measurements of hardness, alkalinity and specific

conductance in the dilution water at test initiation were typical of Wildlife International, Ltd. well water (Table 3). Light intensity at test initia tion was 305 lux at the surface of the water of one representative test chamber.

Daily observations for immobility and signs of toxicity during the test are presented in Table 4. Daphnia in the negative control group app eared normal throughout the test. All daphnids in the 100 mg/L treatment group also appeared normal throughout the test, with no immobility or overt signs of toxicity observed. The no-immobility level and the NOEL were both 100 mg/L. EL50

values at 24 and 48 hours were determined from th e immobility data and were >100 mg/L (Table 5).

CONCLUSIONS The cladoceran, Daphnia magna , was exposed for 48 hours under static conditions to a single limit water accommodated fraction (WAF) of Alcohols, C18-22, distn. residues (CAS No. 1160164- 88-4) at 100 mg/L. The 48-hour EL50 value was >100 mg/L. Daphnids exposed to Alcohols, C18- 22, distn. residues (CAS No. 1160164-88-4) at the nominal WAF loading rate of 100 mg/L appeared normal with no immobility or overt signs of t oxicity. The 48-hour no-immobility level and the NOEL were both 100 mg/L.

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REFERENCES

1 Organisation for Economic C ooperation and Development. 2004. OECD Guidelines for Testing of Chemicals, Guideline 202: Daphnia sp., Acute Immobilization Test . Adopted 13 April 2004.

2 U.S. Environmental Protection Agency. 1996. Series 850 – Ecological Effects Test Guidelines ( draft ), OPPTS Number 850.1010: Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids

.

3 American Society for Testing and Materials. 2007. ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fish es, Macroinvertebrates, and Amphibians

.

4 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.

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Table 1

Measurements of Total Organic Carbon in Test Solution Samples

Nominal WAF Loading Rate (mg/L) Sample Number (527A-102-) Sampling Interval

(Day) Total Organic Carbon (mg C/L) 1

Negative Control TOC-1 0 <LOQ TOC-2 0 <LOQ TOC-5 2 <LOQ TOC-6 2 <LOQ

100 TOC-3 0 <LOQ

TOC-4 0 <LOQ TOC-7 2 <LOQ TOC-8 2 <LOQ

1 LOQ = 1 mg C/L

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Table 2

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

0 Hour 24 Hours 48 Hours Nominal

WAF Loading Rate (mg/L) Replicate Temp. 1 (°C) DO 2 (mg/L) pH Temp. 1 (°C) DO 2 (mg/L) pH Temp. 1 (°C) DO 2 (mg/L) pH

Negative A 20.0 8.1 8.2 20.1 8.8 8.4 20.0 8.4 8.5

Control B 20.0 8.0 8.2 20.1 8.7 8.4 20.0 8.4 8.5

C 19.8 8.1 8.2 20.1 8.8 8.4 20.0 8.5 8.5

100 A 19.9 7.9 8.2 20.1 8.6 8.4 19.9 8.5 8.5

B 20.0 7.9 8.2 20.1 8.7 8.4 20.0 8.5 8.5

C 19.9 8.0 8.2 20.1 8.7 8.4 19.9 8.5 8.5

1 Manual temperature measurements. Temperature measured continuously during the test was approximately

20°C, measured to the nearest 1°C. 2 A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20.0°C in freshwater.

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Table 3

Specific Conductance, Hardness and Alkalinity

Measured in Dilution Water at Test Initiation

Parameter Day 0

Specific Conductance 354 (μS/cm)

Hardness 136 (mg/L as CaCO 3 )

Alkalinity 184 (mg/L as CaCO 3 )Wildlife International, Ltd.Project Number 527A-102- 19 -Table 4Cumulative Immobility and Observations4 Hours 24 Hours 48 HoursNominal WAFLoading Rate(mg/L)Rep.Daphnia/ReplicateNumberImmobile1Observations2NumberImmobile1Observations2NumberImmobile1Observations2PercentImmobileNegative Control A 10 0 10 AN 0 10 AN 0 10 AN 0B 10 0 10 AN 0 10 AN 0 10 ANC 10 0 10 AN 0 10 AN 0 10 AN100 A 10 0 10 AN 0 10 AN 0 10 AN 0B 10 0 10 AN 0 10 AN 0 10 ANC 10 0 10 AN 0 10 AN 0 10 AN1Cumulative number of immobile daphnids.2Observed effects: AN = appear normal.

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Table 5

EL50 Values

Time EL50 1 (mg/L) 95% Confidence Interval (mg/L) Statistical Method

24 Hours >100 -- 2 NA 3

48 Hours >100 -- 2 NA 3

1 EL50 = the theoretical WAF loading rate that produces 50% immobility. 2 95% confidence limits could not be calcu lated with the immobility data obtained. 3 NA = not applicable; <50% immobility preclude d statistical calculation of an EL50 value.

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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 361 356 – 366 (μS/cm) (N = 4)

Hardness 140 136 – 144 (mg/L as CaCO 3 ) (N = 4)

Alkalinity 182 180 – 182 (mg/L as CaCO 3 ) (N = 4)

pH 8.2 8.1 – 8.2 (N = 4)

TOC <1 -- (mg C/L) (N = 1) 1

1 TOC measured once monthly.

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Appendix 2

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

Pesticides and Organics 1

Component Measured Concentration (μg/L) Component Measured Concentration (μg/L)

Aldrin < 0.010 Gamma BHC – Lindane < 0.010 Alpha BHC < 0.010 Gamma Chlordane < 0.010 Alpha Chlordane < 0.010 Guthion (Azinphos-methyl) < 3.0 Beta BHC < 0.010 HCB < 0.010 Bolstar < 2.0 Heptachlor < 0.010 Chlordane < 0.51 Heptachlor Epoxide < 0.010 Coumaphos < 3.0 Kepone < 0.20 Delta BHC < 0.010 Malathion < 2.0 Demeton-O < 2.0 Merphos < 6.1 Demeton-S < 2.0 Methoxychlor < 0.10 Diazinon < 3.0 Methyl Parathion < 2.0 Dichlorvos < 3.0 Mevinphos < 3.0 Dieldrin < 0.020 Mirex < 0.033 Disulfoton < 2.0 Naled < 2.0 Dursban (Chlorpyrifos) < 2.0 o,p-DDD < 0.020 Endosulfan I < 0.010 o,p-DDE < 0.020 Endosulfan II < 0.020 o,p-DDT < 0.020 Endosulfan Sulfate < 0.020 p,p-DDD < 0.020 Endrin < 0.020 p,p-DDE < 0.020 Endrin Aldehyde < 0.10 p,p-DDT < 0.020 Endrin Ketone < 0.020 Phorate < 2.0 EPN < 2.0 Ronnel < 2.0 Ethion < 2.0 Stirophos < 2.0 Ethoprop < 3.0 Telodrin < 0.010 Ethyl Parathion < 2.0 Tokuthion < 2.0 Famphur < 2.0 Toxaphene < 3.0 Fensulfothion < 9.1 Trichloronate < 2.0 Fenthion < 2.0 Trithion < 2.0

1 Analyses performed by Lancaster Laboratories on samples collected on December 22, 2008.

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Appendix 2 (Continued)

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

Metals 1

Component Measured Concentration (mg/L) Component Measured Concentration (mg/L)

Aluminum < 0.200 Magnesium 12.3 Antimony < 0.0200 Manganese < 0.0050 Arsenic < 0.0200 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 7.23 Calcium 32.5 Selenium < 0.0200 Chloride 3.1 Silver < 0.0050 Chromium < 0.0150 Sodium 19.4 Cobalt < 0.0050 Sulfate 5.8 Copper < 0.0100 Thallium < 0.0300 Fluoride 0.52 Vanadium < 0.0050 Iron < 0.200 Zinc < 0.0200 Lead < 0.0150

1 Analyses performed by Lancaster Laboratories on samples collected on December 22, 2008.

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Appendix 3

Personnel Involved in the Study

The following key Wildlife International, Ltd. pers onnel were involved in the conduct or management of this study:

1. Henry O. Krueger, Ph.D., Director of Aquatic, Plant and Insect Toxicology 2. Timothy Ross, Laboratory Supervisor 3. Susan J. Palmer, Senior Biologist 4. Mary Beth Claude, Biologist 5. Amanda L. Weber, Biologist