Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FATHEAD MINNOW ( Pimephales promelas )

FINAL REPORT

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527A-103

OECD GUIDELINE 203 U.S. EPA OPPTS NUMBER 850.1075

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 29, 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 (410) 822-8600

Page 1 of 26

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STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any informati on 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 Gennany GmbH

TITLE: Alcohols, CI8-22, distn. residues (CAS No. 1160164-88-4): A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephales promelas)

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527A-103

STUDY COMPLETION: January 29, 2010

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 (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 perfonned according to Good Laboratory Practice Standards, but were perfonned using a certified laboratory and standard US EP A 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 detennined in accordance with Good Laboratory Practice Standards.

SPONSOR APPROVAL:

Sponsor Representative Date

ApplicantlSubmitter Date

# Wildlife International, Ltd. Project Number 527A-I03



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### 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 (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 13, 2009 October 13, 2009 October 19, 2009

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

Test Substance Preparation November 4, 2009 November 4, 2009 November 9, 2009

Dissolved Oxygen Measurements November 10, 2009 November 10, 2009 November 13, 2009

Data and Draft Report January 17-19,2010 January 19, 2010 January 21, 2010

Final Report January 29, 2010 January 29, 2010 January 29, 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 Germany GmbH

TITLE: Alcohols, CI8-22, distn. residues (CAS No. 1160164-88-4): A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephales promelas)

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527A-I03

STUDY DIRECTOR: 

# cJtOQ,g,A i1AA

PRINCIPAL BIOLOGIST

# ~1l&?1~ Mary Beth laude, B.S. 

# J 

# 1 J;1/f/ ;[0/0

Date Biologist

WILDLIFE INTERNATIONAL. LTD. MANAGEMENT: 

# 914_

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

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TABLE OF CONTENTS

Title/Cover Page............................................................................................................... ...................... 1

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

Good Laboratory Practice Co mpliance Statement ................................................................................. 3

Quality Assuran ce 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

References ..................................................................................................................... ....................... 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............................................................................................................ ..... 19

Table 4. Cumulative Mortality and Clinical Observations............................................................... 20

Table 5. LL50 Values ................................................................................................................... .... 21

APPENDICES

Appendix 1. Specific Conductance, Hardness, Alka linity, pH and Total Organic Carbon of Well Water Measured During the 4-Week Period Immediately Preceding the Test.................................................................................................................... 22

Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water .............................................................................................................. 23

Appendix 3. Changes to the Study Protocol................................................................................ 25

Appendix 4. Personnel Involved in the Study ............................................................................. 26

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SUMMARY

SPONSOR: Sasol Germany GmbH

TITLE: Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): A 96-Hour Static Acute Toxicity Test with the Fathead Minnow ( Pimephales promelas )

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527A-103

TEST DATES: Experimental Start (OECD): November 4, 2009 Experimental Start (EPA): November 6, 2009 Biological Termination: November 10, 2009 Experimental Termination: November 11, 2009

LENGTH OF EXPOSURE: 96 Hours

TEST ORGANISMS: Fathead Minnow ( Pimephales promelas )

SOURCE OF TEST ORGANISMS: Chesapeake Cultures Hayes, Virginia 23072

AGE OF TEST ORGANISMS: Juveniles

MEASUREMENTS OF 10 NEGATIVE CONTROL FISH: Mean Wet Weight: 0.09 g Range: 0.07 – 0.11 g Mean Total Length: 2.3 cm Range: 2.2 – 2.5 cm

WAF LOADING RATE: Nominal Negative Control 100 mg/L

RESULTS: Based on nominal WAF loading rates: 96-Hour LL50: >100 mg/L No-Mortality Level: 100 mg/L 96-Hour NOEL: 100 mg/L

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INTRODUCTION This study was conducted by Wildlife International, Ltd. for Sasol Germany GmbH at the Wildlife International, Ltd. aquatic toxicology fac ility in Easton, Maryland. The in-life phase of the

definitive test was conducted from November 6 to N ovember 10, 2009. Raw data generated by Wildlife International, Ltd. and a copy of the final repor t are filed under Project Number 527A-103 in archives
located on the Wildlife 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 fathead minnow, Pimephales promelas , during a 96-hour exposure period under static test conditions.

EXPERIMENTAL DESIGN Fathead minnows were exposed to a single limit c oncentration of test solution prepared using
water accommodated fractions (WAF) of the test s ubstance and a negative control (dilution water) for 96 hours under static conditions. Three replicate test chambers were maintained in each treatment and control group, with 10 fathead minnows in each test ch amber for a total of 30 minnows 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 achie ve the respective WAF solution. The nominal WAF loading rate selected was 100 mg test substance/L. Sa mples of test water were collected at test initiation and termination for analysis of total organic carbon (TOC).

Fathead minnows were impartially assigned to exposur e chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 3, 24, 48, 75 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups was used to determine LL50 values (i.e., the theoretical WAF loading rate that produces 50% mortality) at 24, 48, 72 and 96 hours. The no-mortality level and the no-observed-effect level (NOEL) were determined by visual interpretation of the mortality and observation data.

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MATERIALS AND METHODS The study was conducted according to the proce dures outlined in the protocol, “Alcohols, C18-
22, distn. residues (CAS No. 1160164-88-4): A 96-Hour Static Acute Toxicity Test with the Fathead
Minnow ( Pimephales promelas )”. The protocol was based on procedures outlined in the OECD Guidelines for Testing of Chemicals, 203: Fish, Acute Toxicity Test (1); the U.S. Environmental Protection Agency Series 850 – Ecological Effects Test Guidelines, OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine (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. identifi cation number 9187 upon receipt and was stored under
ambient conditions. The test substance, a solid, was identified as: Alcohols, C18-22, distn. Residues; Lot number 03585/MA, Spec: 59B1RN2; CAS number 1160164-88- 4. The test substance had a reported purity of 100% and an expi ration date of August 2013.

Test Organism
The fathead minnow, Pimephales promelas , was selected as the test species for this study. Fathead minnows are representative of an important gr oup 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 hatched on approximately September 23, 2009 and were obt ained from Chesapeake Cultures, Inc., Hayes, Virginia. Identification of the species was verified by the supplier.

All fish used in the test were from the same s ource and year class, and the length of the longest fish measured was no more than twice the length of th e shortest. The average total length of 10 negative

control fish measured at the end of the test was 2.3 cm, with a range of 2.2 to 2.5 cm. The average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 0.09 grams, with a range of 0.07 to 0.11 grams. Loading was defined as th e total wet weight of fish per liter of test water and was 0.08 g fish/L.

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The fathead minnows were held for 14 days prior to the test in water from the 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 23.2 to 23.5 ° C, measured with a hand-held liquid-in-glass thermometer. The pH of the water ranged from 8.2 to 8.6, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved oxygen concentrations ranged from 7.5 to
8.2 mg/L ( ≥ 87% of saturation), measured with a Yellow Springs Instruments Model 85 dissolved oxygen meter. Daily during the holding period, the fish were fed a commercially-prepared diet supplied by Zeigler Brothers, Inc., Gardners, Penns ylvania and/or brine shrimp nauplii ( Artemia sp.) supplied by Brine Shrimp Direct, Ogden, Utah. The fish were not fed for at least two days prior to the test or during

the test.

During the 2-week period prior to the test no mort alities occurred and the fish showed no signs of
disease or stress. At test initiation, the 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 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.

The well water is characterized as moderately-har d water. The specific conductance, hardness, alkalinity and pH of the well water during 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 measure the c oncentrations of selected organic and inorganic constituents in the well water are presented in Appendix 2.

Test Apparatus Test chambers were 19-L glass aquaria containing approximately 11 L of test solution. The depth of the test water in a representative test chamber w as 14.0 cm. Each test chamber was labeled with the

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project number, test concentration and replicate. The chambers were indiscriminately positioned in an environmental chamber set to maintain the desired test temperature.

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 prepared by weighing the test substance in a tared beaker and rinsing it with dilution water (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 allo wed to settle for approximately one hour before 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 aspirato r bottle. The negative control solution was prepared using the same procedures, without addition of test substance.

Analytical Sampling Duplicate samples were collected from each test c oncentration and control at the beginning of the test and at 96 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 darkn ess. 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 representa tive test chamber at test initiation using a SPER Scientific Model 840006C light meter.

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 a nd at approximately 24-hour intervals during the test using a liquid-in-glass thermometer. Temperature also was monitored continuously during the test in one

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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 approximately 24-hour intervals during the test. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen mete r and measurements of pH were made using a Thermo Orion Model 525Aplus pH meter.

Specific conductance, hardness and alkalinity in th e dilution water at the beginning of the test were measured. Hardness and alkalinity measurement s 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 CON6 Conductivity-Temperature meter.

Observations Observations were made periodically to determine the number of mortalities in each treatment group. The numbers of individuals exhibiting signs of t oxicity or abnormal behavior also were evaluated. Observations were made approximately 3, 24, 48, 75 and 96 hours after test initiation.

Statistical Analyses
Because this is a limit test, the LL50 value cannot be statistically defined. However, based upon the biological response obtained in the exposure group, the LL50 value was estimated to be greater than the limit loading rate. The no-mortality level and th e no-observed-effect level (NOEL) were determined by visual interpretation 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 app eared clear and colorless during the test with a small amount of white particles on the surface and bottom, a nd suspended in the water column of the solutions. Results of analyses for TOC in the test solutions ar e presented in Table 1. Measurements of TOC in the

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newly prepared test solutions on Day 0 ranged from 1.2 to 1.6 mg C/L. TOC in the 96-hour old solutions on Day 4 ranged from below the limit of quantitation (LOQ = 1 mg C/L) to 1.3 mg C/L.

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 22 ± 1 ° C range established for the test.
Dissolved oxygen concentrations remained ≥ 7.0 mg/L ( ≥ 81% of saturation) throughout the test. Measurements of pH ranged from 8.0 to 8.4. 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). Light intensity at test initiation was 291 lux at the surface of the water of one representative

test chamber.

Daily observations of mortality and signs of t oxicity observed during the test are presented in Table 4. Fathead minnows in the negative control group and in the 100 mg/L treatment group appeared normal throughout the test, with no mortalities or overt signs of toxicity noted. The no-mortality level and the NOEL were both 100 mg /L. LL50 values at 24, 48, 72 and 96 hours were determined from the mortality data and were > 100 mg/L (Table 5).

CONCLUSIONS
Fathead minnows, ( Pimephales promelas ), were exposed for 96 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 96-hour LL50 value was > 100 mg/L . Fathead minnows 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 mortality or overt signs of toxicity. The 96-hour no-mortality level and NOEL were both 100 mg/L.

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REFERENCES

1 Organization for Economic Cooperation and Development . 1992. OECD Guidelines for Testing of Chemicals, Guideline 203: Fish, Acute Toxicity Test.

2 U.S. Environmental Protection Agency. 1996. Series 850 – Ecological Effects Test Guidelines ( draft ), OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine

.

3 American Society for Testing and Materials . 2007. ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Ma terials with Fishes, 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-103-) Sampling Interval

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

Negative Control TOC-1 0 1.4

TOC-2 0 1.6
TOC-5 4 <LOQ
TOC-6 4 <LOQ

100 TOC-3 0 1.2

TOC-4 0 1.4

TOC-7 4 1.3

TOC-8 4 1.0

1 LOQ = 1 mg C/LWildlife International, Ltd.Project Number 527A-103- 17 -Table 2Temperature, Dissolved Oxygen and pH of Water in the Test Chambers0 Hour 24 Hour 48 HourNominal WAFLoading Rate(mg/L)ReplicateTemp.1(oC)DO2(mg/L)pHTemp.1(oC)DO2(mg/L)pHTemp.1(oC)DO2(mg/L)pHNegative Control A 21.9 7.9 8.022.2 7.3 8.2 22.1 7.0 8.3B 21.9 7.8 8.1 22.27.1 8.2 22.1 7.0 8.3C 21.9 8.0 8.1 22.27.1 8.2 22.1 7.2 8.3100 A 22.4 7.9 8.1 22.27.2 8.2 22.1 8.2 8.3B 22.2 7.9 8.1 22.17.2 8.2 22.1 8.1 8.4C 22.0 8.0 8.1 22.17.3 8.3 22.1 8.0 8.41Manual temperature measurements. Temperature monitored continuously during thetest ranged from approximately 22 to 23°C, measured to the nearest 1°C.2A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22oC in freshwater.Wildlife International, Ltd.- 18 -Project Number 527A-103Table 2 (Continued)Temperature, Dissolved Oxygen and pH of Water in the Test Chambers75 Hour 96 HourNominal WAFLoading Rate(mg/L)ReplicateTemp.1(oC)DO2(mg/L)pHTemp.1(oC)DO2(mg/L)pHNegative Control A 22.2 7.4 8.3 22.0 7.5 8.3B 22.1 7.4 8.3 22.1 7.6 8.3C 22.1 7.3 8.4 22.0 7.6 8.4100 A 22.1 7.7 8.4 22.1 7.8 8.4B 22.1 7.6 8.4 22.0 7.8 8.4C 22.1 7.6 8.4 22.2 7.7 8.41Manual temperature measurements. Temperature monitored continuously during thetest ranged from approximately 22 to 23°C, measured to the nearest 1°C.2A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22oC 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 352 (μS/cm)

Hardness 152 (mg/L as CaCO 3 )

Alkalinity 185 (mg/L as CaCO 3 )Wildlife International, Ltd.Project Number 527A-103- 20 -Table 4Cumulative Mortality and Clinical Observations3 Hours 24 Hours 48 Hours75 Hours96 HoursNominal WAFLoading Rate(mg/L)ReplicateNo.ExposedNo.Dead1Obs.2No.Dead1Obs.2No.Dead1Obs.2No.Dead1Obs.2No.Dead1Obs.2CumulativePercentMortalityNegative A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0Control B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 ANC 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN100 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 ANC 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN1Cumulative number of dead fish.2Observations: AN = appear normal.

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Table 5

LL50 Values

Time LL50 1 (mg/L) 95% Confidence Interval (mg/L) Statistical Method

24 Hours > 100 -- 2 NA 3

48 Hours > 100 -- 2 NA 3

72 Hours > 100 -- 2 NA 3

96 Hours > 100 -- 2 NA 3

1 LL50 = the theoretical WAF loading rate that produces 50% mortality.
2 95% confidence limits could not be calculated with the mortality data obtained.
3 NA = not applicable; <50% mortality precluded statistical calculation of an LL50 value.

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Appendix 1

Specific Conductance, Hardness, Alkalinity, pH and Total Organic Carbon of Well Water Measured During the 4-Week Period Immediately Preceding the Test

Mean Range

Specific Conductance (μmhos/cm) 363 (N = 4) 358 – 366

Hardness (mg/L as CaC0 3 ) 141 (N = 4) 140 – 144

Alkalinity (mg/L as CaC0 3 ) 182 (N = 4) 180 – 182

pH 8.2 (N = 4) 8.1 – 8.2

TOC (mg C/L) <1 (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 Laborator ies 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 Laborator ies on samples collected on December 22, 2008.

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Appendix 3

Changes to the Study Protocol

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

1. Observations of mortality and clinical signs of toxicity, as well as water chemistry measurements (DO, pH and temperature) were scheduled for a pproximately 72 hours, but inadvertently were not made until approximately 75 hours. This slight delay had no adverse impact upon the results or interpretation of the study.

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Appendix 4

Personnel Involved in the Study

The following key Wildlife International, Lt d. personnel 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. Christian R. Disque, Biologist