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 
Wildlife International, Ltd. 

 Project Number 527A-103 

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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 527A-103 

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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 
Wildlife International, Ltd. Project Number 527A-103 

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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 

Mary ~1l&?1~ 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 
Wildlife International, Ltd. 

 Project Number 527A-103 

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TABLE OF CONTENTS

Title/Cover 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 

 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, Alkalinity, 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 
Wildlife International, Ltd. 

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INTRODUCTION 

This study was conducted by Wildlife International, Ltd. for Sasol Germany GmbH at the 

Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the 

definitive test was conducted from November 6 to November 10, 2009. Raw data generated by Wildlife 

International, Ltd. and a copy of the final report 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 concentration of test solution prepared using 

water accommodated fractions (WAF) of the test substance 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 chamber for a total of 30 minnows per concentration. 

The nominal WAF loading rate was selected in consultation with the Sponsor based on information 

provided by the Sponsor. For this test, the term loading rate means the total amount of test substance 

added to the dilution water volume (mg/L) to achieve the respective WAF solution. The nominal WAF 

loading rate selected was 100 mg test substance/L. Samples of test water were collected at test initiation 

and termination for analysis of total organic carbon (TOC). 

Fathead minnows were impartially assigned to exposure 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 procedures 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. identification 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 expiration 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 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 hatched 

on approximately September 23, 2009 and were obtained 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 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 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 the 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, Pennsylvania 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 mortalities 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 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 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 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 concentrations 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 was 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 allowed 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 aspirator 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 concentration 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 darkness. 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 22 ± 1oC. Temperature was measured in each 

test chamber at the beginning and end of the test and 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 meter and measurements of pH were made using a 

Thermo Orion Model 525Aplus pH meter. 

Specific conductance, hardness and alkalinity in the dilution water at the beginning of the test 

were measured. Hardness and alkalinity 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 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 toxicity 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 the 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 appeared clear and colorless during the test with a small 

amount of white particles on the surface and bottom, and suspended in the water column of the solutions. 

 Results of analyses for TOC in the test solutions are 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 and 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 toxicity 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 Materials 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/L 
Wildlife International, Ltd. Pr

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Table 2 

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

Nominal WAF 0 Hour 24 Hour 

Loading Rate 

(mg/L) Replicate Temp.1 (oC) DO2 (mg/L) pH Temp.1 (oC) DO2 (mg/L) pH Temp.1 (oC) 

Negative Control A 21.9 7.9 8.0 22.2 7.3 8.2 22.1 

 B 21.9 7.8 8.1 22.2 7.1 8.2 22.1  C 21.9 8.0 8.1 22.2 7.1 8.2 22.1 

100 A 22.4 7.9 8.1 22.2 7.2 8.2 22.1 

 B 22.2 7.9 8.1 22.1 7.2 8.2 22.1  C 22.0 8.0 8.1 22.1 7.3 8.3 22.1 

1 Manual temperature measurements. Temperature monitored continuously during the test ranged from approximately 22 to 23°C, measured to the n2 A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22oC in freshwater. 
Wildlife International, Ltd. 

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Table 2 (Continued) 

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

Nominal WAF 75 Hour 96 Hour 

Loading Rate 

(mg/L) Replicate Temp.1 (oC) DO2 (mg/L) pH Temp.1 (oC) DO2 (mg/L) 

Negative Control A 22.2 7.4 8.3 22.0 7.5 

 B 22.1 7.4 8.3 22.1 7.6  C 22.1 7.3 8.4 22.0 7.6 

100 A 22.1 7.7 8.4 22.1 7.8 

 B 22.1 7.6 8.4 22.0 7.8  C 22.1 7.6 8.4 22.2 7.7 

1 Manual temperature measurements. Temperature monitored continuously during the test ranged from approximately 22 to 23°C, measured t2 A 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 CaCO3) 

 

Alkalinity 185 

 (mg/L as CaCO3) 
Wildlife International, Ltd. Pr

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Table 4 

Cumulative Mortality and Clinical Observations 

Nominal WAF 3 Hours 24 Hours 48 Hours 75 Hours 

Loading Rate 

(mg/L) Replicate 

No. 

Exposed 

No. 

Dead1 Obs.2

No. 

Dead1 Obs.2

No. 

Dead1 Obs.2

No. 

Dead1 Obs.2

N

Dea

Negative A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 

Control B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 

 C 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 

100 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 

 B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0  C 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 

1 Cumulative number of dead fish. 2 Observations: AN = appear normal. 
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Table 5 

LL50 Values 

Time 

LL501

(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 CaC03) 

141 

(N = 4) 

140 – 144 

Alkalinity 

 (mg/L as CaC03) 

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 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

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 approximately 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. 
Wildlife International, Ltd. 

 Project Number 527A-103 

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Appendix 4

Personnel Involved in the Study 

 The following key Wildlife International, Ltd. 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