Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): 

A 72-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA 

(Pseudokirchneriella subcapitata) 

FINAL REPORT 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527A-101 

OECD Guideline 201 

EU Directive 92/69/EEC, Method C.3. 

AUTHORS: 

Tara L. Cartee, Ph.D. 

John R. Porch 

Henry O. Krueger, Ph.D. 

STUDY INITIATION DATE: October 27, 2009 

STUDY COMPLETION DATE: January 11, 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 34
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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, ~td. Prqject Number s27A-1 01 

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

SPONSOR: Sasol Germany GmbH 

TITLE: Alcohols, C18-22, dism. residues (CAS No. 1160164-883): A 72-hour Toxicity Test with 

the Freshwater Alga (Pseudok~rchneuiella subcapitafa) 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527A-101 

STUDY COMPLETION: January 11,2010 

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 (ENVIMCICHEM (98) 17); and Japan MAFF (I I NohSan, 

Notification No. 6283: Agricultural Production Bureau? 1 October 1999): with the following exceptions: 

Periodic analyses of water for potential contaminants were not performed according to Good 

Laborator). Practice Standards, but were performed using a certified laboratory and standard US EPA 

analytical methods. 

The charactermt~on of the test substance and the stabhty of the test substance under the 

condmons of storage at the test slte, were not detcrmmed m accordance n ith Good Laboraton Practm 

Standards 

STUDY DIRECTOR: 

\\ =-wxrq 'Lo16 

Date 

Su ervlsor of Nontarget Plant and Invertebrate Shid~es 

SPONSOR APPROVAL: 

Sponsor Representative Date 

ApplicantiSubmitter Date 
Wildlife Intemtional, Ltd. Project Number 527A-101 

QUALITY ASSURANCE STATEMENT 

This study was examined for compliance with Good Laboratoly 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 (ENVNCICHEM (98) 17); and Japan MAFF (1 1 NohSan, Notification No. 

6283, Agricultural Production Bureau, 1 October 1999). The dates of all inspections and audits and the dates 

that any fmdings were reported to the Study Director and Laboratory Management were as follows: 

ACTIVITY: 

Protocol 

Test Substance Preparation 

pH Measurements 

Biological Data and Draft Report 

Final Report 

DATE CONDUCTED: 

October 9,2009 

October 28,2009 

November 2,2009 

December 29 - 30,2009 

& January4-5.2010 

January 11,2010 

DATE REPORTED TO: 

STUDY DIRECTOR: MANAGEMENT: 

October 9,2009 October 12, 2009 

October 28,2009 October 28,2009 

November 2,2009 November 3,2009 

January 5,2010 Januay 6,2010 

January 11,2010 January 11,2010 

All inspections were study-based unless otherwise noted. 

// ~~~y ad/ 

Linda R Mitchell Date 

Director of Regulatory and Ecotox Operations 
wildlife h~tematio~tal, Ltd. Project Number j27A-101 

-5. 

REPORT APPROVAL 

SPONSOR: Sasol Germany GmbH 

TITLE: Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): A 72-hour Toxicity Test with 

the Freshwater Alga (Pseudokirchneriella subcapitala) 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527A-101 

STUDY DIRECTOR: 

s&wisor of Nontargct Plant and Invertebrate Stud~es 

WILDLlFE INTERNATIONAL. LTD. MANAGEMENT: 

Datc 

Director of Aquatic, Plant and Inscct Toxicology 
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 Project Number 527A-101 

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TABLE OF CONTENTS

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

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

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

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

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

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

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

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

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

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

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

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

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

 Culture Medium....................................................................................................................... 11 

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

 Environmental Conditions....................................................................................................... 11 

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

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

 Inoculation of Test Chambers.................................................................................................. 12 

 Algal Growth Measurements and Observations ...................................................................... 12 

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

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

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

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

 Conditions for the Validity of the Test .................................................................................... 15 

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

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

TABLES AND FIGURES 

Table 1. Measurements of Total Organic Carbon in Freshwater Algal Medium Samples............... 17 

Table 2. Temperature Measurements ............................................................................................... 18 

Table 3. pH Measurements............................................................................................................... 19 

Table 4. Light Intensity Measurements ............................................................................................ 20 

Table 5. Mean Cell Density and Percent Inhibition ......................................................................... 21 

Table 6. Mean Yield and Percent Inhibition..................................................................................... 22 

Table 7. Mean Growth Rate (per Hour) and Percent Inhibition ....................................................... 23 

Table 8. EC50, EyC50, and ErC50 Values Over the 72-hour Exposure Period................................ 24 

Figure 1. Growth of Pseudokirchneriella subcapitata in the Negative Control Over the 72-hour 

Exposure Period, Expressed as Cell Density...................................................................... 25 

Figure 2. Concentration-Response Curve for Pseudokirchneriella subcapitata Exposed to 

 Alcohols, C18-22, distn. Residues for 72 Hours, Expressed as Cell Density .................... 26 

APPENDICES 

Appendix 1. Freshwater AAP Algal Medium ............................................................................... 27 

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

 Well Water................................................................................................................ 28 

Appendix 3. Cell Density By Replicate Over the 72-hour Exposure Period ................................ 30 

Appendix 4. Yield By Replicate at 72-hours................................................................................. 31 

Appendix 5. Growth Rate By Replicate Over the 72-hour Exposure Period ................................ 32 

Appendix 6. Specific Growth Rate of the Negative Control Group.............................................. 33 

Appendix 7. Personnel Involved in the Study............................................................................... 34 
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SUMMARY

SPONSOR: Sasol Germany GmbH 

STUDY: Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): A 72-hour Toxicity Test 

with the Freshwater Alga (Pseudokirchneriella subcapitata) 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527A-101 

TEST DATES: Experimental Start (OECD): October 28, 2009 

 Experimental Start (EPA): October 30, 2009 

 Exposure Termination: November 2, 2009 

 Experimental Termination: November 3, 2009 

LENGTH OF EXPOSURE: 72 Hours 

TEST ORGANISM: Freshwater Alga (Pseudokirchneriella subcapitata) 

SOURCE OF TEST ORGANISMS: Wildlife International, Ltd. Cultures 

 Easton, Maryland 21601 

WAF LOADING RATE: Nominal 

Negative Control 

 100 mg/L 

RESULTS: Based on nominal WAF loading rates: 

CELL DENSITY: 

 72-Hour EC50: >100 mg/L 

 95% Confidence Interval: Not calculable 

 72-Hour NOEC: ≥ 100 mg/L 

YIELD: 

 72-hour EyC50: >100 mg/L 

 95% Confidence Interval: Not calculable 

 72-hour NOEC: ≥ 100 mg/L 

GROWTH RATE: 

 72-Hour ErC50: >100 mg/L 

 95% Confidence Interval: Not calculable 

 72-Hour NOEC: ≥ 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 facility in Easton, Maryland. The in-life phase of the 

definitive test was conducted from October 30 to November 2, 2009, with cell counts completed on 

November 2, 2009. The raw data generated by Wildlife International, Ltd. and a copy of the final report 

are filed under Project Number 527A-101 in archives located on the Wildlife International, Ltd. site. 

OBJECTIVE 

The objective of this study was to determine the toxicity of the test substance, Alcohols, C18-22, 

distn. residues (CAS No. 1160164-88-4), to the freshwater green alga, Pseudokirchneriella subcapitata, 

over a 72-hour exposure period. 

EXPERIMENTAL DESIGN 

The green alga, Pseudokirchneriella subcapitata, was exposed to a single limit concentration 

using water accommodated fractions (WAF) of the test substance and a negative control (culture medium) 

for 72 hours. 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 were collected at test 

initiation for analysis of total organic carbon (TOC). 

Six replicate test chambers were maintained in the single treatment group and in the control 

group. At test initiation, an inoculum of the algal cells was added to each test chamber to achieve a 

nominal concentration of approximately 10,000 cells/mL. Samples were collected from each replicate 

test chamber at approximately 24-hour intervals during the test to determine cell densities, yield, and 

growth rates. Cell densities and growth rates were used to calculate percent inhibition values relative to 

the controls over the 72-hour exposure period. Percent inhibition for yield was calculated at 72 hours. 

EC50 and ErC50 values (i.e., the theoretical toxicant concentrations that would produce a 50% reduction 

in cell density and growth rate, respectively) were calculated at 24, 48, and 72-hours. EyC50 (yield) was 

calculated at 72 hours. No-observed-effect concentrations (NOEC) were determined at 72 hours through 
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statistical evaluation of the cell densities, yield, and growth rates, as well as examination of the 

concentration-response pattern. 

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 72-hour Toxicity Test with the Freshwater Alga 

(Pseudokirchneriella subcapitata).” The protocol was amended to change the study director, otherwise 

there were no changes. The protocol was based on procedures outlined in OECD Guidelines for Testing 

of Chemicals, Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test (1); and the 

Official Journal of the European Communities No. L383, Method C.3.: Algal Inhibition Test (2). 

Test Substance 

The test substance used to prepare the test solutions for the study was received from Sasol on 

September 11, 2009. It was assigned Wildlife International, Ltd. identification number 9187 upon receipt 

and was stored under room temperature (ambient) and dark conditions. The test substance, a solid, was 

identified as: Alcohols, C18-22, distn. Residues; CAS No. 1160164-88-4; lot number 03585/MA. The 

test substance contained 100% active ingredient and had an expiration date of August 2013. 

Test Organism 

The freshwater green alga, Pseudokirchneriella subcapitata, was selected as the test species for 

this study. The species is representative of an important group of freshwater algae, and was selected for 

use in the test based upon a past history of use, and ease of culturing in the laboratory. Original algal 

cultures were obtained from the University of Toronto Culture Collection, and had been maintained in 

culture medium at Wildlife International, Ltd., Easton, Maryland. Algal cells used in this test were 

obtained from Wildlife International, Ltd. cultures that had been actively growing in culture medium for 

at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had 

been transferred to fresh media four days prior to test initiation. The negative control organisms were 

expected to exhibit exponential growth over the 72-hour exposure period. Exponential growth phase, 

defined as the period of growth where the algal cells are dividing at a constant rate, is indicated by the 

linear section of the growth curve (Figure 1). 
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Culture Medium 

The algal cells were cultured and tested in freshwater AAP algal medium (3). Stock nutrient 

solutions were prepared by adding reagent-grade chemicals to purified Wildlife International, Ltd. well 

water (NANOpure® water). The test medium then was prepared by adding appropriate volumes of the 

stock nutrient solutions to NANOpure® water (Appendix 1). The pH of the medium was 7.5 and the 

medium was sterilized by filtration (0.22 μm) prior to use. The results of analyses performed to measure 

the concentrations of selected organic and inorganic constituents in the well water used to prepare the 

algal medium are presented in Appendix 2. 

Test Apparatus 

Test chambers were sterile, 250-mL Erlenmeyer flasks plugged with foam stoppers, and 

contained 100 mL of test or control medium. The test flasks were labeled with the project number, test 

concentration and replicate, and were indiscriminately positioned daily on a mechanical shaker in an 

environmental chamber designed to maintain the desired test temperature throughout the test. The test 

flasks were shaken continuously at 100 rpm. 

Environmental Conditions 

Test chambers were held in an environmental chamber at a temperature of 23 ± 2oC. The 

temperature of a container of water adjacent to the test chambers in the environmental chamber was 

recorded twice daily during the test using a liquid-in-glass thermometer. In addition, temperature in the 

environmental chamber was measured continuously using a minimum-maximum thermometer. 

The algae were held under continuous cool-white fluorescent lighting throughout the test. The 

target light intensity was 4,300 ± 10% lux. Light intensity was measured at test solution level at five 

locations surrounding the test flasks at test initiation using a SPER Scientific 840006C light meter. 

The pH of the medium in each treatment and control group was measured at test initiation and 

exposure termination using a Thermo Orion Model 525Aplus pH meter. At test initiation, pH was 

measured in the individual batches of test solution prepared for each treatment and control group. At 

exposure termination, pH was measured in pooled samples of test solution collected from each of the 

replicates of each treatment and control group. 
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Preparation of Test Concentrations

The test solution was prepared in the form of a water accommodated fraction (WAF) at a nominal 

loading rate of 100 mg/L. The WAF was prepared by weighing the test substance in a tared beaker and 

rinsing it with freshwater algal medium into a 4-L glass aspirator bottle to a final volume of 4 L. The 

solution was stirred with a 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, 

the 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 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. 

Inoculation of Test Chambers 

Prior to test initiation, an inoculum was prepared at approximately 1.0 x 106

 cells/mL. An 

inoculum volume of 1.0 mL was added to each test chamber to achieve a nominal concentration of 

approximately 10,000 cells/mL at test initiation. 

Algal Growth Measurements and Observations 

Test medium samples were collected from each replicate of the treatment and control groups for 

the determination of algal cell densities. Samples were collected at approximately 24-hour intervals 

during the 72-hour exposure and were held for a maximum of two days under refrigerated conditions 

sufficient to inhibit growth until cell counts could be performed. Cell counts were performed using a 

hemacytometer and microscope. The samples were diluted using an electron solution (Isoton®), as 

needed, to maintain counting accuracy. A small amount of each sample was loaded onto a 

hemacytometer and 10 grids were counted. The mean number of cells per grid was calculated and this 
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value was used to calculate the cell density of the sample. Using this technique, the minimum 

quantifiable cell density was 1,000 cells/mL. 

Samples of test solution were collected from each replicate at the end of the test. These samples 

were pooled within their respective treatments, and subsamples were removed and examined 

microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Cells in the 

replicate test chambers also were assessed for aggregations or flocculations of cells, and adherence of the 

cells to the test chamber. 

Statistical Analyses

The calculation of cell densities, yield, growth rates and percent inhibition values, as well as all 

statistical analyses, were conducted using “The SAS System for Windows” (4). 

 Yield was calculated for each treatment and control replicate, and was defined as the cell density 

at the end of the exposure period minus the cell density at the start of the exposure period. 

Growth rate was calculated for each replicate of the control and treatment groups using the 

following formula: 

μ = 

n o

n o

t - t

ln N - ln N

where: μ = Average specific growth rate 

 No = Nominal cell density (cells/mL) at to

Nn = Measured cell density (cells/mL) at tn

 to = Time of beginning of test (hours) 

 tn = Time after beginning of test (hours) 

Inhibition values were calculated for each treatment group as the percent reduction in cell 

density, yield, and growth rate relative to the negative control replicates for each 24-hour interval. The 

following formula was used: 

Percent Inhibition = 

Mean ControlRe sponse

Mean ControlRe sponse − Mean Treatment Re sponse × 100 
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EC50 values in this study represent the theoretical test concentration that would produce a 50% 

reduction in an effect relative to the control. There was less than 50% reduction in cell density (EC50), 

yield (EyC50), and growth rate (ErC50) in the treatment group at all time points. Therefore, EC50 values 

could not be statistically calculated and were estimated by visual observation of the data. 

The 72-hour cell density, yield, and growth rate data were evaluated for normality and 

homogeneity of variance (p = 0.01) using Shapiro-Wilk’s and Levene’s tests, respectively. Since all data 

met the assumptions for normality and homogeneity of variance, the treatment groups were compared to 

the negative control using Dunnett’s test (p = 0.05). The results of the statistical analyses, as well as an 

evaluation of the concentration-response pattern, were used to determine the NOEC relative to each 

parameter at 72 hours. 

RESULTS AND DISCUSSION 

Measurement of TOC in Test Solutions 

The nominal WAF loading rate selected for use in this study was 100 mg/L. At the time of 

preparation the test solution at this nominal WAF loading rate had white particulate matter on the surface 

and suspended throughout the solution. At test termination, no surface slicks or particulate matter was 

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

TOC in the test solution on Day 0 were below that observed in the negative control samples. 

Observations and Measurements 

Measurements of temperature, pH and light intensity are presented in Tables 2, 3 and 4, 

respectively. Temperatures remained within the 23 ± 2°C range established for the test. The pH of all test 

solutions was 7.4 at test initiation and 7.9 and 8.0 at test termination. The pH tended to increase relative 

to increases in algal densities, which is typical for tests conducted with Pseudokirchneriella subcapitata. 

The light intensity ranged from 4,070 to 4,470 lux, which was within the desired range of 4,300 ± 10% 

lux. 

The toxicity of the test substance to Pseudokirchneriella subcapitata was determined by 

evaluating changes in cell density over a 72-hour exposure period. Cell densities were used to determine 

growth rates for each 24-hour interval of exposure, and yield at 72 hours. Mean cell densities, yield, and 
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growth rates with the corresponding percent inhibition values are presented in Tables 5, 6 and 7, 

respectively. EC50, EyC50, and ErC50 values and the corresponding 95% confidence limits for cell 

density, yield and growth rates, respectively, are presented in Table 8. Individual replicate data for each 

growth parameter are presented in Appendices 3, 4 and 5, while mean cell densities are illustrated 

graphically in Figures 1 and 2. Changes in mean cell density in the negative control replicates over the 

72-hour exposure period indicated that exponential growth of cells occurred in those replicates (Figure 1). 

 Appendix 6 provides specific growth rates for the control replicates. 

After 72 hours of exposure, inhibition of cell densities, yield and growth rate in the 100 mg/L 

treatment group was –0.9, -1.0, and –0.1%, respectively, relative to the negative control. Dunnett’s test 

indicated that there was not a significant reduction (p ≤ 0.05) in cell density, yield, or growth rate in the 

100 mg/L treatment group at 72 hours. Consequently, the 72–hour NOEC for cell density, yield, and 

growth rate was ≥100 mg/L. The 72-hour EC50, EyC50, and ErC50 values were >100 mg/L. After 

72 hours of exposure, there were no signs of adherence of cells to the test chambers or noticeable 

aggregations or flocculation in the negative control or in any treatment groups. There were no noticeable 

changes in cell morphology in the 100 mg/L test concentration when compared to the control replicates 

during the microscopic examinations of the cells. 

Conditions for the Validity of the Test 

1) The mean cell density in the control flasks increased by a factor greater than 16 within 

three days. The factor was 124 (Table 5). 

2) The coefficient of variation of average specific growth rate in the control replicates 

during the whole test period did not exceed 7%. It was 3.95% (Appendix 6). 

3) The mean percent coefficient of variation for section-by-section specific growth rates 

(days 0-1, 1-2, and 2-3) did not exceed 35%. It was 10.19% (Appendix 6). 

CONCLUSIONS 

The freshwater green alga, Pseudokirchneriella subcapitata, was exposed to a single limit water 

accommodated fraction (WAF) of Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4) at 

100 mg/L. Effects were evaluated based on cell density, yield, and growth rate. The 72–hour EC50, 

EyC50, ErC50 values were >100 mg/L. The 72-hour NOEC, based on effects on cell density, yield, and 

growth rate, was ≥100 mg/L. 
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REFERENCES 

1 Organisation for Economic Cooperation and Development. 2006. OECD Guidelines for 

Testing of Chemicals, Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition 

Test. Adopted 23 March 2006. 

2 Official Journal of the European Communities. 1992. No. L383. Method C.3. Algal 

Inhibition Test.

3 American Society for Testing and Materials. 1990. ASTM Standard Guide 1218-90E: 

Standard Guide for Conducting Static 96-Hour Toxicity Tests with Microalgae. 

4 The SAS System for Windows. 1999 – 2001. Version 8.2. SAS Institute, Inc., Cary, North 

Carolina. 
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Table 1 

Measurements of Total Organic Carbon in Freshwater Algal Medium Samples 

Nominal WAF 

Loading Rate 

(mg/L) 

Sample 

Number 

(527A-101-) 

Sampling 

Interval 

(Day) 

Total Organic Carbon 

(mg C/L) 1

Negative Control 1 0 3.143 

 2 0 3.012 

 

100 3 0 1.299 

 4 0 1.295 

1

 LOQ = 1 mg C/L 
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Table 2 

Temperature Measurements 

Temperature (°C) 1

Time 

(Days) Measurement 1 Measurement 2 2

 

0 22.4 23.1 

1 22.6 23.1 

2 23.0 23.1 

3 23.2 23.1 

 1

 The minimum and maximum temperatures monitored continuously throughout the study were 23.4 to 23.7°C, 

respectively. 2

 Temperature Measurement 2 was typically taken at least 4 hours after Measurement 1.
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Table 3 

pH Measurements 

Nominal WAF pH Measurements 

Loading Rate 

(mg/L) Day 0 1

 Day 3 2

 

Negative Control 7.4 8.0 

100 7.4 7.9 

 

1

 Day 0 samples were collected from the individual batches of test solution prepared for each treatment and 

control group at test initiation. 2

 Day 3 samples were composites of test solution collected from each of the replicates per treatment and control 

group at test termination.
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Table 4 

Light Intensity Measurements 

Light Intensity (lux)1 

Day Measurement 

No. 1 

Measurement 

No. 2 

Measurement 

No. 3 

Measurement 

No. 4 

Measurement 

No. 5 

0 4100 4180 4470 4070 4290 

1

 Measurements taken at five locations surrounding the test flasks at test solution level. 
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Table 5 

Mean Cell Density and Percent Inhibition 

24 Hours 1 48 Hours 72 Hours Nominal WAF 

Loading Rate 

 (mg/L) 

Mean 

Cell Density 

(cells/mL) 

Percent 

Inhibition 2,3

 Mean 

Cell Density 

(cells/mL) 

Percent 

Inhibition 2,3

 Mean 

Cell Density

(cells/mL) 

I

Negative Control 49,000 -- 252,667 -- 1,236,667 

100 47,667 2.7 260,000 -2.9 1,248,333 

1 The cell density in each replicate at test initiation (0 hours) was approximately 10,000 cells/mL. The mean cell density in the negati

flasks increased by a factor of 124 within three days. 

2 Calculations were performed using SAS Version 8.2. Manual calculations may differ slightly. 3 Percent inhibition was calculated relative to the negative control replicates. 

There was no statistically significant difference from the negative control replicates using Dunnett’s test (p > 0.05).
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Table 6 

Mean Yield and Percent Inhibition 

 72 Hours Nominal WAF 

Loading Rate 

(mg/L) 

 Mean 

Cell Density

(cells/mL) 

Percent 

Inhibition 1,2

Negative Control 1,226,667 -- 

100 1,238,333 -1.0 

1 Calculations were performed using SAS Version 8.2. Manual calculations 

 may differ slightly. 

2 Percent inhibition was calculated relative to the negative control replicates. There was no statistically significant difference from the negative control replicates using Dunnett’s test (p > 0.05).
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Table 7

Mean Growth Rate (per Hour) and Percent Inhibition 

Nominal WAF 0 - 24 Hours 0 - 48 Hours 0 - 72 Hours 

Loading Rate 

 (mg/L) Mean Growth Rate Percent Inhibition 1,2 Mean Growth Rate Percent Inhibition 1,2 Mean Growth Rate Percen

Inhibition 

Negative Control 0.0660 -- 0.0671 -- 0.0667 -- 

100 0.0642 2.6 0.0678 -1.1 0.0668 -0.1 

1 Calculations were performed using SAS Version 8.2. Manual calculations may differ slightly. 2 Percent inhibition was calculated relative to the negative control replicates. 

There was no statistically significant difference from the negative control replicates using Dunnett’s test (p > 0.05).
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Table 8 

EC50, EyC50, and ErC50 Values over the 72-Hour Exposure Period

Cell Density Yield G

Time EC50 

(mg/L) 

95% Confidence Interval 

(mg/L) 

EyC50 

(mg/L) 

95% Confidence Interval 

(mg/L) 

ErC50 

(mg/L) 

95

24 Hours > 100 --1 --2 -- > 100 48 Hours > 100 --1 --2 -- > 100 

72 Hours > 100 --1 > 100 --1 > 100 

1 95% confidence limits could not be calculated with the data obtained. 2 Results for yield calculated at 72 hours only, as per protocol. 
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Figure 1 

Growth of Pseudokirchneriella subcapitata in the Negative Control 

Over the 72-hour Exposure Period, Expressed as Cell Density 

1,000

10,000

100,000

1,000,000

10,000,000

0 24 48 72

Exposure Duration (Hours)

Mean Cell Density (Cells/mL)

Negative Control
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Figure 2

Concentration-Response Curve for Pseudokirchneriella subcapitata

Exposed to Alcohols, C18-22, distn. Residues for 72 Hours, Expressed as Cell Density 

1,000

10,000

100,000

1,000,000

10,000,000

0 24 48 72

Exposure Duration (Hours)

Mean Cell Density (Cells/mL)

Negative Control

100 mg/L
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Appendix 1 

Freshwater AAP Algal Medium 

 Compound 

Nominal 

Concentration 1

MgCl2•6H2O 

CaCl2•2H2O 

H3BO3 

MnCl2•4H2O 

ZnCl2

FeCl3•6H2O 

CoCl2•6H2O 

Na2MoO4•2H2O 

CuCl2•2H2O 

Na2EDTA•2H2O 

NaNO3

MgSO4•7H2O 

K2HPO4

NaHCO3

12.164 mg/L 

 4.410 mg/L 

 0.1855 mg/L 

 0.4154 mg/L 

 3.27 μg/L 

 0.1598 mg/L 

 1.428 μg/L 

 7.26 μg/L 

 0.012 μg/L 

 0.300 mg/L 

25.50 mg/L 

14.70 mg/L 

 1.044 mg/L 

15.00 mg/L 

1

 The pH of the medium was 7.5. 
Wildlife International, Ltd. 

 Project Number 527A-101 

-28- 

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. 
Wildlife International, Ltd. 

 Project Number 527A-101 

-29- 

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. 
Wildlife International, Ltd. 

 Project Number 527A-101 

-30- 

Appendix 3 

Cell Density By Replicate Over the 72-hour Exposure Period 

Nominal WAF Cell Density (cells/mL) 

Loading Rate 

(mg/L) Replicate 24 Hours 1 48 Hours 72 Hours 

Negative Control A 56,000 214,000 900,000 

 B 46,000 276,000 1,460,000 

 C 42,000 325,000 1,480,000 

 D 50,000 226,000 1,100,000 

 E 44,000 239,000 1,310,000 

 F 56,000 236,000 1,170,000 

 

100 A 46,000 241,000 870,000 

 B 48,000 290,000 1,730,000 

C 65,000 245,000 1,250,000 

 D 44,000 277,000 1,110,000 

 E 33,000 252,000 1,300,000 

 F 50,000 255,000 1,230,000 

1 The initial cell density of the stock culture was determined and an inoculum volume was administered to each 

test chamber to yield a cell density of approximately 10,000 cells/mL at test initiation (0 hours). 
Wildlife International, Ltd. 

 Project Number 527A-101 

-31- 

Appendix 4 

Yield By Replicate at 72-Hours 

Nominal WAF Cell Density (cells/mL) 

Loading Rate 

(mg/L) Replicate 72 Hours 

Negative Control A 890,000 

 B 1,450,000 

 C 1,470,000 

 D 1,090,000 

 E 1,300,000 

 F 1,160,000 

 

100 A 860,000 

 B 1,720,000 

C 1,240,000 

 D 1,100,000 

 E 1,290,000 

 F 1,220,000 
Wildlife International, Ltd. 

 Project Number 527A-101 

-32- 

Appendix 5

Growth Rate By Replicate Over the 72-hour Exposure Period 

Nominal WAF Growth Rate (per hour) 

Loading Rate 

(mg/L) Replicate 0 - 24 Hours 0 – 48 Hours 0 – 72 Hours 

Negative Control A 0.0718 0.0638 0.0625 

B 0.0636 0.0691 0.0692 

C 0.0598 0.0725 0.0694 

 D 0.0671 0.0650 0.0653 

 E 0.0617 0.0661 0.0677 

 F 0.0718 0.0659 0.0661 

 

100 A 0.0636 0.0663 0.0620 

B 0.0654 0.0702 0.0716 

C 0.0780 0.0666 0.0671 

 D 0.0617 0.0692 0.0654 

 E 0.0497 0.0672 0.0676 

 F 0.0671 0.0675 0.0668 

 
Wildlife International, Ltd. 

 Project Number 527A-101 

-33- 

Appendix 6

Specific Growth Rate of the Negative Control Group 

Section-by-Section Specific Growth Rates for Negative Controls: 

Control 

Replicate 

0-24 Hour 

Growth Rate 

24-48 Hour 

Growth Rate 

48-72 Hour 

Growth Rate 

0-72 Hour 

% Coefficient of Variation1 

Negative 

Control A 0.0718 0.0559 0.0599 13.26 

Negative 

Control B 0.0636 0.0747 0.0694 8.00 

Negative 

Control C 0.0598 0.0853 0.0632 19.93 

Negative 

Control D 0.0671 0.0629 0.0659 3.34 

Negative 

Control E 0.0617 0.0705 0.0709 7.65 

Negative 

Control F 0.0718 0.0599 0.0667 8.98 

 Mean: 10.19 

1

Coefficient of Variation = (S.D./mean) *100 

 Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ 

slightly. 

Average Specific Growth Rates for the Negative Controls During the Whole Test Period: 

Control Replicate 0-72 Hour Growth Rate 

Negative Control A 0.0625 

Negative Control B 0.0692 

Negative Control C 0.0694 

Negative Control D 0.0653 

Negative Control E 0.0677 

Negative Control F 0.0661 

% Coefficient of Variation: 3.95
Wildlife International, Ltd. 

 Project Number 527A-101 

-34- 

Appendix 7 

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. Willard B. Nixon, Ph.D., Director of Chemistry 

3. John R. Porch, M.S., Supervisor of Nontarget Plant and Invertebrate Studies 

4. Allison M. Jacobs, Biologist 

5. Josh R. Arnie, Biologist 

6. Tara L. Cartee, Ph.D., Senior Biologist