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 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: ____________

# 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

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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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TABLE OF CONTENTS

Title 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 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, E y C50, and E r C50 Values Over the 72-hour Exposure Period................................ 24

Figure 1. Growth of Pseudokirchneriella subcapitata in the Negative Control Over the 72-hour Exposure Period, Expr essed 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 E y C50: >100 mg/L 95% Confidence Interval: Not calculable 72-hour NOEC: ≥ 100 mg/L

GROWTH RATE: 72-Hour E r C50: >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 fac ility 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 locat ed on the Wildlife International, Ltd. site.

OBJECTIVE The objective of this study was to determine the t oxicity 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 sel ected in consultation with the Sponsor based on information provided by the Sponsor. For this test, th e 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 alga l cells was added to each test chamber to achieve a nominal concentration of approximately 10,000 cells/m L. Samples were coll ected from each replicate test chamber at approximately 24-hour intervals duri ng the test to determine cell densities, yield, and growth rates. Cell densities and growth rates were u sed to calculate percent inhibition values relative to the controls over the 72-hour exposure period. Percen t inhibition for yield was calculated at 72 hours.
EC50 and E r C50 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. E y C50 (yield) was
calculated at 72 hours. No-observed-effect concentr ations (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 pro cedures 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 Intern ational, 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. Residu es; CAS No. 1160164-88-4; lot number 03585/MA. The test substance contained 100% active ingredient and had an expirati on 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 impor tant group of freshwater algae, and was selected for use in the test based upon a past history of use, a nd ease of culturing in the laboratory. Original algal cultures were obtained from the University of Toront o 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 ce lls 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 ar e 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 fr eshwater AAP algal medium (3). Stock nutrient solutions were prepared by adding reagent-grade chem icals 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 Erlenm eyer 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 indiscriminat ely positioned daily on a mechanical shaker in an environmental chamber designed to maintain the desi red 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 th e test chambers in the environmental chamber was recorded twice daily during the test using a liquid-in-g lass 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 in tensity was measured at test solution level at five locations surrounding the test flasks at test initia tion 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 Th ermo 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 sam ples 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 th e test solution height. Following the mixing period, the WAF solution was allowed to settle for approxi mately one hour before the solution was decanted from mid-depth into the test chambers through a spi got and tubing placed approximately 2-3 cm from the bottom of the aspirator bottle. The negative contro l 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 to measure concentrations of total orga nic 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 10 6 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 rep licate 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 ma ximum 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 we re diluted using an electron solution (Isoton ® ), as needed, to maintain counting accuracy. A sm all 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., cha nges in cell shape, size or color). Cells in the replicate test chambers also were assessed for aggregations or flocculati ons 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 contro l 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 =

o n o n 

### t

- t N ln

- N ln

where: μ = Average specific growth rate N o = Nominal cell density (cells/mL) at t o N n = Measured cell density (cells/mL) at t n t o = Time of beginning of test (hours) t n = 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 nega tive control replicates for each 24-hour interval. The following formula was used:

Percent Inhibition = sponse Re Control Mean sponse Re Treatment Mean sponse Re Control Mean

− × 100

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EC50 values in this study represent the theoreti cal test concentration that would produce a 50%
reduction in an effect relative to the control. Th ere was less than 50% reduction in cell density (EC50), yield (E y C50), and growth rate (E r C50) 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 ra te 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, we re 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 term ination, no surface slicks or particulate matter was present. Results of analyses for TOC in the test so lutions 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 in tensity 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, whic h is typical for tests conducted with Pseudokirchneriella subcapitata

. The light intensity ranged from 4,070 to 4,470 lux, wh ich 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 expos ure period. Cell densities were used to determine growth rates for each 24-hour interval of exposure, a nd yield at 72 hours. Mean cell densities, yield, and

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growth rates with the corresponding percent inhibiti on values are presented in Tables 5, 6 and 7, respectively. EC50, E y C50, and E r C50 values and the corresponding 95% confidence limits for cell density, yield and growth rates, respectively, are pres ented 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 ce ll 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 de nsities, 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. Consequen tly, the 72–hour NOEC for cell density, yield, and growth rate was ≥ 100 mg/L. The 72-hour EC50, E y C50, and E r C50 values were >100 mg/L. After 72 hours of exposure, there were no signs of adheren ce 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 concen tration 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 sp ecific growth rate in the control replicates during the whole test period did not ex ceed 7%. It was 3.95% (Appendix 6).
3) The mean percent coefficient of variati on for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) did not ex ceed 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 de nsity, yield, and growth rate. The 72–hour EC50,

E y C50, E r C50 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: S tandard Guide for Conducting Static 96-H our 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 c ontinuously 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

pH Measurements Nominal WAF 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.Wildlife International, Ltd.Project Number 527A-101-21-Table 5Mean Cell Density and Percent Inhibition24 Hours148 Hours 72 HoursNominal WAFLoading Rate(mg/L)MeanCell Density(cells/mL)PercentInhibition2,3MeanCell Density(cells/mL)PercentInhibition2,3MeanCell Density(cells/mL)PercentInhibition2,3Negative Control 49,000 -- 252,667 -- 1,236,667 --100 47,667 2.7 260,000 -2.9 1,248,333 -0.91The cell density in each replicate at test initiation (0 hours)was approximately 10,000 cells/mL. The mean cell density in the negative controlflasks increased by a factorof 124 within three days.2Calculations were performedusing SAS Version 8.2. Manualcalculations may differ slightly.3Percent 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).Wildlife International, Ltd.Project Number 527A-101-22-Table 6Mean Yield and Percent Inhibition72 HoursNominal WAFLoading Rate(mg/L)MeanCell Density(cells/mL)PercentInhibition1,2Negative Control 1,226,667 --100 1,238,333 -1.01Calculations were performed usingSAS Version 8.2. Manual calculationsmay differ slightly.2Percent inhibition was calculated relative to the negative control replicates.There was no statistically significant difference from the negative controlreplicates using Dunnett’s test (p> 0.05).Wildlife International, Ltd.Project Number 527A-101-23-Table 7Mean Growth Rate (per Hour) and Percent Inhibition0 - 24 Hours 0 - 48 Hours 0 - 72 HoursNominal WAFLoading Rate(mg/L)MeanGrowth RatePercentInhibition1,2MeanGrowth RatePercentInhibition1,2MeanGrowth RatePercentInhibition1,2Negative Control 0.0660 -- 0.0671 -- 0.0667 --100 0.0642 2.6 0.0678 -1.1 0.0668 -0.11Calculations were performed using SAS Version 8.2. Manual calculations may differ slightly.2Percent 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).Wildlife International, Ltd.Project Number 527A-101-24-Table 8EC50, EyC50, and ErC50 Values over the 72-Hour Exposure PeriodCell Density Yield Growth RateTimeEC50(mg/L)95% Confidence Interval(mg/L)EyC50(mg/L)95% Confidence Interval(mg/L)ErC50(mg/L)95% Confidence Interval(mg/L)24 Hours > 100 --1--2-- > 100 --148 Hours > 100 --1--2-- > 100 --172 Hours > 100 --1> 100 --1> 100 --1195% confidence limits could not becalculated with the data obtained.2Results 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. Residue s 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 MgCl 2 •6H 2 O CaCl 2 •2H 2 O H 3 BO 3 MnCl 2 •4H 2 O ZnCl 2 FeCl 3 •6H 2 O CoCl 2 •6H 2 O Na 2 MoO 4 •2H 2 O CuCl 2 •2H 2 O Na 2 EDTA•2H 2 O NaNO 3 MgSO 4 •7H 2 O K 2 HPO 4 NaHCO 3 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.

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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
Cell Density By Replicate Over the 72-hour Exposure Period

Cell Density (cells/mL) Nominal WAF 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 determin ed 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).

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Appendix 4
Yield By Replicate at 72-Hours

Cell Density (cells/mL) Nominal WAF 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

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

Growth Rate (per hour) Nominal WAF 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

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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 Variation 1

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 Negativ e 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

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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