AN-2: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA ( Pseudokirchneriella subcapitata )

FINAL REPORT

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-119

U.S. EPA OPPTS Number 850.5400
OECD Guideline 201
ASTM Standard E1218-04

AUTHORS:

Joshua R. Arnie, B.S. John R. Porch, M.S. Timothy Z. Kendall, M.S. Henry O. Krueger, Ph.D.

STUDY INITIATION DATE: 15 February 2012

STUDY COMPLETION DATE: 26 June 2012

SUBMITTED TO:

Albemarle Corporation
451 Florida Street Baton Rouge, LA 70801


# Wildlife International, Ltd.

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

Page 1 of 56

# W ildlife International, Ltd. Project Number: 471A-119

-2-

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality, on any basis whatsoever, is made for any information contained in this study. I acknowledge that the information not designated as within the scope of FIFRA section 10(d) (1)(A), (B) or (C) and which pertains to a registered or previously registered pesticide is not entitled to confidential treatment and may be released to the public , subject to the provisions regarding disclosure to multinational entities under FIFRA (10)g.

Company: ________________________________(Typed Name)

Company Agent: ________________________________(Typed Name)

Title: ________________________________

Signature:______________________________Date:______________________

# W ildlife International, Ltd. Project Number: 471A-119

-6-

TABLE OF CONTENTS

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

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

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

Quality Assuran ce Statement ................................................................................................... .............. 4

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

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

Summary ....................................................................................................................... ......................... 9

Introduction .................................................................................................................. ........................ 11

Objective ..................................................................................................................... ......................... 11

Experimental Design ........................................................................................................... ................. 11

Materials and Methods ......................................................................................................... ................ 12
Test Substance ............................................................................................................... .......... 12 Test Organism ................................................................................................................ .......... 12 Test Apparatus ............................................................................................................... .......... 13
Culture Medium ............................................................................................................... ........ 13
Environmental Conditions ..................................................................................................... .. 13 Preparation of Test Concentrations.......................................................................................... 1 4 Analytical Sampling .......................................................................................................... ...... 14 Analytical Method ............................................................................................................ ....... 14
Inoculation of Test Chambers ................................................................................................. . 15 Algal Growth Measurements and Observations ...................................................................... 15
Statistical Analyses ......................................................................................................... ......... 15

Results and Discussion ........................................................................................................ ................. 16
Measurement of Test Concentrations ...................................................................................... 16
Observations and Measurements ............................................................................................. 17 Conditions for the Validity of the Test .................................................................................... 1 8

Conclusions ................................................................................................................... ....................... 19

Refere nces .................................................................................................................... ........................ 20

# W ildlife International, Ltd. Project Number: 471A-119

-7-

TABLE OF CONTENTS (Continued)

TABLES AND FIGURES

Table 1. Measured Concentrations of 14 C AN-2 in Test Solution Samples Analyzed by
LSC ........................................................................................................................... .......... 21

Table 2. Temperature Measurements ............................................................................................... 22

Table 3. pH Measurements .............................................................................................................. . 22

Table 4. Light Intensity Measurements ............................................................................................ 22

Table 5. Mean Cell Density and Percent Inhibition ......................................................................... 23

Table 6. Mean Growth Rate (per Hour) and Percent Inhibition ....................................................... 24

Table 7. Mean Yield and Percent Inhibition ..................................................................................... 25

Table 8. NOEC, EC 50 , E y C 50 and E

r C 50 Values Over the 96-Hour Exposure Period ........................ 26

Figure 1. Growth of P. subcapitata in the Control Groups Over the 96-Hour Exposure
Period ........................................................................................................................ .......... 27

Figure 2. Growth of P. subcapitata Exposed to AN-2 for 96 Hours, Expressed as Cell Density ....................................................................................................................... ......... 27

APPENDICES

Appendix 1. Study Protocol .......................................................................................................... 28

Appendix 2. Certificate of Analysis .............................................................................................. 44

Appendix 3. Freshwater AAP Algal Medium ............................................................................... 45

Appendix 4. Analyses of Pesticides, Organics and Metals in Wildlife International,
Ltd. Well Water ........................................................................................................ 46 Appendix 5. Analysis of AN-2 in Freshwater AAP Algal Medium Analyzed by LSC ................ 48
5.1 Method Outline for the Analysis of 14 C AN-2 in Freshwater AAP Algal Medium ........................................................................................................... 49
5.2 Typical Counting Conditions for Liquid Scintillation Counting .............................. 50
5.3 Calculations for Sample Quantitation ....................................................................... 51

# W ildlife International, Ltd. Project Number: 471A-119

-8-

TABLE OF CONTENTS (Continued)

APPENDICES (Continued)

Appendix 6. Cell Density By Replicate Over the 96-Hour Exposure Period ............................... 52

Appendix 7. Yield By Replicate At 72 and 96 Hours of Exposure ............................................... 53

Appendix 8. Growth Rate By Replicate Over the 96-Hour Exposure Period ............................... 54

Appendix 9. Specific Growth Rate of the Negative Control Group .............................................. 55

Appendix 10. Personnel Involved in the Study ............................................................................... 56

# W ildlife International, Ltd. Project Number: 471A-119

-9-

SUMMARY

SPONSOR: Albemarle Corporation

STUDY: AN-2: A 96-Hour Toxicity Test with the Freshwater Alga ( Pseudokirchneriella subcapitata )

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-119

TEST DATES: Experimental Start (OECD): 20 February 2012 Experimental Start (EPA): 20 February 2012 Exposure Termination: 24 February 2012 Experimental Termination: 27 February 2012

LENGTH OF EXPOSURE: 96 Hours

TEST ORGANISM: Freshwater Alga ( Pseudokirchneriella subcapitata )

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

TEST CONCENTRATIONS: Day 0
Nominal Measured Negative Control < LOQ Solvent Control < LOQ 1.99 ng/L 1.50 ng/L 3.99 ng/L 3.35 ng/L 7.98 ng/L 6.84 ng/L 16.0 ng/L 12.9 ng/L 31.9 ng/L 26.5 ng/L

# W ildlife International, Ltd. Project Number: 471A-119

-10-

SUMMARY (Continued)

RESULTS:

Duration of

Exposure Cell Density Growth Rate Yield

EC 50 (ng/L) 1 95%
Confidence

Interval (ng/L) 1 E
r C 50 (ng/L) 1 95%
Confidence

Interval (ng/L) 1 E
r C 50 (ng/L) 1 95%
Confidence

Interval (ng/L) 1

24 Hours > 26.5 n/a 3 > 26.5 n/a 3

-- 2

-- 2

48 Hours > 26.5 n/a 3 > 26.5 n/a 3

-- 2

-- 2

72 Hours > 26.5 n/a 3 > 26.5 n/a 3 > 26.5 n/a 3

96 Hours > 26.5 n/a 3 > 26.5 n/a 3 > 26.5 n/a 3

NOEC (ng/L) NOEC (ng/L) NOEC (ng/L)

72 Hours 26.5 26.5 26.5

96 Hours 26.5 26.5 26.5

1 EC 50 , E y C 50 and E

r C 50 values and their 95% confidence intervals were calculated using non-linear regression (Bruce and

Versteeg 1992, Environmental Toxicology and Chemistry 11: 1485-1494) with rep licate data (cell density, yield or growth rate) and day 0 meas ured test concentrations.

2 E y C 50 values were only determined at 72 and 96 hours, per protocol.

3 95% confidence intervals could not be calculated with the data obtained.

# W ildlife International, Ltd. Project Number: 471A-119

-11-

INTRODUCTION

This study was conducted by Wildlife Internati onal, Ltd. for Albemarle Corporation at the

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

definitive test was conducted from 20 to 24 February 2012, with cell counts completed on 27 February

2012. Raw data generated by Wild life International, Ltd. and the final report are filed under Project

Number 471A-119 in archives located on the Wildlife International, Ltd. site.

OBJECTIVE

The objective of this study was to determine the toxicity of the test substance, AN-2

(4,4’-Methylenebis (2,6-di-tert-butyl[U- 14 C]phenol), to the freshwater green alga, Pseudokirchneriella

subcapitata , over a 96-hour exposure period.

EXPERIMENTAL DESIGN

The green alga, Pseudokirchneriella subcapitata , was exposed to five test concentrations, a

solvent control (0.1 mL N,N-dimethylformamide pe r L) and a negative control (culture medium) for

96 hours. Nominal test concentrations were selected in consultation with the Sponsor and were based

upon the results of an exploratory range-finding toxicity test. Nominal test concentrations selected were

2.0, 4.0, 8.0, 16.0 and 32.0 nanograms per liter (ng/L) . Measured concentrations were determined from

samples of test medium collected from each treatment and control group at 0 and 96 hours of the test.

Three replicate test chambers were maintained in each treatment group and six replicates were

maintained in the negative control group. At test in itiation, an inoculum of the algal cells was added to

each test chamber to achieve a nominal concentrati on of approximately 10,000 cells/mL. Samples were

collected from each replicate test chamber at approxi mately 24-hour intervals during the test to determine

cell densities, yield and growth rates. Cell densities, yields and growth rates were used to calculate

percent inhibition values relative to the c ontrol over the 96-hour exposure period. EC 50 and E

r C 50 values

(i.e., the theoretical toxicant concentrations that would produce a 50% reduction in cell density and

growth rate, respectively) were calculated, when possible, for each 24-hour interval of the exposure

period. E y C 50 values (i.e., the theoretical toxicant concentrations that would produce a 50% reduction in

yield) were calculated, when possible, at 72 and 96 hours of exposure. No-observed-effect concentrations

# W ildlife International, Ltd. Project Number: 471A-119

-12-

(NOEC) were determined at 72 and 96 hours through st atistical evaluation of cell densities, yields 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, “AN-2: A

96-Hour Toxicity Test with the Freshwater Alga ( Pseudokirchneriella subcapitata )” ( Appendix 1 ). The

protocol was based on procedures outlined in the U.S. Environmental Protection Agency Series 850 –

Ecological Effects Test Guidelines, OPPTS Number 850.5400: Algal Toxicity Tiers I and II (1), ASTM

Standard E1218-04: Standard Guide for Conducting Static Toxicity Test with Microalgae (2), OECD

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

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

Inhibition Test (4).

Test Substance

The 14 C radiolabelled test substance used to prepar e the test solutions for the study was received

from Quotient Bioresearch on May 23, 2011, and was assigned Wildlife International, Ltd. identification

number 10063A, upon receipt. The radiolabelled test s ubstance was stored under frozen conditions. The

test substance, a liquid, was identified as: 4,4’-M ethylenebis (2,6-di-tert-butyl[U-14C]phenol); Batch

number CFQ41115. The test substance had a reported specific activity of 129 μ Ci/mg or 55 mCi/mmol,

and a radiochemical concentration of 968 μ Ci/mL ( Appendix 2 ).

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 three days prior to test initiation. The negative controls organisms were

expected to exhibit exponential growth over the 96-hour exposure period. Exponential growth phase,

# W ildlife International, Ltd. Project Number: 471A-119

-13-

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 ).

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 t est chambers were labeled with the project number,

test concentration and replicate, and were indiscrim inately 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.

Culture Medium

The algal cells were cultured and tested in fresh water AAP algal medium (1-4). Stock nutrient

solutions were prepared by adding reagent-gr ade chemicals to purified well water (NANOpure ® water).

The test medium was then prepared by adding appropriate volumes of the stock nutrient solutions to

NANOpure ® water ( Appendix 3 ). The pH of the medium was adjusted to 7.5 ± 0.1 using

10% hydrochloric acid and was sterilized by filtration (0.22 μm) prior to use. The results of most recent

analyses performed to measure the concentrations of selected contaminants in the well water used to

prepare the algal medium are presented in Appendix 4

.

Environmental Conditions

Test chambers were held in an environmental chamber at a temperature of 24 ± 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 lighti ng throughout the test. The target light intensity

was 6,000 lux ± 10%. 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 525 APlus. 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 th e replicates of each treatment and control group.

# W ildlife International, Ltd. Project Number: 471A-119

-14-

Preparation of Test Concentrations

A primary stock solution was prepared by dissolving 0.213 mL of radiolabelled AN-2 in 50 mL

of N,N-diemthylformamide to achieve a nominal concentration of 32.0 μ g/mL. The primary stock was

mixed by inversion and appeared clear and purple in color upon preparation. A secondary stock was

prepared by diluting 1.0 mL of 32.0 μ g/mL primary stock in 100 mL of N,N-dimethylformamide to

achieve a nominal concentration of 0.319 μ g/mL. Additional stocks were prepared at nominal

concentrations of 0.16, 0.08, 0.04, 0.02 μ g/mL in N,N-dimethylformamide by serial dilution. The 2.0,

4.0, 8.0, 16.0 and 32.0 ng/L test solutions were prepared by diluting 100 μ L of each respective stock

solution in 1000 mL of freshwater AAP medium. The solvent control solution was prepared by diluting

100 μ L of N,N-dimethylformamide in 1000 mL of freshwater AAP medium to achieve a nominal

concentration of 0.1 mL N,N-dimethylformamide per lite r, equivalent to the solvent concentration in all

of the test solutions except the negative control. The negative control solution consisted of freshwater

AAP algal medium without test substance or solvent a dded. All test solutions were mixed by inversion,

and appeared clear and colorless and were otherwise unremarkable after mixing.

Analytical Sampling

Samples of the test solutions were collected at approximately 0 and 96 hours to measure

concentrations of the test substance. Samples at test initiation were collected from the individual batches

of test solution prepared for each tr eatment and control group after distribu tion into the test chambers. At

exposure termination, samples were collected from th e pooled replicates from each treatment and control

group. Samples were processed immediately for LSC analysis.

Analytical Method

The analytical method used to analyze the test samples was based upon methodology developed

by Wildlife International, Ltd. The concentrations of the test substance in freshwater algal medium

samples were verified by liquid scintillation counting (LSC). Samples were extracted two times with

dichloromethane, rotary evaporated to dryness and reconstituted using acetonitrile. Extracts were

quantitatively transferred into scintillation vials containing 10 mL of Ultima Gold XR scintillation

cocktail and submitted for analysis by LSC. A Packard 2500TR Liquid Scintillation Analyzer was used

to determine disintegrations per minute (dpm). In a ddition to the test samples, a 10 mL sample of Ultima

Gold XR scintillation cocktail was an alyzed at each interval to determine background radioactivity.

# W ildlife International, Ltd. Project Number: 471A-119

-15-

Measured concentrations of 14 C-AN-2 are presented in Table 1. A method flowchart is presented in

Appendix 5.1 and instrument parameters are presente d in Appendix 5.2. The calculations for a sample

quantitation are included in Appendix 5.3.

Inoculation of Test Chambers

Prior to test initiation, the concentration of algal cells in the P. subcapitata stock culture (00-1)

was determined using a hemacytometer and micro scope. The density of stock culture 00-1 was

7.35 x 10 5 cells/mL. In order to achieve the desired in itial cell density of approximately 10,000 cells per

milliliter, 1.36 mL of culture was added to each test chamber.

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 96-hour exposure and were held for a ma ximum of three days under refrigerated conditions

sufficient to inhibit growth until cell counts could be performed.

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

were pooled within their respective treatments, and sub-samples 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 aggregation or flocculation 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 Window s, Version 8.2” (5). 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)

# W ildlife International, Ltd. Project Number: 471A-119

-16-

Yield was calculated for each replicate of the control and treatment groups as the final biomass

(cell density) in the exposure period minus the initia l biomass (cell density). Inhibition values were

calculated for each treatment group as the percent reduction in cell density and growth rate relative to the

negative control replicates for each 24-hour interval , and yield at 72 and 96 hours of exposure. The

following formula was used:

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

 × 100

EC 50 values in this study represent the theoretical test concentrations that would produce a 50%

reduction in a variable of interest relative to the negative control. The E y C50 and E

r C50 values and their

95% confidence intervals were calculated, when po ssible, using non-linear regression (6) with replicate

data (cell density, yield or growth rate ) and day 0 measured test concentrations.

The negative and solvent control data was compared using a student’s t-test ( α = 0.05). No

statistically significant differences were detected be tween the negative and solvent control data. The

treatment group responses were compared to the nega tive control response for statistical comparisons and

EC 50 calculations, when possible. The 72 and 96-hour cell density, yield and gr owth rate data were

evaluated for normality and homogeneity of variance ( α = 0.01) using Shapiro-Wilk’s and Levene’s tests,

respectively. All data met assumptions or normal dist ribution and equal variance; therefore, the treatment

groups were compared to the negative c ontrol using Dunnett’s one-tailed t-test ( α = 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 and 96 hours.

RESULTS AND DISCUSSION

Measurement of Test Concentrations

Results of analyses to measure concentrations of 14 C AN-2 by LSC in test solution samples

collected during the study are presented in Table 1 . Nominal test concentrations were 2.0, 4.0, 8.0, 16.0

and 32.0 ng/L. The test solutions appeared clear a nd colorless at the time of preparation, and there was

no evidence of surface slicks or precipitation at expos ure termination. Measured concentrations on Day 0

ranged from 75 to 86% of nominal. Measured concentr ations declined by Day 4, and ranged from 16 to

# W ildlife International, Ltd. Project Number: 471A-119

-17-

24% of nominal. The results of the study are based on day 0 measured concentrations of 1.50, 3.35, 6.84,

12.9 and 26.5 ng/L, representing 75, 84, 86, 81 and 83% of the target nominal test concentrations.

Measured concentrations of 14 C AN-2 by LSC analysis in the negative and solvent controls were less than

the limit of quantitation (LOQ) at both sampling intervals.

Observations and Measurements

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

, 3 and 4

, respectively. Temperatures remained within the 24 ± 2°C range established for the test. The pH of the

test solutions ranged from 7.1 to 7.4 at test initiation and ranged from 8.3 to 8.5 at test termination. An

increase in pH is typical for tests conducted with P. subcapitata and is typically attributed to the

photosynthetic activity of the algae. The light inte nsity ranged from 5,870 to 6,430 lux, which was within

the desired range of 6,000 ± 10% lux.

The toxicity of AN-2 to P. subcapitata was determined by evaluating changes in cell density over

a 96-hour exposure period. Cell densities were used to determine growth rates for each 24-hour interval

of exposure and yields at 72 and 96 hours of exposure. Mean cell densiti es, yields and growth rates and

their corresponding percent inhibition values are presented in Tables 5

, 6 , and 7 , respectively. EC 50 ,

E y C 50 and E

r C 50 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 6

, 7 , and 8

. Figure 1 illustrates changes in mean cell density in the negative

control group throughout the test. Exponential growth, characterized by the linear section of the growth

curve, occurred throughout the 96-hour exposure period. Appendix 9 provides the coefficient of variation

for the specific growth rates of the control replicates. Figure 2 illustrates growth of P. subcapitata

exposed to AN-2, expressed as changes in cell density over the 96- hour exposure period.

After 72 hours of exposure, inhibition of cell dens ity in the 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L

treatment groups was 15, 8, 49, -9 and -3%, respectivel y, relative to the negative control. Inhibition of

yield in the 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L treatme nt groups was 15, 8, 49, -9 and -3, relative to the

negative control. Inhibition of growth rate in th e 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L treatment groups

was 4, 1, 13, -2 and -1%, respectively, relative to th e negative control. Dunnett’s test indicated that there

were significant reductions ( p ≤ 0.05) in cell density, yield and growth rate in the 6.8 ng/L treatment

group. Since this reduction was not dose-responsive and appeared analogous to the data collected from

# W ildlife International, Ltd. Project Number: 471A-119

-18-

the other treatment groups, it was not considered to be treatment related. Consequently, the 72–hour

NOEC for cell density, yield and growth rate was dete rmined to be 26.5 ng/L based on evaluation of the

dose-response.

After 96 hours of exposure, inhibition of cell de nsity in 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L

treatment groups was -23, -22, 12, -10 and 5%, respectiv ely, relative to the negative control. Inhibition of

yield in 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L tr eatment groups was –24, -22, 12, -10 and -5%,

respectively, relative to the negative control. Inhib ition of growth rate in the 1.50, 3.35, 6.84, 12.9 and

26.5 ng/L treatment groups was –3, -4, 2, -2 and -1%, respectively, relative to the negative control. No

significant reductions in cell density, yield or growth rate were detected (Dunnett’s test, p > 0.05) when

the responses of the treatment groups were compared to the negative cont rol response. Consequently, the

96–hour NOEC for cell density, yield and growth rate was 26.5 ng/L.

After 96 hours of exposure, cells present in the 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L treatment

groups appeared normal when compared to cells pres ent in the negative and solvent control groups during

microscopic observations of test solution samples. A dherence to the test chambers and flocculation or

aggregation of cells were not observe d in any of the experimental groups.

Conditions for the Validity of the Test

1) The mean cell density in the control flasks in creased by a factor greater than 16 within three days and by a factor greater than 100 within four days. After three days the mean cell density in the negative control replicates increased by a factor of 189 and after four days the mean cell density increased by a factor of 382.

2) The coefficient of variation of average speci fic growth rates in the control replicates during the first 72 hours did not exceed 7%. It was 5.45% ( Appendix 9 ).

3) The mean coefficient of variation for section- by-section specific growth rates (days 0-1, 1-2, and 2-3) did not exceed 35%. It was 26.3% ( Appendix 9 )

# W ildlife International, Ltd. Project Number: 471A-119

-19-

CONCLUSIONS

The freshwater green alga, Pseudokirchneriella subcapitata , was exposed to a geometric series of

five concentrations of AN-2 ranging from 1. 50 to 26.5 ng/L (based on day 0 measured test

concentrations). Effects were ev aluated based on cell density, yield a nd growth rate. The 72 and 96-hour

EC 50 , E y C 50 and E

r C50 values and corresponding 95% confidence intervals, based on cell density, yield

and growth rate, were all greater than 26.5 ng/L, the highest concentration tested. The 72 and 96-hour

no-observed-effect-concentrations (NOEC), based on e ffects on cell density, yield and growth rate were

26.5 ng/L, for both intervals of exposure.

# W ildlife International, Ltd. Project Number: 471A-119

-20-

REFERENCES

1 U.S. Environmental Protection Agency. 1996. Series 850-Ecological Effects Test Guidelines ( draft ), OPPTS Number 850.5400: Algal Toxicity, Tiers I and II

.

2 American Society for Testing and Materials. 1997. ASTM Standard Guide 1218-04.
S tandard Guide for Conducting Static 96-H our Toxicity Tests with Microalgae

3 Organization 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.

4 . Official Journal of the European Communities. 1992. No. L383. Method C.3. Algal
Inhibition Test.

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

6 Bruce, Robert D. and Donald J. Versteeg. 1992. A Statistical Procedure for Modeling Continuous Toxicity Data. Environmental Toxicology and Chemistry. 11: 1485-1494.Wildlife International, Ltd.Project Number: 471A-119-21-Table 1Measured Concentrations of14C-AN-2 in Test Solution Samples Analyzed by LSCNominalAN-2Concentration(ng/L)SampleNumber(471A-119-)SamplingInterval(Day)SampleVolume(L)SpecificActivity(dpm/g)Total[14C]Found(dpm)AN-2EquivalentsFound(ng/L)1,2,3PercentofNominalMeanAN-2Equivalents(ng/L)MeanPercent ofNominalBackground ----04--------27.3831.06---------- --Negative Control(0.0)18040.3000.30028638028638027.1535.76< LOQ< LOQ------ --Solvent Control(0.0)29040.3000.16028638028638030.4334.57< LOQ< LOQ------ --2.0 310040.3000.150286380286380156.3251.361.500.47375.023.70.987 494.0 411040.3000.150286380286380315.5959.673.350.66683.816.72.01 508.0 521040.2000.140286380286380418.8982.656.841.2985.516.14.07 5116.0 613040.1000.150286380286380396.12137.8612.92.4980.615.67.70 4832.0 714040.1000.150286380286380787.48285.3826.55.9282.818.516.2511AN-2 Equivalents = ({[(Total dpm found – backgroundcontribution)/Sample volume]}/Specific Activity) x 10002The calculated limit of quantitation (LOQ) was 0.582 ng/L.3Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.

# W ildlife International, Ltd. Project Number: 471A-119

-22-

Table 2

Temperature Measurements

Time (Days) Temperature (  C) 1

Measurement 1 Measurement 2 2

0 24.5 24.6
1 24.6 24.9
2 24.9 24.6
3 24.5 24.4
4 24.3 24.4

1 The minimum and maximum temperatures monitored c ontinuously throughout the study were 22.4 to 25.5°C, respectively.

2 Temperature Measurement 2 was typically ta ken at least 4 hours after Measurement 1.

Table 3

pH Measurements
Day 0
Measured Test
Concentration (ng/L) pH Measurements

Day 0 1 Day 4 2

Negative Control 7.1 8.3
Solvent Control 7.2 8.4

1.50 7.2 8.5
3.35 7.2 8.3
6.84 7.3 8.4
12.9 7.4 8.4
26.5 7.4 8.4

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 4 samples were composites of test solution collected from each of the replicates per treatment and control group at test termination.

Table 4

Light Intensity Measurements

Day Light Intensity (lux) 1

Measurement
No. 1 Measurement
No. 2 Measurement
No. 3 Measurement
No. 4 Measurement

No. 5
0 6,190 6,320 6,430 5,870 6,380

1 Measurements taken at five locations surrounding the test flasks at test solution level.Wildlife International, Ltd.Project Number: 471A-119-23-Table 5Mean Cell Density and Percent InhibitionDay 0Measured TestConcentration(ng/L)24 Hours148 Hours172 Hours196 Hours1MeanCell Density(cells/mL)PercentInhibition2MeanCell Density(cells/mL)PercentInhibition2MeanCell Density(cells/mL)PercentInhibition2MeanCell Density(cells/mL)PercentInhibition2Negative Control 56,333 --493,333 -- 1,885,000 -- 3,816,667 --Solvent Control71,333 -- 486,667 -- 1,846,667 -- 4,093,333 --1.5065,000 -15 363,333 26 1,610,000 15 4,713,333 -233.3558,000 -3 343,333 30 1,736,667 8 4,646,667 -226.8463,667 -13 283,333 43 960,000 49* 3,373,333 1212.9100,667 -79 616,667 -25 2,053,333 -9 4,180,000 -1026.587,667 -56 593,333 -20 1,940,000 -3 4,006,667 -51Calculations were performed using the SAS System for Windows, Version 8.2.Manual calculations may differ slightly.2Percent inhibition was calculated relative to the negative control replicates. Negative values indicate an increase relativeto the negative control mean.* Statistically significant reduction from the negativecontrol mean using Dunnett’s one-tailed t-test (p0.05).Wildlife International, Ltd.Project Number: 471A-119-24-Table 6Mean Growth Rate (per Hour) and Percent InhibitionDay 0Measured TestConcentration(ng/L)0-24 Hours10-48 Hours10-72 Hours10-96 Hours1MeanGrowth Rate(hours-1)PercentInhibition2MeanGrowth Rate(hours-1)PercentInhibition2MeanGrowth Rate(hours-1)PercentInhibition2MeanGrowth Rate(hours-1)PercentInhibition2Negative Control 0.0705 -- 0.0803 -- 0.0724 -- 0.0618 --Solvent Control0.0791 -- 0.0799 -- 0.0722 -- 0.0626 --1.500.0770 -9 0.0747 7 0.0696 4 0.0639 -33.350.0729 -3 0.0735 9 0.0714 1 0.0640 -46.840.0748 -6 0.0696 13 0.0626 13* 0.0606 212.90.0961 -36 0.0854 -6 0.0740 -2 0.0628 -226.50.0903 -28 0.0850 -6 0.0730 -1 0.0624 -11Calculations were performed using the SAS System for Windows, Version 8.2.Manual calculations may differ slightly.2Percent inhibition was calculated relative to the negative control replicates. Negative values indicate an increase relativeto the negative control mean.* Statistically significant reduction from the negativecontrol mean using Dunnett’s one-tailed t-test (p0.05).Wildlife International, Ltd.Project Number: 471A-119-25-Table 7Mean Yield and Percent InhibitionDay 0Measured TestConcentration(ng/L)72 Hours196 Hours1MeanCell Density(cells/mL)PercentInhibition2MeanCell Density(cells/mL)PercentInhibition2Negative Control 1,875,000 -- 3,806,667 --Solvent Control1,836,667 -- 4,083,333 --1.501,600,000 15 4,703,333 -243.351,726,667 8 4,636,667 -226.84950,000 49* 3,363,333 1212.92,043,333 -9 4,170,000 -1026.51,930,000 -3 3,996,667 -51Calculations were performed using the SAS Systemfor Windows, Version 8.2.Manual calculations maydiffer slightly.2Percent inhibition was calculated relative to the negative control replicates. Negative values indicate anincrease relative to the negative control mean.* Statistically significant reduction from the negative control mean using Dunnett’s one-tailed t-test(p0.05).Wildlife International, Ltd.Project Number: 471A-119-26-Table 8NOEC, EC50, EyC50and ErC50Values Over the 96-Hour Exposure PeriodDuration ofExposureCell Density Growth Rate YieldEC50(ng/L)195%ConfidenceInterval(ng/L)1ErC50(ng/L)195%ConfidenceInterval(ng/L)1ErC50(ng/L)195%ConfidenceInterval(ng/L)124 Hours > 26.5 n/a3> 26.5n/a3--2--248 Hours > 26.5n/a3> 26.5n/a3--2--272 Hours > 26.5n/a3> 26.5n/a3> 26.5n/a396 Hours > 26.5n/a3> 26.5n/a3> 26.5n/a3NOEC (ng/L) NOEC (ng/L) NOEC (ng/L)72 Hours 26.5 26.5 26.596 Hours 26.5 26.5 26.51EC50, EyC50and ErC50values and their 95% confidence intervals werecalculated using non-linear regression (Bruce andVersteeg 1992, Environmental Toxicologyand Chemistry 11: 1485-1494) with replicate data (cell density, yield orgrowth rate) and day 0 measured test concentrations.2EyC50values were only determinedat 72 and 96 hours, per protocol.395% confidence intervals could not be calculated with the data obtained.

# W ildlife International, Ltd. Project Number: 471A-119

-27-

Figure 1

Growth of P. subcapitata in the Control Groups Over the 96-Hour Exposure Period

Figure 2

Growth of P. subcapitata Exposed to AN-2 for 96 Hours, Expressed as Cell Density 1,000 10,000 100,000 1,000,000 10,000,000

0 24 48 72 96

Exposure Duration (hours)Cell Density (cells/mL) Negative
Control

Solvent
Control

1,000 10,000 100,000 1,000,000 10,000,000

0 24 48 72 96

Exposure Duration (Hours)Cell Density (cells/mL) Negative Control

Solvent Control

1.50 ng/L

3.35 ng/L

6.84 ng/L

12.9 ng/L

26.5 ng/L

# W ildlife International, Ltd. Project Number: 471A-119

-28-

Appendix 1

Study Protocol

# W ildlife International, Ltd. Project Number: 471A-119

-29-

# W ildlife International, Ltd. Project Number: 471A-119

-30-

# W ildlife International, Ltd. Project Number: 471A-119

-31-

# W ildlife International, Ltd. Project Number: 471A-119

-32-

# W ildlife International, Ltd. Project Number: 471A-119

-33-

# W ildlife International, Ltd. Project Number: 471A-119

-34-

# W ildlife International, Ltd. Project Number: 471A-119

-35-

# W ildlife International, Ltd. Project Number: 471A-119

-36-

# W ildlife International, Ltd. Project Number: 471A-119

-37-

# W ildlife International, Ltd. Project Number: 471A-119

-38-

# W ildlife International, Ltd. Project Number: 471A-119

-39-

# W ildlife International, Ltd. Project Number: 471A-119

-40-

# W ildlife International, Ltd. Project Number: 471A-119

-41-

# W ildlife International, Ltd. Project Number: 471A-119

-42-

# W ildlife International, Ltd. Project Number: 471A-119

-43-

# W ildlife International, Ltd. Project Number: 471A-119

-44-

Appendix 2

Certificate of Analysis

# W ildlife International, Ltd. Project Number: 471A-119

-45-

Appendix 3

Freshwater AAP Algal Medium 1

Compound Nominal
Concentration 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 35.0 mg/L

1 The pH of the medium was adjusted to 7.5 ± 0.1 using 10% HCl.

# W ildlife International, Ltd. Project Number: 471A-119

-46-

Appendix 4

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 < 0.0098 Gamma BHC – Lindane < 0.0098 Alpha BHC < 0.0098 Gamma Chlordane < 0.020 Alpha Chlordane < 0.0098 Guthion (Azinphos-methyl) < 5.9

Beta BHC < 0.0098 HCB < 0.0098 Bolstar < 2.0 Heptachlor < 0.0098 Chlordane < 0.49 Heptachlor Epoxide < 0.0098 Coumaphos < 2.9 Kepone < 0.20

Delta BHC < 0.0098 Malathion < 2.9 Demeton-O < 2.0 Merphos < 2.0 Demeton-S < 2.9 Methoxychlor < 0.098 Diazinon < 5.9 Methyl Parathion < 2.0 Dichlorvos < 2.9 Mevinphos < 4.9

Dieldrin < 0.020 Mirex < 0.25

Disulfoton < 2.0 Naled < 5.9 Dursban (Chlorpyrifos) < 2.9 o,p-DDD < 0.020 Endosulfan I < 0.0098 o,p-DDE < 0.74 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 < 0.098 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 < 2.9 Telodrin < 0.0098 Ethyl Parathion < 2.0 Tokuthion < 2.0 Famphur < 2.9 Toxaphene < 2.9

Fensulfothion < 8.8 Trichloronate < 2.0

Fenthion < 2.0 Trithion < 2.0

1 Analyses performed by Lancaster Laborator ies on samples collected on December 29, 2010.

# W ildlife International, Ltd. Project Number: 471A-119

-47-

Appendix 4 (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 13.0 Antimony < 0.0200 Manganese < 0.0050 Arsenic < 0.0200 Mercury < 0.00020

Barium < 0.0050 Nickel < 0.0100 Beryllium < 0.0050 Nitrate Nitrogen 0.55 Bromide < 2.5 Nitrite Nitrogen < 0.50

Cadmium < 0.0050 Potassium 6.77

Calcium 35.1 Selenium < 0.0200

Chloride 3.5 Silver < 0.0050

Chromium < 0.0150 Sodium 18.5

Cobalt < 0.0050 Sulfate 5.6 Copper < 0.0100 Thallium < 0.0300

Fluoride < 0.50 Vanadium < 0.0050

Iron < 0.200 Zinc < 0.0200

Lead < 0.0150

1 Analyses performed by Lancaster Laborator ies on samples collected on December 29, 2010.

# W ildlife International, Ltd. Project Number: 471A-119

-48-

Appendix 5

Analysis of AN-2 in Freshwater AAP Algal Medium Analyzed by LSC

# W ildlife International, Ltd. Project Number: 471A-119

-49-

Appendix 5.1

Method Outline of the Analysis of 14 C-AN-2 in Freshwater AAP Algal Medium



### METHOD OUTLINE FOR THE ANALYSIS OF

14 

### C-AN-2 IN FRESHWATER AAP ALGAL MEDIUM

Rinse separatory funnels and roundbotto m flasks with dichloromethane. 

Using a tiltapetter, add 100 mL of dichloromethane to each sample. Stopper and shake each sample, with venting, for approximately one minute. Allow the organic and aqueous layers to separate. Drain the dichloromethane (lower) layer into a round bottom flask. 

Add 100 mL of dichloromethane to separator y funnel containing the aqueous fraction. Shake and partition as described above and combine each extract in its respective roundbottom flask. ↓

Rotary evaporate each sample to approximately 0.50 – 1.0 mL using a water bath maintained at ~40°C. Do not rotary evaporate to dryness. ↓

Evaporate the samples to dryness under a gentle stream of nitrogen. ↓

Rinse the roundbottom flask with several aliquots of acetonitrile. Transfer the rinsates to a scintillation vial. Add approximately 10 mL of Ultima Gold XR and analyze by LSC.

# W ildlife International, Ltd. Project Number: 471A-119

-50-

Appendix 5.2

Typical Counting Conditions for Liquid Scintillation Counting

INSTRUMENT: Packard 2500TR Liquid Scintillation Analyzer

COUNTING TIME: 3.00 minutes

DATA MODE: DPM

NUCLIDE: 14 C-UG

QUENCH INDICATOR: tSIE/AEC

EXT. STD. TERMINATOR: Count

QUENCH SET: 14 C-UG

# W ildlife International, Ltd. Project Number: 471A-119

-51-

Appendix 5.3

Calculations for Sample Quantitation

Sample Quantitation

The equivalent concentrations of 14 C-AN-2 (ng/L for water) were calculated from the following equation:

Equivalent Concentration (ng/L) of 14 C-AN-2= ({[(Total dpm found - background)/Sample

volume]}/Specific Activity x 1000

Limit of Quantitation for Freshwater

The limit of quantitation (LOQ) was calculated from the following equation:

LOQ (ng/L) = typical background contribution (50 dpm))/Sample volume (0.300 L)/Specific activity

(286380) x 1000

The calculated limit of quantitation (LOQ) was 0.582 ng/L

# W ildlife International, Ltd. Project Number: 471A-119

-52-

Appendix 6

Cell Density By Replicate Over the 96-Hour Exposure Period

Day 0 Measured

Concentration (ng/L) Replicate Cell Density 1 (cells/mL)

24 Hours 48 Hours 72 Hours 96 Hours

Negative Control A 37,000 260,000 1,060,000 2,760,000 B 38,000 510,000 2,200,000 3,600,000 C 57,000 560,000 2,150,000 4,700,000 D 74,000 630,000 2,120,000 3,720,000 E 68,000 390,000 1,690,000 3,840,000 F 64,000 610,000 2,090,000 4,280,000

Solvent Control A 103,000 710,000 2,340,000 4,500,000 B 42,000 340,000 1,370,000 3,820,000 C 69,000 410,000 1,830,000 3,960,000

1.50 A 52,000 320,000 870,000 3,580,000

B 57,000 410,000 1,770,000 5,740,000 C 86,000 360,000 2,190,000 4,820,000

3.35 A 48,000 280,000 1,330,000 4,960,000

B 60,000 370,000 2,010,000 4,320,000 C 66,000 380,000 1,870,000 4,660,000

6.84 A 48,000 270,000 1,040,000 3,260,000

B 48,000 260,000 560,000 3,380,000 C 95,000 320,000 1,280,000 3,480,000

12.9 A 103,000 680,000 2,110,000 4,020,000

B 110,000 720,000 2,040,000 3,900,000 C 89,000 450,000 2,010,000 4,620,000

26.5 A 84,000 550,000 2,320,000 4,480,000

B 82,000 620,000 1,720,000 4,120,000 C 97,000 610,000 1,780,000 3,420,000

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).

# W ildlife International, Ltd. Project Number: 471A-119

-53-

Appendix 7

Yield By Replicate At 72 and 96 Hours of Exposure

Day 0 Measured

Concentration (ng/L) Replicate Yield 1 (cells/mL)

72 Hours 96 Hours

Negative Control A 1,050,000 2,750,000 B 2,190,000 3,590,000 C 2,140,000 4,690,000 D 2,110,000 3,710,000 E 1,680,000 3,830,000 F 2,080,000 4,270,000

Solvent Control A 2,330,000 4,490,000 B 1,360,000 3,810,000 C 1,820,000 3,950,000

1.50 A 860,000 3,570,000

B 1,760,000 5,730,000 C 2,180,000 4,810,000

3.35 A 1,320,000 4,950,000

B 2,000,000 4,310,000 C 1,860,000 4,650,000

6.84 A 1,030,000 3,250,000

B 550,000 3,370,000 C 1,270,000 3,470,000

12.9 A 2,100,000 4,010,000

B 2,030,000 3,890,000 C 2,000,000 4,610,000

26.5 A 2,310,000 4,470,000

B 1,710,000 4,110,000 C 1,770,000 3,410,000

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 in itiation (0 hours). Yield was calculated as the final biomass (cell density) in the exposure period minus the initial biomass (cell density).

# W ildlife International, Ltd. Project Number: 471A-119

-54-

Appendix 8

Growth Rate By Replicate Over the 96-Hour Exposure Period

Day 0 Measured

Concentration (ng/L) Replicate Growth Rate (per hour)

0 - 24 Hours 0 – 48 Hours 0 – 72 Hours 0 – 96 Hours

Negative Control A 0.0545 0.0679 0.0648 0.0585

B 0.0556 0.0819 0.0749 0.0613

C 0.0725 0.0839 0.0746 0.0641

D 0.0834 0.0863 0.0744 0.0617

E 0.0799 0.0763 0.0712 0.0620

F 0.0773 0.0856 0.0742 0.0631

Solvent Control A 0.0972 0.0888 0.0758 0.0636

B 0.0598 0.0735 0.0683 0.0619

C 0.0805 0.0774 0.0724 0.0623

1.50 A 0.0687 0.0722 0.0620 0.0613

B 0.0725 0.0774 0.0719 0.0662

C 0.0897 0.0747 0.0748 0.0644

3.35 A 0.0654 0.0694 0.0679 0.0647

B 0.0747 0.0752 0.0737 0.0632

C 0.0786 0.0758 0.0727 0.0640

6.84 A 0.0654 0.0687 0.0645 0.0603

B 0.0654 0.0679 0.0559 0.0607

C 0.0938 0.0722 0.0674 0.0610

12.9 A 0.0972 0.0879 0.0743 0.0625

B 0.0999 0.0891 0.0739 0.0621

C 0.0911 0.0793 0.0737 0.0639

26.5 A 0.0887 0.0835 0.0756 0.0636

B 0.0877 0.0860 0.0715 0.0627

C 0.0947 0.0856 0.0720 0.0608

# W ildlife International, Ltd. Project Number: 471A-119

-55-

Appendix 9

Specific Growth Rate of the Negative Control Group

Section-by-Section Specific Growth Rat es for the Negative Control Replicates:

Negative
Control Replicate 0-24 Hour
Growth

Rate 24-48 Hour
Growth Rate 48-72 Hour
Growth Rate % Coefficient
of Variation 1

A 0.0545 0.0812 0.0586 22.24

B 0.0556 0.1082 0.0609 38.65

C 0.0725 0.0952 0.0561 26.35

D 0.0834 0.0892 0.0506 28.02

E 0.0799 0.0728 0.0611 13.31

F 0.0773 0.0939 0.0513 28.96

Mean (%) = 26.3

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 fo r the Negative Control Replicates During the Test Period:

Negative Control Replicate 0-72 Hour Growth

Rate

A 0.0648
B 0.0749
C 0.0746
D 0.0744
E 0.0712
F 0.0742

Mean

SD
% Coefficient of Variation 0.0724
0.0039

5.45

# W ildlife International, Ltd. Project Number: 471A-119

-56-

Appendix 10

Personnel Involved in the Study

The following key Wildlife International, Lt d. personnel were involved in the conduct or management of this study:

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

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

3. John R. Porch, M.S., Manager of Plant and Invertebrate Toxicology

4. Timothy Z. Kendall, M.S., Supervisor

5. Joshua R. Arnie, B.S., Biologist