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

FINAL REPORT

WILDLIFE INTERNATIONAL PROJECT NUMBER: 264P-101

OECD Guideline 201 EU Directive 92/69/EEC, Method C.3.

U.S. EPA OCSPP Number 850.4500

AUTHORS:

Allison M. Jacobs, B.S. Timothy Z. Kendall, M.S. John R. Porch, M.S.

STUDY INITIATION DATE: September 19, 2012

STUDY COMPLETION DATE: January 30, 2013

SUBMITTED TO:

American Chemistry Council 700 2 nd Street NE Washington, D.C. 20002 

# Wildlife International

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

Page 1 of 70

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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: __________________

Company Agent: __________________

Title: _________



### ________

Signature:______________________________Date:______________________

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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 ...................................................................... 16 Statistical Analyses ......................................................................................................... ......... 17

Results and Discussion ........................................................................................................ ................. 18 Measurement of Test Concentrations ...................................................................................... 18 Observations and Measurements ............................................................................................. 18 Conditions for the Validity of the Test .................................................................................... 2 0

Conclusions ................................................................................................................... ....................... 20

Refere nces .................................................................................................................... ........................ 21

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TABLE OF CONTENTS (Continued)

TABLES AND FIGURES

Table 1. Non-GLP Range-finding Test Data .................................................................................... 22

Table 2. Measured Concentrations of Benzophe none in Test Solution Samples ............................. 23

Table 3. Temperature Measurements ............................................................................................... 24

Table 4. pH Measurements .............................................................................................................. . 24

Table 5. Light Intensity Measurements ............................................................................................ 24

Table 6. Mean Area Under the Growth Curve and Percent Inhibition ............................................. 25

Table 7. Mean Growth Rate (per Hour) and Percent Inhibition ....................................................... 26

Table 8. Mean Yield and Percent Inhibition ..................................................................................... 27

Table 9. NOEC, E b C 50 , E y C 50 and E r C 50 Values Over the 96-Hour Exposure Period ...................... 28

Figure 1. Growth of P. subcapitata in the Negative Control Over the 96-Hour Exposure Period ............................................................................................................... .. 29

Figure 2. Concentration-Response Curve for P. subcapitata Exposed to Benzophenone for 96 Hours, Expressed as Area Under the Growth Curve ............................................... 30

Figure 3. Concentration-Response Curve for P. subcapitata Exposed to Benzophenone for 96 Hours, Expressed as Growth Rate ........................................................................... 31

Figure 4. Concentration-Response Curve for P. subcapitata Exposed to Benzophenone for 96 Hours, Expressed as Yield ....................................................................................... 32

APPENDICES

Appendix 1. Study Protocol .......................................................................................................... 33

Appendix 2. Certificate of Analysis .............................................................................................. 49

Appendix 3. Freshwater AAP Algal Medium ............................................................................... 50

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TABLE OF CONTENTS (Continued)

APPENDICES (Continued)

Appendix 4. Analyses of Pesticides, Organics and Metals in Wildlife International Well Water ................................................................................................................ 51

Appendix 5. Analysis of Benzophenone in Freshwater Algal Medium ........................................ 53 5.1 Analytical Method Flowchart for the Analysis of Benzophenone in Freshwater Algal Medium ........................................................................................ 54 5.2 Typical HPLC Opera tional Parameters .................................................................... 55 5.3 Analytical Stocks and Standards Preparation ........................................................... 56 5.4 Example Calculations for a Representative Sample ................................................. 57 5.5 Quality Control Samples of Benzophenone in Freshwater Algal Medium ..................................................................................................................... 58 5.6 Representative Calibration Curve for Benzophenone............................................... 59 5.7 Representative Chromatogram of a Low-level Benzophenone Calibration Standard ................................................................................................. 60 5.8 Representative Chromatogram of a High-level Benzophenone Calibration Standard ................................................................................................. 61 5.9 Representative Chromatogram of a Matrix Blank Sample ....................................... 62 5.10 Representative Chromatogram of a Matrix Fortification Sample ............................. 63 5.11 Representative Chromatogram of a Test Sample ...................................................... 64

Appendix 6. Cell Density By Replicate Over the 96-Hour Exposure Period ................................ 65

Appendix 7. Area Under the Growth Curve By Replicate Over the 96-Hour Exposure Period ...................................................................................................... 66

Appendix 8. Yield By Replicate At 72 and 96-Hours of Exposure .............................................. 67

Appendix 9. Growth Rate By Replicate Over the 96-Hour Exposure Period ............................... 68

Appendix 10. Specific Growth Rate of the Negative Control Group .............................................. 69

Appendix 11. Personnel Involved in the Study ............................................................................... 70

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SUMMARY

SPONSOR: 

### American Chemistry Council

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

WILDLIFE INTERNATIONAL PROJECT NUMBER: 264P-101

TEST DATES: Experimental Start (OECD): September 20, 2012 Experimental Start (EPA): September 20, 2012 Exposure Termination: September 24, 2012 Experimental Termination: September 25, 2012

LENGTH OF EXPOSURE: 96 Hours

TEST ORGANISM: Freshwater Alga ( Pseudokirchneriella subcapitata )

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

TEST CONCENTRATIONS:

Nominal Mean, Measured Negative Control <LOQ 0.26 mg/L 0.23 mg/L 0.64 mg/L 0.55 mg/L 1.6 mg/L 1.4 mg/L 4.0 mg/L 3.8 mg/L 10 mg/L 9.1 mg/LWildlife InternationalProject Number: 264P-101-10-SUMMARY (Continued)RESULTS:ExposureDuration(hours)Area Under the Growth Curve(mg/L)Growth Rate(mg/L)Yield(mg/L)NOECEbC50195% CINOECErC50195% CINOEC EyC50195% CI72 0.23 1.7 1.4 to 2.20.23 3.0 2.3 to 4.00.23 1.7 1.2 to 2.496 0.23 1.8 1.3 to 2.40.23 2.9 2.2 to 3.90.23 1.8 0.96 to 3.41EbC50, ErC50and EyC50values and their corresponding 95% confidenceintervals (CI) were calculated using non-linearregression with replicate data (area under the growth curve, growth rate or yield) and mean,measured test concentrations.

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INTRODUCTION This study was conducted by Wildlife Interna tional for American Chemistry Council at the Wildlife International aquatic toxicology facility in Ea ston, Maryland. The in-life phase of the definitive test was conducted from September 20 to 24, 2012, w ith cell counts completed on September 25, 2012. Raw data generated by Wildlife International a nd the final report are filed under Project Number

264P-101 in archives located on the Wildlife International site.

OBJECTIVE The objective of this study was to determine the toxicity of Benzophenone, to the freshwater alga, Pseudokirchneriella subcapitata , over a 96-hour exposure period.

EXPERIMENTAL DESIGN The freshwater alga, Pseudokirchneriella subcapitata , was exposed to five test concentrations and a negative control (culture medium) for 96 hours. Nominal test concentrations were selected in consultation with the Sponsor and were based upon th e results of an exploratory non-GLP range-finding toxicity test with the test substance in culture medium. The range-finding test was conducted with
nominal concentrations of 0.10, 1.0 and 10 mg/L , and yielded -8, 26 and 99% inhibition of mean cell density after 96 hours of exposure, respectively, relative to the mean negative control response ( Table 1 ).
Nominal test concentrations selected for the definitive test were 0.26, 0.64, 1.6, 4.0 and 10 mg/L

. Measured concentrations were determined from sampl es of test medium collected from each treatment and control group at 0 and 96 hours of the test.

Four replicate test chambers were maintained in each treatment group and six replicates were maintained 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 appr oximately 10,000 cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell
densities, which were used to calculate area under the growth curve, yield and growth rates. Area under the growth curve, growth rates and yield were used to calculate percent inhibition values relative to the control at 72 and 96 hours of exposure. E b C 50 , E r C 50 and E y C50 values (i.e., the theoretical concentrations

that would produce a 50% reduction in area under the growth curve, growth rate and yield, respectively) were calculated, when possible, at 72 and 96 hours of exposure. No-observed-effect -concentrations

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(NOEC) were determined at 72 and 96 hours through statistical evaluation of area under the growth curve, growth rate and yield data, as well as ex amination of the concentration-response pattern.

MATERIALS AND METHODS The study was conducted according to the proce dures outlined in the protocol, “Benzophenone: A 96-Hour Toxicity Test with the Freshwater Alga ( Pseudokirchneriella subcapitata )” ( Appendix 1 ). The protocol was based on procedures outlined in OECD Guidelines for Testing of Chemicals,

Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test ( 1 ); Official Journal of the European Communities No. L 383 A, Method C.3.: Algal Inhibition Test ( 2 ); and the U.S. Environmental Protection Agency Series 850 – Ecological Effects Test Guidelines, OCSPP Number 850.4500: Algal
Toxicity ( 3 ).

Test Substance The test substance used to prep are the test solutions, the analytical matrix fortification samples and the analytical calibration standards for the study was received from Sigma Aldrich on July 12, 2012. It was assigned Wildlife International identifi cation number 10615 upon receipt and was stored under room temperature (ambient) and dark conditions. The test substance, a solid, was identified as: Benzophenone; lot number MKBG2920V. An expiration date was not provided by the Sponsor. The test substance contained 99.6% active ingredient ( Appendix 2 ).

Test Organism The freshwater 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, and ha ve been maintained in culture medium at Wildlife International, Easton, Maryland since March 2000. Al gal cells used in this test were obtained from

Wildlife International cultures that had been ac tively growing in culture medium under similar

environmental conditions as used in this test for at l east two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been tr ansferred to fresh medium three days prior to test initiation. The 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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Test Apparatus Test chambers were sterile, 250-mL glass Erlenmeyer flasks plugged with sterile foam stoppers,

and contained 100 mL of test or control medium. The test chambers were labeled with the project number, test concentration and replicate, and were indiscriminately positioned da ily in an environmental chamber designed to maintain the desired test temp erature throughout the test. The test flasks were continuously shaken on a mechanical shaker at 100 rpm throughout the duration of the test.

Culture Medium The algal cells were cultured and tested in freshw ater AAP medium (4). Stock nutrient solutions were prepared by adding reagent-grade 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 contaminan ts in the well water used to prepare the NANOpure ®
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 4Star plus 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 A primary stock solution was prepared by di ssolving 0.0100 g of benzophenone in 1000 mL of

freshwater AAP medium to achieve a nominal concen tration of 10 mg/L. The primary stock was mixed by inversion and was sonicated for ~forty three minutes, as well as stirred with a Teflon ® -coated stirbar and magnetic stirplate for ~fourteen minutes prior to use. The 10 mg/L primary stock appeared clear and

colorless and was otherwise unremarkable. The 10 mg/L stock was proportionately diluted with freshwater AAP medium to prepare four additional test solutions at target nominal concentrations of 0.26, 0.64, 1.6 and 4.0 mg/L. All test solutions were mixed by inversion, and appeared clear and

colorless and were otherwise unremarkable after mixi ng. The negative control solution consisted of

freshwater AAP medium.

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 treatment and contro l group prior to distribution into the test chambers. At exposure termination, samples were collected from the pooled replicates from each treatment and control group. Samples collected at test initiation a nd test termination were collected into glass vessels and were processed immediately for analysis.

Analytical Method The method used for the analysis of benzophenone in freshwater algal medium samples was based upon methodology developed by Wildlife International The analytical method consisted of diluting the samples in freshwater algal medium, as necessary, and analyzing by direct injection high performance liquid chromatography (HPLC) with UV detection at 220 nm.

Concentrations of benzophenone in the samples were determined by HPLC using an Agilent Series 1260 High Performance Liquid Chromatograph (HPLC) e quipped with an Agilent Series 1260 Variable Wavelength Detector. Chromatographic separations were achieved using a YMC-PACK ODS-AM column (150 mm x 4.6 mm, 3-μm particle size). A method fl owchart is provided in Appendix 5.1 and typical instrumental parameters are summarized in Appendix 5.2.

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Five calibration standards of be nzophenone were prepared, ranging in concentration from 0.100 to 1.00 mg/L, using a stock solution of benzophenone in methanol (Appendix 5.3). Fresh standards were prepared and analyzed with each sample set. Linear regression equations were gene rated using the peak area responses versus the respective concentrations of the calibration standards. The concentration of benzophenone in the samples was dete rmined by substituting the peak ar ea responses of the samples into the applicable linear regression equation. An example of the calculations for a representative sample is included in Appendix 5.4.

The method Limit of Quantitation (LOQ) was define d as 0.100 mg/L, calculated as the product of the concentration of the lowest calibration standard (0 .100 mg/L) and the dilution factor of the matrix blank samples (1.00). Two matrix blank samples were an alyzed to determine possible interferences. No

interferences were observed at or above the LOQ during the sample analyses (Appendix 5.5).

Samples of freshwater algal medium were fortif ied at 0.250, 0.750, and 10.0 mg/L using stock solutions of benzophenone in metha nol (Appendix 5.3), and were analyzed concurrently with the test samples. The measured concentrations for the matr ix fortification samples ranged from 93.9 to 110% of fortified concentrations (Appendix 5.5).

A representative calibration curve is presented in Appendix 5.6. Representative chromatograms of low and high-level calibration standards are presen ted in Appendices 5.7 and 5.8, respectively. Representative chromatograms of a freshwater medium matrix blank sample and a matrix fortification sample are presented in Appendices 5.9 and 5.10, resp ectively. A representative chromatogram of a test sample is presented in Appendix 5.11.

Inoculation of Test Chambers Prior to test initiation, the algal cells in the st ock culture (culture identification No. 00-1) were verified in freshwater algal medium at a concentration of approximately 1.36 x 10 6 cells/mL. The concentration of algal cells was verified using a hemacytometer and microscope, and 0.735 mL of the algal culture was added to each test chamber to achieve a nominal concentration of approximately
10,000 cells/mL at test initiation.

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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 four days unde r refrigerated conditions sufficient to inhibit growth until cell counts could be performed. Cell counts were performed using an electronic particle counter (Coulter Electronics, Inc.). Prior to conducting cell c ounts, the linearity of the instrument response was determined at settings previously established for Pseudokirchneriella subcapitata . A primary counting standard containing Pseudokirchneriella subcapitata cells was prepared at a target nominal concentration of 100,000 cells/mL. The density of the primary counting standard was verified using a hemacytometer and a microscope, and the standard was subsequently diluted to provide a series of seven counting standards for the determina tion of instrument linearity. Theoretical densities were assigned to each secondary counting standard based upon the verified density of the primary counting standard and the dilution ratio. The cell densities of the counting standards were measured using the electronic particle counter and were compared to the theoretical densities by performing a least squares regression analysis. Cell counts for samples collected during the test were conducted once instrument linearity was demonstrated (i.e., the R- squared value obtained through the regression analysis was 0.99984). A single aliquot of each sample collected during the test was diluted with an electrolyte solution (Isoton ® ). Three 0.5-mL volumes of the diluted sample were counted, and the resulting counts were averaged. The cell density of the sample w as determined by adjusting the mean cell count (cells/mL) obtained using the particle counter, b ased upon the Y-intercept and slope calculated through the regression analysis, and the dilution factor. The following equation was used:

Cell Density of Sample (cells/mL) = Slope Intercept Y (cells/mL) Count Cell Mean

 × Dilution Factor

Samples of test solution were collected from each re plicate 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 aggreg ation or flocculation of cells, and adherence of the

cells to the test chamber.

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Statistical Analyses The calculation of cell densities, area under the grow th curve, yield, growth rates and percent inhibition values, as well as all statistical analys es, were conducted using “The SAS System for Windows, 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)

Area under the growth curve (biomass) was calcu lated for each replicate of the control and treatment groups at 24, 48, 72 and 96 hours using the following formula:

A = ((N 1 -N 0 )/2)(t 1 )+((N 1 +N 2 -2N 0 )/2)(t 2 -t 1 )+...+((N n-1 +N n -2N 0 )/2)(t n -t n-1 )

Where: A = Area under the growth curve N 0 = Mean nominal number of cells/mL at t 0 N 1 = Mean measured number of cells/mL at t 1 N 2 = Mean measured number of cells/mL at t 2 N n = Mean measured number of cells/mL at t n t 0 = test initiation
t 1 = Time of first measurement after beginning of test (hours)
t 2 = Time of second measurement after beginning of test (hours)
t n = Time of n th measurement after beginning of test (hours)

Yield was calculated for each replicate of the control and treatment groups as the final biomass (cell density) in the exposure period minus the initial biomass (nominal cell density). Inhibition values were calculated for each treatment group as the per cent reduction in area under the growth curve, yield and growth rate relative to the negative cont rol replicates using the following formula:

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

 × 100

EC 50 values, (i.e., the theoretical test concentra tions that would produce a 50% reduction in area under the growth curve (E b C 50 ), growth rate (E r C 50 ) and yield (E y C 50 ), in comparison to the negative control) and the corresponding 95% confidence limits for the 72 and 96 hour intervals were calculated by non-linear regression ( 6 ) with replicate data (area under the gr owth curve, growth rate and yield, respectively) and mean measured test concentrations.

The 72 and 96-hour area under the growth curve, yiel d and growth 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 of normal distribution and homogeneity of variance. The treatment groups were compared to the negative contro l 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 Measured concentrations of benzophenone ra nged from 96 to 105% of nominal in samples collected on Day 0 and from 74 to 83% of nominal at te st termination at 96 hours (Table 2). Test solution appeared clear and colorless after mixing, and no su rface films or particulate matter were observed in the test chambers at study termination. Results of analytical measurements are presented in Table 2 . The results of the study were based on mean, measured concentrations.

Observations and Measurements Measurements of temperature, pH and light intensity are presented in Tables 3, 4 and 5
, respectively. Temperatures remained within the 24 ± 2°C range established for the test. The pH of the test solutions ranged from 7.3 to 7.4 at test initiation and ranged from 8.0 to 9.8 in the test solutions at test

termination. The observed increase in pH is typical for tests conducted with P. subcapitata and is attributed to the photosynthetic activity of the alg ae. The light intensity ranged from 5,590 to 6,520 lux, which was within the desired range of 6000 lux ± 10%.

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The toxicity of Benzophenone to P. subcapitata was determined by evaluating changes in cell density over a 96-hour exposure peri od. Cell densities were used to determine area under the growth curve and growth rates for each 24-hour interval of exposure and yields at 72 and 96 hours of exposure. Mean area under the growth curve, growth rates a nd yields and their corresponding percent inhibition values are presented in Tables 6

, 7 and 8 , respectively. NOEC, E b C 50 , E y C 50 and E r C 50 values and their corresponding 95% confidence limits for area under th e growth curve, yield and growth rates, respectively, are presented in Table 9 . Individual replicate data for each growth parameter are presented

in Appendices 6

, 7
, 8 and 9

. Figure 1 illustrates changes in mean cell density in the negative control group throughout the test and Figure 2 shows changes in mean area under the growth curve in all experimental groups over the 96-hour exposure period. Figures 3 and 4 depict concentration-response

curves for P. subcapitata exposed to benzophenone for 96 hours, expr essed as growth rate and yield, respectively. Exponential growth, characterized by the linear section of the growth curve, occurred throughout the duration of the test in the negative control replicates ( Figure 1 ). Appendix 10 provides the coefficient of variation for the specific growth rates of the control replicates.

After 72 hours of exposure, inhibition of area under the growth curve in the 0.23, 0.55, 1.4, 3.8 and 9.1 mg/L treatment groups was 5, 12, 28, 97 and 98% , respectively, relative to the negative control. Inhibition of growth rate in the 0.23, 0.55, 1.4, 3. 8 and 9.1 mg/L treatment groups was 1, 2, 6, 74 and 78%, respectively, relative to the negative control. Inhibition of yield in the 0.23, 0.55, 1.4, 3.8 and 9.1 mg/L treatment groups was 6, 11, 29, 99 and 99%, re spectively, relative to the negative control. The

72-hour EC 50 values and 95% confidence intervals for area unde r the growth curve, growth rate and yield were 1.7 (1.4 to 2.2) mg/L, 3.0 (2.3 to 4.0) mg/L a nd 1.7 (1.2 to 2.4) mg/L, respectively. Dunnett’s test indicated that there were significant reductions ( p ≤ 0.05) in mean area under the growth curve, growth rate and yield in the 0.55, 1.4, 3.8 and 9.1 mg/L treatment groups. Therefore, the 72-hour NOEC for area under the growth curve, growth rate and yield was 0.23 mg/L.

After 96 hours of exposure, inhibition of area under the growth curve in the 0.23, 0.55, 1.4, 3.8 and 9.1 mg/L treatment groups was 5, 11, 26, 98 and 99% , respectively, relative to the negative control. Inhibition of growth rate in the 0.23, 0.55, 1.4, 3. 8 and 9.1 mg/L treatment gr oups was 1, 2, 4, 76, and 80%, respectively, relative to the negative control. Inhibition of yield in the 0.23, 0.55, 1.4, 3.8 and 9.1 mg/L treatment groups was 5, 11, 23, 99 and 100%, respectively, relative to the negative control. The

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96-hour EC 50 values and 95% confidence intervals for area unde r the growth curve, growth rate and yield were 1.8 (1.3 to 2.4) mg/L, 2.9 (2.2 to 3.9) mg/L a nd 1.8 (0.96 to 3.4) mg/L, respectively. Dunnett’s test indicated that there were significant reductions ( p ≤ 0.05) in mean area under the growth curve, growth rate and yield in the 0.55, 1.4, 3.8 and 9.1 mg/L treatment groups. Therefore, the 96-hour NOEC for area under the growth curve, growth rate and yield was 0.23 mg/L.

At test initiation algal cells appeared norma l. After 96-hours of exposure there were no noticeable changes in cell morphology in any of the te sted concentrations when compared to the control and there were no noticeable aggregations and flocculati ons of cells, or adherence to the test chambers, in any treatment or the control group. The test solution appeared normal with no surface slicks or particulate matter present at the end of exposure.

Conditions for the Validity of the Test 1) The mean cell density in the control replicates increased by a factor greater than 16 within three days and at least 100 within 4 days. Th e cell density increased by a factor of 336 in three days and a factor of 557 in 4 days (Appendix 10).

2) The coefficient of variation of average specific growth rate in the negative control replicates during the first 72 hours did not exceed 7%. It was 1.1% (Appendix 10).

3) The mean percent coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the negative control did not exceed 35%. It was 24% (Appendix 10).

CONCLUSIONS The freshwater alga, Pseudokirchneriella subcapitata , was exposed to a geometric series of five concentrations of Benzophenone rangi ng from 0.23 to 9.1 mg/L, based on mean, measured concentrations of Benzophenone from samples collected at test in itiation and test termination. Toxicity of Benzophenone to P. subcapitata was assessed based on effects on area under the growth curve, yield and growth rate relative to the negative control group. The 72 and 96-hour NOEC was determined to be 0.23 mg/L, for both intervals. The 72-hour EC 50 values and 95% confidence intervals for area under the growth curve, growth rate and yield were 1.7 (1.4 to 2.2) mg/L, 3.0 (2.3 to 4.0) mg/L and 1.7 (1.2 to 2.4) mg/L, respectively. The 96-hour EC 50 values and 95% confidence intervals for area under the growth curve, growth rate and yield were 1.8 (1.3 to 2.4) mg/L, 2.9 (2.2 to 3.9) mg/L and 1.8 (0.96 to 3.4) mg/L, respectively.

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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. L 383 A. Method C.3. Algal Inhibition Test.

3 U.S. Environmental Protection Agency. 2012. Series 850-Ecological Effects Test Guidelines, OCSPP Number 850.4500: Algal Toxicity

.

4 American Society for Testing and Materials. 2004. ASTM Standard Guide E1218-04. S tandard Guide for Conducting Static Toxicity Tests with Microalgae

.

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.

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Table 1

Non-GLP Range-finding Test Data

Nominal Concentration 1 (mg/L) 96-Hour Cell Density

(cells/mL) Percent Inhibition vs. Control

Negative Control 3.63 X 10 6

--

0.10 3.93 X 10 6 -8

1.0 2.70 X 10 6 26

10 1.93 X 10 4 99

1 Appearance of test solutions: clear and colorless, with no precipitation noted.

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Table 2

Measured Concentrations of Benz ophenone in Test Solution Samples

Nominal

Test Concentration (mg/L) Sample Number (264P-101-) Sampling Time (Hours) Measured Concentration (mg/L) 1 Percent of Nominal 2 Mean Measured Concentration (mg/L) Mean Measured Percent of Nominal Negative Control (0.0) 1 7 0 96 < LOQ < LOQ --

-- -- --

0.26 2 8 0 96 0.250 0.200 96.3 77.1 0.23 88

0.64 3 9 0 96 0.626 0.473 97.8 74.0 0.55 86

1.6 4 10 0 96 1.62 1.19 101 74.3 1.4 88

4.0 5 11 0 96 4.19 3.32 105 83.0 3.8 95

10 6 12 0 96 10.4 7.70 104 77.0 9.1 91

1 The limit of quantitation (LOQ) of 0.100 mg/L was calculate d as the product of the concentration of the lowest

calibration standard (0.100 mg/L) and the dilution factor of the matrix blank samples (1.00).

2 Results were generated using Excel 2000 in full preci sion mode. Manual calculations may differ slightly.

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Table 3

Temperature Measurements

Time (Days) Temperature (  C) 1

Measurement 1 Measurement 2

0 23.3 23.7 1 23.7 23.9 2 23.8 23.8 3 23.7 23.9 4 23.6 23.7

1 The minimum and maximum temperatures monitored c ontinuously throughout the study were 23.4 to 23.7°C, respectively.

Table 4

pH Measurements

Mean, Measured Test Concentration (mg/L) pH Measurements

Day 0 1 Day 4 2

Negative Control 7.4 9.7 0.23 7.4 9.8 0.55 7.4 9.8 1.4 7.4 9.8 3.8 7.4 8.3 9.1 7.3 8.0

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 5

Light Intensity Measurements

Shaker Table Day 0 Light Intensity Measurement (lux) 1

No. 1 No. 2 No. 3 No. 4 No. 5 AQL#16 6,260 6,400 5,790 6,520 5,870
AQL#17 5,920 6,200 5,590 5,780 5,610

1 Measurements taken at five locations per table surrounding the te st flasks at test solution level.Wildlife InternationalProject Number: 264P-101-25-Table 6Mean Area Under the Growth Curve and Percent InhibitionMean, MeasuredTestConcentration(mg/L)24 Hours148 Hours172 Hours196 Hours1MeanArea Under theGrowth CurvePercentInhibition2MeanArea Under theGrowth CurvePercentInhibition2MeanArea Under theGrowth CurvePercentInhibition2MeanArea Underthe GrowthCurvePercentInhibition2Negative Control 1,109,778 --11,712,518 -- 61,399,146 -- 168,275,984 --0.231,124,301 -1 11,369,784 358,156,851 5 159,446,538 50.551,016,754 8 10,182,732 13 54,215,319* 12 149,769,297* 111.4912,900 18 8,870,190 24 44,308,878* 28 123,915,819* 263.8288,594 74 931,374 92 1,712,061* 97 2,569,419* 989.1162,240 85 570,864 95 1,130,727* 98 1,759,491* 991Calculations were performed using SAS Version 8.2. Manual calculations may differ slightly.2Percent inhibition was calculated relative to the negative control replicates.* Statistically significant difference (p≤0.05) at 72 and 96 hours from the negative control replicates using Dunnett’stest.Wildlife InternationalProject Number: 264P-101-26-Table 7Mean Growth Rate (per Hour) and Percent InhibitionMean, MeasuredTestConcentration(mg/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 0.0969 -- 0.0912 -- 0.0808 -- 0.0658 --0.230.0974 -1 0.0905 1 0.0799 1 0.0654 10.550.0937 3 0.0881 3 0.0792* 2 0.0647* 21.40.0896 7 0.0851 7 0.0759* 6 0.0631* 43.80.0510 47 0.0286 69 0.0211* 74 0.0159* 769.10.0356 63 0.0232 75 0.0178* 78 0.0134* 801Calculations were performedusing SAS Version 8.2. Manualcalculations may differ slightly.2Percent inhibition was calculated relative to the negative control replicates.* Statistically significant difference (p≤0.05) at 72 and 96 hours from the negative control replicates using Dunnett’stest.Wildlife InternationalProject Number: 264P-101-27-Table 8Mean Yield and Percent InhibitionMean, MeasuredTestConcentration(mg/L)72 Hours196 Hours1MeanYield(cells/mL)PercentInhibition2MeanYield(cells/mL)PercentInhibition2Negative Control 3,349,472 -- 5,556,931 --0.233,138,824 6 5,301,984 50.552,990,280* 11 4,972,551* 111.42,366,192* 29 4,267,720* 233.835,542* 99 35,905* 999.126,123* 99 26,274* 1001Calculations were performedusing SAS Version 8.2. Manualcalculations may differ slightly.2Percent inhibition was calculated relative to the negative control replicates.* Statistically significant difference from the negative control replicates using Dunnett’s test(p0.05).Wildlife InternationalProject Number: 264P-101-28-Table 9NOEC, EbC50, EyC50and ErC50Values Over the 96-Hour Exposure PeriodExposureDuration(hours)Area Under the Growth Curve(mg/L)Growth Rate(mg/L)Yield(mg/L)NOECEbC50195% CINOECErC50195% CINOEC EyC50195% CI72 0.23 1.7 1.4 to 2.20.23 3.0 2.3 to 4.00.23 1.7 1.2 to 2.496 0.23 1.8 1.3 to 2.40.23 2.9 2.2 to 3.90.23 1.8 0.96 to 3.41EbC50, ErC50and EyC50values and their corresponding 95% confidenceintervals (CI) were calculated using non-linearregression with replicate data (area under the growth curve, growth rate or yield) and mean,measured test concentrations.

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Figure 1

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

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

0 24 48 72 96Cell Density (cells/mL)

Exposure Duration (Hours)

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Figure 2

Concentration-Response Curve for P. subcapitata Exposed to Benzophenone for 96 Hours, Expressed as Area Under the Growth Curve

-50000000 0 50000000 100000000 150000000 200000000 250000000 300000000

0.1 1 10Area Under the Growth Curve

Mean, Measured Test Concentration (mg/L ) Data

Means

Predicted

Regression

95% Conf. Int.

50% Inhibition

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Figure 3

Concentration-Response Curve for P. subcapitata Exposed to Benzophenone for 96 Hours, Expressed as Growth Rate

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

0.1

0.1 1 10Growth Rate (hours-1)

Mean, Measured Test Concentration (mg/L ) Data

Means

Predicted

Regression

95% Conf. Int.

50% Inhibition

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Figure 4

Concentration-Response Curve for P. subcapitata Exposed to Benzophenone for 96 Hours, Expressed as Yield

-4000000 -2000000 0 2000000 4000000 6000000 8000000 10000000 12000000 14000000 16000000

0.1 1 10Yield (cells/mL)

Mean, Measured Test Concentration (mg/L) Data

Means

Predicted

Regression

95% Conf. Int.

50% Inhibition

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Appendix 1

Study Protocol

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Appendix 2

Certificate of Analysis

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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 15.0 mg/L

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

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Appendix 4
Analyses of Pesticides, Organics and Me tals in Wildlife International Well Water

Pesticides and Organics 1

Component Measured Concentration (μg/L) Component Measured Concentration (μg/L)

Aldrin < 0.0097 Gamma BHC – Lindane < 0.0097 Alpha BHC < 0.0097 Gamma Chlordane < 0.019 Alpha Chlordane < 0.0097 Guthion (Azinphos-methyl) < 5.8 Beta BHC < 0.0097 HCB < 0.0097 Bolstar < 1.9 Heptachlor < 0.0097 Chlordane < 0.48 Heptachlor Epoxide < 0.0097 Coumaphos < 2.9 Kepone < 0.19 Delta BHC < 0.0097 Malathion < 2.9 Demeton-O < 1.9 Merphos < 1.9 Demeton-S < 2.9 Methoxychlor < 0.097 Diazinon < 5.8 Methyl Parathion < 1.9 Dichlorvos < 2.9 Mevinphos < 4.8 Dieldrin < 0.019 Mirex < 0.24 Disulfoton < 1.9 Naled < 5.8 Dursban (Chlorpyrifos) < 2.9 o,p-DDD < 0.019 Endosulfan I < 0.0097 o,p-DDE < 0.73 Endosulfan II < 0.019 o,p-DDT < 0.019 Endosulfan Sulfate < 0.019 p,p-DDD < 0.019 Endrin < 0.019 p,p-DDE < 0.019 Endrin Aldehyde < 0.097 p,p-DDT < 0.019 Endrin Ketone < 0.019 Phorate < 1.9 EPN < 1.9 Ronnel < 1.9 Ethion < 1.9 Stirophos < 1.9 Ethoprop < 2.9 Telodrin < 0.0097 Ethyl Parathion < 1.9 Tokuthion < 1.9 Famphur < 2.9 Toxaphene < 2.9 Fensulfothion < 8.7 Trichloronate < 1.9 Fenthion < 1.9 Trithion < 1.9

1 Analyses performed by Lancaster Laborator ies on samples collected on December 28, 2011.

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Appendix 4 (Continued)

Analyses of Pesticides, Organics and Me tals in Wildlife International Well Water

Metals 1

Component Measured Concentration (mg/L) Component Measured Concentration (mg/L)

Aluminum < 0.200 Magnesium 13.2 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 7.00 Calcium 34.9 Selenium < 0.0200 Chloride 4.5 Silver < 0.0050 Chromium < 0.0150 Sodium 19.0 Cobalt < 0.0050 Sulfate 5.7 Copper < 0.0100 Thallium < 0.0300 Fluoride 0.85 Vanadium < 0.0050 Iron < 0.200 Zinc < 0.0200 Lead < 0.0150

1 Analyses performed by Lancaster Laborator ies on samples collected on December 28, 2011.

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Appendix 5

Analysis of Benzophenone in Freshwater Algal Medium

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Appendix 5.1

Analytical Method Flowchart for the Analysis of Benzophenone in Freshwater Algal Medium



### METHOD OUTLINE FOR THE ANALYSIS OF BENZOPHENONE IN FRESHWATER ALGAL MEDIUM

Prepare calibration standards in freshwater algal medium using volumetric flasks and gas-tight syringes.



Prepare QC samples in freshwater algal medium us ing volumetric flasks and gas-tight syringes. The matrix blank will be freshwater algal medium.



Dilute samples/QCs with freshwater algal medium (as needed) using volumetric flasks, volumetric pipettes, gas-tight syringes and culture tubes.



Transfer to autosampler vials and submit for HPLC/UV analysis.

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Appendix 5.2

Typical HPLC Operational Parameters

INSTRUMENT: Agilent Series 1260 High Performance Liquid Chromatograph (HPLC) with an Agilent Series 1260 Variable Wavelength Detector

ANALYTICAL COLUMN: YMC-PACK ODS-AM Column ( 150 mm x 4.6 mm, 3-μm particle size)

OVEN TEMPERATURE: 40  C

STOP TIME: 14.00 minutes

FLOW RATE: 1.000 mL/minute

SOLVENT A:

SOLVENT B: 0.1% H 3 PO 4

CH 3 CN

GRADIENT ELUTION PROFILE: Time Flow (min) %A %B (mL/min) 0.00 90.0 10.0 1.000 1.00 90.0 10.0 1.000 9.00 2.0 98.0 1.000 10.00 2.0 98.0 1.000 10.10 90.0 10.0 1.000 14.00 90.0 10.0 1.000

INJECTION VOLUME: 10.00 μL

BENZOPHENONE RETENTION TIME: Approximately 9.5 minutes

PRIMARY ANALYTICAL WAVELENGTH: 220 nm

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Appendix 5.3

Analytical Stocks and Standards Preparation

A stock solution of benzophenone was prepared by weighing 100.21 mg of the test substance on an analytical balance. The test substance was tr ansferred to a 100-mL class-A volumetric flask, and brought to volume using methanol. The primary stock solution (1.00 mg/mL) was diluted in methanol to prepare 0.100 and 0.0100 mg/mL stock solutions. The 0.100 and 0.0100 mg/mL stock solutions were used to prepare the fortification samples. The 0.0100 mg/mL stock solution was used to prepare the calibration standards in freshwater algal medium fo r test sample analyses. The following shows the

dilution scheme for a set of calibration standards:

Stock Concentration (mg/mL) Aliquot (  L) Final Volume

(mL) Standard Concentration (mg/L)

0.0100 0.0100 0.0100 0.0100 0.0100 100 250 500 750 1000 10.0 10.0 10.0 10.0 10.0 0.100 0.250 0.500 0.750 1.00

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Appendix 5.4

Example Calculations for a Representative Sample

The analytical result and percent recovery for sample number 264P-101-4, with a nominal concentration of 1.6 mg/L, were cal culated using the following equations:

Concentration of benzophenone in sample (mg/L) = peak area - (y-intercept) slope x dilution factor

Percent of nominal concentration = Measured concentration of sample (mg/L) Nominal concentration of sample (mg/L) x 100

Peak Area = 16.45356 Y-Intercept = 0.0445 Slope = 20.2969 Dilution Factor = 2.00

Concentration of benzophenone in sample (mg/L) = 16.45356 - 0.0445 20.2969 x 2.00

Concentration of benzophenone in sample (mg/L) = 1.62

Percent of nominal concentration = 1.62 mg/L 1.6 mg/L x 100

Percent of nominal concentration = 101%

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Appendix 5.5

Quality Control Samples of Benzophe none in Freshwater Algal Medium

Sample Number

(264P-101-) Sample Type Fortified Concentration (mg/L) Measured Concentration (mg/L) 1 Percent Recovery 2

MAB-1 MAB-2

MAS-1 MAS-2 MAS-3

MAS-4 MAS-5 MAS-6 Matrix Blank Matrix Blank

Matrix Fortification Matrix Fortification Matrix Fortification

Matrix Fortification Matrix Fortification Matrix Fortification 0.0 0.0

0.250 0.750 10.0

0.250 0.750 10.0 < LOQ < LOQ

0.249 0.742 10.1

0.275 0.752 9.39 --

--

99.4 98.9 101

110 100 93.9

Mean = Standard Deviation = CV = 101 5.25 5.20%

1 The Limit of Quantitation (LOQ) was 0.100 mg/L, calculated as the product of the concentration of the lowest calibration standard (0.100 mg/L) and the dilution factor of the matrix blank samples (1.00).

2 Results were generated using Excel 2000 in the full pr ecision mode. Manual calculations may differ slightly.

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Appendix 5.6

Representative Calibration Curve for Benzophenone

Slope = 20.2969; Y-Intercept = 0.0445; R 2 = 0.9987

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Appendix 5.7

Representative Chromatogram of a Low-level Benzophenone Calibration Standard

min 0 2 4 6 8 10 12 mAU

-10

0 10 20 30 40 VWD1 A, Wavelength=220 nm (X:\HPLC26\1\DATA\BENZ\BENZ 2012-09-20 15-18-49\001-0101.D)9.517

Nominal concentration: 0.100 mg/L.

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Appendix 5.8

Representative Chromatogram of a High-level Benzophenone Calibration Standard

min 0 2 4 6 8 10 12 mAU

-10

0 10 20 30 40 VWD1 A, Wavelength=220 nm (X:\HPLC26\1\DATA\BENZ\BENZ 2012-09-20 15-18-49\005-0501.D)9.521

Nominal concentration: 1.00 mg/L.

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Appendix 5.9

Representative Chromatogram of a Matrix Blank Sample

min 0 2 4 6 8 10 12 mAU

-10

0 10 20 30 40 VWD1 A, Wavelength=220 nm (X:\HPLC26\1\DATA\BENZ\BENZ 2012-09-20 15-18-49\006-0601.D)

Sample number 264P-101-MAB-1. The arrow indicates the approximate retention time of benzophenone.

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Appendix 5.10

Representative Chromatogram of a Matrix Fortification Sample

min 0 2 4 6 8 10 12 mAU

-10

0 10 20 30 40 VWD1 A, Wavelength=220 nm (X:\HPLC26\1\DATA\BENZ\BENZ 2012-09-20 15-18-49\007-0701.D)9.522

Sample number 264P-101-MAS-1; 0.250 mg/L nominal concentration

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Appendix 5.11

Representative Chromatogram of a Test Sample

min 0 2 4 6 8 10 12 mAU

-10

0 10 20 30 40 VWD1 A, Wavelength=220 nm (X:\HPLC26\1\DATA\BENZ\BENZ 2012-09-20 15-18-49\013-1401.D)9.524

Sample number 264P-101-4; Day 0; 1.6 mg/L nominal concentration

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Appendix 6

Cell Density By Replicate Over the 96-Hour Exposure Period

Mean, Measured Test Concentration (mg/L) Replicate Cell Density 1 (cells/mL)

24 Hours 48 Hours 72 Hours 96 Hours

Negative Control A 97,306 677,481 3,321,484 5,585,445 B 117,844 959,554 3,457,267 5,654,488 C 99,087 717,807 3,150,724 5,216,815 D 101,671 816,869 3,765,114 5,783,149 E 100,646 825,703 3,230,224 5,580,882 F 98,335 809,067 3,232,020 5,580,807 Mean 102,482 801,080 3,359,472 5,566,931 S.D. 7,688 98,229 224,534 188,535
0.23 A 107,364 738,565 3,175,199 5,114,335

B 105,946 775,578 3,319,509 5,276,134 C 107,053 793,501 3,270,618 5,674,685 D 94,404 772,750 2,829,969 5,182,780 Mean 103,692 770,099 3,148,824 5,311,984 S.D. 6,222 22,943 220,855 250,730
0.55 A 95,698 642,346 2,902,797 4,961,962

B 92,931 713,948 3,113,083 4,838,986 C 95,571 771,980 3,277,979 5,270,075 D 94,718 628,134 2,707,262 4,859,182 Mean 94,730 689,102 3,000,280 4,982,551 S.D. 1,276 66,806 248,464 199,102
1.4 A 83,543 516,655 2,203,150 4,035,452

B 92,306 683,615 2,731,318 4,539,026 C 80,420 551,124 2,111,651 4,178,849 D 88,031 636,736 2,458,647 4,357,554 Mean 86,075 597,033 2,376,192 4,277,720 S.D. 5,197 76,683 278,591 218,421
3.8 A 31,974 37,980 45,221 42,921

B 32,834 38,567 44,492 43,684 C 37,184 40,681 47,764 51,101 D 34,206 40,834 44,690 45,913 Mean 34,050 39,516 45,542 45,905 S.D. 2,283 1,455 1,513 3,689
9.1 A 24,370 27,699 34,270 32,864

B 22,014 30,852 36,877 38,668 C 23,435 32,078 37,262 36,514 D 24,261 31,499 36,084 37,049 Mean 23,520 30,532 36,123 36,274 S.D. 1,087 1,954 1,329 2,451

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).

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Appendix 7

Area Under the Growth Curve By Replicate Over the 96-Hour Exposure Period

Mean, Measured Test Concentration (mg/L) Replicate Area Under the Growth Curve

24 Hours 48 Hours 72 Hours 96 Hours

Negative Control A 1,047, 672 10,105,116 57,852,696 164,495,844 B 1,294,128 13,982,904 66,744,756 175,845,816 C 1,069,044 10,631,772 56,814,144 156,984,612 D 1,100,052 11,882,532 66,626,328 180,965,484 E 1,087,752 11,963, 940 60,395,064 165,888,336 F 1,060,020 11,708, 844 59,961,888 165,475,812 Mean 1,109,778 11, 712,518 61,399,146 168,275,984 S.D. 92,257 1,341, 532 4,302,846 8,644,676
0.23 A 1,168,368 11,079, 516 57,804,684 157,039,092

B 1,151,352 11,489,640 60,390,684 163,298,400 C 1,164,636 11,731,284 60,260,712 167,364,348 D 1,012,848 11,178,696 54,171,324 150,084,312 Mean 1,124,301 11, 369,784 58,156,851 159,446,538 S.D. 74,660 297,668 2,911,167 7,549,296
0.55 A 1,028,376 9,644, 904 51,946,620 146,083,728

B 995,172 10,437,720 56,122,092 151,306,920 C 1,026,852 11,197,464 59,556,972 161,893,620 D 1,016,616 9,450,840 49,235,592 139,792,920 Mean 1,016,754 10, 182,732 54,215,319 149,769,297 S.D. 15,306 799,922 4,550,228 9,353,698
1.4 A 882,516 7,844,892 40,242,552 114,865,776

B 987,672 10,058,724 50,797,920 137,802,048 C 845,040 8,183,568 39,896,868 115,142,868 D 936,372 9,393,576 46,298,172 127,852,584 Mean 912,900 8,870,190 44,308,878 123,915,819 S.D. 62,370 1,034, 284 5,230,226 1,1063,354
3.8 A 263,688 863,136 1,621,548 2,439,252

B 274,008 890,820 1,647,528 2,465,640 C 326,208 1,020,588 1,841,928 2,788,308 D 290,472 950,952 1,737,240 2,584,476 Mean 288,594 931,374 1,712,061 2,569,419 S.D. 27,395 69,865 99,761 159,010
9.1 A 172,440 557,268 1,060,896 1,626,504

B 144,168 538,560 1,111,308 1,777,848 C 161,220 587,376 1,179,456 1,824,768 D 171,132 600,252 1,171,248 1,808,844 Mean 162,240 570,864 1,130,727 1,759,491 S.D. 13,048 28,076 55,588 90,773

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Appendix 8

Yield By Replicate At 72 and 96-Hours of Exposure

Mean, Measured Test Concentration (mg/L) Replicate Yield 1 (cells/mL)

72 Hours 96 Hours

Negative Control A 3,311,484 5,575,445 B 3,447,267 5,644,488 C 3,140,724 5,206,815 D 3,755,114 5,773,149 E 3,220,224 5,570,882 F 3,222,020 5,570,807 Mean 3,349,472 5,556,931 S.D. 224,534 188,535
0.23 A 3,165,199 5,104,335

B 3,309,509 5,266,134 C 3,260,618 5,664,685 D 2,819,969 5,172,780 Mean 3,138,824 5,301,984 S.D. 220,855 250,730
0.55 A 2,892,797 4,951,962

B 3,103,083 4,828,986 C 3,267,979 5,260,075 D 2,697,262 4,849,182 Mean 2,990,280 4,972,551 S.D. 248464 199,102
1.4 A 2,193,150 4,025,452

B 2,721,318 4,529,026 C 2,101,651 4,168,849 D 2,448,647 4,347,554 Mean 2,366,192 4,267,720 S.D. 278,591 218,421
3.8 A 35,221 32,921

B 34,492 33,684 C 37,764 41,101 D 34,690 35,913 Mean 35,542 35,905 S.D. 1,513 3,689
9.1 A 24,270 22,864

B 26,877 28,668 C 27,262 26,514 D 26,084 27,049 Mean 26,123 26,274 S.D. 1,329 2,451

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 ce lls/mL at test initiation (0 hours). Yield was calculated as the final biomass (cell density) in the exposur e period minus the initial biomass (cell density).

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Appendix 9

Growth Rate By Replicate Over the 96-Hour Exposure Period

Mean, Measured Test Concentration (mg/L) Replicate Growth Rate (per hour)

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

Negative Control A 0.0948 0.0878 0.0806 0.0659 B 0.1028 0.0951 0.0812 0.0660 C 0.0956 0.0890 0.0799 0.0652 D 0.0966 0.0917 0.0824 0.0663 E 0.0962 0.0920 0.0802 0.0659 F 0.0952 0.0915 0.0803 0.0659 Mean 0.0969 0.0912 0.0808 0.0658 S.D. 0.0030 0.0025 0.0009 0.0004

0.23 A 0.0989 0.0896 0.0800 0.0650 B 0.0984 0.0906 0.0806 0.0653 C 0.0988 0.0911 0.0804 0.0661 D 0.0935 0.0906 0.0784 0.0651 Mean 0.0974 0.0905 0.0799 0.0654 S.D. 0.0026 0.0006 0.0010 0.0005

0.55 A 0.0941 0.0867 0.0788 0.0647 B 0.0929 0.0889 0.0797 0.0644 C 0.0941 0.0905 0.0804 0.0653 D 0.0937 0.0863 0.0778 0.0644 Mean 0.0937 0.0881 0.0792 0.0647 S.D. 0.0006 0.0020 0.0012 0.0004

1.4 A 0.0885 0.0822 0.0749 0.0625 B 0.0926 0.0880 0.0779 0.0637 C 0.0869 0.0835 0.0743 0.0629 D 0.0906 0.0865 0.0765 0.0633 Mean 0.0896 0.0851 0.0759 0.0631 S.D. 0.0025 0.0027 0.0016 0.0005

3.8 A 0.0484 0.0278 0.0210 0.0152 B 0.0495 0.0281 0.0207 0.0154 C 0.0547 0.0292 0.0217 0.0170 D 0.0512 0.0293 0.0208 0.0159 Mean 0.0510 0.0286 0.0211 0.0159 S.D. 0.0027 0.0008 0.0005 0.0008

9.1 A 0.0371 0.0212 0.0171 0.0124 B 0.0329 0.0235 0.0181 0.0141 C 0.0355 0.0243 0.0183 0.0135 D 0.0369 0.0239 0.0178 0.0136 Mean 0.0356 0.0232 0.0178 0.0134 S.D. 0.0020 0.0014 0.0005 0.0007

# Wildlife International Project Number: 264P-101

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Appendix 10

Specific Growth Rate of the Negative Control Group

Section-by-Section Specific Growth Rates for the Negative Control Replicates:

Replicate 0-24 Hour Growth Rate 24-48 Hour Growth Rate 48-72 Hour Growth Rate 0-72 Hour % Coefficient of Variation 1

A 0.0948 0.0809 0.0662 17.7

B 0.1028 0.0874 0.0534 31.1

C 0.0956 0.0825 0.0616 21.4

D 0.0966 0.0868 0.0637 20.6

E 0.0962 0.0877 0.0568 25.8

F 0.0952 0.0878 0.0577 24.8

Mean= 23.6

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 Control Replicates During the First 72 hours:

Replicate 0-72 Hour Growth Rate

A 0.0806

B 0.0812

C 0.0799

D 0.0824

E 0.0802

F 0.0803

% Coefficient of Variation: 1.12

Mean 0.0808

Standard Deviation 0.0009

# Wildlife International Project Number: 264P-101

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Appendix 11

Personnel Involved in the Study

The following key Wildlife International 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. Allison M. Jacobs, B.S., Biologist 6. Joshua R. Arnie, B.S., Biologist