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 2nd 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 Assurance 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.......................................................................................... 14 

 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 .................................................................................... 20 

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

References ............................................................................................................................................ 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 Benzophenone 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, EbC50, EyC50 and ErC50 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 Operational 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/L 
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SUMMARY (Continued) 

RESULTS: 

Exposure 

Duration 

(hours) 

Area Under the Growth Curve 

(mg/L) 

Growth Rate 

(mg/L) 

Yield 

(mg/L) 

NOEC EbC501 95% CI NOEC ErC501 95% CI NOEC EyC501 95% CI

72 0.23 1.7 1.4 to 2.2 0.23 3.0 2.3 to 4.0 0.23 1.7 1.2 to 2.496 0.23 1.8 1.3 to 2.4 0.23 2.9 2.2 to 3.9 0.23 1.8 0.96 to 3.4

1 EbC50, ErC50 and EyC50 values and their corresponding 95% confidence intervals (CI) were calculated using non-linear 

regression 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 International for American Chemistry Council at the 

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

test was conducted from September 20 to 24, 2012, with cell counts completed on September 25, 2012. 

Raw data generated by Wildlife International and 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 the 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 samples 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 approximately 10,000 cells/mL. Samples were collected 

from each replicate test chamber at approximately 24-hour intervals during the test to determine cell 

densities, 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. EbC50, ErC50 and EyC50 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 examination of the concentration-response pattern. 

MATERIALS AND METHODS 

The study was conducted according to the procedures 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 prepare 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 identification 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 have been maintained in culture medium at Wildlife 

International, Easton, Maryland since March 2000. Algal cells used in this test were obtained from 

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

environmental conditions as used in this test for at least two weeks prior to test initiation. Algal cells for 

this study were taken from a culture that had been transferred to fresh 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 daily in an environmental 

chamber designed to maintain the desired test temperature 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 freshwater 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 contaminants 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 the test chambers in the environmental chamber was 

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

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

were held under continuous cool-white fluorescent lighting throughout the test. The target light intensity 

was 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 dissolving 0.0100 g of benzophenone in 1000 mL of 

freshwater AAP medium to achieve a nominal concentration 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 mixing. 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 control 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 and 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) equipped 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 flowchart is provided in Appendix 5.1 and typical 

instrumental parameters are summarized in Appendix 5.2. 
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Five calibration standards of benzophenone 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 generated using the peak area 

responses versus the respective concentrations of the calibration standards. The concentration of 

benzophenone in the samples was determined by substituting the peak area 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 defined 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 analyzed 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 fortified at 0.250, 0.750, and 10.0 mg/L using stock 

solutions of benzophenone in methanol (Appendix 5.3), and were analyzed concurrently with the test 

samples. The measured concentrations for the matrix 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 presented 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, respectively. 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 stock culture (culture identification No. 00-1) were 

verified in freshwater algal medium at a concentration of approximately 1.36 x 106

 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 replicate 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 maximum of four days under 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 counts, 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 determination 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 was determined by adjusting the mean cell count 

(cells/mL) obtained using the particle counter, based 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

Mean Cell Count (cells/mL)  Y Intercept × Dilution Factor 

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., changes 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. 
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Statistical Analyses

The calculation of cell densities, area under the growth curve, yield, growth rates and percent 

inhibition values, as well as all statistical analyses, 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 = 

n o

n o

t - t

ln N - ln N

where: μ = Average specific growth rate 

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

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

 to = Time of beginning of test (hours) 

 tn = Time after beginning of test (hours) 

Area under the growth curve (biomass) was calculated for each replicate of the control and 

treatment groups at 24, 48, 72 and 96 hours using the following formula: 

 A = ((N1-N0)/2)(t1)+((N1+N2-2N0)/2)(t2-t1)+...+((Nn-1+Nn-2N0)/2)(tn-tn-1) 

 Where: 

 A = Area under the growth curve 

 N0 = Mean nominal number of cells/mL at t0

 N1 = Mean measured number of cells/mL at t1

 N2 = Mean measured number of cells/mL at t2

 Nn = Mean measured number of cells/mL at tn 

 t0 = test initiation 

 t1 = Time of first measurement after beginning of test (hours) 

 t2 = Time of second measurement after beginning of test (hours) 

 tn = Time of nth 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 percent reduction in area under the growth curve, yield 

and growth rate relative to the negative control replicates using the following formula: 
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Percent Inhibition = 

Mean ControlRe sponse

Mean ControlRe sponse  Mean Treatment Re sponse × 100 

EC50 values, (i.e., the theoretical test concentrations that would produce a 50% reduction in area 

under the growth curve (EbC50), growth rate (ErC50) and yield (EyC50), 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 growth curve, growth rate and yield, 

respectively) and mean measured test concentrations. 

The 72 and 96-hour area under the growth curve, yield 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 control 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 ranged from 96 to 105% of nominal in samples 

collected on Day 0 and from 74 to 83% of nominal at test termination at 96 hours (Table 2). Test solution 

appeared clear and colorless after mixing, and no surface 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 algae. 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 period. 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 and yields and their corresponding percent inhibition 

values are presented in Tables 6, 7 and 8, respectively. NOEC, EbC50, EyC50 and ErC50 values and their 

corresponding 95% confidence limits for area under the 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, expressed 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%, respectively, relative to the negative control. The 

72-hour EC50 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. 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 groups 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 EC50 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. 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 normal. After 96-hours of exposure there were no 

noticeable changes in cell morphology in any of the tested concentrations when compared to the control 

and there were no noticeable aggregations and flocculations 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. The 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 ranging from 0.23 to 9.1 mg/L, based on mean, measured concentrations 

of Benzophenone from samples collected at test initiation 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 EC50 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 EC50 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. 

Standard 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 106 -- 

0.10 3.93 X 106 -8 

1.0 2.70 X 106

 26 

10 1.93 X 104

 99 

1

 Appearance of test solutions: clear and colorless, with no precipitation noted. 
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 Project Number: 264P-101 

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Table 2 

Measured Concentrations of Benzophenone in Test Solution Samples 

Nominal 

 Test 

Concentration 

(mg/L) 

Sample 

Number 

(264P-101-) 

Sampling 

Time 

(Hours) 

Measured 

Concentration 

(mg/L)1 

Percent 

of 

Nominal2

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 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 full precision mode. Manual calculations may differ slightly.
Wildlife International 

 Project Number: 264P-101 

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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 continuously 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 test flasks at test solution level. 
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Table 6 

Mean Area Under the Growth Curve and Percent Inhibition 

Mean, Measured 

Test 

Concentration 

(mg/L) 

24 Hours 1 48 Hours1 72 Hours1 

Mean 

Area Under the 

Growth Curve 

Percent 

Inhibition2

Mean 

Area Under the 

Growth Curve 

Percent 

Inhibition2

Mean 

Area Under the 

Growth Curve 

Percent 

Inhibition2

M

Area 

the G

Cu

Negative Control 1,109,778 -- 11,712,518 -- 61,399,146 -- 168,2

0.23 1,124,301 -1 11,369,784 3 58,156,851 5 159,4

0.55 1,016,754 8 10,182,732 13 54,215,319* 12 149,7

1.4 912,900 18 8,870,190 24 44,308,878* 28 123,93.8 288,594 74 931,374 92 1,712,061* 97 2,569.1 162,240 85 570,864 95 1,130,727* 98 1,75

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

* Statistically significant difference (p≤0.05) at 72 and 96 hours from the negative control replicates using Dunnett’s test.
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Table 7 

 Mean Growth Rate (per Hour) and Percent Inhibition 

Mean, Measured 

Test 

Concentration 

(mg/L) 

0-24 Hours 1 0-48 Hours1 0-72 Hours1 

Mean 

Growth Rate 

(hours-1) 

Percent 

Inhibition2

Mean 

Growth Rate 

(hours-1) 

Percent 

Inhibition2

Mean 

Growth Rate 

(hours-1) 

Percent 

Inhibition2

M

Grow

(ho

Negative Control 0.0969 -- 0.0912 -- 0.0808 -- 0.0

0.23 0.0974 -1 0.0905 1 0.0799 1 0.00.55 0.0937 3 0.0881 3 0.0792* 2 0.0

1.4 0.0896 7 0.0851 7 0.0759* 6 0.03.8 0.0510 47 0.0286 69 0.0211* 74 0.09.1 0.0356 63 0.0232 75 0.0178* 78 0.0

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

* Statistically significant difference (p≤0.05) at 72 and 96 hours from the negative control replicates using Dunnett’s test.
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Table 8 

Mean Yield and Percent Inhibition 

Mean, Measured 

Test 

Concentration 

(mg/L) 

72 Hours 1 96 Hours1

Mean 

Yield 

(cells/mL) 

Percent 

Inhibition2

Mean 

Yield 

(cells/mL) 

Percent 

Inhibition2

Negative Control 3,349,472 -- 5,556,931 -- 

0.23 3,138,824 6 5,301,984 5 0.55 2,990,280* 11 4,972,551* 11 

1.4 2,366,192* 29 4,267,720* 23 3.8 35,542* 99 35,905* 99 9.1 26,123* 99 26,274* 100 

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

* Statistically significant difference from the negative control replicates using Dunnett’s test 

(p  0.05).
Wildlife International Pro

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Table 9 

NOEC, EbC50, EyC50 and ErC50 Values Over the 96-Hour Exposure Period 

Exposure 

Duration 

(hours) 

Area Under the Growth Curve 

(mg/L) 

Growth Rate 

(mg/L) 

Yield 

(mg/L) 

NOEC EbC501 95% CI NOEC ErC501 95% CI NOEC EyC501 95% C

72 0.23 1.7 1.4 to 2.2 0.23 3.0 2.3 to 4.0 0.23 1.7 1.2 to 296 0.23 1.8 1.3 to 2.4 0.23 2.9 2.2 to 3.9 0.23 1.8 0.96 to 

1 EbC50, ErC50 and EyC50 values and their corresponding 95% confidence intervals (CI) were calculated using non-l

regression with replicate data (area under the growth curve, growth rate or yield) and mean, measured test concentrat
Wildlife International 

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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 96

Cell Density (cells/mL)

Exposure Duration (Hours)
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 Project Number: 264P-101 

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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 10

Area Under the Growth Curve

Mean, Measured Test Concentration (mg/L)

Data

Means

Predicted

Regression

95% Conf. Int.

50% Inhibition
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 Project Number: 264P-101 

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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 10

Growth Rate (hours-1)

Mean, Measured Test Concentration (mg/L)

Data

Means

Predicted

Regression

95% Conf. Int.

50% Inhibition
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 Project Number: 264P-101 

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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 10

Yield (cells/mL)

Mean, Measured Test Concentration (mg/L)

Data

Means

Predicted

Regression

95% Conf. Int.

50% Inhibition
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 Project Number: 264P-101 

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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 Medium1

Compound 

Nominal 

Concentration 

MgCl2•6H2O 

CaCl2•2H2O 

H3BO3 

MnCl2•4H2O 

ZnCl2

FeCl3•6H2O 

CoCl2•6H2O 

Na2MoO4•2H2O 

CuCl2•2H2O 

Na2EDTA•2H2O 

NaNO3

MgSO4•7H2O 

K2HPO4

NaHCO3

12.164 mg/L 

 4.410 mg/L 

 0.1855 mg/L 

 0.4154 mg/L 

 3.27 μg/L 

 0.1598 mg/L 

 1.428 μg/L 

 7.26 μg/L 

 0.012 μg/L 

 0.300 mg/L 

25.50 mg/L 

14.70 mg/L 

 1.044 mg/L 

15.0 mg/L 

1

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

 Project Number: 264P-101 

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Appendix 4 

Analyses of Pesticides, Organics and Metals 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 Laboratories on samples collected on December 28, 2011. 
Wildlife International 

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Appendix 4 (Continued) 

Analyses of Pesticides, Organics and Metals 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 Laboratories on samples collected on December 28, 2011. 
Wildlife International 

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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 using 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% H3PO4 

CH3CN 

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 transferred 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 for 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 calculated 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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 Project Number: 264P-101 

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Appendix 5.5 

Quality Control Samples of Benzophenone in Freshwater Algal Medium 

Sample Number 

(264P-101-) Sample 

Type 

Fortified 

Concentration 

(mg/L) 

Measured 

Concentration 

(mg/L) 1

Percent 

Recovery2

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 precision mode. Manual calculations may differ slightly. 
Wildlife International 

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Appendix 5.6 

Representative Calibration Curve for Benzophenone 

Slope = 20.2969; Y-Intercept = 0.0445; R2

 = 0.9987 
Wildlife International 

 Project Number: 264P-101 

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Appendix 5.7 

Representative Chromatogram of a Low-level Benzophenone Calibration Standard 

0 2 4 6 8 10 12 min

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 

0 2 4 6 8 10 12 min

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 

0 2 4 6 8 10 12 min

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 

0 2 4 6 8 10 12 min

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 

0 2 4 6 8 10 12 min

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 
Wildlife International 

 Project Number: 264P-101 

-67- 

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 cells/mL at test initiation (0 hours). Yield was calculated 

as the final biomass (cell density) in the exposure period minus the initial biomass (cell density). 
Wildlife International 

 Project Number: 264P-101 

-68- 

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 

-69- 

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 Variation1 

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 

-70- 

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