AN-2: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA 

(Pseudokirchneriella subcapitata) 

FINAL REPORT 

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

U.S. EPA OPPTS Number 850.5400 

OECD Guideline 201 

ASTM Standard E1218-04 

AUTHORS: 

Joshua R. Arnie, B.S. 

John R. Porch, M.S. 

Timothy Z. Kendall, M.S. 

Henry O. Krueger, Ph.D. 

STUDY INITIATION DATE: 15 February 2012 

STUDY COMPLETION DATE: 26 June 2012 

SUBMITTED TO: 

Albemarle Corporation 

451 Florida Street 

Baton Rouge, LA 70801 

Wildlife International, Ltd.

8598 Commerce Drive 

Easton, Maryland 21601 USA 

1-410-822-8600 

Page 1 of 56 
Wildlife International, Ltd. 

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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: ________________________________(Typed Name) 

 Company Agent: ________________________________(Typed Name) 

 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 ...................................................................... 15 

 Statistical Analyses .................................................................................................................. 15 

Results and Discussion ......................................................................................................................... 16 

 Measurement of Test Concentrations ...................................................................................... 16 

 Observations and Measurements ............................................................................................. 17

 Conditions for the Validity of the Test .................................................................................... 18 

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

References ............................................................................................................................................ 20 
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TABLE OF CONTENTS (Continued) 

TABLES AND FIGURES 

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

LSC ..................................................................................................................................... 21 

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

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

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

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

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

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

Table 8. NOEC, EC50, EyC50 and ErC50 Values Over the 96-Hour Exposure Period ........................ 26 

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

Period .................................................................................................................................. 27 

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

Density ................................................................................................................................ 27 

APPENDICES 

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

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

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

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

Ltd. Well Water ........................................................................................................ 46 

Appendix 5. Analysis of AN-2 in Freshwater AAP Algal Medium Analyzed by LSC ................ 48 

5.1 Method Outline for the Analysis of 14C AN-2 in Freshwater AAP 

Algal Medium ........................................................................................................... 49 

 5.2 Typical Counting Conditions for Liquid Scintillation Counting .............................. 50 

5.3 Calculations for Sample Quantitation ....................................................................... 51 
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TABLE OF CONTENTS (Continued) 

APPENDICES (Continued) 

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

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

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

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

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

SPONSOR: Albemarle Corporation 

STUDY: AN-2: A 96-Hour Toxicity Test with the Freshwater Alga 

(Pseudokirchneriella subcapitata) 

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

TEST DATES: Experimental Start (OECD): 20 February 2012 

 Experimental Start (EPA): 20 February 2012 

 Exposure Termination: 24 February 2012 

 Experimental Termination: 27 February 2012 

LENGTH OF EXPOSURE: 96 Hours 

TEST ORGANISM: Freshwater Alga (Pseudokirchneriella subcapitata) 

SOURCE OF TEST ORGANISMS: Wildlife International, Ltd. Cultures 

 Easton, Maryland 21601 

TEST CONCENTRATIONS: Day 0 

 Nominal Measured 

Negative Control < LOQ 

 Solvent Control < LOQ 

 1.99 ng/L 1.50 ng/L 

 3.99 ng/L 3.35 ng/L 

 7.98 ng/L 6.84 ng/L 

 16.0 ng/L 12.9 ng/L 

 31.9 ng/L 26.5 ng/L 
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SUMMARY (Continued) 

RESULTS: 

Duration of 

Exposure 

Cell Density Growth Rate Yield 

EC50

(ng/L)1

95% 

Confidence 

Interval 

(ng/L)1

ErC50

(ng/L)1

95% 

Confidence 

Interval 

(ng/L)1

ErC50

(ng/L)1

95% 

Confidence 

Interval 

(ng/L)1

24 Hours > 26.5 n/a3 > 26.5 n/a3 --2 --2

48 Hours > 26.5 n/a3 > 26.5 n/a3 --2 --2

72 Hours > 26.5 n/a3 > 26.5 n/a3 > 26.5 n/a3

96 Hours > 26.5 n/a3 > 26.5 n/a3 > 26.5 n/a3

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

72 Hours 26.5 26.5 26.5 

96 Hours 26.5 26.5 26.5 

1

 EC50, EyC50 and ErC50 values and their 95% confidence intervals were calculated using non-linear regression (Bruce and 

Versteeg 1992, Environmental Toxicology and Chemistry 11: 1485-1494) with replicate data (cell density, yield or 

growth rate) and day 0 measured test concentrations. 

2

 EyC50 values were only determined at 72 and 96 hours, per protocol. 

3

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

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

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

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

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

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

OBJECTIVE 

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

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

subcapitata, over a 96-hour exposure period. 

EXPERIMENTAL DESIGN 

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

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

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

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

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

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

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

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

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

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

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

percent inhibition values relative to the control over the 96-hour exposure period. EC50 and ErC50 values 

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

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

period. EyC50 values (i.e., the theoretical toxicant concentrations that would produce a 50% reduction in 

yield) were calculated, when possible, at 72 and 96 hours of exposure. No-observed-effect concentrations 
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(NOEC) were determined at 72 and 96 hours through statistical evaluation of cell densities, yields and 

growth rates, as well as examination of the concentration-response pattern. 

MATERIALS AND METHODS 

The study was conducted according to the procedures outlined in the protocol, “AN-2: A 

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

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

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

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

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

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

Inhibition Test (4). 

Test Substance 

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

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

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

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

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

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

Test Organism 

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

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

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

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

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

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

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

been transferred to fresh media three days prior to test initiation. The negative controls organisms were 

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

Test Apparatus 

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

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

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

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

flasks were shaken continuously at 100 rpm. 

Culture Medium 

The algal cells were cultured and tested in freshwater AAP algal medium (1-4). 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 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 525 APlus. At test initiation, pH was measured in the individual batches of test solution 

prepared for each treatment and control group. At exposure termination, pH was measured in pooled 

samples of test solution collected from each of the replicates of each treatment and control group. 
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Preparation of Test Concentrations

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

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

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

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

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

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

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

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

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

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

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

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

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

Analytical Sampling 

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

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

of test solution prepared for each treatment and control group after distribution into the test chambers. At 

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

group. Samples were processed immediately for LSC analysis. 

Analytical Method 

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

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

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

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

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

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

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

Gold XR scintillation cocktail was analyzed at each interval to determine background radioactivity. 
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Measured concentrations of 14C-AN-2 are presented in Table 1. A method flowchart is presented in 

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

quantitation are included in Appendix 5.3. 

Inoculation of Test Chambers 

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

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

7.35 x 105

 cells/mL. In order to achieve the desired initial cell density of approximately 10,000 cells per 

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

Algal Growth Measurements and Observations 

Test medium samples were collected from each 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 three days under refrigerated conditions 

sufficient to inhibit growth until cell counts could be performed. 

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

were pooled within their respective treatments, and sub-samples were removed and examined 

microscopically for atypical cell morphology (e.g., 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. 

Statistical Analyses

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

statistical analyses, were conducted using “The SAS System for Windows, 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) 
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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 (cell density). Inhibition values were 

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

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

following formula was used: 

Percent Inhibition = 

Mean ControlRe sponse

Mean ControlRe sponse  Mean Treatment Re sponse × 100 

EC50 values in this study represent the theoretical test concentrations that would produce a 50% 

reduction in a variable of interest relative to the negative control. The EyC50 and ErC50 values and their 

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

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

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

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

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

EC50 calculations, when possible. The 72 and 96-hour cell density, 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 or normal distribution and equal variance; therefore, 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 

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

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

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

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

ranged from 75 to 86% of nominal. Measured concentrations declined by Day 4, and ranged from 16 to 
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24% of nominal. The results of the study are based on day 0 measured concentrations of 1.50, 3.35, 6.84, 

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

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

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

Observations and Measurements 

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

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

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

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

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

the desired range of 6,000 ± 10% lux. 

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

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

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

their corresponding percent inhibition values are presented in Tables 5, 6, and 7, respectively. EC50, 

EyC50 and ErC50 values and the corresponding 95% confidence limits for cell density, yield and growth 

rates, respectively, are presented in Table 8. Individual replicate data for each growth parameter are 

presented in Appendices 6, 7, and 8. Figure 1 illustrates changes in mean cell density in the negative 

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

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

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

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

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

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

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

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

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

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

group. Since this reduction was not dose-responsive and appeared analogous to the data collected from 
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the other treatment groups, it was not considered to be treatment related. Consequently, the 72–hour 

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

dose-response. 

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

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

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

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

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

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

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

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

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

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

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

aggregation of cells were not observed in any of the experimental groups. 

Conditions for the Validity of the Test 

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

three days and by a factor greater than 100 within four days. After three days the mean 

cell density in the negative control replicates increased by a factor of 189 and after four 

days the mean cell density increased by a factor of 382. 

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

during the first 72 hours did not exceed 7%. It was 5.45% (Appendix 9). 

3) The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 

1-2, and 2-3) did not exceed 35%. It was 26.3% (Appendix 9) 
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CONCLUSIONS 

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

five concentrations of AN-2 ranging from 1.50 to 26.5 ng/L (based on day 0 measured test 

concentrations). Effects were evaluated based on cell density, yield and growth rate. The 72 and 96-hour 

EC50, EyC50 and ErC50 values and corresponding 95% confidence intervals, based on cell density, yield 

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

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

26.5 ng/L, for both intervals of exposure. 
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REFERENCES 

1 U.S. Environmental Protection Agency. 1996. Series 850-Ecological Effects Test Guidelines 

(draft), OPPTS Number 850.5400: Algal Toxicity, Tiers I and II. 

2 American Society for Testing and Materials. 1997. ASTM Standard Guide 1218-04. 

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

3 Organization for Economic Cooperation and Development. 2006. OECD Guidelines for 

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

Test. Adopted 23 March 2006. 

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

Inhibition Test.

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

Carolina. 

6 Bruce, Robert D. and Donald J. Versteeg. 1992. A Statistical Procedure for Modeling 

Continuous Toxicity Data. Environmental Toxicology and Chemistry. 11: 1485-1494. 
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Table 1 

Measured Concentrations of 14C-AN-2 in Test Solution Samples Analyzed by LSC 

Nominal 

AN-2 

Concentration 

(ng/L) 

Sample 

Number 

(471A-119-) 

Sampling 

Interval 

(Day) 

Sample 

Volume 

(L) 

Specific Activity 

(dpm/g) 

Total 

[14C] 

Found 

(dpm) 

AN-2 

Equivalents 

Found 

(ng/L)1,2,3

Percent 

of 

Nominal 

MeaAN

Equiva

(ng/

Background -- 

-- 

0 4 

-- -- 

-- -- 

27.38 31.06 

-- -- 

-- -- 

-- 

Negative Control 

 (0.0) 

1 8 

0 4 

0.300 0.300 

286380 286380 

27.15 35.76 

< LOQ < LOQ 

-- -- 

-- 

Solvent Control 

 (0.0) 

2 9 

0 4 

0.300 0.160 

286380 286380 

30.43 34.57 

< LOQ < LOQ 

-- -- 

-- 

2.0 3 

10 

0 4 

0.300 0.150 

286380 286380 

156.32 

51.36 

1.50 

0.473 

75.0 23.7 

0.98

4.0 4 

11 

0 4 

0.300 0.150 

286380 286380 

315.59 

59.67 

3.35 

0.666 

83.8 16.7 

2.0

8.0 5 

21 

0 4 

0.200 0.140 

286380 286380 

418.89 

82.65 

6.84 1.29 

85.5 16.1 

4.0

16.0 6 

13 

0 4 

0.100 0.150 

286380 286380 

396.12 137.86 

12.9 2.49 

80.6 15.6 

7.7

32.0 7 

14 

0 4 

0.100 0.150 

286380 286380 

787.48 285.38 

26.5 5.92 

82.8 18.5 

16.

1 AN-2 Equivalents = ({[(Total dpm found – background contribution)/Sample volume]}/Specific Activity) x 1000 2 The calculated limit of quantitation (LOQ) was 0.582 ng/L. 3 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly. 
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Table 2 

Temperature Measurements 

Time 

(Days) 

Temperature (C) 1

Measurement 1 Measurement 2 2

 

0 24.5 24.6 

1 24.6 24.9 

2 24.9 24.6 

3 24.5 24.4 

4 24.3 24.4 

 1

 The minimum and maximum temperatures monitored continuously throughout the study were 22.4 to 25.5°C, 

respectively. 2

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

Table 3 

pH Measurements 

Day 0 

 Measured Test 

Concentration 

(ng/L) 

pH Measurements 

Day 0 1 Day 4 2

 

Negative Control 7.1 8.3 

Solvent Control 7.2 8.4 

1.50 7.2 8.5 

3.35 7.2 8.3 

6.84 7.3 8.4 

12.9 7.4 8.4 

26.5 7.4 8.4 

1

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

control group at test initiation. 2

 Day 4 samples were composites of test solution collected from each of the replicates per treatment and control 

group at test termination.

Table 4 

Light Intensity Measurements 

Day 

Light Intensity (lux)1

Measurement 

No. 1 

Measurement 

No. 2 

Measurement 

No. 3 

Measurement 

No. 4 

Measurement 

No. 5 

0 6,190 6,320 6,430 5,870 6,380 1

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

Mean Cell Density and Percent Inhibition 

Day 0 

Measured Test 

Concentration 

(ng/L) 

24 Hours 1 48 Hours1 72 Hours1 

Mean 

Cell Density 

(cells/mL) 

Percent 

Inhibition 2

Mean 

Cell Density 

(cells/mL) 

Percent 

Inhibition 2

Mean 

Cell Density 

(cells/mL) 

Percent 

Inhibition 2

M

Cell D

(cell

Negative Control 56,333 -- 493,333 -- 1,885,000 -- 3,81

Solvent Control 71,333 -- 486,667 -- 1,846,667 -- 4,09

1.50 65,000 -15 363,333 26 1,610,000 15 4,713.35 58,000 -3 343,333 30 1,736,667 8 4,646.84 63,667 -13 283,333 43 960,000 49* 3,3712.9 100,667 -79 616,667 -25 2,053,333 -9 4,18

26.5 87,667 -56 593,333 -20 1,940,000 -3 4,00

1 Calculations were performed using the SAS System for Windows, Version 8.2. Manual calculations may differ slightly. 2 Percent inhibition was calculated relative to the negative control replicates. Negative values indicate an increase relative to the negative control me

* Statistically significant reduction from the negative control mean using Dunnett’s one-tailed t-test (p  0.05).
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Table 6

Mean Growth Rate (per Hour) and Percent Inhibition 

Day 0 

Measured Test 

Concentration 

(ng/L) 

0-24 Hours1 0-48 Hours1 0-72 Hours1 

Mean 

Growth Rate 

(hours-1) 

Percent 

Inhibition 2 

Mean 

Growth Rate 

(hours-1) 

Percent 

Inhibition 2 

Mean 

Growth Rate 

(hours-1) 

Percent 

Inhibition 2 

M

Grow

(ho

Negative Control 0.0705 -- 0.0803 -- 0.0724 -- 0.0

Solvent Control 0.0791 -- 0.0799 -- 0.0722 -- 0.0

1.50 0.0770 -9 0.0747 7 0.0696 4 0.03.35 0.0729 -3 0.0735 9 0.0714 1 0.06.84 0.0748 -6 0.0696 13 0.0626 13* 0.012.9 0.0961 -36 0.0854 -6 0.0740 -2 0.026.5 0.0903 -28 0.0850 -6 0.0730 -1 0.0

1 Calculations were performed using the SAS System for Windows, Version 8.2. Manual calculations may differ slightly. 2 Percent inhibition was calculated relative to the negative control replicates. Negative values indicate an increase relative to the negative control me

* Statistically significant reduction from the negative control mean using Dunnett’s one-tailed t-test (p  0.05).
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Table 7 

Mean Yield and Percent Inhibition 

Day 0 

Measured Test 

Concentration 

(ng/L) 

72 Hours1 96 Hours1

Mean 

Cell Density 

(cells/mL) 

Percent 

Inhibition 2

Mean 

Cell Density 

(cells/mL) 

Percent 

Inhibition 2

Negative Control 1,875,000 -- 3,806,667 -- 

Solvent Control 1,836,667 -- 4,083,333 -- 

1.50 1,600,000 15 4,703,333 -24 3.35 1,726,667 8 4,636,667 -22 6.84 950,000 49* 3,363,333 12 

12.9 2,043,333 -9 4,170,000 -10 

26.5 1,930,000 -3 3,996,667 -5 

1 Calculations were performed using the SAS System for Windows, Version 8.2. Manual calculations may 

differ slightly. 

2 Percent inhibition was calculated relative to the negative control replicates. Negative values indicate an 

increase relative to the negative control mean. 

* Statistically significant reduction from the negative control mean using Dunnett’s one-tailed t-test 

(p  0.05).
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Table 8 

NOEC, EC50, EyC50 and ErC50 Values Over the 96-Hour Exposure Period 

Duration of 

Exposure 

Cell Density Growth Rate Yield 

EC50

(ng/L)1

95% 

Confidence 

Interval 

(ng/L)1

ErC50

(ng/L)1

95% 

Confidence 

Interval 

(ng/L)1

ErC50

(ng/L)1

95% 

Confidenc

Interval 

(ng/L)1

24 Hours > 26.5 n/a3 > 26.5 n/a3 --2 --248 Hours > 26.5 n/a3 > 26.5 n/a3 --2 --2

72 Hours > 26.5 n/a3 > 26.5 n/a3 > 26.5 n/a3

96 Hours > 26.5 n/a3 > 26.5 n/a3 > 26.5 n/a3

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

72 Hours 26.5 26.5 26.5 96 Hours 26.5 26.5 26.5 

1 EC50, EyC50 and ErC50 values and their 95% confidence intervals were calculated using non-linear regression (Bruce and 

Versteeg 1992, Environmental Toxicology and Chemistry 11: 1485-1494) with replicate data (cell density, yield or 

growth rate) and day 0 measured test concentrations. 

2 EyC50 values were only determined at 72 and 96 hours, per protocol. 3 95% confidence intervals could not be calculated with the data obtained. 
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Figure 1 

Growth of P. subcapitata in the Control Groups 

Over the 96-Hour Exposure Period 

Figure 2

Growth of P. subcapitata Exposed to AN-2 for 

96 Hours, Expressed as Cell Density 

1,000

10,000

100,000

1,000,000

10,000,000

0 24 48 72 96

Exposure Duration (hours)

Cell Density (cells/mL)

Negative

Control

Solvent

Control

1,000

10,000

100,000

1,000,000

10,000,000

0 24 48 72 96

Exposure Duration (Hours)

Cell Density (cells/mL)

Negative Control

Solvent Control

1.50 ng/L

3.35 ng/L

6.84 ng/L

12.9 ng/L

26.5 ng/L
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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 

35.0 mg/L 

1

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

 Project Number: 471A-119 

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Appendix 4 

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water 

Pesticides and Organics 1

Component 

Measured Concentration 

(μg/L) Component 

Measured Concentration 

(μg/L) 

Aldrin < 0.0098 Gamma BHC – Lindane < 0.0098 

Alpha BHC < 0.0098 Gamma Chlordane < 0.020 

Alpha Chlordane < 0.0098 Guthion (Azinphos-methyl) < 5.9 

Beta BHC < 0.0098 HCB < 0.0098 

Bolstar < 2.0 Heptachlor < 0.0098 

Chlordane < 0.49 Heptachlor Epoxide < 0.0098 

Coumaphos < 2.9 Kepone < 0.20 

Delta BHC < 0.0098 Malathion < 2.9 

Demeton-O < 2.0 Merphos < 2.0 

Demeton-S < 2.9 Methoxychlor < 0.098 

Diazinon < 5.9 Methyl Parathion < 2.0 

Dichlorvos < 2.9 Mevinphos < 4.9 

Dieldrin < 0.020 Mirex < 0.25 

Disulfoton < 2.0 Naled < 5.9 

Dursban (Chlorpyrifos) < 2.9 o,p-DDD < 0.020 

Endosulfan I < 0.0098 o,p-DDE < 0.74 

Endosulfan II < 0.020 o,p-DDT < 0.020 

Endosulfan Sulfate < 0.020 p,p-DDD < 0.020 

Endrin < 0.020 p,p-DDE < 0.020 

Endrin Aldehyde < 0.098 p,p-DDT < 0.020 

Endrin Ketone < 0.020 Phorate < 2.0 

EPN < 2.0 Ronnel < 2.0 

Ethion < 2.0 Stirophos < 2.0 

Ethoprop < 2.9 Telodrin < 0.0098 

Ethyl Parathion < 2.0 Tokuthion < 2.0 

Famphur < 2.9 Toxaphene < 2.9 

Fensulfothion < 8.8 Trichloronate < 2.0 

Fenthion < 2.0 Trithion < 2.0 

 

1

 Analyses performed by Lancaster Laboratories on samples collected on December 29, 2010. 
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Appendix 4 (Continued) 

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water 

Metals 1

Component 

Measured Concentration 

(mg/L) Component 

Measured Concentration 

(mg/L) 

 

Aluminum < 0.200 Magnesium 13.0 

Antimony < 0.0200 Manganese < 0.0050 

Arsenic < 0.0200 Mercury < 0.00020 

Barium < 0.0050 Nickel < 0.0100 

Beryllium < 0.0050 Nitrate Nitrogen 0.55 

Bromide < 2.5 Nitrite Nitrogen < 0.50 

Cadmium < 0.0050 Potassium 6.77 

Calcium 35.1 Selenium < 0.0200 

Chloride 3.5 Silver < 0.0050 

Chromium < 0.0150 Sodium 18.5 

Cobalt < 0.0050 Sulfate 5.6 

Copper < 0.0100 Thallium < 0.0300 

Fluoride < 0.50 Vanadium < 0.0050 

Iron < 0.200 Zinc < 0.0200 

Lead < 0.0150 

1

 Analyses performed by Lancaster Laboratories on samples collected on December 29, 2010. 
Wildlife International, Ltd. 

 Project Number: 471A-119 

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Appendix 5 

Analysis of AN-2 in Freshwater AAP Algal Medium Analyzed by LSC
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Appendix 5.1 

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

METHOD OUTLINE FOR THE ANALYSIS OF 

14C-AN-2 IN FRESHWATER AAP ALGAL MEDIUM 

Rinse separatory funnels and roundbottom flasks with dichloromethane. 



Using a tiltapetter, add 100 mL of dichloromethane to each sample. Stopper and shake each 

sample, with venting, for approximately one minute. Allow the organic and 

aqueous layers to separate. Drain the dichloromethane (lower) layer 

into a round bottom flask. 



Add 100 mL of dichloromethane to separatory funnel containing the aqueous fraction. 

Shake and partition as described above and combine each extract in its 

respective roundbottom flask. 

↓

Rotary evaporate each sample to approximately 0.50 – 1.0 mL using a water bath 

maintained at ~40°C. Do not rotary evaporate to dryness. 

↓

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

↓

Rinse the roundbottom flask with several aliquots of acetonitrile. Transfer the rinsates to a 

scintillation vial. Add approximately 10 mL of Ultima Gold XR and analyze by LSC. 
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Appendix 5.2 

Typical Counting Conditions for Liquid Scintillation Counting 

INSTRUMENT: Packard 2500TR Liquid Scintillation Analyzer 

COUNTING TIME: 3.00 minutes 

DATA MODE: DPM 

NUCLIDE: 14C-UG 

QUENCH INDICATOR: tSIE/AEC 

EXT. STD. TERMINATOR: Count 

QUENCH SET: 14C-UG 
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Appendix 5.3 

Calculations for Sample Quantitation 

Sample Quantitation 

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

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

volume]}/Specific Activity x 1000 

Limit of Quantitation for Freshwater

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

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

(286380) x 1000 

The calculated limit of quantitation (LOQ) was 0.582 ng/L 
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Appendix 6 

Cell Density By Replicate Over the 96-Hour Exposure Period 

Day 0 Measured 

Concentration 

(ng/L) Replicate 

Cell Density 1

 (cells/mL) 

24 Hours 48 Hours 72 Hours 96 Hours 

Negative Control A 37,000 260,000 1,060,000 2,760,000 

 B 38,000 510,000 2,200,000 3,600,000 

 C 57,000 560,000 2,150,000 4,700,000 

 D 74,000 630,000 2,120,000 3,720,000 

 E 68,000 390,000 1,690,000 3,840,000 

 F 64,000 610,000 2,090,000 4,280,000 

 

Solvent Control A 103,000 710,000 2,340,000 4,500,000 

 B 42,000 340,000 1,370,000 3,820,000 

 C 69,000 410,000 1,830,000 3,960,000 

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

 B 57,000 410,000 1,770,000 5,740,000 

 C 86,000 360,000 2,190,000 4,820,000 

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

 B 60,000 370,000 2,010,000 4,320,000 

 C 66,000 380,000 1,870,000 4,660,000 

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

 B 48,000 260,000 560,000 3,380,000 

 C 95,000 320,000 1,280,000 3,480,000 

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

 B 110,000 720,000 2,040,000 3,900,000 

 C 89,000 450,000 2,010,000 4,620,000 

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

 B 82,000 620,000 1,720,000 4,120,000 

 C 97,000 610,000 1,780,000 3,420,000 

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

yield a cell density of approximately 10,000 cells/mL at test initiation (0 hours). 
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Appendix 7 

Yield By Replicate At 72 and 96 Hours of Exposure 

Day 0 Measured 

Concentration 

(ng/L) Replicate 

Yield 1

 (cells/mL) 

72 Hours 96 Hours 

Negative Control A 1,050,000 2,750,000 

 B 2,190,000 3,590,000 

 C 2,140,000 4,690,000 

 D 2,110,000 3,710,000 

 E 1,680,000 3,830,000 

 F 2,080,000 4,270,000 

 

Solvent Control A 2,330,000 4,490,000 

 B 1,360,000 3,810,000 

 C 1,820,000 3,950,000 

 

1.50 A 860,000 3,570,000 

 B 1,760,000 5,730,000 

 C 2,180,000 4,810,000 

 

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

 B 2,000,000 4,310,000 

 C 1,860,000 4,650,000 

 

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

 B 550,000 3,370,000 

 C 1,270,000 3,470,000 

 

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

 B 2,030,000 3,890,000 

 C 2,000,000 4,610,000 

 

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

 B 1,710,000 4,110,000 

 C 1,770,000 3,410,000 

1 The initial cell density of the stock culture was determined and an inoculum 

volume was administered to each test chamber to yield a cell density of 

approximately 10,000 cells/mL at test initiation (0 hours). Yield was calculated 

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

(cell density). 
Wildlife International, Ltd. 

 Project Number: 471A-119 

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Appendix 8

Growth Rate By Replicate Over the 96-Hour Exposure Period 

Day 0 Measured 

Concentration 

(ng/L) Replicate 

Growth Rate (per hour) 

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

Negative Control A 0.0545 0.0679 0.0648 0.0585 

 B 0.0556 0.0819 0.0749 0.0613 

 C 0.0725 0.0839 0.0746 0.0641 

 D 0.0834 0.0863 0.0744 0.0617 

 E 0.0799 0.0763 0.0712 0.0620 

 F 0.0773 0.0856 0.0742 0.0631 

 

Solvent Control A 0.0972 0.0888 0.0758 0.0636 

 B 0.0598 0.0735 0.0683 0.0619 

 C 0.0805 0.0774 0.0724 0.0623 

 

1.50 A 0.0687 0.0722 0.0620 0.0613 

 B 0.0725 0.0774 0.0719 0.0662 

 C 0.0897 0.0747 0.0748 0.0644 

 

3.35 A 0.0654 0.0694 0.0679 0.0647 

 B 0.0747 0.0752 0.0737 0.0632 

 C 0.0786 0.0758 0.0727 0.0640 

 

6.84 A 0.0654 0.0687 0.0645 0.0603 

 B 0.0654 0.0679 0.0559 0.0607 

 C 0.0938 0.0722 0.0674 0.0610 

 

12.9 A 0.0972 0.0879 0.0743 0.0625 

 B 0.0999 0.0891 0.0739 0.0621 

 C 0.0911 0.0793 0.0737 0.0639 

 

26.5 A 0.0887 0.0835 0.0756 0.0636 

 B 0.0877 0.0860 0.0715 0.0627 

 C 0.0947 0.0856 0.0720 0.0608 
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Appendix 9

Specific Growth Rate of the Negative Control Group 

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

Negative 

Control 

Replicate 

0-24 Hour 

Growth 

Rate 

24-48 Hour 

Growth Rate 

48-72 Hour 

Growth Rate 

% Coefficient 

of Variation1

A 0.0545 0.0812 0.0586 22.24 

B 0.0556 0.1082 0.0609 38.65 

C 0.0725 0.0952 0.0561 26.35 

D 0.0834 0.0892 0.0506 28.02 

E 0.0799 0.0728 0.0611 13.31 

F 0.0773 0.0939 0.0513 28.96 

 Mean (%) = 26.3 

1

Coefficient of Variation = S.D./mean *100. Results were generated using Excel 

2000 in the full precision mode. Manual calculations may differ slightly. 

Average Specific Growth Rates for the Negative Control Replicates 

During the Test Period: 

Negative Control Replicate 0-72 Hour Growth 

Rate 

A 0.0648 

B 0.0749 

C 0.0746 

D 0.0744 

E 0.0712 

F 0.0742 

Mean 

SD 

% Coefficient of Variation 

0.0724 

0.0039 

5.45 
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Appendix 10 

Personnel Involved in the Study 

 The following key Wildlife International, Ltd. personnel were involved in the conduct or 

management of this study: 

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

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

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

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

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