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PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA (Anabaenaflos-aquae) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-110B 3M LAB REQUEST NO. U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.5400 AUTHORS: Debbie Desjardins Cary A. Sutherland Raymond L. Van Hoven, Ph.D. Henry O. Krueger, Ph.D. STUDY INITIATION DATE: January 28, 2000 STUDY COMPLETION DATE: June 6, 2001 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 Wildlife InternatioLtd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . 2- - PROJECT NO.: 454A -110B GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Anabaenaflos-aquae) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-110B STUDY COMPLETION: June 6, 2001 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice, (ENV/MC/CHEM(98)17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exceptions: The test and reference substances were not characterized in accordance with full GLP compliance prior to its use in the study; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data are being maintained in the 3M archives. The test substance has been recharacterized in accordance with GLP (September 7, 2000). The stability of the test and reference substances under conditions of storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Cary A. Sutherland Laboratory Supervisor SPONSOR: BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -3 - projectno.: 454A-110B QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice, (ENV/MC/CHEM(98)17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: DATE REPORTED TO: DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT: Test Substance Preparation February 24, 2000 February 24, 2000 February 29, 2000 Analytical Standard Preparation February 25, 2000 February 25, 2000 March 7, 2000 Cell Counts February 29, 2000 February 29, 2000 March 8, 2000 Analytical Data and Draft Report July 11 & 12, 2000 July 12, 2000 July 13, 2000 Biological Data and Draft Report July 14, 17 - 19, 2000 July 19, 2000 August 30, 2000 Analytical Data and Second Draft March 9, 2001 Report March 12, 2001 March 12, 2001 Biological Data and Second Draft March 2 0 -2 1 ,2 0 0 1 Report Final Report March 29, 2001 March 21, 2001 June 5, 2001 June 5, 2001 June 5, 2001 James H. Coleman Quality Assurance Representative DATE BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -4 - p r o j e c t n o .: 454A-110B REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Anabaenaflos-aquae) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-110B STUDY DIRECTOR: Cary A. Sutherland Laboratory Supervisor DATE MANAGEMENT: H en iy O . Kruegyceti, Ph D. Director, Aquatic Toxicology and Non-Target Plants BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -5 - p r o j e c t n o .: 454A-110B TABLE OF CONTENTS Title/Cover Page................................................................................................................................................1 Good Laboratory Practice Compliance Statement.........................................................................................2 Quality Assurance Statement........................................................................................................................... 3 Report Approval............................................................................................................................................... 4 Table of Contents.............................................................................................................................................. 5 Summary............................................................................................................................................................7 Introduction....................................................................................................................................................... 9 Objective............................................................................................................................................................9 Experimental Design.........................................................................................................................................9 Materials and Methods....................................................................................................................................10 Results and Discussion....................................................................................................................................15 Conclusions......................................................................................................................................................17 References........................................................................................................................................................18 TABLES T a b le 1 - S u m m ary o f A naly tical C hem istry D a t a ................................................................................................ 19 Table 2 - Temperature Measurements...................................................................................................... 20 Table 3 - Light Intensity Measurements.................................................................................................... 21 Table 4 - pH Measurements....................................................................................................................... 22 Table 5 - Mean Cell Densities and Percent Inhibition for Each 24-Hour Interval During the Test............................................................................................................................ 23 Table 6 - Mean Areas Under the Growth Curve and Percent Inhibition for Each 24-Hour Interval During the T est.............................................................................................. 24 Table 7 - Mean Growth Rates and Percent Inhibition for Each 24-Hour Interval During the Test 25 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . 6- - p r o j e c t n o .: 454a -iio b TABLE OF CONTENTS -Continued- Table 8 - EC Values Based on Cell Density Over the 96-Hour Exposure Period...................................26 Table 9 - EC Values Based on Area Under the Growth Curve Over the 96-Hour Exposure Period .... 27 Table 10 - EC Values Based on Growth Rate Over the 96-Hour Exposure Period...................................28 Table 11 - Cell Densities During the Recovery Phase................................................................................. 29 FIGURES Figure 1 - Negative control algal growth, expressed as cell density, during the 96-hour exposure period............................................................................................................................30 Figure 2 - Concentration-response curve, expressed as cell density, over the 96-hour exposure period............................................................................................................................31 Figure 3 - Recovery-phase response curve, expressed as cell density..................................................... 32 APPENDICES Appendix I - Freshwater Algal Medium Constituents..........................................................................33 Appendix II - Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well W ater..........................................................................................................................34 Appendix III - The Analysis of PFOS in Freshwater Algal Medium in Support of Wildlife International, Ltd. Project No.: 454A-110B.....................................................36 Appendix IV - Cell Density for Each Replicate Per Treatment Over the 96-Hour Exposure Period..................................................................................................................53 Appendix V - Area Under the Growth Curve for Each Replicate Per Treatment Over the 96-Hour Exposure Period...................................................................................54 Appendix VI - Growth Rate for Each Replicate Per Treatment Over the 96-Hour Exposure Period..................................................................................................................55 Appendix VII - Changes to Protocol...........................................................................................................56 Appendix VIII - Personnel Involved in the Study.......................................................................................57 W il d l if e In t e r n a t io n a l , Lt d . -7 - SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: SUMMARY 3M Corporation Rochelle R. Robideau Wildlife International, Ltd. Easton, MD 2 1601 BACK TO MAIN PROJECT NO.: 454A -110B WILDLFE INTERNATIONAL, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF EXPOSURE : 454A-110B PFOS (Perfluorooctanesulfonate, Potassium Salt) IUPAC Name: 1-Octanesulfonic acid,1,1,2,2,3,3,4,4,5,5,6,6, 7,7,8,8,8-heptadecafluoro-potassium salt; CAS #2795-39-3 PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Anabaena flos-aquae) Negative Control, 37.9, 58.6, 88.8, 139, 216 and 331 mg a.i./L Negative Control, 37.9, 63.9, 93.8, 143, 235 and 329 mg a.i./L Experimental Start (OECD) - May 11, 2000 Experimental Start (EPA) - May 12, 2000 Exposure Termination - May 16, 2000 Experimental Termination - June 5, 2000 96 Hours | TEST ORGANISM: SOURCE OF TEST ORGANISMS: Freshwater Alga (Anabaenaflos-aquae) Wildlife International, Ltd. Easton, Maryland 2 1601 W il d l if e In t e r n a t io n a l , Lt d . 8- - CELL DENSITY: 72-HOUR EC50: 95% CONFIDENCE LIMITS: 96-HOUR EC 10: 95% CONFIDENCE LIMITS: 96-HOUR EC50: 95% CONFIDENCE LIMITS: 96-HOUR EC90: 95% CONFIDENCE LIMITS: 72-HOUR NOAEC: 96-HOUR NOAEC: SUMMARY (Continued) 120 mg a.i./L 91.8 - 139 mg a.i./L 82.3 mg a.i./L 29.1 - 123 mg a.i./L 131 mg a.i./L 106 - 142 mg a.i./L 213 mg a.i./L 203 -219 mg a.i./L 37.9 mg a.i./L 93.8 mg a.i./L AREA UNDER THE GROWTH CURVE: 72-HOUR EC50: 95% CONFIDENCE LIMITS: 96-HOUR EC 10: 95% CONFIDENCE LIMITS: 96-HOUR EC50: 95% CONFIDENCE LIMITS: 96-HOUR EC90: 95% CONFIDENCE LIMITS: 72-HOUR NOAEC: 96-HOUR NOAEC: 116 mg a.i./L 49.2 - 142 mg a.i./L 56.1 mg a.i./L 26.3 - 107 mg a.i./L 124 mg a.i./L 104 - 138 mg a.i./L 209 mg a.i./L 197 - 218 mg a.i./L 37.9 mg a.i./L 63.9 mg a.i./L GROWTH RATE: 72-HOUR EC50: 95% CONFIDENCE LIMITS: 174 mg a.i./L 146 - 208 mg a.i./L 96-HOUR EC 10: 95% CONFIDENCE LIMITS: 109 mg a.i./L 84.1 - 125 mg a.i./L 96-HOUR EC50: 95% CONFIDENCE LIMITS: 176 mg a.i./L 169 - 181 mg a.i./L 96-HOUR EC90: 95% CONFIDENCE LIMITS: 225 mg a.i. 220 - 235 mg a.i./L 72-HOUR NOAEC: 96-HOUR NOAEC: 93.8 mg a.i./L 93.8 mg a.i./L All values are mean measured test concentrations. BACK TO MAIN PROJECT NO.: 454A -110B BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -9 - p r o j e c t n o .: 454A-110B INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. Two initial trials of the test were conducted between February 25, 2000 and March 28, 2000. However, the tests were repeated due to high variability in cell counts and lack of a clear dose-response in the first trials. The in-life phase of the definitive test was conducted from May 12, 2000 to May 16, 2000, with the recovery phase completed on May 25, 2000, and cell counts completed on June 5, 2000. Raw data generated by Wildlife International, Ltd and a copy of the final report are filed under Project Number 454A-110B in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of the study was to evaluate the toxicity of PFOS (Perfluorooctanesulfonate, Potassium Salt) to the growth of the freshwater alga, Anabaena flos-aquae, during a 96-hour exposure period. EXPERIMENTAL DESIGN The freshwater alga, Anabaena flos-aquae, was exposed to a geometric series of six test concentrations and a negative (culture medium) control under static conditions for 96 hours. Three replicate test chambers were maintained for each treatment and control group. One additional replicate for each treatment and control group was maintained for analytical sampling at 72 hours. In addition, two "abiotic" replicates (test solution without algae) were prepared for the highest test concentration to determine the stability of the test substance under the conditions of administration. Nominal test concentrations were selected in consultation with the Sponsor and were based upon the results of range finding tests. The nominal test concentrations were 37.9, 58.6, 88.8, 139, 216 and 331 mg active ingredient (a.i.)/L. Measured test concentrations were determined from samples of test medium collected from each treatment and the control group at test initiation, at approximately 72 hours, and at test termination. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -10- p r o j e c t n o .: 454a -iio b At test initiation, an inoculum of the algal cells was prepared at a concentration of approximately 1.0 X 106 cells/mL. The concentration of algal cells in the inoculum was verified and 1.0 mL was added to each test chamber to achieve a nominal concentration of approximately 1.0 X 104 cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell densities. Cell densities were measured for each replicate and were used to calculate areas under the growth curve and growth rates. Percent inhibition values relative to the control were calculated for each parameter over the 96-hour exposure period. EC50 values based upon cell densities, areas under the growth curve and growth rates were calculated for each 24-hour interval. EC 10 and EC90 values were calculated for the 72 and 96-hour intervals. No-observed-adverse-effect-concentrations (NOAEC) were determined based upon statistical evaluation of the 72-hour and 96-hour results and an evaluation of the concentration-response pattern. At the end of the 96-hour exposure, algistatic effects were differentiated from algicidal effects. MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Anabaena flos-aquae)". 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 andII (draft)(l). Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International, Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity of 98.9% and an expiration date of 2008. The test substance was reanalyzed by the Sponsor and the Certificate of Analysis dated September 7, 2000 indicated a purity of 86.9% and an expiration date of August 31, 2001. The test substance was stored at ambient room temperature. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -n - p r o j e c t n o .: 454A-110B Preparation of Test Concentrations Nominal test concentrations were 37.9, 58.6, 88.8, 139, 216 and 331 mg a.i./L, based on a test substance purity of 86.9%. Individual test solutions were prepared in algal medium at each of the six nominal concentrations. The test solutions were stirred with a magnetic stir plate for approximately 18 hours to aid in the solubilization of the test substance. All test solutions appeared clear and colorless. Test Organism The freshwater alga, Anabaena flos-aquae, was selected as the test species for this study. The species is representative of an important group of 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 UTEX - The Culture Collection of Algae at the University of Texas at Austin and have 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. The negative control organisms were expected to exhibit exponential growth over the 96-hour exposure period. Exponential growth, 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). Culture Medium The algal cells were cultured and tested in freshwater algal medium (2). Stock nutrient solutions were prepared by adding reagent-grade chemicals to Wildlife International, Ltd. well water purified by reverse osmosis. The test medium was prepared by adding appropriate volumes of the stock nutrient solutions to purified well water (Appendix I). The pH of the medium was adjusted to 7.5 0.1 using 0.1 N NaOH and 10% HC1, and the medium was sterilized by filtration (0.22 pm) prior to use. Analyses were performed at least once annually to determine the concentrations of selected organic and inorganic constituents in the well water used by Wildlife International, Ltd. The results of the most recent GLP analyses performed to measure the concentrations of selected contaminants in the well water are presented in Appendix II. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 12- p r o j e c t n o .: 454A-110B Test Apparatus Test chambers were sterile, 250-mL glass Erlenmeyer flasks plugged with foam stoppers, and contained 100 mL of test or control medium. The test chambers were labeled with the project number, concentration and replicate, and were indiscriminately positioned daily on mechanical shaker tables in an environmental chamber designed to maintain the desired test temperature throughout the test. The test chambers were shaken continuously at approximately 100 rpm. Environmental Conditions Test flasks were held in an environmental chamber at a temperature of 24 2C. The temperature of a container of water adjacent to the test flasks in the environmental chamber was recorded twice daily during the test using a liquid-in-glass thermometer. The algae were held under continuous cool-white fluorescent lighting throughout the test. The target light intensity was 2200 10% lux. Light intensity was measured at the four comers and the center of each shaker table at test initiation using a SPER Scientific Model 840006 light meter. The pH of the medium prepared for each treatment and control group was measured at test initiation and termination using a Fisher Accumet Model 915 pH meter. Samples for pH measurement at test initiation were collected from the individual batches of test solution prepared for each treatment and control group. At test termination, samples of test solution were collected from pooled replicates of the treatment and control groups for pH measurement. Algal Growth Measurements Test medium samples were collected from the treatment and control groups for the determination of algal cell densities. Single samples were collected from each of the three "biological" replicates per treatment and control group at 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 conducted using a hemacytometer and microscope. Each sample was diluted using an electrolyte solution (Isoton), as needed, to maintain counting accuracy. A small amount of each sample was loaded onto a hemacytometer and 20 grids were counted. The mean number of cells per grid was estimated and this value was used to calculate the cell density of the sample. Using this technique, the minimum quantifiable cell density was 500 cells/mL. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 13 - p r o j e c t n o .: 454a -iio b The samples of test solution collected from each replicate for cell counts were examined microscopically for atypical cell morphology (e.g., changes in cell shape or color). Growth of cells in the replicate test chambers also was assessed for aggregations (clumping) of cells and adherence of the cells to the test chamber. Statistical Analyses Cell densities, areas under the growth curve, growth rates and percent inhibition were calculated using "The SAS System for Windows", Release 6.12 (3). Area under the growth curve was calculated for each replicate of the control and treatment groups using the following formula: A = ((N,-N0)/2)(ti)+((N1+N2-2N0)/2)(t2-t1)+ ... +((Nn.,+Nn-2N0)/2)(tn-tn.1) where: A = Area N0 = Nominal number of cells/mL at t0 Ni = Measured number of cells/mL at ti N2 = Measured number of cells/mL at t2 Nn = Measured number of cells/mL at tn ti = Time of first measurement after beginning of test (hours) t2= Time of second measurement after beginning of test (hours) tn = Time of nthmeasurement after beginning of test (hours) Growth rates were calculated for each replicate of the control and treatment groups using the following formula: lnNn - lnN0 where: p = Average specific growth rate N0 = Nominal number of cells/mL at t0 Nn = Measured number of cells/mL at tn t0 = Time of beginning of the test (hours) tn = Time of nthmeasurement after beginning of test (hours) Percent inhibition was calculated for each treatment group as the percent reduction in cell density, area under the growth curve and growth rate relative to the control replicates. The following formula was used: BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 14 - PROJECT NO.: 454A -110B Percent Inhibition = Mean Responsecontroi - Mean Response-Treatment Mean ResponseControi X 100 Cell densities, areas under the growth curve and growth rates were analyzed statistically to estimate the EC10, EC50 and EC90 values (i.e., the theoretical test concentrations that would produce a 10, 50 or 90% reduction in each parameter, respectively) and 95% confidence limits at 72 and 96 hours. EC50 values were also calculated for the 24 and 48-hour time intervals. The EC values and 95% confidence limits were calculated by linear interpolation with treatment response and exposure concentration data using TOXSTAT Version 3.5 (4). Cell densities, areas under the growth curve and growth rates at 72 and 96 hours were evaluated for normality and homogeneity of variances using the Shapiro-Wilk's test and Levene's test, respectively. When the data were normally distributed with equal variances, the treatment groups were compared to the control using Dunnett's test. In one instance, the data were not normally distributed, and the data were square-root transformed in an attempt to correct the problem. When this did not correct for normality, the data were analyzed using the non-parametric Kruskal-Wallis test. Results of the statistical analyses and evaluation of the concentration-response pattern were used to determine the NOAEC values at 72 and 96 hours. Analytical Chemistry Samples of test medium were collected from the negative control and each treatment group at test initiation, at approximately 72 hours and at test termination to measure concentrations of the test substance. Samples of test medium collected at test initiation were taken from the individual batches of test solution prepared for each treatment and the control group. Samples collected at 72 hours were collected from the additional "analytical" replicates. Samples collected at test termination were a composite of the remaining biotic replicates for each treatment and the control group. The 331 mg a.i./L abiotic replicates were sampled at 72 and 96 hours to determine the stability of the test substance under the conditions of administration. The samples were placed in scintillation vials and were analyzed immediately without storage. Analytical procedures used in the analysis of the samples are presented in Appendix III. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 15 - p r o j e c t n o .: 454a -iio b RESULTS AND DISCUSSION Measurement of Test Concentrations Results of analyses to measure concentrations of PFOS in the test solutions are presented in Table 1 and Appendix III. Nominal concentrations used in this study were 37.9, 58.6, 88.8, 139, 216 and 331 mg a.i./L. Samples collected at the beginning of the test had measured concentrations that ranged from 100 to 112% of nominal. Samples collected at 72 hours and at test termination had recoveries that ranged from 99.0 to 110%, and 99.0 to 109% of nominal, respectively. The abiotic replicates from the 331 mg a.i./L treatment had recoveries of 103 and 107% of nominal at 72 and 96 hours, respectively, which were comparable to the biotic replicate recoveries. When the values obtained at test initiation, at 72 hours and at test termination were averaged, the mean measured test concentrations were 37.9, 63.9, 93.8, 143, 235 and 329 mg a.i./L, representing 100, 109, 106, 103, 109 and 99.4% of nominal concentrations, respectively. Mean measured test concentrations were used in the calculation of EC values. Observations and Measurements Measurements of temperature, light intensity and pH are presented in Tables 2, 3 and 4, respectively. The temperatures ranged from 22.8 to 23.8C and were within the range established for the test (24 2C). The light intensity ranged from 1990 to 2310 lux and was within the desired range for the test (approximately 1980 to 2420 lux). Measurements of pH were 7.4 on Day 0 and ranged from 7.4 to 7.6 at 96 hours. The effect of PFOS upon Anabaena flos-aquae was determined by evaluating differences in cell densities, areas under the growth curve and growth rates. Mean values for each parameter were used to calculate growth inhibition for each 24-hour period. Mean cell densities, areas under the growth curve and growth rates, and the corresponding percent inhibition, are presented in Tables 5, 6 and 7, respectively. Cell density, area under the growth curve and growth rate for each individual replicate are presented in Appendices IV, V and VI, respectively, while cell densities are illustrated graphically in Figures 1 and 2. EC values and 95% confidence limits calculated for each 24-hour interval based on cell density, area under the growth curve and growth rate are presented in Tables 8, 9 and 10, respectively. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 16 - p r o j e c t n o .: 454A-110B Changes in cell density indicated that exponential growth occurred in the negative control replicates (Figure 1). After 72 hours of exposure, cell density percent inhibition in the 37.9, 63.9, 93.8, 143, 235 and 329 mg a.i./L treatment groups was 8.2, 18, 32, 65, 93 and 100%, respectively. Dunnett's test showed that cell density was significantly reduced at concentrations >63.9 mg a.i./L in comparison to the negative control (p<Q.Q5). Consequently, the 72-hour NOAEC for cell density was 37.9 mg a.i./L. After 96 hours of exposure, cell density percent inhibition in the 37.9, 63.9, 93.8, 143, 235 and 329 mg a.i./L treatment groups was -6.3, -2.9, 13, 60, 100 and 99%, respectively. Dunnett's test showed that cell density was significantly reduced in the 143, 235 and 329 mg a.i./L treatment groups (p<0.05). Consequently, the 96-hour NOAEC for cell density was 93.8 mg a.i./L. After 72 hours of exposure, area under the growth curve percent inhibition in the 37.9, 63.9, 93.8, 143, 235 and 329 mg a.i./L treatment groups was 13, 32, 33, 72, 99 and 100%, respectively. Dunnett's test showed that area under the growth curve was significantly reduced at concentrations >63.9 mg a.i./L in comparison to the negative control (p<0.05). Consequently, the 72-hour NOAEC for area under the growth curve was 37.9 mg a.i./L. After 96 hours of exposure, area under the growth curve percent inhibition in the 37.9, 63.9, 93.8, 143, 235 and 329 mg a.i./L treatment groups was 3.0, 13, 24, 66, 99 and 100%, respectively. Dunnett's test showed that area under the growth curve was significantly reduced in the 93.8, 143, 235 and 329 mg a.i./L treatment groups (p<0.05). Consequently, the 96-hour NOAEC for area under the growth curve was 63.9 mg a.i./L. After 72 hours of exposure, growth rate percent inhibition in the 37.9, 63.9, 93.8, 143, 235 and 329 mg a.i./L treatment groups was 2.6, 6.4, 12, 33, 83 and 100%, respectively. The Kruskal-Wallis test showed that growth rate was significantly reduced in the 235 and 329 mg a.i./L treatment groups (p<0.05). While the inhibition of growth rate at 143 mg a.i./L was not statistically significant using the KruskalWallis test, the 33% inhibition in comparison to the control was considered to be treatment-related. Consequently, the 72-hour NOAEC for growth rate was 93.8 mg a.i./L. After 96 hours of exposure, growth rate percent inhibition in the 37.9, 63.9, 93.8, 143, 235 and 329 mg a.i./L treatment groups was -1.3, -0.97, 3.6, 22, 100 and 96%, respectively. Dunnett's test showed that growth rate was significantly reduced in the 143, 235 and 329 mg a.i./L treatment groups (p<0.05). Consequently, the 96-hour NOAEC for growth rate was 93.8 mg a.i./L. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 17- PROJECTNO.: 454A -110B Visual and Microscopic Observations After 96 hours of exposure, there were no signs of aggregation (clumping) or adherence of the algae to the test flasks in the negative control or any PFOS treatment group. When compared to the negative control, there were no noticeable changes in cell morphology in any of the treatment groups in which cells were observed. Reversibility of Growth Inhibition The 235 and 329 mg a.i./L treatment groups were maximally inhibited at the end of the 96-hour exposure period. Aliquots of the test solutions were diluted with algal medium and cultured for nine days. Based on the increase in growth observed by Day 9 of the recovery phase in the 235 mg a.i./L treatment, the effect on algal growth was algistatic, rather than algicidal at a concentration of 235 mg a.i./L. However, no algal cells were detected during the recovery phase in the 329 mg a.i./L treatment, indicating that PFOS was algicidal at that concentration. Cell densities for the recovery phase are presented in Table 11 and are illustrated graphically in Figure 3. CONCLUSIONS The conclusions of this study were based on the most sensitive endpoint measured (i.e., cell density, area under the growth curve and/or growth rate). The 72-hour EC50, based on area under the growth curve, was 116 mg a.i./L, with 95% confidence limits of 49.2 and 142 mg a.i./L. The 96-hour EC50, based on area under the growth curve, was 124 mg a.i./L, with 95% confidence limits of 104 and 138 mg a.i./L. The 72-hour NOAEC, based on cell density and area under the growth curve, was 37.9 mg a.i./L. The 96-hour NOAEC, based on area under the growth curve, was 63.9 mg a.i./L. PFOS was considered to be algistatic at a concentration of 235 mg a.i./L, and algicidal at 329 mg a.i./L. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 18 - p r o j e c t n o .: 454A-110B 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 ASTM Standard Guide 1218-90E. 1990. Standard Guide for Conducting Static 96-Hour Toxicity Tests with Microalgae. American Society for Testing and Materials. Philadelphia, Pennsylvania. 3 The SAS System for Windows. 1996. Release 6.12, TS Level 0020. SAS Institute Inc., Cary, North Carolina. 4 West, Inc. and D.D. Gulley. TOXSTAT Version 3.5. Copyright 1996. Western EcoSystems Technology, Inc., Cheyenne, Wyoming. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 19- p r o j e c t n o .: 454A-110B Table 1 Summary of Analytical Chemistry Data Sponsor: Test Substance: Test Organism: Dilution Water: Nominal Concentration (mg a.i./L) 3M Corporation PFOS Freshwater Alga, Anabaena flos-aquae Freshwater Algal Medium Sampling Time (Hours) Measured Concentration1 (mg a.i./L) Negative Control 37.9 58.6 88.8 02 723 964 0 72 96 0 72 96 0 72 96 < LOQ < LOQ < LOQ 37.9 38.2 37.6 65.6 62.7 63.4 94.3 97.4 89.8 139 0 142 72 146 96 142 216 0 230 72 238 96 236 331 0 331 72 328 96 329 Mean Measured Concentration (mg a.i./L) < LOQ 37.9 63.9 93.8 143 235 329 Percent of Nominal __ 100 109 106 103 109 99.4 331 (abiotic) 72*2345 342 965 356 349 105 ' Limit of Quantitation (LOQ) was 4.80 mg a.i./L. 2 0-hour samples were collected from individual batches of test solution prepared for the treatment and control groups for test initiation. 3 72-hour samples were collected from the additional analytical replicate. 4 96-hour samples were composites of test solution collected from each of the three biological replicates per treatment and control group. 5 72 and 96-hour samples were collected from the additional abiotic replicates. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -20- p r o j e c t n o .. 454A-110B Table 2 Temperature Measurements Sponsor: Test Substance: Test Organism. Dilution Water: Time (Day) 3M Corporation PFOS Freshwater Alga, Anabaenaflos-aquae Freshwater Algal Medium T em p e ra tu re 1 (C) Measurement 1 Measurement 2 0 22.8 1 23.3 2 23.7 3 23.6 4 23.5 5 23.3 6 23.5 7 23.5 8 23.5 9 23.3 10 23.3 11 23.3 12 23.3 13 23.3 23.2 23.5 23.5 23.4 23.3 23.5 23.5 23.5 23.3 23.4 23.3 23.8 23.3 23.4 1 Temperature Measurement 2 was taken at least 4 hours after Measurement 1, with the exception of test initiation and recovery phase termination (Days 0 and 13). BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -21 - p r o j e c t n o .. 454A-110B Table 3 Light Intensity Measurements Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaena flos-aquae Freshwater Algal Medium Test Shaker - -- Light Intensity Measurements1(lux) ..... -- Day Table No. 1 No. 2 No. 3 No. 4 1 2000 1990 0 2 2020 1990 2300 2310 2000 2060 1 Light intensity was measured at five locations over each of two shaker tables. No.5 2020 2290 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -22- p r o j e c t n o .: 454A-110B Table 4 pH Measurements Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaena flos-aquae Freshwater Algal Medium Mean Measured Collocali aliou (mg a.i./L) DayO1 pH Measurements Negative Control 7.4 Day 412 7.6 37.9 7.4 7.6 63.9 7.4 7.6 93.8 7.4 7.5 143 7.4 7.5 235 7.4 7.4 329 7.4 7.4 1 Day 0 samples were collected from the batches of test solution prepared for the treatment and control groups at test initiation. 2 Day 4 samples were collected from the pooled replicates per treatment and control group. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -23 - PROJECT NO.: 454A-110B Table 5 Mean Cell Densities and Percent Inhibition for Each 24-Hour Interval During the Test Sponsor. Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaena Jlos-aquae Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) 24 Hours __________ _____ -___ ___ Mean Cell Density' Percent (cells/mL) Inhibition' 48 Hours ____ ___________ _ ___ -__.. Mean Cell Density' Percent (cells/mL) Inhibition' 72 Hours = ------------------------ --------- Mean Cell Density' Percent (cells/mL) Inhibition' Negative Control 12,833 - 79,667 - 261,500 - 37.9 7,167 44 69,500 13 240,167 8.2 63.9 11,500 10 41,167 48 213,500* 18 93.8 7,667 40 60,833 24 177,000* 32 143 2,167 83 27,500 65 90,333* 65 235 2,333 82 0 100 17,500* 93 329 0 100 0 100 0* 100 1 Values calculated using SAS 6.12. Manual calculations may differ slightly. * Indicates a significant difference from the negative control using Dunnett's test (p < 0.05). 96 Hours , __ Mean Cell Density1 Percent (cells/mL) Inhibition' 569,167 - 605,000 -6.3 585,833 -2.9 492,500 13 228,833* 60 2,333* 100 8,500* 99 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -24 - PROJECT NO.: 454A -110B Table 6 Mean Areas Under the Growth Curve and Percent Inhibition for Each 24-Hour Interval During the Test Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaena flos-aquae Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) 0 - 24 Hours Mean Area1 Percent Inhibition1 0 - 4 8 Hours Mean Area1 Percent Inhibition1 Negative Control 42,000 - 912,000 - 37.9 4,000 90 684,000 25 63.9 52,000 -24 444,000 51 93.8 20,000 52 602,000 34 143 0 100 156,000 83 235 0 100 0 100 329 0 100 0 100 1 Values calculated using SAS 6.12. Manual calculations may differ slightly. * Indicates a significant difference from the negative control using Dunnett's test (p < 0.05). 0 - 7 2 Hours Mean Area1 Percent Inhibition1 4,766,000 - 4,160,000 13 3,260,000* 32 3,216,000* 33 1,330,000* 72 42,000* 99 0* 100 0 - 9 6 Hours Mean Area1 Percent Inhibition1 14,494,000 - 14,062,000 3.0 12,612,000 13 11,010,000* 24 4,920,000* 66 96,000* 99 0* 100 BACK TO MAIN W il d l if e In ter n a tio n a l, Lt d . -25 - PROJECT NO.. 454A-110B Table 7 Mean Growth Rates and Percent Inhibition for Each 24-Hour Interval During the Test Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, AnabaenaJlos-aquae Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) 0 - 2 4 Hours = Mean Growth Rate1 Percent (cells/mL/hr) Inhibition1 0 - 4 8 Hours Mean Growth Rate1 Percent (cells/mL/hr) Inhibition1 0 - 7 2 Hours Mean Growth Rate1 (cells/mL/hr) Percent Inhibition1 0 - 9 6 Hours Mean Growth Rate1 (cells/mL/hr) Percent Inhibition1 Negative Control 0.0113 - 0.0430 - 0.0453 - 0.0420 -- 37.9 0.0013 88 0.0392 8.7 0.0441 2.6 0.0425 -1.3 63.9 0.0116 -2.7 0.0249 42 0.0424 6.4 0.0424 -0.97 93.8 0.0056 50 0.0357 17 0.0398 12 0.0405 3.6 143 0.0000 100 0.0177 59 0.0302 33 0.0326* 22 235 0.0000 100 0.0000 100 0.0079* 83 0.0000* 100 329 0.0000 100 0.0000 100 0.0000* 100 0.0017* 96 ' Values calculated using SAS 6.12. Manual calculations may differ slightly. * Indicates a significant difference from the negative control using the Kruskal-Wallis test (72 hours) or Dunnett's test (96 hours) (p < 0.05). BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -26- PROJECTNO.: 454A -110B Table 8 EC Values Based on Cell Density Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: Time 24 Hours 48 Hours 72 Hours 96 Hours 3M Corporation PFOS Freshwater Alga, Anabaena flos-aquae Freshwater Algal Medium EC10 (mg a.i./L) 95% Confidence Limits (mg a.i./L) Not Determined -- Not Determined - 42.6 34.2-83.6 82.3 29.1-123 EC50 (mg a.i./L) 105 117 120 131 95% Confidence Limits (mg a.i./L) <0-152 <0-194 91.8 - 139 106 - 142 EC90 (mg a.i./L) Not Determined Not Determined 224 213 95% Confidence Limits (mg a.i./L) - - 193 -275 203 - 219 BACK TO MAIN W il d l if e In te r n a t io n a l, Lt d . -27- PROJECT NO.: 454A -110B Table 9 EC Values Based on Area Under the Growth Curve Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: Time 3M Corporation PFOS Freshwater Alga, Anabaena Jlos-aquae Freshwater Algal Medium EC10 (mg a.i./L) 95% Confidence Limits (mg a.i./L) 24 Hours Not Determined -- 48 Hours 72 Hours Not Determined <37.9 - __1 96 Hours 56.1 26.3-107 1Confidence limits could not be calculated with the data obtained. EC50 (mg a.i./L) 90.1 103 116 124 95% Confidence Limits (mg a.i./L) 40.0 - 150 <0-141 49.2-142 104 - 138 EC90 (mg a.i./L) Not Determined Not Determined 204 209 95% Confidence Limits (mg a.i./L) - - 134-226 197-218 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -28 - PROJECT NO.: 454A -110B Table 10 EC Values Based on Growth Rate Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: Time 24 Hours 48 Hours 72 Hours 96 Hours 3M Corporation PFOS Freshwater Alga, Anabaena flos-aquae Freshwater Algal Medium EC10 (mg a.i./L) 95% Confidence Limits (mg a.i./L) Not Determined -- Not Determined -- 82.2 49.0- 116 109 84.1 - 125 EC50 (mg a.i./L) 93.8 128 174 176 95% Confidence Limits (mg a.i./L) 32.6 - 145 <0-216 146 - 208 169- 181 EC90 (mg a.i./L) Not Determined Not Determined 275 225 95% Confidence Limits (mg a.i./L) - -- 162 - 330 220 - 235 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -29- PROJECT NO.: 454A -110B Table 11 Cell Densities During the Recovery Phase Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaena flos-aquae Freshwater Algal Medium Mean Measured Concentration1 (mg a.i./L) ;-- =-- Day 02 Cell Densities (cells/mL) - - --- Day 3 Day 6 -- Day 9 Negative Control 9,500 275,000 2,270,000 3,855,000 235 0 0 2,000 111,000 329 00 00 1 Each treatment group was diluted to a concentration of the test substance that theoretically would not inhibit growth. 2 Due to the method defined in protocol used to prepare recovery phase test solutions, initial cell densities were not equivalent in all groups. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -30- p r o j e c t n o .: 454A-110B Figure 1. Negative control algal growth, expressed as cell density, during the 96-hour exposure period. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -31 - PROJECT NO.: 454A -110B Figure 2. Concentration-response curve, expressed as cell density, over the 96-hour exposure period. Negative Control -H -- 37.9 mg a.i./L -A--63.9 mg a.i./L - X 93.8 mg a.i./L -X-- 143 mg a.i./L - --235 mg a.i./L -B--329 mg a.i./L W il d l if e In t e r n a t io n a l , Lt d . - 32- Figure 3. Recovery-phase response curve, expressed as cell density. BACK TO MAIN p r o j e c t n o .. 454a -iio b BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -33 - PROJECT NO.: 454A-110B APPENDIX I Freshwater Algal Medium Constituents' Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaena flos-aquae Freshwater Algal Medium Nominal Compound___________________ Concentration MgCl2*6H20 CaCl2*2H20 H 3BO 3 MnCl2*4H20 ZnCl2 FeCl3*6H20 CoC12*6H20 Na2M o04*2H20 CuCl2*2H20 N a 2EDTA*2H20 NaN03 M gS04*7H20 k 2h p o 4 NaHC03 12.16 4.40 0.1856 0.416 3.28 0.1598 1.428 7.26 0.012 0.300 25.50 14.70 1.044 15.0 mg/L mg/L mg/L mg/L Pg/L mg/L Pg/L Pg/L Pg/L mg/L mg/L mg/L mg/L mg/L 1 The pH was adjusted to 7.5 0.1 using 10% HC1 and 0.1 N NaOH. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 34- PROJECT NO.: 454A-11 OB APPENDIX II Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Component Measured Concentration Component Pesticides and Organics Measured Concentration Aclonifen Alachlor Ametryn Atrazin Azinphos-ethyl Azinphos-methyl Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophos-ethyl Broompropylaat Bupirimaat Carbaryl Carbofuran Carboxin Chlorfenvinphos Chloridazon Chlorpropham Chlorpyriphos Chlorpyriphos-methyl Chlorthalonil Coumaphos Cyanazin Cyfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-o Desethylatrazin Desisopropylatrazin Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.08 pg/L <0.25 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.25 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.03 pg/L <0.02 pg/L <0.01 pg/L <0.03 pg/L Dimethomorf Disulfoton DMST Dodemorf Endosulfan-a Endosulfan- Endosulfan-sulfaat Epoxiconazole Eptam Esfenvaleraat Ethion Ethofumesaat Ethoprophos Etridiazole Etrimphos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropimorf Fenthion Fenvaleraat Fluazifop-butyl Fluoroglycofen-ethyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane Malathion Metalaxyl Metamitron Metazachlor Methidathion <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.03 pg/L <0.03 pg/L <0.05 pg/L <0.25 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999. Continued BACK TO MAIN W il d l if e In t e r n a t io n a l, Lt d . -35 - PROJECT NO.: 454A-110B APPENDIX II Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Page 2 Component Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalon Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazin Propham Propiconazool Propoxur Propyzamide Prosulfocarb Pyrazophos Pesticides And Organics (Page 2) Measured Concentration <0.01 pg/L <0.25 pg/L <0.02 pg/L <0.03 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.03 pg/L Component Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifenox-2 Pyrimethanil Quizalofop-ethyl Simazin Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbutylazin Tetrachlorvinphos Tetrahydroftaalimide Tetramethrin Thiabendazole Thiometon Tolclophos-methyl Tolylfluanid Triadimefon Triadimenol Triallaat Triazophos Trifluralin Vamidothion Vinchlozolin Measured Concentration <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.05 pg/L <0.04 pg/L <0.01 pg/L <0.04 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 11.0 mg/L 18.0 mg/L 29 mg/L <0.015 mg/L 1.1 mg/L <0.02 mg/L <0.1 pg/L <0.2 pg/L <0.5 pg/L <0.2 pg/L Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium <1.1 pg/L <0.7 pg/L <0.25 pg/L <0.3 pg/L <0.2 pg/L <0.1 pg/L <0.5 pg/L <0.025 pg/L <0.5 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 36- PROJECT NO.: 454A-110B APPENDIX III THE ANALYSIS OF PFOS IN FRESHWATER ALGAL MEDIUM IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-110B BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 37- projectno.: 454a-iiob REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Anabaena flos-aquae) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-110B MANAGEMENT: Willard B. Nixon, Ph.D' Director, Analytical Chemistry i/W DAT] BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 38- p r o j e c t n o .: 4 5 4 A -110B Introduction Freshwater algal medium samples were collected from a four-day toxicity test designed to determine the effects of PFOS (Perfluorooctanesulfonate, Potassium Salt) to the freshwater alga (Anabaena flos-aquae). This study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A-110B. The analyses of these water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis on May 12, May 15, and May 16, 2000 and were analyzed on each sample receipt day. Test Substance and Internal Standard The test substance used for this study was Wildlife International, Ltd. identification number 4675. The test substance was used to prepare calibration and matrix fortification samples. The internal standard was received from 3M Corporation on July 2, 1998 and was assigned Wildlife International, Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The standard (hereafter referred to as 4H PFOS) was stored under ambient conditions. Analytical Method The method used for the analysis of the freshwater algal medium samples was developed at Wildlife International, Ltd. and entitled "Analytical Method for the Determination of PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix II of Wildlife International; Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Samples were diluted in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range of the PFOS methodology. Concentrations of the PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Qg analytical column (50 mm x 2 mm ID ., 3-jim particle size). The instrument parameters are summarized in Table 1. A method flowchart is provided in Figure 1. W il d l if e In t e r n a t io n a l , Lt d . -39- BACK TO MAIN PROJECTNO.: 454A-110B Calibration Curve and Limit of Quantitation Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.0480 to 0.480 mg a.i./L, were analyzed with the samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) of the calibration standards. A typical calibration curve is presented in Figure 2. The concentration of PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Figures 3 and 4, respectively. The method limit of quantitation (LOQ) for these analyses was set at 4.80 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.0480 mg a.i./L) and the dilution factor of the matrix blank samples (100). Matrix Blank and Fortification Samples Three matrix blank samples were analyzed to determine possible interference. No interferences were observed at or above the LOQ during samples analyses (Table 2). A representative ion chromatogram of a matrix blank is presented in Figure 5. Freshwater algal medium was fortified at 19.2, 96.0 and 384 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected for the mean procedural recovery of 103%. A representative ion chromatogram of a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-110B-5, nominal concentration of 139 mg a.i./L in freshwater algal medium. Peak Area Ratio = Analyte Peak Area/Internal Standard Peak Area Concentration Ratio = Concentration of Analyte/Concentration of Internal Standard Internal Standard Concentration: 0.100 mg/L BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -40- PROJECT NO.: 454A-110B Initial Volume: 0.100 mL Final Volume: 50.0 mL Dilution Factor: 500 PFOS Peak Area: 7379800 Internal Standard Peak Area: 2046853 Peak Area Ratio: 3.6054 Calibration curve equation. Slope: 1.1378 Intercept: 0.3720 Curve is weighted (1/x) Peak area ratio - (Y-intercept) PFOS (mg a.i./L) at Instrument = Slope x Internal Standard Concentration = 0.284 PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at Instrument x Dilution Factor = 0.284 x 500 = 142 PFOS (mg a.i./L) in sample Percent of Nominal Concentration = PFOS (mg a.i./L) nominal x = 102% RESULTS Sample Analysis Freshwater algal medium samples were collected from the four-day toxicity test with the freshwater alga (Anabaena flos-aquae) at test initiation, May 12, 2000 (Day 0), on May 15, 2000 (Day 3) and at test termination, BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -41 - p r o j e c t n o .. 4 5 4 A -i i o b May 16, 2000 (Day 4). The measured concentrations of PFOS in the samples collected at initiation of exposure of the test organisms (Hour 0) ranged from 100 to 112% of the nominal concentrations. Samples collected at Day 3 had a measured concentration range of 99.0 to 110% of nominal values. Samples collected at test termination (Day 4) had a measured concentration range of 99.0 to 109% of nominal values (Table 4). Samples from the abiotic 331 mg a.i./L treatment group were comparable to samples from the 331 mg a.i./L treatment group with the freshwater alga present (Table 4). A representative ion chromatogram of a test sample is shown in Figure 7. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -42 - p r o j e c t n o .: 454a -iio b INSTRUMENT: Table 1 Typical HPLC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: Keystone Betasil C]8 column (50 mm x 2 mm I.D., 3-pm particle size) 30C 5.00 minutes 220 pL/minute 72.0% Methanol : 28.0% NANOpure Water containing 0.1% Formic Acid - Day 0 analysis 70.0% Methanol : 30.0% NANOpure Water containing 0.1% Formic Acid - Days 3 and 4 analyses 5.0 pL Approximately 4.2 minutes - Day 0 analysis Approximately 3.3 minutes - Days 3 and 4 analyses INTERNAL STANDARD RETENTION TIME: PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: Approximately 3.0 minutes - Day 0 analysis Approximately 2.1 minutes - Days 3 and 4 analyses 498.6 amu 426.7 amu BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -43 - PROJECT NO.: 454A-110B Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-110B-) MAB-1 Sample Type Matrix Blank Measured Concentration of PFOS1 (mg a.i./L) < LOQ MAB-2 Matrix Blank < LOQ MAB-3 Matrix Blank < LOQ 1 The limit of quantitation (LOQ) was 4.80 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0480 mg a.i./L) and the dilution factor of the matrix blank samples (100)._____________________ BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -44- p r o j e c t n o .: 454a -iio b Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis*12 Sample Number (454A-110B-) MAS-1 MAS-4 MAS-7 Concentrations of PFOS ____________________(mg a.i./L)____________________ Fortified1 Measured1 19.2 19.2 19.2 22.2 19.2 22.0 Percent Recovered' 100 116 115 MAS-2 MAS-5 MAS-8 96.0 96.0 96.0 91.4 95.2 97.9 95.2 99.1 102 MAS-3 MAS-6 MAS-9 384 384 384 380 392 372 98.9 102 96.8 Mean = 103 Standard Deviation =7.54 CV =7.34% N =9 1 Concentrations were corrected for change in test substance purity (90.49% to 86.9%) per Certificate of Analysis dated September 7, 2000. 2 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since fortified and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes.________________________________________________________________________________ BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -45 - p r o j e c t n o .: 454a -iio b Table 4 Measured Concentrations of PFOS in Freshwater Algal Medium Samples from a Freshwater Alga Four-Day Toxicity Test Nominal Test Concentration1 (mg a.i./L) 0.0 (Negative Control) 37.9 Sample Number (454A-110B-) 1 8 16 2 9 17 Sampling Time (Day) 0 3 4 0 3 4 PFOS Measured Concentration1,2 (mg a.i./L) < LOQ < LOQ < LOQ 37.9 38.2 37.6 Percent of Nominal23 -- -- 100 101 99.0 58.6 3 0 10 3 18 4 65.6 112 62.7 107 63.4 108 88.8 4 0 11 3 19 4 94.3 106 97.4 110 89.8 101 139 5 0 12 3 20 4 142 102 146 105 142 102 216 6 0 13 3 21 4 230 106 238 110 236 109 331 7 0 14 3 22 4 331 100 328 99.0 329 99.2 331 (Abiotic) 15 23 3 4 342 103 356 107 1 Concentrations were corrected for change in test substance purity (90.49% to 86.9%) per Certificate of Analysis dated September 7, 2000. 2 The limit of quantitation (LOQ) was 4.80 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0480 mg a.i./L) and the dilution factor of the matrix blank samples (100). 3 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since nominal and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. _____________________ W il d l if e In t e r n a t io n a l , Lt d . -46- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -110B METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER ALGAL MEDIUM Prepare matrix fortification samples by spiking the requisite volume of PFOS stock solutions directly into freshwater algal medium using gas-tight syringes and Class A volumetric flasks. Dilute matrix fortification and test samples into the range of the calibration standards by partially filling Class A volumetric flasks with 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v). Add the appropriate volume of sample and bring the flask to volume with the dilution solvent. Process the matrix blank sample using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. I Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in freshwater algal medium. W il d l if e In t e r n a t io n a l , Lt d . -47 - BACK TO MAIN p r o j e c t n o .: 4 5 4 A -110B Figure 2. A typical calibration curve for PFOS. Slope = 1.1378; Intercept = 0.3720; r = 0.9959. Curve is weighted (1/x). W il d l if e In t e r n a t io n a l , Lt d . -48 - BACK TO MAIN PROJECT NO.: 454A-110B intensity: 6 0 0 0 0 0 cps Figure 3. A representative ion chromatogram of a low-level (0.0480 mg a.i./L) PFOS standard. W il d l if e In t e r n a t io n a l , Lt d . -49 - BACK TO MAIN PROJECT NO.: 454A-110B intensity: 600000 cps Figure 4. A representative ion chromatogram of a high-level (0.480 mg a.i./L) PFOS standard. W il d l if e In t e r n a t io n a l , Lt d . -50- BACK TO MAIN p r o j e c t n o .. 4 5 4 A -110B intensity: 6 0 0 0 0 0 cps Figure 5. A representative ion chromatogram of a matrix blank sample (454A-110B-MAB-1). The arrow indicates the retention time of PFOS. W il d l if e In t e r n a t io n a l , Lt d . -51 - BACK TO MAIN PROJECT NO.: 454A-110B intensity: 6 0 0 0 0 0 cps Figure 6. A representative ion chromatogram of a matrix fortification sample (454A-110B-MAS-3). W il d l if e In t e r n a t io n a l , Lt d . -52- BACK TO MAIN PROJECTNO.: 454A-110B intensity: 6 0 0 0 0 0 cps Figure 7. A representative ion chromatogram of a test sample (454A-110B-5). BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -53 - PROJECT NO.: 454A-110B APPENDIX IV Cell Density for Each Replicate Per Treatment Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaena jlos-aquae Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) Replicate 24 Hours Cell Densities (Cells/mL)1 48 Hours 72 Hours 96 Hours Negative Control A 12,500 96,000 258,500 460,000 B 18,000 65,500 254,000 597,500 C 8,000 77,500 272,000 650,000 37.9 A 5,500 41,500 243,000 450,000 B 11,000 94,500 235,000 627,500 C 5,000 72,500 242,500 737,500 63.9 A 7,500 20,500 184,500 562,500 B 4,000 21,500 235,000 592,500 C 23,000 81,500 221,000 602,500 93.8 A 15,000 72,000 186,000 590,000 B 0 81,500 202,500 450,000 C 8,000 29,000 142,500 437,500 143 A 0 47,500 109,500 234,500 B 4,500 27,000 95,500 202,000 C 2,000 8,000 66,000 250,000 235 A 3,000 0 18,000 4,000 B 0 0 4,000 2,000 C 4,000 0 30,500 1,000 329 A 00 0 9,000 B 0 0 0 16,500 C 00 0 0 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). BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -54- PROJECTNO.: 454A-110B APPENDIX V Area Under the Growth Curve for Each Replicate Per Treatment Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaena flos-aquae Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) Replicate Cumulative Area Under the Growth Curve 0 - 2 4 Hours 0 - 4 8 Hours 0 - 72 Hours 0 - 96 Hours Negative Control A B C 30,000 96,000 0 1,092,000 858,000 786,000 5,106,000 4,452,000 4,740,000 13,488,000 14,430,000 15,564,000 37.9 A 0 324,000 3,498,000 11,574,000 B 12,000 1,038,000 4,752,000 14,862,000 C 0 690,000 4,230,000 15,750,000 63.9 A 0 96,000 2,316,000 11,040,000 B 0 66,000 2,904,000 12,594,000 C 156,000 1,170,000 4,560,000 14,202,000 93.8 A 60,000 864,000 3,720,000 12,792,000 B 0 738,000 3,906,000 11,496,000 C 0 204,000 2,022,000 8,742,000 143 A 0 330,000 1,974,000 5,862,000 B 0 138,000 1,368,000 4,698,000 C 0 0 648,000 4,200,000 235 A 0 0 0 24,000 B 0 00 0 C 0 0 126,000 264,000 329 A 0 0 0 0 B 0 00 0 C 0 00 0 BACK TO MAIN W il d l if e In t e r n a t io n a l, Lt d . -55 - p r o j e c t n o .: 4 5 4 A -110B APPENDIX VI Growth Rate for Each Replicate Per Treatment Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaena flos-aquae Freshwater Algal Medium Mean Measured Growth Rate (mg a.i./L) Replicate 0 - 2 4 Hours 0 - 4 8 Hours 0 - 7 2 Hours Negative Control A 0.0093 0.0471 0.0452 B 0.0245 0.0392 0.0449 C 0.0000 0.0427 0.0459 37.9 A 0.0000 0.0296 0.0443 B 0.0040 0.0468 0.0438 C 0.0000 0.0413 0.0443 63.9 A 0.0000 0.0150 0.0405 B 0.0000 0.0159 0.0438 C 0.0347 0.0437 0.0430 93.8 A 0.0169 0.0411 0.0406 B 0.0000 0.0437 0.0418 C 0.0000 0.0222 0.0369 143 A 0.0000 0.0325 0.0332 B 0.0000 0.0207 0.0313 C 0.0000 0.0000 0.0262 235 A 0.0000 0.0000 0.0082 B 0.0000 0.0000 0.0000 C 0.0000 0.0000 0.0155 329 A 0.0000 0.0000 0.0000 B 0.0000 0.0000 0.0000 C 0.0000 0.0000 0.0000 0 - 9 6 Hours 0.0399 0.0426 0.0435 0.0397 0.0431 0.0448 0.0420 0.0425 0.0427 0.0425 0.0397 0.0394 0.0329 0.0313 0.0335 0.0000 0.0000 0.0000 0.0000 0.0052 0.0000 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -56- PROJECT NO.: 454A-110B APPENDIX VII Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to add the proposed experimental start and termination dates for the first trial and the test concentrations, and to add the test substance identification number. 2. The protocol was amended to clarify that the nominal concentration may exceed 100 mg/L. 3. The protocol was amended to change the experimental start and termination dates for the two repeat trials 4. The protocol was amended to increase the number o f grids to be counted on the hemacytometer in the determination o f cell density, and to change the test substance purity to 90.49%. 5. Test substance purity was changed to 86.9%, and nominal concentrations were recalculated based on the new purity. 6. The protocol was amended to change the test apparatus from plastic to glass. 7. The protocol was amended to correct typographical errors in the units o f measurement for some o f the components o f the algal medium. 8. Two abiotic replicates, rather than one, were prepared at the highest test concentration, with one replicate sampled on each o f Days 3 and 4. 9. Assessments o f cell morphology, aggregation, and flocculation were made on samples collected for cell counts at each measurement interval, rather than on pooled samples collected at exposure termination. 10. The protocol was amended to change test substance name from Perfluorooctane Sulfonic Acid, Potassium Salt to Perfluoroocatanesulfonate, Potassium Salt. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 57- APPENDIX VIII Personnel Involved in the Study p r o j e c t n o .: 4 5 4 A -110B The following key personnel were involved in the conduct or management o f this study: 1. Henry 0 . Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 3. Raymond L. Van Hoven, Ph.D., Scientist 4. Cary A. Sutherland, Laboratory Supervisor 5. Debbie Desjardins, Biologist