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BACK TO MAIN 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 0.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 International, Ltd. 8598 Commerce Drive Easton, Maryland 2 1601 (410) 822-8600 Page 1 of 57 ~~ BACK TO MAIN WILDLIFE INTERNATIONLATLD, . -2- PROJECT NO. 454A-11OB GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (AnabaenaJlos-quae) 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 conhtions of storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: v- Cary A. Suherland Laboratory Supervisor SPONSOR: BACK TO MAIN WILDLIFE INTERNATIONAL,LTD. -3- PROJECT NO.: 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 SubstancePreparation February 24,2000 February 24, 2000 February 29,2000 Analytical Standard Preparation FebruaIy 25, 2000 February 25,2000 March 7, 2000 Cell Counts February 29,2000 February 29,2000 March 8, 2000 Analytical Data and Draft Report Biological Data and Draft Report July 11 & 12,2000 July 14, 17 - 19, 2000 July 12, 2000 July 19, 2000 July 13, 2000 August 30,2000 Analytical Data and Second Draft March 9,2001 Report March 12, 2001 March 12, 2001 Biological Data and Second Draft Report March 20 - 2 1, 200 1 March 2 1, 200 1 June 5,2001 Final Report March 29, 2001 June 5, 2001 June 5,2001 J h e s H. Coleman Quality Assurance Representative 6-c-01 DATE BACK TO MAIN WILDLI FE INTERNATIONALT, D. -4- PROJECT NO.: 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. gutherland Laboratory Supervisor . MANAGEMENT: HehMO. K/rUegd, Ph.D. Director, Aquatic Toxicology and Non-Target Plants WILDLIFE INTERNATIONAL. LTD. -5- BACK TO MAIN PROJECT NO.. 454A-110B TABLE OF CONTENTS TitleKover 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 Table 1 . Summary of Analytical Chemistry Data ........................................................................... 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 Test ..................................................................................... 24 Table 7 . Mean Growth Rates and Percent Inhibition for Each 24-Hour Interval During the Test................................................................................................................. 25 ~ ~~ BACK TO MAIN WILDLI FE INTERNATIONAL,LTD. -6- PROJECT NO.: 454A-110B 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 .... 2 7 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-responsecurve, expressed as cell density, over the 96-hour exposure period..............,......................................................................................,,.,,...... 3 1 Figure 3 - Recovery-phase response curve, expressed as cell density ................................................. 32 APPENDICES Appendix I - Freshwater Algal Medium Constituents ................................................................... 33 Appendix I1 - Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water ............................................................................................................. 34 Appendix 111- 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 VI1 - Changes to Protocol ................................................................................................ 56 Appendix VI11 - Personnel Involved in the Study..............................,.. ............................................._57 WILDLIFE INTERNATIONALT, 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 WILDLIFE 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: 1-0ctanesulfonic acid,1,1,2,2,3,3,4,4,5,5,6,6, 7,7,8,8,8-heptadecafluoro-potassiusmalt; CAS #2795-39-3 PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Anabaena$os-aquae) Negative Control, 37.9, 58.6,88.8,139, 216 and 33 1 mg a.i./L Negative Control, 37.9, 63.9, 93.8, 143, 235 and 329 mg a.i./L Experimental Start (OECD) - May 11,2000 Experimental Start (EPA) - May 12, 2000 Exposure Termination - May 16,2000 Experimental Termination - June 5,2000 96 Hours TEST ORGANISM: SOURCE OF TEST ORGANISMS: Freshwater Alga (Anabaena$os-aquae) Wildlife International, Ltd. Easton, Maryland 2 1601 WILDLI FE INTERNATIONAL, LTD. -8- CELL DENSITY: 72-HOUR EC50: 95% CONFIDENCE LIMITS: 96-HOUR EC 10: 95% CONFIDENCELIMITS: 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 rng 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 EC10: 95% CONFIDENCE LIMITS: 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 96-HOUR EC50: 95% CONFIDENCELEVITS: 96-HOUR EC90: 95% CONFIDENCELIMITS: 72-HOUR NOAEC: 96-HOUR NOAEC: 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 I GROWTHRATE: 72-HOUR EC50: ' 95% CONFIDENCE LIMITS: 96-HOUR EC 10: ' 95% CONFIDENCE LIMITS: 96-HOUR EC50: 95% CONFIDENCE LIMITS: 174 mg a.i./L 146 - 208 mg a.i./L 109 mg a.i./L 84.1 - 125 mg a.i./L 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 WILD LIFE INTERNATIoNAL, LTD. -9- PROJECT NO.: 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 $os-aquae, during a 96-hour exposure period. EXPERIMENTAL DESIGN The freshwater alga, Anabaena $os-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 WILDLI FE NTERNATIONAL, LTD. - 10- PROJECT NO.: 454A-110B At test initiation, an inoculum of the algal cells was prepared at a concentration of approximately 1.O X lo6cells/mL. The concentration of algal cells in the inoculum was verified and 1.O mL was added to each test chamber to achieve a nominal concentration of approximately 1.O X lo4cells/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. EClO 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 $os-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 ToxiciQ, Tiers Zand ZZ (draft)(1). 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 3 1, 2001. The test substance was stored at ambient room temperature. BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 11 - PROJECT NO.: 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 f 0.1 using 0.1 N NaOH and 10% HCl, 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 11. BACK TO MAIN WILDLIFE INTERNATIONAL,LTD. -12- PROJECT NO.: 454A-1lOB 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 f2C. 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 f 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 - 13 - PROJECT NO.: 454A-110B 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: where: A = Area No = Nominal number of cells/mL at to N1= Measured number of cells/mL at tl N2 = Measured number of cells/mL at tZ N, = Measured number of cells/mL at t, t l = Time of first measurement after beginning of test (hours) tz= Time of second measurement after beginning of test (hours) t, = Time of n* measurement after beginning of test (hours) Growth rates were calculated for each replicate of the control and treatment groups using the following formula: lnN, - lnN, = tn - to where: p = Average specific growth rate No = Nominal number of cells/mL at to N, = Measured number of cells/mL at t, to = Time of beginning of the test (hours) t, = Time of n` measurement 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 WILDLIFE INTERNATIONALL, TD. -14- PROJECT NO.: 454A-1lOB x Mean response^^,,,,^ - Mean ResponseTreatment Percent Inhibition = Mean Responsecontrol 100 Cell densities, areas under the growth curve and growth rates were analyzed statistically to estimate the EC10, EC50 and EC90 values (Le.,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 33 1 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 111. ~~~ BACK TO MAIN - 15 - PROJECT NO.: 454A-110B 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 111. 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 1lo%, 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 23.8"C and were within the range established for the test (24 f 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 WILDLIFE INTERNATIONAL,LTD. - 16- PROJECT NO.: 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 loo%, respectively. Dunnett's test showed that cell density was significantly reduced at concentrations 263.9 mg a.i./L in comparison to the negative control (p<0.05). 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 loo%, respectively. Dunnett's test showed that area under the growth curve was significantly reduced at concentrations 263.9 mg a.i./L in comparison to the negative control (pc0.05). 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 loo%, 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 loo%, respectively. The Kruskal-Wallis test showed that growth rate was significantly reduced in the 235 and 329 mg a.i./Ltreatment 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 WILDLIFE INTERNATIONAL,LTD. - 17- PROJECT NO.: 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. Reversibilitv 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 andor 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 mga.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 WILDLIFE INTERNATIONAL,LTD. - 18 - PROJECT NO.: 454A-110B REFERENCES U.S. Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (draft),OPPTS Number 850.5400: Algal Toxicity, Tiers Z and ZZ. ASTM Standard Guide 1218-903. 1990. Standard Guide for Conducting Static 96-Hour Toxicity Tests with Microalgae. American Society for Testing and Materials. Philadelphia, Pennsylvania. The SAS System for Windows. 1996. Release 6.12, TS Level 0020. SAS Institute Inc., Cary, North Carolina. West, Inc. and D.D. Gulley. TOXSTAT Version 3.5. Copyright 1996. Western Ecosystems Technology, Inc., Cheyenne, Wyoming. ~~ BACK TO MAIN WILDLI FE INTERNATIONALL, TD. - 19- PROJECT NO.: 454A-110B Table 1 Summary of Analytical Chemistry Data Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaenaflos-aquae Freshwater Algal Medium Nominal Concentration (mg a.i./L) Sampling Time (Hours) Measured Concentration' (mg a.i./L) Negative Control O2 723 964 < LOQ LOQ < LOQ 37.9 0 37.9 72 38.2 96 37.6 58.6 0 65.6 72 62.7 96 63.4 88.8 0 94.3 72 97.4 96 89.8 139 0 142 72 146 96 142 216 0 230 72 238 96 236 33 1 0 33 1 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 33 1 (abiotic) 72' 342 96' 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 WILDLIFE INTERNATIONLATLD, . - 20 - PROJECT NO.: 454A-110B Table 2 Temperature Measurements Sponsor: Test Substance: Test Organism: Dilution Water: Time (Day) 3M Corporation PFOS Freshwater Alga, AnabaenaJlos-aquae Freshwater Algal Medium Temperature ("C) Measurement 1 Measurement 2 0 22.8 23.2 1 23.3 23.5 2 23.7 23.5 3 23.6 23.4 4 23.5 23.3 5 23.3 23.5 6 23.5 23.5 7 23.5 23.5 8 23.5 23.3 9 23.3 23.4 10 23.3 23.3 11 23.3 23.8 12 23.3 23.3 13 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 WILDLIFE INTERNATIONAL, LTD. -21 - PROJECT NO.: 454A-110B Table 3 Light Intensity Measurements Sponsor: Test Substance: Test Organism: Dilution Water: Test Shaker Day Table 3M Corporation PFOS Freshwater Alga, Anabaenaflos-aquae Freshwater Algal Medium Light Intensity Measurements ' (lux) NO. 1 No. 2 No. 3 No. 4 1 2000 1990 0 2 2020 1990 2300 23 10 2000 2060 1 Light intensity was measured at five locations over each of two shaker tables. No.5 2020 2290 I WILDLI FE INTERNATIONAL, LTD. - 22 - BACK TO MAIN PROJECT NO.: 454A-110B Table 4 pH Measurements Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaena $03-aquae Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) Day 0' pH Measurements Day Negative Control 7.4 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 23 5 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. Day 4 samples were collected from the pooled replicates per treatment and control group. BACK TO MAIN WILDLI FE I NTERNATI ONAL, LTD. - 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, Anabaenaflos-aquae Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) 24 Hours Mean Cell Density' (cells/mL) Percent Inhibition' Negative Control 12,833 -- 48 Hours Mean Cell Density' (cells/mL) Percent Inhibition' 79,667 -- 72 Hours Mean Cell Density' (cells/mL) Percent Inhibition' 261,500 -- 37.9 7,167 44 69,500 13 240,167 8.2 63.9 11,500 10 41,167 48 2 13,500* 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 1 Values calculatedusing SAS 6.12. Manual calculations may differ slightly. * Indicates a significantdifferencefrom the negative control using Dunnett's test (p< 0.05). O* 100 96 Hours Mean Cell Density' (cells/mL) Percent 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 WILDLIFE INTERNATIONAL, LTD. - 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 Area' Percent Inhibition' Negative Control 42,000 -- 0 - 48 Hours Mean Area' Percent Inhibition' 912,000 -- ~ ~~~ 0 - 72 Hours Mean Area' Percent Inhibition' 4,766,000 -- 0 - 96 Hours _______ Mean Percent Area' Inhibition' 14,494,000 -- 37.9 4,000 90 684,000 25 4,160,000 13 63.9 52,000 -24 444,000 51 3,260,000* 32 93.8 20,000 52 602,000 34 3,2 16,000* 33 143 0 100 156,000 83 1,330,000* 72 235 0 100 0 100 42,000* 99 329 0 100 0 100 1 Values calculated using SAS 6.12. Manual calculations may differ slightly. * Indicates a significantdifference from the negative control using Dunnett's test J(J < 0.05). O* 100 14,062,000 3.O 12,612,000 13 11,010,000* 24 4,920,000* 66 96,000* 99 O* 100 BACK TO MAIN WILDLI FE I NTERNATIONAL, LTD. - 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,Anabaenaflos-aquae Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) 0 - 24 Hours Mean Growth Rate' (cells/mL/hr) Percent Inhibition' Negative Control 0.0113 -- 0 - 48 Hours Mean Growth Rate' Percent (cells/mL,/hr) Inhibition' 0.0430 -- 0 - 72 Hours Mean Growth Rate' (cells/mL/hr) Percent Inhibition' 0.0453 _- 0 - 96 Hours Mean Growth Rate' (cells/mL/hr) Percent Inhibition' 0.0420 -- 37.9 0.0013 88 0.0392 8.7 0.0441 2.6 0.0425 -1.3 63.9 0.01 16 -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 o.oooo* 100 0.0017* 96 I 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 WILDLIFE INTERNATIONAL, LTD. - 26 - PROJECT NO.: 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, Anabaenaflos-aquae Freshwater Algal Medium EClO (mg a.i./L) Not Determined Not Determined 95% Confidence Limits (mg a.i./L) __ __ 42.6 34.2 - 83.6 82.3 29.1 - 123 EC50 (mg a.i./L) 105 117 120 13 1 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 ... WI LDLlFE I NTERNATlONAL, LTD . - 27 - BACK TO MAIN 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 24 Hours 48 Hours 72 Hours ~ ~~ ~ ~ 3M Corporation PFOS Freshwater Alga, AnabaenaJlos-aquae Freshwater Algal Medium EClO (mg a.i./L) Not Determined Not Determined 95% Confidence Limits (mga.i./L) __ __ < 37.9 --1 EC50 (mg a.i./L) 90.1 103 116 95% Confidence Limits (mg a.i./L) 40.0 - 150 < 0 - 141 49.2 - 142 EC90 (mg a.i./L) Not Determined Not Determined 204 96 Hours 56.1 26.3 - 107 124 104 - 138 209 ~ 1 Confidence limits could not be calculated with the data obtained. -~ 95% Confidence Limits (mg a.i./L) -- -- 134 - 226 197 - 218 BACK TO MAIN WILDLIFE INTERNATIONAL, LTD. - 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, Anabaenaflos-aquae Freshwater Algal Medium EClO (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 WILDLIFE INTERNATIONALT, 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, Anabaenaflos-aquae Freshwater Algal Medium Mean Measured Concentration' (mg a.i./L) Day 0' 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 0 0 0 0 1 Each treatment group was diluted to a concentration of the test substance that theoretically would not inhibit growth. 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 WILDLIFE INTERNATIONAL,LTD. - 30 - PROJECT NO.: 454A-110B Figure 1. Negative control algal growth, expressed as cell density, during the 96-hour exposure period 1,000,000 100,000 +Negative Control 1,000 0 24 48 72 Exposure Duration (Hours) __1 96 BACK TO MAIN WILDLIFE INTERNATIONALT, D. -31 - PROJECT NO.: 454A-110B Figure 2. Concentration-response curve, expressed as cell density, over the 96-hour exposure period. 1,000,000.0 100,000.0 ! 0 / / 24 48 72 Exposure Duration (Hours) +Negative Contro +37.9 mg a X . *63.9 mg a.i./L *93.8 mg a.i./L +143 mg a.i./L +235 mg a.i./L. -6- 329 mg a.i./L 96 WILDLIFE INTERNATIONALL, TD. - 32 - Figure 3 . Recovery-phase response curve, expressed as cell density. 10,000,000 1,000,000 100,000 10,000 1,000 100 10 0 3 6 Recovery Duration (Days) BACK TO MAIN PROJECT NO.: 454A-110B +Negative Control +235 mg a.i./L -A- 329 mg a.i./L 9 BACK TO MAIN WILDLIFE INTERNATIONALT, D. -33 - PROJECT NO.: 454A-110B APPENDIX I Freshwater Algal Medium Constituents' Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Anabaenaflos-aquae Freshwater Algal Medium Compound Nominal Concentration MgC12*6H20 CaC12-2H20 H3B03 MnC12.4H20 ZnClz FeC13*6H20 CoC12.6HzO Na2Mo04*2H20 CuC12*2HzO NazEDTA-2H20 NaN03 MgS 04*7H20 KzHP04 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 The pH was adjusted to 7.5 f 0.1 using 10% HC1 and 0.1 N NaOH. BACK TO MAIN WILDLIFE INTERNATIONAL,LTD. - 34 - PROJECT NO.: 454A-110B APPENDIX I1 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water' Component Measured Concentration Component Measured Concentration Pesticides and Organics Aclonifen Alachlor hew 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 C yfluthrin 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 p g 5 <0.05 pg/L <0.05 pg/L C0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L C0.05 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 <0.04pg/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 p g n <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 E sfenvaleraat 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 C0.02 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 pgrL <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 p g L <0.01 pg/L <0.03 pg/L <0.03 pg/L <0.05 p g L <0.25 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.02 p g / L <0.02 pgiL <0.05 pgL. <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.02 p p n <0.01 pglL <0.05 pg/L <0.02 pgrL <0.05 p g L <0.02 pg/L <0.02 pg/L <0.05 pgK. <0.05 pg/L <0.02 pg/L <0.02 pgiL 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999. Continued BACK TO MAIN WILDLIFE INTERNATIONAL,LTD. - 35 - PROJECT NO.: 454A-110B APPENDIX I1 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water' Page 2 Component Dichlorvos Dicofol Diethyltoluamide Di fenoconazole 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 p g 5 <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 co.01 pg/L <0.02 pg/L <0.05 pg/L <0.03 pg/L C0.02 pg/L <0.02 pg/L <0.03 pg/L Component Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropy1 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 . 0 5pg/L <0.05 pg/L <0.02 pglL <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 11.0mg/L 18.0 mg/L 29 m g 5 <0.015 mg/L 1.1 mg/L <0.02 mg/L <0.1 pg/L <0.2 pg/L, <0.5 pg1, <0.2 p g 5 Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium 4 . 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 WILDLIFE INTERNATIONALL, TD. - 36 - PROJECT NO.: 454A-110B APPENDIX 111 T H E A J. LYSIS OF PFOS IN FRESHWATER LG, MEDIUM IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-110B BACK TO MAIN WILDLIFE INTERNATIONALLT, D. - 37 - PROJECT NO.: 454A-110B REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Anabaenaflos-aquae) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-110B PRINCIPAL INVESTIGATOR: MANAGEMENT: Willard B. Nixon, Ph.D! Director, Analytical Chemistry DATE DATB BACK TO MAIN WILDLI FE INTERNATIONAL, LTD. - 38 - PROJECT NO.: 454A-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: IH, IH, 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 I1 of Wildlife International; Ltd. protocol number 4541011299/MVAL/SUB454.It was based upon methodologyprovidedby 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 CISanalytical column (50 mm x 2 mm I.D., 3-pm particle size). The instrument parameters are summarized in Table 1. A method flowchart is provided in Figure 1. WILDLIFE INTERNATIONALL, TD. - 39 - BACK TO MAIN PROJECT NO.: 454A-110B Calibration Curve and Limit of Ouantitation 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 concentrationof 139 mg a.i./L in freshwateralgal medium. Peak Area Ratio = Analyte Peak AredInternal Standard Peak Area ConcentrationRatio = Concentrationof Analyte/Concentrationof Internal Standard Internal Standard Concentration:0,100 m a BACK TO MAIN WILDLIFE INTERNATIONALLT, D. ~ - 40 - 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 (l/x) PROJECT NO.: 454A-110B PFOS (mg a.i.L)at Instrument = Peak area ratSiolope(Y-intercept) x Internal Standard Concentration - 3.6054 - 0.3720 x 0.100 1.1378 = 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 m a.i./L insam le Percent of Nominal Concentration = pFos((m~ nomiI:l x 100 = 102% RESULTS Sample Analysis Freshwater algal medium samples were collected from the four-day toxicity test with the freshwater alga (Anabaenaflos-aquae) at test initiation, May 12, 2000 (Day 0), on May 15, 2000 (Day 3) and at test termination, BACK TO MAIN WILDLI FE INTERNATIONAL, LTD. -41 - PROJECT NO.: 454A-110B 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./Ltreatment group were comparableto samples from the 33 1 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 WILDLI FE INTERNATIONAL, LTD. - 42 - PROJECT NO.: 454A-110B INSTRUMENT: Table 1 Typical HPLCMS Operational Parameters Hewlett-PackardModel 1100High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a PerkinElmer TurboIonSprayion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: Keystone Betas11 CI8column (50 mm x 2 mm I.D., 3-pm particle size) 30C 5.00 minutes 220 pLIminute 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 Approximately4.2 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 WILDLIFE INTERNATIONAL, LTD. - 43 - PROJECT NO.: 454A-110B Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-l10B-) MAB-1 Sample Type Matrix Blank Measured Concentration of PFOS' (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 BACK TO MAIN WILDLIFE INTERNATIONALT, D. - 44 - PROJECT NO.: 454A-110B Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-l10B-) MAS- 1 MAS-4 MAS-7 Concentrations of PFOS (mg a.i./L) Fortified' Measured' 19.2 19.2 19.2 22.2 19.2 22.0 Percent Recovered2 100 116 115 MAS-2 96.0 91.4 95.2 MAS-5 96.0 95.2 99.1 MAS-8 96.0 97.9 102 MAS-3 384 380 98.9 MAS-6 384 3 92 102 MAS-9 3 84 372 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 WILDLI FE INTERNATIONAL, LTD. -45 - PROJECT NO.: 454A-110B Table 4 Measured Concentrations of PFOS in Freshwater Algal Medium Samples from a Freshwater Alga Four-Day Toxicity Test Nominal Test Concentration' (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-l10B-) 1 8 16 Sampling Time (Day) 0 3 4 PFOS Measured Concentration"* (mg a.i./L) < LOQ < LOQ < LOQ Percent of Nominal3 -- _-_- 37.9 2 0 9 3 17 4 37.9 100 38.2 101 37.6 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 33 1 100 328 99.0 329 99.2 33 1 15 3 342 103 (Abiotic) 23 4 356 107 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. WILDLIFE INTERNATIONALL, TD. -46- BACK TO MAIN PROJECT NO.: 454A-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. 1 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 0,100mg 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. Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in freshwater algal medium. WILDLIFE INTERNATIONALL, TD. - 47 - BACK TO MAIN PROJECT NO.: 454A-110B 14 6'oo i 5.00 d 1 4.00 1 1 0 / / 1 2.00 1 0.00 0.00 -- m 1.oo 2.00 3.00 4.00 5.00 Concentration *ti.) Figure 2. A typical calibration curve for PFOS. Slope = 1.1378; Intercept = 0.3720; r = 0.9959. Curve is weighted (Ux). WILDLIFE INTERNATIONAL, LTD. - 48 - BACK TO MAIN PROJECT NO.: 454A-110B intensity: 600000 cps 9 10 0 41 81 121 161 201 241 281 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4.70Time Figure 3. A representative ion chromatogram of a low-level(O.0480mg a.i./L)PFOS standard. . WILDLI FE INTERNATIONAL, LTD. BACK TO MAIN PROJECT NO.: 454A-110B 1oc go 80 70 60 50 40 30 20 10 0 intensity: 600000 cps 247 I\ 41 8 1 1 2 1 1 6 1 2 0 1 241 281 Scan 0.69 1.36 2.02 2.69 3.36 4.03 4.70Time Figure 4. A representative ion chromatogram of a high-level (0.480 mg a.i./L) PFOS standard. WILDLI FE INTERNATIONALT, D. - 50 - BACK TO MAIN PROJECT NO.: 454A-110B 100 90 80 70 60 50 40 30 20 1 0- O! intensity: 600000 cps -1 33 60 1 I I I I 41 81 0.69 1.36 122 I I 121 2.03 168193 I I I I 161 201 2.69 3.36 248 I I 241 4.03 1 2 8 1 Scan 4.70Time Figure 5 . A representative ion chromatogram of a matrix blank sample (454A-1IOB-MAB-1). The arrow indicates the retention time of PFOS. WILDLIFE INTERNATIONALL, TD. -51 - BACK TO MAIN PROJECT NO.: 454A-110B intensity: 600000 cps 41 0.69 8 1 121 1 6 1 201 2 4 1 2 8 1 Scan 1.36 2.02 2.69 3.36 4.03 4.70Time Figure 6. A representative ion chromatogram of a matrix fortification sample (454A-1IOB-MAS-3). WILDLIFE INTERNATIONALT, D. -52- BACK TO MAIN PROJECT NO.: 454A-110B "9'90 intensity: 600000 cps 0.69 1.36 2.02 2.69 3.36 4.03 can 4.70Time Figure 7. A representative ion chromatogram of a test sample (454A-110B-5). ~~ ~ BACK TO MAIN WILDLIFE INTERNATIONAL, LTD. - 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 flos-aquae Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) Replicate 24 Hours Cell Densities (Cells/mL)' 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 0 0 0 9,000 B 0 0 0 16,500 C 0 0 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 WILDLIFE INTERNATIONAL, LTD. - 54 - PROJECT NO.: 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, Anabaenaflos-aquae Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) Replicate Cumulative Area Under the Growth Curve 0 - 24 Hours 0 - 48 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 2,3 16,000 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 0 0 0 C 0 0 126,000 264,000 329 A 0 0 0 0 B 0 0 0 0 C 0 0 0 0 BACK TO MAIN Wi L D L ~FE I NTERNATIONAL, LTD. - 55 - PROJECT NO.: 454A-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,Anabaenaflos-aquae Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) Replicate 0 - 24 Hours Growth Rate 0 - 48 Hours 0 - 72 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 0.0 150 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 - 96 Hours 0.0399 0.0426 0.0435 0.0397 0.043 1 0.0448 0.0420 0.0425 0.0427 0.0425 0.0397 0.0394 0.0329 0.03 13 0.0335 0.0000 0.0000 0.0000 0.0000 0.0052 0.0000 BACK TO MAIN WILDLIFE INTERNATIONAL, LTD. - 56 - APPENDIX VI1 Changes to Protocol PROJECT NO.: 454A-110B 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 of grids to be counted on the hemacytometer in the determination of 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 of measurement for some of the components of the algal medium. 8. Two abiotic replicates, rather than one, were prepared at the highest test concentration, with one replicate sampled on each of Days 3 and 4. 9. Assessments of 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 WILDLIFE INTERNATIONALL, TD. - 57 - APPENDIX VI11 Personnel Involved in the Study PROJECT NO.: 454A-110B The following key personnel were involved in the conduct or management of 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