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BACK TO MAIN PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER DIATOM (NaviculapeZZicuZosa) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A- 112 3M LAB REQUEST NO. U2723 - U.S. EnvironmentalProtection Agency Series 850 Ecological Effects Test Guidelines OPPTS Number 850.5400 AUTHORS: Cary A. Sutherland Henry 0.Krueger, Ph.D. STUDY INITIATION DATE: January 28,2000 STUDY COMPLETION DATE: March 26,200 1 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 2 1601 (4 10) 822-8600 Page 1 of 56 WILDLIFE INTERNATIONALLT, D. -2- BACK TO MAIN PROJECT NO.: 454A-112 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Diatom (Naviculapelliculosa) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-112 STUDY COMPLETION: March 26,200 1 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 substance was 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 substance 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 DATE SPONSOR: WILDLIFE INTERNATIONAL, LTD. -3- BACK TO MAIN PROJECT NO.: 454A-112 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Prac,tice 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: Test Substance Preparation Analytical Standard Preparation Recovery Phase Cell Counts Analytical Data and Draft Report Biological Data and Draft Report Analytical Report, Second Draft Biological Report, Second Draft Final Report DATE REPORTED TO: DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT: February 24,2000 February 24,2000 February 29,2000 February 25,2000 February 25,2000 March 7, 2000 March 7, 2000 March 9, 2000 March 14, 2000 May 12 and 15,2000 May 16,2000 May 16,2000 June 5 - 8,2000 June 8,2000 June 23,2000 March 12,2001 March 12, 2001 March 16,2001 March 14 -15,2001 March 15, 2001 March 20, 2001 March 2 1,2001 March 21, 2001 March 26, 2001 James H!Coleman Quality Assurance Representative 3-26-0 I DATE WILDLIFE INTERNATIONALLT, D. -4- BACK TO MAIN PROJECT NO.: 454A-112 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Diatom (Naviculapelliculosa) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-112 STUDY DIRECTOR: VI Cary A. Sutherland Laboratory Supervisor MANAGEMENT: Director, Aquatic Toxicology and Non-Target Plants WILDLIFE INTERNATIONAL. LTD. -5- BACK TO MAIN PROJECT NO.. 454A-112 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. PROJECT NO.: 454A-112 -6TABLE OF CONTENTS -ContinuedTable 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-response curve, 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 with Silica Constituents .................................................. 33 Appendix I1 - Analyses of Pesticides, Organics, Metals and Other Inorganics 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- 112 ....,........................................... 35 Appendix IV - Cell Density for Each Replicate Per Treatment Over the 96-Hour Exposure Period........................................................................................ 52 Appendix V - Area Under the Growth Curve for Each Replicate Per Treatment Over the 96-Hour Exposure Period........................................................................................ 53 Appendix VI - Growth Rate for Each Replicate Per Treatment Over the 96-Hour Exposure Period. 54 Appendix VI1 - Changes to Protocol ................................................................................................ 55 Appendix VI11 - Personnel Involved in the Study ............................................................................... 56 WILDLIFE INTERNATIONALT, D. -7SUMMARY SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: 3M Corporation Rochelle R. Robideau Wildlife International, Ltd. Easton, MD 2 1601 BACK TO MAIN PROJECT NO.: 454A-112 WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMWAL TEST CONCENTRATIONS: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF EXPOSURE: 454A-112 PFOS (Perfluorooctanesulfonate,Potassium Salt) IUPAC Name: 1-Octanesulfonicacid,1,1,2,2,3,3,4,4,5,5,6,6, 7,7,8,8,8-heptadecafluoro-potassiumsalt; CAS #2795-39-3 PFOS: A 96-Hour Toxicity Test with the Freshwater Diatom (Navicula pelliculosa) Negative Control, 61.5, 81.3, 110, 147, 198, 264 and 347 mg a.i./L Negative Control, 62.3, 83.2, 111, 150, 206, 266 and 335 mg a.i./L Experimental Start (OECD) - February 24,2000 Experimental Start (EPA) - February 25,2000 Exposure Termination - February 29,2000 Experimental Termination - March 7, 2000 96 Hours TEST ORGANISM: SOURCE OF TEST ORGANISMS: Freshwater Diatom (Navicula Wildlife International, Ltd. Easton, Maryland 2 1601 WILDLIFE INTERNATIONAL, LTD. -8SUMMARY (Continued) 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: 242 mg a.i./L 200 - 276 mg a.i./L <62.3 mg a.i./L Not Calculable 263 mg a.i./L 217 - 299 mg a.i./L 322 mg a.i./L 310 - 328 mg a.i./L <62.3 mg a.i./L 150 mg a.i./L AREA UNDER THE GROWTH CURVE: ~ 72-HOUR EC50: ' 95% CONFIDENCE LIMITS: 96-HOUR EC10: 95% CONFIDENCELIMITS: 96-HOUR EC50: 95% CONFIDENCE LIMITS: 96-HOUR EC90: 95% CONFIDENCE LIMITS: 72-HOUR NOAEC: 96-HOUR NOAEC: 246 mg a.i./L 2 10 - 277 mg a.i./L <62.3 mg a.i./L Not Calculable 252 mg a.i./L 220 - 285 mg a.i./L 3 19 mg a.i./L 308 - 326 mg a.i./L <62.3 mg a.i./L <62.3 rng a.i./L GROWTH RATE: 72-HOUR EC50: 95% CONFIDENCE LIMITS: 295 mg a.i./L 288 - 305 mg a.i./L 96-HOUR EC10: 95% CONFIDENCE LIMITS: 243 mg a.i./L 209 - 295 mg a.i./L 96-HOUR EC50: 95% CONFIDENCE LIMITS: 305 mg a.i./L 295 - 316 mg a.i./L 96-HOUR EC90: 95% CONFIDENCE LIMITS: >335 mg a.i./L Not Calculable 72-HOUR NOAEC: 96-HOUR NOAEC: 206 mg a.i./L 206 mg a.i./L All values are mean measured test concentrations. BACK TO MAIN PROJECT NO.: 454A-112 BACK TO MAIN WILDLI FE INTERNATIONALT, D. -9INTRODUCTION PROJECT NO.: 454A-112 This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the test was conducted from February 25, 2000 to February 29, 2000, with the recovery phase completed on March 7, 2000. Raw data generated by Wildlife International, Ltd and a copy of the final report are filed under Project Number 454A-112 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 diatom, Navicula pelliculosa, during a 96-hour exposure period. EXPERIMENTAL DESIGN The freshwater diatom, Navicula pelliculosa, was exposed to a geometric series of seven 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. 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 61.5, 81.3, 110, 147, 198, 264 and 347 mg active ingredient (a.i.)/L. Mean 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. At test initiation, an inoculum of the algal cells was prepared at a concentration of approximately 1.O X 1O6 cells/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 1.0 X lo4 cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine BACK TO MAIN WILD LIFE INTER NATIONAL, LTD. PROJECT NO.: 454A-112 - 10- 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, if possible, for the 72 and 96-hour intervals. The no-observed-adverse-effect-concentration (NOAEC) was determined based upon statistical evaluation of the 72-hour and 96-hour results. At the end of the 96-hour exposure, algistatic effects were differentiatedfrom 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 Diatom (Navicula pelliculosa)". The protocol was based on procedures outlined in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.5400: Algal Toxicity, Tiers I and II (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 expirationdate of August 3 1, 200 1 . The test substance was stored at ambient room temperature. Preparation of Test Concentrations Nominal test concentrations were 61.5, 81.3, 110, 147, 198, 264 and 347 mg a.i./L, based on a test substance purity of 86.9%. All materials which came into contact with the test substance during preparation of test concentrations were constructed of plastic or were Teflon@-lined. Individual test solutions were prepared in algal medium at each of the seven nominal concentrations. The test solutions were stirred with a magnetic stir plate for approximately 24 hours to aid in the solubilization of the test substance. All test solutions appeared clear and colorless. BACK TO MAIN WILDLI FE INTERNATIONALT, D. PROJECT NO.: 454A-112 - 11 - Test Organism The freshwater diatom, Navicula pelliculosa, was selected as the test species for this study. The species is representative of an important group of freshwater algae, and was selected for use in the test based upon a past history of use and ease of culturing in the laboratory. Original algal cultures were obtained from 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 with silica and selenium (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. WILDLIFE INTERNATIONAL, LTD. - 12- BACK TO MAIN PROJECT NO.: 454A-112 Test Apparatus Test chambers were sterile, 250-mL plastic 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 f 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 4300 f 10% lux. Light intensity was measured at the four comers and the middle 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 WILDLIFE INTERNATIONAL, LTD. - 13 - BACK TO MAIN PROJECT NO.: 454A-112 sample was loaded onto a hemacytometer and 10 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 1.O X lo3cells/mL. Samples of test solution were collected from each replicate per treatment and control group at the end of the test. These samples were pooled within their respective treatment, and subsamples were removed and 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 the control and treatment groups using the following formula: where: A = Area No = Mean nominal number of cells/mL at to N1= Mean measured number of cells/mL at tl N2= Mean measured number of cells/mL at t2 N, = Mean measured number of celldml at tn tl = Time of first measurement after beginning of test (hours) t2= Time of second measurement after beginning of test (hours) t, = Time of n* measurement after beginning of test (hours) Growth rates were calculated for the control and each treatment group using the following formula: lnN, - lnN, p = t, -to where: p = Average specific growth rate No = Mean nominal number of cells/mL at to N, = Mean measured number of cells/mL at t, to = Time of beginning of the test (hours) tn = Time of n* measurement after beginning of test (hours) WILDLI FE INTERNATIONALT, D. - 14- BACK TO MAIN PROJECT NO.: 454A-112 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: x Mean Responsecontro-lMean 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 (ie., 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. The treatment groups were then compared to the control using Dunnett's test. Results of the statistical analyses and evaluation of the concentration-response pattern were used to determine the NOAEC values. 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 335 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 plastic centrihge tubes and were analyzed immediately without storage. The 72 and 96-hour samples were centrifuged approximately 5 minutes at approximately 1500 rpm prior to analysis. Analytical procedures used in the analysis of the samples are presented in Appendix 111. WILDLIFE INTERNATIONAL, LTD. - 15 - BACK TO MAIN PROJECT NO.: 454A-112 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 61.5, 81.3, 110, 147, 264 and 347 mg a.i./L. Samples collected at the beginning of the test had measured concentrations that ranged from 96.2 to 106% of nominal. Samples collected at 72 hours and at test termination had recoveries that ranged from 98.5 to 106%, and 94.8to 101% of nominal, respectively. The abiotic replicates from the 347 mg a.i./L treatment had recoveries of 98.2 and 96.8% 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 62.3, 83.2, 111, 150, 206, 266 and 335 mg a.i./L, representing 101, 102, 101, 102, 104, 101 and 96.5% 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 23.1 to 24.6"C and were within the range established for the test (24 f2C). The light intensity ranged from 3910 and 45 10 lux and was within the desired range for the test (approximately 3870 to 4730 lux). Measurements of pH were 7.5 on Day 0 and ranged from 7.7 to 8.9 at 96 hours. The pH of the abiotic replicate at test termination was 7.7, which was comparable to the biotic replicates at the same concentration. The effect of PFOS upon Navicula pelliculosa 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. 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. WILDLIFE INTERNATIONALT, D. - 16- BACK TO MAIN PROJECT NO.: 454A-112 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 62.3, 83.2, 111, 150,206,266 and 335 mg a.i./L treatment groups was 34, 30, 34, 23, 25, 64 and 99%, respectively. Dunnett's test showed that cell density was significantly reduced in the 62.3, 83.2, 111, 266 and 335 mg a.i./L treatment groups in comparison to the negative control (pF0.05). Consequently,the NOAEC for 72-hour cell density was < 62.3 mg a.i./L, the lowest concentration tested. After 96 hours of exposure, cell density percent inhibition in the 62.3, 83.2, 111, 150, 206, 266 and 335 mg a.i./L treatment groups was 13, 13, 13, 9.3, 23, 5 1 and 99%, respectively. Dunnett's test showed that cell density was significantly reduced in the 206, 266 and 335 mg a.i./Ltreatment groups (p10.05). Consequently, the NOAEC for 96-hour cell density was 150 mg a.i./L. After 72 hours of exposure, area under the growth curve percent inhibition in the 62.3, 83.2, 111, 150, 206, 266 and 335 mg a.i./L treatment groups was 28, 27, 29, 20, 23, 62 and 99%, respectively. After 96 hours of exposure, percent inhibition was similar to the inhibition at 72 hours and was 24, 22, 24, 16, 24, 58 and 99%, respectively. Dunnett's test showed that area under the growth curve was significantly reduced (p10.05) in all treatments except the 150 mg a.i./L treatment group at both 72 and 96 hours. Consequently, the NOAEC for 72 and 96-hour area under the growth curve was < 62.3 mg a.i./L, the lowest concentration tested. After 72 hours of exposure, growth rate percent inhibition in the 62.3, 83.2, 111, 150, 206, 266 and 335 mg a.i./L treatment groups was 8.1, 6.7, 7.8, 5.2, 5.5, 20 and 90%, respectively. Dunnett's test showed that growth rate was significantly reduced in the 266 and 335 mg a.i./Ltreatment groups (p10.05). Consequently, the NOAEC for 72-hour growth rate was 206 mg a.i./L. M e r 96 hours of exposure, growth rate percent inhibition in the 62.3, 83.2, 111, 150, 206, 266 and 335 mg a.i./L treatment groups was 2.5, 2.7, 2.5, 1.7, 4.7, 13 and 79%, respectively. Dunnett's test showed that growth rate was significantly reduced in the 266 and 335 mg a.i./L treatment groups (p10.05).Consequently, the NOAEC for 96-hour growth rate was 206 mg a.i./L. WILDLI FE INTERNATIONAL, LTD. - 17- BACK TO MAIN PROJECT NO.: 454A-112 Visual and Microscopic Observations AAer 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. At 72 and 96 hours of exposure, some cells in the 335 mg a.i./L treatment group appeared small in comparison to the control. All other treatment groups showed no noticeable changes in cell morphology. Reversibility of Growth Inhibition The 335 mg a.i./L treatment group was maximally inhibited at the end of the 96-hour exposure period. Aliquots of the test solution were diluted with algal medium and cultured for seven days. Based on the increase in growth observed in the recovery phase, the effect on algal growth was found to be algistatic, rather than aligicidal. 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 (Le., cell density, area under the growth curve and/or growth rate). The 72-hour EC50, based on cell density, was 242 mg a.i./L, with 95% confidence limits of 200 and 276 mg a.i./L. The 96-hour EC50, based on area under the growth curve, was 252 mg a.i./L, with 95% confidence limits of 220 and 285 mg a.i./L. The 72-hour NOAEC, based on cell density and area under the growth curve, was 4 2 . 3 mg a.i./L, the lowest concentration tested. The 96-hour NOAEC, based on area under the growth curve, was G 2 . 3 mg a.i./L, the lowest concentration tested. Based on the presence of visible algal growth at recovery termination, PFOS was considered to be algistatic, rather than algicidal, at the concentrations tested. WILDLIFE INTERNATIONALL, TD. - 18 - BACK TO MAIN PROJECT NO.: 454A-112 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 1218-903. 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. WILDLIFE INTERNATIONAL,LTD. - 19- BACK TO MAIN PROJECT NO.: 454A-112 Table 1 Summary of Analytical Chemistry Data Sponsor: Test Substance: Test Organism: Dilution Water: Nominal Concentration (mg a.i./L) 3M Corporation PFOS Freshwater Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium Sampling Time (Hours) Measured Concentration' (mg a.i./L) Mean Measured Concentration (mg a.i./L) Negative Control O2 723 964 61.5 0 72 96 < LOQ < LOQ < LOQ 62.3 63.6 61.1 < LOQ 62.3 81.3 0 83.8 83.2 72 84.7 96 81.0 110 0 109 111 72 113 96 110 147 0 147 150 72 154 96 149 198 0 209 206 72 209 96 199 264 0 27 1 266 72 268 96 258 Percent of Nominal 101 102 101 102 104 101 347 0 334 72 342 96 329 335 96.5 347 (abiotic) 72' 341 96' 336 339 97.7 1 Limit of Quantitation (LOQ) was 4.39 mg a.i./L. 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 replicates per treatment and control group. 5 72 and 96-hour samples were collected from the additional abiotic replicates. WILDLIFE INTERNATIONALT, D. - 20 - BACK TO MAIN PROJECT NO.: 454A-112 Table 2 Temperature Measurements Sponsor: Test Substance: Test Organism: Dilution Water: Time (Day) 3M Corporation PFOS Freshwater Diatom, Naviculapelliculosa Freshwater Algal Medium with Silica and Selenium Temperature ("C) Measurement 1 Measurement 2 0 23.6 23.9 1 24.5 24.2 2 24.6 24.2 3 24.2 24.1 4 24.0 23.3 5 23.7 23.7 6 23.8 23.4 7 23.3 23.3 8 23.2 23.5 9 23.2 23.1 10 23.5 23.7 11 23.2 23.3 1 Temperature Measurement 2 was taken at least 4 hours after Measurement 1, with the exception of test initiation and termination (Days 0 and 11). WILDLIFE INTERNATIONALLT, D. -21 - BACK TO MAIN PROJECT NO.: 454A-112 Table 3 Light Intensity Measurements Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Diatom, Navicula pellzculosa Freshwater Algal Medium with Silica and Selenium Test Shaker Day Table No. 1 Light Intensity Measurements (lux) No. 2 No. 3 No 4 1 0 2 3910 4500 4370 4050 4490 45 10 4230 3930 1 Light intensity was measured at five locations over each of two shaker tables. No 5 4320 3920 WILDLI FE INTERNATIONAL, LTD. - 22 - BACK TO MAIN PROJECT NO.: 454A-112 Table 4 pH Measurements Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium Mean Measured Concentration (mg a.i./L) 0 Hours' pH Measurements 96 Hours2 Negative Control 7.5 8.6 62.3 7.5 8.6 83.2 7.5 8.8 111 7.5 8.9 150 7.5 8.8 206 7.5 8.3 266 7.5 8.2 335 7.5 7.7 339 --3 7.7 (abiotic) 1 O-hour samples were collected from the batches of test solution prepared for the treatment and control groups at test initiation. 2 96-hour samples were collected from the pooled replicates per treatment and control group. The abiotic replicates were prepared from the test solution used to prepare the 335 mg a.i./L test solution on Day 0. BACK TO MAIN WILDLI FE INTERNATIONALT, D. -23 - PROJECT NO.: 454A-112 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 Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium Mean Measured Concentration (mg a.i./L) Negative Control 24 Hours Mean Cell Density' (cells/mL) Percent Inhibition' 44,333 -_ 48 Hours Mean Cell Density' (cells/mL) Percent Inhibition' 269,667 -- 72 Hours Mean Cell Density' (cells/mL) Percent Inhibition' 1,696,667 -- 62.3 44,333 0 235,000 13 1,126,667* 34 83.2 35,333 20 229,333 15 1,190,000* 30 111 39,333 11 227,667 16 1,123,333* 34 150 37,667 15 248,333 7.9 1,306,667 23 206 42,333 4.5 2 17,000 20 1,276,667 25 266 26,333 41 126,667 53 606,667* 64 335 7,000 84 15,333 94 17,333* 99 ' Values calculated using SAS 6.12. Manual calculations may differ slightly. * Indicates a significant difference from the negative control using Dunnett's test (p 5 0.05). ~ ~~ 96 Hours Mean Cell Density' (cells/mL) Percent Inhibition' 2,726,667 -- 2,366,667 3 2,366,667 3 2,373,333 3 2,473,333 9.3 2,093,333* 23 1,330,000* 51 35,333* 99 WILDLIFE INTERNATIONAL, LTD. - 24 - BACK TO MAIN PROJECT NO.: 454A-112 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 Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium Mean Measured Concentration (mg a.i./L) 0 - 24 Hours Mean Area' Percent Inhibition' 0 - 48 Hours Mean Area' Percent Inhibition' Negative Control 412,000 _- 3,940,000 -- ~ ~~ 0 - 72 Hours Mean Area' Percent Inhibition' 27,296,000 -- 0 - 96 Hours ~ Mean Area' Percent Inhibition' 80,136,000 _- 62.3 412,000 0.0 3,524,000 11 19,624,000* 28 83.2 304,000 26 3,240,000 18 20,032,000* 27 111 352,000 15 3,3 16,000 16 19,288,000* 29 150 332,000 19 3,524,000 11 21,944,000 20 206 388,000 5.8 3,260,000 17 20,944,000* 23 266 196,000 52 1,792,000 55 10,352,000* 62 335 0 100 32,000 99 184,000* 99 ~ ~ ~ 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 5 0.05). 6 1,304,000* 24 62,472,000* 22 6 1,008,000* 24 67,064,000 16 6 1,144,000* 24 33,352,000* 58 576,000* 99 BACK TO MAIN WILDLIFE INTERNATIONAL, LTD. - 25 - PROJECT NO.: 454A-112 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 Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium Mean Measured Concentration (mg a.i./L) 0 - 24 Hours Mean Growth Rate' (cells/mL/hr) Percent Inhibition' 0 - 48 Hours Mean Growth Rate' Percent (cells/mL/hr) Inhibition' Negative Control 0.0620 -- 0.0686 __ 62.3 0.0618 0.28 0.0657 4.3 83.2 0.0525 15 0.0652 4.9 111 0.0569 8.3 0.0650 5.2 150 0.0544 !2 0.0669 2.5 206 0.0600 3.1 0.0640 6.7 266 0.0383 38 0.0518 24 335 0.0000 100 0.0086 87 ' Values calculatedusing SAS 6.12. Manual calculations may differ slightly. * Indicates a significantdifference from the negative control using Dunnett'stest (p I0.05). 0 - 72 Hours Mean Growth Rate` (cells/mL/hr) Percent Inhibition' 0.0711 -- 0.0653 8.1 0.0663 6.7 0.0656 7.8 0.0674 5.2 0.0672 5.5 0.0569* 20 0.0068* 90 0 - 96 Hours Mean Growth Rate' (cells/mL/hr) 0.0584 Percent Inhibition' __ 0.0569 2.5 0.0568 2.7 0.0570 2.5 0.0574 1.7 0.0556 4.7 0.0506* 13 0.0125* 79 WILDLI FE INTERNATIONAL, LTD. BACK TO MAIN PROJECT NO.: 454A-112 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 3M Corporation PFOS Freshwater Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium EClO (mg a.i./L) Not Determined 95% Confidence Limits (mg a.i./L) __ EC50 (mg a.i./L) 28 1 Not Determined -- 26 1 72 Hours < 62.3 _-1 242 96 Hours < 62.3 _-1 263 1 Confidence limits could not be calculated with the data obtained. 95% Confidence Limits (mg a.i./L) 214 - 312 219 - 306 200 - 276 217 - 299 EC90 (mg a.i./L) Not Determined Not Determined 3 17 322 ~~ ~_____~ 95% Confidence Limits (mg a.i./L) -- -- 306 - 326 310 - 328 BACK TO MAIN WILDLIFE INTERNATIONAL, LTD. - 27 - PROJECT NO.: 454A-112 Table 9 EC Values Based on Area Under the Growth Curve Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium Time 24 Hours 48 Hours EClO (mg a.i./L) Not Determined Not Determined 95% Confidence Limits (mg a.i.L) __ __ EC50 (mg a i.L) 262 259 72 Hours < 62.3 __I 246 96 Hours < 62.3 __1 252 -1 C_o_nfidence limits could not be calculated with the data obtained. 95% Confidence Limits (mg a.i./L) 205 - 308 227 - 303 210 - 277 220 - 285 ~ EC90 (mg a.i./L) Not Determined Not Determined 3 18 3 19 95% Confidence Limits (mg a.i.L) --- 307 - 325 308 - 326 BACK TO MAIN WILDLIFE INTERNATIONALL, TD. - 28 - PROJECT NO.: 454A-112 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 3M Corporation PFOS Freshwater Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium EClO (mg a.i./L) Not Determined 95% Confidence Limits (mg a.i./L) __ Not Determined -- 72 Hours 96 Hours 22 1 190 - 252 243 209 - 295 1Confidence limits could not be calculatedwith the data obtained. EC50 (mg a.i./L) 279 294 295 305 95% Confidence Limits (mg a.i./L) 212 - 306 271- 307 288 - 305 295 - 316 EC90 (mg a.i./L) Not Determined Not Determined 335 > 335 95% Confidence Limits (mg a.i./L) -- _- 323 - 335 --1 WILDLIFE INTERNATIONALTL,D. - 29 - BACK TO MAIN PROJECT NO.: 454A-112 Table 11 Cell Densities During the Recovery Phase Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium Mean Measured Concentration (mg a.i./L) Day O2 Cell Densities (cells/ml,) Day 3 Day 6 Day 7 Negative Control 15,000 2,280,000 2,560,000 2,520,000 335l 5,000 7,000 179,000 780,000 1 The treatment group was diluted to a concentration of the test substance that theoretically would not inhibit growth. 2 Due to the method defined in the protocol used to prepare recovery phase test solutions, initial cell densities were not equivalent in both groups. WILDLIFE INTERNATIONALT, D. BACK TO MAIN PROJECT NO.: 454A-112 Figure 1. Negative control algal growth, expressed as cell density, during the 96-hour exposure period. 10,000,000 T 1,000,000 100,000 10,000 1,000 0 +Negative Control 24 48 12 Exposure Duration (Hours) __1 96 WILDLIFE INTERNATIONAL,LTD. -31 - BACK TO MAIN PROJECT NO.: 454A-112 Figure 2. Concentration-response curve, expressed as cell density, over the 96-hour exposure period. 10,000,000 .3 1,000,000 E m 3 8 v Q.3- m 100,000 n 3 d E m 2 10,000 1,000 ' 0 24 48 72 Exposure Duration (Hours) __1 96 +Negative Control +62.3 mg a.i./L +83.2 mg a.i./L +1 1 1 mg a . i . 5 +150 mg a.i./L +206 mg a.i./L +266 mg a.i./L -335 mg a.i./L WILDLIFE INTERNATIONALT, D. - 32 - Figure 3. Recovery-phase response curve, expressed as cell density. 10,000,000 BACK TO MAIN PROJECT NO.: 454A-112 1,000,000 3 U 10,000 -0- Negative Control +335 mg a.i./L 1,000 0 3 6 Recovery Duration (Days) 7 7 WILDLIFE INTERNATIONAL,LTD. - 33 - BACK TO MAIN PROJECT NO.: 454A-112 APPENDIX I Freshwater Algal Medium with Silica Constituents' Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium Compound Nominal Concentration MgC12*6H20 CaC12*2H20 MnC12*4H20 ZnC12 FeC13*6H20 CoC12*6H20 Na2Mo04*2Hz0 CuC12.2H20 Na2EDTA*2H20 NaN03 MgS 04*7H20 K2HP04 NaHCO3 Na2Si03*9H20 Na2Se03*5H20 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 20.0 0.010 ' The pH was adjusted to 7.5 f 0.1 using 10% HC1 and 0.1 N NaOH. WILDLI FE INTERNATIONAL, LTD. - 34 - BACK TO MAIN PROJECT NO.: 454A-112 APPENDIX I1 Analyses of Pesticides, Organics, Metals and Other Inorganics in Wildlife International, Ltd. Well Water' ANALYSIS Miscellaneous Measurements Total Dissolved Solids Ammonia Njtrogen Total Organic Carbon' Total Cyanide MEASURED CONCENTRATION 286 < 0.050 < 1.o < 10.0 Organochlorines and PCBs Alilrin Alpha BHC Beta BHC Delta BHC Gamma BHC (Lindane) Chlordane DDD, PP,' EEkK DDE, PPI Endosulfan, A Endosulfan, B Endosulfan Sulfate < 0.005 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < 0.005 < 0.005 < 0.007 < 0.005 < 0.500 < 0.260 < 0.260 < 0.260 < 0.720 < 0.720 < 0.720 < 0.720 Metals an4 Other Inorganics Aluminym Arsenic ~~~11j~~ Ca m i 9 Calcium ~hroqum~ Cobalt copper3 Iron Lead3 Magnesiunf Manganese Mercu Molyb$num3 Nickel Iron' selenium3 Silver Sodiym3 Zinc < 100 < 25.0 < 0.50 < 1.0 35.0 < 2.0 < 1 .o < 20.0 < 100 < 10.0 < 113..o5 < 0.20 < 2.0 < 2.0 6.62 < 25.0 < 1.0 21.3 < 20.0 * Analyses performed by QST Environmental, Gainesville, Florida for samples collected on November 3 through November 7, 1997. Analyses performed by Wildlife International, Ltd. for the sample collected on November 5, 1997. Analyses performed by Wildlife International, Ltd. for samples collected on November 5 through 7, 1997. WILDLIFE INTERNATIONAL, LTD. - 35 - BACK TO MAIN PROJECT NO.: 454A-112 APPENDIX I11 THE ANALYSIS OF PFOS IN FRESHWATER ALGAL MEDIUM IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-112 WILDLI FE INTERNATI ONAL, LTD. - 36 - BACK TO MAIN PROJECT NO.: 454A-112 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Diatom (Naviculapelliculosa) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-112 PRINCIPAL INVESTIGATOR: " MANAGEMENT: ' Willard B. Nixon, Ph.D. Director, Analytical Chemistry 3 i h hI DATE DA ~~ WILDLIFE INTERNATIONALL, TD. - 37 - BACK TO MAIN PROJECT NO.: 454A-112 Introduction Freshwater algal medium samples were collected from a 96-hour toxicity test designed to determine the effects of PFOS (Perfluorooctanesulfonate, Potassium Salt) to the freshwater diatom (Navicula pelliculosa). This study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A-112. 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 between February 25 and 29,2000 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: lH, lH, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 2761997-2. The standard was stored under ambient conditions. Analvtical 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 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Samples were centrifuged, as necessary, and 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. WILDLI FE INTERNATIONALL, TD. - 38 - BACK TO MAIN PROJECT NO.: 454A-112 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 lOOLC Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil CI8 analytical 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. Calibration Curve and Limit of Ouantitation Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpure@ water solution containing 0,100mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.0439 to 0.879 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 4.39 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.0439 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 17.6, 176 and 351 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 3). Sample concentrations were not WILDLIFE INTERNATIONAL,LTD. - 39 - BACK TO MAIN PROJECT NO.: 454A-112 corrected for the mean procedural recovery of 108%. A representative ion chromatogram of a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-112-5, nominal concentration of 147 mg a.i./L in freshwater algal medium. Peak Area Ratio = Analyte Peak Areahternal Standard Peak Area Concentration Ratio = Concentration of Analyte/Concentration of Internal Standard Internal Standard Concentration: 0,100 mg/L Initial Volume: 0,100 mL Final Volume: 50.0 mL Dilution Factor: 500 PFOS Peak Area: 8477265 Internal Standard Peak Area: 1849815 Peak Area Ratio: 4.58276 Calibration curve equation. Slope: 1.44418 Intercept: 0.32839 Curve is weighted (l/x) PFOS (mg a.i./L) at instrument = Peak area raStiloop- e(Y-intercept) x Internal Standard Concentration - 4.58276 - 0.32839 o,loo 1.44418 = 0.2946 WILDLIFE INTERNATIONALLT, D. -40 - ~ ~~ BACK TO MAIN PROJECT NO.: 454A-112 PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at instrument x Dilution Factor = 0.2946 x 500 = 147 PFOS (mg a.i./L) in sample Percent of Nominal Concentration = PFOS (mg a.i./L)nominal x 100 --147 - x 100 147 = 100% Quantitation software for recoveries: MacQuan, version 1.6. RESULTS Sample Analysis Freshwater algal medium samples were collected from the 96-hour toxicity test with the freshwater diatom (Navicula pelliculosa) at test initiation, February 25, 2000 (Day 0), on February 28, 2000 (Day 3 ) and at test termination, February 29, 2000 (Day 4). The measured concentrations of PFOS in the samples collected at initiation of exposure of the test organisms (Hour 0) ranged from 96.2 to 106% of the nominal concentrations. Samples collected at Day 3 had a measured concentration range of 98.2 to 106% of nominal values. Samples collected at test termination (Day 4) had a measured concentration range of 94.8 to 101% of nominal values (Table 4). Samples from the abiotic 347 mg a.i./L treatment group were comparable to samples from the 347 mg a.i./L treatment group with the freshwater diatom present (Table 4). A representative ion chromatogram of a test sample is shown in Figure 7. WILDLIFE INTERNATIONALL, TD. -41 - ~~ BACK TO MAIN PROJECT NO.: 454A-112 INSTRUMENT: Table 1 Typical HF'LCMS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API lOOLC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSprayion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: INTERNAL STANDARD RETENTION TIME: PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: Keystone Betasil CIScolumn (50 mm x 2 mm I.D., 3-pm particle size) 30C 5.00 minutes 0.220 mL/minute 72.0%Methanol : 28.0% NANOpure@Water containing 0.1% Formic Acid 5.0 pL Approximately 3.7 minutes Approximately 2.6 minutes 498.6 amu 426.7 amu WILDLI FE INTERNATIONAL, LTD. - 42 - BACK TO MAIN PROJECT NO.: 454A-112 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-112-) 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 4.39 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0439 mg a.i./L) and the dilution factor of the matrix blank samples (100). WILDLIFE INTERNATIONALL, TD. - 43 - BACK TO MAIN PROJECT NO.: 454A-112 Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-112-) Concentrations of PFOS (mg a.i./L) Fortified' Measured' Percent Recovered2 MAS-1 17.6 20.9 119 MAS-4 17.6 20.3 115 MAS-7 17.6 20.4 116 MAS-2 176 192 109 MAS-5 176 187 106 MAS-8 176 185 105 MAS-3 351 359 102 MAS-6 351 349 99.2 MAS-9 351 352 100 Mean = 108 Standard Deviation = 7.29 CV = 6.76% N =9 ' Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate of Analysis dated September7, 2000. 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. WILDLI FE INTERNATIONAL, LTD. - 44 - BACK TO MAIN PROJECT NO.: 454A-112 Table 4 Measured Concentrations of PFOS in Freshwater Algal Medium Samples from a Freshwater Diatom (Navicula pelliculosu) 96-Hour Toxicity Test Nominal Test Concentration' (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-112-) 1 9 18 Sampling Time (Day) 0 3 4 PFOS Measured (mg a.i./L) Percent of Nominal3 61.5 2 0 10 3 19 4 62.3 101 63.6 103 61.1 99.3 81.3 3 0 11 3 20 4 83.8 103 84.7 104 81.0 99.7 110 4 0 12 3 21 4 109 99.6 113 103 110 99.7 147 5 0 13 3 22 4 1,47 100 154 105 149 101 198 6 0 14 3 23 4 209 106 209 106 199 101 264 7 0 15 3 24 4 2'7 1 103 268 102 258 97.7 347 8 0 16 3 25 4 334 96.2 342 98.5 329 94.8 347 17 3 (Abiotic) 26 4 34 1 98.2 336 96.8 I Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate of Analysis dated September 7,2000. The limit of quantitation (LOQ) was 4.39 mg a.i.iL based upon the product of the lowest calibration standard analyzed (0.0439 mg a.i./L) and the dilution factor of the matrix blank samples (100). 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. WILDLI FE INTER NATIONAL, LTD. -45 - BACK TO MAIN PROJECT NO.: 454A-112 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. J. Centrifuge all samples, as necessaq, for approximatelyfive minutes at approximately 1500 rpm. .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% NANOpureBwater 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. .1 Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in freshwateralgal medium. WILDLIFE INTERNATIONALL, TD. -46 - BACK TO MAIN PROJECT NO.: 454A-112 Figure 2. 0.00 2.00 4.00 6.00 8.00 10.00 ~ ' Concentration(Ratio) I A typical calibration curve for PFOS. Slope = 1.44418; Intercept = 0.32839;r = 0.9975. Curve is weighted (l/x). WILDLI FE I NTERNATIONAL, LTD. - 47 - BACK TO MAIN PROJECT NO.: 454A-112 100- 90- 80- 70- 60- 50- 40- 30- 20- 10- 10 36 67 102 1 2 6 158 01......1...111.1. 21 7 Figure 3. A representative ion chromatogram of a low-level (0.0439 mg a.i./L) PFOS standard. WILDLIFE INTERNATIONALL, TD. BACK TO MAIN PROJECT NO.: 454A-112 intensity: 800000 cps 10 'K 7 6 - 12 37 66 11.. 31 61 83 91 111 121 0.52 1.03 1.53 2.04 .2.54 3.05 3.55 4.06 4.56 Time Figure 4. A representative ion chromatogram of a high-level (0.879 mg a.i./L) PFOS standard. WILDLIFE INTERNATIONALL, TD. -49 - BACK TO MAIN PROJECT NO.: 454A-112 100 90 80 70 60 50 40 30 20 10 10 0 37 31 0.52 71 61 1.03 91 1.53 116 121 2.04 150 151 2.54 intensity: 800000 cps -1 186. 181 3.05 218 211 3.55 ,243 241 4.06 272 271 4.56 scan Time Figure 5 . A representative ion chromatogram of a matrix blank sample (454A-112-MAB-1). The arrow indicates the retention time of PFOS. WILDLIFE INTERNATIONALL, TD. -50- BACK TO MAIN PROJECT NO.: 454A-112 intensity: 800000 cps 10 7 6 54 21 8 n 1 8 4 31 0.52 61 1.03 91 1.53 121 2.04 151 2.54 181 3.05 211 3.55 241 4.06 271 4.56 Scan Time Figure 6 . A representativeion chromatogramof a matrix fortification sample (454A-112-MAS-1). WILDLIFE INTERNATIONALLT, D. -51 - BACK TO MAIN PROJECT NO.: 454A-112 100 90 80 70 60 50 40 30 2 1 intensity: 800000 cps 218 31 0.52 61 1.03 91 1.53 121 2.04 2.54 3.05 3.55 4.06 4.56 can Time Figure 7. A representative ion chromatogram of a test sample (454A-112-2). WILDLI FE INTERNATIONAL, LTD. BACK TO MAIN PROJECT NO.: 454A-112 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 Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium Mean Measured Concentration (mg a.i.4,) Replicate 24 Hours Cell Densities (Cells/mL)' 48 Hours 72 Hours 96 Hours Negative Control A 47,000 276,000 1,370,000 2,980,000 B 45,000 249,000 1,620,000 2,500,000 C 4 1,000 284,000 2,100,000 2,700,000 62.3 A 46,000 271,000 1,120,000 2,200,000 B 38,000 2 18,000 1,410,000 2,600,000 C 49,000 2 16,000 850,000 2,300,000 83.2 A 33,000 220,000 1,320,000 2,580,000 B 39,000 25 1,000 1,190,000 2,640,000 C 34,000 217,000 1,060,000 1,880,000 111 A 42,000 216,000 1,090,000 2,500,000 B 34,000 212,000 1,070,000 2,440,000 C 42,000 255,000 1,210,000 2,180,000 150 A 39,000 259,000 1,630,000 2,480,000 B 28,000 240,000 1,020,000 2,460,000 C 46,000 246,000 1,270,000 2,480,000 206 A 46,000 246,000 1,550,000 2,140,000 B 42,000 207,000 1,130,000 1,900,000 C 39,000 198,000 1,150,000 2,240,000 266 A 34,000 172,000 670,000 1,710,000 B 29,000 78,000 520,000 890,000 C 16,000 130,000 630,000 1,390,000 335 A 9,000 18,000 14,000 53,000 B 7,000 12,000 26,000 3 1,000 C 5,000 16,000 12,000 22,000 1 The initial cell density of the stock culture was determined and an inoculum volume was administered to each test chamber to yield a cell density of approximately 10,000 cells/mL at test initiation (0 hours). WILDLIFE INTERNATIONALL, TD. - 53 - BACK TO MAIN PROJECT NO.: 454A-112 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 Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium 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 444,000 4,080,000 23,592,000 75,552,000 B 420,000 3,708,000 25,896,000 75,096,000 C 372,000 4,032,000 32,400,000 89,760,000 62.3 A 432,000 3,996,000 20,448,000 60,048,000 B 336,000 3,168,000 22,464,000 70,344,000 C 468,000 3,408,000 15,960,000 53,520,000 83.2 A 276,000 3,072,000 21,3 12,000 67,872,000 B 348,000 3,588,000 20,640,000 66,360,000 C 288,000 3,060,000 18,144,000 53,184,000 111 A 384,000 3,240,000 18,672,000 61312,000 B 288,000 3,000,000 18,144,000 60,024,000 C 384,000 3,708,000 2 1,048,000 61,488,000 150 A 348,000 3,684,000 26,112,000 75,192,000 B 216,000 3,192,000 18,072,000 59,592,000 C 432,000 3,696,000 21,648,000 66,408,000 206 A 432,000 3,696,000 25,008,000 69,048,000 B 384,000 3,132,000 18,936,000 55,056,000 C 348,000 2,952,000 18,888,000 59,328,000 266 A 288,000 2,520,000 12,384,000 40,704,000 B 228,000 1,272,000 8,208,000 24,888,000 C 72,000 1,584,000 10,464,000 34,464,000 335 A 0 84,000 228,000 792,000 B 0 0 216,000 660,000 C 0 12,000 108,000 276,000 WILDLI FE INTERNATIONAL, LTD. - 54 - BACK TO MAIN PROJECT NO.: 454A-112 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 Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium Mean Measured Concentration (mg a.i./L) Replicate 0 - 24 Hours Growth Rate 0 - 48 Hours 0 - 72 Hours Negative Control A 0.0645 0.0691 0.0683 B 0.0627 0.0670 0.0707 C 0.0588 0.0697 0.0743 62.3 A 0.0636 0.0687 0.0655 B 0.0556 0.0642 0.0687 C 0.0662 0.0640 0.0617 83.2 A 0.0497 0.0644 0.0678 B 0.0567 0.0671 0.0664 C 0.0510 0.0641 0.0648 111 A 0.0598 0.0640 0.0652 B 0.05 10 0.0636 0.0649 C 0.0598 0.0675 0.0666 150 A 0.0567 0.0678 0.0707 B 0.0429 0.0662 0.0642 C 0.0636 0.0667 0.0673 206 A 0.0636 0.0667 0.0700 B 0.0598 0.063 1 0.0657 C 0.0567 0.0622 0.0659 266 A 0.0510 0.0593 0.0584 B 0.0444 0.0428 0.0549 C 0.0196 0.0534 0.0575 335 A 0.0000 0.0122 0.0047 B 0.0000 0.0038 0.0133 C 0.0000 0.0098 0.0025 0 - 96 Hours 0.0593 0.0575 0.0583 0.0562 0.0579 0.0566 0.0578 0.0581 0.0545 0.0575 0.0573 0.0561 0.0574 0.0573 0.0574 0.0559 0.0547 0.0564 0.0536 0.0468 0.0514 0.0174 0.01 18 0.0082 WILDLIFE INTERNATIONAL,LTD. - 55 - BACK TO MAIN PROJECT NO.: 454A-112 APPENDIX VI1 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, test concentrations and test substance identificationnumber. 2. The protocol was amended to clarify that the nominal concentration may exceed 100 mgL. 3 . The protocol was amended to add a recovery phase to the study 4. The nominal test concentrations were a series of seven concentrations, and were recalculated based on a test substance purity of 90.49%. 5 . The nominal test concentrations were a series of seven concentrations, and were recalculated based on a test substance purity of 86.9%. 6. The test solutions were prepared individually, rather than by dilution of a primary or secondary stock solution. 7. Two abiotic replicates, rather than one, were prepared at the highest concentration, with one replicate sampled on each of Days 3 and 4. 8. The protocol was amended to correct typographical errors in the units of measurement for some of the components of the algal medium. 9. The protocol was amended to change the test substance name from Perfluorooctane Sulfonic Acid, Potassium Salt to Perfluorooctanesulfonate, Potassium Salt. WILDLIFE INTERNATIONAL, LTD. -56- BACK TO MAIN PROJECT NO.: 454A-112 APPENDIX VI11 Personnel Involved in the Study 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