Document LpkGXoorJLwo8MJEvYv3zbjmw

BACK TO MAIN PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER DIATOM {Navcula pelliculosa) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-112 3M LAB REQUEST NO. U2723 U.S. Environmental Protection 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, 2001 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 o f 56 W il d l if e In t e r n a t io n a l , Lt d . -2 - BACK TO MAIN PR O JE C T NO.: 454A -112 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Diatom {Navculapelliculosd) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-112 STUDY COMPLETION: March 26, 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 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 SPONSOR: W il d l if e In t e r n a t io n a l , Lt d . -3 - BACK TO MAIN PR O JE C T NO.: 454A -112 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice, (ENV/MC/CHEM(98)17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: DATE REPORTED TO: DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT: Test Substance Preparation February 24, 2000 February 24, 2000 February 29, 2000 Analytical Standard Preparation February 25, 2000 February 25, 2000 March 7, 2000 Recovery Phase Cell Counts March 7, 2000 March 9, 2000 March 14, 2000 Analytical Data and Draft Report May 12 and 15, 2000 May 16, 2000 May 16, 2000 Biological Data and Draft Report June 5 - 8 , 2000 June 8, 2000 June 23, 2000 Analytical Report, Second Draft March 12, 2001 March 12, 2001 March 16, 2001 Biological Report, Second Draft March 14-15, 2001 March 15, 2001 March 20, 2001 Final Report March 2 1, 2001 March 21, 2001 March 26, 2001 James H "Coleman Quality Assurance Representative 3-A' I DATE W il d l if e In t e r n a t io n a l , Lt d . -4- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -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: Cary A. Sutherland Laboratory Supervisor DATE MANAGEMENT: and Non-Target Plants BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -5 TABLE OF CONTENTS Title/Cover Page................................................................................... Good Laboratory Practice Compliance Statement............................. Quality Assurance Statement............................................................... Report Approval................................................................................... Table of Contents.................................................................................. Summary............................................................................................... Introduction........................................................................................... Objective............................................................................................... Experimental Design............................................................................. Materials and Methods......................................................................... Results and Discussion......................................................................... Conclusions........................................................................................... References............................................................................................. PR O JE C T NO.: 454A -112 .. 1 ..2 ,,3 ..4 ..5 ,,7 .9 .9 .9 10 15 17 18 TABLES Table 1 - Summary of Analytical Chemistry D ata...................................................................................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 T est.............................................................................................. 23 Table 6 - Mean Areas Under the Growth Curve and Percent Inhibition for Each 24-Hour Interval During the T est...............................................................................................24 Table 7 - Mean Growth Rates and Percent Inhibition for Each 24-Hour Interval During the Test..............................................................................................................................25 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . p r o j e c t n o .: 4 54 A -112 6- TABLE 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 .... 27 Table 10- EC Values Based on Growth Rate Over the 96-Hour Exposure Period................................. 28 Table 11 - Cell Densities During the Recovery Phase................................................................................ 29 FIGURES Figure 1 - Negative control algal growth, expressed as cell density, during the 96-hour exposure period..........................................................................................30 Figure 2 - Concentration-response curve, expressed as cell density, over the 96-hour exposure period.............................................................................................................31 Figure 3 - Recovery-phase response curve, expressed as cell density..................................................... 32 APPENDICES Appendix I - Freshwater Algal Medium with Silica Constituents.......................................................33 Appendix II - Analyses of Pesticides, Organics, Metals and Other Inorganics in Wildlife International, Ltd. Well Water...................................................................... 34 Appendix I I I - The A nalysis o f PFO S in Freshw ater Algal M edium in Support o f 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 VII - Changes to Protocol......................................................................................................... 55 Appendix VIII - Personnel Involved in the Study...................................................................................... 56 W il d l if e In t e r n a t io n a l , Lt d . -7 SUMMARY 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 P R O JE C T NO.: 454A -112 WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF EXPOSURE: 454A-112 PFOS (Perfluorooctanesulfonate, Potassium Salt) IUPAC Name: 1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6, 7,7,8,8,8-heptadecafluoro-potassium salt; CAS #2795-39-3 PFOS: A 96-Hour Toxicity Test with the Freshwater 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 {Navcula pelliculosa) Wildlife International, Ltd. Easton, Maryland 2 1601 W il d l if e In t e r n a t io n a l , Lt d . 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: 8- - SUMMARY (Continued) 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 EC 10: 95% CONFIDENCE LIMITS: 96-HOUR EC50: 95% CONFIDENCE LIMITS: 96-HOUR EC90: 95% CONFIDENCE LIMITS: 72-HOUR NOAEC: 96-HOUR NOAEC: 246 mg a.i./L 210 - 277 mg a.i./L <62.3 mg a.i./L Not Calculable 252 mg a.i./L 220 - 285 mg a.i./L 319 mg a.i./L 308 - 326 mg a.i./L <62.3 mg a.i./L <62.3 mg a.i./L GROWTH RATE: 72-HOUR EC50: 95% CONFIDENCE LIMITS: 295 mg a.i./L 288 - 305 mg a.i./L 96-HOUR EC 10: 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 P R O JE C T NO.: 454A -112 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -9INTRODUCTION p r o j e c t n o .: 4 5 4 A -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 1.0 X 106 cells/mL. The concentration of algal cells in the inoculum was verified and 1.0 mL was added to each test chamber to achieve a nominal concentration of approximately 1.0 X 104 cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . p r o j e c t n o .: 4 5 4 A -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 EC 10 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 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 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 11 (draft)(l). Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International, Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity o f 98.9% and an expiration date o f 2008. The test substance was reanalyzed by the Sponsor and the Certificate of Analysis dated September 7, 2000 indicated a purity of 86.9% and an expiration date of August 31, 2001. The test substance was stored at ambient room temperature. 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 W il d l if e In t e r n a t io n a l , Lt d . p r o j e c t n o .: 4 5 4 A -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 0. 1 using 0.1 N NaOH and 10% HC1, and the medium was sterilized by filtration (0.22 pm) prior to use. Analyses were performed at least once annually to determine the concentrations of selected organic and inorganic constituents in the well water used by Wildlife International, Ltd. The results of the most recent GLP analyses performed to measure the concentrations of selected contaminants in the well water are presented in Appendix II. W il d l if e In t e r n a t io n a l , Lt d . - 12- BACK TO MAIN P R O JE C T N O .: 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 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 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 o f test solution prepared for each treatm ent 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 W il d l if e In t e r n a t io n a l , Lt d . - 13 - BACK TO MAIN p r o j e c t n o .: 4 5 4 A -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 1.0 X 103cells/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: A = ((N,-N0)/2)(t, )+((Ni+N2-2N0)/2)(t2-ti)+... +((Nn.1+Nn-2N,,)/2)(tn-tn,1) where: A = Area N0 = Mean nominal number of cells/mL at t0 Ni = Mean measured number of cells/mL at ti N 2= Mean measured number of cells/mL at t2 Nn = Mean measured number of cells/mL at tn ti = Time of first measurement after beginning of test (hours) t2= Time of second measurement after beginning of test (hours) L = Time of nthmeasurement after beginning of test (hours) Growth rates were calculated for the control and each treatment group using the following formula: lnNn - lnNp tn -to where: p. = Average specific growth rate N0= Mean nominal number of cells/mL at t0 Nn= Mean measured number of cells/mL at tn t0 = Time of beginning of the test (hours) tn = Time of nthmeasurement after beginning of test (hours) W il d l if e In t e r n a t io n a l , Lt d . - 14- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -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: Percent Inhibition = Mean ResponseControi - Mean ResponseTreatment Mean ResponseControi X 100 Cell densities, areas under the growth curve and growth rates were analyzed statistically to estimate the EC10, EC50 and EC90 values (i.e., the theoretical test concentrations that would produce a 10, 50 or 90% reduction in each parameter, respectively) and 95% confidence limits at 72 and 96 hours. EC50 values were also calculated for the 24 and 48-hour time intervals. The EC values and 95% confidence limits were calculated by linear interpolation with treatment response and exposure concentration data using TOXSTAT Version 3.5 (4). Cell densities, areas under the growth curve and growth rates at 72 and 96 hours were evaluated for normality and homogeneity of variances using the Shapiro-Wilk's test and Levene's test, respectively. 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 o f test medium were collected from the negative control and each treatm ent 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 centrifuge 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 III. W il d l if e In t e r n a t io n a l , Lt d . - 15 - BACK TO MAIN p r o j e c t n o .: 4 54 A -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 III. 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 94.8 to 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 24.6C and were within the range established for the test (24 2C). The light intensity ranged from 3910 and 4510 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 o f the abiotic replicate at test term ination w as 7.7, which w as com parable 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. W il d l if e In t e r n a t io n a l , Lt d . - 16- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -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 (p<0.05). 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, 51 and 99%, respectively. Dunnett's test showed that cell density was significantly reduced in the 206, 266 and 335 mg a.i./L treatment groups (p<0.05). 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 (p<0.05) 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./L treatment groups (p<0.05). Consequently, the NOAEC for 72-hour growth rate was 206 mg a.i./L. After 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 (/?<0.05). Consequently, the NOAEC for 96-hour growth rate was 206 mg a.i./L. W il d l if e In t e r n a t io n a l , Lt d . - 17- BACK TO MAIN PR O JE C T NO.: 454A -112 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. 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 (i.e., 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 grow th curve, w as 252 m g a.i./L, with 95% confidence limits o f 220 and 285 mg a.i./L. The 72-hour NOAEC, based on cell density and area under the growth curve, was <62.3 mg a.i./L, the lowest concentration tested. The 96-hour NOAEC, based on area under the growth curve, was <62.3 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. W il d l if e In t e r n a t io n a l , Lt d . - 18 - BACK TO MAIN p r o j e c t n o .: 4 5 4 A -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 1218-90E. 1990. Standard Guide fo r 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. W il d l if e In t e r n a t io n a l , Lt d . - 19- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -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 Concentration1 (mg a.i./L) Mean Measured Concentration (mg a.i./L) Negative Control 61.5 81.3 110 147 198 264 02 723 964 0 72 96 0 72 96 0 72 96 0 72 96 0 72 96 0 72 96 < LOQ < LOQ < LOQ 62.3 63.6 61.1 83.8 84.7 81.0 109 113 110 147 154 149 209 209 199 271 268 258 < LOQ 62.3 83.2 111 150 206 266 Percent of Nominal ,, 101 102 101 102 104 101 347 0 334 72 342 96 329 335 96.5 347 (abiotic) 7212345 341 965 336 339 97.7 1 Limit of Quantitation (LOQ) was 4.39 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 replicates per treatment and control group. 5 72 and 96-hour samples were collected from the additional abiotic replicates. W il d l if e In t e r n a t io n a l , Lt d . - 20- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -112 Table 2 Temperature Measurements Sponsor: Test Substance: Test Organism: Dilution Water: Time (Day) 3M Corporation PFOS Freshwater Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium T em p e ra tu re 1 (C) Measurement 1 Measurement 2 0 23.6 1 24.5 2 24.6 3 24.2 4 24.0 5 23.7 6 23.8 7 23.3 8 23.2 9 23.2 10 23.5 11 23.2 23.9 24.2 24.2 24.1 23.3 23.7 23.4 23.3 23.5 23.1 23.7 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). W il d l if e In t e r n a t io n a l , Lt 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 pelliculosa Freshwater Algal Medium with Silica and Selenium Test Shaker Day Table 1 0 2 No. 1 3910 4500 Light Intensity Measurements1(lux) - No. 2 No. 3 No. 4 4370 4490 4230 4050 4510 3930 1 Light intensity was measured at five locations over each of two shaker tables. No.5 4320 3920 W il d l if e In t e r n a t io n a l , Lt d . -22 - BACK TO MAIN PR O JE C T NO.: 454A -112 Table 4 pH Measurements Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Diatom, Ncivicula pelliculosa Freshwater Algal Medium with Silica and Selenium Mean Measured Concentration (mg a.i./L) 0 Hours1 pH Measurements Negative Control 7.5 96 Hours2 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 8.2 335 339 (abiotic) 7.5 __3 7.7 7.7 1 0-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. 3 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 W il d l if e In t e r n a t io n a l , Lt d . -23 - PRO JECT 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) 24 Hours = Mean Cell Density1 Percent (cells/mL) Inhibition1 48 Hours Mean Cell Density1 (cells/mL) Percent Inhibition1 72 Hours Mean Cell Density1 (cells/mL) Percent Inhibition1 Negative Control 44,333 - 269,667 - 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 4.5 217,000 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 1 Values calculated using SAS 6.12. Manual calculations may differ slightly. * Indicates a significant difference from the negative control using Dunnett's test (p < 0.05). 96 Hours Mean Cell Density1 (cells/mL) Percent Inhibition1 2,726,667 - 2,366,667 13 2,366,667 13 2,373,333 13 2,473,333 9.3 2,093,333* 23 1,330,000* 51 35,333* 99 W il d l if e In t e r n a t io n a l , Lt d . -24- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -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 - 2 4 Hours Mean Area1 Percent Inhibition1 0 - 4 8 Hours Mean Area1 Percent Inhibition1 Negative Control 412,000 - 3,940,000 ~ 62.3 412,000 0.0 3,524,000 11 83.2 304,000 26 3,240,000 18 111 352,000 15 3,316,000 16 150 332,000 19 3,524,000 11 206 388,000 5.8 3,260,000 17 266 196,000 52 1,792,000 55 335 0 100 32,000 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 < 0.05). 0 - 7 2 Hours Mean Area1 Percent Inhibition1 27,296,000 - 19,624,000* 28 20,032,000* 27 19,288,000* 29 21,944,000 20 20,944,000* 23 10,352,000* 62 184,000* 99 0 - 9 6 Hours Mean Area1 Percent Inhibition1 80,136,000 - 61,304,000* 24 62,472,000* 22 61,008,000* 24 67,064,000 16 61,144,000* 24 33,352,000* 58 576,000* 99 W il d l if e In t e r n a t io n a l , Lt d . -25 - BACK TO MAIN PRO JECT 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 - 2 4 Hours - Mean Growth Rate' Percent (cells/mL/hr) Inhibition1 0 - 4 8 Hours Mean Growth Rate' Percent (cells/mL/hr) Inhibition' 0 - 7 2 Hours Mean Growth Rate' (cells/mL/hr) Percent Inhibition1 Negative Control 0.0620 - 0.0686 - 0.0711 - 62.3 0.0618 0.28 0.0657 4.3 0.0653 8.1 83.2 0.0525 15 0.0652 4.9 0.0663 6.7 111 0.0569 8.3 0.0650 5.2 0.0656 7.8 150 0.0544 12 0.0669 2.5 0.0674 5.2 206 0.0600 3.1 0.0640 6.7 0.0672 5.5 266 0.0383 38 0.0518 24 0.0569* 20 335 0.0000 100 0.0086 87 0.0068* 90 1 Values calculated using SAS 6.12. Manual calculations may differ slightly. * Indicates a significant difference from the negative control using Dunnett's test (p < 0.05). 0 - 9 6 Hours Mean Growth Rate' (cells/mL/hr) Percent Inhibition' 0.0584 - 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 W il d l if e In t e r n a t io n a l , Lt d . -26- BACK TO MAIN PRO JECT 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 3M Corporation PFOS Freshwater Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium EC10 (mg a.i./L) 95% Confidence Limits (mg a.i./L) EC50 (mg a.i./L) 24 Hours Not Determined - 281 48 Hours 72 Hours 96 Hours Not Determined <62.3 <62.3 - __1 __1 261 242 263 1Confidence 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 317 322 95% Confidence Limits (mg a.i./L) - - 306 - 326 310-328 W il d l if e In t e r n a t io n a l , Lt d . -27- BACK TO MAIN P R O JE C T N O .: 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: Time 3M Corporation PFOS Freshwater Diatom, Navicula pelliculosa Freshwater Algal Medium with Silica and Selenium EC10 (mg a.i./L) 95% Confidence Limits (mg a.i./L) EC50 (mg a.i./L) 24 Hours Not Determined - 262 48 Hours 72 Hours 96 Hours Not Determined <62.3 <62.3 -- __1 __1 259 246 252 1Confidence 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 318 319 95% Confidence Limits (mg a.i./L) - -- 307 - 325 308 - 326 W il d l if e In t e r n a t io n a l , Lt d . -28 - BACK TO MAIN PR O JE CT 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 3M Corporation PFOS Freshwater Diatom, Navcula pelliculosa Freshwater Algal Medium with Silica and Selenium EOO (mg a.i./L) 95% Confidence Limits (mg a.i./L) 24 Hours Not Determined - 48 Hours Not Determined -- 72 Hours 221 190 - 252 96 Hours 243 209 - 295 1Confidence limits could not be calculated with 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 W il d l if e In t e r n a t io n a l , Lt d . -29- BACK TO MAIN p r o j e c t n o .. 4 5 4 A -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 v 0 lT C d U t dll0 IT (mg a.i./L) DayO2 Cell Densities (cclls/mL) Day 3 Day 6 Day 7 Negative Control 15,000 2,280,000 2,560,000 2,520,000 3351 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. W il d l if e In t e r n a t io n a l , Lt d . -30- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -112 Figure 1. Negative control algal growth, expressed as cell density, during the 96-hour exposure period. W il d l if e In t e r n a t io n a l , Lt d . -31 - BACK TO MAIN p r o j e c t n o .: 4 5 4 A -112 Figure 2. Concentration-response curve, expressed as cell density, over the 96-hour exposure period. Exposure Duration (Hours) Negative Control 62.3 mg a.i./L &--83.2 mg a.i./L K-- 111 mg a.i./L *-- 150 mg a.i./L *--206 mg a.i./L H-- 266 mg a.i./L -- 335 mg a.i./L W il d l if e In t e r n a t io n a l , Lt d . -32- Figure 3. Recovery-phase response curve, expressed as cell density. BACK TO MAIN p r o j e c t n o .: 4 5 4 A -112 W il d l if e In t e r n a t io n a l , Lt d . -33 - BACK TO MAIN p r o j e c t n o .: 4 5 4 A -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 Nominal Compound___________________ Concentration MgCl2*6H20 CaCl2*2H20 H3BO3 MnCl2*4H20 ZnCl2 FeCl3*6H20 CoC12`6H20 Na2M o04*2H20 CuC12*2H20 Na2EDTA*2H20 NaN03 M gS04*7H20 k 2h p o 4 NaHC03 Na2S i03*9H20 N a 2Se0 3 *5H 20 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 mg/L mg/L mg/L mg/L Pg/L mg/L pg/L Pg/L Pg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L ' The pH was adjusted to 7.5 0.1 using 10% HC1 and 0.1 N NaOH. W il d l if e In t e r n a t io n a l , Lt d . - 34- BACK TO MAIN PROJECTNO.: 454A-112 APPENDIX II Analyses of Pesticides, Organics, Metals and Other Inorganics in Wildlife International, Ltd. Well Water1 ANALYSIS Miscellaneous Measurements Total Dissolved Solids Ammonia Nitrogen Total Organic Carbon*2 Total Cyanide MEASURED CONCENTRATION 286 < 0.050 < 1.0 < 10.0 m g/L m g/L m g/L Mg/L Organochlorines and PCBs Aldrin Alpha BHC Beta BHC Delta BHC Gamma BHC (Lindane) Chlordane DDD, pp' DDE, pp' DDT, pp' Dieldrm Endosulfan, A Endosulfan, B Endosulfan Sulfate Endrin Endrin Aldehyde Heptachlor Methoxychlor Heptachlor Epoxide Toxaphene PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 < 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 Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Metals and Other Inorganics Aluminum3 Arsenic3 Beryllium: Cadmium Calcium3 Chromium3 Cobalt3 Copper3 Iron3, Lead3 Magnesium3 Manganese3 Mercury Molybdenum3 Nickel3 Iron3 Selenium3 Silver3 Sodium3 Zinc3 < 100 < 25.0 < 0.50 < 1.0 35.0 < 2.0 < 1.0 < 20.0 < 100 < 10.0 13.5 < 1.0 < 0.20 < 2.0 < 2.0 6.62 < 25.0 < 1.0 21.3 < 20.0 Mg/L Mg/L Mg/L Mg/L m g/L Mg/L Mg/L Mg/L Mg/L Mg/L m g/L Mg/L Mg/L Mg/L Mg/L m g/L Mg/L Mg/L m g/L Mg/L Analyses performed by QST Environmental, Gainesville, Florida for samples collected on November 3 through November 7, 1997. 2 Analyses performed by Wildlife International, Ltd. for the sample collected on November 5, 1997. 3 Analyses performed by Wildlife International, Ltd. for samples collected on November 5 through 7, 1997. W il d l if e In t e r n a t io n a l , Lt d . -35 - BACK TO MAIN p r o j e c t n o .. 454A-112 APPENDIX III THE ANALYSIS OF PFOS IN FRESHWATER ALGAL MEDIUM IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-112 W il d l if e In t e r n a t io n a l , Lt d . -36- BACK TO MAIN p r o j e c t n o .: 4 54 A -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 DATE DA' W il d l if e In t e r n a t io n a l , Lt d . -37- BACK TO MAIN PROJECTNO.: 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: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619 97-2. The standard was stored under ambient conditions. Analytical Method The method used for the analysis o f the freshw ater algal medium sam ples w as developed at W ildlife International, Ltd. and entitled "Analytical Method for the Determination of PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix II of Wildlife International, Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Samples were 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. W il d l if e In t e r n a t io n a l , Lt d . -38- BACK TO MAIN PROJECTNO.: 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 100LC Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil C]8 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 Quantitation Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.0439 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 W il d l if e In t e r n a t io n a l , Lt d . -39- BACK TO MAIN PR O JE C T 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 Area/Intemal 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 (1/x) Peak area ratio - (Y-intercept) PFOS (mg a.i./L) at instrument = Slope x Internal Standard Concentration 0.2946 W il d l if e In t e r n a t io n a l , Lt d . -40- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -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 100 147 147 x 100 = 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 (Navcula 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. W il d l if e In t e r n a t io n a l , Lt d . -41 - BACK TO MAIN p r o j e c t n o .: 4 5 4 A -112 INSTRUMENT: Table 1 Typical HPLC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: INTERNAL STANDARD RETENTION TIME: PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: Keystone Betasil Ci8 column (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 W il d l if e In t e r n a t io n a l , Lt d . -42- BACK TO MAIN p r o j e c t n o .: 4 54 A -112 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis1 Number (454A-112-) MAB-1 Sample Type Matrix Blank Measured Concentration of rrU (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). W il d l if e In t e r n a t io n a l , Lt d . -43 - BACK TO MAIN p r o j e c t n o .: 4 5 4 A -112 Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-112-) MAS-1 MAS-4 MAS-7 Concentrations of PFOS (mg a.i./L) Percent Fortified1 Measured1 Recovered2 17.6 20.9 17.6 20.3 17.6 20.4 119 115 116 MAS-2 MAS-5 MAS-8 176 176 176 192 187 185 109 106 105 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 12 1 Concentrations were corrected for change in test substance purity (98.9% 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. W il d l if e In t e r n a t io n a l , Lt d . - 44- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -112 Table 4 Measured Concentrations of PFOS in Freshwater Algal Medium Samples from a Freshwater Diatom (Navicula pelliculosa) 96-Hour Toxicity Test123 Nominal Test Concentration1 (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-112-) 1 9 18 Sampling Time Pay) 0 3 4 PFOS Measured Concentration1,2 (mg a.i./L) < LOQ < LOQ < LOQ Percent of Nominal' -- -- 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 4 147 100 154 105 149 101 198 6 0 14 3 23 4 209 106 209 106 199 101 264 7 0 15 3 24 4 271 103 268 102 258 97.7 347 8 0 16 3 25 4 334 96.2 342 98.5 329 94.8 347 (Abiotic) 17 26 3 4 341 98.2 336 96.8 1 Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate of Analysis dated September 7, 2000. 2 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). 3 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since nominal and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. W il d l if e In t e r n a t io n a l , Lt d . -45 - BACK TO MAIN P R O JE CT 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. I Centrifuge all samples, as necessary, for approximately five minutes at approximately 1500 rpm. 4 Dilute matrix fortification and test samples into the range of the calibration standards by partially filling Class A volumetric flasks with 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v). Add the appropriate volume of sample and bring the flask to volume with the dilution solvent. Process the matrix blank sample using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. 4 Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in freshwater algal medium. W il d l if e In t e r n a t io n a l , Lt d . -46 - BACK TO MAIN PR O JE C T NO.: 454A -112 Concentration (Ratio) Figure 2. A typical calibration curve for PFOS. Slope = 1.44418; Intercept = 0.32839; r = 0.9975. Curve is weighted (1/x). W il d l if e In t e r n a t io n a l , Lt d . -47 - BACK TO MAIN P R O JE C T NO.: 454A -112 intensity: 8 0 0 0 0 0 cps Figure 3. A representative ion chromatogram of a low-level (0.0439 mg a.i./L) PFOS standard. W il d l if e In t e r n a t io n a l , Lt d . -48 - BACK TO MAIN PR O JE C T NO.: 454A -112 intensity: 8 0 0 0 0 0 cps Figure 4. A representative ion chromatogram of a high-level (0.879 mg a.i./L) PFOS standard. W il d l if e In t e r n a t io n a l , Lt d . -49- BACK TO MAIN P R O J E C T N O : 454A -112 Intensity: 8 0 0 0 0 0 cps Figure 5. A representative ion chromatogram of a matrix blank sample (454A-112-MAB-l). The arrow indicates the retention time of PFOS. W il d l if e In t e r n a t io n a l , Lt d . -50- BACK TO MAIN P R O JE C T N O .: 454A -112 Intensity: 8 0 0 0 0 0 cps Figure 6. A representative ion chromatogram of a matrix fortification sample (454A -112-M AS-l). W il d l if e In t e r n a t io n a l , Lt d . -51 - BACK TO MAIN p r o j e c t n o .: 4 5 4 A -112 intensity: 8 0 0 0 0 0 cps Figure 7. A representative ion chromatogram of a test sample (454A -112-2). W il d l if e In t e r n a t io n a l , Lt d . -52- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -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./L) Replicate 24 Hours Cell Densities (Cells/mL)1 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 41,000 284,000 2,100,000 2,700,000 62.3 A 46,000 271,000 1,120,000 2,200,000 B 38,000 218,000 1,410,000 2,600,000 C 49,000 216,000 850,000 2,300,000 83.2 A 33,000 220,000 1,320,000 2,580,000 B 39,000 251,000 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 31,000 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). W il d l if e In t e r n a t io n a l , Lt d . -53 - BACK TO MAIN p r o j e c t n o .: 4 54 A -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 i/ouceiiirauon (mg a.i./L) Replicate Cumulative Area Under the Growth Curve 0 - 2 4 Hours 0 - 4 8 Hours 0 - 7 2 Hours 0 - 9 6 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 21,312,000 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 61,512,000 B 288,000 3,000,000 18,144,000 60,024,000 C 384,000 3,708,000 21,048,000 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 W il d l if e In t e r n a t io n a l , Lt d . - 54- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -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 Growth Rate (mg a.i./L) Replicate 0 - 2 4 Hours 0 - 4 8 Hours 0 - 7 2 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 0.0510 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 0.0631 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 - 9 6 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 0.0118 0.0082 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -55 - p r o j e c t n o .: 4 5 4 A -112 APPENDIX VII Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to add the proposed experimental start and termination dates, test concentrations and 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 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 o f measurement for some o f 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. W il d l if e In t e r n a t io n a l , Lt d . - 56- BACK TO MAIN p r o j e c t n o .: 4 5 4 A -112 APPENDIX VIII 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