Document mmyJw4kYQXp8pZdgxvBmGV5nZ

PFOS: A 96-HOUR TOXICITY TEST WITH THE MARINE DIATOM (Skeletonema costatum) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-113A 3M LAB REQUEST NO. U2723 U S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.5400 AUTHORS: Debbie Desjardins Cary A. Sutherland Raymond L. Van Hoven, Ph D. Henry O. Krueger, Ph D. STUDY INITIATION DATE: January 28, 2000 STUDY COMPLETION DATE: March 27, 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 of 54 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -2 - PROJECT NO.: 454A-113A GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Marine Diatom (Skeletonema costatum) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-113A STUDY COMPLETION: March 27, 2001 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice, (ENV/MC/CHEM(98)17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exceptions: The test and reference substances were not characterized in accordance with lull GLP compliance prior to its use in the study; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data are being maintained in the 3M archives. The test substance has been recharacterized in accordance with GLP (September 7, 2000). The stability of the test and reference substances under conditions of storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Cary A. Sutherland Laboratory Supervisor SPONSOR: DATE BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -3 - p r o j e c t n o .: 454a - i i 3a 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 Initial Trial 454A-113 Test Substance Preparation March 2, 2000 March 2, 2000 March 2, 2000 Matrix Fortifications March 3, 2000 March 6, 2000 March 14, 2000 Definitive Trial 454A-113A Analytical Data and Draft Report July 11 & 12, 2000 July 12, 2000 July 14, 2000 Biological Data and Draft Report July 17 & 18, 2000 July 18, 2000 August 28, 2000 Analytical Data and Second Draft Report March 12, 2001 March 12, 2001 March 13, 2001 Biological Data and Second Draft Report Final Report March 26, 2001 March 27, 2001 March 26, 2001 March 27, 2001 March 27, 2001 March 27, 2001 Quality Assurance Program Supervisor BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -4 - p r o j e c t n o .: 454a - i i 3a REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Marine Diatom (Skeletonema costatum) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER. 454A-113A STUDY DIRECTOR: Cary A. Sutherland Laboratory Supervisor DATE MANAGEMENT: HcnrV O.vXrueger, Director, Aquatic Toxicology and Non-Target Plants DATI BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -5 - p r o j e c t n o .: 454a - i i 3a TABLE OF CONTENTS Title/Cover Page................................................................................................................................................1 Good Laboratory Practice Compliance Statement.........................................................................................2 Quality Assurance Statement........................................................................................................................... 3 Report Approval............................................................................................................................................... 4 Table of Contents.............................................................................................................................................. 5 Summary............................................................................................................................................................7 Introduction....................................................................................................................................................... 9 Objective............................................................................................................................................................9 Experimental Design.........................................................................................................................................9 Materials and Methods....................................................................................................................................10 Results and Discussion....................................................................................................................................14 Conclusions..................................................................................................................................................... 16 References........................................................................................................................................................17 TABLES T ab le 1 - S u m m ary o f A naly tical C hem istry D a t a .............................................................................................. 18 Table 2 - Temperature Measurements.......................................................................................................19 Table 3 - Light Intensity Measurements....................................................................................................20 Table 4 - pH Measurements.......................................................................................................................21 Table 5 - Mean Cell Densities and Percent Inhibition forEach 24-Hour Interval During the Test............................................................................................................................ 22 Table 6 - Mean Areas Under the Growth Curve andPercentInhibition for Each 24-Hour Interval During the T est.............................................................................................. 23 Table 7 - Mean Growth Rates and Percent Inhibition for Each 24-Hour Interval During the Test 24 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . 6- - p r o j e c t n o .: 454a - i d a TABLE OF CONTENTS -Continued- Table 8 - EC Values Based on Cell Density Over the 96-Hour Exposure Period................................. 25 Table 9 - EC Values Based on Area Under the Growth Curve Over the 96-Hour Exposure Period.......................................................................................................................... 26 Table 10- EC Values Based on Growth Rate Over the 96-Hour Exposure Period................................. 27 FIGURES Figure 1 - Negative control algal growth, expressed as cell density, during the 96-hour exposure period........................................................................................................................... 28 Figure 2 - Treatment response, expressed as cell density, over the 96-hour exposure period............... 29 APPENDICES Appendix I - Saltwater Algal Medium Constituents.............................................................................30 Appendix II - Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well W ater......................................................................................................................... 31 Appendix III- The Analysis of PFOS in Saltwater Algal Medium in Support of Wildlife International, Ltd. Project No.: 454A-113A.................................................... 33 Appendix IV - Cell Density for Each Replicate Per Treatment Over the 96-Hour Exposure Period................................................................................................................. 50 Appendix V - Area Under the Growth Curve for Each Replicate Per Treatment Over the 96-Hour Exposure Period.................................................................................. 51 Appendix VI - Growth Rate for Each Replicate Per Treatment Over the 96-Hour Exposure Period................................................................................................................. 52 Appendix VII - Changes to Protocol............................................................................................................53 Appendix VIII - Personnel Involved in the Study......................................................................................... 54 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -7 - PROJECT NO.: 454A-113A SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: SUMMARY 3M Corporation Rochelle R. Robideau Wildlife International, Ltd. Easton, MD 21601 WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF EXPOSURE: 454A-113A 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-heptadecafluoropotassium salt; CAS #2795-39-3 PFOS: A 96-Hour Toxicity Test with the Marine Diatom (Skeletonema costatum) Negative Control and 3.46 mg a.i./L Negative Control and 3.20 mg a.i./L Experimental Start (OECD) - March 2, 2000 Experimental Start (EPA) - May 19, 2000 Exposure Termination - May 23, 2000 Experimental Termination - May 23, 2000 96 Hours TEST ORGANISM: SOURCE OF TEST ORGANISMS: Marine Diatom (Skeletonema costatum) Wildlife International, Ltd. Easton, Maryland 21601 W il d l if e In t e r n a t io n a l , Lt d . 8- - SUMMARY (Continued) CELL DENSITY: 72-H O U R EC50: 9 5 % CONFIDENCE LIMITS: > 3.20 mg a.i./L Not calculable 96-H O U R EC 10, EC50 and EC90: 9 5 % CONFIDENCE LIMITS: > 3.20 mg a.i./L Not calculable 72-HOUR NOAEC: 96-HOUR NOAEC: 3.20 mg a.i./L 3.20 mg a.i./L AREA UNDER THE GROWTH CURVE: 72-H O U R EC50: 9 5 % CONFIDENCE LIMITS: 96-H O U R EC10, EC50 and EC90 : 9 5 % CONFIDENCE LIMITS: > 3.20 mg a.i./L Not calculable > 3.20 mg a.i./L Not calculable 72-HOUR NOAEC: 96-HOUR NOAEC: 3.20 mg a.i./L 3.20 mg a.i./L GROWTH RATE: 72-H O U R EC50: 9 5 % CONFIDENCE LIMITS: 9 6 -H O U R EC 10, EC50 and EC90: 9 5 % CONFIDENCE LIMITS: > 3.20 mg a.i./L Not calculable > 3.20 mg a.i./L Not calculable 72-HOUR NOAEC: 96-HOUR NOAEC: 3.20 mg a.i./L 3.20 mg a.i./L All values are mean measured test concentrations. BACK TO MAIN PROJECT NO.: 454A-113A BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -9 - p r o j e c t n o .: 454A -113A INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. An initial trial of the test was conducted from March 3, 2000 to March 7, 2000. However, the test was repeated due to low analytical recoveries in the first trial. The in-life phase of the definitive test was conducted from May 19, 2000 to May 23, 2000. Raw data generated by Wildlife International, Ltd and a copy of the final report are filed under Project Number 454A-113A 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 marine diatom, Skeletonema costatum, during a 96-hour exposure period. EXPERIMENTAL DESIGN The marine diatom, Skeletonema costatum, was exposed to a single test concentration and a negative (culture medium) control under static conditions for 96 hours. Six replicate test chambers were maintained in the treatment and control groups. 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 maintained in the treatment group to determine the stability of the test substance under the conditions of administration. The nominal test concentration was selected in consultation with the Sponsor and was based upon the results of range finding tests and the approximate solubility of PFOS in saltwater algal medium. The nominal test concentration was 3.46 mg active ingredient (a.i.)/L. The mean measured test concentration was determined from samples of test medium collected from the treatment and control groups at test initiation, at approximately 72 hours, and at test termination. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -10- p r o j e c t n o .: 454A -113A At test initiation an inoculum of the algal cells was added to each test chamber to achieve an initial nominal concentration of approximately 77,000 cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell densities. Cell densities were measured for each replicate and were used to calculate areas under the growth curve and growth rates. Percent inhibition values relative to the control were calculated for each parameter over the 96-hour exposure period. EC10, EC50 and EC90 values could not be calculated using statistical methods because observed effects were below these levels. Because there were no statistically significant effects at the test concentration (3.20 mg a.i./L) EC10, EC50 and EC90 values can be taken as an estimate of the no observed-adverse-effect-concentration (NOAEC). The no-observed-adverse-effect-concentration (NOAEC) was determined based upon statistical evaluation of the 72-hour and 96-hour results. MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: A 96-Hour Toxicity Test with the Marine Diatom (Skeletonema costatum)". 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 expiration date of August 31, 2001. The test substance was stored at ambient room temperature. Preparation of Test Concentrations The nominal test concentration was 3.46 mg a.i./L, based on a test substance purity of 86.9%. A single test solution was prepared in saltwater algal medium. The test solution was sonicated for approximately 30 minutes and was stirred with a magnetic stir plate for approximately 43 hours to aid in the solubilization of the test substance. The test solution appeared clear and colorless. W il d l if e In t e r n a t io n a l , Lt d . - li - BACK TO MAIN PROJECT NO.: 454A-113A Test Organism The marine diatom, Skeletonema costatum, was selected as the test species for this study. The species is representative of an important group of algae, and was selected for use in the test based upon a past history of use and ease of culturing in the laboratory. Original algal cultures were obtained from the Culture Collection of Algae and Protozoa, Dunstaffnage Marine Laboratory at Oban Argyll, Scotland, 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 saltwater algal medium (2). Stock nutrient solutions were prepared by adding reagent-grade chemicals to Wildlife International, Ltd. well water purified by reverse osmosis. The test medium was prepared by adding appropriate volumes of the stock nutrient solutions to artificial saltwater (Appendix I). The pH of the medium was 8.1, 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. Test Apparatus Test chambers were sterile, 250-mL glass Erlenmeyer flasks plugged with foam stoppers, and contained 100 mL of test or control medium. The test chambers were labeled with the project number, concentration and replicate, and were indiscriminately positioned daily on a mechanical shaker table in an environmental chamber designed to maintain the desired test temperature throughout the test. The test chambers were shaken continuously at approximately 100 rpm. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 12 - p r o j e c t n o .: 454A -113A Environmental Conditions Test flasks were held in an environmental chamber at a temperature of 20 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 a photoperiod of 14 hours of cool-white fluorescent lighting at a target light intensity of 4300 430 lux, and 10 hours of darkness. Light intensity was measured at the four comers and the middle of the shaker table at test initiation using a SPER Scientific Model 840006 light meter. The pH of the medium prepared for the treatment and control groups 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 the treatment and control groups. 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 six "biological" replicates per treatment and control group at 24-hour intervals during the 96-hour exposure, and were held for a maximum of two days under refrigerated conditions sufficient to inhibit growth until cell counts could be performed. Cell counts were conducted using a hemacytometer and microscope. Each sample was diluted using an electrolyte solution (Isoton), as needed, to maintain counting accuracy. A small amount of each sample was loaded onto a hemacytometer and 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. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 13 - PROJECT NO.: 454A-113A Statistical Analyses Cell densities, areas under the growth curve, growth rates and percent inhibition were calculated using "The SAS System for Windows", Release 6.12 (3). Area under the growth curve was calculated for each replicate of the control and treatment groups using the following formula: A = ((Nr N0)/2)(t1)+((N1+N2-2N0)/2)(t2-t1)+ ...+((Nn.,+Nn-2N0)/2)(tn-tn.1) where: A = Area N0= Nominal number of cells/mL at t0 Nj = Measured number of cells/mL at ti N2= Measured number of cells/mL at t2 Nn = Measured number of cells/mL at tn ti = Time of first measurement after beginning of test (hours) t2= Time of second measurement after beginning of test (hours) tn = Time of nthmeasurement after beginning of test (hours) Growth rates were calculated for each replicate of the control and treatment groups using the following formula: lnNn - lnNp where: p = Average specific growth rate N0 = Nominal number of cells/mL at t0 Nn = Measured number of cells/mL at tn t0 = Time of beginning of the test (hours) tn = Time of nthmeasurement after beginning of test (hours) Percent inhibition was calculated for the treatment group as the percent reduction in mean cell density, area under the growth curve and growth rate relative to the control. The following formula was used: Mean Responsecontroi - Mean Responseireatment Percent Inhibition = Mean ResponseControi X 100 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 for cell BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 14- p r o j e c t n o .: 454A -113A densities, area under the growth curve and growth rates could not be calculated using statistical methods. Cell densities, areas under the growth curve and growth rates at 72 and 96 hours were evaluated for normality using the Shapiro-Wilk's test and for equality of variance using an F-test. Since the data was normally distributed with equal variances, the treatment group was compared to the control group using analysis of variance (ANOVA) and a 2-sample t-test. Results of the statistical analyses were used to determine the NOAEC values at 72 and 96 hours. Analytical Chemistry Samples of test medium were collected from the negative control and the 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 the treatment and control groups. 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 control group. The 3.46 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 glass scintillation vials and were analyzed immediately without storage. The 72 and 96-hour samples were centrifuged approximately 10 minutes at approximately 2000 rpm prior to analysis. Analytical procedures used in the analysis of the samples are presented in Appendix III. 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. The nominal concentration used in this study was 3.46 mg a.i./L. The sample collected at the beginning of the test had a measured concentration that was 94.2% of nominal. Samples collected at 72 hours and at test termination had recoveries of 92.2 and 91.2% of nominal, respectively. The abiotic replicates from the 3.46 mg a.i./L treatment had recoveries of 97.1 and 86.8% 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 concentration BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 15 - p r o j e c t n o .: 454A -113A was 3.20 mg a.i./L, representing 92.5% of the nominal concentration. The mean measured test concentration was used in the determination 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 20.2 to 21.4C and were within the range established for the test (20 2C). The light intensity ranged from 3880 to 4710 lux and was within the desired range for the test (approximately 3870 to 4730 lux). Measurements of pH were 8.0 on Day 0 and 8.4 on Day 4. The effect of PFOS upon Skeletonema costatum was determined by evaluating differences in cell densities, areas under the growth curve and growth rates. Mean values for each parameter were used to calculate growth inhibition for each 24-hour period. Mean cell densities, areas under the growth curve and growth rates, and the corresponding percent inhibition, are presented in Tables 5, 6 and 7, respectively. Cell density, area under the growth curve and growth rate for each individual replicate are presented in Appendices IV, V and VI, respectively, while cell densities are illustrated graphically in Figures 1 and 2. EC values and 95% confidence limits calculated for each 24-hour interval based on cell density, area under the growth curve and growth rate are presented in Tables 8, 9 and 10, respectively. Changes in cell density indicated that exponential growth occurred in the negative control replicates (Figure 1). At 72 and 96 hours, growth in the 3.20 mg a.i./L treatment group was comparable to growth in the control group. After 72 hours of exposure, percent inhibition in the 3.20 mg a.i./L treatment group in relation to cell density, area under the growth curve and growth rate was -16, -4.8 and -4.6%, respectively. After 96 hours, percent inhibition for the three parameters was -4.8, -7.3 and -1.3%, respectively. There were no significant differences (p>0.05) in cell density, area under the growth curve or growth rate between the 3.20 mg a.i./L treatment group and the control group at 72 and 96 hours. Consequently, the NOAEC at 72 and 96 hours was 3.20 mg a.i./L. Visual and Microscopic Observations After 96 hours of exposure, there were no noticeable changes in cell morphology, nor were there signs of aggregation (clumping) or adherence of the algae to the test flasks in the 3.20 mg a.i./L treatment BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 16- p r o j e c t n o .: 454A-113A group. After 96 hours of exposure, adherence of cells to the test chamber was observed in the negative control group. However, this was not observed in the PFOS treatment group. Reversibility of Growth Inhibition After 96 hours of exposure, there was no significant inhibition of growth in the 3.20 mg a.i./L treatment group, the single concentration tested. Therefore, a recovery phase was not conducted. CONCLUSIONS A single concentration of PFOS was tested at the approximate solubility of the test substance in saltwater algal medium. The 72 and 96-hour EC50, based on cell density, area under the growth curve and growth rate, was > 3.20 mg a.i./L, the single concentration tested. Since there was no significant inhibition of growth in the treatment group during the test, the 72 and 96-hour NOAEC was 3.20 mg a.i./L. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 17 - p r o j e c t n o .: 454A -113A REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines {draft), OPPTS Number 850.5400: Algal Toxicity, Tiers I and II. 2 ASTM Standard Guide 1218-90E. 1990. Standard Guide for Conducting Static 96-Hour Toxicity Tests with Microalgae. American Society for Testing and Materials. Philadelphia, Pennsylvania. 3 The SAS System for Windows. 1996. Release 6.12, TS Level 0020. SAS Institute Inc., Cary, North Carolina. 4 West, Inc. and D.D. Gulley. TOXSTAT Version 3.5. Copyright 1996. Western EcoSystems Technology, Inc., Cheyenne, Wyoming. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 18 - p r o j e c t n o .: 454A-113A Table 1 Summary of Analytical Chemistry Data Sponsor: Test Substance: Test Organism: Dilution Water: Nominal Concentration (mg a.i./L) 3M Corporation PFOS Marine Diatom, Skeletonema costatimi Saltwater Algal Medium Sampling Time (Hours) Measured Concentration1 (mg a.i./L) Negative Control 02 723 964 < LOQ < LOQ < LOQ Mean Measured Concentration (mg a.i./L) < LOQ Percent of Nominal __ 3.46 0 3.26 72 3.19 96 3.15 3.20 92.5 3.46 (abiotic) 72123*5 3.36 965 3.00 3.18 91.9 1 Limit of Quantitation (LOQ) was 0.480 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. 96-hour samples were composites of test solution collected from each of the six replicates per treatment and control group. 5 72 and 96-hour samples were collected from the additional abiotic replicates. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 19 - p r o j e c t n o .: 454A-113A Table 2 Temperature Measurements Sponsor: Test Substance: Test Organism: Dilution Water: Time (Day) 3M Corporation PFOS Marine Diatom, Skeletonema costatum Saltwater Algal Medium T em p e ra tu re 1 (C) Measurement 1 Measurement 2 0 21.2 1 20.4 2 21.3 3 20.2 4 20.3 21.1 21.4 20.8 21.3 21.2 1 Temperature Measurement 2 was taken at least 4 hours after Measurement 1, with the exception of test termination (Day 4). BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 20- p r o j e c t n o .: 454a - i i 3a Table 3 Light Intensity Measurements Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Marine Diatom, Skeletonema costatum Saltwater Algal Medium Test Day No. 1 Light Intensity Measurements1(lux) No. 2 No. 3 No. 4 0 3970 3880 4710 4450 No.5 4130 1 Light intensity was measured at five locations surrounding the test flasks on the shaker table. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -21 - p r o j e c t n o .: 454A -ii3A Table 4 pH Measurements Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Marine Diatom, Skeletonema costatum Saltwater Algal Medium Mean Measured Concentration (mg a.i./L) DayO1 pH Measurements Negative Control 8.0 Day 42 8.4 3.20 8.0 8.4 1 Day 0 samples were collected from the batches of test solution prepared for the treatment and control groups at test initiation. 2 Day 4 samples were collected from the pooled replicates per treatment and control group. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -22 - PROJECT NO.: 454A-113A 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 Marine Diatom, Skeletonema costatum Saltwater Algal Medium 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 Density1,2 (cells/mL) Percent Inhibition1 Negative Control 297,333 - 1,064,167 - 1,848,333 - 3.20 275,167 7.5 1,041,667 2.1 2,140,000 -16 1 Values calculated using SAS 6.12. Manual calculations may differ slightly. 2 The treatment group response was not significantly different from the negative control using an F-test (p < 0.05). 96 Hours Mean Cell Density1'2 (cells/mL) Percent Inhibition1 2,481,667 - 2,601,667 -4.8 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -23 - PROJECT NO.. 454A-113A 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 Marine Diatom, Skeletonema costatum Saltwater Algal Medium Mean Measured Concentration (mg a.i./L) 0 - 2 4 Hours Mean Area1 Percent Inhibition1 0 - 4 8 Hours Mean Area1 Percent Inhibition1 0 - 7 2 Hours Mean Area1,2 Percent Inhibition1 Negative Control 2,644,000 - 17,134,000 - 50,236,000 - 3.20 2,378,000 10 16,332,000 4.7 52,664,000 -4.8 1 Values calculated using SAS 6.12. Manual calculations may differ slightly. 2 The treatment group response was not significantly different from the negative control using an F-test (p < 0.05). 0 - 96 Hours Mean Area1,2 Percent Inhibition1 100,348,000 - 107,716,000 -7.3 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -24- PROJECT NO.: 454A-113A 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 Marine Diatom, Skeletonema costatum Saltwater Algal Medium Mean Measured Concentration (mg a.i./L) 0 - 2 4 Hours 0 - 4 8 Hours _________________ _____________________ _____________ Mean Growth Mean Growth Rate1 Percent Rate1 Percent (cells/mL/hr) Inhibition1 (cells/mL/hr) Inhibition1 0 - 7 2 Hours Mean Growth Rate1,2 (cells/mL/hr) Percent Inhibition1 Negative Control 0.0561 - 0.0546 - 0.0441 -- 3.20 0.0530 5.5 0.0542 0.84 0.0461 -4.6 1 Values calculated using SAS 6.12. Manual calculations may differ slightly. 2 The treatment group response was not significantly different from the negative control using an F-test (p < 0.05). 0 - 9 6 Hours Mean Growth Rate1,2 (cells/mL/hr) Percent Inhibition1 0.0362 0.0366 -1.3 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -25 - PROJECT NO.: 454A-113A 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 Marine Diatom, Skeletonema costatum Saltwater Algal Medium EC10 (mg a.i./L) 95% Confidence Limits (mg a.i./L) 24 Hours Not Determined - 48 Hours Not Determined - 72 Hours > 3.20 __1 96 Hours >3.20 __1 1Confidence limits could not be calculated with the data obtained. EC50 (mg a.i./L) >3.20 >3.20 >3.20 >3.20 95% Confidence Limits (mg a.i./L) __1 __1 __1 EC90 (mg a.i./L) Not Determined Not Determined >3.20 >3.20 95% Confidence Limits (mg a.i./L) - __1 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -26- PROJECTNO.: 454A-113A 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 Marine Diatom, Skeletonema costatum Saltwater Algal Medium EC10 (mg a.i./L) 95% Confidence Limits (mg a.i./L) 24 Hours Not Determined - 48 Hours Not Determined ~ 72 Hours 96 Hours >3.20 >3.20 __1 1Confidence limits could not be calculated with the data obtained. EC50 (mg a.i./L) >3.20 >3.20 >3.20 > 3.20 95% Confidence Limits (mg a.i./L) --1 __1 EC90 (mg a.i./L) Not Determined Not Determined >3.20 >3.20 95% Confidence Limits (mg a.i./L) - -__1 __1 BACK TO MAIN W il d l if e In t e r n a t io n a l , L t d . -27 - PROJECT NO.: 454A-113A 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 Marine Diatom, Skeletonema costatum Saltwater Algal Medium EC10 (mg a.i./L) 95% Confidence Limits (mg a.i./L) 24 Hours Not Determined - 48 Hours 72 Hours 96 Hours Not Determined >3.20 >3.20 --1 __1 1Confidence limits could not be calculated with the data obtained. EC50 (mg a.i./L) >3.20 >3.20 >3.20 >3.20 95% Confidence Limits (mg a.i./L) --1 __1 --1 EC90 (mg a.i./L) Not Determined Not Determined >3.20 >3.20 95% Confidence Limits (mg a.i./L) - __1 __1 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -28 - p r o j e c t n o .: 454A -113A Figure 1. Negative control algal growth, expressed as cell density, during the 96-hour exposure period. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -29 - PROJECT NO.: 454A-113A Figure 2. Treatment response, expressed as cell density, over the 96-hour exposure period. Exposure Duration (Hours) BACK TO MAIN W il d l if e In t e r n a t io n a l , L t d . -30- p r o j e c t n o .: 454A -ii3A APPENDIX I Saltwater Algal Medium Constituents12 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Marine Diatom, Skeletonema costatum Saltwater Algal Medium Component1 Nominal Concentration2 FeCl3*6H20 MnCl2*4H20 Z n S 0 4 *7H20 CuS 0 4*5H20 CoC12*6H20 H3BO3 N a 2 EDTA*2H20 k 3p o 4 NaN03 Na2S i0 3*9H20 Thiamine Hydrochloride Biotin B 12 0.72 mg/L 2.16 mg/L 0.675 mg/L 2.36 pg/L 6.06 pg/L 17.1 mg/L 15.0 mg/L 3.0 mg/L 50.0 mg/L 20.0 mg/L 0.25 mg/L 0.05 pg/L 0.5 pg/L 1 The components were added to instant saltwater at a salinity of approximately 30 parts per thousand. 2 The pH of the medium was 8.1. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -31 - PROJECT NO.: 454A-113A APPENDIX II Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Component Aclonifen Alachlor Ametryn Atrazin Azinphos-ethyl Azinphos-methyl Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophos-ethyl Broompropylaat Bupirimaat Carbaryl Carbofuran Carboxin Chlorfenvinphos Chloridazon Chlorpropham Chlorpyriphos Chlorpyriphos-methyl Chlorthalonil Coumaphos Cyanazin Cyfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-o Desethylatrazin Desi sopropylatrazin Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid Measured Concentration Component Pesticides and Organics <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.08 pg/L <0.25 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.25 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.03 pg/L <0.02 pg/L <0.01 pg/L <0.03 pg/L Dimethomorf Disulfoton DMST Dodemorf Endosulfan-a Endosulfan- Endosulfan-sulfaat Epoxiconazole Eptam Esfenvaleraat Ethion Ethofumesaat Ethoprophos Etridiazole Etrimphos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropimorf Fenthion Fenvaleraat Fluazifop-butyl Fluoroglycofen-ethyl Fluroxypyr-meptyl Flutolanil Fonophos Furaiaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane Malathion Metalaxyl Metamitron Metazachlor Methidathion Measured Concentration <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.03 pg/L <0.03 pg/L <0.05 pg/L <0.25 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L `Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999. Continued BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 32- PROJECT NO.: 454A-113A APPENDIX II Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Page 2 Component Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalon Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazin Propham Propiconazool Propoxur Propyzamide Prosulfocarb Pyrazophos Pesticides And Organics (Page 2) Measured Concentration <0.01 pg/L <0.25 pg/L <0.02 pg/L <0.03 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.03 pg/L Component Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifenox-2 Pyrimethanil Quizalofop-ethyl Simazin Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbutylazin Tetrachlorvinphos Tetrahydroftaalimide Tetramethrin Thiabendazole Thiometon Tolclophos-methyl Tolylfluanid Triadimefon Triadimenol T ria lla at Triazophos Trifluralin Vamidothion Vinchlozolin Measured Concentration <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.05 pg/L <0.04 pg/L <0.01 pg/L <0.04 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 11.0 mg/L 18.0 mg/L 29 mg/L <0.015 mg/L 1.1 mg/L <0.02 mg/L <0.1 pg/L <0.2 pg/L <0.5 pg/L <0.2 pg/L Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium <1.1 pg/L <0.7 pg/L <0.25 pg/L <0.3 pg/L <0.2 pg/L <0.1 pg/L <0.5 pg/L <0.025 pg/L <0.5 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999. W il d l if e In t e r n a t io n a l , Lt d . -33 APPENDIX III BACK TO MAIN p r o j e c t n o .: 4 5 4 A -H 3 A THE ANALYSIS OF PFOS IN SALTWATER ALGAL MEDIUM IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-113A BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 34- projectno.: 454A - ii3A REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Marine Diatom (Skeletonema costatum) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-113A MANAGEMENT: Director, Analytical Chemistry BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -35 - projectno.: 454a -h 3a Introduction Saltwater algal medium samples were collected from a 96-hour toxicity test designed to determine the effects of PFOS (Perfluorooctanesulfonate, Potassium Salt) to the marine diatom (Skeletonema costatum). This study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A-113A. The analyses of these water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis on May 19, May 22, and 23, 2000 and were analyzed on each sample receipt day. Test Substance and Internal Standard The test substance used for this study was Wildlife International, Ltd. identification number 4675. The test substance was used to prepare calibration and matrix fortification samples. The internal standard was received from 3M Corporation on July 2, 1998 and was assigned Wildlife International, Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The standard (hereafter referred to as 4H PFOS) was stored under ambient conditions. Analytical Method The method used for the analysis of the saltwater algal medium samples was developed at Wildlife International, Ltd. and entitled "Analytical Method for the Determination of PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix II of Wildlife International, Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Samples were 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. Concentrations of the PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil C]8 analytical column (50 mm x 2 mm I.D., 3-pm particle size). The instmment parameters are summarized in Table 1. A method flowchart is provided in Figure 1. W il d l if e In t e r n a t io n a l , Lt d . - 36- BACK TO MAIN p r o j e c t n o .: 454a - i i 3a 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 0.00480 to 0.0480 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 0.480 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.00480 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. Saltwater algal medium was fortified at 2.40 and 4.80 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected for the mean procedural recovery of 88.5%. A representative ion chromatogram of a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-113A-4, nominal concentration of 3.46 mg a.i./L in saltwater algal medium. Peak Area Ratio = Analyte Peak Area/Internal Standard Peak Area Concentration Ratio = Concentration of Analyte/Concentration of Internal Standard Internal Standard Concentration: 0.100 mg/L BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 37- p r o j e c t n o .. 454A-113A Initial Volume: 0.100 mL Final Volume: 10.0 mL Dilution Factor: 100 PFOS Peak Area: 1242173 Internal Standard Peak Area: 2202273 Peak Area Ratio: 0.5640 Calibration curve equation. Slope: 1.6984 Intercept: 0.0227 Curve is weighted (1/x) _ Peak area ratio - (Y-intercept) PFOS (mg a.i./L) at Instrument Slope x Internal Standard Concentration 0 . 5 6 4 0 - 0 .0 2 27 1.6984 x 0.100 = 0.0319 PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at Instrument x Dilution Factor = 0.0319 x 100 = 3.19 PFOS (mg a.i./L) in sample Percent of Nominal Concentration = PFOS (mg a.i./L) nominal x 100 3,19 3.46 x 100 = 92.2% RESULTS Sample Analysis Saltwater algal medium samples were collected from the 96-hour toxicity test with the marine diatom (,Skeletonema costatum) at test initiation, May 19, 2000 (Day 0), on May 22, 2000 (Day 3) and at test termination, BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -38 - p r o j e c t n o .: 454A -113A May 23, 2000 (Day 4). The measured concentration of PFOS in the sample collected at initiation of exposure of the test organisms (Hour 0) was 94.2% of the nominal concentration. Samples collected at Day 3 had a measured concentration range of 92.2 to 97.1% of nominal values. Samples collected at test termination (Day 4) had a measured concentration range of 86.8 to 91.2% of nominal values (Table 4). Samples from the abiotic 3.46 mg a.i./L treatment group were comparable to samples from the 3.46 mg a.i./L treatment group with the marine diatom present (Table 4). A representative ion chromatogram of a test sample is shown in Figure 7. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -39- p r o j e c t n o .: 454a - i i 3a INSTRUMENT: Table 1 Typical HPLC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: Keystone Betasil C!8 column (50 mm x 2 mm I.D., 3-pm particle size) 30C 5.00 minutes 220 pL/minute 70.0% Methanol : 30.0% NANOpure Water containing 0.1% Formic Acid 5.0 pL Approximately 3.5 minutes INTERNAL STANDARD RETENTION TIME: PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: Approximately 2.3 minutes 498.6 amu 426.7 amu BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -40 - PROJECT NO.: 454A-113A Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-113A-) MAB-1 Sample Type Matrix Blank Measured Concentration of PFOS1 (mg a.i./L) < LOQ MAB-2 Matrix Blank < LOQ MAB-3 Matrix Blank < LOQ 1 The limit of quantitation (LOQ) was 0.480 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.00480 mg a.i./L) and the dilution factor of the matrix blank samples (100).____________________ BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -41 - PROJECT NO.: 454A-113A Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-113A-) MAS-1 MAS-3 MAS-5 Concentrations of PFOS ____________________(mg a.i./L)____________________Percent Fortified1*2 Measured1 Recovered' 2.40 2.02 84.3 2.40 2.21 2.40 2.05 92.1 85.4 MAS-2 MAS-4 MAS-6 4.80 4.80 4.80 4.34 4.52 4.05 90.4 94.2 84.3 Mean = 88.5 Standard Deviation =4.33 CV =4.90% __________________________ N =6 1 Concentrations were corrected for change in test substance purity (90.49% to 86.9%) per Certificate of Analysis dated September 7, 2000. 2 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since fortified and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes.________________________________________________________________________________ BACK TO MAIN W il d l if e In t e r n a t io n a l , L t d . -42- PROJECTNO.: 454A-113A Table 4 Measured Concentrations of PFOS in Saltwater Algal Medium Samples from a Marine Diatom 96-hour Toxicity Test Nominal Test Concentration1 (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-113A-) 1 3 6 Sampling Time (Day) 0 3 4 PFOS Measured Concentration1,2 (mg a.i./L) < LOQ < LOQ < LOQ Percent of Nominal3 -- ~ - 3.46 2 0 43 74 3.26 94.2 3.19 92.2 3.15 91.2 3.46 5 3 3.36 97.1 (Abiotic) 8 4 3.00 86.8 1 Concentrations were corrected for change in test substance purity (90.49% to 86.9%) per Certificate of Analysis dated September 7, 2000. 2 The limit of quantitation (LOQ) was 0.480 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.00480 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 . -43 - BACK TO MAIN p r o j e c t n o .: 454a - i i 3a METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN SALTWATER ALGAL MEDIUM Prepare matrix fortification samples by spiking the requisite volume of PFOS stock solutions directly into saltwater algal medium using gas-tight syringes and Class A volumetric flasks. 4 Centrifuge all samples, as necessary, for approximately ten minutes at approximately 2000 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. F ig ure 1. Analytical method flowchart for the analysis of PFOS in saltwater algal medium. 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 .: 454A -ii3A Concentration (Ratio) F ig u re 2. A typical calibration curve for PFOS. Slope = 1.6984; Intercept = 0.0227; r = 0.9981. Curve is weighted (1/x). W il d l if e In t e r n a t io n a l , L t d . -45 - BACK TO MAIN PROJECT NO.: 454A-113A intensity: 150000 cps Figure 3. A representative ion chromatogram of a low-level (0.00480 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 . - 46- BACK TO MAIN PROJECT NO.: 454A-113A intensity: 150000 cps Figure 4. A representative ion chromatogram of a high-level (0.0480 mg a.i./L) PFOS standard. W il d l if e In t e r n a t io n a l , L t d . -47 - BACK TO MAIN p r o j e c t n o .: 454a - i i 3a intensity: 1 5 000 0 cps Figure 5. A representative ion chromatogram of a matrix blank sample (454A-113A-MAB-2). 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 . -48 - BACK TO MAIN PROJECT NO.: 454A-113A intensity: 150000 cps Figure 6. A representative ion chromatogram of a matrix fortification sample (454A-113A-MAS-4). W il d l if e In t e r n a t io n a l , Lt d . -49- BACK TO MAIN PROJECT NO.. 454A-113A intensity: 150000 cps Figure 7. A representative ion chromatogram of a test sample (454A-113A-4). BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -50- p r o j e c t n o .: 454a - i d a 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 Marine Diatom, Skeletonema costatum Saltwater Algal Medium Mean Measured Concentration (mg a.i./L) Replicate 24 Hours Cell Densities (Cells/mL)1 48 Hours 72 Hours 96 Hours Negative Control 3.20 A 296,000 1,085,000 1,880,000 2,610,000 B 316,000 1,130,000 1,860,000 2,370,000 C 356,000 970,000 1,740,000 2,500,000 D 267,000 925,000 2,100,000 2,410,000 E 259,000 1,215,000 1,880,000 2,530,000 F 290,000 1,060,000 1,630,000 2,470,000 A 290,000 1,140,000 2,160,000 2,220,000 B 263,000 1,135,000 2,360,000 2,670,000 C 250,000 1,110,000 2,140,000 2,450,000 D 288,000 875,000 1,940,000 2,740,000 E 255,000 945,000 1,900,000 2,690,000 F 305,000 1,045,000 2,340,000 2,840,000 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 77,000 cells/mL at test initiation (0 hours). BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -51 - p r o j e c t n o .: 454A-113A 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 Marine Diatom, Skeletonema costatum Saltwater Algal Medium Mean Measured ^onuenirauon (mg a.i./L) Replicate Cumulative Area Under the Growth Curve 0 - 2 4 Hours 0 - 4 8 Hours 0 - 72 Hours 0 - 9 6 Hours Negative Control A 2,628,000 17,352,000 51,084,000 103,116,000 B 2,868,000 18,372,000 52,404,000 101,316,000 C 3,348,000 17,412,000 48,084,000 97,116,000 D 2,280,000 14,736,000 49,188,000 101,460,000 E 2,184,000 18,024,000 53,316,000 104,388,000 F 2,556,000 16,908,000 47,340,000 94,692,000 3.20 A 2,556,000 17,868,000 55,620,000 106,332,000 B 2,232,000 17,160,000 57,252,000 115,764,000 C 2,076,000 16,548,000 53,700,000 106,932,000 D 2,532,000 14,640,000 46,572,000 100,884,000 E 2,136,000 14,688,000 46,980,000 100,212,000 F 2,736,000 17,088,000 55,860,000 116,172,000 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -52- p r o j e c t n o .: 454A -113A 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 Marine Diatom, Skeletonema costatum Saltwater Algal Medium Mean Measured Concentration (mg a.i./L) Replicate 0 - 2 4 Hours Growth Rate ...... 0 - 4 8 Hours 0 - 7 2 Hours Negative Control A 0.0561 0.0551 0.0444 B 0.0588 0.0560 0.0442 C 0.0638 0.0528 0.0433 D 0.0518 0.0518 0.0459 E 0.0505 0.0575 0.0444 F 0.0553 0.0546 0.0424 3.20 A 0.0553 0.0561 0.0463 B 0.0512 0.0561 0.0475 C 0.0491 0.0556 0.0462 D 0.0550 0.0506 0.0448 E 0.0499 0.0522 0.0445 F 0.0574 0.0543 0.0474 0 - 9 6 Hours 0.0367 0.0357 0.0363 0.0359 0.0364 0.0361 0.0350 0.0369 0.0360 0.0372 0.0370 0.0376 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -53 - p r o j e c t n o .: 454A-H3A 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 and the test concentrations for the first trial, and to add the test substance identification number. 2. The protocol was amended to change the experimental design to a limit test with six biological replicates in each of the treatment and control groups, and to correct a typographical error in the initial cell density. 3. The protocol was amended to change the experimental start and termination dates and the test concentrations for the repeat test, and to change the test apparatus from plastic to glass. 4. Two abiotic replicates, rather than one, were prepared at the test concentration, with one replicate sampled on each of Days 3 and 4. 5. The test chambers were inoculated with a calculated volume of the algal stock culture, rather than with 1 mL of an inoculum prepared from the stock culture. 5. The nominal concentration was calculated based on a test substance purity of 90.49%. 5. The nominal concentration was calculated based on a test substance purity of 86.9%. 5. The protocol w as am ended to change the test substance nam e from Perfluorooctane Sulfonic Acid, Potassium Salt to Perfluorooctanesulfonate, Potassium Salt. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 54- p r o j e c t n o .: 454A -113A APPENDIX VIII Personnel Involved in the Study The following key personnel were involved in the conduct or management of this study: 1. Henry O. Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Manager, Analytical Chemistry 3. Raymond L. Van Hoven, Ph.D., Scientist 4. Cary A. Sutherland, Laboratory Supervisor 5. Debbie Desjardins, Biologist