Document zoo3wGK2VNd9wN72zjRrNaJD3

2^071 'n P " >- - . .. it PFOS: A 96-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST WITH THE SPRING PEEPER TADPOLE (Pseudacris crucifer) FINAL REPORT P-1 WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER : 454A-251 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0083 ASTM Standard E729-96 AUTHORS : Tui Minderhout, Ph.D. Jon A. MacGregor, B.S. Henry O. Krueger, Ph.D. STUDY INITIATION DATE : March 15, 2007 STUDY COMPLETION DATE : April 12, 2007 SUBMITTED TO : 3M Corporation Environmental Laboratory 3M Center Building 0260-05-N-17 Maplewood, MN 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 40 CONTAINS NO CE P-2 Wildlife International, Ltd. Project Number 454A-251 2- GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS A 96-Hour Static-Renewal Acute Toxicity Test with the Spring Peeper Tadpole (Pseudacris crucifer) WILDLIFE INTERNATIONAL, LTD PROJECT NUMBER: 454A-251 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER E07-0083 STUDY COMPLETION: April 12, 2007 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U S. Environmental Protection Agency (40 CFR Parts 160 and 792, 17 August 1989) with the following exception: Periodic analyses of well water for potential contaminants were performed using a certified laboratory and standard U S EPA analytical methods STUDY DIRECTOR: Senior Biologist SPONSOR APPROVAL Date D aiv 7 P-3 Wildlife International, Ltd. Project Number 454A-251 -3QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency (40 CFR Parts 160 and 792, 17 August 1989). The dates o f all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Protocol Test Substance Preparation Matrix Fortification and Observations Analytical Data and Draft Report Biological Data and Draft Report Final Report DATE CONDUCTED: February 27 to 28, 2007 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: Februaiy 28, 2007 March 8, 2007 March 16, 2007 March 16, 2007 March 21,2007 March 23, 2007 March 23, 2007 March 28, 2007 April 3, 2007 April 2 to 3, 2007 April 12,2007 April 3, 2007 April 3, 2007 April 12, 2007 April 4, 2007 April 4, 2007 April 12, 2007 All inspections were study-based unless otherwise noted. Date Wildlife International, Ltd. Project Number 454A-251 -4REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static-Renewal Acute Toxicity Test with the Spring Peeper Tadpole (Pseudacris crucifer) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-251 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0083 STUDY DIRECTOR: W kt( P1/`) ^ ^ ____________ TuiMinderhout, Ph.D. Senior Biologist 3l April .^00} Date PRINCIPAL INVESTIGATOR: WILDLIFE INTERNATIONAL. LTD. MANAGEMENT: Director o f Aquatic Toxicology/Terrestrial Plants and Insects Willard B. Nixon, Ri.D Director of Chemistry P-5 Wildlife International, Ltd. Project Number 454A-251 -5 TABLE OF CONTENTS Title/Cover Page...............................................................................................................................................1 Good Laboratory Practice Compliance Statement........................................................................................ 2 Quality Assurance Statement..........................................................................................................................3 Report Approval.............................................................................................................................................. 4 Table of Contents............................................................................................................................................ 5 S u m m a ry .......................................................................................................................................................... 7 Introduction......................................................................................................................................................8 O b je c tiv e .......................................................................................................................................................... 8 Experimental Design....................................................................................................................................... 8 Materials and Methods....................................................................................................................................9 Test Substance....................................................................................................................................9 Test Organism..................................................................................................................................... 9 Dilution Water.................................................................................................................................. 10 Test Apparatus.................................................................................................................................. 10 Preparation of Test Concentrations.................................................................................................11 Analytical Sampling.........................................................................................................................11 Analytical Method............................................................................................................................11 Environmental Conditions............................................................................................................... 13 Observations..................................................................................................................................... 13 Statistical Analyses...........................................................................................................................14 Results and Discussion.................................................................................................................................. 14 Measurement of Test Concentrations..............................................................................................14 Observations and Measurements.....................................................................................................14 Conclusions.................................................................................................................................................... 15 References...................................................................................................................................................... 16 / P-6 ildl^C International, Ltd. Project Number 454A-251 6- TABLE OF CONTENTS (Continued) TABLES AND FIGURES Table 1. Measured Concentrations of PFOS in Freshwater Samples.....................................................17 Table 2. Temperature, Dissolved Oxygen and pH of Water in the Test Chambers............................... 18 Table 3. Specific Conductance, Hardness and Alkalinity Measured in Dilution Water At Test Initiation and Termination.............................................................................................19 Table 4. Cumulative Mortality and Clinical Observations...................................................................... 20 Table 5. LC50 Values.................................................................................................................................21 Figure 1. Concentration-Response Curve (96-Hour Mortality D ata).....................................................22 APPENDICES Appendix 1. Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test......................23 Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water..................................................................................................................... 24 Appendix 3. 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 The Analysis of PFOS in Freshwater..............................................................................26 Analytical Method Flowchart for the Analysis of PFOS in Freshwater..................... 27 Typical HPLC/MS/MS Operations Parameters........................................................... 28 Analytical Stocks and Standards Preparation................................................................29 Example Calculations for a Representative Sample.................................................... 30 Quality Control Samples of PFOS in Freshwater........................................................ 32 Representative Calibration Curve for PFOS.................................................................33 Representative Chromatogram of a Low-level PFOS Calibration Standard...............34 Representative Chromatogram of a High-level PFOS Calibration Standard.............. 35 Representative Chromatogram of a Matrix Blank Sample.......................................... 36 Representative Chromatogram of a Matrix Fortification Sample................................ 37 Representative Chromatogram of a Test Sample...........................................................38 Appendix 4. Changes to Protocol.........................................................................................................39 Appendix 5. Personnel Involved in the Study.................................................................................... 40 // p.7 Wildlife International, Ltd. Project Number 454A-251 -7SUMMARY SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static-Renewal Acute Toxicity Test with the Spring Peeper Tadpole {Pseudacris crucifer) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-251 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0083 TEST DATES: Experimental Start: Biological Termination: Experimental Termination: March 20, 2007 March 24, 2007 March 26, 2007 LENGTH OF EXPOSURE: 96 Hours TEST ORGANISMS: Spring peeper tadpole {Pseudacris crucifer) SOURCE OF TEST ORGANISMS: Carolina Biological Supply Company Burlington, North Carolina AGE OF TEST ORGANISMS: Juveniles MEASUREMENTS OF 10 REPRESENTATIVE TADPOLES: AT TEST INITIATION: AT TEST TERMINATION: Mean Wet Weight: 0.030 g Range: 0.010-0.044 g Mean Wet Weight: 0.040 g Range: 0.023-0.063 g TEST CONCENTRATIONS: Nominal Negative Control 3.2 mg a.i./L 6.3 mg a.i./L 13 mg a.i./L 25 mg a.i./L 50 mg a.i./L Mean Measured < LOQ 3.6 mg a.i./L 7.0 mg a.i./L 14 mg a.i./L 27 mg a.i./L 51 mg a.i./L RESULTS: Based on mean measured concentrations: 96-Hour LC50: 95% Confidence Interval: No-Mortality Concentration: No-Observed-Effect Concentration: 38 mg a.i./L 2 7 -5 1 mg a.i./L 3.6 mg a.i./L 3.6 mg a.i./L /Z p.8 Wildlife International, Ltd. Project Number 454A-251 8- - INTRODU CTION 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 and a back-up trial, with a slightly lower test concentration, were conducted from March 19 to 23, 2007 and March 20 to 24, 2007, respectively. The two tests resulted in similar analytical concentrations and LC50 values. However, unlike that observed in the initial trial, both the no-mortality and no-observed-effect concentration were clearly determined in the back-up test. Therefore only the results from the back up trial are reported. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-251 in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of this study was to evaluate the acute effects of perfluorooctane sulfonate potassium salt (PFOS) on the spring peeper tadpole (Pseudacris crucifer) during a 96-hour exposure period under static-renewal test conditions. EXPERIMENTAL DESIGN Spring peeper tadpoles were exposed to a geometric series of five test concentrations and a negative control (dilution water) for 96 hours under static-renewal conditions. Two replicate test chambers were maintained in each treatment and control group, with 10 tadpoles in each test chamber for a total of 20 tadpoles per concentration. Nominal test concentrations were selected in consultation with the Sponsor. Nominal test concentrations selected were 3.2, 6.3, 13, 25 and 50 mg active ingredient (a.i.)/L. Test solutions were renewed at approximately 48 hours by transferring the test organisms to freshly prepared solutions. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning of the test, prior to and after renewal at 48 hours, and at test termination. Spring peeper tadpoles were impartially assigned to exposure chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 4, 24, 48, 72 and 96 hours after test initiation. The cumulative percent mortality observed in the treatment groups was used to determine LC50 values at 24, 48, 72 and 96 hours. The no-mortality concentration and the no-observed-effect-concentration (NOEC) were determined by visual interpretation of the mortality and observation data. /3 P-9 Wildlife International, Ltd. -9- Project Number 454A-251 MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A 96Hour Static-Renewal Acute Toxicity Test with Spring Peeper Tadpole (Pseudacris crucifer)". The protocol was based on procedures outlined in ASTM Standard E729-96 Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates and Amphibians ( 1). Test Substance The test substance used to prepare the test solutions, analytical calibration standards and the analytical matrix fortification samples for the study was received from 3M Corporation on October 29, 1998. It was assigned Wildlife International, Ltd. identification number 4675 upon receipt and was stored under ambient conditions. The test substance, a white powder, was identified as: FC-95, Lot number 217. The test substance contained 86.9% active ingredient and had an expiration date of August 31, 2016. Test Organism Spring peeper tadpole, Pseudacris crucifer, was selected as the test species for this study. Spring peeper tadpoles are representative of an important group of aquatic vertebrates, and were selected for use in the test based upon past history of use in the laboratory. Spring peeper tadpoles used in the test were obtained from Carolina Biological Supply Company, Burlington, North Carolina. Identification of the species was verified by the supplier. All tadpoles used in the test were from the same source and year class. The average wet weight (blotted dry) of 10 representative tadpoles was 0.030 grams with a range of 0.010 to 0.044 grams at test initiation and 0.040 grams with the range of 0.023 to 0.063 grams at test termination. Loading was defined as the total wet weight of tadpole per liter of test water and was 0.12 g tadpole/L at test start and 0.16 g tadpole/L at test termination. The tadpoles were held for 5 days prior to the test in water from the same source and at approximately the same temperature as used during the test. During the 5 days period immediately preceding the test, water temperatures in the cultures ranged from 20.4 to 21.4C, measured with a Wildlife International, Ltd. Project Number 454A-251 - 10- hand-held liquid-in-glass thermometer. The pH of the water ranged from 7.8 to 8.2, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved oxygen concentrations ranged from 7.0 to 9.0 mg/L (> 81% of saturation), measured with a Yellow Springs Instruments Model 5 IB dissolved oxygen meter. Daily, the tadpoles were fed a commercially-prepared diet supplied by Carolina Biological Supply Company, Burlington, North Carolina, during the holding period. During the test the tadpoles were fed at test initiation on day 0 and at test renewal on day 2 with 25 mg ground commercially supplied tadpole food. During the 5 days period prior to the test the tadpoles showed no signs of disease or stress. At test initiation, the tadpoles were collected from the culture tank and impartially distributed one and two at a time to the test chambers until each contained 10 tadpoles. Dilution Water The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity and pH of the well water during the four-week period immediately preceding the test are presented in Appendix 1. The well water was passed through a sand filter to remove particles greater than approximately 25 pm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 pm and passed through an ultraviolet sterilizer to remove fine particles and microorganisms. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the well water used by Wildlife International, Ltd. are presented in Appendix 2. Test Apparatus Test chambers were approximately 3-L plastic vessel containing 2.5 L of test solution. The depth of the test water in a representative test chamber was approximately 9.3 cm. Each test chamber was labeled with the project number, test concentration and replicate. The chambers were indiscriminately positioned by treatment group in an environmental chamber. p. 11 Wildlife International, Ltd. Project Number 454A-251 -11 - Preparation of Test Concentrations A stock solution was prepared at a nominal concentration of 50 mg a.i./L, the highest concentration tested, by mixing a calculated amount of PFOS into dilution water (UV sterilized well water). The primary stock was stirred overnight using a top-down electric mixer. The primary stock solution prepared at test initiation appeared clear and colorless with foam on the surface. The primary stock solution prepared for test solution renewal on day 2 appeared clear and colorless. Test solutions were prepared at nominal concentrations of 3.2, 6.3, 13 and 25 mg a.i./L by adding the appropriate volume of primary stock to dilution water in a calibrated plastic container to achieve a final volume of 5 L. Each solution was mixed by stirring with a whisk. All test solutions were adjusted to 100% active ingredient during preparation, based on the test substance purity (86.9%). New test solutions were prepared for renewal at approximately 48 hours. All test solutions appeared clear and colorless at test initiation and termination. Analytical Sampling At test initiation on day 0 and at the renewal of the test solutions on day 2, samples of new solutions were collected from the newly prepared batches of test solution and dilution water (for the control group) to determine concentrations of the test substance. Prior to renewal at approximately 48 hours and at test termination, samples of old test solutions were collected from each test chamber of each treatment and control group. All samples were collected at mid-depth and placed in plastic vials. Samples collected at test initiation and at renewal on day 2 were processed immediately for analysis. The samples collected at test termination were stored under refrigeration (at approximate 4C) until analyzed. Analytical Method The analytical method used for the analysis of PFOS in freshwater was developed at Wildlife International, Ltd. The analytical method consisted of dilution of the samples 1:1, v/v with acetonitrile, followed by secondary dilution using acetonitrile: HPLC-grade bottled water (50:50, v/v), and analysis by direct injection high performance liquid chromatography with mass spectrometric (LC/MS/MS) detection. Concentrations of PFOS in the samples were determined by LC/MS/MS using an Agilent 1100 Series High Performance Liquid Chromatograph interfaced with an Applied Biosystems / MDS Wildlife International, Ltd. Project Number 454A-251 - 12 - Sciex API 3000 mass spectrometer (MS/MS) operated in negative ion multiple-reaction monitoring (MRM) detection mode. The mass spectrometer was equipped with a Turboion Spray ion source. Chromatographic separations were achieved using an Agilent Zorbax RX-Cg column (150 mm x 2.1 mm, 5pm particle size). A flow chart for the analysis of PFOS is provided in Appendix 3.1 and typical instrumental parameters are summarized in Appendix 3.2. Calibration standards of PFOS, ranging in concentration from 0.0500 to 1.00 pg a.i./mL, were prepared in acetonitrile: HPLC-grade bottled water solution (50:50, v/v) using a stock solution of PFOS in methanol (Appendix 3.3). Quadratic (weighted 1/x) regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards using Analyst Version 1.4.1 software of the Applied Biosystems/MDS Sciex API 3000 mass spectrometer system. The concentration of PFOS in the samples was determined by substituting the peak area responses of the samples into the applicable regression equation. An example of the calculations for a representative sample is included in Appendix 3.4. The method limit of quantitation (LOQ) for these analyses was set at 1.00 mg a.i./L, calculated as the product of the lowest calibration standard (0.0500 pg a.i./mL) and the dilution factor of the matrix blank samples (20.0). Three matrix blank samples were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during the sample analyses (Appendix 3.5). Matrix fortification samples were prepared fresh on each sampling day and were analyzed concurrently with the samples. Samples of freshwater were fortified with a stock solution of the test substance in methanol at nominal PFOS concentrations of 2.00, 15.0 and 50.0 mg a.i./L. The measured concentrations for the matrix fortification samples ranged from 99.4 to 106% of nominal concentrations (Appendix 3.5) A representative calibration curve is presented in Appendix 3.6. Representative chromatograms of low and high-level calibration standards are presented in Appendices 3.7 and 3.8, respectively. A representative chromatogram of a matrix blank sample is presented in Appendix 3.9 and a representative chromatogram of a matrix fortification sample is presented in Appendix 3.10. A representative chromatogram of a test sample is presented in Appendix 3.11. Wildlife International, Ltd. - 13 - Project Number 454A-251 Environmental Conditions Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone 50) were used for illumination of the cultures and test chambers. A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30-minute transition period of low light intensity was provided at the beginning and end of the 16-hour light period to avoid sudden changes in lighting. Light intensity at test initiation, measured using a SPER Scientific Model 840006C light meter, was 120 lux at the surface of the water of one representative test chamber. The target test temperature during the study was 22 1C. Temperature was measured in each test chamber at the beginning and end of the test and at approximately 48-hour before and after renewals, using a liquid-in-glass thermometer. Temperature also was measured continuously during the test in one negative control test chamber using a Fulscope ER/C Recorder, which was verified prior to test initiation with a liquid-in-glass thermometer. Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at approximately 48-hour before and after renewals. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter and measurements of pH were made using a Thermo Orion Model 525Aplus pH meter. Specific conductance, hardness and alkalinity were measured in the dilution water at test initiation and termination. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods fo r the Examination o f Water and Wastewater (2). Observations Observations were made periodically to determine the number of mortalities in each treatment group. The number of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 4, 24, 48, 72 and 96 hours after test initiation. p. 14 Wildlife International, Ltd. Project Number 454A-251 - 14- Statistical Analyses The mortality data were analyzed using the computer program of C. E. Stephan (3). The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation (4, 5, 6). In this study, the Binomial probability method was used to calculate the 24, 48, 72 and 96-hour LC50 values. The no-mortality concentration and NOEC were determined by visual interpretation of the mortality and observation data. RESULTS AND DISCUSSION Measurement of Test Concentrations Nominal concentrations selected for use in this study were 3.2, 6.3, 13, 25 and 50 mg a.i./L. Results of analyses to measure concentrations of PFOS in the test solution samples collected during the test are presented in Table 1. Samples collected at test initiation had measured concentrations that ranged from 104 to 118% of the nominal concentrations. Samples collected prior to and after renewal of the test solutions at 48 hours had measured concentrations that ranged from 99.2 to 107% and 105 to 131%, respectively, of the nominal concentrations. Samples collected at test termination had measured concentrations that ranged from 96.8 to 101% of the nominal concentrations. When the measured concentrations of samples collected at 0, 48 and 96 hours were averaged, the mean measured concentrations for the study were 3.6, 7.0, 14, 27 and 51 mg a.i./L, representing 113, 111, 108, 108 and 102% of nominal concentrations, respectively. The results of the study were based on the mean measured test concentrations. Observations and Measurements Measurements of temperature, dissolved oxygen and pH of the water in each test chamber are presented in Table 2. Water temperatures were within the 22 1C range established for the test. Dissolved oxygen concentrations remained >7.2 mg/L (>83% of saturation) throughout the test. Measurements of pH ranged from 8.3 to 8.6. The measurements of hardness, alkalinity and specific conductance in the dilution water at test initiation and termination were typical of Wildlife International, Ltd. well water (Table 3). Daily observations of mortality and signs of toxicity observed during the test are presented in Table 4. All tadpoles in the negative control group and in the 3.6 mg a.i./L treatment group appeared Wildlife International, Ltd. Project Number 454A-251 - 15 - normal throughout the test with no mortalities or overt signs of toxicity observed. At test termination, mortality in the 7.0, 14, 27 and 51 mg a.i./L treatment group was 15, 15, 15 and 80%, respectively. While the tadpoles that survived to test termination in the 7.0 mg a.i./L treatment appeared normal, the toxicity signs observed in the affected tadpole from the other treatment groups included lethargy and curved tail and spine. The no-mortality concentration and the NOEC were both 3.6 mg a.i./L. LC50 values at 24, 48, 72 and 96 hours were determined from the mortality data and are shown in Table 5. A graph of the concentration-response curve is included in Figure 1. CONCLUSIONS Tadpoles of spring peeper, Pseudacris crucifer, were exposed under static-renewal conditions for 96 hours to five mean measured concentrations of PFOS ranging from 3.6 to 51 mg a.i./L. The 96-hour LC50 value was 38 mg a.i./L, with a 95% confidence interval of 27 to 51 mg a.i./L. Tadpoles exposed to PFOS at a concentration of 3.6 mg a.i./L appeared normal with no mortality or overt signs of toxicity. The no-mortality concentration and NOEC were both 3.6 g a.i./L. 0 Wildlife International, Ltd. - 16REFERENCES Project Number 454A-251 1 ASTM Standard E729-96. 1996. Standard Guide fo r Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. 2 APHA, AWWA, WPCF. 1985. Standard Methods fo r the Examination o f Water and Wastewater. 16th Edition, American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York. 3 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 4 Thompson, W.R. 1947. Bacteriological Reviews. Vol. II, No. 2. Pp. 115-145. 5 Stephan, C.E. 1977. "Methods for Calculating an LC50," Aquatic Toxicology and Hazard Evaluations. American Society for Testing and Materials. Publication Number STP 634, pp 65-84.6 6 Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London. Wildlife International, Ltd. Project Number 454A-251 - 17 Table 1 Measured Concentrations of PFOS in Freshwater Samples Nominal Test Concentration (mg a.i./L) 0.00 (Negative Control) 3.2 6.3 13 25 50 Sample Number (454A-251-) 1 7 13 19 2 8 14 20 3 9 15 21 4 10 16 22 5 11 17 23 6 12 18 24 Sampling Time (Hours) Measured Concentration PFOS (mg a.i./L)1 Percent of Nominal1 Mean Measured Concentration (mg a.i./L) Mean Measured Percent of Nominal 0(new) 48(old) 48(new) 96(old) 0(new) 48(old) 48(new) 96(old) < LOQ2 < LOQ < LOQ < LOQ 3.77 3.17 4.18 3.24 -- -- - 118 99.2 131 101 < LOQ 3.6 113 0(new) 48(old) 48(new) 96(old) 0(new) 48(old) 48(new) 96(old) 0(new) 48(old) 48(new) 96(old) 0(new) 48(old) 48(new) 96(old) 7.15 6.62 8.28 6.10 13.8 13.4 15.0 12.9 28.1 26.8 28.0 25.2 51.9 51.7 52.3 49.2 113 105 131 96.8 106 103 115 99.4 113 107 112 101 104 103 105 98.5 7.0 14 27 51 111 108 108 102 Results generated using Analyst version 1.4.1 software. Manual calculations may vary. 2 The limit of quantitation (LOQ) was 1.00 mg a.i./L calculated as the product of the lowest calibration standard (0.0500 pg a.i./mL) and the dilution factor of the matrix blanks (20.0). 2Z Wildlife International, Ltd Project Number 454A-251 - 18 Table 2 Temperature, Dissolved Oxygen and pH of Water in the Test Chambers Mean Measured Concentration (mg a.i./L) Negative Control 0 Hour Temp.1 (C) 21.7 21.5 DO2 (mg/L) 8.6 8.5 pH 8.5 8.5 48 Hours (Old solution Prior to Renewal) Temp.1 DO2 (C) (mg/L) pH 22.5 7.6 8.3 22.6 7.6 8.3 48 Hours (New Solution At Renewal) Temp.1 DO2 (C) (mg/L) pH 21.6 8.6 8.5 21.9 8.5 8.5 96 Hours Temp.1 (C) 22.5 22.6 DO2 (mg/L) 7.7 7.6 pH 8.4 8.4 3.6 21.4 8.5 8.5 22.6 7.6 8.3 22.0 8.5 8.5 22.6 7.6 8.4 21.3 8.5 8.5 22.7 7.6 8.4 22.1 8.5 8.5 22.6 7.8 8.4 7.0 21.3 8.4 8.5 22.6 7.6 8.4 22.0 8.4 8.5 22.6 7.7 8.4 21.2 8.4 8.6 22.6 7.6 8.4 22.3 8.4 8.5 22.6 7.7 8.4 14 21.5 8.6 8.6 22.6 7.6 8.4 22.6 8.5 8.6 22.5 7.7 8.4 21.6 8.6 8.6 22.6 7.7 8.4 22.7 8.5 8.5 22.6 7.8 8.4 27 21.7 8.5 8.6 22.6 7.6 8.4 22.3 8.6 8.6 22.6 7.6 8.4 21.8 8.3 8.6 22.6 7.7 8.4 22.2 8.7 8.6 22.6 7.4 8.4 51 22.3 8.2 8.6 22.6 7.6 8.4 21.9 8.6 8.6 22.5 7.5 8.5 22.4 8.2 8.6 22.6 7.2 8.4 21.8 8.5 8.6 22.6 7.5 8.5 Manual temperature measurements. Temperature measured continuously during the test ranged from approximately 21.0 to 22.5C, measured to the nearest 0.5C. 2 A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22C in freshwater. Wildlife International, Ltd. Project Number 454A-251 - 19- Table 3 Specific Conductance, Hardness and Alkalinity Measured in Dilution Water at Test Initiation and Termination Parameter Specific Conductance (pmhos/cm) Hardness (mg/L as CaC03) Alkalinity (mg/L as CaC03) Day 0 305 128 178 Day 4 295 124 169 24 Wildlife International, Ltd. -20Table 4 Cumulative Mortality and Clinical Observations Project Number 454A-251 Mean Measured Concentration (mg a.i./L) Negative Control No. Replicate Exposed A 10 B 10 4 Hours No. Dead1 Obs.23 0 10 AN 0 10 AN 24 Hours No. Dead Obs. 0 10 AN 0 10 AN 48 Hours3 (T) No. Dead Obs. 0 10 AN 0 10 AN 3.6 A 10 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 0 10 AN 0 10 AN 7.0 A 10 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 0 10 AN 0 10 AN 14 A 10 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 1 9 AN 1 9 AN 27 A 10 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 0 9AN; 1C, V 0 9AN; 1C,V 51 A 10 0 10 AN 1 6AN; 3C, V 3 3AN; 4C, V B 10 0 10 AN 1 6AN; 2C; 1C,V 6 2AN; 2C, V 1 Cumulative number of dead tadpoles. 2 Observations: AN = appear normal; C = lethargy; V = curved spine and tail. 3 Test solutions were renewed at approximately 48 hours. T = transferred to new test solutions. 72 Hours No. Dead Obs. 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 1 8 AN; 1 C 1 9 AN 0 10 AN 0 9AN; 1C,V 5 3AN; 2C,V 7 IAN; 2C,V 96 Hours No. Dead Obs. 0 10 AN 0 10 AN Cumulative Percent Mortality 0 0 10 AN 0 10 AN 0 1 9 AN 2 8 AN 15 2 7 AN; 1 C 1 9 AN 15 2 8 AN 1 6AN; 3C, V 15 7 IAN, 2C,V 8+1V 1 C, V 80 p. 20 Wildlife International, Ltd. -21 Table 5 LC50 Values Project Number 454A-251 Time 24 Hours LC50 (mg a.i./L) >51 95% Confidence Interval (mg a.i./L) 1 Statistical Method Binomial Probability 48 Hours >27 2 Binomial Probability 72 Hours 47 2 Binomial Probability 96 Hours 38 27-51 Binomial Probability 1 At a confidence level of 95% the binomial test shows that the LC50 is above 51 mg a.i./L.2 2 At a confidence level of 95% the binomial test shows that the LC50 is above 27 mg a.i./L. Z6 Wildlife International, Ltd. Project Number 454A-251 -22Figure 1 Concentration-Response Curve (96-Hour Mortality Data) Concentration (mg a.i./L) 27 Wildlife International, Ltd. Project Number 454A-251 -23 - Appendix 1 Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test Parameter Specific Conductance (pmhos/cm) Hardness (mg/L as CaC03) Alkalinity (mg/L as CaC03) pH Mean 291 (N = 4) 136 (N = 4) 183 (N = 4) 8.1 (N = 4) Range 290 - 295 132-140 182-184 8.1 -8 .1 Wildlife International, Ltd. Project Number 454A-251 -24Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Component Aldrin Alpha BHC Alpha Chlordane Beta BHC Bolstar Chlordane Coumaphos Delta BHC Demeton-O Demeton-S Diazinon Dichlorvos Dieldrin Disulfoton Dursban (Chlorpyrifos) Endosulfan I Endosulfan II Endosulfan Sulfate Endrin Endrin Aldehyde Endrin Ketone EPN Ethion Ethoprop Ethyl Parathion Famphur Fensulfothion Fenthion Gamma BHC - Lindane Gamma Chlordane Guthion (Azinphos-methyl) HCB Pesticides and Organics Measured Concentration (Pg/L) Component <0.021 <0.011 <0.011 < 0.026 <2.1 <0.53 <3.2 < 0.026 <2.1 <3.2 <4.3 <3.2 <0.021 <2.1 <2.1 <0.011 <0.021 < 0.043 <0.021 <0.11 < 0.043 <4.3 <2.1 <3.2 <2.1 <3.2 < 16 <2.1 <0.011 <0.011 <4.3 <0.11 Heptachlor Heptachlor Epoxide Kepone Malathion Merphos Methoxychlor Methyl Parathion Mevinphos Mirex Naled o,p-DDD o,p-DDE o,p-DDT p,p-DDD p,p-DDE p,p-DDT PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 Phorate Ronnel Stirophos Telodrin Tokuthion Toxaphene Trichloronate Trithion Measured Concentration (Pg/L) <0.011 < 0.026 <0.21 <3.2 <6.4 <0.11 <2.1 <4.3 <0.12 <3.2 <0.021 <0.021 <0.021 <0.021 <0.021 <0.021 <0.53 <0.53 <0.53 <0.53 <0.53 <0.53 <0.53 <2.1 <2.1 <2.1 <0.011 <2.1 < 1.1 <2.1 <2.1 1 Analyses performed by Lancaster Laboratories on samples collected on December 13, 2006. 21 Wildlife International, Ltd, Project Number 454A-251 -25 Appendix 2 (Continued) Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Component Aluminum Antimony Arsenic Barium Beryllium Bromide Cadmium Calcium Chloride Chromium Cobalt Copper Fluoride Iron Lead Metals Measured Concentration (mg/L) Component < 0.200 < 0.0200 < 0.0200 0.0065 < 0.0050 55.9 < 0.0050 236 12100 <0.0150 < 0.0050 <0.0100 < 10.0 < 0.200 <0.0150 Magnesium Manganese Mercury Nickel Nitrate Nitrogen Nitrite Nitrogen Potassium Selenium Silver Sodium Sulfate Thallium Vanadium Zinc Measured Concentration 740 < 0.0050 < 0.00020 <0.0100 <0.50 < 10.0 290 < 0.0200 < 0.0050 5600 1630 < 0.0200 < 0.0050 < 0.0200 Analyses performed by Lancaster Laboratories on samples collected on December 13, 2006. 30 p. 26 Wildlife International, Ltd. Project Number 454A-251 -26 Appendix 3 The Analysis of PFOS in Freshwater 5! Wildlife International, Ltd. Project Number 454A-251 -27Appendix 3.1 Analytical Method Flowchart for the Processing of PFOS in Freshwater METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER Prepare calibration standards in acetonitrile : HPLC-grade bottled water (50:50,v/v) using volumetric flasks and gas-tight syringes, STORE REFRIGERATED. I Prepare matrix fortification samples in well water using volumetric flasks, volumetric pipettes, 15-mL tubes and gas-tight syringes. Dilute all samples initially 1:1 with 100% acetonitrile using 15-mL culture tubes or equivalent, gas-tight syringes and/or class A volumetric pipettes. Mix well. Volumetrically dilute solutions further, if necessary, with acetonitrile : HPLC-grade bottled water (50:50,v/v) so that the final sample concentrations fall within the calibration standard range. Mix well. I Transfer aliquots of final sample dilutions and calibration standards to autosampler vials for analysis by LC/MS/MS. 31 Wildlife International, Ltd. Project Number 454A-251 -28 Appendix 3.2 Typical HPLC/MS/MS Operational Parameters INSTRUMENT: ION SOURCE: ANALYTICAL COLUMN: STOP TIME: FLOW RATE: OVEN TEMPERATURE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: PFOS MONITORED MASS: Agilent Series 1100 High Performance Liquid Chromatograph (HPLC) coupled with an Applied Biosystems/MDS Sciex API 3000 Mass Spectrometer (MS/MS) operated in the negative ion multiplereaction monitoring (MRM) mode. Turboion Spray Agilent Zorbax RX-C8 (150 mm x 2.1 mm, 5 pm particle size) 5.00 minutes 0.300 mL/minute 40C 80% MeOH : 20% H20 containing 0.1% formic acid 10.0 pL Approximately 2.8 minutes 499 --+99 amu 33 Wildlife International, Ltd. Project Number 454A-251 -29Appendix 3.3 Analytical Stocks and Standards Preparation A stock solution of PFOS was prepared by weighing 1.1507 g (corrected for purity) of the test substance on an analytical balance. The test substance was transferred to a 100-mL volumetric flask and brought to volume using methanol. This primary stock solution contained 10.0 mg a.i./mL of PFOS. Secondary stocks of PFOS in methanol (1.00 and 0.100 mg a.i./mL) were prepared from the primary stock by volumetric dilution. The 10.0 and 1.00 mg a.i./mL stock solutions were used to prepare concurrent matrix fortification samples (QC) for this study. The 0.100 mg a.i./mL stock solution was used to prepare calibration standards. The calibration standards were prepared in acetonitrile: HPLC-grade bottled water (50:50, v/v). The following shows the dilution scheme for the set of calibration standards. Stock Concentration (mg a.i./mLl 0.100 0.100 0.100 0.100 0.100 Aliquot (mL) 0.0500 0.150 0.250 0.500 1.00 Final Volume (mL) 100 100 100 100 100 Standard Concentration (ug a.i./mLl 0.0500 0.150 0.250 0.500 1.00 Wildlife International, Ltd. Project Number 454A-251 -30Appendix 3.4 Example Calculations for a Representative Sample The analytical result and percent recovery for sample number 454A-251-2, an exposure sample prepared at a nominal concentration of 3.2 mg a.i./L, was calculated as follows using the software algorithms of Analyst Version 1.4.1 of the Applied Biosystems/MDS Sciex API 3000 mass spectrometer system. Regression was used to generate calibration equations for each analytical sequence relating the measured peak areas of reference standard solution injections of PFOS with their known concentrations. The curve was weighted 1/x with respect to concentration and expressed as a quadratic function as follows: y = ax2+ bx + c where: y = instrumental peak area response of concentration x of PFOS in mg a.i./L a = quadratic coefficient b = linear coefficient c =constant coefficient (y intercept) Concentrations of PFOS in samples were determined by substituting peak area responses of the samples into the applicable rearranged regression equation as follows: PFOS (mg a.i./L) = Dilution Factor - Linear Coefficient + ^/(Linear Coefficien t) 2 - [4 (Quadratic Coefficien t) (Y Interce pt - Peak Area)] 2 (Quadratic Coefficien t) where the Dilution Factor compensates for dilution of the water sample so that the peak response was bracketed by the standard calibration curve. Data used for quantitation of PFOS in Sample Number 454A-251-2 are summarized below: Peak area = 1878700 Constant Coefficient = 4990.54 Linear Coefficient = 10046500 Quadratic Coefficient = -505910 Dilution Factor (Vfmai/Vjnitiai): = 20.0 Wildlife International, Ltd. Project Number 454A-251 -31 Appendix 3.4 (Continued) Example Calculations for a Representative Sample -10046500 + 7(10046500)2 - [(4* (-505910))* (4990.54-1878700)] PFOS = 20.0 2 (-505910) PFOS = 20.0 0.188285 mg a.i./L PFOS = 3.77 mg a.i./L The measured concentration was compared to the nominal concentrations as follows: _____ PFOS in sample (mg a.i./L)_____ Percent of nominal concentration = PFOS nominal concentration (mg a.i./L) .77 mg/L .2 mg/L = 118% Wildlife International, Ltd. Project Number 454A-251 -32Appendix 3.5 Quality Control Samples of PFOS in Freshwater Sample Number (454A-251-) MAB-1 MAB-2 MAB-3 MAS-1 MAS-2 MAS-3 MAS-4 MAS-5 MAS-6 MAS-7 MAS-8 MAS-9 Sampling Time (Hours) 0 48 96 0 0 0 48 48 48 96 96 96 Concentration (mg a.i./L) Fortified 0.0 0.0 0.0 2.00 15.0 70.0 2.00 15.0 70.0 2.00 15.0 70.0 Measured 1,2 < LOQ < LOQ < LOQ 2.08 15.9 71.2 2.09 15.6 69.6 2.04 15.1 69.8 Percent Recovery 1 _ -- - 104 106 102 104 104 99.4 102 101 99.8 X=102 S.D.=2.20 C.V.=2.16 % 1 Results generated using Analyst version 1.4.1 software. Manual calculations may vary. 2 The limit of quantitation (LOQ) was 1.00 mg a.i./L calculated as the product of the lowest calibration standard (0.0500 pg a.i./mL) and the dilution factor of the matrix blanks (20.0). 7 Wildlife International, Ltd. Project Number 454A-251 -33Appendix 3.6 Representative Calibration Curve for PFOS 0B2007_DOidb(FFOE " 0 0 * ^ Ffegresscr("1 / / wejting>y =-506eE * 2 + 1et007x +4.99sO03(r=1.0000) aas6: 95e6: 9036' a5e6j S0e6^ 7.5e6: 7.036: 65e6: 6C66- 6.536 so-; 4.5e6~ 4.0e6; a5e6; a0e6i 25e6; 9 20e6- 1.5e6; 1.066' / 50asj*'' Q05 Q10 Q15 020 025 30 Q36 040 Q45 Q3D 056 0.60 0.66 0.70 075 080 O aT OSO 095 1.00 Cbmertricrv u j aiM t Linear coefficient= 10046500; constant coeffcient= 4990.54; quadratic coefficient= -505910; r=0.99997 Wildlife International, Ltd, Project Number 454A-251 -34Appendix 3.7 Representative Chromatogram of a Low-level PFOS Calibration Standard Nominal concentration: 0.0500 mg a.i./L 31 Wildlife International, Ltd. Project Number 454A-251 -35Appendix 3.8 Representative Chromatogram of a High-level PFOS Calibration Standard ISin*Hit* VKB S SarrctalCr'STDl OOLflil/rrl' F<* Tl Carrot *ffC6ff *ra*xrr*e Nominal concentration: 1.00 mg a.i./L Wildlife International, Ltd. Project Number 454A-251 -36Appendix 3.9 Representative Chromatogram of a Matrix Blank Sample Sample number 454A-251-MAB-1. Dilution factor = 20.OX. The arrow indicates the retention time of PFOS. Wildlife International, Ltd. Project Number 454A-251 ISr06*iracT>K_r Gofnrart'4ASr7 i -37Appendix 3.10 Representative Chromatogram of a Matrix Fortification Sample Sample number: 454A-251-MAS-1, nominal concentration 2.00 mg a.i./L. Dilution factor = 20.0 X. Wildlife International, Ltd Project Number 454A-251 -38Appendix 3.11 Representative Chromatogram of a Test Sample Sample number: 454A-251-2, Day 0, nominal concentration 3.2 mg a.i./L. Dilution factor = 20.0 X. Wildlife International, Ltd. Project Number 454A-251 -39Appendix 4 Changes to Protocol This study was conducted in accordance with the approved protocol with the following changes: 1. The protocol was amended to state that spring peeper tadpoles {Pseudacris crucifer) will be used as the test organism in this test, rather than the leopard frog tadpole (Ranapipiens). 2. The protocol was amended to state that a back-up study at slightly lower concentrations will be initiated soon after the definitive test is started in the event the definitive study not provide a NOEC or fails. Criteria for failure will be based on not meeting temperature and dissolved oxygen criteria as stated in the protocol. Additional criteria include >10% mortality in the controls or poor analytical data (recoveries much less than 70% of nominal). If the first test is judged acceptable the second test will be stopped, not reported and filed as data not used. If the first test is not acceptable, the data will not be reported and filed as data not used. The protocol was amended to add the proposed test dates, the test concentrations and the test substance identification. 3. The protocol was amended to add the Environmental Laboratory Project Number E07-0083 as assigned by the Sponsor after the protocol was executed. 4. Dilution water was passed through a UV sterilizer prior to use. This had no adverse impact on the study results. 5. Tadpoles were held for 5 days, rather than 14 days, prior to the test. This had no adverse impact on the study results. 6. The acceptance and selection criteria used to determine which of the two tests would be reported were slightly modified to include an evaluation of the dose response pattern and an evaluation of the quality of the no-observed-effect concentration (NOEC) estimate. This had no adverse impact on the study. p. 40 Wildlife International, Ltd. Project Number 454A-251 -40Appendix 5 Personnel Involved in the Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study: 1. Henry O. Krueger, Ph.D., Director of Aquatic Toxicology/Terrestrial Plants and Insects 2. Willard B. Nixon, Ph.D., Director of Chemistry 3. Tui Minderhout, Ph.D., Senior Biologist 4. Amy S. Blankinship, Laboratory Supervisor 5. Jon A. Macgregor, Scientist i