Document xjGNkmONBqxMLXdgBQgJzxdmE

P-1 ^ 2Z-377 L PFOS: A 96-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST WITH Lumbriculus variegatus FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-252A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0084 ASTM Standard E729-96 AUTHORS: Tui Minderhout, Ph.D. Jon A. MacGregor, B.S. Henry O. Krueger, Ph.D. STUDY INITIATION DATE: February 1, 2007 STUDY COMPLETION DATE: March 15, 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 USA 1-410-822-8600 Page 1 of 42 CONTAINS NO CBi Wildlife International, Ltd. Project Number 454A-252A 2- GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR 3M Corporation TITLE PFOS: A 96-Hour Static-Renewal Acute Toxicity Test with Lumbriculus variegatus WILDLIFE INTERNATIONAL, LTD PROJECT NUMBER 454A-252A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER E07-0084 STUDY COMPLETION: March 15, 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 exceptions: Periodic analyses o f 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 P-3 Wildlife International, Ltd. Project Number 454A-252A -3 QUALITY 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 Initial Trial 454A-252 Test Substance Preparation Observations Matrix Fortification Definitive Test 454A-252A Observations Matrix Fortification Analytical Data, Biological Data and Draft Report Final Report DATE REPORTED TO: DATE CONDUCTED: STUDY DIRECTOR. MANAGEMENT: January 22, 2007 January 22, 2007 February 12,2007 February 2, 2007 February 8, 2007 February 9, 2007 February 5,2007 February 8, 2007 February 9, 2007 February 9,2007 February 12, 2007 February 14, 2007 March 2, 2007 March 2, 2007 March 7 - 8 , 2007 March 2,2007 March 2, 2007 March 8, 2007 March 5, 2007 March 5,2007 March 9, 2007 March 14, 2007 March 14, 2007 March 15,2007 All inspections were study-based unless otherwise noted. Linda R. Mitchell Date Director o f Regulatory and Ecotox Operations P-4 Wildlife International, Ltd. Project Number 454A-252A -4REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static-Renewal Acute Toxicity Test with Lumbriculus variegatus WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-252A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0084 STUDY DIRECTOR: fL (jbat f)i^b\)\o^J fui Minderhout, Ph.D. Senior Biologist PRINCIPAL INVESTIGATOR: Afrrd1 I f >o? Date -- i t * ----iM A . MacGregor, B.S Scientist Date WILDLIFE INTERNATIONAL. LTD. MANAGEMENT: /r Henry O.TCrueger, Ph.D. Date Director of Aquatic Toxicology/Terrestrial Plants and Insects Willard B. Nixon, Ph.D Director o f Chemistry i/f/ff Date /60 P-5 Wildlife International, Ltd. Project Number 454A-252A -5TABLE OF CONTENTS Title 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 jectiv 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.............................................................................................10 Analytical Sampling.....................................................................................................................11 Analytical Method........................................................................................................................11 Environmental Conditions........................................................................................................... 12 Observations................................................................................................................................. 13 Statistical Analyses..................................................................................................................... 13 Results and Discussion.............................................................................................................................. 14 Measurement of Test Concentrations..........................................................................................14 Observations and Measurements.................................................................................................14 C onclusions................................................................................................................................................ 15 R eferences.................................................................................................................................................. 16 P-6 Wildlife International, Ltd. Project Number 454A-252A 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 in Composite of Negative Control at Test Termination..................19 Table 4. Cumulative Mortality and Observations...............................................................................20 Table 5. LC50 Values.......................................................................................................................... 23 Figure 1. Concentration-Response Curve (96-Hour Mortality Data)................................................24 APPENDICES Appendix 1. Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test..................25 Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water................................................................................................................. 26 Appendix 3. The Analysis of PFOS in Freshwater..........................................................................28 3.1 Analytical Method Flowchart for the Processing of PFOS inFreshwater................29 3.2 Typical HPLC/MS/MS Operational Parameters....................................................... 30 3.3 Analytical Stocks and Standards Preparation............................................................ 31 3.4 Example Calculations for a Representative Sample.................................................32 3.5 Quality Control Samples of PFOS in Freshwater.....................................................34 3.6 Representative Calibration Curve for PFOS............................................................. 35 3.7 Representative Chromatogram of a Low-level PFOS Calibration Standard........... 36 3.8 Representative Chromatogram of a High-level PFOS Calibration Standard........... 37 3.9 Representative Chromatogram of a Matrix Blank Sample....................................... 38 3.10 Representative Chromatogram of a Matrix Fortification Sample.............................39 3.11 Representative Chromatogram of a Test Sample...................................................... 40 Appendix 4. Changes to Protocol.....................................................................................................41 Appendix 5. Personnel Involved in the Study.................................................................................42 /X p.7 Wildlife International, Ltd. Project Number 454A-252A -7SUMMARY SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static-Renewal Acute Toxicity Test with Lumbriculus variegatus WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-252A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0084 TEST DATES: LENGTH OF EXPOSURE: Experimental Start: Biological Termination: Experimental Termination: 96 Hours February 26, 2007 March 2, 2007 March 2, 2007 TEST ORGANISMS: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: Oligochaete (Lumbriculus variegatus) Wildlife International, Ltd. Cultures Easton, Maryland 21601 Adult at test start TEST CONCENTRATIONS: Nominal Negative Control 0.80 mg a.i./L 1.5 mg a.i./L 3.0 mg a.i./L 6.0 mg a.i./L 12 mg a.i./L Mean Measured < LOQ 0.71 mg a.i./L 1.4 mg a.i./L 2.8 mg a.i./L 5.6 mg a.i./L 11 mg a.i./L RESULTS: Based on mean measured concentrations: 96-Hour LC50: 95% Confidence Interval: No-Mortality Concentration: No-Observed-Effect Concentration: 5.6 mg a.i./L 2 .8 -1 1 mg a.i./L 2.8 mg a.i./L 2.8 mg a.i./L (01 p.8 Wildlife International, Ltd. Project Number 454A-252A 8- - 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 was conducted from February 5 to 9, 2007 but was repeated at lower concentrations to identify a no-observed-effect concentration (NOEC). The in-life phase of the definitive toxicity test was conducted from February 26 to March 2, 2007. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-252A in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of this study was to determine the acute effects of perfluorooctanesulfonate, potassium salt (PFOS) on the oligochaete, Lumbriculus variegatus, during a 96-hour exposure period under static-renewal test conditions. EXPERIMENTAL DESIGN Adult oligochaetes were exposed to a geometric series of five test concentrations and a negative control (dilution water) for 96 hours under static-renewal conditions. Ten replicate test chambers were maintained in each treatment and control group, with a single organism in each test chamber for a total of 10 oligochaetes per concentration. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results of exploratory range finding toxicity data and the initial trial. Nominal test concentrations selected were 0.80, 1.5, 3.0, 6.0 and 12 mg PFOS active ingredient (a.i.)/L. Test solutions were renewed at approximately 48 hours. 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 following renewal at 48 hours, and at the end of the test. Oligochaetes were impartially assigned to test chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 3, 24, 48, 72 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups were 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. P-9 Wildlife International, Ltd. Project Number 454A-252A -9- MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A 96Hour Static-Renewal Acute Toxicity Test with Lumbriculus variegatus". The protocol was based on procedures outlined in the U.S. Environmental Protection Agency Report number 600/R-99/064 Methods fo r Measuring the Toxicity and Bioaccumulation o f Sediment-Associated Contaminants with Freshwater Invertebrates (1) and ASTM Standard E729-96 Standard Guide fo r Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates and Amphibians (2). 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 on October 29, 1998. It was assigned Wildlife International, Ltd. identification number 4675A 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 The oligochaete, Lumbriculus variegatus, was selected as the test species for this study. Oligochaetes are representative of an important group of aquatic invertebrates and were selected for use in the test based upon past history of use and ease of culturing in the laboratory. Adult oligochaetes used in the test were from Wildlife International, Ltd. cultures. The organisms were originally obtained from Environmental Consulting and Testing (ECT), Superior, Wisconsin. Prior to the test, the organisms were held in a container with paper toweling and overlying water from the same source and at approximately the same temperature as that used in the test. Oligochaetes were fed with a mixture of yeast, cereal grass media, and trout chow (YCT), during the holding time but were not fed during the test. During the 2-week period immediately preceding the test, water temperatures in the holding container ranged from 23.3 to 24.4C, measured with a hand-held liquid-in-glass thermometer. The pH of the water ranged from 8.0 to 8.4, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved oxygen ranged from 7.2 to 7.9 mg/L (>85% of saturation), measured with a Yellow Springs Instruments Model 5IB dissolved oxygen meter. Wildlife International, Ltd. Project Number 454A-252A - 10- The organisms showed no signs of disease or stress during the holding period. At test initiation, oligochaetes were collected from the holding aquaria and indiscriminately transferred one at a time to each test chamber. All transfers were made below the water surface using wide-bore pipettes. 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 to remove fine particles. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the well water are presented in Appendix 2. Test Apparatus Test chambers were 30-mL Nalgene polypropylene plastic beakers filled with approximately 20 mL of water with no substrate provided. The depth of the test water in a representative chamber was 2.2 cm. Test chambers were positioned in a temperature-controlled chamber to maintain a temperature of 23 1C. Test chambers were covered with plastic wrap and were labeled with the project number, test concentration and replicate. Preparation of Test Concentrations A stock solution was prepared at a nominal concentration of 12 mg a.i./L, the highest concentration tested, by mixing a calculated amount of PFOS into dilution water (Wildlife International, Ltd. well water). The stock solution was mixed by sonication for 15 minutes followed by inversion. The stock appeared clear and colorless. Aliquots of the 12 mg a.i./L stock solution were proportionally diluted with well water to prepare 300 mL of test solution at nominal concentrations of 0.80, 1.5, 3.0 and 6.0 mg a.i./L. The solutions were mixed by inversion. All test solutions were adjusted to 100% active ingredient during preparation, based on the test substance Wildlife International, Ltd. Project Number 454A-252A - 11 - purity (86.9%). Test solutions were prepared for test initiation and renewal on day 2. All surviving oligochaetes were transferred from old to new solutions at approximately 48 hours. At test initiation and termination, all solutions appeared clear and colorless. Analytical Sampling At the beginning of the test and on day 2, samples were collected from the newly prepared batches of test solution to determine concentrations of the test substance. Prior to renewal at approximately 48 hours and at test termination, samples of test solution were collected from each test chamber and pooled by treatment group for analysis of test substance concentrations. All samples were collected at mid-depth, placed in plastic vials, and processed immediately for analysis. 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 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 a 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 p. 12 Wildlife International, Ltd. Project Number 454A-252A - 12- 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 0.200 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 (4.00). 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 0.500, 3.00 and 15.0 mg a.i./L. The measured concentrations for the matrix fortification samples ranged from 96.4 to 108% of nominal concentrations (Appendix 3.5). A representative calibration curve is presented in Appendix 3.6. Representative chromatograms oflow 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. 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 oflow light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity at test initiation, measured using a SPER Scientific Model 840006C light meter, was 648 lux at the surface of the water of one representative test chamber. The target test temperature during the study was 23 1C. Temperature was measured in two alternate replicates at the beginning of the test, prior to and after each renewal (old and new solutions) and at the end of the test (old solution) using a liquid-in-glass thermometer. Temperature was also Wildlife International, Ltd. Project Number 454A-252A - 13 - monitored daily in a container of water adjacent to the test chambers in the environmental chamber using a continuous temperature recorder. Dissolved oxygen and pH were measured in samples collected from batches of new test solutions or were measured in composite samples of old solutions. Old solutions from the replicates of each test group were combined and a composite sample collected for analysis. 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 meter. Hardness, alkalinity and specific conductance were measured in the dilution water at test initiation and in the composite of negative control at test 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 Methodsfo r the Examination o f Water and Wastewater (3). Observations Observations of mortality were made periodically in each treatment group. Lethality is defined as the lack of visible movement in the oligochaete. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 3, 24, 48, 72 and 96 hours after test initiation. Statistical Analyses The mortality data were analyzed using the computer program of C. E. Stephan (4). 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 (5, 6 and 7). In this study, the binomial 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. Wildlife International, Ltd. Project Number 454A-252A - 14- RESULTS AND DISCUSSION Measurement of Test Concentrations Nominal concentrations selected for use in this study were 0.80, 1.5, 3.0, 6.0 and 12 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 92 to 97% of the nominal concentrations. Samples collected prior to and after renewal of the test solutions on day 2 had measured concentrations that ranged from 88 to 93% and 85 to 94%, respectively, of the nominal concentrations. Samples collected at test termination had measured concentrations that ranged from 88 to 97% of the nominal concentrations. When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations for this study were 0.71, 1.4, 2.8, 5.6 and 11 mg a.i./L, representing 89, 93, 93, 93 and 92% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations. Observations and Measurements Measurements of temperature, dissolved oxygen and pH of the water in the test chambers are presented in Table 2. Water temperatures were within the 23 1C range established for the test. Dissolved oxygen concentrations remained >7.4 mg/L (>87% of saturation) throughout the test. Measurements of pH ranged from 8.2 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 for mortality and signs of toxicity during the test are presented in Table 4. Oligochaetes in the negative control group appeared normal throughout the test. All oligochaetes in the 0.71, 1.4 and 2.8 mg a.i./L treatment groups also appeared normal throughout the test, with no mortality or overt signs of toxicity observed. Percent mortality at test termination in the 5.6 and 11 mg a.i./L treatment groups was 50 and 100%, respectively. The no-mortality concentration and the NOEC were both 2.8 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. Wildlife International, Ltd. Project Number 454A-252A - 15 - CONCLUSIONS The oligochaete, Lumbriculus variegatus, was exposed for 96 hours under static-renewal conditions to five mean measured concentrations of PFOS ranging from 0.71 to 11 mg a.i./L. The 96-hour LC50 value was 5.6 mg a.i./L, with a 95% confidence interval of 2.8 to 11 mg a.i./L. The no-mortality concentration and the NOEC were both 2.8 mg a.i./L. /// Wildlife International, Ltd. Project Number 454A-252A - 16REFERENCES 1 U.S. Environmental Protection Agency. 2000. Methods fo r Measuring the Toxicity and Bioaccumulation o f Sediment-Associated Contaminants with Freshwater Invertebrates. EPA 600/R-99/064. 2 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. 3 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. 4 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 5 Thompson, W.R. 1947. Bacteriological Reviews. Vol. II, No. 2. Pp. 115-145. 6 Stephan, C.E. 1977. "Methods for Calculating an LC50," Aquatic Toxicology and Flazard Evaluations. American Society for Testing and Materials. Publication Number STP 634, pp 65-84. 7 Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London. Wildlife International, Ltd. Project Number 454A-252A - 17Table 1 Measured Concentrations of PFOS in Freshwater Samples Nominal Test Concentration (mg a.i./L) Sample Number (454A-252A-) Sampling Time (Hours) Measured Concentration PFOS (mg a.i./L) 1 Percent of N om inal1 Mean Measured Concentration (mg a.i./L) Mean Measured Percent of Nominal 0.00 1 0(new) < LOQ2 < LOQ (Negative Control) 7 48(new) < LOQ - 13 48(old) < LOQ - 19 96(old) < LOQ -- 0.80 2 0(new) 0.749 93.7 0.71 8 48(new) 0.683 85.4 14 48(old) 0.707 88.4 20 96(old) 0.700 87.5 1.5 3 0(new) 1.37 91.6 1.4 9 48(new) 1.39 92.4 15 48(old) 1.35 90.1 21 96(old) 1.36 90.5 3.0 4 0(new) 2.87 95.7 2.8 10 48(new) 2.73 91.0 16 48(old) 2.80 93.3 22 96(old) 2.88 95.9 6.0 5 0(new) 5.80 96.7 5.6 11 48(new) 5.64 94.0 17 48(old) 5.33 88.9 23 96(old) 5.80 96.7 12 6 0(new) 11.4 94.9 11 12 48(new) 11.3 93.8 18 48(old) 11.0 91.5 24 96(old) 11.6 96.8 88.8 93.3 93.3 93.3 91.7 Results generated using Analyst version 1.4.1 software. Manual calculations may vary. 2 The limit o f quantitation (LOQ) was 0.200 mg a.i./L calculated as the product o f the lowest calibration standard (0.0500 pg a.i./mL) and the dilution factor o f the matrix blanks (4.00). //3 Wildlife International, Ltd. Project Number 454A-252A - 18- Table 2 Temperature, D issolved O xygen and pH o f Water in the Test Chambers Mean Measured Concentration (mg a.i./L) Negative Control OHour Temp.1 (C) 22.4 22.4 DO2 (mg/L) 8.3 -- pH2 8.2 -- 48 Hours (Old Solution Prior to Renewal) Temp.1 D O 2 (C) (mg/L) pH2 23.8 8.0 8.6 23.8 48 Hours (New Solution At Renewal) Temp. ' D O 2' (C) (mg/L) pH2 23.4 8.2 8.2 23.8 -- -- 96 Hours Temp.11 D O 2 (C) (mg/L) 23.3 8.3 23.3 -- pH2 8.6 -- 0.71 22.9 8.3 8.2 23.8 8.0 8.6 23.1 8.4 8.3 23.5 8.3 8.6 22.7 -- -- 23.9 -- -- 23.2 -- -- 23.5 -- -- 1.4 22.9 8.4 8.2 23.9 8.0 8.6 23.0 8.4 8.3 23.4 8.4 8.6 22.9 - - 23.8 - - 22.8 - - 23.5 - - 2.8 23.2 8.4 8.2 23.8 8.0 8.6 22.5 8.3 8.3 23.4 8.3 8.6 22.7 - - 24.0 - - 22.5 - - 23.7 - - 5.6 23.2 8.4 8.2 24.0 7.9 8.5 22.8 8.2 8.3 23.5 8.3 8.6 23.2 - - 23.8 - - 23.1 - - 23.645 - - 11 23.1 8.3 8.2 23.5 7.4 8.5 23.13 8.2 8.2 23.1s 8.2 8.6 22.9 -- -- 23.5 -- -- 23.23 -- -- 23.3s -- -- Manual temperature measurements replicate: day 0 (A, B); 48-hr old (C, D); 48-hr new (E, F); 96-hr old (G, H). Temperature measured continuously during the test ranged from 22.9 - 24.3C. 2 Dissolved oxygen and pH were measured in samples collected from batches o f new test solution or measured in composite samples o f old solutions. A dissolved oxygen concentration o f 5.1 mg/L represents 60% saturation at 23.0C in freshwater. 3 Manual temperature measurements were taken at 48 hours in replicates H and I due to mortality in replicate C. 4 Manual temperature measurements were taken at 96 hours in replicate I due to 100% mortality in replicate H. 5 Manual temperature measurements were taken at 96 hours in replicate D and H due to 100% mortality in replicate G.________ p. 18 Wildlife International, Ltd. Project Number 454A-252A - 19- Table 3 Specific Conductance, Hardness and Alkalinity Measured in Dilution Water at Test Initiation and in Composite of Negative Control at Test Termination Parameter Specific Conductance (pmhos/cm) Hardness (mg/L as CaC03) Alkalinity (mg/L as CaC03) Day 0 300 128 180 Day 4 320 138 190 Wildlife International, Ltd, -20- Table 4 Cumulative Mortality and Observations Mean Measured Concentration (mg a.i./L) Negative Control Replicate1 A B C D E F G H I J 3 Hours No. Dead2 Obs.3 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0.71 A B C D E F G H I J 0 0 0 0 0 0 0 0 0 0 1 A single midge was exposed in each replicate. 2 Cumulative number of dead midges. 3 Observations: AN = appear normal. 4 T = transferred to new test solution. AN AN AN AN AN AN AN AN AN AN 24 Hours No. Dead2 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN acO* 48 Hours (T)4 No. Dead2 Obs.3 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN Project Number 454A-252A 72 Hours No. Dead2 Obs.3 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 96 Hours No. Dead2 Obs.3 Cumulative Percent Mortality 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 p. 20 Wildlife International, Ltd X) Mean Measured Concentration (mg a.i./L) 1.4 Replicate1 A B C D E F G H I J 3 Hours No. Dead2 Obs.3 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 2.8 A B C D E F G H I J 0 0 0 0 0 0 0 0 0 0 A single midge was exposed in each replicate. 2 Cumulative number o f dead midges. 3 Observations: AN = appear normal. 4 T = transferred to new test solution. AN AN AN AN AN AN AN AN AN AN -21 - Table 4 (Continued) Cumulative Mortality and Observations 24 Hours No. Dead2 Obs.3 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 48 Hours (T)4 No. Dead2 Obs.3 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN Project Number 454A-252A 72 Hours No. Dead2 Obs.3 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 96 Hours No. Dead2 Obs.3 Cumulative Percent Mortality 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 p. 21 Wildlife International, Ltd, -22- Table 4 (Continued) Cumulative Mortality and Observations Mean Measured Concentration (mg a.i./L) 5.6 Replicate1 A B C D E F G H I J 3 Hours No. Dead2 Obs.3 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 11 A 0 D B 0D C 0W D 0 AN E 0D F 0 D, W G 0D H 0D I 0 AN J 0 D, W 1 A single midge was exposed in each replicate. 2 Cumulative number o f dead midges. 3 Observations: AN = appear normal, D = pale, W = fragmentation. 4 T = transferred to new test solution. 24 Hours No. Dead2 Obs.3 0 AN 0 D, W 0 AN 0D 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 1-- 10 D, W 0 AN 1~ 10D 0D 0 AN 1- 48 Hours (T)4 No. Dead2 Obs.3 0 AN 10 AN 10D 0 AN 0 AN 10 AN 0 AN 1110D 1110D 0D 1- Project Number 454A-252A 72 Hours No. Dead2 Obs.3 0D 10D 10D 0 AN 0 AN 10D 0 AN 1111111111- 96 Hours No. Dead2 Obs.3 Cumulative Percent Mortality 0D 10D 11-- 0 AN 0 AN 10D 1-- 50 1111111111-- 100 p. 22 Wildlife International, Ltd. Project Number 454A-252A -23 Table 5 LC50 Values Time LC50 (mg a.i./L) 95% Confidence Interval (mg a.i./L) Statistical Method 24 Hours 11 >5.6` Binomial Probability 48 Hours 7.9 >2.82 Binomial Probability 72 Hours 6.5 2.8-11 Binomial Probability 96 Hours 5.6 2.8-11 Binomial Probability 1 At a confidence level of 95% the binomial test shows that the LC50 is above 5.6 mg a.i./L. 2 At a confidence level of 95% the binomial test shows that the LC50 is above 2.8 mg a.i./L. Wildlife International, Ltd. Project Number 454A-252A -24Figure 1 Concentration-Response Curve (96-Hour Mortality Data) Concentration (mg a.i./L) /zo Wildlife International, Ltd. Project Number 454A-252A -25 - 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) 182 (N = 4) 8.1 (N = 4) Range 285-300 132-140 180-184 7.9-8.1 /V Wildlife International, Ltd Project Number 454A-252A -26- Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Component Pesticides and Organics Measured Concentration (Pg/L) Component Aldrin Alpha BHC Alpha Chlordane Beta BHC Bolstar Chlordane Coumaphos Delta BHC Demeton-0 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 <0.019 < 0.0096 < 0.0096 <0.038 < 1.9 <0.48 <2.9 < 0.0096 < 1.9 < 1.9 < 1.9 < 1.9 < 0.029 < 1.9 < 1.9 < 0.0096 <0.019 <0.019 <0.019 < 0.096 <0.019 <3.8 < 1.9 < 1.9 < 1.9 < 1.9 <3.8 < 1.9 < 0.0096 < 0.096 <3.8 < 0.096 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 (PS/L) < 0.0096 < 0.0096 <0.19 < 1.9 < 1.9 < 0.096 < 1.9 < 1.9 <0.11 <2.9 <0.019 <0.019 <0.019 <0.019 <0.019 <0.019 <0.48 <0.48 <0.48 <0.48 <0.48 <0.48 <0.48 < 1.9 < 1.9 < 1.9 < 0.0096 < 1.9 <0.96 < 1.9 < 1.9 1Analyses performed by Lancaster Laboratories on samples collected on December 15, 2005. Wildlife International, Ltd Project Number 454A-252A -27- Appendix 2 (Continued) Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Metals Measured Concentration Measured Concentration Component__________________ (mg/L)_________________Component__________________(mg/L) Aluminum Antimony Arsenic Barium Beryllium Bromide Cadmium Calcium Chloride Chromium Cobalt Copper Fluoride Iron Lead < 0.200 < 0.0200 < 0.0200 < 0.0050 < 0.0050 <2.5 < 0.0050 33.1 2.7 <0.0150 <0.0050 <0.0100 0.56 < 0.200 < 0.0200 Magnesium Manganese Mercury Nickel Nitrate Nitrogen Nitrite Nitrogen Potassium Selenium Silver Sodium Sulfate Thallium Vanadium Zinc 13.3 < 0.0050 < 0.00020 <0.0100 <0.50 <0.50 7.65 < 0.0200 < 0.0050 19.1 <5.0 < 0.0200 < 0.0050 < 0.0200 1Analyses performed by Lancaster Laboratories on samples collected on December 15, 2005. Wildlife International, Ltd. Project Number 454A-252A -28 Appendix 3 The Analysis of PFOS in Freshwater Wildlife International, Ltd. Project Number 454A-252A -29Appendix 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. Prepare matrix fortification samples in well water using volumetric flasks, volumetric pipettes, 15-mL culture 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. Transfer aliquots of final sample dilutions and calibration standards to autosampler vials for analysis by LC/MS/MS. /25 Wildlife International, Ltd. Project Number 454A-252A -30Appendix 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 multiple-reaction monitoring (MRM) mode. Turboion Spray Zorbax RX-C8(150 mm x 2.1 mm, 5 pm particle size) 5.00 minutes 0.300 mL/minute 40C 80% CH3OH : 20% H20 containing 0.1% formic acid 10.0 pL Approximately 2.8 minutes 499 --*99 amu /ZC, Wildlife International, Ltd. Project Number 454A-252A -31 Appendix 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 1.00 mg a.i./mL secondary stock solution was used to prepare concurrent matrix fortification samples (QC) for this study. The 0.100 mg a.i./mL secondary stock solution was used to prepare calibration standards. The calibration standards were prepared in acetonitrile: FIPLC-grade bottled water (50:50, v/v). The following shows the dilution scheme for the set of calibration standards. Stock Concentration (mg a.i./mL) 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 fug a.i./mL) 0.0500 0.150 0.250 0.500 1.00 n7 Wildlife International, Ltd. Project Number 454A-252A -32- Appendix 3.4 Example Calculations for a Representative Sample The analytical result and percent recovery for sample number 454A-252A-2, an exposure sample prepared at a nominal concentration of 0.80 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./F 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./F) = Dilution Factor - Finear 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-252A-2 are summarized below: Peak area = 1066000 Constant Coefficient = -6561.21 Finear Coefficient = 5757530 Quadratic Coefficient = -174737 Dilution Factor (Vfmal/Vimtial): = 4.00 Wildlife International, Ltd. Project Number 454A-252A -33 Appendix 3.4 (Continued) Example Calculations for a Representative Sample 5757530 + V(5757530)2 - [(4 (-174737)) (-6561.21-1066000)] PFOS = 4.00 2 .(-174737) PFOS = 4.00 0.18735 mg a.i./L PFOS = 0.749 mg a.i./L The measured concentration was compared to the nominal concentrations as follows: Percent of nominal concentration = _____ PFOS in PFOS nominal sample (mg a.i./L)_____ concentration (mg a.i./L) X 100 _ 0.749 mg/L 0.800 mg/L X 100 = 93.7% Wildlife International, Ltd. Project Number 454A-252A -34Appendix 3.5 Quality Control Samples of PFOS in Freshwater Sample Number (454A-252A-) 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 0.500 3.00 15.0 0.500 3.00 15.0 0.500 3.00 15.0 M easu red 1,2 < LOQ < LOQ < LOQ 0.540 3.11 14.9 0.486 3.01 14.5 0.490 3.07 14.6 Percent Recovery 1 __ -- - 108 104 99.3 97.2 100 96.4 98.0 102 97.3 X=100 S.D.=3.81 C.V.=3.81% 1 Results generated using Analyst version 1.4.1 software. Manual calculations may vary. 2 The limit o f quantitation (LOQ) was 0.200 mg a.i./L calculated as the product o f the lowest calibration standard (0.0500 pg a.i./mL) and the dilution factor o f the matrix blanks (4.00). Wildlife International, Ltd. Project Number 454A-252A -35- Appendix 3.6 Representative Calibration Curve for PFOS ( /x" y= *2+ x+-6 (r=C22607_DQrtb FFCS): "OBjtkf Rsg^saon("1 westing): -1.75&KE5 5.76&KE6 56et003 0:9998) 5S6 &5s6 5L0B6 4.5e6- 4.0 e6 - 3506- _,y'' 's ' a0e6 : 25e6 20e6 1.5b6 - 1.006- y ,' aces r 0.05 0.10 Q15 Q20 Q25 030 Q36 040 045 050 055 Q60 066 070 0.75 080 085 090 095 1X) Cbrrertcticn, irjaiVni. Linear coefficient=5757530; constant coefficient=-6561.21; quadratic coefficient= -174737; r=0.9998 Wildlife International, Ltd, Project Number 454A-252A -36Appendix 3.7 Representative Chromatogram of a Low-level PFOS Calibration Standard /3 l Wildlife International, Ltd Project Number 454A-252A -37Appendix 3.8 Representative Chromatogram of a High-level PFOS Calibration Standard Nominal concentration: 1.00 mg a.i./L t33 Wildlife International, Ltd. Project Number 454A-252A -38Appendix 3.9 Representative Chromatogram of a Matrix Blank Sample Sample number 454A-252A-MAB-1. Dilution factor = 4.00X. The arrow indicates the retention time of PFOS. Wildlife International, Ltd. Project Number 454A-252A -39Appendix 3.10 Representative Chromatogram of a Matrix Fortification Sample Sample number: 454A-252A-MAS-1, nominal concentration 0.500 mg/L. Dilution factor = 4.00X. p. 40 Wildlife International, Ltd, Project Number 454A-252A -40Appendix 3.11 Representative Chromatogram of a Test Sample Sample number: 454A-252A-2, Day 0, nominal concentration 0.80 mg a.i./L. Dilution factor = 4.00X. i3 y p. 41 Wildlife International, Ltd. Project Number 454A-252A -41 Appendix 4 Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to include measurement of the temperature in two alternate replicates, rather than in each replicate, at the beginning of the test, prior to and after each renewal (old and new solutions) and at the end of the test (old solution) using a liquid-in-glass thermometer. 2. The protocol was amended to include monitoring of the temperature daily in a container of water adjacent to the test chambers in the environmental chambers using a continuous temperature recorder, rather than in the negative control chamber. 3. The protocol was amended to include measurement of dissolved oxygen and pH in samples collected from batches of new test solutions or measured in composite samples of old solutions. Old solutions from the replicates of each test group were combined and a composite sample collected for analysis. 4. The protocol was amended to include the Environmental Laboratory Project Number of E070084. 5. The protocol was amended to change the test concentrations for the repeat test to 0.80, 1.5, 3.0, 6.0 and 12 mg a.i./L. 6. The test organisms were held in a container with paper toweling and overlying water, rather than in container with sand and overlying water. During the holding period test organisms were fed with a mixture of YCT only without the supplement of green algae. This had no adverse impact on the study results. p. 42 Wildlife International, Ltd. Project Number 454A-252A -42 Appendix 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, Aquatics 5. Jon A. MacGregor, Scientist