Document MJaMg7md0NQ1Qv8K7KQpXLZ4V

P-1 PFOS: A 96-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST WITH Hyalella azteca FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-253A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0082 ASTM Standard E729-96 AUTHORS: Tui Minderhout, Ph.D. Jon A. MacGregor, B.S. Henry O. Krueger, Ph.D. STUDY INITIATION DATE: February 15, 2007 STUDY COMPLETION DATE: March 29, 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 43 CONTAINS NO CB: so P-2 Wildlife International, Ltd. Project Number 454A-253A 2- GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR 3M Corporation TITLE PFOSi A 96-Hour Static-Renewal Acute Toxicity Test with Hyalella azteca WILDLIFE INTERNATIONAL, LTD PROJECT NUMBER 454A-253A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER E07-0082 STUDY COMPLETION March 29, 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 o f well water for potential contaminants were performed using a certified laboratory and standard U S EPA analytical methods STUDY DIRECTOR: V Senior Biologist SPONSOR APPROVAL Date 5/ P-3 Wildlife International, Ltd. Project Number 454A-253A -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 of 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-253A Test Substance Preparation Definitive Test: 454A-253A Matrix Fortification Observations Analytical Data and Draft Report Biological Data and Draft Report Final Report DATE REPORTED TO: DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT: February 19, 2007 February 19,2007 February 20,2007 February 16,2007 February 16,2007 February 21,2007 March 9, 2007 March 9, 2007 March 9, 2007 March 9, 2007 March 20 and 21, 2007 March 21, 2007 March 19-21,2007 March 21,2007 March 29, 2007 March 29,2007 March 15, 2007 March 15, 2007 March 22, 2007 March 26, 2007 March 29, 2007 All inspections were study-based unless otherwise noted. c :lyvV> James H. Coleman Quality Assurance Representative 3-J4-Q 7 Date 52 P-4 Wildlife International, Ltd. project Number 454A-253A -4REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static-Renewal Acute Toxicity Test with Hyalella azteca WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-253A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0082 STUDY DIRECTOR: ui /lifu U U ^ Tui Mindeihout, Ph D. Senior Biologist 93 ja g J ~co'? Date PRINCIPAL INVESTIGATOR: (//is i/i A, t kin A. MacGregor, B. A''Sirciientist Date WILDLIFE INTERNATIONAL. LTD MANAGEMENT 0 24 Henr^'UD."Krueger, Ph.D. Date Director o f Aquatic Toxicology/Terrestrial Plants and Insects Willard B. Nixon,'Ph.D. Director of Chemistry m Date 07 Wildlife International, Ltd. Project Number 454A-253A -5TABLE OF CONTENTS Title Page...................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement.................................................................................... 2 Quality Assurance Statement..................................................................................................................... 3 Report Approval.......................................................................................................................................... 4 Table of Contents........................................................................................................................................ 5 Summary......................................................................................................................................................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.............................................................................................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 References 16 P-6 Wildlife International, Ltd. Project Number 454A-253A 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 DilutionWater at Test Initiation and in Composite of Negative Control at Test Termination..................19 Table 4. Cumulative Mortality and Clinical Observations................................................................ 20 Table 5. LC50 Values.......................................................................................................................... 24 Figure 1. Concentration-Response Curve (96-Hour Mortality Data)................................................25 APPENDICES Appendix 1. Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test..................26 Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water................................................................................................................. 27 Appendix 3. The Analysis of PFOS in Freshwater..........................................................................29 3.1 Analytical Method Flowchart for the Processing of PFOS in Freshwater...............30 3.2 Typical HPLC/MS/MS Operational Parameters....................................................... 31 3.3 Analytical Stocks Preparation.................................................................................... 32 3.4 Example Calculations for a Representative Sample.................................................33 3.5 Quality Control Samples of PFOS in Freshwater..................................................... 35 3.6 Representative Calibration Curve for PFOS............................................................. 36 3.7 Representative Chromatogram of a Low-level PFOS Calibration Standard............37 3.8 Representative Chromatogram of a High-level PFOS Calibration Standard.......... 38 3.9 Representative Chromatogram of a Matrix Blank Sample.......................................39 3.10 Representative Chromatogram of a Matrix Fortification Sample............................40 3.11 Representative Chromatogram of a Test Sample......................................................41 Appendix 4. Changes to Protocol.....................................................................................................42 Appendix 5. Personnel Involved in the Study.................................................................................43 55 p.7 Wildlife International, Ltd. Project Number 454A-253A -7SUMMARY SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static-Renewal Acute Toxicity Test with Hyalella azteca WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-253A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0082 TEST DATES: LENGTH OF EXPOSURE: Experimental Start: Biological Termination: Experimental Termination: 96 Hours March 5,2007 March 9, 2007 March 9, 2007 TEST ORGANISMS: Amphipod (Hyalella azteca) SOURCE OF TEST ORGANISMS: Environmental Consulting and Testing Superior, Wisconsin AGE OF TEST ORGANISMS: 12 days old at test initiation TEST CONCENTRATIONS: Nominal Negative Control 13 mg a.i./L 25 mg a.i./L 50 mg a.i./L 100 mg a.i./L 200 mg a.i./L Mean Measured < LOQ 13 mg a.i./L 25 mg a.i./L 51 mg a.i./L 102 mg a.i./L 199 mg a.i./L RESULTS: Based on mean measured concentrations: 96-Hour LC50: 95% Confidence Interval: No-Mortality Concentration: No-Observed-Effect Concentration 15 mg a.i./L 4.5 - 24 mg a.i./L <13 mg a.i./L <13 mg a.i./L p.8 Wildlife International, Ltd. Project Number 454A-253A 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 19 to 22, 2007 but was terminated due to unacceptable percent survival in the negative control possibly caused by stress of organisms during shipment. The in-life phase of the definitive toxicity test was conducted from March 5 to 9, 2007. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-253A 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 amphipod, Hyalella azteca, during a 96-hour exposure period under static-renewal test conditions. EXPERIMENTAL DESIGN Amphipods 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 groups while twenty replicates were maintained in the control group. A single organism was placed in each test chamber for a total of 10 amphipods per treatment concentration and twenty amphipods per negative control. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results of exploratory range finding toxicity data. Nominal test concentrations selected were 13, 25, 50, 100 and 200 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 renewal at 48 hours, and at the end of the test. Amphipods were impartially assigned to test chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 4.5, 24, 48, 72 and 96 hours after test initiation. 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. S '7 P-9 Wildlife International, Ltd. Project Number 454A-253A -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 Hyalella azteca". The protocol was based on procedures outlined in the U.S. Environmental Protection Agency Report number 600/R-99/064 Methods for Measuring the Toxicity and Bioaccumulation of Sediment-Associated Contaminants with Freshwater Invertebrates (1) and ASTM Standard E729-96 Standard Guide for 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 amphipod, Hyalella azteca, was selected as the test species for this study. Amphipods 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. Amphipods used in the test were obtained from Environmental Consulting and Testing (ECT), Superior, Wisconsin. Amphipods were hatched on February 21, 2007 and were 12 days old at test initiation. Prior to the test, the organisms were held for 6 days in a container with pieces of gauze and overlying water from the same source and at approximately the same temperature as that used in the test. Amphipods were fed YCT (1.8 g/L) and Tetramin flake food during the holding period and YCT on days 0 and 2 of the test. During the 6 days immediately preceding the test, water temperatures in the holding container ranged from 21.8 to 23.4C, measured with a hand-held liquid-in-glass thermometer. The pH of the water ranged from 8.1 to 8.5, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved oxygen ranged from 6.4 to 8.0 mg/L (>75% of saturation), measured with a Yellow Springs Instruments Model 5IB dissolved oxygen meter. Wildlife International, Ltd. Project Number 454A-253A - 10- The organisms showed no signs of disease or stress during the holding period. At test initiation, amphipods were collected from the holding container and placed in one or more transfer containers, then 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 and passed through an ultraviolet (UV) 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 are presented in Appendix 2. Test Apparatus Test chambers were 30-mL Nalgene plastic beakers filled with approximately 20 mL of water. The depth of the test water in a representative chamber was 1.9 cm. The cups had a piece of Nitex screen placed in each of the cup as substrate for the organisms. Test chambers were positioned in a temperature-controlled chamber to maintain a temperature of 23 1C. Test chambers were covered with plastic 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 200 mg a.i./L, the highest concentration tested, by mixing a calculated amount of PFOS into dilution water (Wildlife International, Ltd. UV sterilized well water). The stock solution for day 0 was mixed by stirring overnight and was sonicated for approximately 5 to 10 minutes the following day. For the day 2 solution, the stock solution was mixed by stirring overnight and was sonicated for 5 minutes the next Wildlife International, Ltd. Project Number 454A-253A - 11 - day. Both stock solutions appeared clear and colorless. Aliquots of the 200 mg a.i./L stock solution were proportionally diluted with well water to prepare 300 mL of test solution at nominal concentrations of 13, 25, 50 and 100 mg a.i./L. The solutions were mixed by inversion and all appeared clear and colorless. All test solutions were adjusted to 100% active ingredient during preparation, based on the test substance purity (86.9%). Test solutions were prepared for test initiation and renewal. All surviving amphipods 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 old test solutions were collected from each test chamber and pooled 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 Series 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 an Agilent Zorbax RX-CS 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 Wildlife International, Ltd. Project Number 454A-253A - 12- 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 2.00 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 (40.0). 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 5.00, 50.0 and 250 mg a.i./L. The measured concentrations for the matrix fortification samples ranged from 98.4 to 114% 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. 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 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 668 lux at the surface of the water of one representative test chamber. Wildlife International, Ltd. Project Number 454A-253A - 13 - 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 monitored daily in a container of water adjacent to the test chambers in the environmental chambers 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 a composite sample of negative control water at test termination. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-ConductivityTemperature meter. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methodsfor 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 amphipod. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 4.5, 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 probability was used to calculate the 24-hour LC50 value and the probit analysis was used to calculate the 48, 72 and 96-hour LC50 values. The no-mortality concentration and NOEC were determined by visual interpretation of the mortality and observation data. p. 14 Wildlife International, Ltd. Project Number 454A-253A - 14- RESULTS AND DISCUSSION Measurement of Test Concentrations Nominal concentrations selected for use in this study were 13, 25, 50, 100 and 200 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 100 to 105% of the nominal concentrations. Samples collected prior to renewal of the test solutions at 48 hours (old solutions), at renewal (new solutions) and at test termination had measured concentrations that ranged from 96.4 to 103%, 95 to 100% and 100 to 102% of the nominal concentrations, respectively. When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations for this study were 13, 25, 51, 102 and 199 mg a.i./L, representing 100, 100, 102, 102 and 99.5% 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 each test chamber are presented in Table 2. Water temperatures were within the 23 1C range established for the test. Dissolved oxygen concentrations remained >8.1 mg/L (>95% of saturation) throughout the test. Measurements of pH ranged from 8.1 to 8.6. The measurements of hardness, alkalinity and specific conductance in the dilution water at test initiation 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. Amphipods in the negative control group appeared normal throughout the test, with the exception of two lethargic amphipods that resulted in mortality at test termination. This was well within an acceptable level as indicated in the guideline. Percent mortality at test termination in the 13, 25 and 51 mg a.i./L treatment groups was 40, 80, and 80%, respectively. There was 100% mortality in the 102 and 199 mg a.i/L PFOS treatment groups. The no-mortality concentration and the NOEC were both <13 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-253A - 15 - CONCLUSIONS The amphipod, Hyalella azteca, was exposed for 96 hours under static-renewal conditions to five mean measured concentrations of PFOS ranging from 13 to 199 mg a.i./L. The 96-hour LC50 value was 15 mg a.i./L, with a 95% confidence interval of 4.5 to 24 mg a.i./L. The slope of the concentration-response curve was 2.4. The no-mortality concentration and the NOEC were both <13 mg a.i./L. Wildlife International, Ltd. Project Number 454A-253A - 16REFERENCES 1 U.S. Environmental Protection Agency. 2000. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-Associated Contaminants with Freshwater Invertebrates. EPA 600/R-99/064. 2 ASTM Standard E729-96. 1996. Standard Guide for 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 for the Examination of 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 Hazard Evaluations. American Society for Testing and Materials. Publication Number STP 634, pp 7 Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London. Wildlife International, Ltd Project Number 454A-253A - 17 Table 1 Measured Concentrations of PFOS in Freshwater Samples Nominal Test Concentration (mg a.i./L) Sample Number (454A-253A-) 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 o f Nominal 0.00 (Negative Control) 13 25 50 100 200 1 7 13 19 2 8 14 20 3 9 15 21 4 10 16 22 5 11 17 23 6 12 18 24 0(new) 48(new) 48(old) 96(old) 0(new) 48(new) 48(old) 96(old) 0(new) 48(new) 48(old) 96(old) 0(new) 48(new) 48(old) 96(old) 0(new) 48(new) 48(old) 96(old) 0(new) 48(new) 48(old) 96(old) < LOQ2 < LOQ < LOQ < LOQ 13.4 12.3 13.5 13.0 25.1 24.3 24.9 25.4 52.6 49.4 50.2 50.8 102 100 103 101 204 3 193 3 -- - - 103 95.0 103 100 100 97.0 99.6 101 105 98.9 100 102 102 100 103 101 102 ___3 96.4 3 < LOQ 13 25 51 102 199 100 100 102 102 99.5 Results generated using Analyst version 1.4.1 software. Manual calculations may vary. 2 The limit o f quantitation (LOQ) was 2.00 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 (40.0). 3 Study level not sampled due to 100% mortality. Value not used in statistical calculations. Wildlife International, Ltd. Project Number 454A-253A - 18 - Table 2 Temperature, Dissolved Oxygen and pH of Water in the Test Chambers Nominal Concentration (mg a.i./L) Negative Control 0 Hour Temp.1 (C) 22.4 22.5 DO2 (mg/L) 8.3 - 13 22.2 8.4 22.4 -- 25 22.6 8.5 22.7 -- 50 22.7 8.5 23.1 -- pH 8.2 -- 8.2 -- 8.2 - 8.2 -- 48 Hours (Old solution Prior to Renewal) Temp.1 DO 2 (C) (mg/L) pH 23.3 8.2 8.6 23.4 -- -- 23.4 8.1 8.6 23.3 " 23.4 8.1 8.6 23.3 - - 23.0 8.2 8.6 23.3 - -- 48 Hours (New Solution At Renewal) Temp.1 DO 2 (C) (mg/L) pH 23.4 8.4 8.2 23.7 -- -- 23.4 8.4 8.2 23.7 -- -- 23.4 8.5 8.3 23.5 - -- 23.4 8.4 8.2 23.8 -- -- 96 Hours T em p .1 (C) 23.1 23.3 DO2 (mg/L) 8.5 -- 23.4 8.4 23.1 " 23.1 8.4 23.1 - 23.3 8.3 23.4 - pH 8.5 -- 8.5 -- 8.5 -- 8.5 -- 100 22.9 8.5 8.2 23.1 8.2 8.6 23.6 8.4 8.3 23.44 8.3 8.5 23.1 -- -- 23.2 -- -- 23.7 -- -- 23.34 -- - 200 23.2 8.4 8.1 23.2 8.1 8.6 23.73 ___ __ ___ __ __ 23.4 -- -- 23.3 ---- 23.73 -- -- -- ---- 1 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 23.5 to 23.8C. 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 when appropriate volume o f solutions were available. A dissolved oxygen concentration of 5.1 mg/L represents 60% saturation at 23.0C in freshwater. 3 Manual temperature measurements were taken at 48 hours prior to observation in replicates D and J, due to 100% mortality, and discontinued. 4 Manual temperature measurements were taken at 96 hours in replicates A and H due to 100% mortality. p. 18 Wildlife International, Ltd. Project Number 454A-253 - 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 0 295 132 182 Day 4 320 136 186 Wildlife International, Ltd, Ni -20Table 4 Cumulative Mortality and Clinical Observations Mean Measured Concentration (mg a.i./L) Replicate.1 4.5 Hours No. Dead2 Obs.3 Negative Control A 0 AN B 0 AN C 0 AN D 0 AN E 0 AN F 0 AN G 0 AN H 0 AN I 0 AN J 0 AN K 0 AN L 0 AN M 0 AN N 0 AN 0 0 AN P 0 AN Q 0 AN R 0 AN S 0 AN T 0 AN Cumulative No. Dead 0 1 A single amphipod was exposed in each replicate. 2 Cumulative number of dead amphipods. 3 Observations: AN = appear normal; C = lethargy. 4 T = transferred to new test solution. 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 0 48 Hours (T) No. Dead2 Obs.3 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0C 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 Project Number 454A-253A 72 Hours No. Dead2 Obs.3 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0C 0 AN 0 AN 10 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 1 96 Hours Cumulative No. Dead2 Percent Obs.3 Mortality 0 AN 10 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 10 AN 0 AN 10 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 2 p. 20 Wildlife International, Ltd -21 Table 4 (Continued) Cumulative Mortality and Clinical Observations Mean Measured Concentration (mg a.i./L) Replicate1 13 A B C D E F G H I J Cumulative No. Dead 4.5 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 25 A B C D E F G H I J 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN Cumulative No. Dead 0 1 A single amphipod was exposed in each replicate. Cumulative number of dead amphipods. J Observations: AN = appear normal; C = lethargy. T = transferred to new test solution. 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 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 48 Hours (T) No. Dead2 Obs.3 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0C 0 AN 0 0 AN 0C 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0C 0 AN Project Number 454A-253A 72 Hours No. Dead2 Obs.3 0 AN 0C 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 10 AN 1 0C 10 AN 0C 10 AN 0C 1-- 10 AN 4 96 Hours Cumulative No. Dead2 Percent Obs.3 Mortality 0 AN 40 110 AN 0 AN 0 AN 0 AN 0 AN 114 1 .... 80 10 AN 1111110 AN 8 p. 21 Wildlife International, Ltd. Project Number 454A-253A -22Table 4 (Continued) Cumulative Mortality and Clinical Observations Mean Measured Concentration (mg a.i./L) Replicate1 51 A B C D E F G H I J Cumulative No'. Dead 4.5 Hours No. Dead2 Obs.34 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 102 A B C D E F G H I J 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN Cumulative No. Dead 0 1 A single amphipod was exposed in each replicate. 2 Number of dead amphipods at time of observation. 3 Observations: AN = appear normal; C = lethargy. 4 T = transferred to new test solution. 24 Hours No. Dead2 0 0 0 0 0 0 0 0 0 0 0 Obs.3 AN AN AN AN AN AN AN AN AN AN 0 AN 0 AN 0 AN 0 AN 0C 0 AN 0 AN 0 AN 0 AN 0 AN 0 48 Hours (T) No. Dead2 Obs.3 0C 10 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0C 0C 1 0 0 c c 1- 0c 1- 0c 1- 0c 1- 1- 5 72 Hours No. Dead2 Obs.3 1 __ 10 AN 11110 AN 1 18 1_ 11111 111 1- 10 96 Hours Cumulative No. Dead2 Percent 0 b s 3 Mortality 1 80 10 AN 1111~ 0 AN 118 1 100 11 1111~ 111- 10 p. 22 Wildlife International, Ltd, -23 Table 4 (Continued) Cumulative Mortality and Clinical Observations Mean Measured Concentration (mg a.i./L) Replicate1 4.5 Hours No. Dead2 Obs.3 199 A 0 AN B 0 AN C 0 AN D 0 AN E 0 AN F 0 AN G 0 AN H 0 AN I 0 AN J Cumulative No. Dead 0 AN 0 1 A single amphipod was exposed in each replicate. 2 Cumulative number o f dead. 3 Observations: AN = appear normal. 4 T = transferred to new test solution. 24 Hours No. Dead2 1 1 1 0 1 1 1 1 1 0 8 Obs.3 AN AN 48 Hours (T) No. Dead2 1 1 1 1 1 1 1 1 1 1 10 Obs.3 Project Number 454A-253A 72 Hours No. Dead2 1 1 1 1 1 1 1 1 1 1 10 Obs.3 -- - 96 Hours Cumulative Percent Mortality No. Dead2 Obs.3 1 - 100 11111111110 p. 23 Wildlife International, Ltd. Project Number 454A-253A -24Table 5 LC50 Values Time 24 Hours 48 Hours 72 Hours 96 Hours LC50 (mg a.i./L) 161 93 29 15 95% Confidence Interval (mg a.i./L) >102' 70 - 125 20-40 4.5 - 24 Statistical Method Binomial Probability Probit Analysis Probit Analysis Probit Analysis l At a confidence level of 95% the binomial test shows that the LC50 is above 102 mg a.i./L. Wildlife International, Ltd. Project Number 454A-253A -25 Figure 1 Concentration-Response Curve (96-Hour Mortality Data) Concentration (mg a.i./L) 1000 p. 26 Wildlife International, Ltd. Project Number 454A-253A -26- 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 290 (N = 4) 136 (N = 4) 182 (N = 4) 8.1 (N = 4) Range 285 - 295 132-140 180-184 8.1 75 Wildlife International, Ltd. Project Number 454A-253A -27 - 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 (Pg/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-253A -28Appendix 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 < 0.0050 < 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 Measured Concentration (mg/L) 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-253A -29 Appendix 3 The Analysis of PFOS in Freshwater p. 30 Wildlife International, Ltd. Project Number 454A-253A -30Appendix 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. I 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. Wildlife International, Ltd. Project Number 454A-253A -31 Appendix 3.2 Typical HPLC/MS/MS Operational Parameters INSTRUMENT: ION SOURCE: ANALYTICAL COLUMN: STOP TIME: PLOW 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 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 to p. 32 Wildlife International, Ltd. Project Number 454A-253A -32Appendix 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 irne 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 lug a.i./mL) 0.0500 0.150 0.250 0.500 1.00 8/ Wildlife International, Ltd. Project Number 454A-253A -33 Appendix 3.4 Example Calculations for a Representative Sample The analytical result and percent recovery for sample number 454A-253A-2, an exposure sample prepared at a nominal concentration of 13 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-253A-2 are summarized below: Peak area = 1460100 Constant Coefficient = -3901.34 Linear Coefficient = 4406500 Quadratic Coefficient = -80517.7 Dilution Factor (Vtinai/Vinitiai): = 40.0 p. 34 Wildlife International, Ltd. Project Number 454A-253A -34Appendix 3.4 (Continued) Example Calculations for a Representative Sample - 4406500 + 7 (4406500)2 - [(4 (-80517.7)) (-3901.34 -1460100)] PFOS = 40.0 2 *(-80517.7) PFOS = 40.0 0.33425 mg a.i./F PFOS = 13.4 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./F)_____ concentration (mg a.i./F) X 100 13.4 mg/L 13.0 mg/L X 100 = 103% Wildlife International, Ltd. Project Number 454A-253A -35Appendix 3.5 Quality Control Samples of PFOS in Freshwater Sample Number (454A-253A-) 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 5.00 50.0 250 5.00 50.0 250 5.00 50.0 250 M easu red 1,2 < LOQ < LOQ < LOQ 5.51 51.5 251 5.71 52.4 246 4.98 49.5 246 Percent Recovery 12 _ -- - 110 103 100 114 105 98.4 99.5 99.0 98.5 X=103 S.D-5.62 C.V.=5.46% 1 Results generated using Analyst version 1.4.1 software. Manual calculations may vary. 2 The limit of quantitation (LOQ) was 2.00 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 (40.0). Wildlife International, Ltd. Project Number 454A-253A -36Appendix 3.6 Representative Calibration Curve for PFOS hCOE C507JID.n>(FK): "Q jadricf R s g e s s o n fl weighing): y = -3 C6eKX)4)^2 M 41 eK I]6 x + -3S&KXX3(r=QS99G) 4.4e6 4.6 4.GB6 38e6 36e6 34e6 3 3fe6 28e6 26s6 24e6 2&6 2CE6 1.8s6 1.606 1.4e6 1.236 1.096 a0e5 6Ce5 4.065 20e5 Q05 Q10 0.15 Q20 Q25 Q30 035 Q40 Q45 050 055 Q60 _____________________________________________CbriBrtrcfoT, ugajVrrL Q65 0.70 Q75 Q60 085 030 096 1.00 Linear coefficient=4406500; constant coefficient=-3901.34; quadratic coefficient= -80517.7; r=0.9999 Wildlife International, Ltd, Project Number 454A-253A -37Appendix 3.7 Representative Chromatogram of a Low-level PFOS Calibration Standard Nominal concentration: 0.0500 mg a.i./L Wildlife International, Ltd Project Number 454A-253A -38Appendix 3.8 Representative Chromatogram of a High-level PFOS Calibration Standard Nominal concentration: 1.00 mg a.i./L Wildlife International, Ltd Project Number 454A-253A -39Appendix 3.9 Representative Chromatogram of a Matrix Blank Sample 25*-, 22l*A5*** 2233** 22* 2z221i*** 2D* 19*- 1M 17*1.7*IIBS** 15* 13* o 12* 10* 95 85 765 6M 55* 1*0S30710BXg) 096 1051.19.12? 138 150 1*175 06 08 10 12 IS 1. 105 2052V 23 236 2 * . 262 Z74202 299 913 125 3 344352180 371180 20 22 24 28 28 30 32 1* 16 38 39T 40B 428,01*41 0 *2 4.4 _____________ kaum_______________ _________________________________ 4B 4 48 Sample number 454A-253A-MAB-1. Dilution factor = 40.0X. The arrow indicates the retention time of PFOS. p. 40 Wildlife International, Ltd Project Number 454A-253A -40Appendix 3.10 Representative Chromatogram of a Matrix Fortification Sample Sample number: 454A-253A-MAS-1, nominal concentration 5.00 mg/L. Dilution factor = 40.0X. 0? p. 41 Wildlife International, Ltd, Project Number 454A-253A -41 Appendix 3.11 Representative Chromatogram of a Test Sample Sample number: 454A-253A-2, Day 0, nominal concentration 13 mg a.i./L. Dilution factor = 40.0X. p. 42 Wildlife International, Ltd. Project Number 454A-253A -42 Appendix 4 Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. The test chambers had a piece of Nitex screen placed in each of the beakers as substrate. 2. The temperature was measured 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. 3. The temperature was monitored 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. 4. Dissolved oxygen and pH were measured 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. 5. A tray of clean water was placed in the environmental chamber to maintain the moisture content of the air in the environmental chamber and thus decrease the rate of evaporative loss of water from the test solutions in the test chambers. 6. Test organisms were obtained from Environmental Consulting and Testing, Superior, WI. 7. The Environmental Laboratory Project Number assigned by the Sponsor for this study was E07082. 8. Twenty replicate test chambers were maintained in the negative control group while ten replicate test chambers were maintained in each treatment group. A single organism was placed in each test chamber for a total of twenty organisms per negative control group, and ten organisms per each treatment group. 9. Dilution water was passed through a UV sterilizer prior to use. This had no adverse impact on the study results. 10. The test solution level in each chamber was not examined and brought up to the 20 mL mark daily with reverse osmosis water. This had no adverse impact on the study results. V p. 43 Wildlife International, Ltd. Project Number 454A-253A -43 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 r> n