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P. z z6 -37773 ~~- PFOA : A 96-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST WITH Chironomus tentans AMENDED FINAL REPOR T WILDLIFE INTERNATIONAL, LTD . PROJECT NUMBER : 454A-250 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER : E07-0081 ASTM Standard E729-9 6 AUTHORS : Tui Minderhout, Ph .D . Jon A . MacGregor, B .S . Henry O . Krueger, Ph .D . STUDY INITIATION DATE : February 7, 2007 STUDY COMPLETION DATE : March 2, 2007 AMENDED FINAL REPORT DATE : March 8, 2007 SUBMITTED TO : 3M Corporation Environmental Laboratory 3M Center Building 0260-05-N-17 Maplewood, MN 55144 Wildlife Intey-national, Ltd. 8598 Commerce Drive Easton, Maryland 21601 USA 1-410-822-860 0 Page 1 of 45 CONTAINS NO CB I , ~~ AMENDED p. 2 Wildlife International, Ltd. Project Number 454A-25 0 -2GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR 3M Corporatio n TITLE PFOA A 96-Hour Static-Renewal Acute Toxicity Test with Chironomus tentans WILDLIFE INTERNATIONAL, LTD PROJECT NUMBER 454A-25 0 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER E07-0081 STUDY COMPLETION March 2 . 200 7 AMENDED REPORT DATE March 8, 200 7 This study was conducted in compliance with Good Laboratory Practice Stan dards as published by the U S Environmental Protection Agency ( 40 CFR Parts 160 and 792, 17 August 1989) with the following exception s Periodic analyses of well water for potential contamin ants were performed using a certified laboratory and standard U S EPA an alytical methods The test substan ce w as characterized in compli ance with Good Laborato ry Practice St an dards prior to the start of the test However, test solutions were prepared an d used, an d the test w as carried out, after the test substance expiration date of October 31, 2006 while the Certificate of Anal ysis was being updated STUDY DIRECTOR . XV L ~(~" Tui Minderhout, Ph D Date Senior Biologis t SPONSOR APPROVAL .._.--~_---- ; ~ ?Ss'or~Reprc sentative Date '~~L,~ AMENDED p. 3 Wildlife International, Ltd. Project Number 454A-25 0 -3QUALITY ASSURANCE STATEMEN T 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 : DATE REPORTED TO : ACT[VITY : DATE CONDUCTED : STUDY DIRECTOR: MANAGEMENT : Protocol February 12, 2007 February 12, 2007 February 15, 2007 Test Substance Preparation February 12, 2007 February 12, 2007 February 21, 2007 Matrix Fortification February 16, 2007 February 16 . 2007 February 21, 2007 Water Chemistry February 16, 2007 February 16, 2007 February 21, 2007 Analytical Data and Draft Report Februarv 22 - 23 . 2007 February 23 . 2007 March 1, 2007 Biological Data and Draft Report February 22 - 23, 2007 February 23 . 2007 February 27, 2007 Final Report March 2 . 2007 March 2, 2007 March 2, 2007 Amended Final Report March 8, 2007 March 8, 2007 March 8, 200 7 All inspections were study-based unless otherwise noted . J s H . Coleman Date Quality Assurance Representative /~~ AMENDED p. 4 Wildlife International, Ltd. -4REPORT APPROVAL Project Number 454A-25 0 SPONSOR: 3M Corporatio n TITLE : PFOA : A 96-Hour Static-Renewal Acute Toxicity Test with Chironomus lentans WILDLIFE INTERNATIONAL . LTD . PROJECT NUMBER : 454A-25 0 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER : E07-0081 STUDY DIRECTOR : ~u,i 1'y ; n~~,, ~u.~,,~ ~7 ui Minderhout. Ph .D . Date Senior Biologist PRINCIPAL INVEST GATOR : Jon X. MacGregor. B .S . Sc i ntis t Date WILDLIFE INTERNATIONAL . LTD . MANAGEMENT : o Henry . Krueger . Ph .D . Date Director of Aquatic Toxicology/Terrestrial Plants and Insects 411ltllpl~o. Willard B . Nixon, Pfi .D . Director of Chemistn- 1011f Date /~ I AMENDED p. 5 Wildlife International, Ltd. -5TABLE OF CONTENT S Project Number 454A-250 Title Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Good Laboratory Practice Compliance Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Quality Assurance Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Report Approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Measurement of Test Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Observations and Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 1~47 p. 6 Wildlife International, Ltd. Project Number 454A-250 -6TABLE OF CONTENTS (Continued ) TABLES Table 1 . Measured Concentrations of PFOA in Freshwater Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Table 2 . Temperature, Dissolved Oxygen and pH of Water in the Test Chambers . . . . . . . . . . . . . . . . . . . . . . . . 18 Table 3 . Specific Conductance, Hardness and Alkalinity Measured in Dilution Water at Test Initiation and Termination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Table 4 . Cumulative Mortality and Clinical Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Table 5 . LC50 Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 APPENDICE S Appendix 1 . Certificate of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Appendix 2 . Specific Conductance, Hardness, Alkalinity and pH of Well Wate r Measured During the 4-Week Period Immediately Preceding the Test . . . . . . . . . . . . . . . . . . 27 Appendix 3 . Analyses of Pesticides, Organics and Metals in Wildlife Intern ational, Ltd . Well Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Appendix 4 . The Analysis of PFOA in Freshwater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 4 .1 Analytical Method Flowchart for the Processing of PFOA in Freshwater . . . . . . . . . . . . . 31 4 .2 Typical HPLC/MS/MS Operational Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 4 .3 Analytical Stocks and Standards Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4 .4 Example Calculations for a Representative Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4 .5 Quality Control Samples of PFOA in Freshwater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4 .6 Representative Calibration Curve for PFOA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4 .7 Representative Chromatogram of a Low-level PFOA Calibration Standard . . . . . . . . . . 38 4 .8 Representative Chromatogram of a High-level PFOA Calibration Standard . . . . . . . . . 39 4 .9 Representative Chromatogram of a Matrix Blank Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 4 .10 Representative Chromatogram of a Matrix Fortification Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 4 .11 Representative Chromatogram of a Test Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Appendix 5 . Changes to Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Appendix 6 . Personnel Involved in the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Appendix 7. Report Amendment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 /~~ AMENDED p. 7 Wildlife International, Ltd. Project Number 454A-250 -7SUMMARY SPONSOR : 3M Corporatio n TITLE : PFOA : A 96-Hour Static-Renewal Acute Toxicity Test with Chironomus tentans WILDLIFE INTERNATIONAL, LTD . PROJECT NUMBER : 454A-25 0 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER : E07-0081 TEST DATES : LENGTH OF EXPOSURE : Experimental Start : Februa ry 12, 2007 Biological Termination : February 16, 2007 Experimental Termination : February 16, 2007 96 Hours TEST ORGANISMS : SOURCE OF TEST ORGANISMS : AGE OF TEST ORGANISMS : Freshwater Midge (Chironomus tentans) Environmental Consulting and Testing Superior, WI 54880 2d to 3`d instar larvae (approximately 10-day old) at test sta rt TEST CONCENTRATIONS : Nominal Me an Measure d Negative Control <LOQ 63 mg a .i ./L 74 mg a .i ./L 125 mg a .i ./L 153 mg a .i ./L 250 mg a .i ./L 277 mg a .i ./L 500 mg a .i ./L 579 mg a .i ./L 1000 mg a .i ./L 1090 mg a .i ./L RESULTS : Based on mean measured concentrations : 96-Hour LC50 : No-Mortality Concentration : No-Observ ed-Effect Concentration : >1090 mg a .i ./L 277 mg a .i ./L 277 mg a .i ./L ~~ ~ p. 8 WZIdlZfe International, Ltd. Project Number 454A-250 -8- INTRODUCTIO N This study was conducted by Wildlife International, Ltd . for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland . The in-life phase of the definitive toxicity test was conducted from February 12 to 16, 2007 . Raw data generated by Wildlife International, Ltd . and a copy of the final report are filed under Project Number 454A-250 in archives located on the Wildlife International, Ltd . site . OBJECTIVE The objective of this study was to determine the acute effects of perfluorooctanoate, ammonium salt (PFOA) on the freshwater midge, Chironomus tentans, during a 96-hour exposure period under static-renewal test conditions . EXPERIMENTAL DESIGN Midge larvae 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 midges per concentration . 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 63, 125, 250, 500 and 1000 mg 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 after renewal at 48 hours, and at the end of the test . Midge larvae were impartially assigned to test chambers at test initiation . Observations of mortality and other signs of toxicity were made approximately 2 .5, 25, 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/immobility concentration and the noobserved-effect concentration (NOEC) were determined by visual interpretation of the mortality and observation data. l~ p. 9 W ildl fe International, Ltd. Project Number 454A-250 -9- MATERIALS AND METHOD S The study was conducted according to the procedures outlined in the protocol, "PFOA : A 96-Hour Static-Renewal Acute Toxicity Test with Chironomus tentans" . 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 Substanc e 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 Decmber 27, 2006 . It was assigned Wildlife International, Ltd . identification number 7864 upon receipt and was stored under frozen conditions . The test substance, a solid, was identified as : FC-143 ; PFOA ; Lot number 332 ; CAS number 3825-26-1 . The test substance contained 95% active ingredient and had an expiration date of February 27, 2017 (Appendix 1) . Test Organism The midge, Chironomus tentans, was selected as the test species for this study . Midges 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 . Midge larvae used in the test were second- to third-instar larvae (approximately 10 days old) at test initiation . Midge larvae were obtained from Environmental Consulting and Testing (ECT), Superior, Wisconsin and were 7 days old when received . During the 3-day period prior to the test, the organisms were held in an aquarium with sand and overlying water from the same source and at approximately the same temperature as that used in the test . Midges were fed a 56 g/L suspension of flake food during the holding period and a 4 g/L suspension of Tetramin flake food on days 0 and 2 of the test . During the 3-day acclimation period immediately preceding the test, water temperatures in the holding aquarium ranged from 22 .0 to 22 .8C, measured with a hand-held liquid-in-glass thermometer . The pH of the water ranged from 8 .1 to 8 .4, measured with a Fisher Scientific Accumet Model 915 pH meter . Dissolved oxygen ranged from 7 .2 to 8 .6 mg/L (>_85% of saturation), measured with a Yellow Springs Instruments Model 51 B dissolved oxygen meter . ~S~ AMENDED P. 10 Wildlife International, Ltd. Project Number 454A-250 - 10- The organisms showed no signs of disease or stress during the holding period . At test initiation, midge larvae were collected from the holding aquarium 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 holding 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 2 . The well water was passed through a sand filter to remove particles greater than approximately 25 m, 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 m 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 3 . Test Apparatu s Test chambers were 30-mL Nalgene plastic beakers filled with approximately 20 mL of water . The cups had approximately one-eighth of a teaspoon of sand on the bottom of the cup to provide a monolayer of burrowing substrate for the midges . 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 1000 mg a .i ./L, the highest concentration tested, by mixing a calculated amount of PFOA into dilution water . The stock solution was sonicated for five minutes and mixed by inversion, and appeared clear and colorless . Aliquots of the 1000 mg a .i ./L stock solution were proportionally diluted with well water to prepare 300 mL of test solutions at nominal concentrations of 63, 125, 250 and 500 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 purity (95%) . Test solutions were prepared on day 0 and day 2 . All surviving _15._Cz P. 11 Wildlife International, Ltd. Project Number 454A-250 -11- midge larvae were transferred from old to new solutions at approximately 48 hours . At test initiation and termination, all solutions appeared clear and colorless in the test chambers . Analytical Samplin g 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 PFOA 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 PFOA 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 Turbolon Spray ion source . Chromatographic separations were achieved using a Zorbax RX-C8 column (150 mm x 2 .1 mm, 5m particle size) . A flow chart for the analysis of PFOA is provided in Appendix 4 .1 and typical instrumental parameters are summarized in Appendix 4 .2 . Calibration standards of PFOA, ranging in concentration from 0 .100 to 1 .00 g a .i ./mL, were prepared in acetonitrile : HPLC-grade bottled water solution (50 :50, v/v) using a stock solution of PFOA in acetonitile (Appendix 4 .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 PFOA in the samples was determined by substituting the peak area I ,49k-.-3 p. 1 2 WZrdllfe International, Ltd. Project Number 454A-250 -12- responses of the samples into the applicable regression equation . An example of the calculations for a representative sample is included in Appendix 4 .4 . The method limit of quantitation (LOQ) for these analyses was set at 10 .0 mg a.i ./L, calculated as the product of the lowest calibration standard (0 .100 g a .i ./mL) and the dilution factor of the matrix blank samples (100). Three matrix blank samples were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during the sample analyses (Appendix 4 .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 PFOA concentrations of 25 .0, 250 and 1200 mg a .i ./L . The measured concentrations for the matrix fortification samples ranged from 97 .0 to 107% of nominal concentrations (Appendix 4 .5 ) A representative calibration curve is presented in Appendix 4 .6 . Representative chromatograms of low and high-level calibration standards are presented in Appendices 4 .7 and 4.8, respectively . A representative chromatogram of a matrix blank sample is presented in Appendix 4 .9 and a representative chromatogram of a matrix fortification sample is presented in Appendix 4 .10 . A representative chromatogram of a test sample is presented in Appendix 4 .11 . Environmental Condition s Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone 50) were used for illumination of the holding 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 776 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 of each treatment and control group at the beginning of the test, prior to and after the renewal (old and new solutions) and at the end of the test (old solution) using a liquid-in-glass /'45-~/ p . 13 Wildlife International, Ltd. Project Number 454A-25 0 -13- thermometer. Temperature was also 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 the batches of new test solution at 0 and 48 hours or were measured in composite samples of old solutions at 48 and 96 hours . 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 O ri on Model 850Aplus dissolved oxygen meter, and measurements of pH were made using a Thermo O rion Model 525Aplus meter . Hardness, alkalinity and specific conductance were measured in the dilution water at test initiation and termination . Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity -Conductivi ty-Temperature meter . Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods for the Examination of Water and Wastewater (3) . Observations Observ ations of mo rtali ty were made periodically in each treatment group . Lethality was defined as the lack of visible movement in the midge. The numbers of individuals exhibiting signs of toxici ty or abnormal behavior also were evaluated . Obse rv ations were made approximately 2 .5, 25, 48, 72 and 96 hours after test initiation . Statistical Analyse s Less than 50% mo rt ality in any of the PFOA treatment groups was obse rv ed during the test, which precluded the statistical calculation of LC50 values . Therefore, the 24-, 48-, 72- and 96-hour LC50 values were estimated to be greater than the highest concentration tested . The nomortali ty/immobili ty concentration and the no-observed-effect concentration (NOEC) were determined by visual interpretation of the mo rtality , immobili ty and obse rv ation data. /5S p. 1 4 Wildlife International, Ltd. Project Number 454A-250 -14- RESULTS AND DISCUSSION Measurement of Test Concentra tion s Nominal concentrations selected for use in this study were 63, 125, 250, 500 and 1000 mg a .i ./L . Results of analyses to measure concentrations of PFOA 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 102 to 106% of the nominal concentrations . Samples collected prior to and after renewal of the test solutions at 48 hours had measured concentrations that ranged from 120 to 141% and 102 to 104%, respectively, of the nominal concentrations . Samples collected at test termination had measured concentrations that ranged from 107 to 160% of the nominal concentrations . The concentration of the solutions collected after 48 and 96 hours were slightly higher than those of newly prepared solutions due in part to the evaporative loss of water from the tests solutions even though the test chambers were covered with plastic . The high surface area in comparison to the small volume of the solution and the position of the fan that circulated the air in the environmental chamber might have contributed to the evaporation rate . When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations for this study were 74, 153, 277, 579 and 1090 mg a .i ./L, representing 118, 123, 111, 116 and 109% of nominal concentrations, respectively . The mean measured concentrations indicated no reduction in test concentrations in the test system during the study . 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 f 1C range established for the test . Dissolved oxygen concentrations remained ?7 .6 mg/L (>_89% of saturation) throughout the test . Measurements of pH ranged from 7 .9 to 8 .5 . 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 . A 10% mortality rate was observed in both the negative control group and in the 74 mg a .i ./L 1'!5%V1 p. 1 5 Wildlife International, Ltd. Project Number 454A-250 -15- treatment group after 72 and 96 hours of exposure, respectively . Since the mortality in the 74 mg a.i ./L treatment group was comparable to the negative control, and was not dose-responsive, it was not considered to be treatment-related . No mortalities or signs of toxicity were observed among midges in the 153 and 277 mg a.i ./L treatment groups during the test . Percent mortality at test termination in the 579 and 1090 mg a .i ./L treatment groups was 30 and 40%, respectively . All surviving midges in the PFOA treatment groups appeared normal throughout the test. The nomortality concentration and the NOEC were both 277 mg a .i ./L . LC50 values at 24, 48, 72 and 96 hours were estimated to be >1090 mg a .i ./L, the highest concentration tested (Table 5) . CONCLUSIONS The midge, Chironomus tentans, was exposed for 96 hours under static-renewal conditions to five mean measured concentrations of PFOA ranging from 74 to 1090 mg a .i ./L . The 96-hour LC50 value was >1090 mg a .i ./L, the highest concentration tested . Based on the mortality seen in the 579 and 1090 mg a .i ./L treatment groups, the 96-hour no-mortality concentration and the NOEC were both 277 mg a .i ./L . ~~~ p . 16 WZldllfe International, Ltd. -16REFERENCES Project Number 454A-25 0 1 U .S . Environmental Protection Agency . 2000 . Methods for Measuring the Toxicity an d 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 . 11!!~S p. 1 7 Wildlife International, Ltd . Project Number 454A-250 - 17- Table 1 Measured Concentrations of PFOA in Freshwater Samples Measured Mean Mean Nominal Test Sample Sampling Concentration Percent Measured Measured Concentration Number Time PFOA of Concentration Percent o f (mg a .i ./L) (454A-250-) (Hours) (mg a.i ./L) ~ Nominal ~ (mg a .i ./L) Nominal 0 .00 1 0(new) < LOQZ -- < LOQ -(Negative Control) 7 48(new) < LOQ -- 13 48(old) < LOQ -19 96(old) < LOQ -- 63 2 0(new) 65 .4 104 74 117 8 48(new) 65 .1 10 3 14 48(old) 75 .5 120 20 96(old) 91 .2 14 5 125 3 0(new) 130 104 9 48(new) 129 10 3 15 48(old) 154 123 21 96(old) 200 16 0 153 122 250 4 0(new) 259 104 10 48(new) 259 10 4 16 48(old) 302 121 22 96(old) 289 11 6 277 111 500 5 0(new) 532 106 11 48(new) 511 10 2 17 48(old) 707 141 23 96(old) 566 11 3 579 116 1000 6 0(new) 1020 102 12 48(new) 1020 10 2 18 48(old) 1260 126 24 96(old) 1070 107 1090 109 Results generated using Analyst version 1 .4 .1 software . Manual calculations may vary. 2 The limit of quantitation (LOQ) was 10.0 mg a .i ./L calculated as the product of the lowest calibration standard (0 .100 g a .i ./mL) and the dilution factor of the matrix blanks (100) . 16 l p . 18 Q R 00 00 00 00 00 ' 00 0 bA o , O O o 00 00 00 00 ~O l~ ~ cC ^ i..~ ~O ~ ,-r r Vl 00 [~ --00 00 U o N N N N N N N N N N N N , '-' N~..~ N N N N N N N N N N N N i- : ~ '~" M ; M 1 M 1 N , N O , 00 00 00 00 00 0 0 U a t- v p a~ O on 06 00 , 00 , 00 , 0 0 C, Cd I=. ^ cn ~',:,' U N N N N N N N N N N N N yy U.~ N N N N N N N N N N N N 3 W y 3 Cy..~ ~ U 00 o M Cu. 00 ' 00 00 ' 00 ' 0 0 00 ' Q 00 -,z; 3 ^ .. 0 cz z ~ rn o o - -~ o zb oo ' 00 00 ' oo ' oo ' O ct 0 ~ ~ 0 Ul V) 6) ~ - ~T N 0 o O~ 00 [ --~ 00 N O E U N N N N -~ --N N N N N N N N N N N N N N N NN E n O o t" cd N Q~ O oo ' 00 ' x A i 00 ' oo a cd y N~ a - U 0 d 0 O6 C 00 ^ O C V 1 'J . 0c 0 00n 00 U C V A. ^ ~n M l~ O v~ ~n v1 ~n ~n M o N N N M N N N N N N NM N N N N N N N N N N N N Ln v, S." CO ~ Q0 4 'b cl G) (L) ~ > ~ --~ N vl ~ O C,3O U o d *-.,A "t *v_ ~GD P. 19 Wildlife International, Ltd. Project Number 454A-250 -19- Table 3 Specific Conductance, Hardness and Alkalinity Measured in Dilution Water at Test Initiation and Terminatio n Parameter Day 0 Day 4 Specific Conductance 290 310 (mhos/cm ) Hardness 132 136 (mg/L as CaCO3) Alkalinity 180 182 (mg/L as CaCO3) ~~O/' p . 20 ~ [ . _ E NO 7 U O N N ~ Z z Z z Z Z Z Z Z Z Z Z Z Z ~ Z Z Z Z o O d Q Q ~ Q Q Q Q Q Q Q Q d d d d d kn .; fl E:3 z U b z Av o 0 0~ o 0 0 o O o 0 0 0 0 0~ o 0 0 0 p`", ~ ~ d Q d~ d d Q Q d Q Q d Q d d A Q Q x~ O N r- 6 1 Z~ O O COD CD O O Cl O O O O O O O O O O O O Fr' o Z QZ z QZ Z Z z z z z z z Z Z Z Z Z Z Z N N rA O x v Z Iq o 0 0 0 0 0 0 0 0 0 ~ A C. 0 0 0 0 0 0 0 0 0 V3 Z Z z z Z Z Z Z Z z z Z z z Z Z z z z Z Cd O Q Q Q Q Q Q Q d d d d Q Q Q d d O '~ .. c; 6 Z~ O O O O O O O O O O O O O O O O O O O O A U r~ ~ ~O d d z Q Z d Z Q d Q z Qz 1a ~ v' N y O O O O O O O O O O O O O O O O O O O O y yj in, rt' A a -,d :3 ~ Q w U A w cL . ~ x~-~ d w U A w w c7 x '~ v Ec .~, 0 9~ N 14Q u= w z a ~a y ~- . ., o U b E ~ai ~ > .~w CIS 0 u z QuoH ~~ ~ p . 21 kn ZZZZZZZZZZZZZZZZZZZ Z N ~ d Q d d d d Q Q Q Q Q d Q Q Q d d d Q~. ao ~ z u N Z adi O o 0 0 0 0 0 0 0 o O o 0 0 0 0 0 0 0 0 zzzzzZZZzzzZzzzZzzz z y ~ d d Q Q Q Q Q Q d Q d N ~ O O O O O O O O O O O O O O O O O O O O O cd ~ ^O ~ cl U cv U T Z Q Z d Z QZdZQZQZ dZ QZ Z QZQZQZQZZZZ ZzQz z z Z Z Z Z Z Z z z z z z z z Z Z z z C,3 ~ d d Q F" ~ x G) N C ,-~- y O O O O O O O O O O O O O O O O O O O O ~ Q U Q z d d z d d d d `la~ ~ kn N z o 0 0 0 0 0 0 0 0 0 0 0 o O o 0 0 0 0 } ; ~Ai U y c O ^ d CQ U~1 w L~ ( 7 x CA U Q - W LL b (,~J x i"'"' ~' O, N A ~ .N a~ ~ .s >\l _ ~" A c,3 E cC ~ bc~ ~ ~ r ~d C UOE- 163 p . 22 ~? ~ . ., C) E o U U O N v-~ 0 ~ z z ~ ~ ' o 0 0 0 0- o o-- -- o- o- . O o o O-- U ~ ~ Z Z z z Z z z z z z ZZ Z Qy ~ Q A d 0 N n b z cz A O O O O O O --O O----O O O --O O O O--- S"-r O Cd ~ a p z z ZzZz ZZ z z z ZZZ Z ~ ~ O Q Q Q ' d d d ~ Q Q Q Q ON ct o ~ C N U 0v 0 o 0 0 0 0 0-- o- o 0 0 0- o 0 0 0 U zA ZZZZZZ ' ZZ ; z z z z ZZZ Z ~ O d d Q Q Q Q Q d d d Q d d d d 0 N .-~ Im O o 0 0 0 0 -- o oo 0 0 0 o 0 0 0-- ~ U I mN z z Z z z z ' Z Z z z z z z z z z z z z ~ d Q Q d d d d d I~ ^ 0 vx ai ' 1 \ !1 i ,- N -~ 0 o C. O O O Oc O --O O ~ C. 0 ai V A. O O Cl O 0 O O O ti U m y cd G y~ ~ aa . U A w w O x-- ti d w U w w C7 a ~e 'o ~~bz C to < ~ ~ U dUOE-~ M a /~~ p. 23 Wildlife International, Ltd. Project Number 454A-250 - 23 Table 5 LC50 Value s Time LC50 95% Confidence Interval Statistical (mg a .i ./L) (mg a .i ./L) Method 24 Hours > 1090 NA 2 48 Hours > 1090 --' NA Z 72 Hours > 1090 ' NA' 96 Hours > 1090 --' NA z ' 95% confidence limits could not be calculated with the mo rt ali ty/immobili ty data obtained . 2 NA = not applicable ; <50% mo rt ality precluded statistical calculation of an LC50 value . & p . 24 Wlldl fe International, Ltd. -24Appendix 1 Certificate of Analysi s Project Number 454A-25 0 /4/~:7 ji~ Wildlife International, Ltd. -25- p. 25 Project Number 454A-250 INTERIM CER TIFICA TE OF ANAL YSIS Revision 3 purity, Centre Analytical Laboratories COA Reference # : 023-034 3M Product : PFOA, Prima ry Standard Test Control Reference # : TCR-99030-30 Puri : 95.0 % Test Name Specifications Pesult 95.0% Appearance White, crystalline solid Conforms Identification NMR Positi ve Metals (ICPIMS) 1 . Calcium 2 . Magnesium 3 . Sodium 4. Potassium 1 . 0 .001 wt ./wt "/o 2 . <0.001 wt./wt% 3 . 0 .001 wtlwt .% 4 . <0.001 wt./wt.% 5 . Nickel 5 . <0.001 wt./wt.% 6. Iron 7 . Manganese 6 . <0 .001 wtJwt.% 7 . <0 .001 wtJwt. % Total % Impurity (NMR) 0 .36 wtJwt .% Total % Impurity (LC/MS) 4 .68wtJwt .% Total "/o Impurity (GC/MS) None Quantified.% Residual Solvents GA) None Detected Purity by DSC 99 .8% Inorganic Anions (IC ) l . Chloride 1 . 2 . Fluori de 2. 3 . Bromide 3. 4 . Nitrate 4. 5 . Nitri te 5. 6 . Phosphate 6. 7 . Sulfate 7 Organic Acids2 (IC) 1. TFA 2 . PFPA 1. 2. 3 . HFBA 3 . 4 . NFPA 4. <0 .015 wtiwt.% <0 .005 wt ./wt.% <0 .040 wtJwt .% <0 .009 wtJwt .% <0 .006 wt ./wt .% <0 .006 wt ./wt.% <0 .040 wt ./wt .% <0 .1 wt ./wt .% <0 .1 wtlwt .% <0 .1 wtJwt .% <0 .25 wtJwt.% Elemental Analysis' : 1 . Carbon 1 . Theoretical Value = 22.3% 1 . 18.9 wtJwt% 2 . Hydrogen 2 . Theoretical Value = 0 .935% 2 . 1 .27 wtJwt.% 3 . Nitrogen 3 . Theoretical Value = 3 .25% 3 . 3 .76 wtJwt.% 4 . Sulfur 4. Theoretical Value = 0% 4 . 4.38 wtJwt.% 5 . Fluorine 5 . Theoretical Value = 66.1 "/0 5 . 62.1 wtJwt.% Ammonium Analysis Ion Selective Electrode Theoretical Value = 4 .18% 2 .94 wtJwt. % COA023034 REVISION 3 .doc Page 1 of 2 147 AMENDED Wildlife International, Ltd. -26- p. 26 Project Number 454A-250 INTERIM CERTIFICATE OF ANAL YSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-034 3M Product : PFOA, Prima ry Standard Test Control Reference # : TCR-99030-30 Date of Last Analysis : 2/27/0 7 Expiration Date : 2/27/17 Storage Conditions : <-10C Re-assessment Date : 2/27/1 7 'Purity = 100% - (total metal impurities, 0 .002% + Total NMR impurities, 0 .36 + Total LC/MS impurities, 4 .68 %) Total impurity from all tests = 5 .042% Purity = 100% - 5 .042% = 95 .0 % 2 TFA Trifluoroacetic acid HFBA Heptafluorobutyric acid NFPA Nonafluoropentanoic acid PFPA Pentafluoropropanoic aci d 3 Theoretical value calculations based on the empirical formula, CgFi50Z(-)mna1+1 (MW=43 1 . 1 ) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160) . LC/MS Purity Profile : Peak # Retention Time (min) Mass(s) Identity Area /. Area 1 12 .144 269.0 C6 205859 0 .87 2 13 .533 331,319 F1,/C7 903329 3 .81 3 14 .238 369 PFOA 2263060 0 Total 23739788 4.68 Prepared By : ~~ Charles Exygen Resear h Si ns -A - 1 Date Reviewed By : Kevin Lloyd Date Exygen Researc h COA023034 REVISION 3 .doc Page 2 of 2 AC, 10 AMENDED p. 27 W lldl fe International, Ltd. Project Number 454A-250 -27- Appendix 2 Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test Parameter Mean Range Specific Conductance 293 285 - 30 0 (mhos/cm) (N = 4) Hardness 136 132 - 140 (mg/L as CaCO3) (N = 4 ) Alkalinity 183 182 - 184 (mg/L as CaCO3) (N = 4 ) pH 8 .0 (N = 4 ) 7 .9-8 .1 147 p . 28 Wildlife Intemational, Ltd. Project Number 454A-25 0 -28- Appendix 3 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water' Pestic i des and Organics Measured Concentration Measured Concentration Component (g/L) Component (g/L) Aldrin < 0 .019 Heptachlor < 0 .009 6 Alpha BHC < 0 .0096 Heptachlor Epoxide < 0 .0096 Alpha Chlordane < 0 .0096 Kepone < 0 .19 Beta BHC < 0 .038 Malathion < 1 .9 Bolstar < 1 .9 Merphos < 1 .9 Chlordane < 0 .48 Methoxychlor < 0 .096 Coumaphos < 2 .9 Methyl Parathion < 1 .9 Delta BHC < 0 .0096 Mevinphos < 1 .9 Demeton-O < 1 .9 Mirex < 0 .11 Demeton-S < 1 .9 Naled < 2 .9 Diazinon < 1 .9 o,p-DDD < 0 .019 Dichlorvos < 1 .9 o,p-DDE < 0 .019 Dieldrin < 0 .029 o,p-DDT < 0 .019 Disulfoton < 1 .9 p,p-DDD < 0 .019 Dursban (Chlorpyrifos) < 1 .9 p,p-DDE < 0 .019 Endosulfan I < 0 .0096 p,p-DDT < 0 .019 Endosulfan II < 0.019 PCB-1016 < 0 .48 Endosulfan Sulfate < 0 .019 PCB-1221 < 0 .48 Endrin < 0 .019 PCB-1232 < 0 .48 Endrin Aldehyde < 0 .096 PCB-1242 < 0 .48 Endrin Ketone < 0 .019 PCB-1248 < 0 .48 EPN < 3 .8 PCB-1254 < 0 .48 Ethion < 1 .9 PCB-1260 < 0 .48 Ethoprop < 1 .9 Phorate < 1 .9 Ethyl Parathion < 1 .9 Ronnel < 1 .9 Famphur < 1 .9 Stirophos < 1 .9 Fensulfothion < 3 .8 Telodrin < 0 .0096 Fenthion < 1 .9 Tokuthion < 1 .9 Gamma BHC - Lindane < 0 .0096 Toxaphene < 0 .96 Gamma Chlordane < 0 .096 Trichloronate < 1 .9 Guthion (Azinphos-methyl) < 3 .8 Trithion < 1 .9 HCB < 0 .09 6 Analyses performed by Lancaster Laboratories on samples collected on December 15, 2005 . /b p. 29 Wlldl fe International, Ltd. Project Number 454A-250 -29- Appendix 3 (Continued ) Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd . Well Water' Metals Measured Concentration Measured Concentratio n Component (mg/L) Component (mg/L) Aluminum < 0 .200 Magnesium 13 .3 Antimony < 0 .0200 Manganese < 0 .0050 Arsenic < 0 .0200 Mercury < 0 .00020 Barium < 0 .0050 Nickel < 0.0100 Beryllium < 0 .0050 Nitrate Nitrogen < 0 .50 Bromide < 2 .5 Nitrite Nitrogen < 0 .50 Cadmium < 0 .0050 Potassium 7 .65 Calcium 33 .1 Selenium < 0 .0200 Chloride 2 .7 Silver < 0 .0050 Chromium < 0 .0150 Sodium 19 .1 Cobalt < 0 .0050 Sulfate < 5 .0 Copper < 0 .0100 Thallium < 0.0200 Fluoride 0 .56 Vanadium < 0 .0050 Iron < 0.200 Zinc < 0 .0200 Lead < 0 .020 0 Analyses performed by Lancaster Laborato ri es on samples collected on December 15, 2005 . ~~/ p . 30 Wildlife International, Ltd. Project Number 454A-25 0 -30Appendix 4 The Analysis of PFOA in Freshwater I7~ p. 31 Wildlife International, Ltd. Project Number 454A-250 -31Appendix 4 . 1 Analytical Method Flowchart for the Processing of PFOA in Freshwater METHOD OUTLINE FOR THE ANALYSIS OF PFOA IN FRESHWATE R 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 . 173 p. 32 Wildlife International, Ltd. Project Number 454A-250 -32Appendix 4 . 2 Typical HPLC/MS/MS Operational Parameters INSTRUMENT : 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 . ION SOURCE : Turbolon Spray ANALYTICAL COLUMN : Zorbax RX-Cg (150 mm x 2 .1 mm, 5 m particle size) STOP TIME : 5 .00 minutes FLOW RATE : 0 .300 mL/minute OVEN TEMPERATURE : 40 C MOBILE PHASE : 80% CH3OH : 20% H2O containing 0 .1% formic acid INJECTION VOLUME : 5 .00 L PFOA RETENTION TIME : Approximately 2 .6 minutes PFOA MONITORED MASS : 413 ~ 369 amu -_7Z Z 1-7 p. 33 Wlldlafe International, Ltd. Project Number 454A-250 -33Appendix 4 .3 Analytical Stocks and Standards Preparation A calibration standard stock solution of PFOA was prepared by weighing 0 .1053 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 acetonitile . This primary stock solution contained 1 .00 mg a .i ./mL of PFOA . A secondary stock of PFOA in acetonitile (0 .100 mg a .i ./mL) was prepared from the primary stock by volumetric dilution . The 0 .100 mg a.i ./mL secondary 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 Final Standard Concentration Aliquot Volume Concentration (mg a .i ./mL) (ML) mL (p.g a .i ./mL) 0 .100 0 .100 100 0 .100 0 .100 0 .250 100 0 .250 0 .100 0 .500 100 0 .500 0 .100 0 .750 100 0 .750 0 .100 1 .00 100 1 .00 A fortification standard stock solution of PFOA was prepared by weighing 1 .0526 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 PFOA and was used to prepare concurrent matrix fortification (QC) samples . 175 p. 34 Wildlife International, Ltd. Project Number 454A-250 -34Appendix 4 .4 Example Calculations for a Representative Sampl e The analytical result and percent recovery for sample number 454A-250-2, an exposure sample prepared at a nominal concentration of 63 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 PFOA 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 PFOA in mg a .i ./L a = quadratic coefficient b = linear coefficient c =constant coefficient ( y_intercept) Concentrations of PFOA in samples were determined by substituting peak area responses of the samples into the applicable rearranged regression equation as follows : PFOA (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 curv e . Data used for quantitation of PFOA in Sample Number 454A-250-2 are summarized below : Peak area = 15456000 Constant Coefficient = 32311 .9 Linear Coefficient = 26602900 Quadratic Coefficient = -4615070 Dilution Factor ( VfnaiNininal) : = 100 ~~~ p. 35 Wildlife International, .Ltd. Project Number 454A-250 -35Appendix 4 .4 (Continued) Example Calculations for a Representative Sampl e - 26602900 + (-26602900)2 - [(4 (-4615070)) (32311 .9 -15456000)] PFOA = 100 2 (-4615070) PFOA = 100 0 .654 mg a .i ./ L PFOA = 65 .4 mg a .i ./L The measured concentration was compared to the nominal concentrations as follows : PFOA in sample (mg a .i ./L ) Percent of nominal concentration = PFOA nominal concentration (mg a .i ./L) X 100 _65 .4m L X 10 63 .0 mg/L 0 = 104% /7~' p . 36 WZldllfe International, Ltd. Project Number 454A-25 0 -36Appendix 4. 5 Quality Control Samples of PFOA in Freshwater Sample Sampling Concentration (mg a .i ./L) Number Time (454A-250-) (Hours) Fortified Measured 1,2 Percent Recove ry MAB-1 0 0 .0 MAB-2 48 0 .0 MAB-3 96 0 .0 < < < LOQ LOQ LOQ ---- MAS-1 0 25 .0 26 .9 107 MAS-2 0 250 258 103 MAS-3 0 1200 1190 99 .1 MAS-4 48 25 .0 25 .3 101 MAS-5 48 250 254 102 MAS-6 48 1200 1200 99 .7 MAS-7 96 25 .0 25 .9 104 MAS-8 96 250 256 102 MAS-9 96 1200 1160 97 . 0 Mean = 102 S .D . = 2 .93 C .V . = 2 .87 % Results generated using Analyst version 1 .4 .1 software . Manual calculations may vary . 2 The limit of quantitation (LOQ) was 10 .0 mg a .i ./L calculated as the product of the lowest calibration standard (0 .100 g a .i ./mL) and the dilution factor of the matrix blanks (100) . l73 p . 37 Wlldl fe International, Ltd. Project Number 454A-25 0 -37- Appendix 4.6 Representative Calibration Curve for PFOA 021207 _DO .rdb (PFOA) : "Quadratic" Regression ("1/x" weighting) : y=4 .62e+0p6 x^2 + 2.66e+007 x+ 3.23e+004 ( . .. 2.3e 7 2 .2e7 2 .1e7 2 .Oe7 1 .9e7 1 .8e7 1 .7e7 1 .6e7 1 .5e71 .4e7CD 1 .3e7 0 0 1 .2e7 1 .1e7 1 .Oe7 9 .Oe6 8 .Oe 6 7 .Oe6 6 .Oe6 5 .oe6 4 .0e6 3 .Oe6 ! i 0 .1 02 0 .3 0 .4 0S 0.6 0 .7 0.8 0.9 1 .0 Concentration, ug a.i ./m L Linear coefficient=26602900 ; constant coefficient=32311 .9 ; quadratic coefficient=-4615070 ; r=0 .9999 177 p. 38 Wildlife International, Ltd. Project Number 454A-250 -38Appendix 4 . 7 Representative Chromatogram of a Low-level PFOA Calibration Standard ~ . ~ .~ . . Po#Nare'fiaA' AY6sps)'4130[169Uamf ~mt .m1rg,c'.~c: w.t a,m~am - q ime ~~ o/` oo~u 2b6 ?4 { s c~ .F zs.u 24e6 23e& ~eexeiauve av no 91o zz .6+ a n .E 21 I x _~ 6 ,ys~c 2e& E.d iime . _. 1o mi~ 19e6' 1BaE 11e6{ tba6 i 3 1~1 1 Ae6~ $ >BH 12ee 1186'i 1e6 9eS 8eY ]fbYl BDeS,,, SMSI <e5l 2R 30e5 IOeS~ 1M 500 u5 10 15 _ 20 25 10 3$ < 45 Nominal concentration : 0 .100 g a .i ./mL /SO p . 39 W lldl fe International~ Ltd. Project Number 454A-25 0 -39Appendix 4 . 8 Representative Chromatogram of a High-level PFOA Calibration Standard ~a. iOfiNane'iFOA I.Ywpf)'s13Aq890am1 Minnnf SFMb' hMrd3m ' *s~x . e~ 3~zBiazoa~e ., imc zeacl .,,e Y)xn ctas,,~ zs .c zos = . ssac-, ~. a w ~ . naj cxp . v ~o . a.s 2]e6- { z n ~eeaeiacave ax . no m~~^ 2b6 TYPa= 21a6: on 'c z 2f6! ~a ttme _ s~ mae t~j 1 Be&~ 1 )e6 ~ I ~ _ t~i 3 t5081 1Aa6. g 1]a6 13eB tta6 t0e6 I 9De5- I B oe5~ I 10e5 60e5~ I SIbSi <Da51 3 oe5~ 30e5 tlb5~ a o ~-----~.._ _ OS 1D 15 2D 25 3D 3.5 _ l0 (5 Nominal concentration : 1 .00 g a .i ./mL ~ ~~ p. 40 Wildlife International, Ltd. Project Number 454A-250 -40Appendix 4 . 9 Representative Chromatogram of a Matrix Blank Sample Po#Na~mTiOR' A6sM7~13U3890amI mmm~t'6<At54' Mrq.m' sampte [ g .~ /mt 2Be6 g .me . 39 aJ m 2 -d classac os p 2 2~ I I u e aetac~ve vi . 3 11 ~xo m,, -12 a r~we 2 .5] m~n 2 3~ 21a6 20e6 ;~ 1)efi t6e6 ~ 15e6 14e6 1 tafi 9Ae ~ 6 pa5{{ 60e5 4Ce5 ] 3 16D ~ 0 25 Ub14 6i 2E ]19 4b1 05 1D 15 2. IS 3D ].5 40 45 Sample number 454A-250-MAB-1 . Dilution factor = 100X . The arrow indicates the retention time of PFOA. 1~~ p. 41 WZrdrZfe International, Ltd. Project Number 454A-250 -41Appendix 4 .1 0 Representative Chromatogram of a Matrix Fortification Sample h#Naim'(TOR' IAnpa)'4t30/3690am1 mrment'~IA256' Mrtlaim' Oc 350 9 ime- oc '~SI/9a0 ~19 a/m. aty:- Cta::~~ 2 { 2]eBl 26eB1 ~0 P 25a6~ ZZ : e~knc~a _ 2~ ~T Window .i ., e nela ..~e a'r . xo 2- Tan 2 on ia a10ao15 Ttefn 20e6+ tlTame . 2 13 ~ .ll man 1~~' 18e6 tbe61 a 15e6i 13e6i t2e6{ 11e6~ 1 Ab6-' 9De5~ BIleS)OeSI 6Da5~ 50 .5. 4 Oe5' 3De5 2DwF tDa S 0D 2]5 OS tD 15 2A 35 3D 3 .5 40 45 Sample number : 454A-250-MAS-2, nominal concentration 250 mg a .i ./L . Dilution factor = 100X . 133 p . 42 Wildlife International, Ltd. Project Number 454A-25 0 -42Appendix 4 .1 1 Representative Chromatogram of a Test Sampl e Pa9~Narte"PfOA' 1.!IKl'<I]OLK90amI (bmRnt'61A'156' Fn~t3'ni ' -r Is~ a O< ./ml 2~I eL 1~/300'r~9 a inN~q ~me . 9 .1 ._0 Of 2-j ------- B _ ..~ass~_ 2 ~ .O5 p 2 Expa P Exp . AG eap . V 01a cao . 2ae6 2 ~eeaela[ive 0.i vo ~r 22 tYPe' 2 21efi a r~me e .~3 man ~~ 1B.G 76 2 6W -.__ I 1-2~e6i11 11e61 tDe6~ 90e5~ BOeS{ 10e 6D~111 SOeS~ <Oe5 10e OD OS 1D ~t5 2A 35 30 35 l0 4 5 Sample number : 454A-250-3, Day 0, nominal concentration 125 mg a .i ./L . Dilution factor = 200X . Of p. 43 Wildlife International, Ltd. -43Appendix 5 Changes to Protocol Project Number 454A-250 This study was conducted in accordance with the approved Protocol with the following changes : 1 . The protocol was amended to add that the cups will have approximately one-eighth of a teaspoon of sand on the bo ttom of the cup to provide a monolayer of burrowing substrate for the midges . 2 . The protocol was amended to state that the temperature will be measured in two alte rnate 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 protocol was amended to add that the temperature will also be 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 . The protocol was amended to state that dissolved oxygen and pH will be measured in samples collected fr om batches of new test solutions or measured in composite samples of old solutions . 5 . The protocol was amended to state that the test concentrations will be 63, 125, 250, 500 and 1000 mg a .i ./L rather than 16, 31, 63, 125 and 250 mg a.i ./L as requested by the Sponsor to accommodate a no-effect study . 6 . The protocol was amended to add the Environmental Laborato ry Project Number of E07-0081 as assigned by the Sponsor after the protocol was executed . 7 . The protocol was amended to state that test organisms will be obtained fr om a commercial supplier to complete the protocol as required by GLP . 8 . Midge larvae used in the test were obtained from a commercial supplier and held until used in the test rather than hatched from egg masses . This had no adverse impact on the study results . 9 . Plastic wrap rather than plastic or glass lids was used to cover the test chambers . This had no adverse impact on the study results . 10 . The 24-hour obse rv ations were made after 25 .25 hours rather than within 24 1 hour of test initiation . This had no adverse impact on the study results . / ~5 p. 44 Wildlife International, Ltd. Project Number 454A-250 -44Appendix 6 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 Biologis t 4 . Amy S . Blankinship, M .S ., Laboratory Supervisor, Aquatics 5 . Jon A . MacGregor, B .S ., Scientist 6 . Susan T . Thomas, B .S ., Biologist I ~~ p . 45 Wildlife International, Ltd. Project Number 454A-25 0 -45- Appendix 7 Report Amendmen t 1 . Original Repo rt : Pages 1, 2, 3, 4 and 6 Amendment : The pages we re ch anged to include the amended repo rt date, the amended report audit date, revised page numbers, an d/or new signatures and dates due to the addition of the report amendment as Appendix 7 . Reason : To re flect the issuing of an amended report . 2 . Original Repo rt : Page 9 Amendment: The expiration date was changed in th e Test Substance section of the repo rt . Reason : A new Ce rtificate of Analysis was supplied after the issuance of the original final report. 3 . O riginal Repo rt : Pages 25 an d 2 6 Amendment : The Certificate of Analysis was replaced . Reason : A new Certi fi cate of Analysis was supplied after the issuance of the o ri ginal final report. AMENDMENT SIGNATURES: j'b ;~W N ~~'~>~ 31 ~JjX G-J Study Director Date l Laboratory Management 1 Date REVIEWED BY : G&,ou, , (bkity Assurance Date 13 7 AMENDED