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STUDY TITLE Extraction of Potassium Perfluorooctanesulfonate from Mallard Serum and Mallard Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry DATA REQUIREMENTS Analytical Method Requirements STUDY DIRECTOR Sean Gallagher STUDY COMPLETED ON September 06,2001 PERFORMING LABORATORY / TESTING FACILITY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 Phone: 814-231-8032 STUDY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 POBox 33331 St. Paul, MN 55133-3331 PROJECT Centre Protocol Number: 00P-023-042 Centre Study Number: 023-042 Total Pages: 104 000479 Centre Study No.: 023-042 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Centre Study Num ber 023-042, entitled "Extraction o f Potassium Pcrfluorooctanesulfonate from M allard Serum and M allard Liver fa r A nalysis U sin g HPLC-Elactrospray/M ass Spectrom etry," conducted for 3M Environm ental Technology and Safety Services, w as perform ed in com pliance with U S EPA TSC A G ood Laboratory Practice Standards (40 CFR Part 7 92) by Centre Analytical Laboratories, Inc, fr l k c k # '. Principal Investigator Centre A nalytical Laboratori, la c. Sean Gallagher Study D irector W ildlife International, Ltd. Jfciei D ate Jo 0 D ate vironm enul T echnology and Safety Services Cantre Analytical Laboratories, Inc. Page 2 of 104 000480 Centre Study No.: 023-042 QUALITY ASSURANCE STATEMENT Centre Analytical Laboratories' Quality Assurance Unit reviewed Centre Study Number 023-042, entitled, "Extraction of Potassium Perfluorooctanesulfonate from Mallard Serum and Mallard Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry". All reviewed phases were inspected for conduct according to Centre Analytical Laboratories' Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Study Director and to management. Phase Date Insnected Date Reported to Date Reported to Centre Study Director and Management Snonsor Management 1. Protocol Review 11/20/00 11/30/00 12/28/00 2. Extraction, Fortification 11/20/00 11/30/00 12/28/00 3. Raw Data Review 12/12/00 12/21/00 12/28/00 4. Raw Data and Draft Report Review 3/18-21/00 3/29/01 5/7/01 5. Final Report Review 9/6/01 9/6/01 9/6/01 LL_X -- '* c^ v OL-CL'--' Naomi Lovallo Sr. Quality Assurance Auditor ______(gjo I Date Centre Analytical Laboratories, Inc. Page 3 of 104 C/04S1 CERTIFICATION OFA t Centre Study No.: 023-042 mux T h is report, for Centre Study Num ber 023-042, Is a true and com plete representation of the raw data for the study. Subm itted by: Centre Analytical Laboratories, Inc. 3048 Research D rive State C ollege, PA 16801 (814)231-8032 Principal Investigator, Centre: ffi- T te g V .g / 1. Decker Seiender Centre A nalytical Laboratories, Lie. -llk jp l D ate Centre A nalytical Laboratories, Inc. Facility Management: 7 R ichard A . Q fazzinkT h.D , P r e sid e n t Centre A nalytical Laboratories, Inc. -J B P .O } D ate Study D irector, W ild life International: Sean G allagher W ild life International, Ltd. f Sponsor R epresentative, 3M: \ l l 0 i. Dale lobideau "3M Environm ental Technology and Safety Services Centre Analytical Laberatonai, hie. Page 4 of 104 C Q 0482 Centre Study No.: 023-042 STUDY IDENTIFICATION Extraction of Potassium Perfluorooctanesulfonate from Mallard Serum and Mallard Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry CENTRE PROTOCOL NUMBER: 00P-023-042 CENTRE STUDY NUMBER: 023-042 TYPE OF STUDY: Analytical SAMPLE MATRIX: Mallard Serum and Mallard Liver TEST SUBSTANCE: Perfluorooctanesulfonate (PFOS) SPONSOR: 3M Environmental Technology and Safety Services Building 2-3E-09 POBox 33331 St. Paul, MN 55133-3331 STUDY DIRECTOR: Sean Gallagher Wildlife International, Ltd. 8598 Commerce Drive Easton, MD 21601 SPONSOR REPRESENTATIVE: Rochelle Robideau 3M Environmental Technology and Safety Services Building 2-3E-09 POBox 33331 St. Paul, MN 55133-3331 TESTING FACILITY: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: Experimental Start Date: Experimental Termination Date: Study Completion Date: 11/10/00 11/17/00 03/13/01 09/06/01 Centre Analytical Laboratories, Inc. Page 5 of 104 C04S3 Centre Study No.: 023-042 PROJECT PERSONNEL The Study Director for this project was Sean Gallagher at Wildlife International, Ltd. The folowing personnel from Centre Analytical Laboratories, Inc., were associated with various phases of the study: Name Emily Stauffer Dave Bell Melissa Kennedy Whitsel Tiffany Proctor Angela Morgan Michelle Aijmand Lawrence Ord Rickey Keller Karen Risha Title Scientist Scientist Group/Team Leader Technician Technician Technician Sample Custodian Sample Custodian Scientist Centre Analytical Laboratories, Inc. Page 6 of 104 000484 Centre Study No.: 023-042 TABLE OF CONTENTS Page TITLE PAGE.......................................................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT.............................. 2 QUALITY ASSURANCE STATEMENT............................................................................3 CERTIFICATION OF AUTHENTICITY............................................................................ 4 STUDY IDENTIFICATION.................................................................................................. 5 PROJECT PERSONNEL....................................................................................................... 6 TABLE OF CONTENTS....................................................................................................... 7 LIST OF TABLES..................................................................................................................8 LIST OF FIGURES...............................................................................................................10 LIST OF APPENDICES.......................................................................................................11 1.0 SUMMARY................................................................................................................... 12 2.0 OBJECTIVE..................................................................................................................12 3.0 INTRODUCTION..........................................................................................................12 4.0 TEST SYSTEM.............................................................................................................13 5.0 REFERENCE MATERIAL.......................................................................................... 13 6.0 DESCRIPTION OF ANALYTICAL METHOD......................................................... 14 6.1 Extraction Procedure.................................................................................................. 14 6.2 Preparation o f Standards and Fortification Solutions................................................ 14 6.3 Chromatography..........................................................................................................15 6.4 Instrument Sensitivity................................................................................................. 15 6.5 Description of Instrument and Operating Conditions................................................ 15 6.6Quantitation and Example Calculation.......................................................................16 7.0 EXPERIMENTAL DESIGN........................................................................................ 18 8.0 RESULTS...................................................................................................................... 18 9.0 CONCLUSIONS............................................................................................................18 10.0 RETENTION OF DATA AND SAMPLES............................................................... 19 Centre Analytical Laboratories, Inc. Page 7 o f 104 0OG455 Centre Study No.: 023-042 Table I. LIST OF TABLES Page Summary of PFOS in Reagent Blanks.............................................................. 21 Table II. Summary of PFOS in Mallard Liver Blanks................ 22 Table III. Summary of PFOS in Mallard Serum Blanks...................................................22 Table IV. Summary of PFOS Recoveries in Mallard Liver..............................................23 Table V. Summary of PFOS Recoveries in Mallard Serum............................................24 Table VI. Summary of PFOS in Adult Liver Samples at 0 ppm a.i. (Test Termination 19 W eeks)....................................................... 25 Table VII. Summary of PFOS in Adult Liver Samples at 1.8ppm a.i. (Test Termination 6W eeks)......................................................................................26 Table VIII. Summary of PFOS in Adult Liver Samples at 6.2 ppm a.i. (Test Termination 6W eeks)......................................................................................27 Table IX. Summary o f PFOS in Adult Liver Samples at 17.6 ppm a.i. (Test Termination 19 W eeks)....................................................................................28 Table X. Summary of PFOS in Juvenile Liver Samples at 0 ppm a.i. (Test Termination 12 Weeks)....................................................................................29 Table XI. Summary of PFOS in Juvenile Liver Samples at 1.8ppm a.i. (Test Termination 12 W eeks)....................................................................................30 Table XII. Summary of PFOS in Juvenile Liver Samples at 6.2 ppm a.i. (Test Termination 12 Weeks)....................................................................................31 Table XIII. Summary o f PFOS in Juvenile Liver Samples at 17.6 ppm a.i. (Test Termination 12 W eeks)....................................................................................32 Table XIV. Summary of PFOS in Control Adult Serum Samples.....................................33 Table XV. Summary o f PFOS in Adult Serum Samples at 0 ppm a.i. (Test Termination 19 Weeks)....................................................................................34 Table XVI. Summary o f PFOS in Adult Serum Samples at 1.8 ppm a.i. (Test Termination 6W eeks)......................................................................................35 Table XVII. Summary of PFOS in Adult Serum Samples at 6.2 ppm a.i. (Test Termination 6W eeks)......................................................................................36 Table XVIII. Summary of PFOS in Adult Serum Samples at 17.6 ppm a.i. (Test Termination 19 W eeks)....................................................................................37 Centre Analytical Laboratories, Inc. Page 8 o f 104 G004S6 Centre Study No.: 023-042 LIST OF TABLES (Continued) Table XIX. Summary o f PFOS in Adult Serum Samples at 17.6 ppm a.i. (6Week Samples)........................................................................................................... 38 Table XX. Summary of PFOS in Juvenile Serum Samples at 0 ppm a.i. (Test Termination 12 Weeks)....................................................................................39 Table XXI. Summary of PFOS in Juvenile Serum Samples at 1.8 ppm a.i. (Test Termination 12 Weeks)....................................................................................40 Table XXII. Summary o f PFOS in Juvenile Serum Samples at 6.2 ppm a.i. (Test Termination 12 W eeks)....................................................................................41 Table XXIII. Summary of PFOS in Juvenile Serum Samples at 17.6 ppm a.i. (Test Termination 12 Weeks)....................................................................................42 Table XXTV. Summary of Percent Moistures for Mallard Liver Samples....................... 43 Centre Analytical Laboratories, Inc. Page 9 of 104 0004-37 Centre Study No.: 023-042 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFOS............................................................... 46 Figure 2. Chromatogram Representing a 0.1 ng/mL standard for PFOS......................47 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PFOS......................48 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Centre ID Reagent Blank A, Set: 112000A )................................................................... 49 Figure 5. Chromatogram Representing Control Mallard Serum for PFOS (Centre ID: 0003803 Blank A, Set: 112100B).................................................................... 50 Figure 6. Chromatogram Representing Control Mallard Liver for PFOS, (Centre ID: 0003804 Blank A, Set: 112000A)................................................................... 51 Figure 7. Chromatogram Representing Control Mallard Serum fortified with 10 ppb of PFOS (Centre ID: 0003803 Spk A, Set: 112100B)............................. 52 Figure 8. Chromatogram Representing Control Mallard Liver Fortified at 1000 ppb with PFOS (Centre ID: 0003804 Spk A, Set: 112100A)................................ 53 Figure 9. Chromatogram Representing Juvenile Mallard Serum Sample (Centre ID: 0008143, Sponsor ID: 2332, Set: 112700A).................................................... 54 Figure 10. Chromatogram Representing Juvenile Mallard Liver Sample (Centre ID: 0008108, Sponsor ID: 2337, Set: 112100A).................................................... 55 Centre Analytical Laboratories, Inc. Page 10 of 104 G004S8 Centre Study No.: 023-042 LIST OF APPENDICES Page A p p en d ix A Study Protocol 00P-023-042 (Centre Study No. 023-042) and Amendments and Deviation........................................................................ 56 Centre Analytical Laboratories, Inc. Page 11 o f 104 000489 Centre Study No.: 023-042 1.0 SUMMARY Centre Analytical Laboratories, Inc. (Centre) extracted mallard liver samples and mallard serum samples for the determination of perfluorooctanesulfonate (PFOS) in mallard serum and mallard liver according to protocol 00P-023-042 (Appendix A). The limit o f quantitation for mallard liver was 10 ppb and 10 ng/mL for mallard serum. The LOQ for each matrix was determined in a method verification study performed at Centre (Centre Study: 023-044). Residues o f PFOS in the mallard liver samples ranged from non-quantifiable levels (a peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) to 22.8 pg/g. These results are reported on a wet weight basis. PFOS in the mallard serum samples ranged from non-quantifiable levels to 218 pg/g. The average percent recovery standard deviation for PFOS in liver samples was 95% 12%. The average percent recovery standard deviation for PFOS in serum samples was 93% 10%. 2.0 OBJECTIVE The objective o f this study was to determine levels o f perfluorooctanesulfonate (PFOS) in specimens o f mallard liver and mallard serum using the analytical methods described in protocol 00P-023-042. 3.0 INTRODUCTION This report details the results o f the analysis for the determination o f PFOS in mallard liver and mallard serum, using the analytical methods entitled, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" and "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry". The study was initiated on November 10, 2000, when the study director signed Centre protocol number 00P-023-042. The experimental start date was November 17,2000, and the experimental termination date was March 13,2001. Centre Analytical Laboratories, Inc. Page 12 o f 104 000490 Centre Study No.: 023-042 4.0 TEST SYSTEM The control mallard liver and serum used for the matrix blanks and matrix fortifications was received on May 18,2000 from Wildlife International Ltd., Easton, MD. The control mallard liver was assigned the Centre ID o f 0003804 and the control mallard serum was assigned the Centre ID of 0003803. Sixty mallard liver samples and sixty mallard serum samples were received at Centre on August 10, 2000. The samples were stored frozen until they were logged in by Centre personnel on August 16, 2000 and stored frozen. An additional twenty mallard liver samples and forty mallard serum samples were received on February 24,2001 and stored frozen until they were logged in by Centre personnel on March 1,2001. Sample log-in and chain o f custody information can be found in the raw data package associated with this study. Storage records will be kept at Centre Analytical Laboratories, Inc. and a true copy of the storage records can be found in the raw data package associated with this study. 5.0 REFERENCE MATERIAL The analytical standard PFOS was received at Centre on June 3, 2000 from 3M Environmental Technology and Services. The available information for the reference material is listed below. The reference material was stored frozen. Compound PFOS Centre Control No.. TCR No. Purity (%) Expiration Date 00-023-042 TCR 00017-46 97.9 08/31/01 The m olecular structure o f PFOS is given below. PFOS Chemical Name Molecular weight = = Perfluorooctanesulfonate 499 (CgFnSOj") O CaFi7S -- O- O Note: The neutral molecule and standard form from which PFOS (anion) is derived, is potassium perfluorooctanesulfonate [C8F17SO3K], molecular weight 538. Centre Analytical Laboratories, Inc. Page 13 of 104 000491 Centre Study No.: 023-042 6.0 DESCRIPTION OF ANALYTICAL M ETHOD Analytical methods entitled "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" and "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" were used for this study. 6.1 Extraction Procedure A ~ 0.50 g sample o f liver and a 100 pL aliquot o f the serum was used for the extraction procedure. After fortification of appropriate samples, the samples were vortexed for ~ 15 seconds. One milliliter o f 0.5 M TBA was added to the liver samples and 0.5 milliliter was added to the serum samples. Two milliliters o f 0.25 M sodium carbonate/sodium bicarbonate was added to the liver samples and 1 milliliter was added to the serum samples. Five milliliters of MTBE was added to both the liver and serum samples. Each sample was placed on wrist-action shaker for ~ 20 min. and then centrifuged for ~ 15 min. Four milliliters of the organic layer was taken and dried on a nitrogen evaporator and then reconstituted with 1 milliliter o f methanol. Each sample was analyzed by LC/MS/MS electrospray. 6.2 Preparation of Standards and Fortification Solutions Standard solutions were prepared on September 18, 2000 as specified in Centre Analytical Laboratories' protocol 00P-023-042. An individual stock standard solution of PFOS was prepared at a concentration of 100 pg/mL by dissolving 10 mg o f the standard (corrected for purity and salt content) in methanol. From this solution, a 1.0 pg/mL fortification standard solution was prepared by taking 1 mL o f the stock and bringing the volume up to 100 mL with methanol. A 0.1 pg/mL fortification standard was prepared by taking 10 mL o f the 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol. A 0.01 pg/mL standard was prepared by taking 10 mL o f the 0.1 pg/mL standard and bringing to 100 mL with methanol. A set o f standards containing PFOS was prepared by serial dilution of the 0.1 pg/mL and 0.01 pg/mL solutions in the following manner: Initial Cone. (pg/mL)1 Volume (mL) 0.1 5 0.1 2 0.1 1 0.01 5 0.01 2 0.01 1 1of PFOS Diluted to (mL) 100 100 100 100 100 100 Final Cone. (pg/mL) 0.005 0.002 0.001 0.0005 0.0002 0.0001 Centre Analytical Laboratories, Inc. Page 14 o f 104 000492 Centre Study No.: 023-042 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation can be found in the raw data associated with this report. 6.3 Chromatography Quantification o f PFOS was accomplished by LC/MS/MS electrospray. The retention time o f PFOS was ~ 4.4 min. Peaks were detected in the control matrices corresponding to the analyte retention time, but the amounts detected were only significant enough to alter several fortification recoveries and the rest were less than the lowest calibration standard (0.0001 pg/mL). 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 0.0001 pg/mL of PFOS. 6.5 Description of Instrument and Operating Conditions Instrument: PE SCIEX API 3000 Biomolecular Mass Analyzer Interface: SCIEX Turboion Spray Liquid Introduction Interface Computer: Dell UltraScan P 1110 Software: HPLC: PE SciexAnalyst version 1.1 Windows NT, version 4 Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column:Genesis Cg (Jones Chromatography), 2.1 mm x 50 mm, 4p Column Temp.: 35 C Injection Voi.: 10 pL Mobile Phase (A): 2 mM Ammonium Acetate in ASTM type I water Mobile Phase (B): Methanol Flow Rate: 0.3 mL/min. Centre Analytical Laboratories, Inc. Page 15 o f 104 C00493 Time % A % B 0 60 40 1.0 0 100 7.0 0 100 7.5 40 60 11.0 40 60 Centre Study No.: 023-042 Ions monitored: Analvte PFOS Mode negative Transition Monitored 499 99 Approximate Retention Time (mini 4.20 Tune File Parameters Controls IS-Iospray DP-Declustering Potential FP-Focusing Potential EP-Entrance Potential CE-Collision Energy CXP-Collision Cell Exit Potential DF-Defiector CEM-Channel Electron Multiplier Set -4200.0 -51.0 -230.0 10.0 -70 -5 300.0 2800.0 Gas Flows Nebulizer Gas Curtain Gas Collision Gas TIS Temperature Set 12 13 4 350C 6.6 Quantitation and Example Calculation Ten microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations o f standards. The concentration for mallard liver (pg/g) and mallard serum (pg/mL) was determined from the equations below. Equation 1 calculated the amount of analyte found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Then Equation 2 calculated the amount o f analyte found in pg/mL (the equations for serum are shown). Centre Analytical Laboratories, Inc. Page 16 o f 104 C00494 Centre Study No.: 023-042 Equation 1: Analyte found (ng/mL) = (Peak area - intercept! slope Equation 2: Analyte found (pg/mL) = ((anal, found (ng/mL) x FV (mL) x DF x EV (mL)) x 1 pg (AV (mL) x sample vol. (mL)) 1000 ng Where: FV = Final Volume DF = Dilution Factor. EV = Extraction Volume AV = Aliquot Volume For samples fortified with known amounts of PFOS prior to extraction, Equation 3 calculated the percent recovery. Equation 3: Recovery (%) = ((anal, found (ng/mL) - avg. anal, in Ctrl (pg/mL)) x (1000 ng/1 pg)) x 100 amount added (ng/mL) An example o f a calculation using an actual sample follows: Mallard serum sample Centre ID 0003803 Spk A (Set: 112100B), fortified at 10 ng/mL with PFOS, where: peak area = 6558 intercept = 515 slope = 7740 dilution factor =1 ng added (fort level) = 1ng avg. amt in controls = 0 (Not quantifiable) final volume = 1 mL extraction volume = 5 mL aliquot volume = 4 mL sample weight (volume) = 0.1 mL From equation 1: Analyte found (ng/mL) = r6558- 5151 7740 From equation 2: Analyte found (pg/mL) 0.8 ng/mL = (0.8 ng/mL x 1mL x 1 x 5 mL) x 1ug (4mLx0.1mL) 1000 ng 0.00976 pg/mL Centre Analytical Laboratories, Inc. Page 17 o f 104 C00495 Centre Study No.: 023-042 From equation 3: % Recovery = ((0,00976 itg/mL x - 0 ng/mLi x 1000) x 100 lOng/mL = 98% 7.0 EXPERIMENTAL DESIGN Each set of samples (liver or serum) consisted of one reagent blank, one matrix blank, two matrix blanks fortified at known concentrations, and ~ 20 samples. Each sample was extracted using the appropriate method and then analyzed in duplicate. 8 0 RESULTS The PFOS found in the reagent blanks are listed in Table I. The PFOS in the mallard liver blanks and mallard serum blanks are given in Tables II & III. Individual recoveries for PFOS in the liver samples are detailed in Table IV. The average percent recovery standard deviation for PFOS in liver samples was 95% 12%. Individual recoveries for PFOS in the serum samples are detailed in Table V. The average percent recovery relative standard deviation for PFOS in serum samples was 93% 10%. PFOS in the mallard liver samples ranged from non-quantifiable levels (a peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) to 22.8 pg/g. Individual results are listed in Tables VI-XIII. The results are reported on a wet weight basis. PFOS in the mallard serum samples ranged from non-quantifiable levels (a peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) to 218 pg/g. Individual results are listed in Tables XIV-XXIII. The percent moistures for the mallard liver samples are given in Table XXIV. 9.0 CONCLUSIONS The mallard liver and serum samples were successfully extracted and analyzed according to protocol 00P-023-042. Centre Analytical Laboratories, Inc. Page 18 of 104 C00496 Centre Study No.: 023-042 10.0 RETENTION OF DATA AND SAM PLES When the final report is complete, all original paper data generated by Centre Analytical Laboratories, Inc. will be shipped to the sponsor. This does not include facility-specific raw data such as instrument logs, however exact copies of temperature logs will be submitted. Exact copies o f all raw data, as well as a signed copy o f the final analytical report and all original facility-specific raw data, will be retained in the Centre Analytical Laboratories, Inc. archives for the period of time specified in 40 CFR Part 792. Retained samples o f reference substances are archived by the sponsor. Centre Analytical Laboratories, Inc. Page 19 o f 104 C00497 Centre Study No.: 023-042 TS Centre Analytical Laboratories, Inc. Page 20 o f 104 C00498 Centre Study No.: 023-042 Table I. Summary o f PFOS in Reagent Blanks Sponsor ID na na na na na na na na na na na na na na na na na na Centre ID Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Set Number 111700A 111700A 112000A 112000A 112100A 112100A 112100B 112100B 112700A 112700A 112700B 112700B 030701A 030701A 03070IB 030701B 030801A 030801A Extraction Analysis Analyte Date Date Found (pg/g) 11/17/00 11/18-19/00 NQ 11/17/00 11/20/00 11/18-19/00 11/20-21/00 NQ NQ 11/20/00 11/20-21/00 NQ 11/21/00 11/22-23/00 NQ 11/21/00 11/21/00 11/22-23/00 11/23/00 NQ NQ 11/21/00 11/23/00 NQ 11/27/00 11/27/00 11/27/00 11/27/00 3/7/01 3/7/01 3/7/01 11/28-29/00 11/28-29/00 11/29/00 11/29/00 3/9/01 3/9/01 3/9-10/01 NQ NQ NQ NQ NQ NQ NQ 3/7/01 3/9-10/01 NQ 3/8/01 3/11/01 NQ 3/8/01 3/11/01 ______ NQ___ AVERAGE: NQ STANDARD DEVIATION: NQ * Duplicate injection NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) For values recorded as NQ, 0.00005 pg/g (half the value o f the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 21 of 104 000499 Centre Study No.: 023-042 Table II. Summary of PFOS in Mallard Liver Blanks Sponsor ID na na na na na na na na Centre ID 0003804 Blank A 0003804 Blank A* 0003804 Blank A 0003804 Blank A* 0003804 Blank A 0003804 Blank A* 0003804 Blank A 0003804 Blank A* Set Number 111700A 111700A 112000A 112000A 112100A 112100A 030801A 030801A Extraction Date Analysis Date 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 3/8/01 3/11/01 3/8/01 3/11/01 AVERAGE: STANDARD DEVIATION: Analyte Found (uk/e) 0.000294 0.000300 NQ NQ NQ NQ NQ NQ NQ NQ Table III. Summary of PFOS in M allard Serum Blanks Sponsor ID na na na na na na na na na na Centre ID 0003803 Blank A 0003803 Blank A* 0003803 Blank A 0003803 Blank A* 0003803 Blank A 0003803 Blank A* 0003803 Blank A 0003803 Blank A* 0003803 Blank A 0003803 Blank A* * Duplicate injection Set Number 112100B 112100B 112700A 112700A 112700B 112700B 030701A 030701A 030701B 030701B Extraction Analysis Analyte Date Date Found (ne/mL) 11/21/00 11/23/00 11/21/00 11/23/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/29/00 11/27/00 11/29/00 3/7/01 3/9/01 3/7/01 3/9/01 3/7/01 3/9-10/01 3/7/01 3/9-10/01 AVERAGE: STANDARD DEVIATION: NQ NQ NQ NQ NQ 0.00159 0.00826 0.00825 0.00159 0.00397 0.00239 0.00333 NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. Centre Analytical Laboratories, Inc. Page 22 of 104 Centre Study No.: 023-042 Table IV. Summary of PFOS Recoveries in Mallard Liver Sponsor ID na na na na na na na na na na na na na na na na Centre ID Set Number 0003804 Spk A 0003804 Spk A* 0003804 Spk B 0003804 Spk B* 0003804 Spk A 0003804 Spk A* 0003804 Spk B 0003804 Spk B* 0003804 Spk A 0003804 Spk A* 0003804 Spk B 0003804 Spk B* 0003804 Spk A 0003804 Spk A* 0003804 Spk B 0003804 Spk B* 111700AD 111700AD 111700A 111700A 112000AR 112000AR 112000A 112000A 112100A 112100A 112100AD 112100AD 030801A 030801A 030801AD 030801AD Extraction Analysis Amt Date Date Added (ne/e) 11/17/00 11/19/00 10 11/17/00 11/19/00 11/17/00 11/18-19/00 10 50 11/17/00 11/20/00 11/20/00 11/20/00 11/20/00 11/21/00 11/18-19/00 11/22/00 11/22/00 11/20-21/00 11/20-21/00 11/22-23/00 50 100 100 4000 4000 1000 11/21/00 11/21/00 11/21/00 11/22-23/00 11/27/00 11/27/00 1000 20000 20000 3/8/01 3/11/01 20 3/8/01 3/8/01 3/11/01 3/11-12/01 20 200 3/8/01 3/11-12/01 200 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: % Recovery 79 77 85 84 114 115 106 107 89 88 90 89 92 92 107 101 95 11 12 * Duplicate injection LOQ = 0.01 ng/g for liver and 0.01 ng/mL for serum Centre Analytical Laboratories, Inc. Page 23 o f 104 000501 Centre Study No.: 023-042 Table V. Summary of PFOS Recoveries in Mallard Serum Sponsor ID na na na na na na na na na na na na na na na na na na na na Centre ID 0003803 Spk A 0003803 Spk A* 0003803 Spk B 0003803 SpkB* 0003803 Spk A 0003803 Spk A* 0003803 SpkB 0003803 Spk B* 0003803 Spk A 0003803 Spk A* 0003803 SpkB 0003803 Spk B* 0003803 Spk A 0003803 Spk A* 0003803 SpkB 0003803 SpkB* 0003803 Spk A2 0003803 Spk A2* 0003803 SpkB2 0003803 Spk B2* Set Number 112100B 112100B 112100B 112100B 112700A 112700A 112700A 112700A 112700B 112700B 112700B 112700B 030701A 030701A 030701A 030701A 030701B 030701B 03070IB 03070IB * Duplicate injection Extraction Analysis Amt Date Date Added Ine/mL) 11/21/00 11/21/00 11/21/00 11/21/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 11/23/00 11/23/00 11/23/00 11/23/00 11/28-29/00 11/28-29/00 11/28-29/00 11/28-29/00 11/29/00 11/29/00 11/29/00 11/29/00 3/9/01 3/9/01 3/9/01 3/9/01 3/9-10/01 3/9-10/01 3/9-10/01 3/9-10/01 10 10 500 500 10 10 10000 10000 500 500 100000 100000 100 100 1000 1000 100 100 1000 1000 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: % Recovery 98 98 84 86 81 81 91 91 97 97 89 89 79 77 89 93 90 90 118 119 92 II 12 LOQ = 0.01 fig/g for liver and 0.01 (ig/mL for serum Centre Analytical Laboratories, Inc. Page 24 of 104 000502 Centre Study No.: 023-042 Table VI. Summary of PFOS in Adult Liver Samples at 0 ppm a.i. (Test Termination 19 Weeks) Sponsor ID 1961 ^ 1961* 1962 1962* 1963 * 1963* 1964 c 1964* 1965 ^ 1965* 1966 f 1966* 1967 A' 1967* 1968 f 1968* 1969 ^ 1969* 1970 J1970* Centre ID 0008006 0008006 0008007 0008007 0008008 0008008 0008009 0008009 0008010 0008010 0008011 0008011 0008012 0008012 0008013 0008013 0008014 0008014 0008015 0008015 Set Number 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700ADD 111700ADD 111700AD 111700AD 111700AD 111700AD 111700AD 111700AD Extraction Analysis Date Date 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/17/00 11/18-19/00 11/17/00 11/20/00 11/17/00 11/20/00 11/17/00 11/19/00 11/17/00 11/19/00 11/17/00 11/19/00 11/17/00 11/19/00 11/17/00 11/19/00 11/17/00 11/19/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 0.000813 0.000763 0.000509 0.000631 0.000937 0.000887 0.00155 0.00157 0.00110 0.00111 0.00446 0.00432 0.662 0.684 0.238 0.246 0.115 0.106 0.355 0.343 0.138 0.219 * Duplicate injection LOQ = 0.01 ng/g for liver and 0.01 (ig/rnL for serum Centre Analytical Laboratories, Inc. Page 25 o f 104 C00503 Centre Study No.: 023-042 Table VII. Summary of PFOS in Adult Liver Samples at 1.8 ppm a.i. (Test Termination 6 Weeks) Sponsor ID E01-0263-21065 ^ E01-0263-21065* E01-0263-21066 ^ EOI-0263-21066* EOI-0263-21067 M EOI-0263-21067* EOI-0263-21068 4' EOI-0263-21068* EOl-0263-21069 A* E01-0263-21069* EOI-0263-21075 r-" EOI-0263-21075* EOI-0263-21076 i E01-0263-21076* EOI-0263-21077 ? E01-0263-21077* EOI-0263-21078 f E01-0263-21078* E01-0263-21079 <' EOI-0263-21079* Centre ID 0103870 0103870 0103871 0103871 0103872 0103872 0103873 0103873 0103874 0103874 0103880 0103880 0103881 0103881 0103882 0103882 0103883 0103883 0103884 0103884 Set Number 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD Extraction Date Analysis Date 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 3/8/01 3/11-12/01 AVERAGE: STANDARD DEVIATION: Analyte Found he/s) 0.359 0.350 0.647 0.631 0.886 0.880 2.11 2.04 0.222 0.232 0.317 0.326 0.219 0.230 0.194 0.197 0.346 0.342 0.0893 0.0888 0.535 0.574 * Duplicate injection LOQ = 0.01 ng/g for liver and 0.01 fig/mL for serum Centre Analytical Laboratories, Inc. Page 26 o f 104 600504 Centre Study No.: 023-042 Table VIII. Summary of PFOS in Adult Liver Samples at 6.2 ppm a.i. (Test Termination 6 Weeks) Sponsor ID EOl-0263-21070 -i EOI-0263-21070* EOI-0263-21071 EOI-0263-21071* E01-0263-21072 E01-0263-21072* EOI-0263-21073 EOI-0263-21073* EOI-0263-21074 EOI-0263-21074* v EOI-0263-21080 EOI-0263-21080* E01-0263-21081 E01-0263-21081* E01-0263-21082 E01-0263-21082* E01-0263-21083 E01-0263-21083* ] EOI-0263-21084 , EOI-0263-21084* ' Centre ID 0103875 0103875 0103876 0103876 0103877 0103877 0103878 0103878 0103879 0103879 0103885 0103885 0103886 0103886 0103887 0103887 0103888 0103888 0103889 0103889 Set Number 030801AD 030801AD 030801AD 030801 AD 030801 AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD Extraction Analysis Date Date 3/8/01 3/8/01 3/8/01 3/8/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/8/01 3/8/01 3/8/01 3/8/01 3/8/01 3/8/01 3/8/01 3/8/01 3/8/01 3/8/01 3/8/01 3/8/01 3/8/01 3/8/01 3/8/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/11-12/01 3/8/01 3/11-12/01 AVERAGE: STANDARD DEVIATION: Analyte Found (na/a) 1.04 1.01 0.963 0.907 1.92 1.81 0.791 0.827 1.28 1.34 1.01 1.03 0.0614 0.0578 0.473 0.517 1.66 1.52 0.0862 0.0878 0.920 0.576 * Duplicate injection LOQ = 0.01 ng/g for liver and 0.01 ng/mL for serum Centre Analytical Laboratories, Inc. Page 27 of 104 000505 Centre Study No.: 023-042 Table IX. Summary of PFOS in Adult Liver Samples at 17.6 ppm a.i. (Test Termination 19 Weeks) Sponsor ID 1991 a- 1991* 1992 f= 1992* 1993 ^ 1993* 1994 1994* 1995 1995* 1996 r 1996* 1997 / 1997* 1998 r 1998* 1999 M 1999* 2000 r 2000* Centre ID 0008016 0008016 0008017 0008017 0008018 0008018 0008019 0008019 0008020 0008020 0008021 0008021 0008022 0008022 0008023 0008023 0008024 0008024 0008025 0008025 Set Number 111700ADD 111700ADD 111700ADD 111700ADD 111700ADD 111700ADD 111700AD 111700AD 111700AD 111700AD 111700ADD 111700ADD 111700AD 111700AD 111700AD 111700AD 111700ADD 111700ADD 111700AD 111700AD Extraction Analysis Date Date 11/17/00 11/17/00 11/17/00 11/17/00 11/17/00 11/17/00 11/17/00 11/17/00 11/17/00 11/17/00 11/17/00 11/17/00 11/17/00 11/17/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/19/00 11/19/00 11/19/00 11/19/00 11/20/00 11/20/00 11/19/00 11/19/00 11/17/00 11/19/00 11/17/00 11/19/00 11/17/00 11/20/00 11/17/00 11/20/00 11/17/00 11/19/00 11/17/00 11/19/00 AVERAGE: STANDARD DEVIATION: Analyte Found (njj/g) 15.9 16.4 22.8 21.9 7.02 6.69 2.82 2.93 3.53 3.60 13.2 13.5 5.62 5.58 3.72 3.86 15.5 16.1 5.97 6.10 9.64 6.57 * Duplicate injection LOQ = 0.01 ng/g for liver and 0.01 jag/mL for serum Centre Analytical Laboratories, Inc. Page 28 of 104 000506 Centre Study No.: 023-042 Table X. Summary of PFOS in Juvenile Liver Samples at 0 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 2301 2301* 2302 c 2302* 2303 ^ 2303* 2305 A' 2305* 2307 ^ 2307* 2308 2308* 2314 * 2314* 2316 ^ 2316* 2317 ^ 2317* 2320 ^ 2320* Centre ID 0008086 0008086 0008087 0008087 0008088 0008088 0008089 0008089 0008090 0008090 0008091 0008091 0008092 0008092 0008093 0008093 0008094 0008094 0008095 0008095 Set Number 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A Extraction Analysis Date Date 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 AVERAGE: STANDARD DEVIATION: Analyte Found ( h r /r ) NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ * Duplicate injection NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. Centre Analytical Laboratories, Inc. Page 29 o f 104 000507 Centre Study No.: 023-042 Table XI. Summary of PFOS in Juvenile Liver Samples at 1.8 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 2322 r: 2322* 2323 !* 2323* 2325 < 2325* 2326 2326* 2328 2328* 2330 2330* 2331 ^ 2331* 2332 f 2332* 2333 2333* 2334 /V\ 2334* Centre ID 0008096 0008096 0008097 0008097 0008098 0008098 0008099 0008099 0008100 0008100 0008101 0008101 0008102 0008102 0008103 0008103 0008104 0008104 0008105 0008105 Set Number 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A Extraction Analysis Date Date 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 AVERAGE: STANDARD DEVIATION: Analyte Found (ub/b) 0.00454 0.00458 0.00124 0.00129 0.00150 0.00154 0.00236 0.00245 0.00167 0.00165 0.00185 0.00182 0.000719 0.000763 0.000425 0.000430 0.000969 0.000966 0.00356 0.00355 0.00189 0.00126 * Duplicate injection LOQ = 0.01 ng/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 30 of 104 000508 Centre Study No.: 023-042 Table XII. Summary of PFOS in Juvenile Liver Samples at 6.2 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 2335 ? 2335* 2336 f 2336* 2337 ? 2337* 2338 2338* 2339 A" 2339* 2341 ^ 2341* 2342 f 2342* 2343 f i 2343* 2344 ' 2344* 2346 <y ' 2346* Centre ID 0008106 0008106 0008107 0008107 0008108 0008108 0008109 0008109 0008110 0008110 0008111 0008111 0008112 0008112 0008113 0008113 0008114 0008114 0008115 0008115 Set Number 112100A 112100A 112100A 112I00A 112100A 112100A 112100A 112100A 112100A 112100A 112100A 112100A 112100A 112100A 112100A 112100A 112100A 112100A 112100AD 112100AD Extraction Analysis Date Date 11/21/00 11/21/00 11/21/00 11/22-23/00 11/22-23/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/27/00 11/21/00 11/27/00 AVERAGE: STANDARD DEVIATION: Analyte Found iwj/R) 0.00197 0.00200 0.00377 0.00378 0.00380 0.00363 0.00555 0.00535 0.00540 0.00518 0.00306 0.00313 0.00312 0.00320 0.00329 0.00332 0.00402 0.00405 0.0141 0.0146 0.00482 0.00341 * Duplicate injection LOQ = 0.01 p.g/g for liver and 0.01 jag/mL for serum Centre Analytical Laboratories, Inc. Page 31 of 104 000509 Centre Study No.: 023-042 Table XIII. Summary of PFOS in Juvenile Liver Samples at 17.6 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 2347 ^ 2347* 2348 F 2348* 2353 P 2353* 2354 ^ 2354* 2355 ^ 2355*/C 2357 rTV 2357* 2358 i? 2358* 2359 P 2359* 2360^ 2360* 2363 2363* Centre ID 0008116 0008116 0008117 0008117 0008118 0008118 0008119 0008119 0008120 0008120 0008121 0008121 0008122 0008122 0008123 0008123 0008124 0008124 0008125 0008125 Set Number 112100AD 112100AD 112100AD 112100AD 112100AD 112100AD 112100AD 112100AD 112100A 112100A 112100AD 112100AD 112100AD 112100AD 112100AD 112100AD 112100A 112100A 112100AD 112100AD Extraction Analysis Date Date 11/21/00 11/27/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/22-23/00 11/22-23/00 11/27/00 11/27/00 11/27/00 11/21/00 li/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/27/00 11/27/00 11/27/00 11/22-23/00 11/22-23/00 11/27/00 11/21/00 11/27/00 AVERAGE: STANDARD DEVIATION: Analyte Fonnd (iu /i ) 0.0383 0.0370 0.0456 0.0465 0.0234 0.0227 0.0184 0.0189 0.0110 0.0105 0.0616 0.0629 0.0175 0.0189 0.0211 0.0211 0.00623 0.00628 0.0172 0.0191 0.0262 0.0168 * Duplicate injection LO Q --0.01 |ig /g for liv er and 0 .01 n g/m L fo r serum Centre Analytical Laboratories, Inc. Page 32 o f 104 000510 Centre Study No.: 023-042 Table XIV. Summary o f PFOS in Control A dult Serum Sam ples Sponsor ID E01-0263-21005 -M E01-0263-21005* E01-0263-21006 ft E01-0263-21006* E01-0263-21007^\ E01-0263-21007* EOI-0263-210084 EOl-0263-21008* E01-0263-21009 * E01-0263-21009* E01-0263-21025 P E01-0263-21025* E01-0263-21026 f E 0 1-0263-21026* E01-0263-21027i E01-0263-21027* E01-0263-21028 f' EOI-0263-21028* EOI-0263-21029 Y EOl-0263-21029* Centre ID 0103810 0103810 0103811 0103811 0103812 0103812 0103813 0103813 0103814 0103814 0103830 0103830 0103831 0103831 0103832 0103832 0103833 0103833 0103834 0103834 Set Number 030701A 030701A 030701A 030701A 030701A 030701A 030701A 030701A 030701A 030701A 030701B 030701B 030701B 030701B 030701B 03070IB 030701B 03070IB 030701B 030701B Extraction Analysis Analyte Date Date Found (ne/mL) 3/7/01 3/9/01 3/7/01 3/9/01 3/7/01 3/9/01 3/7/01 3/9/01 3/7/01 3/9/01 3/7/01 3/9/01 3/7/01 3/9/01 3/7/01 3/9/01 3/7/01 3/9/01 3/7/01 3/9/01 3/7/01 3/9-10/01 3/7/01 3/9-10/01 3/7/01 3/9-10/01 3/7/01 3/9-10/01 3/7/01 3/9-10/01 3/7/01 3/9-10/01 3/7/01 3/9-10/01 3/7/01 3/9-10/01 3/7/01 3/9-10/01 3/7/01 3/9-10/01 AVERAGE: STANDARD DEVIATION: 0.0463 0.0455 0.00828 0.009280.00954 0.0103 0.00319-V 0.00452^ 0.00423^ 0.00616 0.00285 0.00207 NQ NQ NQ NQ 0.00428 0.00265 NQ NQ 0.00797 0.0134 * Duplicate injection NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but w as less than the low est concentration o f the calibration standards (0.0001 pg/m L)) For values recorded as NQ, 0.00005 pg/mL (halfthe value o f the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 33 o f 104 000511 mm Centre Study No.: 023-042 Table XV. Summary of PFOS in Adult Serum Samples at 0 ppm a.i. (Test Termination 19 Weeks) Sponsor ID 1961 M 1961* 1962 F 1962* . 1963 1963* 1964 1964* 1965 *0 1965* 1966 C 1966* 1967 m 1967* 196* f 1968* 1969 ^ 1969* 1970 P' 1970* e n tre ID 0008026 0008026 0008027 0008027 0008028 0008028 0008029 0008029 0008030 0008030 0008031 0008031 0008032 0008032 0008033 0008033 0008034 0008034 0008035 0008035 Set Number 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100BD 112100BD 112100BD 112100BD 112100BD 112100BD 112100BD 112100BD Extraction Analysis Analyte Date Date Found (w/m L) 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/23/00 11/23/00 11/23/00 11/23/00 11/23/00 11/23/00 11/23/00 11/23/00 11/23/00 11/23/00 11/23/00 11/23/00 11/27-28/00 11/27-28/00 11/27-28/00 11/27-28/00 0.00605 0.00594 0.00494 0.00501 0.00844 0.00825 0.00798 0.00761 0.0104 0.0103 0.0358 0.0357 6.11 6.02 3.80 3.81 11/21/00 11/27-28/00 11/21/00 11/27-28/00 11/21/00 11/27-28/00 11/21/00 11/27-28/00 AVERAGE: STANDARD DEVIATION: 0.843 0.849 1.41 1.38 1.22 2.03 * Duplicate injection L O Q " 0 .0 1 n g /g for liv e r and 0.01 (j-g/mL for serum Centre Analytical Laboratories, Inc. Page 34 o f 104 000512 000513 C00514 Centre Study No.: 023-042 Table XVIJ3. Summary o f PFOS in Adult Serum Samples at 17.6 ppm a.i. (Test Termination 19 Weeks) Sponsor ID 1991 M 1991* 1992 F 1992* 1993 1993* 1994 F 1994* 1995 1995* 1996 P 1996* 1997 r* 1997* 1998 P 1998* 1999 * \ 1999* 2000 2000* Centre ID 0008036 0008036 0008037 0008037 0008038 0008038 0008039 0008039 0008040 0008040 0008041 0008041 0008042 0008042 0008043 0008043 0008044 0008044 0008045 0008045 * Duplicate injection Set Number 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD Extraction Analysis Analyte Date Date Found (ite/mL) 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 11/21/00 11/28/00 AVERAGE: STANDARD DEVIATION: 41.0 41.1 47.0 49.5 199 198 93.9 93.0 182 180 218 217 214 212 156 156 156 161 200 205 151 65.0 LOQ = 0.01 itg/g for liver and 0.01 jtg/mL for serum Centre Analytical Laboratories, Inc. I Page 37 o f 104 000515 Centre Study No.: 023-042 Table XIX. Summary of PFOS in Adult Serum Samples at 17.6 ppm a.i. (6 Week Samples) Sponsor ID Centre ID E01-0263-21020 4 0103825 E01-0263-21020* 0103825 E01-0263-21021 ^ 0103826 E01-0263-21021* 0103826 E01-0263-21022 <v\ 0103827 E01-0263-21022* E01-0263-21023 M E01-0263-21023* E01-0263-21024 X 1 0103827 0103828 0103828 0103829 E1-0263-21024* 0103829 E01-0263-21040 C 0103845 E01-0263-21040' 0103845 E01-0263-21041 P 0103846 E01-0263-21041* 0103846 E01-0263-21042 p 0103847 E01-0263-21042* 0103847 E01-0263-21043 G 0103848 E01-0263-21043* E01-0263-21044 / 0103848 0103849 E01-0263-21044* 0103849 Set Number 030801AD 030801AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030801AD 030801AD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD * Duplicate injection Extraction Analysis Date Date 3/7/01 3/11-12/01 3/7/01 3/7/01 3/11-12/01 3/10/01 3/7/01 3/7/01 3/10/01 3/10/01 3/7/01 3/10/01 3/7/01 3/7/01 3/10/01 3/10/01 3/7/01 3/7/01 3/11-12/01 3/11-12/01 3/7/01 3/10-11/01 3/7/01 3/10-11/01 3/7/01 3/10-11/01 3/7/01 3/10-11/01 3/7/01 3/10-11/01 3/7/01 3/7/01 3/10-11/01 3/10-11/01 3/7/01 3/10-11/01 3/7/01 3/10-11/01 3/7/01 3/10-11/01 AVERAGE: STANDARD DEVIATION: Analyte Found frig/mL) 18.6 18.1 152 151 156 157 137 138 42.2 39.1 55.9 62.1 49.8 43.2 15.8 17.7 18.5 17.3 14.1 14.4 65.9 57.5 LOQ - 0.01 (tg/g for liver and 0.01 iig/mL for serum 1 Centre Analytical Laboratories, Lie. Page 38 o f 104 C00516 Centre Study No.: 023-042 Table XX. Summary of PFOS in Juvenile Serum Samples at 0 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 2301 C 2301* 2302 ? 2302*i 2303 2303* 2305 Z*' 2305* 2307/h 2307* 2308 ^ 2308* 2314^ 2314* 2316 /V> 2316* 2317 t=2317* 2320 r 2320* Centre ID 0008126 0008126 0008127 0008127 0008128 0008128 0008129 0008129 0008130 0008130 0008131 0008131 0008132 0008132 0008133 0008133 0008134 0008134 0008135 0008135 Set Number 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A Extraction Date Analysis Date Analyte Found (iia/mL) 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 AVERAGE* STANDARD DEVIATION: NQ NQ NQ NQ 0.00458 0.00453 NQ NQ 0.00278 0.00272 NQ NQ NQ NQ 0.00381 0.00379 0.00626 0.00601 0.00141 0.00153 0.00190 0.00222 * Duplicate injection N Q - N o t Quantifiable (A peak w as detected at the corresponding analyte retention tim e but was less than the lowest concentration o f the calibration standards (0.0001 pg/mL)) For values recorded as NQ, 0.00005 pg/mL (halfthe value o f the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 39 o f 104 000517 Centre Study No.: 023-042 Table XXL Summary of PFOS in Juvenile Serum Samples at 1.8 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 2322 ^ 2322* 2323 W 2323* 2325 F 2325* 2326 2326* 2328 2328* 2330 <= 2330* 2331 2331* 2332 ? 2332* 2333 2333* 2334 ^ 2334*- Centre ID 0008136 0008136 0008137 0008137 0008138 0008138 0008139 0008139 0008140 0008140 0008141 0008141 0008142 0008142 0008143 0008143 0008144 0008144 0008145 0008145 Set Number 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A 112700D 112700D 112700A 112700A 112700A 112700A 112700A 112700A 112700A 1I2700A 112700A 112700A Extraction Analysis Analyte Date Date Found (uu/mLl 11/27/00 11/27/00 11/27/00 11/28-29/00 11/28-29/00 11/28-29/00 0.0464 0.0472 0.0274 11/27/00 11/28-29/00 0.0281 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/28-29/00 11/28-29/00 11/28-29/00 11/28-29/00 11/29-30/00 11/29-30/00 0.0497 0.0487 0.0160 0.0156 0.249 0250 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/28-29/00 11/28-29/00 11/28-29/00 11/28-29/00 11/28-29/00 0.00788 0.00747 0.0101 0.00998 0.0113 11/27/00 11/27/00 11/28-29/00 11/28-29/00 0.0110 0.0339 11/27/00 11/28-29/00 0.0339 11/27/00 11/27/00 11/28-29/00 11/28-29/00 AVERAGE: 0.0554 0.0555 0.0507 STANDARD DEVIATION: 0.0701 * Duplicate injection LOQ = 0.01 |ig/g for liver and 0.01 (ig/m L for serum Centre Analytical Laboratories, Inc. Page 40 o f 104 000518 Centre Study No.: 023-042 Table XXII. Summary of PFOS in Juvenile Serum Samples at 6.2 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 2335 r 2335* 2336 F 2336* 2337 f 2337* 2338 <vA 2338*' 2339 2339* 2341 A 2341* 2342 ^ 2342* 2343 W 2343* 2344 f 2344* 2346 T 2346* Centre ID 0008146 0008146 0008147 0008147 0008148 0008148 0008149 0008149 0008150 0008150 0008151 0008151 0008152 0008152 0008153 0008153 0008154 0008154 0008155 0008155 Set Number 112700B 112700B 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700B 112700B 112700D 112700D 112700D 112700D 112700B 112700B Extraction Analysis Analyte Date Date Found (u&/mL) 11/27/00 11/29/00 11/27/00 11/29/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29/00 11/27/00 11/29/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29/00 11/27/00 11/29/00 AVERAGE: STANDARD DEVIATION: 0.0159 0.0162 0.118 0.124 0.181 0.183 0.0584 0.0592 0.0889 0.0840 0.0556 0.0558 0.0380 0.0383 0.0700 0.0717 0.0676 0.0688 0.0371 0.0373 0.0734 0.0466 * Duplicate injection LOQ = 0.01 p g/g for liver and 0.01 pg/m L for serum Centre Analytical Laboratories, Inc. Page 41 o f 104 000519 Centre Study No.: 023-042 T able X X III. Sum m ary o f PFO S in Juvenile Serum Sam ples at 17.6 ppm a.!. (Test Term ination 12 W eeks) Sponsor n> 2347 F 2347* 2348 F 2348* 2353 F 2353* 2354 /H 2354* 2355 2355* 2357/^ 2357* 2358 F 2358* 2359 F 2359* 2360 ^ 2360* 2363 A 2363* Centre ID 0008156 0008156 0008157 0008157 0008158 0008158 0008159 0008159 0008160 0008160 0008161 0008161 0008162 0008162 0008163 0008163 0008164 0008164 0008165 0008165 Set Number 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D Extraction Analysis Analyte Date Date Found (ua/mL) 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 11/27/00 11/29-30/00 AVERAGE: STANDARD DEVIATION: 0.490 0.496 0.150 0.156 0.894 0.908 0.591 0.621 0.399 0.384 0.547 0.547 0.512 0.517 0.402 0.404 0.521 0.514 0.439 0.434 0.496 0.184 * Duplicale injection LOQ = 0.01 n g/g for liver and 0.01 pg/m L for serum Centre Analytical Laboratories, Inc. Page 42 o f 104 000520 Centre Study No.: 023-042 Table XXTV. Summary o f Percent M oistures for M allard Liver Sam ples Sponsor Centre Average m ID % Moisture 1961 1962 0008006 0008007 69.7 73.4 1963 1964 1965 0008008 0008009 0008010 75.7 69.2 71.9 1966 1967 1968 1969 1970 1991 1992 1993 0008011 0008012 0008013 0008014 0008015 0008016 0008017 0008018 75.0 72.8 70.4 69.5 72.9 75.3 71.8 71.9 1994 1995 0008019 0008020 71.8 71.5 1996 0008021 72.4 1997 0008022 70.5 1998 1999 2000 2301 2302 2303 2305 2307 2308 2314 0008023 0008024 0008025 0008086 0008087 0008088 0008089 0008090 0008091 0008092 71.3 69.8 72.0 76.6 77.3 72.7 72.5 72.3 72.0 75.0 2316 2317 2320 2322 2323 2325 2326 2328 0008093 0008094 0008095 0008096 0008097 0008098 0008099 0008100 69.6 71.8 71.6 71.6 72.5 732 72.6 72.0 2330 2331 2332 2333 2334 2335 0008101 0008102 0008103 0008104 0008105 0008106 68.9 71.4 71.7 69.8 68.1 71.7 Centre Analytical Laboratories, Inc. Page 43 o f 104 000521 Centre Study No.: 023-042 Table X X IV (cont'). Summary o f P ercent M oistures for M allard Liver Sam ples Spouor Centre Average ID ___________ID______ % Moisture 2336 2337 2338 2339 2341 2342 2343 2344 2346 2347 2348 2353 2354 2355 2357 2358 2359 2360 2363 E01-0263-21065 E 0 1-0263-21066 E01-0263-21067 E01-0263-21068 E01-0263-21069 E01-0263-21070 E01-0263-21071 EO1-0263-21072 E01-0263-2I073 E 0 1-0263-21074 E01-0263-21075 E01-0263-21076 E01-0263-21077 E 0 1-0263-21078 E01-0263-21079 E01-0263-21080 E01-0263-21081 E01-0263-21082 E01-0263-21083 E01-0263-21084 0008107 0008108 0008109 0008110 0008111 0008112 0008113 0008114 0008115 0008116 0008117 0008118 0008119 0008120 0008121 0008122 0008123 0008124 0008125 0103870 0103871 0103872 0103873 0103874 0103875 0103876 0103*77 0103878 0103879 0103880 0103881 0103882 0103883 0103884 0103885 0103886 0103887 0103888 0103889 69.7 69.7 69.8 70.6 72.6 782 70.9 69.2 70.9 69.7 70.3 73 2 68.5 67.8 74.5 72.0 73.6 69.0 74.1 73.8 81.0 82.4 75.8 78.2 T1A 77.5 74.8 77.5 82.7 71.7 77.9 77.5 76.0 63.8 78.2 73.6 76.7 81.4 72.3 Centre Analytical Laboratories, Inc. Page 44 o f 104 Centre Study No.: 023*042 FIGURES Centre Analytical Laboratories, Inc. Page 45 o f 104 600523 rV n tm Sfii(?v N n (IW JU ?. Figure 1. Typical Calibration Curve for FFUS .O r ti m m RmhmI m m iea " a K9 m m iea \ \ \ \. \ \ . .- . \\ \\ V\ \ \ m m m m \m HHM !ll=a ISMW iwsa ==a!1 \tsja lasm mxaa IsU9 nma iMJ ICMA 'WKJM ISSXi t*S* mam w **/ cn - XT Ul w ro TT w 4/ CM P a h 4 i -a A n n l i I A k A M t A f t A S In/"* *\ \ V w- CM %u **' v-- m i m A A (a a Tt* <u _ V-- rLP f 1*0 L c I ?. tc . ' F ! C |.'-i c i f r- i e__n n r-\ -- O in Maoa A A At MIA Centre Study No.: 023-042 Figure 2. Chromatogram Representing a 0.1 ng/mL standard for PFOS Sample Index: Sim ple Type: ! C oncentration: j Acq. Data: Acq. Time: S ta n d a rd M odified: I Bunching F actort ; Hois* Threshold: [ Area Threshold: - Nun. Smooths: I RT Window: : E x p e c te d RT: Sep. W idth: Sep. H eight: Exp. Peak R atio: Exp. Ad*). R a tio : Exp. V al. R atio: Use R e l a tiv e RT: I n t. Type: , R e te n tio n Time: 4.54 Centre Analytical Laboratories, Inc. Page 47 of 104 C00525 Centre Study No.: 023-042 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PFOS . Sample Index: ! Sample Type: C oncentration: Acq. Data: Acq. Tina: 4 Standard 1.000 11/22/00 12:49:51 m M odified: Bunching F actor: Noise Threshold: Area Threshold: Non. Smooths: AT Window: E x p ected AT: j Sep. Width: ; Sep. H eight: Exp. Peak R atio: Exp. Adj. R atio: Exp. V al. R atio: Use R e la tiv e AT: Yes 1 2,82 1 4 .OS 1 30.0 4*52 0.20 0.01 5.00 4.00 3.00 Wo sec min i ? I In t. Type: R eten tio n Time: A rea: Manual 4.S4 S3S5.5 min counts 2895 2500 *00 | 1500 ! 1000 500 fVl <54 4 Tim, min Centre Analytical Laboratories, Inc. Page 48 o f 104 G0052 Centre Study No.: 023-042 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Centre ID Reagent Blank A , Set.* 112000A) Sample Index: Sample Type: C oncentration: Acq. Date: Acq. Time: 8 ....... Blank O.-OQQ 11/ 20/00 0 9 :2 1 :1 6 PM M odified; Bunching r e c to r: N oise Threshold: Ares Threshold: Nun. Smooths; RT Window: E x p e c te d RT: Sep. W idth: Sep. H eight: Exp. Peak R atio: Exp. Ad). R atio: Exp. v a l. R atio: Dee R e l a tiv e RT: Yes 1 2.82 14. Q8 130.0 4.52 0.20 0.01 5.00 4.00 3.00 NO sec min 5 i I * I n t. Type: R e te n tio n Time: A res: Manual 4.57 432.0 min counts 133 . 120 j i 100 J J 80 ] ! 60: i I 40j I 2) o' 4.87 4.8 "/'A ! > ***' 4 Tim, min SO \\ ?1.81W?0. \ TOO' J. . . Centre Study No.: 023-042 Figure 5. Chromatogram Representing Control Mallard Serum for FFOS (Centre ID: 0003803 Blank A, Set: 112100B) i&M'r-L I p [>; " p ' ` ... 4,;:. :.f i. " *. i : Sample Index: Sample Type; C oncentration: Acq. Data: Acq. Time: 10 Blanle 0.000 11/23/00 0 3 :3 8 :5 2 AM 200 `M odified: Bunching Factor: Noise Threshold: Area Threshold: Hum. Smooths: RT Window: E x p ected RT: Sep. W idth: Sep. H eight: Exp. Peak Ratio: Exp. A dj. Ratio: Exp. V al. Ratio: Use R e l a tiv e RT: Yes 1 2 . 2 1 4 .OS 1 30.0 4.52 0 .2 0 0.01 5.00 4.00 3.00 Ho eec min 160 ; F 100 | | * 50 I n t. Type: R etention Tine: A rea: Manual 4.54 612.8 min counts * 1 2 4.53 4 .4.66 4.45 y `% * .* * ? 8 f i + a.a7 7.70 ' v -r ./ 'VeVr' - * A-. 3456 7 Time, min Centre Analytical Laboratories, Inc, Page 50 o f 104 000528 Centre StudyNo.: 023-042 Figure 6. Chromatogram Representing Control M allard Liver for PFOS, (Centre ID: 0003804 Blank A, Set: 112000A) t; i"I> '''-,,Vl'.V't'M* 'r ,'vv Hi'- i Sajnple Index: ; S .a p l. Typ: i C oncentration: 1 Acq. Date: Acq. Tiaw: 10 Blank 0.000 11/20/00 05:46:39 n i 227 200 | M odified: yes ; Bunching ra e to r: Boise Threshold: 1 2.82 150- Ares Threshold:, Mum. Smooths: RT Window; E x p ected RT: Sep. W idth: Sep. Height*. 14.08 1 t I l3 0 . 0 4.52 aec in 0.20 0.01 c 100 Exp. Peak R atio: 5.00 Exp. AdJ. R atio: Exp. V al. R atio: 4.00 3.00 50 Use R e l a tiv e RT: i In t. Type: | R eten tio n Time: ; Axes: Wo Manual 4.56 829.8 in counts 0 12 4.5 4.66 74.43 m V ;5-00.-s -# 8.10 6 S B Time, min 7.58 Centre Analytical Laboratories, Inc, Page SI of 104 000529 C00530 C00531 000532 c 00533 Centre Study No.: 023-042 APPENDIX A Study Protocol 00P-023-042 (Centre Study No. 023-042) and Amendments and Deviation Centre Analytical Laboratories, Inc. Page 56 o f 104 000534 Centre Study No.: 023-042 Centre Protocol No. 00P-023-042 STUDY PROTOCOL STUDY TITLE EXTRACTION OFPOTASSIUM PERFLUOROOCTANESULFONATEFROM MALLARD SERUM AND MALLARD LIVERFOR ANALYSIS USING HPLC- ELECTROSPRAY/MASS SPECTROMETRY SPONSOR 3MEnvironmental Technology andSafety Services Building 2-3E-09 PO Box 33331 SL.Phul.MN 55133-3331 DATA REQUIREMENTS Analytical Method Requirements TESTING LABORATORY Centre Analytical Laboratories. Inc. (Centre) 3048 Research Drive State College, PA 16801 Phone 814-231-8032 PROTOCOL IDENTIFICATION NUMBER OOP-023-042 Centre Analytical Laboratories. Inc. Centre Analytical Laboratories, Inc. Page 1 of 43 Page 57 o f 104 000535 Centre Study No.: 023-042 Centre Protocol No. 00P-O23-O42 Title: EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM MALLARD SERUM AND MALLARD LIVER FOR ANALYSIS USING HPLCELECTROSPRAY/MASS SPECTROMETRY TABLE OF CONTENTS _______________________________________________________________________________________________ TABLE OFCONTENTS ...................................................................... ................. 2 1. PURPOSE...................................... -....................................................... -..... 3 2. TEST MATERIALS................... 3 3. SPONSOR......................................................... 4 4. TESTING FACILITY AND PERFORMINGLABORATORY..........................4 5. PROPOSEDEXPERIMENTALTIME-ERAME........................................ 4 6. JUSTIFICATIONFORTHESELECTION OFTHETEST SYSTEM................4 7. SAMPLEPROCESSING. STORAGE AND IDENTIFICATION.......................5 8. ANALYTICALMETHOD............................................................................... 5 9. EXPERIMENTALDESIGN........... .......................... 10 10. PROTOCOLAMENDMENTS AND DEVIATIONS....................................... 10 11. RECORDS.....................................................................................................11 12. QUALITYASSURANCE.............................................................................. 11 13. DATA ANDREPORT.................................................................................... 12 14. ARCHIVESTATEMENT...............................................................................12 15. REFERENCES................................................................... 13 16. PROTOCOLAPPROVAL............................................................................... 14 APPENDIX: ANALYTICALMETHODS.................................................................. 15 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 2 of43 Page 58 o f 104 G00536 Centre Study No.: 023-042 / Centre Protocol No. OOP-023-042 SlwdyTitk; EXTRACTIONOF POTASSIUMPERFLUOROOCTANESULFONATEFROM MALLARD SERUM AND MALLARD LIVERFORANALYSIS USING HPLC- ELECTROSPRAY/MASS SPECTROMETRY 1. PURPOSE The purpose of this study is to analyze mallard serum samples and mallard liver samples for residues of perfluorooctanesulfonate (PFOS) using methods entitled, '"Extraction Of Potassium Perfluorooctanesulfonate Or Other Fluorochemical Compounds From Serum For Analysis Using HPLC-Elecoospray/Mass Spectrometry" (3M method number ETS-8-4.1) and "Extraction Of Potassium Perfluorooctanesulfonate Or Other Fluorochemical Compounds From liver For Analysis Using HPLC-Electrospray/Mass Spectrometry" (3M method number ETS-8-6.0) with'the modifications listed in Section 8. The Quality Assurance Unit of Centre Analytical Laboratories, Inc., will audit the study for compliance with EPA TSCA Good Laboratory Practice standards 40 CFR Part 792(1). 2. TEST MATERIALS The following analytical standards will be used: Test Material PFOS TCR Number TCR00017-46 Purity (%) 97.9 Expiration Date 08/31/01 Chemical name and structure of the compound is presented below. PFOS Chemical Name: IUPAC Name: CAS Number Molecular Weight; Perfluorooctanesulfonate 1-Octanesulfonic acid,l,1,2^3,4,4,5^,6,6.7,7,8,8,8heptadecafluoco-, potassiumsalt 2795-39-3' 499 (CjF tSOT) O II C gF 17S ----0 O Note: The neutral molecule and standard form that PFOS (anion) is derived from is perfluorooctanesulfonate potassium salt [CsFhSOjK], molecular weight 538. CentreAnalytical Laboratories, Inc. I Page 3 of 43 Centre Analytical Laboratories, Inc. Page 59 o f 104 000537 Centre Study No.: 023-042 CentreProtocol No. 0OP-23-O42 A record of test and reference substance receipt, storage conditions, and a record of use will be maintained at Centre Analytical Laboratories, Inc. Forms documenting chain-of-custody and shipping records for tracking of the test substances will be included as part of the rawdatapackage. All standards/test substances and any preparedsolutions must be identified with a unique label or numberon the containerorcross-referenced to the container. HAZARD INFORMATION A current MSDS for the chemical(s).u$ed in this study will be maintain.) at the testing facility. 3. SPONSOR 3MEnvironmental Technology andSafety Services Building 2-3E-09 PO Box 33331 St Paul, MN 55133-3331 STUDY DIRECTOR: Rochelle Robideau TELEPHONE: 651-778-7065 FAX NUMBER: 651-778-6176 4. TESTING FACILITY AND PERFORMING LABORATORY Centre Analytical Laboratories, he. (Centre) 3048 Research Drive State College, PA 16801 PRINCIPALINVESTIGATOR: Emily R. Stauffer, Centre TELEPHONE: 814-231-8032 FAX NUMBER: 814-231-1580 5. PROPOSEDEXPERIMENTALTIME-FRAME Experimental Start Date Experimental TerminationDate Report Issued Nov. 6,2000 Nov. 24,2000 Dec. 30,2000 6. JUSTIFICATION FORTHE SELECTION OF THE TEST SYSTEM Mallard serumsamples andliver samples collected by Wildlife International, Ltd. will be used as the test systems for this study. Mallards represent wild bird populations and they are anEPA recommended species because they do well in a laboratory environment. Mallard habitats include ponds, lakes and marshes, and CentreAnalytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 4 of 43 Page 60 o f 104 G00538 Centre Study No.: 023-042 CentreProtocolNo. OOP-023-042 they areconsidered aquatic grazers. The use of these matrices for the study was to determine if PFOS was deposited in the liver and serum of mating adults that were fedPFOS. 7. SAMPLEPROCESSING, STORAGE AND IDENTIFICATION No processing will be required for the serum samples. Due to the small amount of sample available, the liver samples will not be homogenized priorto extraction; however, they will be homogenized by lying the samples on a flat surface and mashing themby hand as best as possible. Each sample will be assigned a unique sample identification number at Centre, which will be used for tracking and identification of the samples. The samples will be stored in a temperature-monitored freezer, maintained at < >10*C, except when removed for extraction and analysis as described in the method. The sampleswill be keptisolated fromthe test substance duringstorage. Sample receipt and storage location and conditions during the study will be documented. All samples and any resulting sample extracts will be identified with a unique label or sample number. Such identification will be either on the containerorcross-referenced to the container. 8. ANALYTICAL METHOD The serum samples will be analyzed according to the analytical method titled "Extraction Of Potassium Perfluorooctanesulfonate Or Other Fluorochentical Compounds From Serum For Analysis Using HPLC-Electrospray/Mass Spectrometry" (3M method number ETS-8-4.1) and the control liver samples will be analyzed according to the method titled "Extraction Of Potassium Peifluorooctanesulfonate Or Other Fluorochemical Compounds From Liver For Analysis Using HPLC-Electrospray/Mass Spectrometry" (3M method number ETS-8-6.0). The following modifications w ill be applied to each method: 1. There'will be no surrogate standard used in the extraction procedure. The stock, fortification, andcalibration standards will be preparedas follows: Stock Solution Prepare a stock solution of PFOS at 100 pg/mL by weighing out 10.0 m g of analytical standard(corrected for percent salt and percent purity). Adjust final volume to 100 mL with methanol in a 100-mL volumetric flask. Store this stock solution (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximumperiodof 6 months from the date'ofpreparation. Fortification Solutions a. 1.0 mt/mLFortification Solution - Pipette 1.0 mL of the 100 pg/mL stock solution into a 100 mL volumetric flask. Bring up to volume with methanol. Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 5 of 43 Page 61 of 104 C 'o sa a Centre Study No.: 023-042 Centre Protocol N o. 00P-023-042 b. Q.t ug/mL Fortification. Solution - Pipette 10.0 mL of the 1.0 pg/ml fortification solution into a 100-mL volumetric flask and bring up to volume with methanol. c. 0.01 ue/mL Fortification Solution - Pipette 10.0 mL of the 0.1 pgfmL fortification solution into a 100-mL volumetric flask and bring up to volume with methanol Store all fortification standard solutions (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximum period of 6 months from the date ofpreparation. Calibration Standards Prepare six IX7MS/MS calibration standards in methanol via dilution of the 0.1 |ig/mT. and the 0.01 pg/mLfortification solutions. This is a typical example; additional concentrations may be prepared as needed. Initial Cone. (us/mL) 0.1 0.1 0.1 0.01 0.01 0.01 Volume (mL) 5. 2. 15. 2 1 Diluted to (mL) 100 100 100 - 100 100 100 final Cone, ipe/ml.) 0.005 0.002 0.001 0.0005 0.0002 0.0001 Store all calibration standard solutions (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximum period of 6 months from the date ofpreparation. 2. The liver samples will not be processed to make the homogenate described in the method Section 12.2 to.12.12. Due to limited sample available, samples will be mashed on a flat surface by handprior to extraction. 3. The sample weight for liverwill be0.50 g ( 0.05) and the sample volume for scram will be 100 pL The amount of 0.5 M TBA added to the serum sample will be 0.5 mL and the amount of 0.25 M sodium carbonate/sodium bicarbonate buffer added wiJl be 1.0 mL. Samples will be placed on a wristaction shaker for - 20 min. at level 10 and then centrifuged for - 15 min @ -3000 rpra. The samples will onlybe filtered with 0.2 pm nylon mesh filter if necessary. Samples will be reconstituted in a final volume of. 1 mL of methanol for both matrices. Centre Analytical Laboratories, be. Centre Analytical Laboratories, Inc. Page 6 of 43 Page 62 o f 104 G00540 Centre Study No.: 023-042 Cent ProtocolNa OpP-023-042 4. The samples will be analyzed under the following conditions: LC/MS/MS System and Operating Conditions (Turbolonspray) Mass Spec: . PE SCIEX API 3000 BiomolecularMass Analyzer Interface:. SCIEX Turboion SprayLiquid Introduction Interface Harvard infusion pump Computen PowerMacintosh C3 Software: PESciexAnalysr 1.1 Windows NT HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degass' HP Autosampler HP Column Oven HPLCColumn:Genesis C, (Tones Chromatography). 2.1 mm x 50 mm, 4|i Column Temp.: 35 C Injection Voi.: 10 pL Mobile Phase (A): 2 mM Ammonium Acetate in ASTM type I water MobilePhase (B): Methanol Eow Rate: 0.3 mL/min. Time %A 0 60 40 1.0 0 100 7.0 0 100 7.5 40 60 11.0 40 60 It may be necessary to adjust the HPLC gradient in order to optimize instrument performance. Columns with different dimensions (e.g., 2.1 mm x 30 mm) and columns from different manufacturers (Keystone Betasii Co etc.) can be used, provided equivalent chromatography is obtained. Ions monitored: Analyte PFOS Mads negative Transition Monitored 499-99 Approximate Retention Tim* 4.20 Centn AnalyticalLaboratories, Inc Centre Analytical Laboratories, Inc. Page 7 of43 Page 63 o f 104 CO0541 Centre Study No.; 023-042 CentreProtocol No. 00P-023-O42 On a day-torday basis, the retention times may vary slightly depending on the batch of mobile phase, etc. Example Tune File Parameters The following values are provided as an example. Actual values may vary from instrument to instrument Also, these values may be changed from time to time in orderto optimize for greatest sensitivity. The spectrometer is tuned using a 0.5pg/mL PFOS solution, prepared via dilution of the stock solution in methanol. The solution is infused (using a *T* connector) at 10 pL/min into a 0.2 mL/min stream of mobile phase consisting of 40% methanol and 60% 2 mM ammonium acetate. The analytes are initially tuned for the parent ion and then tuned for the product ion. Once the instrument is tuned, the optimized parameters are saved as a "tune file". This tune file is then used during routine analysis. The tuning procedure may be repeated as necessary to ensure optimal sensitivity. Controls IS-Iospray OR-Orifice RNG-Focus Ring QO-QuadO Rod Offset IQ1-Interquad 1 lens ST-Stubbies ROl-Quadl Rod Offset IQ2-Interquad21ens R02-Quad 2 rodoffset ST3-Stubbies R03- Quad3 rod offset DF-CEMDeflection Plate CEM-Channel Electron Multiplier Sa -4000.0 -61.0 -270.0 10.0 9.3 15.0 9.3 20.0 84.0 100.0 86.0 300.0 2800.0 SasJBffiss Nebulizer Gas Curtain Gas Collision Gas US Temperature Ssi 12 13 4 350C Calibration Procedures x Inject the same volume (between 10 to 20 pL) of each calibration standard (preparedin MeOH) into theLC/MSJMS. b. Use linear, 1/x weighted standardcurves for quantification. Linear standard curves are generated for each set by linear regression using the appropriate software system. Any calibration standards falling outside i 30%, based on its calculated concentration, must CentreAnalytical Laboratories,Inc. Centre Analytical Laboratories, Inc. Page 8 of43 Page 64 of 10^ 0*00542 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 be excluded fromthe calibration curve. However, the total number of calibration standards thatmaybe excluded must not exceed30% of the total numberof standards injected. c. The correlation coefficient (r) for calibration curves generated must be 20.9925 (1*20.985). If calibration results fall outside these limits, then appropriate steps should be taken to adjust instrument operation, and the relevant set of samples must be reanalyzed. i Sample Analysis Inject the same volume used for the calibration standards (between 10 to 20 pL) of each sample, fortification, control, etc. into the LC/MS/MS. Each sample must be analyzed in duplicate. a. Standards corresponding to at least six concentrations must be included in ananalytical set b. Inject an entire set of standards (six) at the beginning of the run and inject standards interspersed about every 5-10 samples. All sample injections mustbe bracketed bystandard injections. c. Each set of samples analyzed (not to exceed 25) must include at least one reagent control (extraction solvents only), at least one matrix control, and two matrix control samples fortified at known concentrations andcarried through the procedureto verify recovery. d. The concentratitin of each sample, fortification, control, etc. is determined from the standard curve based on the peak area of the analyte in all standards injected during a set. The standard responses must bracket responses of the residue found in the sample set If necessary, dilute the samples and re-analyze to give a response within the standard curve range. e. Fortification recoveries within 80 to 120% are acceptable for fortifications at the LOQ level. Recoveries between 80 to 120% are acceptable for fortifications at levels greater than the LOQ. Failure to . meet these criteria requires an investigation of cause and a full reanalysis of the affected samples. f. Simples in which no peaks are detected at the corresponding analyte retention times will be repotted as ND (not detected). Samples in which peaks are detected at the corresponding analyte retention times but are less than 3:1 signalmoise will be reported as NQ (not quantifiable). Centre Analytical Laboratories, be. Centre Analytical Laboratories, Inc. Page 9 of 43 Page 65 o f 104 000543 Centre Study No.: 023-042 CentreProtocolNo. 0OP-Q23-O42 e Background levels of analyte found in control/blank samples that correspond to values below the LOQ, but are still quantifiable, will be traH tocorrectfortification recoveries. h. If samples are not loaded on the instrumentto be analyzed the day they are extracted, samples must be stored refrigerated at approximately 2C to 6C until analysis andanalyzed preferably withina week. 9. EXPERIMENTAL DESIGN Samples obtained by Wildlife Internationa] Ltd. have been dripped to Centre Analytical Laboratories, Inc. for analysis. All samples will be analyzed according to methods, "Extraction of Potassium Perfluorooctanesulfonate or Other Fiuorochemical Compounds Prom Serum for Analysis Using HPLC-Eectrospray/Mass Spectrometry* and "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochenucal Compounds Prom Liver for Analysis Using HOPLC-EIectrospray/Mass Spectrometry* with the modifications detailed in Section 8. Methods to control bias will include assay of untreated control samples, fortification of untreated control samples to obtain recovery data, and replicate analysis of fortified samples to provide an indication of reproducibility. The average recovery and relative standard deviation of the fortified samples will be calculated. Also, the average residue found and standard deviation for each matrix will be calculated 10. PROTOCOL AMENDMENTS AND DEVIATIONS Any deviations from the protocol or from the analytical method as provided will be documented and reported promptly to the Study Director. Planned changes to this protocol or to the analytical method will be made in writing as an amendment and approved by the Principal Investigator and the Study Director. Any amendments or deviations will be appended to this protocol and included in the final report 1. Protocol amendments Planned changes to the approved protocol shall be documented by amendments that clearly describe the change, justification for the change, andimpacton the study. Amendments will be signed and dated by the Principal Investigator and Study Director. Copies of amendments will be sent to the quality assurance unit. Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 10 of43 Page 66 of 104 000544 Centre Study-No.: 023-042 Centre Protocol No. OOP-023-042 2. Protocol delations: Protocol deviations, which are one time and unplanned deviations from the protocol, shall be documented in the studyrecords, noting the nature of the deviation, potential effect or impact on the study, and corrective action if required. Protocol deviations are signed and dated by the Principal InvestigatorandStudy Director andreviewed by QAU. 11. RECORDS t- Records to be maintained include, but are not limited the following (as appropriate): 1. Sampletrackingsheets) 2. Sample receipt records, storage history, andchains of custody 3. History andpreparation ofstandards (stock, fortification, calibration) 4. Description of any modifications to the method 5. Instrumentrunstreets, bench-sheets or logs 6. Analytical datatables 7. All chromatographic andinstrumental conditions 8. Sample extraction and analysis dates 9. A completelisting of studypersonnel,signatures andinitials 10. Chronological presentation of all study correspondence 11. Any other datanecessary for the reconstruction ofthe study All chromatograms will contain the following: a. Sample identification, date, snow or other indication of the area of interest, andinjection numberconesponding to the run. b. Additionally, fortifications will include the fortification level of the analyte. c. Analytical standard chromatograms will additionally include the concentration (e-g., pg/ml, ng/mL, ppb, ppt, etc.). Each data set will contain information on temperatures, flow rates, column parameters, gases, instrument parameters, and instrument type, etc. The Centre study number will also be recorded on the first chromatogram of each daily analytical run. 12. QUALITYASSURANCE Centre QA Unit will review the protocol, audit the study conduct and the study documentation (including raw data and final report) to ascertain that all QA/GLP procedures are adhered to. Centre QA Unit will inspect the study at intervals adequate to assure compliance to GLP's, andwill report the findings of the audits to thePrincipal Investigator, Centre Management, and the StudyDirector. Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 11 of 43 Page 67 o f 104 OC0545 Centre Study No.: 023-042 CentreProtocol No. OQP-023-042 13. DATAAND REPORT 1. AHtaw data and the original signed protocol will be maintained in the study file. This data includes the protocol amendments, protocol deviations, laboratory notebooks, analytical standard solution preparation, sample chain of custody sheets, sample work sheets, chromatograms, calibration curves, andany other appropriate data generated. Raw data not used will be stored in the studyfile. The reason forexclusion will bedocumented. 2. A final report will be issued by Centre, approved and signed by the Study Director and sufficient for submission to EPA. The report contents should include, butnot limited to: 1. Objectives andprocedures stated in theprotocol 2. Analytical andstatistical methods used 3. Test materials identified by name, lot,purity, and othercharacteristics 4. Name of testing facility and studyinitiation, andcompletion dates 5. Statement prepared and signed by the quality assurance unit 6. Tables containing all applicable data 7. AHchromatographicandinstrumental conditions 8. A complete listing of Centre study personnel Centre will send a copy of the draft report to the Study Director, who will return the repott with comments. Centre Analytical will make revisions and finalize the report with the approval of the Study Director.' The Centre QA unit will conduct an audit of the final draft report and data files to assure accuracy and GLP compliance. The final report must be approved by the Study Director prior' to signature. A statement of QAU inspections and a statement of GLP compliance will be included in the final report. Any corrections or additions to the final report shall be in the form of an amendment by the Principal Investigator. The amendment shall clearly identify the part of the final report that is being corrected and the justification for correction. The amendment shall be signed and dated by the Principal Investigatorandthe Study Director. REPORTDISTRIBUTION: Original: Study Direct Copy: CentreArchives 14. ARCHIVESTATEMENT Study records to be maintained: Records to be maintained for the study include all raw rfqr, observations recorded during the conduct of the study, documentation, chromatograms, specimen tracking information, and study related correspondence. Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 12 of 43 Page 68 of 104 00054.6 Centre Study No.: 023-042 CentreProtocol No. OOP-023-042 This includes a description of equipment used during the conduct of the study. All characterization data and anyshipping records shall be retained. Document archives: Upon completion of the study, the study records, protocol and amendments, and the final report and amendments shall be retained in the 3M document archives. If it is necessary to substitute a copy for an original record, it will be certified as an enact copy. Centre will retain facility-related originals and a copy of the final report and raw data package. IS. REFERENCES 1 U.S. Environmental Protection Agency. Toxic Substances Control Act (TSCA). Good LaboratoryPractice Standards. naJ Rule, 40 CFRPart 792. CentreAnalytical Laboratories, Inc Centre Analytical Laboratories, Inc. Page 13 of43 Page 69 of 104 00054.7 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 lfi. PROTOCOL APPROVAL This protocol was audited by the Quality Assurance Unit of Centre Analytical Laboratories, Inc. Quality Assurance Reviewer, Centre Quality Assurance Auditor ------------------------ Principal Investigator, Centre iW '/ Study Director, 3M Centre Study No.: 023-042 CentreProtocol No. OOP-023-042 APPENDIX: ANALYTICAL METHODS A. "Extraction of Potassium Perfluorooctanesulfonate or OtherFluorochemical Compounds From Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" B. "Extraction of Potassium Perfluorooctanesulfonate or OtherFluorochemical Compounds Fromliver for Analysis Using HPLC-Electrospray/Mass Spectrometry" Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 15 of 43 Page 71 o f 104 000549 000550 C00551 C90552 000553 BEST COPY AVAILABLE Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 " ilrl-'cv "V* -.1 rT i, ; ` ' V ` < v-.v-*.. r i'.-.y 8-11.J Dilute working standard J with hiethanol for a working standard 3 solution r ^ ^ S iW ro x .O J O p p m . `. . .not S u m g a te s t c k rt n d ^ preparation .: - aO *v 'V.--'i'T8.1*Z.IV.WM*ak miM ! >1i*r*O-Ama "Vhiihvm I .\/.vw S& -Centra-Analytical-Laboratories, In e.------- Centre Analytical Laboratories, Inc. -Page 20 of 43 Page 76 o f 104 000554 BEST CO PY AVAILABLE Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 c V-JtrSV .Centre Analytical Laboratories! Inc. Centre Analytical Laboratories, Inc. Page 21 of 43 Page 77 o f 104 000555 BEST CO PY AVAILABLE Centre Study No.; 023-042 Centre Protocol No. OOP-023-042 ^Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 22 of 43 Page 78 o f 104 000556 BEST COPY AVAILABLE Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 Centre Analytical^barateries, Inc. Centre Analytical Laboratories, Inc. Page 23 of 43 Page 79 o f 104 000557 BEST COPY AVAILABLE Centre Study No.: 023-042 Centre Protocol No. Q0P-O23-42 C00558 BEST COPY AVAILABLE Centre Study No.: 023-042 Cernie Protocol No. OOP-023-042 -fePircAnajytical Laboratories. Ine.________ . ___ Centre Analytical Laboratories, Inc. Page 25 of 43 Page 81 of 104 600559 000560 GG0561 BEST COPYavailable Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 ComponntR BTOS&OH c ' *>..Preparedrangt ,. LCRfiom %Reeovnj `^^tSrui ! ^rfataKtanli,''. '- airve % -j. Singe.;:' r - BT*'; f e p f ^Mnp'oel.) : <y<ppt>)i? * s fc s a a fc s -i: (ng/mL) -* iX * S ;v X ; USD Range >> ^*F\7-U7*:lIoVl 5 ; X'97;IotX %Hi-2 : JtyJp .V < I9 S & ? j9.7S7t; iiiX-'-M-'in-'i f u i ' i 7-, t~'r ` - '** r Rabbit Sctht' ~W *r J XCR.&oni.' Recovery . ' *SD ,r ^ o r r e ^ ? ; - .>VRange^- Range;.. C*'-'Z*. ;v. v > d^kS^Xfl rCng/mlX . > f: / ' * ^ R in g e ^ i Vr. V-C^.n/< -j; sw m X :. 101-142 C- LowCurved ^^-110 J .11.-19J.;. C High curwSf :-,,*-.J 0 f t <\t ; m 3u ;:W ;&r.yuv*`?1"j`w.v'.'- "s>Vdi"t im-*i ? * 10;l-19.l '. ''7 i CompoundMS56r i? 5 fv i^ t & m % r-> - .Preparedrange. LOt4om^ Rabbit Scruni' ITc*?...'. *.* Tofjhtadard*:; l'j'ciirre ^ .(ppb)(ngtal,) _* - (ng/mL) t % ReereiyJ; ?T,:RSDv ^'RangeWl' ?A`Rmge;-` fiR.vl'r:53^ rto.-.V' Full Binge : 24. .J.-9*7.6.Wt' ?{ M K ^ :i- '-ow.CrvTv-*5' ; .;-t, 3igh arnesi '^'-9tfM' -.976 j.76\-rie f ;-i , :-'T 5r 1t**.;n*..-iV9K. .% ?iK'iw t * ; i I ;ftB->^>J-7rr *'f-ci7-a*f'-a5: } 7iv.,.i0V-v:* 't* ft ---------- Centre Analytical Laboratories. Inc. Centre Analytical Laboratories, Inc. Page 28 of43 Page 84 of 104 CG05G2 BEST COPYAVAILABLE Centre Study No.; 023-042 Centre Protocol No. OOP-023-042 W O S .j S&coBe S f& & fr ab '. 'jS ? X ' ';F n u l voi W n g tiL 5 & J p t t d n t i i . . ' m r- ivOLSO/^P * 7 * 5 3 2 1 * 0501*3 l< * 0 3 2 1 i; 0501% : * O 0 1 0 ,i OJOO.- A 0 J0 7 r 1 ^ 0 .5 3 2 ; j p i0 1 V IS .0 J2 1 * 5.00. n s m n JW 5 JX V s 3 T M i w 3 i t & O 5 0 I Sfc U 20 ,V S?5.0IvS{; ? 0.005?, 7 - 1-Q25 "1 010 5-00 " v lJ U a * 5 3 2 : E 3 3 i 3 H ^ 3 I > c " t U l '- .;; a o io -Jt % 1415 K s j t n i i .'-i3 2 r> c ,\.-0 .1 ? (' |* S 3 l- .-J&-&01 & 1* 8.020 .W V: 1.025 . 50.0?-. s f c ja j.t X .5 1 2 S i ~ S U t'f [w s m j s ' s a x - s r . : 50.0 -. jo l i; s i:0 2 T * ' - 5 0 0 -, 3 2 E ,'5 0 .1 s t f e t t l * 5 0 .1 i'i. i ;,5 o . n r ^ 5 i i i . V V 'JO .l.^ rn s c n s tS x G S ^ x H 5 o .x - 7 v .'jx u r , a s 5 o ,i% . v r 0.005 *.- .0 0 1 0 : : 01015 0 .0 2 0 ,. -u n o ; 1.015 V 1.020? ! 025 " Centre Analytical Laboratories, Inc. 000563 BEST CO PY AVAILABLE Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 -Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 30 of 43 Page 86 of 104 000564 8 E s r ^ u B l Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 -i2?^na!ytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 31 of 43 Page 87 of 104 C00565 BEST COPY AVAILABLE Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 C * , . . '* ''V.'. vn``V.`-.a vY" * * ? '** *'* ' j`i . . ' - 6 S ; Ntrogei vaponilci; Organomylion . - X jpAyMa>(iiiaijftfaCJ00g ) **!'?:'-tai..?:: \ . -W~"m S W W J >'"*- >* * : : itw yafa. yW m i Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 32 of 43 Page 88 of 104 0-00566 b e st co p y a va ila ble Centre Study No.: 023-042 Centre Protocol No. 00P-O23-O42 ' .. / , mll , ,, ,, ___________________ . t ION soiuqoninioiiw i ^voiuiBcny jmsie spa fMutptovofamngTi (~ ; o \ ' r ? ' l f T B A m l a l I , v o l u m t . m ^ ^ g 5 p O i n L ' M i f f i ; Q ^ w t ! A d m s t t ' * . ' ; , V~ (W % adding tielastm L ' - _ ' ;:i';-.;`I.,-...V-ofNaOH,addslowlybecause(Sepfitchangesabraptly).Mate tovolarnewith ; T \ } - ' / ;U13JTBArwttirert'cb^B kcw ffi lO.'Admtas .'/' '" >. .t.-i'i.ii' -.--1a-.TM-- f. ;.. . -:. '.> .r.S-.m* 0.3SMMdhimboMlrtodfaniMeri>anttbdfcfiptee0u!tW ' ' ^ io a m ^ 2 6 ^ g o f s o d I r im < M f c p n ie ^ C X ^ i 2 0 io fi< ) d h n ^ - 7" Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. S?sps3?fi Page 33 of43 Page 89 o f 104 000567 BEST COPYAVAILABLE Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 1 i * t rV L W a > L . o l l l t i / l f l f r i ll m r i t l i w i t liW 9 t t l n f L m f t i t . i I I 4 - _ 1 . .* .. '4 .<... l1i Pi iii i^i i ipi i iai i l5i i ieMhI 8I I ^ ^ t o t o . r -v:.-.AS^^tei^^y-ryS^aJ^^^^^fe-gCTaaj^ftglftSMLedSBi t a mnmmmyonecqg L 8 $.^ii`V-55/j*-J fgQ&fBrJ&l?-: Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 34 of 43 Page 90 of 104 000568 BEST COPY AVAILABLE Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 35 of 43 Page 91 o f 104 BEST COPY AVAILABLE Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 'k ApproximateSpfldnfAmountsJbr.CalfbradonStandan -* f fifJVoridneStandardg Approx, final cone, of v;.'-i i(Appioi.,Conc.)cii *?' PFOSmKwii'r . * i: *.0 V ' r $ 5 X ^ * - ; ; .' TM_ wBlankA* - Ww?-2.'ti*'V 0.00ppni:c:.` ; v ?-->' i ' ^ , a30pp<?*4'r. Sr" * ? * >C'. 0.010 ppn-ftv d i a r i o r,ji. :.\v 0.02 ppm\-. '.'/y.-: ` W a j S f r f g f c S g L -n 2 0 y.i- mCf >0.050ppm.? >. oyr^.OJO pgn'/Si^ YSS40 > -.r 0.100 ppni '.'L * ii-.k^'S-OppiB B ^ ' - I A ^ S I O * > '&2S0ppa'-v * - t 'S.Oppm-\^el!-rJP iiii- ' \ v OJOOppm A.-a-iSS.O h 2 9 & 3 0 .?$ ' : v 0.750ppm " V ' . 1.00 ppm Centre Analytical Laboratories. Inc. Centre Analytical Laboratories, Inc. Page 36 of 43 Page 92 of 104 BEST COPY AVAILABLE Centre Study No.: 023-042 000571 GQ0S72 000573 000574 000575 000576 000577 000578 000579 C005S0 600581 C0552