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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 PO Box 33331 St. Paul, MN 55133-3331 PROJECT Centre Protocol Number: 00P-023-042 Centre Study Number: 023-042 Total Pages: 104 Centre Study No.: 023-042 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Centre Study Number 023-042, entitled "Extraction o f Potassium Perfluorooctanesulfonate from Mallard Serum and Mallard Liver far Analysis Using HPLC-Electrospray/Mass Spectrometry," conducted for 3M Environmental Technology and Safety Services, was performed in compliance with US EPA TSCA Good Laboratory Practice Standards (40 CFR Part 792) by Centre Analytical Laboratories, Inc, Principal Investigator Centre Analytical Laboratories, Inc. Seamn GGaallllfalcgrhheerr Study Director Wildlife International. Ltd. J Date lo 0 Date rideau ior Representath 3M'Environmental Technology and Safety Services Centre Analytic al Laboratories, Inc. Page 2 of 104 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 1. Protocol Review 2. Extraction, Fortification 3. Raw Data Review 4. Raw Data and Draft Report Review 5. Final Report Review Date Inspected Date Reported to Date Reported to Centre Study Director and Management Sponsor Management 11/20/00 11/30/00 12/28/00 11/20/00 11/30/00 12/28/00 12/12/00 3/18-21/00 12/21/00 3/29/01 12/28/00 5/7/01 9/6/01 9/6/01 9/6/01 LJi (kjy-- t Naomi Lovallo Sr. Quality Assurance Auditor 9/(,fo I Date Centre Analytical Laboratories, Inc. Page 3 of 104 Centre Study No.: 023-042 CERTIFICATION OF AUTHENTICITY This report, for Centre Study Number 023-042, is a true and complete representation of the raw data for the study. Submitted by: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 (814) 231-8032 Principal Investigator, Centre: L Dancer Scientist Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, foe. Facility Management: Centre Analytical Laboratories, Inc. Study Director, Wildlife International: Date 'JB?-e Date Seaann GH uaHllaafgthhfeiirr W ildlife International, Ltd. I Centre Analytical Laboratories, Inc. Page 4 of 104 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 PO Box 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 PO Box 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 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 Arjmand 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 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 of 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 of 104 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 Weeks)................................................................................. 25 Table VII. Summary of PFOS in Adult Liver Samples at 1.8 ppm a.i. (Test Termination 6Weeks)................................................................................... 26 Table VIII. Sunmary of PFOS in Adult Liver Samples at 6.2 ppm a.i. (Test Termination 6Weeks)................................................................................... 27 Table IX. Sunmary of PFOS in Adult Liver Samples at 17.6 ppm a.i. (Test Termination 19 Weeks)................................................................................. 28 Table X. Sunmary of PFOS in Juvenile Liver Samples at 0 ppm a.i. (Test Termination 12 Weeks)................................................................................. 29 Table XI. Sunmary of PFOS in Juvenile Liver Samples at 1.8ppm a.i. (Test Termination 12 Weeks)................................................................................. 30 Table XII. Sunmary of PFOS in Juvenile Liver Samples at 6.2 ppm a.i. (Test Termination 12 Weeks)................................................................................. 31 Table XIII. Sunm ary o f PFOS in Juvenile Liver Sam ples at 17.6 ppm a.i. (Test Termination 12 Weeks)................................................................................. 32 Table XTV. Sunmary of PFOS in Control Adult Serum Samples................................... 33 Table XV. Sunmary of PFOS in Adult Serum Samples at 0 ppm a.i. (Test Termination 19 Weeks).............................................................. 34 Table XVI. Sunmary of PFOS in Adult Serum Samples at 1.8 ppm a.i. (Test Termination 6Weeks)................................................................ 35 Table XVII. Sunmary of PFOS in Adult Serum Samples at 6.2 ppm a.i. (Test Termination 6Weeks)................................................................................... 36 Table XVIII. Summary of PFOS in Adult Serum Samples at 17.6 ppm a.i. (Test Termination 19 Weeks)................................................................................. 37 Centre Analytical Laboratories, Inc. Page 8 of 104 Centre Study No.: 023-042 LIST OF TABLES (Continued) Page Table XIX. Summary of 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 of PFOS in Juvenile Serum Samples at 6.2 ppm a.i. (Test Teimination 12 Weeks)................................................................................. 41 Table XXIII. Summary of PFOS in Juvenile Serum Samples at 17.6 ppm a.i. (Test Teimination 12 Weeks)................................................................................. 42 Table XXIV. Summary of Percent Moistures for Mallard Liver Samples...................... 43 Centre Analytical Laboratories, Inc, Page 9 of 104 Centre Study No.: 023-042 LIST OF FIGURES Page Figure 1. 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 Centre Study No.: 023-042 LIST OF APPENDICES Page Appendix A Study Protocol 00P-023-042 (Centre Study No. 023-042) and Amendments and Deviation...................................................................... 56 Centre Analytical Laboratories, Inc Page 11 of 104 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 of 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 of 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 of this study was to determine levels of perfluorooctanesulfonate (PFOS) in specimens of mallard liver and mallard serum using the analytical methods described in protocol 00P-023-042. 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFOS in mallard liver and mallard serum, using the 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". 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 of 104 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 of 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 of 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 O = = Perfluorooctanesulfonate 499 (CgFnSOs-) c 8Fi7s -- 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 Centre Study No.: 023-042 6,0 DESCRIPTION OF ANALYTICAL METHOD 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 of liver and a 100 pL aliquot of the serum was used for the extraction procedure. After fortification of appropriate samples, the samples were vortexed for ~ 15 seconds. One milliliter of 0.5 M TBA was added to the liver samples and 0.5 milliliter was added to the serum samples. Two milliliters of 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 of 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 of 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 of 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 of 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 of the 0.1 pg/mL standard and bringing to 100 mL with methanol. A set of 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 of 104 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 of PFOS was accomplished by LC/MS/MS electrospray. The retention time of 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 P1110 Software: HPLC: PE SciexAnalyst version 1.1 Windows NT, version 4 H ewlett 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 of 104 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 (min) 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-Deflector 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 of 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 of analyte found in pg/mL (the equations for serum are shown). Centre Analytical Laboratories, Inc. Page 16 of 104 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 (pg/mL) - avg. anal, in Ctrl (pg/mL)) x (1000 ng/1 pg)) x 100___ amount added (ng/mL) An example of 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 (N ot 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) = f6558- 5151 7740 From equation 2: Analyte found (pg/mL) 0.8 ng/mL = (0,8 ng/mL x 1 mL x 1 x 5 mL) x 1 ug (4mLx0.1mL) 1000 ng = 0.00976 pg/mL Centre Analytical Laboratories, Inc. Page 17 of 104 Centre Study No.: 023-042 From equation 3: % Recovery = ((0.00976 pg/mL x - 0 ng/mL) x 1000) x 100 10 ng/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 Centre Study No.: 023-042 10.0 RETENTION OF DATA AND SAMPLES 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 of all raw data, as well as a signed copy of 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 of reference substances are archived by the sponsor. Centre Analytical Laboratories, Inc Page 19 of 104 Centre Study No.: 023-042 TABLES Centre Analytical Laboratories, Inc. Page 20 of 104 Centre Study No.: 023-042 Table I. Summary of 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* Recigent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reaigent 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 Date Date 11/17/00 11/17/00 11/20/00 11/20/00 11/21/00 11/21/00 11/21/00 11/21/00 11/18-19/00 11/18-19/00 11/20-21/00 11/20-21/00 11/22-23/00 11/22-23/00 11/23/00 11/23/00 11/27/00 11/27/00 11/27/00 11/27/00 3/7/01 3/7/01 3/7/01 3/7/01 3/8/01 3/8/01 11/28-29/00 11/28-29/00 11/29/00 11/29/00 3/9/01 3/9/01 3/9-10/01 3/9-10/01 3/11/01 3/11/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 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)) 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 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 (pg/g) 0.000294 0.000300 NQ NQ NQ NQ NQ NQ NQ NQ Table III. Summary of PFOS in Mallard 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* 0003 803 Blank A 0003803 Blank A* 0003803 Blank A 0003803 Blank A* 0003803 Blank A 0003803 Blank A* Set Number 112100B 112100B 112700A 112700A 112700B 112700B 030701A 030701A 030701B 03070IB Extraction Analysis Analyte Date Date Found (pg/mL) 11/21/00 11/21/00 11/27/00 11/27/00 11/27/00 11/27/00 3/7/01 3/7/01 3/7/01 3/7/01 11/23/00 11/23/00 11/28-29/00 11/28-29/00 11/29/00 11/29/00 3/9/01 3/9/01 3/9-10/01 3/9-10/01 NQ NQ NQ NQ NQ 0.00159 0.00826 0.00825 0.00159 0.00397 AVERAGE: 0.00239 STANDARD DEVIATION: 0.00333 * 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 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 (ng/g) 11/17/00 11/17/00 11/17/00 11/17/00 11/20/00 11/20/00 11/20/00 11/20/00 11/21/00 11/21/00 11/21/00 11/21/00 3/8/01 11/19/00 11/19/00 11/18-19/00 11/18-19/00 11/22/00 11/22/00 11/20-21/00 11/20-21/00 11/22-23/00 11/22-23/00 11/27/00 11/27/00 3/11/01 10 10 50 50 100 100 4000 4000 1000 1000 20000 20000 20 3/8/01 3/8/01 3/8/01 3/11/01 3/11-12/01 3/11-12/01 20 200 200 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 jig/g for liver and 0.01 gg/mL for serum Centre Analytical Laboratories, Inc. Page 23 of 104 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 Set Number 0003803 Spk A 0003803 Spk A* 0003803 Spk B 0003803 Spk B* 0003803 Spk A 0003803 Spk A* 0003803 Spk B 0003803 SpkB* 0003803 Spk A 0003803 Spk A* 0003803 Spk B 0003803 SpkB* 0003803 Spk A 0003803 Spk A* 0003803 Spk B 0003803 Spk B* 0003803 Spk A2 0003803 Spk A2* 0003803 Spk B2 0003803 Spk B2* 112100B 112100B 112100B 112100B 112700A 112700A 112700A 112700A 112700B 112700B 112700B 112700B 030701A 030701A 030701A 030701A 030701B 03070IB 030701B 030701B * Duplicate injection Extraction Analysis Amt Date Date Added (ng/mL) 11/21/00 11/21/00 11/21/00 11/23/00 11/23/00 11/23/00 10 10 500 11/21/00 11/23/00 11/27/00 11/28-29/00 500 10 11/27/00 11/27/00 11/27/00 11/28-29/00 11/28-29/00 11/28-29/00 10 10000 10000 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 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 3/9-10/01 500 500 100000 100000 100 100 1000 1000 100 100 3/7/01 3/7/01 3/9-10/01 3/9-10/01 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 11 12 LOQ = 0.01 ng/g for liver and 0.01 |ig/mL for serum Centre Analytical Laboratories, Inc. Page 24 of 104 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 A' 1963* 1964 c 1964* 1965 ^ 1965* 1966 f 1966* 1967 " 1967* 1968 F 1968* 1969 ^ 1969* 1970 i 1970* Centre ID 0008006 0008006 0008007 0008007 0008008 0008008 0008009 0008009 0008010 0008010 0008011 0008011 0008012 0008012 0008013 0008013 0008014 0008014 0008015 0008015 * Duplicate injection Set Number 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700A 111700ADD 111700ADD 111700AD 111700AD 111700AD 111700AD 111700AD 111700AD Extraction Date Analysis 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 LOQ = 0.01 ng/g for liver and 0.01 |rg/mL for serum Centre Analytical Laboratories, Inc. Page 25 of 104 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 A' E01-0263-21066* EOI-0263-21067 M EOI-0263-21067* EOI-0263-21068 /L' EOl-0263-21068* EOI-0263-21069 A* E01-0263-21069* E01-0263-21075 iE01-0263-21075* E01-0263-21076 E01-0263-21076* E01-0263-21077 ? E01-0263-21077* E01-0263-21078 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 (ng/g) 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 |xg/g for liver and 0.01 pg/m L for serum Centre Analytical Laboratories, Inc. Page 26 of 104 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 E01-0263-21070 ,1EOI -0263-21070* EOI-0263-21071 EOI-0263-21071* EOI-0263-21072 EOI-0263-21072* EOI-0263-21073 E01-0263-21073* EOI-0263-21074 EOI-0263-21074* v EOI-0263-21080 EOI-0263-21080* EOI-0263-21081 EOI-0263-21081* E01-0263-21082 E01-0263-21082* EOI-0263-21083 EOI-0263-21083* : EOI-0263-21084 J, E01-0263-21084* S 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 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 (pg/g) 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 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 27 of 104 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 a1991* 1992 p 1992* 1993 ^ 1993* 1994 1994* 1995 *j 1995* 1996 f 1996* 1997 / 1997* 1998 f 1998* 1999 . 1999* 2000 ,< 2000* Centre ID 0008016 0008016 0008017 0008017 0008018 0008018 0008019 0008019 0008020 0008020 0008021 0008021 0008022 0008022 0008023 0008023 0008024 0008024 0008025 0008025 * Duplicate injection Set Number 111700ADD 111700ADD 111700ADD 111700ADD 111700ADD 111700ADD 111700AD 111700AD 111700AD 111700AD 111700ADD 111700ADD 111700AD 111700AD 111700AD 111700AD 111700ADD 111700ADD 111700AD 111700AD Extraction Date Analysis Date 11/17/00 11/20/00 11/17/00 11/20/00 11/17/00 11/20/00 11/17/00 11/20/00 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 11/20/00 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 11/20/00 11/17/00 11/20/00 11/17/00 11/19/00 11/17/00 11/19/00 AVERAGE: STANDARD DEVIATION: Analyte Found (ng/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 LOQ = 0.01 jAg/g for liver and 0.01 fxg/mL for serum Centre Analytical Laboratories, Inc. Page 28 of 104 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 /V' 2305* 2307 ^ 2307* 2308 M 2308* 2314 * 2314* 2316 ^ 2316* 2317 2317* 2320 1* 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 Date Analysis Date 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 11/20/00 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 11/20-21/00 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 (pg/g) 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 of 104 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 A1' 2328* 2330 ^ 2330* 2331 ^ 2331* 2332 f 2332* 2333 ri 2333* 2334 pi\ 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 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 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 |4.g/g for liver and 0.01 |_ig/mL for serum Centre Analytical Laboratories, Inc. Page 30 of 104 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 V 2339* 2341 ^ 2341* 2342 v 2342* 2343 A1' 2343* 2344 2344* 2346 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 112100A 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 11/21/00 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 11/22-23/00 11/22-23/00 11/22-23/00 11/22-23/00 11/22-23/00 11/22-23/00 11/22-23/00 11/22-23/00 11/22-23/00 11/22-23/00 11/22-23/00 11/22-23/00 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 11/27/00 11/21/00 11/27/00 AVERAGE: STANDARD DEVIATION: Analyte Found (ng/g) 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 pg/m L for serum Centre Analytical Laboratories, Inc. Page 31 of 104 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 F 2353* 2354 ^ 2354* 2355 ^ 2355*rC 2357 /A 2357* 2358 1? 2358* 2359 p 2359* 2360 2360* 2363 /A 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 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 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 11/21/00 11/27/00 11/21/00 11/27/00 li/21/00 11/27/00 11/21/00 11/27/00 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 (jig/g) 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 LOQ = 0.01 p,g/g for liver and 0.01 pg/m L for serum Centre Analytical Laboratories, Inc. Page 32 of 104 Centre Study N o.: 023-042 Table XIV. Summary of PFOS in Control Adult Serum Samples Sponsor ID E01-0263-21005 'V) E01-0263-21005* E01-0263-21006 E01-0263-21006* E01-0263-21007 'A EOI-0263-21007* EOl-0263-21008^ E01-0263-21008* E01-0263-21009 * E01-0263-21009* E01-0263-21025 ^ E01-0263-21025* E01-0263-21026 ^ E01-0263-21026* E01-0263-21027 EOI-0263-21027* EOI-0263-21028 f E01-0263-21028* EOI-0263-21029 ^ E01-0263-21029* 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 030701B 030701B 030701B 030701B 030701B Extraction Analysis Analyte Date Date Found (ue/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 0.00928 0.00954 0.0103 0.00319 \ 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 (ag/mL)) For values recorded as NQ, 0.00005 pg/mL (halfthe value of 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 A nalytical Laboratories, Inc. Page 33 o f 104 Centre Study N o.: 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 ^ 1965* 1966 1966* 1967 /iA 1967* 1968 f 1968* 1969 1969* 1970 1970* Centre 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 (ne/mL) 11/21/00 11/23/00 11/21/00 11/23/00 11/21/00 11/23/00 11/ 2 1 / 0 0 11/23/00 11/21/00 11/23/00 11/21/00 11/23/00 11/2 1 / 0 0 11/23/00 11/21/00 11/23/00 11/21/00 11/23/00 11/ 2 1 / 0 0 11/23/00 11/ 2 1 / 0 0 11/23/00 11/2 1 / 0 0 11/23/00 11/21/00 11/27-28/00 11/ 2 1 / 0 0 11/27-28/00 11/ 2 1 / 0 0 11/27-28/00 11/21/00 11/27-28/00 11/21/00 11/27-28/00 11/ 2 1 / 0 0 11/27-28/00 11/ 2 1 / 0 0 11/27-28/00 11/2 1 / 0 0 11/27-28/00 AVERAGE: STANDARD DEVIATION: 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 0.843 0.849 1.41 1.38 1.22 2.03 * Duplicate injection LOQ = 0.01 p.g/g for liver and 0.01 ng/m L for serum Centre A nalytical Laboratories, Inc. Page 34 o f 104 Centre Study N o.: 023-042 Table XVI. Summary of PFOS in Adult Serum Samples at 1.8 ppm a.i. (Test Termination 6 Weeks) Sponsor ID E 0 1-0263-21010 E01-0263-21010* E01-0263-21011 A) E01-0263-21011* E01-0263-21012 * E01-0263-21012* E01-0263-21013 ^ E01-0263-21013* E01-0263-21014 1 E01-0263-21014* E01-0263-21030 C E01-0263-21030* E01-0263-21031 ^ E01-0263-21031* E01-0263-21032 E01-0263-21032* E01-0263-21033 f E01-0263-21033* E01-0263-21034 r E01-0263-21034* Centre ID 0103815 0103815 0103816 0103816 0103817 0103817 0103818 0103818 0103819 0103819 0103835 0103835 0103836 0103836 0103837 0103837 0103838 0103838 0103839 0103839 Set Number 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD Extraction Analysis Analyte Date Date Found (jig/mL) 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/10/01 3/10/01 3/10/01 3/10/01 3/10/01 3/10/01 3/10/01 3/10/01 3/10/01 3/10/01 3/10-11/01 3/10-11/01 3/10-11/01 12.8 12.1 18.2 20.1 22.5 24.0 20.4 20.4 10.6 10.6 0.870 0.854 6.70 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 3/10-11/01 3/7/01 3/10-11/01 AVERAGE: STANDARD DEVIATION: 6.43 0.443 0.468 8.88 8.38 1.68 1.74 10.4 8.15 * Duplicate injection LOQ = 0.01 |ig /g for liver and 0.01 i^g/mL for serum Centre A nalytical Laboratories, Inc. Page 35 o f 104 Centre Study No.: 023-042 Table XVII. Summary of PFOS in Adult Serum Samples at 6.2 ppm a.i. (Test Termination 6 Weeks) Sponsor ID E01-0263-21015 ^ E01-0263-21015* E01-0263-21016 *\ E01-0263-21016* E01-0263-21017 ^ E01-0263-21017* E01-0263-21018 W E01-0263-21018* E01-0263-21019 M E01-0263-21019* E01-0263-21035 F E 0 1-0263-21035* E01-0263-21036 r E01-0263-21036* E01-0263-21037 P E01-0263-21037* E01-0263-21038^ E01-0263-21038* E01-0263-21039 ? E01-0263-21039* Centre ID 0103820 0103820 0103821 0103821 0103822 0103822 0103823 0103823 0103824 0103824 0103840 0103840 0103841 0103841 0103842 0103842 0103843 0103843 0103844 0103844 Set Number 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD Extraction Analysis Analyte Date Date Found (ue/mL) 3/7/01 3/10/01 3/7/01 3/10/01 3/7/01 3/10/01 3/7/01 3/10/01 3/7/01 3/10/01 3/7/01 3/10/01 3/7/01 3/10/01 3/7/01 3/10/01 3/7/01 3/10/01 3/7/01 3/10/01 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 3/10-11/01 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 AVERAGE: STANDARD DEVIATION: 47.1 44.7 40.4 35.5 45.5 44.4 23.5 24.7 20.3 20.8 17.8 17.3 2.83 2.87 3.48 4.12 61.9 60.3 3.69 2.79 26.2 20.0 * Duplicate injection LOQ = 0.01 (xg/g for liver and 0.01 fig/m L for serum Centre A nalytical Laboratories, Inc. Page 36 o f 104 Centre Study N o.: 023-042 Table XVIII. Summary of PFOS in Adult Serum Samples at 17.6 ppm a.i. (Test Termination 19 Weeks) Sponsor ID 1991 M 1991* 1992 F 1992* 1993 M 1993* 1994 F 1994* 1995 1995* 1996 P 1996* 1997 ^ 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 (pg/mL) 11/21/00 11/ 2 1 / 0 0 11/ 2 1 / 0 0 11/2 1 / 0 0 11/28/00 11/28/00 11/28/00 11/28/00 41.0 41.1 47.0 49.5 11/ 2 1 / 0 0 11/ 2 1 / 0 0 11/2 1 / 0 0 11/ 2 1 / 0 0 11/2 1 / 0 0 11/21/00 11/2 1 /0 0 11/28/00 11/28/00 11/28/00 11/28/00 11/28/00 11/28/00 11/28/00 199 198 93.9 93.0 182 180 218 11/ 2 1 / 0 0 11/2 1 / 0 0 11/ 2 1 / 0 0 11/ 2 1 / 0 0 11/28/00 11/28/00 11/28/00 11/28/00 217 214 212 156 11/ 2 1 / 0 0 11/28/00 156 11/ 2 1 / 0 0 11/28/00 156 11/ 2 1 / 0 0 11/21/00 11/28/00 11/28/00 161 200 11/21/00 11/28/00 205 AVERAGE: STANDARD DEVIATION: 151 65.0 LOQ = 0.01 iig/g for liver and 0.01 [xg/mL for serum Centre A nalytical Laboratories, Inc. Page 37 o f 104 Centre Study N o.: 023-042 Table XIX. Summary of PFOS in Adult Serum Samples at 17.6 ppm a.i. (6 Week Samples) Sponsor ID E01-0263-21020 ^ E01-0263-21020* E01-0263-21021 E01-0263-21021* EOI-0263-21022 M E01-0263-21022* E01-0263-21023 4 E01-0263-21023* E01-0263-21024 y ' E01-0263-21024* EOI-0263-21040 C E01-0263-21040* E01-0263-21041 P E01-0263-21041* E01-0263-21042 p E01-0263-21042* E01-0263-21043 C E01-0263-21043* EOI-0263-21044 f E01-0263-21044* Centre ID 0103825 0103825 0103826 0103826 0103827 0103827 0103828 0103828 0103829 0103829 0103845 0103845 0103846 0103846 0103847 0103847 0103848 0103848 0103849 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 Date Analysis Date 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/11-12/01 3/11-12/01 3/10/01 3/10/01 3/10/01 3/10/01 3/10/01 3/10/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/7/01 3/11-12/01 3/11-12/01 3/10-11/01 3/10-11/01 3/10-11/01 3/10-11/01 3/10-11/01 3/7/01 3/7/01 3/10-11/01 3/10-11/01 3/7/01 3/7/01 3/7/01 3/10-11/01 3/10-11/01 3/10-11/01 AVERAGE: STANDARD DEVIATION: Analyte Found Oig/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 jig/g for liver and 0.01 |tg/mL for serum Centre A nalytical Laboratories, Inc. Page 38 o f 104 Centre Study N o.: 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 A' 2305* 2307/b 2307* 2308 ^ 2308* 2314 ^1 2314* 2316 /A 2316* 2317 i r 2317* 2320 T 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 Analysis Analyte Date Date Found Oig/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 Q uantifiable (A peak w as detected at the corresponding analyte retention tim e but was less than the lowest concentration of the calibration standards (0.0001 jig/mL)) For values recorded as NQ, 0.00005 iig/mL (halfthe value of the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. LOQ = 0.01 |ig/g for liver and 0.01 pg/mL for serum Centre A nalytical Laboratories, Inc. Page 39 o f 104 Centre Study N o.: 023-042 Table XXI. 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 1' 2326* 2328 /A 2328* 2330 2330* 2331 /v\ 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 112700A 112700A 112700A Extraction Analysis Analyte Date Date Found (|ig/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 11/29-30/00 11/27/00 11/29-30/00 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: 0.0464 0.0472 0.0274 0.0281 0.0497 0.0487 0.0160 0.0156 0.249 0.250 0.00788 0.00747 0.0101 0.00998 0.0113 0.0110 0.0339 0.0339 0.0554 0.0555 0.0507 0.0701 * Duplicate injection LOQ = 0.01 p.g/g for liver and 0.01 pg/m L for serum Centre A nalytical Laboratories, Inc. Page 40 o f 104 Centre Study N o.: 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 aA 2338*' 2339 2339* 2341 aA 2341* 2342 ^ 2342* 2343 2343* 2344 F 2344* 2346 ! 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 (jtg/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 n g/g for liver and 0.01 (ag/mL for serum Centre A nalytical Laboratories, Inc. Page 41 o f 104 Centre Study N o.: 023-042 Table XXIII. Summary of PFOS in Juvenile Serum Samples at 17.6 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 2347 F 2347* 2348 IF" 2348* 2353 F 2353* 2354 2354* 2355 2355* 2357 2357* 2358 1' 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 Date Analysis Date Analyte Found (ue/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 * Duplicate injection LOQ = 0.01 |ig /g for liver and 0.01 |xg/mL for serum Centre A nalytical Laboratories, Inc. Page 42 o f 104 Centre Study N o.: 023-042 Table XXTV. Summary of Percent Moistures for Mallard Liver Samples Sponsor ID Centre ID Average % Moisture 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2301 2302 2303 2305 2307 2308 2314 2316 2317 2320 2322 2323 2325 2326 2328 2330 2331 2332 2333 2334 2335 0008006 0008007 0008008 0008009 0008010 0008011 0008012 0008013 0008014 0008015 0008016 0008017 0008018 0008019 0008020 0008021 0008022 0008023 0008024 0008025 0008086 0008087 0008088 0008089 0008090 0008091 0008092 0008093 0008094 0008095 0008096 0008097 0008098 0008099 0008100 0008101 0008102 0008103 0008104 0008105 0008106 69.7 73.4 75.7 69.2 71.9 75.0 72.8 70.4 69.5 72.9 75.3 71.8 71.9 71.8 71.5 72.4 70.5 71.3 69.8 72.0 76.6 77.3 72.7 72.5 72.3 72.0 75.0 69.6 71.8 71.6 71.6 72.5 73.2 72.6 72.0 68.9 71.4 71.7 69.8 68.1 71.7 Centre A nalytical Laboratories, Inc. Page 43 o f 104 Centre Study N o.: 023-042 Table XXIV (cont'). Summary of Percent Moistures for Mallard Liver Samples Sponsor ID Centre ID Average % Moisture 2336 2337 2338 2339 2341 2342 2343 2344 2346 2347 2348 2353 2354 2355 2357 2358 2359 2360 2363 E01-0263-21065 E01-0263-21066 E01-0263-21067 E01-0263-21068 E01-0263-21069 E01-0263-21070 E01-0263-21071 E01-0263-21072 EO1-0263-21073 E01-0263-21074 E01-0263-21075 E01-0263-21076 E01-0263-21077 E01-0263-21078 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 0103877 0103878 0103879 0103880 0103881 0103882 0103883 0103884 0103885 0103886 0103887 0103888 0103889 69.7 69.7 69.8 70.6 72.6 78.2 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 77.4 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 A nalytical Laboratories, Inc. Page 44 o f 104 Centre Study N o.: 023-042 FIGURES Centre A nalytical Laboratories, Inc. Page 45 o f 104 Centre Study N o.: 023-042 Figure 1. Typical Calibration Curve for PFOS C nnrpntratirm pi/mU Centre A nalytical Laboratories, Inc. Page 46 o f 104 Centre Study N o.: 023-042 Figure 2. Chromatogram Representing a 0.1 ng/mL standard for PFOS j Sample Index: | Sample Type: i Concentration: !Acq. Date: * Acq. Time: 1 Standard 0.100 1 1 /22/00 12:11:52 PM i Modified: | Bunching factor: Noise Threshold: Area Threshold: Num. Smooths: RT Window: Expected RT: Sep. Width: Sep. Height: Exp. Peak Ratio: Exp. Adj. Ratio: Exp. Val. Ratio: Use Relative RT: Yes 1 2.82 14.08 1 30.0 4.52 0.20 0 .0 1 5.00 4.00 3.00 No 'M 8- sec min c '-- - Int. Type: Retention Time: | Area: Manual 4.55 1548.4 min counts 450 400'350 300 ; 250 ; 200150 100 50 | au ' -, \ ; . . 1 ...A ''j- y ' -**, z y ; *-. - /> .;-y, l f,yv * 4.54 1. 2 -. 34 Time, min 5 Vov*`t Centre A nalytical Laboratories, Inc. Page 47 o f 104 Centre Study N o.: 023-042 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PFOS ________ Si C ^w ie^^M >gii^P O T O 09t8 I ! Sample Index: 4 Sample Type: Standard Concentration: 1.000 Acq. Date: 1 1 /22/00 Acq. Time: 12:49:51 PM Modified: Bunching Factor: Noise Threshold: Area Threshold: Num. Smooths: RT Window: Expected RT: Sep. Width: Sep. Height: Exp. Peak Ratio: Exp. Adj. Ratio: Exp. Val. Ratio: Use Relative RT: Yes 1' 2,82 14.08 1 30.0 4,52 0.20 0.01 5.00 4.00 3.00 Mo sec min & o c c Int. Type: Retention Time: Area: Manual 4.54 min 8355.5 counts 2695 2500 2000 1500 1000 500 nu 4.54 Time, min Centre Analytical Laboratories, Inc, Page 48 o f 104 Centre Study N o.: 023-042 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Centre ID Reagent Blank A, Set: 112000A) Sample Index: Sample Type: Concentration: Acq. Date: Acq. Time: 8 Blank 0.-QQ0 11/20/00 05:21:16 PM Modified: Bunching Factor: Noise Threshold: Area Threshold: Num. Smooths: RT Window: Expected RT: Sep. Width: Sep. Height: Exp. Peak Ratio: Exp. Adj. Ratio: Exp. Val. Ratio: Use Relative RT: Yes 1 2.82 14.08 1* 30.0 4.52 0.20 0 .0 1 5.00 4.00 3.00 NO sec min u $<0 Int. Type: Retention Time: Area: Manual 4.57 432.0 min counts 133 120 100 J 80 1 60< 40 i 20 i ! 4.57 2345 6 7 Time, min - Centre A nalytical Laboratories, Inc. Page 49 o f 104 Centre Study N o.: 023-042 Figure 5. Chromatogram Representing Control Mallard Serum for PFOS (Centre ID: 0003803 Blank A, Set: 112100B) Sample Index: Sample Type: Concentration: Acq. Date: Acq. Time: 10 Blank 0.000 11/23/00 03:38:52 AM `Modified: Yes Bunching Factor: 1 Noise Threshold: 2.82 Area Threshold: 14.08 M Nura. Smooths: 1 u RT Window: Expected RT: 30.0 4.52 sec min *M Sep. Width: 0.20 & Sep. Height: 0 .0 1 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 Use Relative RT: No Int. Type: Retention Time: Area: Manual 4.54 612.8 min counts 200 150 100 50 4.53 ' r X t.4.66 r 4 Time, min 6:?4' 6-87 7.70 Centre A nalytical Laboratories, Inc, Page 50 o f 104 Centre Study N o.: 023-042 Figure 6. Chromatogram Representing Control Mallard Liver for PFOS, (Centre ID: 0003804 Blank A, Set: 112000A) ; Sample Type: i Concentration: j Acq, Date: 1 Acq. Time: Blank 0.000 1 1 /20/00 05:46:39 PM Modified: Bunching Factor: Noise Threshold: Area Thresholds Num. Smooths: RT Window: Expected RT: Sep. Width: Sep. Height: Exp. Peak Ratio: Exp. Adj. Ratio: Exp. Val. Ratio: Use Relative RT: Yes 1 2.82 14.08 1 30.0 4.52 0.20 0.01 5.00 4.00 3.00 No sec min so. M Int. Type: Retention Time: Area: Manual 4.56 min 829.8 counts 227 200 i 150100 50 0- 4.56 4.66 4.43 J '!',.;5 00,5.4 610. 6.59 _' _____V1;' "'V 4 Time, min 7.58 Centre Analytical Laboratories, Inc, Page 51 o f 104 Centre Study N o.: 023-042 Figure 7. Chromatogram Representing Control Mallard Serum fortified with 10 ppb of PFOS (Centre ID: 0003803 Spk A, Set: 112100B) Sample Index: Sample Type: Concentration: Acq. Date: Acq. Time: QC * . 0.800 11/23/00 04:04:15 AM Modified: Yes Bunching Factor: ` 1 Noise Threshold: 2.82 Area Threshold: ' 14.08 Num. Smooths: 1 RT Window: 30.0 Expected RT: - 4.52 Sep. Width: 0.20 Sep. Height: 0 .0 1 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 Use Relative RT: No sec min M o 2<0? -- Int. Type: Retention Time: Area: Manual 4.53 6557.7 min counts 2150 2000 I ! 1500 j i ii 1000 { j 1 \ 500 ii 1 1 4.53 4* Time, min Centre A nalytical Laboratories, Inc. Page 52 o f 104 Centre Study N o.: 023-042 Figure 8. Chromatogram Representing Control Mallard Liver Fortified at 1000 ppb with PFOS (Centre ID: 0003804 Spk A, Set: 112100A) Sample Index: I Sample Type: I Concentration: j Acq. Date: ! Acq. Time: QC 1.600 1 1 /22/00 02:31:00 PM j Modified: Bunching Factor: | Noise Threshold: , Area Threshold: 1 Num. Smooths: ' RT Window: Expected RT: . Sep. Width: . Sep. Height: ' Exp. Peak Ratio: i Exp. Adj. Ratio: ; Exp. Val. Ratio: ' Use Relative RT: Yes 1 2.82 .14.08 1 30.0 4.52 0.20 0.01 5.00 4.00 3.00 No sec min o (A C -- ' Int. Type: Manual : Retention Time: 4.-54 min ; Area: - 10563.0 counts 3251 3000 2500 2000 1500 1900 .500 nV 4.53 4 Time, min Centre A nalytical Laboratories, Inc. Page 53 o f 104 Centre Study N o.: 023-042 Figure 9. Chromatogram Representing Juvenile Mallard Serum Sample (Centre ID: 0008143, Sponsor ID: 2332, Set: 112700A) jftiBWBHiiiffSatlgT Mass(esH CwSmenSoOMtii Sample Index: Sample Type: Concentration: Acq. Date: Acq. Time: 55 ' Unknown N/A 11/29/00 04:07:5 Modified: Bunching Factor: Noise Threshold: Area Threshold: Nun. Smooths: RT Window: Expected RT: Sep. Width: Sep. Height: Exp. Peak Ratio: Exp. Adj, Ratio: Exp. Val! Ratio: U se Rel ati ve RT: Yes 1 2. 2 14.08 1 30.0 4.52 0.20 0.01 5.00 4.00 3.00 No f Int. Type: Retention Time: Area: Height: Start Tine: End Time: Manual 4.50 4677.3 count* cps1 . 4 1 e + 0 0 3 4.40 4.58 1JO0. 1200J 1100' 1000 00 eoo 700 4 500 i 400 4 200 100- 0.5 ___________________ . . . Co M 2~0 Ti TO To i l S.Q Time, min - 5.5 6.0 . ` ".5 " 7.cT'' " ^ 7 5 Centre Analytical Laboratories, Inc. Page 54 o f 104 Centre Study N o.: 023-042 Figure 10. Chromatogram Representing Juvenile Mallard Liver Sample (Centre ID: 0008108, Sponsor ID: 2337, Set: 112100A) Sample Index: Sample Type: Concentration: Acq. Date: Acq. Time: 21 Unknown N/A 11/ 2 2 /0 0 04:25:14 PM | Modified: | Bunching Factor: j Noise Threshold: | Area Threshold: j Num. Smooths: RT Window: 1 Expected RT: i Sep. Width: | Sep. Height: | Exp.Peak Ratio: j Exp.Adj. Ratio: | Exp.Val. Ratio: ! Use Relative RT: Yes 1 2".82 14.08 1 30.0 4.52 0.20 0.01 5.00 4.00 3.00 No sec min 3498 3000 2500 &I 2000 2? c 1500 1000 500 I Int. Type: Manual T Retention Time: 4.54 i Area: 11166.9 min counts 4.54 3 '4 5 Time, min 6 -7 ' Centre A nalytical Laboratories, Inc. Page 55 o f 104 Centre Study N o.: 023-042 APPENDIX A Study Protocol 00P-023-042 (Centre Study No. 023-042) and Amendments and Deviation Centre A nalytical Laboratories, Inc. Page 56 o f 104 Centre Study N o.: 023-042 Centre Protocol No. OOP-023-042 . STUDY PROTOCOL STUDY TITLE EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM MALLARD SERUM AND MALLARD LIVER FOR ANALYSIS USING HPLC- ELECTROSPRAY/MASS SPECTROMETRY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St-P&ul, 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 t 00P-023-042 Centre Analytical Laboratories, Inc. Centre A nalytical Laboratories, Inc. Page 1 o f 43 Page 57 o f 104 Centre Study N o.: 023-042 Centre Protocol No. OOP-023-042 Title: EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM MALLARD SERUM AND MALLARD LIVER FOR ANALYSIS USING HPLCELECTROSPRAY/MASS SPECTROMETRY TABLE OF CONTENTS ________ ____________ _________________________________ _________ Page TABLE OF CONTENTS.................................................................................................2 1. p u r p o se ................................... ......:................................................................... .. 2. TEST MATERIALS................................................................................................ 3. SPONSOR...................................................................................... 4 4. TESTING FACILITY AND PERFORMING LABORATORY............................4 5. PROPOSED EXPERIMENTAL TIME-FRAME........................................... 4 6. JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM.................4 7. SAMPLE PROCESSING, STORAGE AND IDENTIFICATION........................ 5 8. ANALYTICAL M E TH O D ....................................................................................................5 9. EXPERIMENTAL DESIGN............ .....................................................................10 10. PROTOCOL AMENDMENTS AND DEVIATIONS........................................... 10 11. RECORDS..................................................................................................... 11 12. QUALITY ASSURANCE.....................................................................................11 13. D A T A A N D REPORT.......................................................................................................... 12 14. ARCHIVE STATEMENT.................................................................................... 12 15. REFERENCES...................................................................................................... 13 16. PROTOCOL APPROVAL.................................................................................... 14 APPENDIX: ANALYTICAL METHODS.......................................................................15 Centre Analytical Laboratories, Inc. Centre A nalytical Laboratories, Inc. Page 2 of 43 Page 58 o f 104 Centre Study N o.: 023-042 i Centre Protocol No. OOP-023-042 Study Title: , EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM MALLARD SERUM A N D MALLARD LIVER FOR ANALYSIS 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-Electiospray/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,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-, potassium salt 2795-39-3' 499(C*F|7S O n O C8F 17S -- 0 o Note: The neutral molecule and standard form that PFOS (anion) is derived from is perfluorooctanesulfonate potassium salt [C8F17SO3K], molecular weight 538. Centre Analytical Laboratories, Inc. Centre A nalytical Laboratories, Inc. Page 3 o f 43 Page 59 o f 104 Centre Study N o.: 023-042 Centre Protocol No. OOP-023-042 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 raw data package. All standards/test substances and any prepared solutions must be identified with a unique label or number on the container or cross-referenced to the container. HAZARD INFORMATION ' A current MSDS for the chemical(s) used in this study will be maintained at the testing facility. 3. SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 . . STUDY DIRECTOR: Rochelle Robideau TEUEPHONE: 651-778-7065 FAX NUMBER: 651-778-6176 4. -TESTING FACILITY AND PERFORMING LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 PRINCIPAL INVESTIGATOR: Em ily R. Stauffer, Centre TELEPHONE: 814-231-8032 FAX NUMBER: 814-231-1580 . 5. PROPOSED EXPERIMENTAL TIME-FRAME Experimental Start Date Experimental Termination Date Report Issue4 Nov. 6,2000 Nov. 24,2000 Dec. 30,2000 6. JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM Mallard serum samples and liver samples collected by Wildlife International, Ltd. will be used as the test systems for this study. Mallards represent wild bird populations and they are an EPA recommended species because they do well in a laboratory environment. Mallard habitats include ponds, lakes and marshes, and Centre Analytical Laboratories, Inc. Centre A nalytical Laboratories, Inc. Page 4 o f 43 Page 60 o f 104 Centre Study N o.: 023-042 Centre Protocol No. OOP-023-042 they are considered aquatic grazers. The use of these matrices for the study was to determine if PFOS was deposited in the liver and senim of mating adults that were fed PFOS. 7. SAMPLE PROCESSING, 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 prior to extraction; however, they will be homogenized by lying the samples on a flat surface and mashing them by 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 samples will be kept isolated from the test substance during storage. 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 contain-or cross-referenced to the container. 8. ANALYTICAL METHOD ' The serum samples will be analyzed according to the analytical method titled "Extraction Of Potassium Perfluorooctanesuifonate Or Other Fluorochemical 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 Perfluorooctanesuifonate 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, and calibration standards will be prepared as follows: Stock Solution Prepare a stock solution of PFOS at 100 pg/mL by weighing out 10.0 mg 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 maximum period of 6 months from the date of preparation. Fortification Solutions a. 1.0 ug/mL Fortification 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 A nalytical Laboratories, Inc. Page 5 o f 43 Page 61 o f 104 Centre Study N o.: 023-042 Centre Protocol No. 00P-023-042 b. 0.1 ue/mL Fortification Solution - Pipette 10.0 mL of the 1.0 pg/ml fortification solution into a 100-raL volumetric flask and bring up to volume with methanol. c. 0,01 ug/mL Fortification Solution - Pipette 10.0 mL of the 0.1 pg/mL fortification solution into a 100-mL volumetric flask and bring up to volume with methanol i` 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 of preparation. Calibration Standards - . Prepare six LC/MS/MS calibration standards in methanol via dilution of the 0.1 pg/mL and the 0.01 pg/mL fortification solutions. This is a typical example; additional concentrations may be prepared as needed. Initial Cone. (pg/mL) 0.1 0.1 0.1 0.01 0.01 0.01 Volume (mL) Diluted to (mL) 5 . 100 2 . 100 1 - 100 5 . 100 2 ' 100 1 100 Final Cone. (ug/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 of preparation. 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 hand prior to extraction. 3. The sample weight for liverwill be 0.50 g ( 0.05) and the sample volume for serum 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 will 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 rpm. The samples will only be 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, Inc. Centre A nalytical Laboratories, Inc. Page 6 o f 43 Page 62 of 104 Centre Study N o.: 023-042 Centre Protocol No. 00P-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 Biomolecular Mass Analyzer Interface:. SCIEX Turboion Spray Liquid Introduction Interface Harvard infusion pump Computer: PowerMacintosh G3 Software: HPLC: . PE SciexAnalyst 1.1 Windows NT Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column:Genesis C8 (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 ' Row Rate: 0.3mL/min. ' Time %A %B 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 Betasil C|8 etc.) can be used, provided equivalent chromatography is obtained. Ions monitored: Analvte PFOS Mode negative Transition Monitored 499 -> 99 Approximate Retention Time finin') 4.20 Centre Analytical Laboratories, Inc. Centre A nalytical Laboratories, Inc. Page 7 o f 43 Page 63 o f 104 Centre Study N o. : 023-042 Centre Protocol No. OOP-023-042 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 order to optimize for greatest sensitivity. The mass spectrometer is tuned using a 0.5|ig/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-Quad 0 Rod Offset IQ1-Inter quad 1 lens ST-Stubbies ROl-Quadl Rod Offset IQ2-Interquad21ens R02-Quad 2 rod offset ST3-Stubbies R03- Quad 3 rod offset DF-CEM Deflection Plate CEM-Channel Electron Multiplier Gas Flows Nebulizer Gas . Curtain Gas Collision Gas TIS Temperature - Set -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 Set 12 13 4 350C Calibration Procedures a. Inject the same volume (between 10 to 20 pL) of each calibration standard (prepared in MeOH) into the LC/MS/MS. b. Use linear, 1/x weighted standard curves for quantification. Linear standard curves are generated for each set by linear regression using the appropriate software system. Any calibration standards falling outside 30%, based on its calculated concentration, must Centre Analytical Laboratories, Inc. Centre A nalytical Laboratories, Inc. Page 8 o f 43 Page 64 o f 104, Centre Study N o.: 023-042 Cintre Protocol No. OOP-023-042 be excluded from the calibration curve. However, the total number of calibration standards that may be excluded must not exceed 30% of the total number of standards injected. c. The correlation coefficient (r) for calibration curves generated must be >0.9925 (r2>0.985). 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. 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 an analytical 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 must be bracketed by standard 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 and carried through the procedure to verify recovery. d. The concentration 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. Samples in which no peaks are detected at the corresponding analyte retention times will be reported as ND (not detected). Samples in which peaks are detected at the corresponding analyte retention times but are less than 3:1 signaknoise will be reported as NQ (not quantifiable). Centre Analytical Laboratories, Inc. Centre A nalytical Laboratories, Inc. Page 9 of 43 Page 65 o f 104 Centre Study N o.: 023-042 Centre Protocol No. OOP-023-042 g. Background levels of analyte found in control/blank samples that correspond to values below the LOQ, but are still quantifiable, will be used to correct fortification recoveries. h. If samples are not loaded on the instrument to be analyzed the day they are extracted, samples must be stored refrigerated at approximately 2C to 6C until analysis and analyzed preferably within a week. 9. EXPERIMENTAL DESIGN Samples obtained by Wildlife International Ltd. have been shipped to Centre Analytical Laboratories, Inc. for analysis. All samples will be analyzed according to methods, "Extraction, of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" and `Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC-Electrospray/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. PROTOCOLAMENDMENTSANDDEVIATIONS 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, and impact on 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 A nalytical Laboratories, Inc. Page 10 o f 43 Page 66 o f 104 Centre Study N o.: 023-042 Centre Protocol No. OOP-023-042 2. Protocol deviations: Protocol deviations, which are one time and unplanned deviations from the protocol, shall be documented in the study records, notin'* 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 Investigator and Study Director and reviewed by QAU. 11. RECORDS Records to be maintained include, but are not limited the following (as appropriate): 1. Sample tracking sheet(s) 2. Sample receipt records, storage history, and chains of custody 3. History and preparation of standards (stock, fortification, calibration) 4. Description of any modifications to the method 5. Instrument run sheets, bench-sheets or logs . 6. Analytical data tables 7. All chromatographic and instrumental conditions 8. Sample extraction and analysis dates 9. A complete listing of study personnel, signatures and initials 10. Chronological presentation of all study correspondence 11. Any other data necessary for the reconstruction of the study All chromatograms will contain the following: a. Sample identification, date, arrow or other indication of the area of . interest, and injection number corresponding 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. QUALITY ASSURANCE i 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, and will report the findings of the audits to the Principal Investigator, Centre Management, and the Study Director. Centre Analytical Laboratories, Inc. Centre A nalytical Laboratories, Inc. Page 11 o f 43 Page 67 o f 104 Centre Study N o.: 023-042 Centre Protocol No. OOP-023-042 13. DATA AND REPORT 1. All raw 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, and any other appropriate data generated. Raw data not used will be stored in the study file. The reason for exclusion will be documented. 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, but not limited to: 1. Objectives and procedures stated in the protocol 2. Analytical and statistical methods used 3. Test materials identified by name, lot, purity, and other characteristics 4. Name of testing facility and study initiation and completion dates 5. Statement prepared and signed by the quality assurance unit 6. Tables containing all applicable data ' 7. All chromatographic and instrumental 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 report 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 Investigator and the Study Director. REPORT DISTRIBUTION: Original: Study Director Copy: Centre Archives 14. ARCHIVE STATEMENT Study records to be maintained: Records to be maintained for the study include all raw data, observations recorded during the conduct of the study, documentation, chromatograms, specimen tracking information, and study related correspondence. Centre Analytical Laboratories, Inc. Centre A nalytical Laboratories, Inc. Page 12 o f 43 Page 68 o f 104 Centre Study N o.: 023-042 Centre Protocol No. OOP-023-042 This includes a description of equipment used during the conduct of the study. All characterization data and any shipping 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 exact copy. Centre will retain facility-related originals and a copy of the final report and raw data package. 15. REFERENCES 1 U.S. Environmental Protection Agency. Toxic Substances Control Act (TSCA). Good Laboratory Practice Standards. Final Rule, 40 CFR Part 792. Centre Analytical Laboratories, Inc. Centre A nalytical Laboratories, Inc, Page 13 o f 43 Page 69 o f 104 Centre Study N o.: 023-042 Centre Protocol No. OOP-023-042 16. PROTOCOL APPROVAL This protocol was audited by the Quality Assurance Unit of Centre Analytical Laboratories, Inc. Quality Assurance Reviewer, Centre Naomi Lovallo, Ph.D. Quality Assurance Auditor Principal Investigator, Centre ;// Date ` ----- ^ -------- :-- Scientisti) Facility Management, Centre .John Flaherty / Laboratory Manager ////f/ O Date If- fiIW O Date -- CentreAnalytical Laboratories, Inc. Centre A nalytical Laboratories, Inc. Page 14 o f 43 Page 70 o f 104 Centre Study N o.: 023-042 Centre Protocol No. OOP-023-042 APPENDIX: ANALYTICAL METHODS A. "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" B. "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" . Centre Analytical Laboratories, Inc. Centre A nalytical Laboratories, Inc. Page 15 o f 43 Page 71 o f 104 Centre Study N o.: 023-042 Centre Protocol No. OOP-023-042 ! Centre Analytical Laboratories, Inc.---------------------Centre A nalytical Laboratories, Inc. ----------------- Page 16 of 43 Page 72 o f 104 Centre Study No.: 023-042 * CentreProtocol No. 00P-023-042 ^ y` ' " ' ' V ' r : ^X ^jfT 'o-^';-" . , S ^ f f S ? i P S p g l h i w o o c t a n e s a l f n n a t ' r: serum, or other fluids, usmg an .. < -- :i. v ^ g p g ^ a i ta ^ oe e t h y l- / r t ^ t i l e 4 r ( M f f lE ) : lhthis'method,seta - * Q- -: ' i c/JSS.sample and the analvte ion pair is partitioned into MtBR-.Tie n/toc -- . "Centre Analytieai-Laboratories, Ine-.Centre Analytical Laboratories, Inc, Page 17 of 43 Page 73 o f 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 y I- /- - A '<*\ -- 1 _ /.V ' r , 6.1.4,. Nitrogen evaporator, Organomation ,6.1.5.,?Balance(0.100gj",; :: - . , ' ? -2 A S upplies andMattriai'.s : ; ~ ` ' v " /ut'' * . 7J bottiesTcapable ofholding 250 mL and 1 L NNo0t6e: PPnrinorr ftno tci ^ngV gAll-ascfsrvnr^srric'arn<d4jKjorttktllcMs, nrinnsce* 3^ ftrimimeass wwiitthhmmpefthhaannAoll an<d4 "3i times with *>* V:--.' ' ". '. ' ' MtllLnlMiiMtnmniM''! minrmttm nf>QlimM wtrh '`^ - ,L.?*V:Vy.*T:eIxeaj^^e^CT;MilH:Q^Veq^^ . be Milli-O1?4water and mav be oroyided bv Mli-0 TOC Plus1*?s^ei^> v::':`7s '.. : ; W ^TetbSaMmnKn&todi^smQteCIBAL'Kod&weqmvalent -8 ' ,i ..--....8.4-- ASodium caibonte (Na,COj), J.T. Baker.or " equivalent c ,, , 'I < -...5 v SotumWcarixnatNaHCOO.'J.T. Baker or equivalent, ' :. 8.0 x.Methl-T-Bu^lEe^OiimisoIy,^'glaadisdUed^orHPLCgrade.j-n-srA 8.7 _!. MeSmi f ^ i m t v ' vliS'disiiUedorOTLC rd -=:^ . K -h. '/"u '*.* -. .* Centre Analytical-Laboratories,-Inc-r Centre Analytical Laboratories, Inc. ------------ Page 18 of 43 Page 74 of 104 Centre Study No.: 023-042 Centre Protocol No. 00P-023-042 n .' ' . .8J J PFOSAA (3M Specialty Chemical Division), molcula- weight = 585. . . 8-M . EtFOSE-OH (3M SpecialtyChemical Division), molecular weight = 579 PROSEA (3M Specialty Chemical Division), molecular weight = 527' -> ^erjyoiume^inaii iisicam reagent, standard, orsurrogate -* : V. .v r-'V's- ^ :r - " .7. .-'.*> ' i . ,8'10.1;l^Nsomn'htoHde^aOH): W ei^^pfo:di^ly2(X)gN aOEPo(irjnio a * '**- 1000mLb5 w^htinig5TOmLMilE^TMwto,mmuntaEsbBds are - " / ' , dissblved^torema 1 L N a lg m eb o ttle .^ ^ W.-y;.. ' '. ; v ---8.10.2 1 Nso^ummydroyde(NaO: Dilute IONNaOH 1:10. Measure 10 mTnf ; q .. /* .. ,NaHsoiltionmto.a 00n i volumetric flaskanddiluteto volume using ` c^rMilh-QTMwatCT^Store^al25'i^NalgenejttIe.>i,.' . - v v . A i r ' . .. 8.10.3^0.5 Mtei&utylammoniim^^rogensulfite(FB): Weigh^riimately69 e ' - ' -V. ' ` ' '^-^ofraAmtoaJLTOlmetnc.cOTtainmgSOOmLhfilKAj^water.AdjusttopH6 - ri: 10 sing approximtel^ijo 54 m lpf 10 NNaOH (While adding the last mL o f ' ' ' V ->. ^MonU `AA cAarh/hi*n<ft'tift'nTT:rhnp ahnmflv *TS1*>^TM*kL2Tr'_ . * : Centre-Analytical Laboratoriesrlne- Centre Analytical Laboratories, Inc --Page 19 o f 43 Page 75 of 104 Centre Study No.: 023-042 Centre Protocol No. 00P-023-042 C . : . . 8-11.7 Dilute working standard 1 with ethanol for a working standard 3 solution of ^ .-approx 0.50 ppni. ... ! - : . ... S u rro g a te stock standard preparation - ' J ' 8.12.1 W ei^ approximately 50hS0 mg ofsurrogate standard 1-H.l-E "t I '- 't r c V - !C,F,,SO,Hirito'a 50 mLvolmnetric'flask and record the actua Centre-Analytical-Laboratories, Inc. Centre Analytical Laboratories, Inc. --Page 20 o f 43 Page 76 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 ` 10.3.4 The expected concentrations will fall within the mid-range of th initial. calibration curve. Additional spikes may be included that M in the low-rane* f - . the initial calibration curve. This is necessity if the analyst must quantitate S i T ' 'r ' only the low rad of the calibration curve (for example,j ppb -100 ppb rather ^ 5ppb-1000ppb) " - 1 : ..,lli^LreiuTMNi^STirffiARDrzATioN:ir><-.- 2 sV-. i"''- . ' :/ . . ..Centre Analytical Laboratories! Inc. Centre Analytical Laboratories, Inc. Page 21 o f 43 Page 77 of 104 Centre Study No.: 023-042 Centre Protocol No. 00P-023-042 si-/ vfvr.i":; ;/.V. 5: ppfoimte spiking amoMto.for standards and spikes f i i m L i t ntaix'&-$~ :; ;v" ^ W o rk in g standard Approx, final cone, of Hs,7(approx.*cone.) i S w J iS S s f c :Vanalyt in matrix* ' -* :*V-*>Vl Blank r,: Jv ^i0 .5 0 0 ppm'jSi.t.'S' t ^ lO v C ^ : : f - i f. 0.005 ppm l^'v; S K 0.500 ppm }V4->iV ?35i20.-SSiV ^ 0.010 ppm > S i / ^ 5,-00 ppm -SSir l^ ,# 5 i)p .p p m YSSSf 0.025 ppm ;^- W:*:0.050-ppmSvi'.i5 "?*'-'./'v' .>?i-S3iS00 ppjnSiSS& ^ 5 2 0 ^ ^ ? b S .0.100 ppm 3r:-.'.i- *$ 4 & r50.0 p p m ^ i i . ;f.0250 ppm Sii/;- * K**'- "w;,- . :-i;fr/:'.~;50.^.ppn vS^.Sr 10 ti v!S.0.500 ppm~.>;-; 3 S s . 505 ppmj?,?.? w m i s 0.750 ppm ; i v . ggiSOlO ppin'tSP? yX -20 ii";?vl.00 ppm r ;'<' " .'*`C -^polypropylenec e n trifu g e tu b e .^ v ^ ,V ^ ~ P r:y \]T " ^ r ^ r ' _ . , * ' *,^' * samplesjo fre c r after traction amounts have been removed -:- : . -, .^^ .y R ^ o r d fee initial vnlnm enrith e ^ r a c t i o n ^ w o n c s h e e L * 1 '*- '* . Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc Page 22 of. 43 Page 78 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 V - O * - ' . - r.. C ~ _/ . 12.14. 1 5 ^ COTttifuge'^e with the same information as in 12^.' -.^ .1 5 Remove 4.0 inL of the^rganic laytr to'this clean 15 mL centrifuge tube.- . ' Yr'J-iii -PSuytWea-ch'samnle nn thft*na1vv\tJ-i:eal rnytrnpen eva-oovra'l'or until tirv ann.m.vim.;nf.i... . . \ 1 ' Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc Page 23 o f 43 Page 79 of 104 Centre Study No.: 023-042 Z- \ Centre Protocol No. OQP-023-042 _5i , ; -L > * . l j -0 REConns' ___ _________ If*?,: .;5?mpiete theextraction worksheet attached to this method, and tape in the study r \ . hotebookor include in the 3-ring study binder, as appropriate. 'H .ir itB, MDL/LOQ values and summary - ^ - : C ^ c S ^ o n ito d ^ co n cen tratio n w o rk sh t"i : '. / ' V 'ri A v . IV ^ - . O ^ -18.1: ' .>?/H-*:P--L.C-" * 1i24nLATFECTED DorTlMFJfTSt Vi i .-. i- Seriim or Other Fluid Extracts for Fluorocheinicals romtry^ -ill./ M ;,;' *1 .>/.!>* ;v \ j ; E X ^ . l ^ A p ^ . . Fluid Extracts forFluorochemicals using' : c Z--.' ' :Vi, -!',HP^-Electrosp'ray Mass opecrromeuy M a . . ;c.v viv-:-.'.--; .v . . v , . Revision - - Bate; 04/02/99 ' -ft .Z-.' Section 12.17 Final volume is l.OmL; not adjstedor initial volumes 0; - - : .j ... ; - ~* -CsiLtjeAnalytical Laboratories, Inc. Centre Analytical Laboratories, Inc. . _5g?_24of 43 Page 80 of 104 Centre Study No.: 023-042 Centre Protocol No. 00P-02.3-042 ----- Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 25 of 43 Page 81 of 104 Centre Study No.: 023-042 i-:.' ' Centre Protocol No. 00P-023-042 ..v.** .tl* :w -- ---------- > V - : v 1 \ : - - Compound MDL LOQ Linear Calibration Bange (LCR) ` -------- -- - (ppb) '.' (ppb) rrOS Standard Calibration Curve ' ` ' . Pt ^ ^ - 1.74 ' i . i i 5 ppb - 1000 ppb v- ' S ; ; . . . . . ~ ---------------- - J.PSA\"v 4:79- 5 ppb- 1000p p b .-.. ..... . j^UAA&vv* : 3.46.'. 20.5 5 ppb - 1000 ppb / * r- fc*: ; %.. . ,. ------- *.11.4 >. 362 5ppb-1000ppb .v.v.iiv, M656^T?y?j f: 6.03 .- .192 5 ppb -1000 ppb.vs . .-- -,v .... ,. : ---- PFOSHAv^i .182- 5ppb-1000ppb s K & ' V i . - f . t r ; , - . .-. : ^ U L O q valt in d o v in e , monkey, andhumanscrum^d monkeyplasmawere no725SSrnr-- c ' r .. ;.*--*-'.'. .1r,- -V :T.V .- .} .-;r.r.Vv^-.*--*r : :' " . - - r .:: : S ? S I S S i v \ \ ; : V;:1. ^ yv;:i:;0: ; j % * :.:'u . V ` :v . ' U - ^.v'^Sifa . ^;v- Sv- iti ---------- -----Centre Analytical Laboratories," Inc. Centre Analytical Laboratories, Inc. Page 26 o f 43 Page 82 of 104 Centre Study No.: 023-042 Centre Protocol No. 00P-023-042 C; fJ_. .-. *.. r > .. .... . ....7*;" -. -..* ... Componnd:PFOS " --"V* : - Prepared range LCR from % Recovery ,, JRabfcit Serrar 'if istandards 3 - curve /. Range . `ISlSS;'(ppbj (ng/mL) *</:. (PPb).;, s2i?si':^ i :S' * (ng/mL) : .' RSD Range . .. ljt s5 ^ 9 7 8 ^ 2 0 -9 7 8 '7 i 83;108 -.1.4767-11.0 <,'*'VT SJw.<Ct_'.n rS/v U4*-24S-7 ^ 85-104 \ '-`5M-I S gS ^ mm* > ? 7 4 j 3 > 85-106_ '- ;4*.8-4**-9-*jJv0 > 4 ^ -9 7 8 4 : '94 -ll^ j-76r0<-'.lvO- J .'Co'mwMd: T f'y . ' ;,' "f .--: * Rabbit Serum ; Preparedrange > ir ofstandards^ .V4-V-.v.iL- 'cCAuRr-vfrteomV.' . i Recovery J^Rnge_ f j.-RSD^J -j . Range C- $ a & i8 ! M <PPb)if: ! - '> c FaRang1*|; fJ^oS37>7S;^ ^ 4 5 3 ^ 7 $ r*r| .^.8..-.10. "3'-:r- ? 5.IO-M.7 '; Low'Chiryc^' 'VZ&'-Jff-. i & m ^S 7 -I0 5w-A-.. S-14.7 ^~V, ' ffigh.iirre _. . X ^ ^5 ^y-224'."8.?r<28j:t # 3-102. '* 508-133 f:4 U W 4 7 5.10-14J V .. r. . . - Vf--."v'i'-. v - f-fj* Compomdi'PFOSAAT-^'v. X -& S 'Z 'fs y*-*' iWsx7jfS-y'rifSr^v:'1', x '-V - ^ **' rr-O''pi1 Prepared rangej -i LCRfrom *: % Recovery Rabbit Scrum ^.fstindads';;. ^ ^ c u rre j.^ r. s\ ** " . (ppbj (ngfeiL)"' V. (ppb).-/' ; (ng/mL) 'C 5 rv v. . v ; .. . Full Range iW C brn^ 0,991 - 574-r V itr ^ s w il ? W U 7 : Aor'/ ' - 247>;J *?9.74^247^ ^ 9 i 7 j S- 6 jl s ' High'ctffve X492:974^V, .&''S7]4-974*: r .y *fp >43S-12_5.^ 7 . j>;o^'y74 t-.?.74.:974i.r 95.115& c S u i(s 3 * f ..*.'*";~i#4'.*. .. . a . "* V *" ""-. V - U,', ... CentreAnalytical Laboratories, Inc. Centre Analytical Laboratories, Inc Page 27 o f 43 Page 83 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 - V* ^P rep aid range L C R fro m % Rccovoy ; RSD it)~$faL)* ( p p i> ) y r S T m m s K:fnW i- :f & s f 1-2S.5 . - -J* " ;-W W & l w t m aSgig.y*?j m s m /I9T,ZS- - 9 76 'i V: . :v. ' X 0 $ l ZnsiZ S i- l '~i -4 .4 .1 -2 U : 5 $ ; ' "/ Raiibit Soum ^ L O tW r tf# S S ' KC0393 ,;9 7 6 '| ^ 4 # f 9 7 ^ % Rccovoy . RSD . -A `-"V *'. - : 10.1-16^ ; . v ^ -:- c t tj; i '** ; ri '' 8 ? 9.'76^ 2t? '' 8 ^ - 1 1 0 8 - :i*-- tV -r- .. . 9.76 - 976^ .11-5-19.5 ... 7 2-SJ- ''\ 'yj Rabbit Scrim ' '8 i. . Prepared range . ,,L C R f i o m f . / . R e c o v ^ ? o f ^ r d s -.V.'-cuive r m & r n L ) ? v p p b ) 8 ? ' (ngtoL) : m \ Fun S ig e b 'i Lo'.Cut^:C m m8 24-s. - 8 8 8 88: I0 r -, 9.76 - 97.6;-:4*-s* .'.' i; ,' . 818Vv* ffig h c u r i^ : r :? V?-7 : 97 ; 9 7 - 1'io r S ' %"v.A-Jv7'- iv.ssr&j. . V " .r: - 8 H . ../ ;;y- Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 28 of 43 Page 84 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 r ' Ion Pair Standard Curves- Fluids P rep date(s): _AnaIvteisl:, ? ' i` -" * S ta n d a r d n u m b e r : E quipm ent nnm ben cimcs!^Stdoex^\Std con'c.^Std conc;^'Std cene** $ 8 & 4 ^ ,?* -'V,-'. rd u i vi spied mL';^ rmT/t-.' 0.500.?', ' 0.507.--r ?70J32S* >'O JO t;;|.>O j2I:; .T0.501 *: '> 0.010 ; : 1.015 . 0.500V * 0.507?* 0.532^ leK S O iV -p.asa* >M^0_50I Sfc vL 0.020 5.1* 7 1.025 -, TV5.07 JRA32-X.- M.01 S fliiii2 is g . -i-5.or.5i-. 0.005", ." 1.010 - -5.00 ir :a5.07v-. >_;532 t i o i a i w r s i -sa&oi:- i; 0.010 i> ' 1.015 ; 5 00 i S 5.07.-i :-f5J2 ,N5.015'|2252r<5'- .*5.01 is? St 0.020,vi i 1.025 v ~50.0>v s.50.1.~ir V.53.2 s i :1:50.1 ? d & S 2 .l-& i M50.1 *rt 0.005 ?, i.' 1.010 ; " 50.0 ' **50.1 ; i . 5 l2 sr. i?50.1W ~ 52.1Jai; Vv"50.1,'5' .0.010 : 71.015 ' 50.0 ic50.1 y 'e53.2 `ri- f t 50.1 m,>C42.1> ^ ,5 0 .1 . u: 0.015.;? "* 1.020 _50.0 *.504.? iC 'S iiis . -;>50.1 |?t'^2:13re Tfe0.020,. | f .1.025 . 4J3 ^ V<*SjOO*< ~ - k $ 2 A s * '. >*4.94 t5.or> <;iS.l3Sl ^ too. . '.*3 0.005 . 9.76 -v ..9J9..-V ii* 10.4:45 9.78 T- .Ci 953 >;J0.2 X 2U - `',25.1 :* .r~ 26J-: -24.S :* 3J2.2*/ '`Surrogate 49J . 50.0 - .-.514 vv .*;49.4 .s:50.1:;*v ^ 5 I J * Finiiconci -97.6 *919 & 4*-. 104 v i : ,-.97.8*4 >,99.3>5- yT:102-1~- ingnL^w '24 I V 2 5 1 :<?. i 263t*. .Ti248ii: *252* 4 iis ? S i 493: K vi'500 :-*= ?:.524`>.V a v-494 a'. A'513 7.W S g b # 735:j>- tifi- 746^;* 97(5- .' >?*i737t-' ^749j^ V5V.M1 Tl- .inn -i s#-'SlseSr.-r$Vi ai w & y & -Centre Analytical Laboratories. Inc. ; Centre Analytical Laboratories, Inc Page 29 of 43 Page 85 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 . . i - ' . .... ^^* ':--?-^jJaiEm7y._>,I_RON MENTALLABORATORY -Centre. Analytical Laboratories, Inc._ Centre Analytical Laboratories, Inc. Page 30 o f 43 Page 86 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 Mdmethyl-tert-btyl etheiMtBl.-Int& method sevei fln X ^ J L . pa? B8 V. " ? - J i Surrogate standard: lH-lH-2H-2Hperfluorooctane^fom^ac1d :" -v 5.*fL V A R N iw t amp r m o w -. Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 31 o f 43 Page 87 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 . . ; ` W-5 . Nitrogen Evaporator, Organomation ' ; . . . . . - 3 ^ir.fi'l-fi^BalahceisaisitivitytoO.lOOg)^^".- ;. ' -.V.';.; . V j ' . . ' . 'f g f a f c - A O T i Mtbi' ! "_____ -V- > ' . Note: Prior touring times ___;______ Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 32 o f 43 Page 88 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 HI C . PFOSA (3M Specialty Chemical Division), molecular weight = 499 SpecialtyChanicalDivision),ml:a]arweight = 585 : ' ^ ^ i p 4 EffOS^OH (3M Specialty Chemical Division), moleoilarweight = 570 PM Specialty Chemical Division), molecular weiiht = s ? 7 - . jT- y . .. J ^ ".j> *'-v"v" **v iriMSurc iy rn/(of - ION NaOH solution into a 100.mL volumetric flask and dilute tn vni,,TM... - ^ ^ M ll W ^ w a t e n ; StoreinafSTmL;Nalgehebottle:' ' A " - ; ; . - ; - - . ' - . 8 ff 11-3. 0.5 M tetrabutylammommn hydrogWailfete (TBA): nM ' \ . ' ofTBAintoa 1 L"voluine^.cpritanng500mL'Mi]ii-QTMwata,JAdjustto ' * V* " PS 10using"approxmkelyiyi5j_^_qiONNaOH(Whileaddinthe W ' . ofNaOH aHHslowlvhecaiKfi thftnTTrhanoM nimTnMwS *2..? - H1L_ .' '' Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 33 o f 43 Page 89 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 . ' " W ' - ^ v W ; * : r- - C / . ' : ' . .j, 8;.'l_l6 bilutcthcrtocksolution"witmethanolforaworidngriandarti2 solutionof '':* -:. - . ``'-i+tr appir'5.0ppinl;< y ' ' ' \=.. : v . i "- " .' ""/. ' i v iV* >'' * * ' . ... ., . > > ',;L**.S_17 7 TVItt* f U * 'A1nf4rtnTin*Vi mftV*ejnnI fnr a ut/nririntr ^ - .. - i^8.UX^a^^prox^teiy.5W0mg pfsunpg^stanri " ,.. . ^^^'^?^nSO,H'mt7i 50i.vqlumetncfl^'mdiSr4theac6ialwH^:>^^ : ' ` - '- a# Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 34 of 43 Page 90 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 35 o f 43 Page 91 of 104 Centre Study No.: 023-042 Centre Protocol No. 00P-023-042 &Ap^proximate Spiking Amai2To*uVrn*ts for.Calibratifon S t^dards{,*v1* {S.Woridng Standard .-j; s p t & v M if^ p ro i" Cone.) Approx.; finaJconc. of J-.' PFS in liver V f ..-xsu `i'-Bhnkc-y - vitg'ifffflSO ppm * W v 2 . ^ v i 0.005 ppm Jlv 1 ^ 0 . 5 0 ppffi'C-1^ Z%x:f.4 k& * . *ti~. 0.010 ppm - " t P4?pm JH SiA"10 .'-- 0.025ppm*,:' . \ 0 . 5 0 'p ^ ^ y ^ 5-%20>-i" ?<C: -0.050 ppm > -, rc-'it, 0.50 vpt-jssK- V S S40 s,-. 0.100 ppm 5.0 ppt W%ir- A i U O .- y . '0.250 ppm'< N-itsrS.O p p m > \ii '. v 0.500 ppm . J- ' - 5 0 ppm ~ ^ 3 0 ; ^ : 0.750 ppm " ' -?-S~sr.50 ppm ^ 4 iA ' 1.00 ppm Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 36 o f 43 Page 92 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 ^ ipc.ra. 1:.mL' or otter appropne yolum<ofomgenate into a 15 m L poIyproDvlene ' ' ; ' . - .' r 1 C C H Infilo^ tllKo T tk +Tk viti* t k * 11 .t.` .. * * * ' . !: - ; each sample and putonm e s h ^ a `at'a setting o f300 ipml for 20 minutW^ : O': : ^* - -f - N- - 4?-31 ^ e la 'fi^ i5 mL'rab^get&iwiAe'sTOeii^orinafi6nasini2 lO ''>'-'1 ' . ' __________Centre Analytical Laboratories, Inc. ~r ' i Centre Analytical Laboratories, Inc. Page 37 of 43 Page 93 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 r ^ '- .' C _ .: ''-i^JL^ATAAiVALyST.S AND C ALCULATIONS ' "` ' ` V ________ ' - v - ' ' .. . ' A ^ r > ^ ;U 3 .1 .k Calculate the average density of the liverhomogenate by recording each separate l.OmL aliquots ofhomogenate .3= . .. . . . . - 1-4^ Average density (mg/mL) = Average mass - -V ? mLaliqt s -... s n*i ? #ue amount.ofkver (mg) per 1.0 mL of:' : thK/l fUiSM5 Ui iCV nU^Clr UmLLT>/Unif UJtUnUmlnUaK^CnUiiltUiC*ciUi i U t iU l U i il USU1Z f b l l w i n o ; '. r-x g of Water) <i 'V.:: UUCalcuiateractuali ' "standards using the i ?&;'< ;/. ?:; :,'"i\ V': nf=Final ConciliationCug/gorifig/kg):: ( ' - ; ' " mg Liver/1 mL - j .v" t-g'-nr. ofPFOS mLiver 7 TM -/ IX 1 "refe to 13.1.2 for details.- i :*i -ft*--.. __ . 1 M _ M e t h o d PERrOnMANTE 14.1 :Ts hu eo 'u iC U"lU U` UClCCuO'H l im n \ j x x u i * j Jo o lia i j v * < ^ e r tMDL report for * .specific MDLand limit ofquantitation (LOQ)------ --- ---------------------- -'-B^ ,,i udu ,Q. .. 14.2 ThefoUowmgquahtycontrolsamplKarecttiadrfwi^battoraimpies'toevaluate -' .'the quality of the traction and analjrsh.-g'ig:; ' r" ' -5'1 **x ~ " ='"........... !/i .j1prcisnofthe"extract Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page-38 of 43 Page 94 of 104 (. : >< Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 ^ 'PiK.:, ..- v.;;-:.-: Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 39 o f 43 Page 95 of 104 Centre Study No.: 023-042 Centre Protocol No. 00P-023-042 .... Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc Page 40 o f 43 Page 96 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 r MPL/LOQ vaines for rabbit iiver Compound MDL ; LOQ Linear Cabbration Range (LCR) .(ppb)/ (ppb) Approamate concentrations to be used for preparino the ;! *7 ' Standard Calibration Curve ' , " PWjS;^ -T8.45..W 26-9.- 3 0 ppb-: 1200ppb - . -- I 't P S A V * : '; 3 i 0 11.1 12 ppb-.1200 ppb-.` -v OSAA^r 24.6 i .7 Z3 .30ppbr 1200ppb : . .; ~_ JbtFOSE^OH.V 108 V T;345 60 ppb-r900 ppb* ' ..; 3:82.3 :: 262- 60ppb-1200ppb p r o s a v a i i & r & z ',108;. | 3 0 ppb-1200ppb v:- . . MOl/LOQ values inrat, Bovine,'and monkey liver were not statistically determined. Two - curves meacbqi^hesemati^ and analyzed vdth tile rabbit liver curves to : . d e te r g e ^ v ^ m c e .'- Responses in'SenC bovin^ and troikey liver curves were equivalent to fce nibbit responses, fherlfore,;i3eir MDLand LOQ will be assumed to be equivalent to those .valuqsas t t e e n t r i D e d *: v`.S;?a;;'.> v _;. V R efe r to IXX} S u h m a y L d &7 J0 A M lo r b r i b e r n f o n n t i o n " * EtfOSH-OHestimates only forMDCahdLOQSDid notmeet criteria forvalidation. . ' Compound: PFQS .Liver;*', 'nutr*' ..'^Prepared* ^rangc'of Vstandards(ppb)fttiAaL) ;*.Raavenr?eaegoef r^LCR-izonirb ^ayccnnrp; r-Rngeof,.^CRfdm^ ^Jowjtd i*Sovsfi^ . curve /... curve_\.*Scvftrir= (ppb) (ngjW.) *:(ppb)7(ngtl-r (ppb) (egM55^^ng5EB :.**"*** r.LCRW; highad .^ u s S S . curve -* Kiv*-qjygjE Rabbit :V 19-1237 ^2- '1200^ ^ 2^ ^ ^<j-i2qq;^^g Compound:PFOSA;H ^ ^ i v .=' v*C. ; . V. / ; . Prepared Liver" rangeof ; Rangeof- U'LCR^fiom^i Range . average; Vj^ve^ve^; Y. lowstd ::^^w.std3r 01; VLCR6oni vbfriM y-^asas nutria: * .*standards *- 'curve y`cosvt (ppb) (n*/mU (ppb) (ntfwL)VpjWrfngAnLV (ppb)(drArL) -pbV*(i?pmL)^ita*) (tata k*i.S3Sv Rabbit .19-1237 ;12-12.0. 0*/rj4i712fiV-AI'2s0.v0i*;i .12-300 Compound:PFOSAA'S&tfjF- t ,,-- Prepared : Rangeof " RangeofiTt^LCR^fiomt.^ : Liver Vrenge'ofv ..avenge "a'. avecurve.-, -j. ;matrix^. !;standards'* ri. curve . aan:}i^cury^i (ppb) (nftW.) (pekHnsfaU ~(p*) (taLV (ppb)(BgAnL) 'faw(ttiiml iprt) Rabbit '6.1-1232 V12- 1200 *V~30VlbOi ...307 .y, . ` ' __________ Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 41 o f 43 Page 97 of 104 Centre Study No.: 023-042 Centre Protocol No. 00P-023-042 .eJ^e Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc, Page 42 o f 43 Page 98 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 r // IonPairStandardCurves- Tissue f Anifyijs): ` I SampWmatrii: -/ - Mrthqd/jreviiion' 7 ' '- - S ta n d.a r d n u m.b e r: v;. . E quipm ent num ber: ' V F inal solvent and TN: .. B lank Irver/idendC en . : ' > ; FCmi^appri.'^OOppm: " ' " " "i- ` ' . ' -\..:^n^itd pprox. 50.0ppm - ` ^ " ......- A ctffid c o B c e n h r tio n 't'n f in th e F C m il . * PFOS^ Std cone ugAnL .PFOSA vPJF+OXSTAAP J* (EtFOSE Std cone'; Std conc i Std cone PFOSEA. . M556 . Std cone Std cone * Stdeoa: ng/mL ; ngnlf? ',ug/m S ug/mL' i ug/mL ' ug/mL ; au AJJ Ami spiked Density mL *' " 0.500 - 0300 : 0.500 ' tOJOO ,, 0500 0300 . 0.002 . 0.167 . 0300 ? : 0300 0300 . % 0300,6$ 0300 0300 . ' 0.004 0.167 0.500 : 0.500 .* 0.500 , 'o io o -r 0.500 0300 - 0.010 0.167 OJSOO 0300 L - -0300 . 0300 J 0-0.500 . 0300 ' - . 0.020 0.167 0.500 ojoo i: 0.500 OJOO'' 0 5 0 0 '. 5 ,0 5 0 0 0.040 0.167 r 5.00 - 5.00 > - 1. 5.00 . - 5.00 V , 5 : 0 0 ^ ''-'5.00 - 0.010 ... 0.167 a mN _ 5.00.O -.5.00 5.00.-: . 5.00 - , i - 5.00 - 0.020 0.167 5.00 - 5.00 : V-- .5.00 !t 5.00 - .- 5 .0 0 : ---5.003.' 0.030 . 0.167 50.0 i : 50.0 T 50.0 : 50.0 r 5 0 .0 > d 'i - 5 0 .0 ',t:K -: / 0.004 . 0.167 pros : Final . cone _; ng/g'7- -PFQSA : Final : / cone ' ng/g . PFOSAA Final cone 'og/g E tFO S E ^ Final ^ `cone vsr 'ng/g - PFOSEA /F in al.'.' I P ! cone. . concj-. ng/g ' " . ng/g Surrogate ^ A Sta cone t Std conc.'L V A m i , v h g /m L :',spiked ' ? L ':.v i 5.99 12.0 5.99 5 5 9 5 5 9 . 5 5 9 ' 5 5 9 :: 12.0 : . .12.0 12.0 . 12.0 . 12.0 *1t r.V.V JOp. iV- ,-.0.005 - 2 9 3 ; ... 29.9 ` . 2 9 5 . 293 2 9 5 V 2 9 3 ~ .vSoiTOgiitXk -- 5 9 5 . 59.9 * 5 9 3 . - 5 9 5 595 - .-593,- V Finii arac t e - : 120 / 120. : / V 120 :U 2 0 - .120 . " 120 > 299 - - 299 : * 299 . 299 s.-299 v , - '299 -V --0506^35 5 9 9 .= 98 119* , ~ 599 i 599 - 898 _:. 898 1198 - 119* 399 - 599 - 599 ... 898 - . 8 9 8 ' - : " 898 r.1198 r. .1198 - 1198 ot \ c Validated ranges ^annmTmife concentrations'j^v\ >4^ U v c r .v ; ` Kabit ''. o y i - PFOS - . ; PFOSA e | r PFOSAA -. 5-IQ00ppb 5-1000 ppb 1 5-1000 ppb : Estimates only, use rabbit values. E tF O S E O H , 5-ipp | .PAArt;-: 1 -5-1000 ppb v : PFOSEA \ "5-1000 ppb. 1 ' R ar.; Estimates only, use rabbit values. :* -V -- Monkey .. :. F a m ru aihr m e rabbit values. ..r . Jf-.-N V .:- ;: . *' :r- > `/ - . v - ^ v : ^ Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc Page 43 o f 43 Page 99 of 104 Centre Study No.: 023-042 Centre Analytical Laboratories, Inc. 3048 Research Drive, State College, PA 16801. Phone: (814)231-8032, Facsimile: (814)231-1253 PROTOCOL AMENDMENT Page 1 of 1 Amendment Number: I Effective Date: 11/21/00 Centre Study Number. 023-042 Centre Protocol Number 00P-023-042 DESCRIPTION OF AMENDED SECTION 1. 6: Justification for the Selection of the Test System AMENDED TO 1. Add the following: . A complete description of the test system along with all of the in-life parameters is documented in the sponsor's protocol no. 454/010600/MP/SUB454, Project No. 454 105 that is associated with this analytical protocol. __________ RATIONALE 1. At the request of the sponsor, a description of the in-life parameters for the test system was not to be included in this protocol that covers the analytical phase for the analysis of mallard liver and serum. IMPACT ON THE STUDY 1. No negative impact on the study. 2 o/L Date lentative (Study Director) Signature Date ' ' Centre QAU Review U6 <- nheb,r,_____ ____________________ ,_________ February 12.1998/1 Centre Analytical Laboratories, Inc. Page 100 of 104 Centre Study No.: 023-042 09 / 0 8 / 01 _ H l0 9 _ F ,U 410 822 0632 WILDLIFE INTERNATIONAL SEP. 6.2031 3*2301 EXVGEM RESPRCH @ 003 NO.5 9 5 P .3 jigSSSV Centre Analytical wS^BOBk. Laboratories, Inc. 3048ScseanfeDrive,StatsCollege,PA M W , Phone (814)231.8032,Isotonic:<S14)231-I2S3 PROTOCOLAMENDMENT Page 1 of X Amendment Number: 2 Effective Date: 11/29/00 CentroStudyNumber: 023-042 DF.SCRTFTIONOPAMENDEDSECTION 1. 9: ExperimentalDesign AMENDEDTO 1. Adddiefollowing: ' Moisture determinationwill beperformedonallliversamplesaccordingtoAOAC OfficialMethod950.46SectionB.(a), withthefollowingmodifications: . 1. Thesampleweightwillbe- 0.5g. 2. Thedryingfiniewillbe- 12-14hours. RATIONALE . 1. Attherequestofthesponsor,moisturedeterminationswillbedoneforeveryliver sample. Duetolimitedsamplesize,thesampleweightusedwillbe~0_5ginsteadof -10g. Thedryingtimerequiredforsuchasmallsamplesizedoesnothavetobe16-18 hours,sothesampleswillbedriedfor-12-14hours IMPACTONTHESTUDY 1. Nonegativeimpactonthestudy. ipalInvestigatorSignature Date A-Lw . ? SponsorRepresentative(StudyDirector))Signaatiure Date CentreQAUReview _________ f `/c/a'a/ FFeehbnriuffarr-y 12. 190/1 JtoT- T>U6 AmAhShAkTYl*5 (L-vKuiD N^/c#|tJl h A ^ t OlLUHM LwAS COST $& p m < b LPSr T ^ r s i) ^ hJ^/o RECEIVED TIME SE P . 6 . 4 * 06PM Centre Analytical Laboratories, Inc. 3 PRINT TIME SEP. 6. 4:08PM Page 101 of 104 Centre Study No.: 023-042 $ * 09/01 _ 16:09 fat 410 822 0632 1 WILDLIFE INTERNATIONAL SEP, 5.3601 3=2SP(1 EXVGEN RESEARCH @ 004 N O .5 9 5 P .4 Centre Analytical Laboratories, Inc. 3048ReMardiPriva. gErtgOntlm. PA HTML______ Phaaei (814g31-aQ32. Faalmllt; PROTOCOLAMENDMENT Amendment-Number 2 EffectiveDaiK03/22/01 Page 1 of 1 Cemie Study Number 023-042 CentreProtocol Number: OQP-023-042 rfflsrRtPTTON OF AMENDED SECTION 1. $8; Sample Analysis (f) . :L 9: Experimental Design 3. 8: T.C/MS/MS System and Operating Conditions (Tnrbolonspriy) AMENDED TO 1. Changethelastsentencetoreadthefollowing: Samples in which peaks arc detected at the cuiiBspondfag analyte retention times but aie less tfwn the lowest concentration of the calibration standards (0.0001 pg/mL) w in be reported as NQ (not quantifiable). 2. Addthefollowing: __ The following equations will be nsed for qnantiiabon. of the samples: 1-AnalytePbdnd(ng/mL)==(peakarea--intercept)/ slope 2 _ A a a l y t e F o u n d / j . g / g ) = /a n a ly t e f o u n d ftia f t r i U x F V f o i T .) t n i t x E V / m T . I t I f y v i p g Av(mL)'xtanglewt (g)x1pg WhereFV=finalvolume,DF=dilntionfactor,EV=extractionvdhmc,AV =aliquot volume 3-Recovtsy(%) =(upM-fmmd w .analyteinetri(ug/mLHxIcvywtinner, amountadded(ngfaLJxljig 3. Change the computer listed to Hell LUtxaScanP1110 rationale 1-3. farihowtheamplesarequantitatedandreponedandtheimnumaitsusedmanpitp data. IMPACT ON THB STUDY 1-3. No negative impact on the study. StudyDTilrweJcitiorrSCiiuginuafltIuIrSe. * i16' DA-T%f\ i 'S ttUO 0 ^ jo ' i i/h `/l /ai Date4 4 L l o\ D a te CentreQAUReview. ^ m )*?/ /6J ' ` February IX 1898/1 otX&t#*LW*s LCT$ ffrertt Lr i RECEIVE! TIME S E P . 6 . 4:05P M Centre Analytical Laboratories, Inc. PRINT TIME SEP. S. 4:08PM Page 102 of 104 Centre Study No.: 023-042 Centre Analytical Laboratories, Inc. 3048 Research Drive, State College, PA 16801. Phone: (814)231-8032, Facsimile: (814)231-1253 CentreStudyNumber: 023-042 PROTOCOLAMENDMENT AmendmentNumber: 4 DateofOccurrence: 08/24/01 Page 1of CentreProtocol Number 00P-023-042 DESCRIPTIONOFAMENDEDSECTION 1. 3: SPONSOR AMENDEDTO 1. Change the study director from Rochelle Robideau at 3M Environmental Technology and Safety Services to Sean Gallagher at Wildlife International, LTD, 8598 Commerce Drive, Easton, M D 21601, phone: 410-822-8600, fax: 410-822-0632. 2. Change Rochelle Robideau to the Sponsor Representative. RATIONALE 1-2. ByrequestofRochelleRobideau(faxdated08/24/01includedincorrespondencesectionof therawdataassociatedwiththisstudy) ' IMPACTONTHESTUDY 1-2. Nonegativeimpactonthestudy. OTngFacilityManagement Signature Date IncomingStudyDirectorSignature m)oi Date CentreQAUReview &P)|6 stl3lol __________________________February12, 1998/1 Centre Analytical Laboratories, Inc. Page 103 of 104 Centre Study No.: 023-042 _09/06/Ol__16:08 Fill 410 822 0602 WILDLIFE INTERNATIONAL SEP. 6.2001 3:29PM EXVGEN RESEARCH @002 N0.555 P.2 Centre Analytical Laboratories. Inc. 3048 Research P rix, Stato r n l la t . PA 1 O 0 L P h o n e T O n i-SH M . F a g iir ilE (214031-1353 PROTOCOL DEVIATION D eviation N um ber: 1 DataofOccurrence: (1) 11/19/00C) 11/19/00,(3)11/23/00(4) 11/29-30/00(5)3/10/01 CatreStudyNumber: 023-042 CentreProtocolNumber 00P-Q23-042 DESCRIPTION OF DEVIATION 1. 8 Sample Analysis (a) Page 9: Accepted recoveries of 79% and 77% for Centre saamls 0003804 Spk A sad! Its duplicate rnj^-r!rm in Set 111700AD. 2. 8CalibrarionProcedures(b)Pages8-9: Includedastandardinliecalibrationcurvethatfell outside305b ofitscalculatedconcentrationinSet1H700AD(C091800-5). 3. 8 Calibration Procedures (b) Pages 8*9: Excluded a standard firm the calibration curve that fell vdrhiiii 3056 of its calculated eancennsrioain Set 11210QB (C091800-3). 4. 8C aB brarinn Procedures(b ) Pages8-9: Ihelndfldastandardinthecalibrationcurvethatfell outside3056 ofitspalm iercancootrarianinSet11270QD(C091800-6), 5. 5 8 Sample Analysis (b) Page 9: Accepted recoveries of 79% and 77% for Centre Ezmjlo 0003803 Spk A and if HupHeatr, Tnjrrtirm in Set 03070IA. 5 &Gib iblei 1- i Protocoldeviationiss RecordedBy/Dan ACTIONS TAKEN -, . i M nendm m t l e u a i SOP rev id en . a te _ Tkd/y *lh>i IMPACT ONTHE STUDY IfS. Nonejjatlve^^nparronthestndybecametheoverallaverageofthespikerecoveriesforthestudy 2,4f.eNllawuitehj'izntxyfrvwe*impactontrhaensgteudoyfSbQec%a^u1s2e0t%bs. curvewasstillwithinacceptableHoear requirements 3. Nonegativeinqiactonthestudybecausethetotalmimbcrofstandardscscdmledfiomthecurve was less thtm 20% of ths number of standards iqjcctcd. jt&A.zS - ' -PnnappaalKJwvesstriggBatiaorrSSigrraamnirrce jiijfL Date Lg-^v- Py StudyDirectorSignature / Date Hk Centre. Ji^/l ftU>*kfo1 Date CAL QAReview. j s 4_ W 0 T&5 *>M t-ZsSu0 <?!(#{{!! S C CXJSJSAL fWCrK o t M l D jty) (,|tn Wgtary12. man U6f de RECEIVED Ti c e SE P . 6 . 4:06P M Centre Analytical Laboratories, Inc. PRINT TIME SEP. 6. 4:08PM Page 104 of 104