Document O3YaKrQnMdB9L3V9718jxEYnv

AR226-1744 STUDY TITLE Extraction of Potassium Perfluorooctanesulfonate from Red Blood Cells and Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry DATA REQUIREMENTS Analytical Method Requirements STUDY DIRECTOR Sean Gallagher ANALYTICAL REPORT DATE June 2, 2003 PERFORMING LABORATORY / TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 PROJECT Exygen Protocol Number: 01P-023-066 Exygen Study Number: 023-066 Total Pages: 98 Exygen Study No.: 023-066 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number 023-066, entitled "Extraction of Potassium Perfluorooctanesulfonate from Red Blood Cells and Serum for Analysis Using HPLCElectrospray/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 Exygen Research. Wildlife International, Ltd. fr/2M Date Date Date Exygen Research Page 2 of 98 Exygen Study No.: 023-066 QUALITY ASSURANCE STATEMENT Exygen's Quality Assurance Unit reviewed Exygen Study Number 023-066, entitled, "Extraction of Potassium Perfluorooctanesulfonate from Red Blood Cells for Analysis Using HPLC-Electrospray/Mass Spectrometry". All reviewed phases were inspected for conduct according to Exygen's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Study Director and to management. Phase Date Inspected Date Reported to Date Reported to Exygen Study Director and Manaeement Sponsor Manaeement 1. Protocol Review 5/22/01 6/4/01 6/15/01 2. Extraction, Fortification 5/22/01 6/4/01 6/15/01 3. Raw Data Review 6/4,6,8/01 7/13/01 9/7/01 4. Raw Data Review 8/7-9/01 8/21/01 9/7/01 5. Draft Report Review 11/15,16,19/01 12/05/01 02/21/02 6. Final Report Review 04/11,21/03 06/02/03 06/02/03 Date Exygen Research Page 3 of 98 Exygen Study No.: 023-066 CERTIFICATION OF AUTHENTICITY This report, for Exygen Study Number 023-066, is a true and complete representation of the raw data for the study. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Principal Investigator, Exygen: Date Exygen Facility Management: i-jy/Oe'-oZ Date Study Director, Wildlife International: Sean Gallagher Wildlife International, Ltd. ' Date Exygen Research Page 4 of 98 Exygen Study No.: 023-066 STUDY IDENTIFICATION Extraction of Potassium Perfluorooctanesulfonate from Red Blood Cells and Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry EXYGEN PROTOCOL NUMBER: 01P-023-066 EXYGEN STUDY NUMBER: 023-066 TYPE OF STUDY: Analytical SAMPLE MATRIX: Mallard and Quail Red Blood Cells and Serum 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: John Newsted Entrix, Inc. 4295 Okemos Road, Suite 101 Okemos, MI 48864 TESTING FACILITY/PRINCIPAL: Emily R. Decker INVESTIGATOR Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: Experimental Start Date: Experimental Termination Date: Analytical Report Date: 04/23/01 05/18/01 07/28/01 06/02/03 Exygen Research Page 5 of 98 Exygen Study No.: 023-066 PROJECT PERSONNEL The Study Director for this project was Sean Gallagher at Wildlife International, Ltd. The following personnel from Centre Analytical Laboratories, Inc., were associated with various phases of the study: Name Emily Decker Dave Bell Karen Risha Ling Ling Liu Marcia Swartzwelder Xiaoming Zhu Mitra Arjmand Lawrence Ord Ed Carnes Shawn Robb Title Scientist Scientist Scientist Technician Technician Technician Technician Sample Custodian Sample Custodian Sample Custodian Exygen Research Page 6 of 98 Exygen Study No.: 023-066 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 6.6 Quantitation and Example Calculation.................................................................... 16 7.0 EXPERIMENTAL DESIGN...................................................................................... 18 8.0 RESULTS................................................................................................................... 18 9.0 CONCLUSIONS........................................................................................................ 19 10.0 RETENTION OF DATA AND SAMPLES............................................................. 19 Exygen Research Page 7 of 98 Exygen Study No.: 023-066 Table I. LIST OF TABLES Page Summary of PFOS in Reagent Blanks........................................................... 21 Table EL Summary of PFOS in Mallard Red Blood Cell Blanks.................................. 22 Table HI. Summary of PFOS in Mallard Serum Blanks................................................ 22 Table IV. Summary of PFOS in Quail Red Blood Cell Blanks..................................... 23 Table V. Summary of PFOS in Quail Serum Blanks.................................................... 23 Table VI. Summary of PFOS Recoveries in Mallard Red Blood C ells.........................24 Table VII. Summary of PFOS Recoveries in Mallard Serum......................................... 25 Table VIH. Summary of PFOS Recoveries in Quail Red Blood Cells............................. 26 Table IX. Summary of PFOS Recoveries in Quail Serum............................................. 27 Table X. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 5 ........................................................................................................... 28 Table XI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 5 ........................................................................................................... 29 Table XII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for Week 5 ........................................................................................................... 30 Table XIII. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 10......................................................................................................... 30 Table XIV. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 10......................................................................................................... 31 Table XV. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 15......................................................................................................... 32 Table XVI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 15......................................................................................................... 33 Table XVII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for TERM............................................................................................................. 33 Table XVIII. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 5 ... 34 Table XIX. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 5.... 35 Table XX. Summary of PFOS in Mallard Serum Samples at 50 ppm a.i. for Week 5.... 35 Table XXI. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 10.... 36 Exygen Research Page 8 of 98 Exygen Study No.: 023-066 Table XXII. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 10................................................................................................................... 37 Table XXITT. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 15...................................................................................................... 38 Table XXIV. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 15..................................................................................................... 39 Table XXV. Summary of PFOS in Mallard Serum Samples at 50 ppm a.i. for TERM......................................................................................................... 39 Table XXVI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 5 .................................................................................................. 40 Table XXVII. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 5 ........................................................................................................ 41 Table XXVIII. Summary of PFOS in Quail Red Blood Cell Samples at 50 ppm a.i. for Week 5....................................................................................................... 42 Table XXIX. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 10...................................................................................................... 42 Table XXX. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 10......................................................................................................43 Table XXXI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 15...................................................................................................... 44 Table XXXII. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 15...............................................................................................45 Table XXXm. Summary of PFOS in Quail Red Blood Cell Samples at 50 ppm a.i. for TERM.................................................................................................. 45 Table XXXIV. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 5.... 46 Table XXXV. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 5 ... 47 Table XXXVI. Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for Week 5 ..................................................................................................... 47 Table XXXVII. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 10...................................................................................................48 Table XXXVIII. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 10.................................................................................................. 49 Table XXXIX. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 15.. 50 Table XL. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 15.....51 Table XLI. Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for TERM.......51 Exygen Research Page 9 of 98 Exygen Study No.: 023-066 LIST OF FIGURES Page Figure 1. Typical Calibration Curve for PFOS...............................................................53 Figure 2. Chromatogram Representing a 0.1 ng/mL standard for PFOS.......................54 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PFOS.......................55 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Exygen ID Reagent Blank A, Set: 051801A ).............................................. 56 Figure 5. Chromatogram Representing Control Quail Red Blood Cells for PFOS (Exygen ID: 0107376 Blank A, Set: 051801A)............................................ 57 Figure 6. Chromatogram Representing Control Quail Serum for PFOS, (Exygen ID: 0107375 Blank A, Set: 072301C)............................................ 58 Figure 7. Chromatogram Representing Control Quail Red Blood Cells fortified with 10 ng/mL of PFOS (Exygen ID: 0107376 Spk A, Set: 051801DR).....59 Figure 8. Chromatogram Representing Control Quail Serum fortified with 10 ng/mL of PFOS (Centre ID: 0107375 Spk A, Set: 072301D).........60 Figure 9. Chromatogram Representing Quail Red Blood Cell Sample (Exygen ID: 0107084, Sponsor ID: 454-108-401 Wk5, Set: 051801DR)......................... 61 Figure 10. Chromatogram Representing Quail Serum Sample (Exygen ID: 0107207, Sponsor ID: 454-108-407-Wkl5, Set: 072301DD)...................................... 62 Exygen Research Page 10 of 98 Exygen Study No.: 023-066 LIST OF APPENDICES Page Appendix A Study Protocol 01P-023-066 (Exygen Study No. 023-066) and Amendments and Deviations.................................................................... 63 Exygen Research Page 11 of 98 Exygen Study No.: 023-066 1.0 SUMMARY Exygen Research (Exygen) extracted mallard and quail red blood cell samples and serum samples for the determination of perfluorooctanesulfonate (PFOS) according to protocol 01P-023-066 (Appendix A). The limit of quantitation for mallard red blood cells and serum was 10 ng/mL. The limit of quantitation for quail red blood cells and serum was 10 ng/mL. The LOQ for each matrix was determined in a method verification study performed at Centre (Centre Study: 023-065). PFOS in the mallard red blood cell samples ranged from non-quanitifiable levels to 418 pg/mL. PFOS in the mallard serum samples ranged from non-detected levels to 685 pg/mL. PFOS in the quail red blood cell samples ranged from non-quantifiable levels to 361 pg/mL. PFOS in the quail serum samples ranged from non-quantifiable levels to 514 pg/mL. The average percent recoveries standard deviation for PFOS in mallard red blood cell samples and serum samples were 90% 9% and 99% 13%, respectively. The average percent recovery standard deviation for PFOS in quail red blood cell samples and serum samples were 100% 10% and 102% 20%, respectively. 2.0 OBJECTIVE The objective of this study was to determine levels of perfluorooctanesulfonate (PFOS) in specimens of mallard and quail red blood cell samples and serum samples using the analytical method described in protocol 01P-023-066. 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFOS in mallard and quail red blood cell samples and serum samples, using the analytical method entitled, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry". The study was initiated on April 23, 2001, when the study director signed Exygen protocol number 01P-023-066. The experimental start date was May 18, 2001, and the experimental termination date was July 28, 2001. Exygen Research Page 12 of 98 Exygen Study No.: 023-066 4.0 TEST SYSTEM The control mallard and quail red blood cells and serum used for the matrix blanks and matrix fortifications were received chilled on blue ice on May 17, 2001 from Wildlife International Ltd., Easton, MD. They were assigned the following Exygen ID numbers: Matrix Quail Serum Quail Blood Mallard Serum Mallard Blood ExygenID 0107375 0107376 0107377 0107378 Seventy-seven quail red blood cell samples and seventy-six quail serum samples were received chilled on blue ice at Exygen on May 17, 2001 and logged in by Exygen personnel and placed in frozen storage. Seventy-three mallard red blood cell samples and seventy-two mallard serum samples were received chilled on blue ice at Exygen on May 17, 2001 and logged in by Exygen personnel and placed in frozen storage. 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 Exygen 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 Exygen 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 Exygen Control No. TCR No. Purity (%) Expiration Date 00-023-042 TCR 00017-46 97.9 08/31/01 Exygen Research Page 13 of 98 Exygen Study No.: 023-066 The molecular structure of PFOS is given below. PFOS Chemical Name Molecular weight Perfluorooctanesulfonate 499 (C8F17S 03~) o II C8Fi7S -- O' o Note: The neutral molecule and standard form from which PFOS (anion) is derived, is potassium perfluorooctanesulfonate [CgFi7S0 3K], molecular weight 538. 6.0 DESCRIPTION OF ANALYTICAL METHOD Analytical method entitled "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" was used for this study. 6.1 Extraction Procedure A 100 pL aliquot of the red blood cells and serum was used for the extraction procedure. After fortification of appropriate samples, the samples were vortexed for ~ 15 seconds. An aliquot of 0.5 milliliter of 0.5 M tetrabutylammonium hydrogen sulfate was added to the samples. One milliliter of 0.25 M sodium carbonate/sodium bicarbonate was added to the samples. Five milliliters of MTBE were added to the samples. Each sample was placed on wrist-action shaker for ~ 20 min. and then centrifuged for ~ 15 min. Four milliliters of the organic layer were 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 March 14, 2001 as specified in Exygen protocol 01P-023-066. 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 Exygen Research Page 14 of 98 Exygen Study No.: 023-066 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) Diluted to (mL) Final Cone. (pg/mL) 0.1 5 100 0.005 0.1 2 100 0.002 0.1 1 100 0.001 0.01 5 100 0.0005 0.01 2 100 0.0002 0.01 1 100 0.0001 1of PFOS 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 PI 110 Software: HPLC: PE SciexAnalyst version 1.1 Windows NT, version 4 Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser Exygen Research Page 15 of 98 Exygen Study No.: 023-066 HP Autosampler HP Column Oven HPLC ColummGenesis 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. Time % A %B 0 60 40 1.0 0 100 7.0 0 100 7.5 60 40 11.0 60 40 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 Exygen Research Page 16 of 98 Exygen Study No.: 023-066 linear regression) by Analyst software using six concentrations of standards. The concentration 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). 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) = 1 ng avg. amt in controls = 0 (Not quantifiable) final volume - 1 mL extraction volume = 5 mL aliquot volume = 4 mL sample weight (volume) - 0.1 mL Exygen Research Page 17 of 98 Exygen Study No.: 023-066 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 pg (4m L x0.1m L ) 1000 ng = 0.00976 pg/mL From equation 3: % Recovery = ((0.00976 ug/mL x - 0 ug/mL) x 1000) x 100 10 ng/mL = 98% 7.0 EXPERIMENTAL DESIGN Each set of samples (red blood cells 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 amount of PFOS found in the reagent blanks are listed in Table I. The amount of PFOS in the mallard red blood cells and serum blanks are given in Tables II & III. The amount of PFOS in the quail red blood cells and serum are given in Tables IV & V. Peaks were detected in the control matrices corresponding to the analyte retention time. The amounts detected were only significant enough to alter several fortification recoveries and as a result are reported. The rest of the samples were less than the lowest calibration standard (0.0001 pg/mL) and not quantifiable. Individual recoveries for PFOS in the mallard red blood cell and serum samples are detailed in Tables VI & VII. The average percent recoveries standard deviations for PFOS in mallard red blood cell and serum samples were 90% 9% and 99% 13%, respectively. Individual recoveries for PFOS in the quail red blood cell and serum samples are detailed in Tables VIII & IX. The average percent recoveries standard deviations for PFOS in mallard red blood cell and serum samples were 100% 10% and 102% 20%, respectively. PFOS in the mallard red blood cell samples ranged from non-detected levels to 418 pg/mL. Individual results are listed in Tables X-XVII. PFOS in the mallard serum Exygen Research Page 18 of 98 Exygen Study No.: 023-066 samples ranged from non-detected levels to 685 pg/mL. Individual results are listed in Tables XVIII-XXV. PFOS in the quail red blood cell samples ranged from non-detected levels to 361 pg/mL. Individual results are listed in Tables XXVI-XXXIII. PFOS in the quail serum samples ranged from non-detected levels to 514 pg/mL. Individual results are listed in Tables XXXIV-XLI 9.0 CONCLUSIONS The mallard and quail red blood cell and serum samples were successfully extracted and analyzed according to protocol 01P-023-066. 10.0 RETENTION OF DATA AND SAMPLES When the final report is complete, all original paper data generated by Exygen 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 Exygen archives for the period of time specified in 40 CFR Part 792. Retained samples of reference substances are archived by the sponsor. Exygen Research Page 19 of 98 Exygen Study No.: 023-066 TABLES Exygen Research Page 20 of 98 Exygen Study No.: 023-066 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 na na na na na na na na Centre ID Reagent Blank A Reagent Blank A* Reagent Blank A2 Reagent Blank A2* 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 A3 Reagent Blank A3* Reagent Blank A Reagent Blank A* Reagent Blank A2 Reagent Blank A2* Reagent Blank A Reagent Blank A* Reagent Blank A2 Reagent Blank A2* Reagent Blank A3 Reagent Blank A3* Set Number 051801A 051801A 051801B 051801B 052101A 052101A 052101B 052101B 052201A 052201A 05220IB 05220IB 052201C 052201C 071901A 071901A 072001A 072001A 07200IB 07200IB 072301A 072301A 072301B 072301B 072301C 072301C Extraction Date 5/18/01 5/18/01 5/18/01 5/18/01 5/21/01 5/21/01 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 7/19/01 7/19/01 7/20/01 7/20/01 7/20/01 7/20/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 Analysis Analyte Date Found (pg/mL) 5/18-19/01 NQ 5/18-19/01 NQ 5/19/01 5/19/01 5/22/01 5/22/01 5/22/01 NQ NQ NQ NQ NQ 5/22/01 5/23/01 NQ NQ 5/23/01 NQ 5/23/01 NQ 5/23/01 NQ 5/23/01 NQ 5/23/01 7/19-20/01 7/19-20/01 7/21/01 7/21/01 7/21-22/01 NQ ND ND ND ND NQ 7/21-22/01 ND 7/25/01 NQ 7/25/01 NQ 7/24/01 NQ 7/24/01 7/24-25/01 7/24-25/01 AVERAGE: STANDARD DEVIATION: 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)) ND = Not Detected (A peak was not detected at the corresponding analyte retention time) 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. LOQ = 0.01 pg/mL for red blood cells and serum Exygen Research Page 21 of 98 Exygen Study No.: 023-066 Table II. Summary of PFOS in Mallard Red Blood Cell Blanks Sponsor ID control control control control control control control control Centre ID 0107378 Blank A 0107378 Blank A* 0107378 Blank A 0107378 Blank A* 0107378 Blank A2 0107378 Blank A2* 0107378 Blank B 0107378 Blank B* Set Number 052101B 052101B 052201A 052201A 052201B 052201B 052201C 052201C Extraction Analysis Analyte Date Date Found (pg/mL) 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 NQ NQ NQ NQ 0.00224 0.00184 5/22/01 5/23/01 NQ 5/22/01 5/23/01 NQ AVERAGE: STANDARD DEVIATION: 0.000548 0.000927 Table III. Summary of PFOS in Mallard Serum Blanks Sponsor ID control control control control control control Centre ID 0107377 Blank A3 0107377 Blank A3* 0107377 Blank A 0107377 Blank A* 0107377 Blank A2 0107377 Blank A2* Set Number 071901A 071901A 072001A 072001A 07200IB 07200IB Extraction Analysis Analyte Date Date Found (pg/mL) 7/19/01 7/19-20/01 7/19/01 7/19-20/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 AVERAGE: STANDARD DEVIATION: 0.00325 0.00331 0.00277 0.00312 0.00188 0.00214 0.00275 0.000605 * 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/mL for red blood cells and 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. Exygen Research Page 22 of 98 Exygen Study No.: 023-066 Table IV. Summary of PFOS in Quail Red Blood Cell Blanks Sponsor ID control control control control control control control control Centre ID 0107376 Blank A 0107376 Blank A* 0107376 Blank A2 0107376 Blank A2* 0107376 Blank A 0107376 Blank A* 0107376 Blank A 0107376 Blank A* Set Number 051801A 051801A 051801B 051801B 052101A 052101A 052201C 052201C Extraction Analysis Analyte Date Date Found (pg/mL) 5/18/01 5/18-19/01 NQ 5/18/01 5/18-19/01 NQ 5/18/01 5/19/01 NQ 5/18/01 5/19/01 NQ 5/21/01 5/21/01 5/22/01 5/22/01 NQ NQ 5/22/01 5/23/01 NQ 5/22/01 5/23/01 NQ AVERAGE: STANDARD DEVIATION: NQ NQ Table V. Summary of PFOS in Quail Serum Blanks Sponsor ID control control control control control control Centre ID 0107375 Blank A 0107375 Blank A* 0107375 Blank A2 0107375 Blank A2* 0107375 Blank A3 0107375 Blank A3* Set Extraction Analysis Analyte Number Date Date Found (pg/mL) 072301 AD 072301AD 072301B 072301B 7/23/01 7/23/01 7/23/01 7/23/01 7/26/01 7/26/01 7/24/01 7/24/01 0.00309 0.00272 NQ NQ 072301C 072301C 7/23/01 7/24-25/01 7/23/01 7/24-25/01 AVERAGE: STANDARD DEVIATION: NQ NQ 0.00100 0.00148 * 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/mL for red blood cells and 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. Exygen Research Page 23 of 98 Exygen Study No.: 023-066 Table VI. Summary of PFOS Recoveries in Mallard Red Blood Cells Sponsor ID control control control control control control control control control control control control control control Centre ID 0107378 Spk A 0107378 Spk A* 0107378 Spk B 0107378 Spk B* 0107378 Spk A 0107378 Spk A* 0107378 Spk B 0107378 Spk B* 0107378 Spk A2 0107378 Spk A2* 0107378 Spk B2 0107378 Spk B2* 0107378 Spk B 0107378 Spk B* Set Number 052101B 052101B 052101B 052101B 052201A 052201A 052201A 052201A 05220IB 05220IB 05220IB 05220IB 052201C 052201C Extraction Analysis Amt Date Date Added (ng/mL) 5/21/01 5/21/01 5/22/01 5/22/01 10 10 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 50000 50000 10 10 50000 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 50000 10 10 50000 50000 5/22/01 5/23/01 50000 5/22/01 5/23/01 50000 AVERAGE STANDARD DEVIATION RELATIVE STANDARD DEVIATION % Recovery 85 88 91 95 84 82 84 82 80 76 101 105 102 101 90 9 11 * Duplicate injection LOQ = 0.01 |ug/mL for red blood cells and serum Exygen Research Page 24 of 98 Exygen Study No.: 023-066 Table VII. Summary of PFOS Recoveries in Mallard Serum Sponsor ID control control control control control control control control control control control control Centre ID 0107377 Spk A3 0107377 Spk A3* 0107377 SpkB3 0107377 Spk B3* 0107377 Spk A 0107377 Spk A* 0107377 Spk B 0107377 Spk B* 0107377 Spk A2 0107377 Spk A2* 0107377 Spk B2 0107377 Spk B2* Set Number 071901A 071901A 071901A 071901A 072001A 072001A 072001A 072001A 07200IB 07200IB 07200IB 07200IB Extraction Analysis Amt Date Date Added (ng/mL) 7/19/01 7/19/01 7/19-20/01 7/19-20/01 10 10 7/19/01 7/19/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/19-20/01 7/19-20/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 500000 500000 10 10 500000 500000 10 10 500000 500000 AVERAGE STANDARD DEVIATION RELATIVE STANDARD DEVIATION % Recovery 90 89 111 112 76 82 117 97 107 110 92 _99___ 99 13 13 * Duplicate injection LOQ = 0.01 [ig/mL for red blood cells and serum Exygen Research Page 25 of 98 Exygen Study No.: 023-066 Table VIII. Summary of PFOS Recoveries in Quail Red Blood Cells Sponsor ID control control control control control control control control control control control control control control Centre Set ID Number 0107376 Spk A 051801DR 0107376 Spk A* 051801DR 0107376 Spk B 051801DR 0107376 Spk B* 051801DR 0107376 Spk A2 051801DR 0107376 Spk A2* 051801DR 0107376 Spk B2 051801DR 0107376 Spk B2* 051801DR 0107376 Spk A 0107376 Spk A* 0107376 Spk B 0107376 Spk B* 052101A 052101A 052101A 052101A 0107376 Spk A 052201C 0107376 Spk A* 052201C Extraction Analysis Amt Date Date Added (ng/mL) 5/18/01 5/21-22/01 10 5/18/01 5/21-22/01 10 5/18/01 5/21-22/01 50 5/18/01 5/21-22/01 50 5/18/01 5/21-22/01 10 5/18/01 5/21-22/01 10 5/18/01 5/21-22/01 50 5/18/01 5/21-22/01 50 5/21/01 5/22/01 10 5/21/01 5/22/01 10 5/21/01 5/22/01 50000 5/21/01 5/22/01 50000 5/22/01 5/23/01 10 5/22/01 5/23/01 10 AVERAGE STANDARD DEVIATION RELATIVE STANDARD DEVIATION % Recovery 102 99 111 111 113 110 109 104 87 89 91 91 90 94 100 10 10 * Duplicate injection LOQ = 0.01 |ig/mL for red blood cells and serum Exygen Research Page 26 of 98 Exygen Study No.: 023-066 Table IX. Summary of PFOS Recoveries in Quail Serum Sponsor ID control control control control control control control control control control control control Centre Set ID Number 0107375 Spk A 072301AR 0107375 Spk A* 072301AR 0107375 Spk B 072301AR 0107375 Spk B* 072301AR 0107375 Spk A2 072301D 0107375 Spk A2* 072301D 0107375 Spk B2 072301B 0107375 Spk B2* 072301B 0107375 Spk A3 07230ID 0107375 Spk A3* 072301D 0107375 Spk B3 072301C 0107375 Spk B3* 072301C Extraction Analysis Amt Date Date Added (ng/rnL) 7/23/01 7/25/01 10 7/23/01 7/25/01 10 7/23/01 7/25/01 500000 7/23/01 7/25/01 500000 7/23/01 7/26-27/01 10 7/23/01 7/26-27/01 10 7/23/01 7/24/01 500000 7/23/01 7/24/01 500000 7/23/01 7/26-27/01 10 7/23/01 7/26-27/01 10 7/23/01 7/24-25/01 500000 7/23/01 7/24-25/01 500000 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: % Recovery 64 62 104 107 112 113 124 126 106 105 100 101 102 20 20 * Duplicate injection LOQ = 0.01 (ig/mL for red blood cells and serum Exygen Research Page 27 of 98 Exygen Study No.: 023-066 Table X. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-109-3249-Wk5 454-109-3249-Wk5* 454-109-3251-Wk5 454-109-3251-Wk5* 454-109-3252-Wk5 454-109-3252-Wk5* 454-109-3253-Wk5 454-109-3253-Wk5 * 454-109-3254-Wk5 454-109-3254-Wk5* 454-109-3255-Wk5 454-109-3255-Wk5* 454-109-3256-Wk5 454-109-3256-Wk5* Centre ID 0107230 0107230 0107231 0107231 0107232 0107232 0107233 0107233 0107234 0107234 0107235 0107235 0107236 0107236 Set Number 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B Extraction Analysis Date Date 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) NQ NQ 0.00197 0.00167 NQ NQ NQ NQ NQ NQ 0.00143 NQ NQ NQ 0.000401 0.000706 * 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/mL for red blood cells and 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. Exygen Research Page 28 of 98 Exygen Study No.: 023-066 Table XI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-109-3289-Wk5 454-109-3289-Wk5* 454-109-3290-Wk5 454-109-3290-Wk5* 454-109-3291-Wk5 454-109-3291-Wk5* 454-109-3292-Wk5 454-109-3292-Wk5* 454-109-3293-Wk5 454-109-3293-Wk5* 454-109-3294-Wk5 454-109-3294-Wk5* 454-109-3295-Wk5 454-109-3295-Wk5 * 454-109-3296-Wk5 454-109-3296-Wk5* Centre ID 0107237 0107237 0107238 0107238 0107239 0107239 0107240 0107240 0107241 0107241 0107242 0107242 0107243 0107243 0107244 0107244 Set Number 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD Extraction Analysis Analyte Date Date Found (pg/mL) 5/21/01 5/23/01 24.4 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 24.7 11.2 10.0 11.8 12.3 15.1 5/21/01 5/23/01 13.1 5/21/01 5/23/01 30.1 5/21/01 5/23/01 30.8 5/21/01 5/21/01 5/21/01 5/23/01 5/23/01 5/23/01 4.88 3.95 18.9 5/21/01 5/23/01 18.8 5/21/01 5/23/01 26.4 5/21/01 5/23/01 AVERAGE: 28.5 17.8 STANDARD DEVIATION: 8.80 * Duplicate injection LOQ = 0.01 |ag/mL for red blood cells and serum Exygen Research Page 29 of 98 Exygen Study No.: 023-066 Table XII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for Week 5 Sponsor ID 454-109-3 329-Wk5 454-109-3329-Wk5* 454-109-3330-Wk5 454-109-3330-Wk5* Centre ID 0107245 0107245 0107246 0107246 Set Number 052201CD 052201CD 052201CD 052201CD Extraction Analysis Date Date 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 58.9 62.2 112 123 89 33 Table XIII. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-109-3249-Wkl0 454-109-3249-Wkl0* 454-109-3250-WklO 454-109-3250-WklO* 454-109-3251-Wkl0 454-109-3251-Wkl0* 454-109-3252-WklO 454-109-3252-WklO* 454-109-3253-Wkl0 454-109-3253-Wkl0* 454-109-3254-WklO 454-109-3254-Wkl0* 454-109-3255-Wkl0 454-109-3255-Wkl0* 454-109-3256-WklO 454-109-3256-WklO* Centre ID 0107250 0107250 0107251 0107251 0107252 0107252 0107253 0107253 0107254 0107254 0107255 0107255 0107256 0107256 0107257 0107257 Set Number 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A Extraction Analysis Analyte Date Date Found (pg/mL) 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 NQ NQ NQ NQ NQ NQ NQ NQ 5/22/01 5/23/01 NQ 5/22/01 5/23/01 5/22/01 5/23/01 5/22/01 5/23/01 5/22/01 5/23/01 5/22/01 5/23/01 5/22/01 5/23/01 5/22/01 5/23/01 AVERAGE: STANDARD DEVIATION: NQ NQ NQ NQ NQ NQ 0.00245 0.000200 0.000600 * 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/mL for red blood cells and 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. Exygen Research Page 30 of 98 Exygen Study No.: 023-066 Table XIV. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-109-3289-Wkl0 454-109-3289-Wkl0* 454-109-3290-WklO 454-109-3290-WklO* 454-109-3291-Wkl0 454-109-3291-Wkl0* 454-109-3292-W kl 0 454-109-3292-W kl 0* 454-109-3293-Wkl0 454-109-3293-Wkl0* 454-109-3294-WklO 454-109-3294-Wkl0* 454-109-3295-Wkl0 454-109-3295-Wkl0* 454-109-3296-Wkl0 454-109-3296-WklO* Centre ID 0107258 0107258 0107259 0107259 0107260 0107260 0107261 0107261 0107262 0107262 0107263 0107263 0107264 0107264 0107265 0107265 * Duplicate injection Set Number 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD 052201 AD Extraction Analysis Date Date 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 19.3 19.6 20.4 20.2 14.1 14.9 67.3 66.0 33.5 34.2 60.8 58.9 16.1 16.0 11.6 11.7 30.3 20.7 LOQ = 0.01 |ig/mL for red blood cells and serum Exygen Research Page 31 of 98 Exygen Study No.: 023-066 Table XV. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 15 Sponsor ID 454-109-3249-Wkl5 454-109-3249-W kl5* 454-109-3250-W kl5 454-109-3250-Wkl5* 454-109-3251-Wkl5 454-109-3251 -Wkl 5* 454- 109-3252-Wkl5 454-109-3252-Wkl5* 454-109-3253-Wkl5 454-109-3253-Wkl5* 454-109-3254-W kl5 454-109-3254-Wkl5* 454-109-3255-Wkl5 454-109-3255-Wkl5* 454-109-3256-W kl5 454-109-3256-Wkl5* 454-109-3236-Wkl5 454-109-3236-W kl5* Centre ID 0107266 0107266 0107267 0107267 0107268 0107268 0107269 0107269 0107270 0107270 0107271 0107271 0107272 0107272 0107273 0107273 0107274 0107274 Set Number 052201B 05220IB 052201B 05220IB 05220IB 05220IB 05220IB 05220IB 052201B 052201B 05220IB 05220IB 05220IB 05220IB 052201B 05220IB 05220IB 052201B Extraction Analysis Analyte Date Date Found (pg/mL) 5/22/01 5/23/01 NQ 5/22/01 5/23/01 NQ 5/22/01 5/23/01 NQ 5/22/01 5/23/01 NQ 5/22/01 5/23/01 0.00179 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 0.00188 NQ NQ NQ NQ ND 5/22/01 5/23/01 ND 5/22/01 5/23/01 NQ 5/22/01 5/23/01 NQ 5/22/01 5/23/01 NQ 5/22/01 5/23/01 5/22/01 5/23/01 5/22/01 5/23/01 AVERAGE: STANDARD DEVIATION: NQ NQ NQ 0.000243 0.000613 * Duplicate injection ND = No peak detected 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/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 32 of 98 Exygen Study No.: 023-066 Table XVI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-109-3289-W kl5 454-109-3289-Wkl5* 454-109-3290-W kl5 454-109-3290-Wkl5* 454-109-3291-Wkl5 454-109-3291 -W kl 5* 454-109-3292-Wk 15 454-109-3292-Wkl 5* 454-109-3293-Wkl5 454-109-3293-Wkl5* 454-109-3294-W kl5 454-109-3294-Wkl5* 454-109-3295-Wkl5 454-109-3295-Wkl5* 454-109-3296-W kl5 454-109-3296-W kl5* Centre ID 0107275 0107275 0107276 0107276 0107277 0107277 0107278 0107278 0107279 0107279 0107280 0107280 0107281 0107281 0107282 0107282 Set Number 052201BD 052201BD 052201BD 052201BD 052201BD 052201BD 052201BD 052201BD 05220 IB DR 05220 IB DR 052201BDR 052201BDR 052201BDR 052201BDR 052201BD 052201BD Extraction Analysis Analyte Date Date Found (pg/mL) 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/24/01 5/24/01 5/24/01 5/24/01 5/24/01 5/24/01 5/23-24/01 5/23-24/01 AVERAGE: 53.5 48.2 3.98 3.44 60.9 61.4 9.65 8.79 113 116 1.54 1.61 8.03 6.56 0.623 0.505 31.1 STANDARD DEVIATION: 39.8 Table XVII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for TERM Sponsor ID 454-109-3297-TERM 454-109-3297-TERM* 454-109-3298-TERM 454-109-3298-TERM* Centre ID 0107283 0107283 0107284 0107284 * Duplicate injection Set Number 052201 CD 052201CD 052201BDR 052201BDR Extraction Date Analysis Date Analyte Found (jig/mL) 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: 275 272 406 418 343 80 LOQ = 0.01 |ug/mL for red blood cells and serum Exygen Research Page 33 of 98 Exygen Study No.: 023-066 Table XVIII. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-109-3249-Wk5 454-109-3249-Wk5* 454-109-3251-Wk5 454-109-3251-Wk5* 454-109-3252-Wk5 454-109-3252-Wk5* 454-109-3253-Wk5 454-109-3253-Wk5* 454-109-3254-Wk5 454-109-3254-Wk5* 454-109-3255-Wk5 454-109-3255-Wk5* 454-109-3256-Wk5 454-109-3256-Wk5 * Centre ID 0107303 0107303 0107304 0107304 0107305 0107305 0107306 0107306 0107307 0107307 0107308 0107308 0107309 0107309 * Duplicate injection Set Number 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A Extraction Analysis Date Date 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 7/20/01 7/20/01 7/21/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.00414 0.00417 0.00566 0.00599 0.00878 0.00943 0.00516 0.00499 0.00432 0.00444 0.00462 0.00392 0.00544 0.00516 0.00544 0.00167 LOQ = 0.01 |ig/mL for red blood cells and serum Exygen Research Page 34 of 98 Exygen Study No.: 023-066 Table XIX. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-109-3289-Wk5 454-109-3289-Wk5* 454-109-3290-Wk5 454-109-3290-Wk5* 454-109-3291-Wk5 454-109-3291-Wk5* 454-109-3292-Wk5 454-109-3292-Wk5* 454-109-3293-Wk5 454-109-3293-Wk5* 454-109-3294-Wk5 454-109-3294-Wk5* 454-109-3295-Wk5 454-109-3295-Wk5* Centre ID 0107310 0107310 0107311 0107311 0107312 0107312 0107313 0107313 0107314 0107314 0107315 0107315 0107316 0107316 Set Number 072001 AD 072001 AD 072001 AD 072001 AD 072001AD 072001AD 072001 AD 072001 AD 072001AD 072001 AD 072001 AD 072001 AD 072001AD 072001AD Extraction Analysis Date Date 7/20/01 7/22/01 7/20/01 7/20/01 7/22/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 128 120 79.6 94.2 104 110 83.4 85.8 59.4 76.6 60.6 57.5 73.3 76.9 86.4 22.2 Table XX. Summary of PFOS in Mallard Serum Samples at 50 ppm a.i. for Week 5 Sponsor ID 454-109-3329-Wk5 454-109-3329-Wk5 * 454-109-3330-Wk5 454-109-3330-Wk5* Centre ID 0107317 0107317 0107318 0107318 * Duplicate injection Set Number 072001 AD 072001AD 072001AD 072001 AD Extraction Analysis Date Date 7/20/01 7/20/01 7/20/01 7/20/01 7/22/01 7/22/01 7/22/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 376 400 320 290 347 50 LOQ = 0.01 fag/mL for red blood cells and serum Exygen Research Page 35 of 98 Exygen Study No.: 023-066 Table XXI. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-109-3249-Wkl0 454-109-3249-Wkl0* 454-109-3250-WklO 454-109-3250-WklO* 454-109-3251-WklO 454-109-3251-Wkl0* 454-109-3252-Wkl0 454-109-3252-Wkl0* 454-109-3253-Wkl0 454-109-3253-Wkl0* 454-109-3254-WklO 454-109-3254-Wkl0* 454-109-3255-Wkl0 454-109-3255-Wkl0* 454-109-3256-Wkl0 454-109-3256-WklO* Centre ID 0107322 0107322 0107323 0107323 0107324 0107324 0107325 0107325 0107326 0107326 0107327 0107327 0107328 0107328 0107329 0107329 Set Number 07200IB 07200IB 07200IB 072001B 07200IB 07200IB 07200IB 07200IB 07200IB 07200IB 07200IB 072001B 07200IB 07200IB 07200IB 07200IB Extraction Analysis Date Date 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.00271 0.00372 0.00726 0.00666 0.00659 0.00530 0.00899 0.00940 0.00517 0.00482 ND ND 0.00337 0.00403 0.00419 0.00384 0.00476 0.00266 * Duplicate injection ND = No peak detected 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/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 36 of 98 Exygen Study No.: 023-066 Table XXII. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-109-3289-WklO 454-109-3289-WklO* 454-109-3290-WklO 454-109-3290-WklO* 454-109-3291 -W kl 0 454-109-3291-Wkl0* 454-109-3292-WklO 454-109-3292-WklO* 454-109-3293-Wkl0 454-109-3293-Wkl0* 454-109-3294-WklO 454-109-3294-WklO* 454-109-3295-WklO 454-109-3295-Wkl0* 454-109-3296-Wkl0 454-109-3296-Wkl0* Centre ID 0107330 0107330 0107331 0107331 0107332 0107332 0107333 0107333 0107334 0107334 0107335 0107335 0107336 0107336 0107337 0107337 Set Number 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD Extraction Analysis Date Date 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/20/01 7/20/01 7/22/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 145 147 32.7 32.5 93.5 102 107 111 132 136 85.3 86.4 123 120 114 112 105 33.8 * Duplicate injection LOQ = 0.01 |ug/mL for red blood cells and serum Exygen Research Page 37 of 98 Exygen Study No.: 023-066 Table XXIII. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 15 Sponsor ID 454-109-3236-W kl5 454-109-3236-Wkl5* 454-109-3249-Wkl5 454-109-3249-Wk 15* 454-109-3250-W kl5 454-109-3250-W kl5* 454-109-3251-Wkl5 454-109-3251 -Wk 15* 454-109-3252-Wk 15 454-109-3252-WM5* 454-109-3253-WM5 454-109-3253-Wkl5* 454-109-3254-Wkl5 454-109-3254-Wkl5* 454-109-3255-Wkl5 454-109-3255-Wkl5* 454-109-3256-Wkl5 454-109-3256-W kl5* Centre ID 0107338 0107338 0107339 0107339 0107340 0107340 0107341 0107341 0107342 0107342 0107343 0107343 0107344 0107344 0107345 0107345 0107346 0107346 Set Number 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A Extraction Analysis Date Date 7/19/01 7/19-20/01 7/19/01 7/19/01 7/19/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19/01 7/19-20/01 7/19/01 7/19-20/01 7/19/01 7/19-20/01 7/19/01 7/19-20/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) ND ND 0.00729 0.0102 ND ND 0.00466 0.00441 ND ND 0.00649 0.00723 ND ND 0.0233 0.00699 0.0195 ND 0.000503 0.00616 * Duplicate injection ND = No peak detected 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/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 38 of 98 Exygen Study No.: 023-066 Table XXIV. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-109-3289-Wkl5 454-109-3289-Wkl5* 454-109-3290-W kl5 454-109-3290-W kl5* 454-109-3291-Wkl5 454-109-3291-Wkl5* 454-109-3292-W kl 5 454-109-3292-W kl 5* 454-109-3293-Wkl5 454-109-3293-Wkl5* 454-109-3294-W kl5 454-109-3294-Wkl5* 454-109-3295-Wk 15 454-109-3295-Wkl5* 454- 109-3296-Wkl5 454-109-3296-W kl5* Centre ID 0107347 0107347 0107348 0107348 0107349 0107349 0107350 0107350 0107351 0107351 0107352 0107352 0107353 0107353 0107354 0107354 Set Number 071901AD 071901AD 071901AD 071901 AD 071901AD 071901AD 071901AD 071901AD 071901AD 071901AD 071901AD 071901AD 071901 AD 071901 AD 071901 AD 071901AD Extraction Analysis Date Date 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 152 162 10.1 8.75 152 142 12.1 11.6 128 132 6.73 4.52 117 116 9.52 11.3 73.5 67.3 Table XXV. Summary of PFOS in Mallard Serum Samples at 50 ppm a.i. for TERM Sponsor ID 454-109-3297-TERM 454-109-3297-TERM* 454-109-3298-TERM 454-109-3298-TERM* Centre ID 0107355 0107355 0107356 0107356 * Duplicate injection Set Number 072001 AD 072001 AD 072001 AD 072001 AD Extraction Analysis Date Date 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/rnL) 685 601 664 601 638 43 LOQ = 0.01 |ig/m L for red blood cells and serum Exygen Research Page 39 of 98 Exygen Study No.: 023-066 Table XXVI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-108-361-Wk5 454-108-361-Wk5* 454-108-362-Wk5 454-108-362-Wk5* 454-108-363-Wk5 454-108-363-Wk5* 454-108-364-Wk5 454-108-364-Wk5* 454-108-365-Wk5 454-108-365-Wk5* 454-108-366-Wk5 454-108-366-Wk5* 454-108-368-Wk5 454-108-368-Wk5* Centre ID 0107077 0107077 0107078 0107078 0107079 0107079 0107080 0107080 0107081 0107081 0107082 0107082 0107083 0107083 Set Number 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A Extraction Analysis Date Date 5/18/01 5/18-19/01 5/18/01 5/18/01 5/18/01 5/18-19/01 5/18-19/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 0.00292 0.00264 NQ NQ 0.000440 0.000993 * 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/mL for red blood cells and 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. Exygen Research Page 40 of 98 Exygen Study No.: 023-066 Table XXVII. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-108-401-Wk5 454-108-401-Wk5* 454-108-402-Wk5 454- 108-402-Wk5 * 454-108-403-Wk5 454-108-403-Wk5* 454-108-404-Wk5 454-108-404-Wk5* 454-108-405-Wk5 454-108-405-Wk5* 454-108-406-Wk5 454-108-406-Wk5* 454-108-407-Wk5 454-108-407-Wk5* 454-108-408-Wk5 454-108-408-Wk5* Centre ID 0107084 0107084 0107085 0107085 0107086 0107086 0107087 0107087 0107088 0107088 0107089 0107089 0107090 0107090 0107091 0107091 Set Number 051801DR 051801DR 051801DRD 051801DRD 051801DR 051801DR 051801DR 051801DR 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD Extraction Analysis Date Date 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/21-22/01 5/18/01 5/18/01 5/21-22/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/22/01 5/18/01 5/18/01 5/18/01 5/18/01 5/22/01 5/22/01 5/22/01 5/22/01 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 113 107 353 351 30.5 30.4 26.9 25.8 415 447 1050 1040 495 501 451 471 369 324 * Duplicate injection LOQ = 0.01 |ig/mL for red blood cells and serum Exygen Research Page 41 of 98 Exygen Study No.: 023-066 Table XXVIII. Summary of PFOS in Quail Red Blood Cell Samples at 50 ppm a.i. for Week 5 Sponsor ID 454-108-441-Wk5 454-108-441-Wk5* 454-108-442-Wk5 454-108-442-Wk5* Centre ID 0107092 0107092 0107093 0107093 Set Number 052201CD 052201CD 052201CD 052201CD Extraction Analysis Date Date 5/22/01 5/24/01 5/22/01 5/22/01 5/24/01 5/24/01 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 166 163 108 103 135 34 Table XXIX. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-108-361-Wkl0 454-108-361-Wkl0* 454-108-362-Wkl0 454-108-362-Wkl0* 454-108-363-Wkl0 454-108-363-Wkl0* 454-108-364-WklO 454-108-364-WklO* 454-108-365-Wkl0 454-108-365-Wkl0* 454-108-366-Wkl0 454-108-366-WklO* 454-108-367-WklO 454-108-367-Wkl0* 454- 108-368-Wkl0 454-108-368-Wkl0* Centre ID 0107100 0107100 0107101 0107101 0107102 0107102 0107103 0107103 0107104 0107104 0107105 0107105 0107106 0107106 0107107 0107107 * Duplicate injection Set Number 051801B 051801B 051801B 05180IB 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 05180IB 051801B Extraction Analysis Date Date 5/18/01 5/19/01 5/18/01 5/18/01 5/18/01 5/19/01 5/19/01 5/19/01 5/18/01 5/18/01 5/19/01 5/19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/18/01 5/18/01 5/18/01 5/18/01 5/19/01 5/19/01 5/19/01 5/19/01 5/18/01 5/19/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 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/mL for red blood cells and 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. Exygen Research Page 42 of 98 Exygen Study No.: 023-066 Table XXX.Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-108-401-Wkl0 454-108-401-Wkl0* 454-108-402-Wkl 0 454-108-402-WklO* 454-108-403-Wkl0 454-108-403-Wkl0* 454-108-404-Wk 10 454-108-404-WklO* 454-108-405-Wkl0 454-108-405-Wkl0* 454-108-406-Wkl0 454-108-406-Wkl0* 454-108-407-WklO 454-108-407-WklO* 454-108-408-Wkl0 454-108-408-Wkl0* Centre ID 0107108 0107108 0107109 0107109 0107110 0107110 0107111 0107111 0107112 0107112 0107113 0107113 0107114 0107114 0107115 0107115 * Duplicate injection Set Number 051801DRD 051801DRD 051801DRD 051801DRD 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR Extraction Analysis Date Date 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/18/01 5/21-22/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 672 671 203 190 23.0 20.7 19.0 21.2 18.0 20.0 48.9 50.5 22.8 22.3 30.4 30.2 129 220 LOQ = 0.01 (ig/mL for red blood cells and serum Exygen Research Page 43 of 98 Exygen Study No.: 023-066 Table XXXI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 15 Sponsor ID 454-108-361-Wkl5 454-108-361-Wkl5* 454-108-362-W kl5 454-108-362-Wkl5* 454-108-363-Wkl5 454-108-363-Wkl5* 454-108-364-W kl5 454-108-364-W kl5* 454-108-365-Wkl5 454-108-365-Wkl5* 454-108-366-W kl5 454-108-366-W kl5* 454-108-367-Wkl5 454-108-367-Wkl5* 454-108-368-Wkl5 454-108-368-Wkl5* 454-108-333-Wkl5 454-108-333-WM5* Centre ID 0107116 0107116 0107117 0107117 0107118 0107118 0107119 0107119 0107120 0107120 0107121 0107121 0107122 0107122 0107123 0107123 0107124 0107124 Set Number 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A Extraction Date Analysis Date Analyte Found (pg/mL) 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 AVERAGE: STANDARD DEVIATION: NQ NQ NQ NQ 0.00226 0.00328 0.00127 NQ 0.00705 0.00391 NQ NQ 0.00795 0.00816 NQ NQ 0.00130 NQ 0.00198 0.00304 * Duplicate injection ND = No peak detected 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/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 44 of 98 Exygen Study No.: 023-066 Table XXXII. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-108-401-Wkl5 454-108-401 -Wkl 5* 454-108-402-Wkl 5 454-108-402-Wk 15* 454-108-403-Wkl 5 454-108-403-Wkl 5* 454-108-404-W kl5 454-108-404-W kl5* 454-108-405-Wkl5 454- 108-405-Wkl5 * 454-108-406-W kl5 454-108-406-Wkl5* 454-108-407-Wk 15 454-108-407-Wkl 5* 454-108-408-Wkl5 454-108-408-Wkl5* 454-108-383-Wkl5 454-108-383-Wkl5* Centre ID 0107125 0107125 0107126 0107126 0107127 0107127 0107128 0107128 0107129 0107129 0107130 0107130 0107131 0107131 0107132 0107132 0107133 0107133 Set Number 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052201CD 052201CD 052101AD 052101AD Extraction Analysis Analyte Date Date Found (pg/mL) 5/21/01 5/22-23/01 72.2 5/21/01 5/22-23/01 69.1 5/21/01 5/21/01 5/21/01 5/22-23/01 5/22-23/01 5/22-23/01 2.36 1.65 21.2 5/21/01 5/21/01 5/22-23/01 5/22-23/01 18.8 1.30 5/21/01 5/22-23/01 1.22 5/21/01 5/22-23/01 15.8 5/21/01 5/22-23/01 17.9 5/21/01 5/22-23/01 3.61 5/21/01 5/21/01 5/21/01 5/22-23/01 5/22-23/01 5/22-23/01 3.23 13.3 9.60 5/21/01 5/24/01 1.24 5/21/01 5/24/01 1.55 5/21/01 5/22-23/01 56.7 5/21/01 5/22-23/01 AVERAGE: STANDARD DEVIATION: 54.1 20.3 24.8 Table XXXIII. Summary of PFOS in Quail Red Blood Cell Samples at 50 ppm a.i. for TERM Sponsor ID 454-108-409-TERM 454-108-409-TERM* 454-108-410-TERM 454-108-410-TERM* Centre ID 0107134 0107134 0107135 0107135 Set Number 052201CD 052201CD 052201CD 052201CD Extraction Date Analysis Date Analyte Found (pg/mL) 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: 361 356 106 114 234 144 * Duplicate injection LOQ = 0.01 |ig/mL for red blood cells and serum Exygen Research Page 45 of 98 Exygen Study No.: 023-066 Table XXXIV. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-108-361-Wk5 454-108-361-Wk5* 454-108-362-Wk5 454-108-362-Wk5 * 454-108-363-Wk5 454-108-363-Wk5* 454-108-364-Wk5 454-108-364-Wk5* 454-108-366-Wk5 454-108-366-Wk5* 454-108-368-Wk5 454-108-368-Wk5* Centre ID 0107154 0107154 0107155 0107155 0107156 0107156 0107157 0107157 0107159 0107159 0107160 0107160 * Duplicate injection Set Number 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR Extraction Analysis Date Date 7/23/01 7/25/01 7/23/01 7/23/01 7/25/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/23/01 7/25/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.0282 0.0295 0.0149 0.0134 0.0198 0.0166 0.00510 0.00535 0.0135 0.0120 0.0138 0.0156 0.0156 0.00746 LOQ = 0.01 (ug/mL for red blood cells and serum Exygen Research Page 46 of 98 Exygen Study No.: 023-066 Table XXXV. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-108-402-Wk5 454-108-402-Wk5 * 454-108-403-Wk5 454-108-403-Wk5 * 454-108-404-Wk5 454-108-404-Wk5* 454-108-405-Wk5 454-108-405-Wk5* 454-108-406-Wk5 454-108-406-Wk5 * 454-108-407-Wk5 454-108-407-Wk5 * 454-108-408-Wk5 454-108-408-Wk5* Centre ID 0107162 0107162 0107163 0107163 0107164 0107164 0107165 0107165 0107166 0107166 0107167 0107167 0107168 0107168 Set Number 072301AD 072301 AD 072301AD 072301AD 072301AD 072301AD 072301 AD 072301 AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301 AD Extraction Analysis Date Date 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/23/01 7/26/01 7/26/01 7/23/01 7/26/01 7/23/01 7/23/01 7/26/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/23/01 7/26/01 7/26/01 7/23/01 7/23/01 7/26/01 7/26/01 7/23/01 7/26/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 45.8 45.6 115 128 104 98.3 138 129 106 107 42.4 36.3 77.6 84.4 89.8 35.0 Table XXXVI. Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for Week 5 Sponsor ID 454-108-441-Wk5 454-108-441-Wk5* 454-108-442-Wk5 454- 108-442-Wk5 * Centre ID 0107092 0107092 0107093 0107093 Set Number 072301AD 072301AD 072301AD 072301AD Extraction Analysis Date Date 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 AVERAGE: STANDARD DEVIATION: Analyte Found (fig/mL) 551 562 529 520 541 19.4 * Duplicate injection LOQ = 0.01 |ug/mL for red blood cells and serum Exygen Research Page 47 of 98 Exygen Study No.: 023-066 Table XXXVII. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-108-361-Wkl0 454-108-361-Wkl0* 454-108-362-WklO 454-108-362-WklO* 454-108-363-Wkl0 454-108-363-Wkl0* 454-108-364-WklO 454-108-364-Wkl0* 454-108-365-Wkl0 454- 108-365-Wkl0* 454-108-366-WklO 454-108-366-Wkl0* 454-108-367-Wkl0 454-108-367-Wkl0* 454-108-368-Wkl0 454-108-368-Wkl0* Centre ID 0107177 0107177 0107178 0107178 0107179 0107179 0107180 0107180 0107181 0107181 0107182 0107182 0107183 0107183 0107184 0107184 * Duplicate injection Set Number 072301B 07230IB 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 07230IB 072301B 072301B 07230IB 072301B Extraction Analysis Date Date 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/23/01 7/24/01 7/24/01 7/23/01 7/23/01 7/23/01 7/24/01 7/24/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.00483 0.00489 0.00295 0.00249 0.00467 0.00521 0.00390 0.00375 0.00271 0.00292 0.00439 0.00437 0.00563 0.00542 0.00537 0.00526 0.00430 0.00105 LOQ = 0.01 |ug/mL for red blood cells and serum Exygen Research Page 48 of 98 Exygen Study No.: 023-066 Table XXXVIII. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-108-401 -Wk 10 454-108-401-Wkl0* 454-108-402-Wkl 0 454-108-402-Wk 10* 454-108-403-Wkl0 454-108-403-Wkl0* 454-108-404-WklO 454-108-404-Wkl0* 454-108-405-Wkl0 454-108-405-Wkl0* 454-108-407-Wkl0 454-108-407-WklO* 454-108-408-Wk 10 454-108-408-Wkl0* Centre ID 0107185 0107185 0107186 0107186 0107187 0107187 0107188 0107188 0107189 0107189 0107190 0107190 0107191 0107191 Set Number 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301D 072301D 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD Extraction Analysis Date Date 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (fig/mL) 213 207 53.6 53.7 148 177 19.4 20.3 157 147 105 110 97.0 93.7 114 63.4 * Duplicate injection LOQ = 0.01 pig/mL for red blood cells and serum Exygen Research Page 49 of 98 Exygen Study No.: 023-066 Table XXXIX. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 15 Sponsor ID 454-108-361-Wkl5 454-108-361-Wkl5* 454-108-362-Wkl5 454-108-362-WW5* 454-108-363-Wkl5 454-108-363-Wkl5* 454-108-364-W kl5 454-108-364-Wkl5* 454- 108-365-Wkl5 454-108-365-Wkl5* 454-108-366-W kl5 454-108-366-Wkl5* 454- 108-367-Wkl5 454-108-367-Wkl5* 454-108-368-Wkl5 454-108-368-Wkl5* 454-108-333-Wkl5 454-108-333-Wkl5* Centre ID 0107192 0107192 0107193 0107193 0107194 0107194 0107195 0107195 0107196 0107196 0107197 0107197 0107198 0107198 0107199 0107199 0107200 0107200 Set Number 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C Extraction Analysis Date Date 7/23/01 7/24-25/01 7/23/01 7/23/01 7/23/01 7/24-25/01 7/24-25/01 7/24-25/01 7/23/01 7/23/01 7/23/01 7/23/01 7/24-25/01 7/24-25/01 7/24-25/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/23/01 7/24-25/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/23/01 7/24-25/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) NQ NQ NQ NQ 0.00154 0.00160 NQ NQ NQ NQ NQ NQ 0.00227 0.00249 0.00253 0.00255 0.00162 0.00160 0.000928 0.00105 * 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/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 50 of 98 Exygen Study No.: 023-066 Table XL. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-108-401-Wk 15 454-108-401 -Wk 15* 454-108-402-Wkl 5 454-108-402-Wkl5* 454-108-403-Wkl5 454-108-403-Wkl5* 454-108-404-W kl5 454-108-404-W kl5* 454-108-405-Wkl5 454-108-405-Wkl5* 454-108-406-W kl5 454-108-406-Wkl 5* 454-108-407-W kl 5 454- 108-407-Wkl5 * 454-108-408-Wkl5 454-108-408-Wkl5* 454-108-383-Wkl5 454-108-383-Wkl5* Centre ID 0107201 0107201 0107202 0107202 0107203 0107203 0107204 0107204 0107205 0107205 0107206 0107206 0107207 0107207 0107208 0107208 0107209 0107209 Set Number 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD Extraction Analysis Date Date 7/23/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 181 180 8.21 8.45 246 239 11.1 10.7 141 156 7.04 6.98 107 114 10.2 8.91 131 143 95.0 86.6 Table XLI. Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for TERM Sponsor ID 454-108-410-TERM 454-108-410-TERM* Centre ID 0107211 0107211 * Duplicate injection Set Number 072301DD 072301DD Extraction Analysis Date Date 7/23/01 7/23/01 7/27-28/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 476 514 495 26.9 LOQ = 0.01 |ig/mL for red blood cells and serum Exygen Research Page 51 of 98 Exygen Study No.: 023-066 FIGURES Exygen Research Page 52 of 98 Exygen Study No.: 023-066 Figure 1. Typical Calibration Curve for PFOS 051801A.rdb (PFOS): "Linear" Regression ("1 / x" weighting): y = 7822.67 x + 1176.24 (r = 0.9989155) Exygen Research Page 53 of 98 Exygen Study No.: 023-066 Figure 2. Chromatogram Representing a 0.1 ng/mL standard for PFOS Sample Name: '051801A-101' Sample ID: 'Standard' File: "quail blood wifi* Comment: *0.1 nmL PFOS (C031401-6)' Annotation: ' Sample Index: 1 Sample Type: Standard Concentration: 0.100 ng/mL31! Calculated Cone : 0.101 ng/mL Acq. Date: 0055//1188//0011 Acq. Time 04:15:25 PM Modified : Bunching Factor: Noise Threshold: Area Threshold: Num. Smooths: RT Window: Expected RT: Sep. Width: Sep. Height : Exygen Research Page 54 of 98 Exygen Study No.: 023-066 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PFOS ISample Name. '051801A-104* Sample ID:'Standard* Peak Name: 'PFOS* Maasfes): '499.0/99.0 amu* File:'quail blood.witf' Comment: '1.0 nmL. PFOS (C031401-3)' Annotation: " Sample Index: 4 Sample Type: Standard n g /t Modified: Bunching F< Noise Threi RT Window: Expected RT: Sep. Width: Sep. Height: Exp. Peak Rat Exp. Adj. Rat Exp. Val. Rat Use Relative Int. Type: tart Tim :nd Time: 1 . 51e*Q03 1500 1460 1460 1440 1420 1400 1380 1360 1340 1320 1300 1280 1260 1240 1220 1200 1080 1060 1040 1020 torn Exygen Research 1.38421.4723 2177(12.2777 3 .1 9 6 3 ^ _ 3 .6 6 1 9 ^ 4 05^ > . 7'"-. A '*\ .-4.6937 '/W , -.5,0209 5 722!56 6 39256.7071- 7.4495,S 6 ^ Page 55 of 98 Exygen Study No.: 023-066 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Exygen ID Reagent Blank A, Set: 051801A) ISample Name: '051801 A-110' Sample ID. 'Reagent Blank' Peak Name: 'PFOS' Mass(e8): ` 499.0/99.0 amu' Comment. ` Reagent Blank A' Annotation: " Sample Index: 10 Sample Type: Unknown Vlft N/A Calculated Cot Acq. Date: 205 File: ` quail blood.witt' Modified: Bunching Factor: Noise Threshold: Area Threshold: Num. Smooths: RT Window: Expected RT: Sep. Width: Sep. Height: Exp. Peak Ratio: Exp. A d j . Ratio : Exp. Val. Ratio: Use Relative RT: 5.00 4.00 3 .00 No Int. Type: Height : Start Time End Time: Baae To Baae 4.42 min 1099.9 counl 1.81e*002 cp: 4.29 min 200 195 190 165 160 175 170 4.4169 95 0.6921- 90 85 80 !75 70 65 60 55 SO 45 40 35 30 -5.0461 7.7136 7.5880- fl 7.5125- I fl fv 20 15 10 1.3591 Ai 0 0.5 1.0 1.5 2.0 2 5 3.0 3.5 4.0 4.5 5.0 5.5 $.0 6 5 7.0 7.5 Exygen Research Page 56 of 98 Exygen Study No.: 023-066 Figure 5. Chromatogram Representing Control Quail Red Blood Cells for PFOS (Exygen ID: 0107376 Blank A, Set: 051801A) Exygen Research Page 57 of 98 Exygen Study No.: 023-066 Figure 6. Chromatogram Representing Control Quail Serum for PFOS, (Exygen ID: 0107375 Blank A, Set: 072301C) Exygen Research Page 58 of 98 Exygen Study No.: 023-066 Figure 7. Chromatogram Representing Control Quail Red Blood Cells fortified with 10 ng/mL of PFOS (Exygen ID: 0107376 Spk A, Set: 051801DR) ISample Name: '051001 DR-408' Sample ID: ` Spk A'* File: `quail blood.wiff* Peak Name: 'PFOS' Mass(ee): '499.0/99.0 amu` Comment: '0107376 Spk A 10 ng/m l` Annotation: " Sample Index: 21 Sample Type: Concentration: QC 0.900 ng/mL 4356 Calculated Cone: 0.021 ng/mL Acq. Date: 05/21/01 4300- Acq. Time: 05:15:09 PM 4200- Modified: Bunching Factor: Noise Threshold: 10.04 4100- Area Threshold: 50.20 Nun. Smooths: RT Window: 2 30.0 4000- Expec ted R T : 4.36 Sep. Width: Sep. Height: 0. 20 0.01 3900- Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: Exp. Val. Ratio: 4.00 3 .00 3000- Use Relative RT: No 3700- Int. Type: Manual Retention Time: 4.43 Area: 34434.8 3600- Height: 4.27e*003 Start Time: End Time: 4.30 4.78 3500 3400 3300 3200 3100 3000 2900 1.4295 2700 2500 2400 2300 2200 2100 2000 1900 1800 1700 Exygen Research Page 59 of 98 Exygen Study No.: 023-066 Figure 8. Chromatogram Representing Control Quail Serum fortified with 10 ng/mL of PFOS (Exygen ID: 0107375 Spk A, Set: 072301D) Exygen Research Page 60 of 98 Exygen Study No.: 023-066 Figure 9. Chromatogram Representing Quail Red Blood Cell Sample (Exygen ID: 0107084, Sponsor ID: 454-108-401 Wk5, Set: 051801DR) Exygen Research Page 61 of 98 Exygen Study No.: 023-066 Figure 10. Chromatogram Representing Quail Serum Sample (Exygen ID: 0107207, Sponsor ID: 454-108-407-W kl5, Set: 072301DD) 0107207 (454-108-407-Wk15) DF = 5000 072301DD-635 Sm (Mn, 3x2) 28-Jul-2001 00:11:27 LC/MS/MS #7 MRM of 1 Channel ES- Exygen Research Page 62 of 98 Exygen Study No.: 023-066 APPENDIX A Study Protocol 01P-023-066 (Centre Study No. 023-066) and Amendments and Deviations Exygen Research Page 63 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 ANALYTICAL PHASE PROTOCOL ANALYTICAL PHASE TITLE EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM RED BLOOD CELLS FOR ANALYSIS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 DATA REQUIREMENTS Analytical Method Requirements PERFORMING LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 Phone 814-231-8032 PROTOCOL IDENTIFICATION NUMBER 01P-023-066 | i ! | Centre Analytical Laboratories, Inc. Exygen Research Page 1 of 29 Page 64 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 Title: EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM RED BLOOD CELLS FOR ANALYSIS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY TABLE OF CONTENTS TABLE OF CONTENTS................................................................................................. . 2 1. PURPOSE............................................................................................................ ,3 2. REFERENCE MATERIAL....................................................................................3 3. SPONSOR.............................................................................................................4 4. TESTING FACILITY......................................................................................... ..4 5. PERFORMING LABORATORY....................................................................... ..4 6 . PROPOSED EXPERIMENTAL TIME-FRAME............................................... ..4 7. JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM..............." 5 8 . SAMPLE PROCESSING, STORAGE AND IDENTIFICATION...................... ..5 9. ANALYTICAL METHOD.................................................................................... 5 10. EXPERIMENTAL DESIGN............................................................................... 10 11. PROTOCOL AMENDMENTS AND DEVIATIONS........................................ 11 12. RECORDS......................................................................................................... 11 13. QUALITY ASSURANCE.................................................................................. . 1 2 14. DATA AND REPORT....................................................................................... . 1 2 15. ARCHIVE STATEMENT.................................................................................. .13 16. REFERENCES................................................................................................... .13 17. PROTOCOL APPROVAL................................................................................ .14 APPENDIX: ANALYTICAL METHOD...................................................................... .15 b& If |! [ i i ; i I Centre Analytical Laboratories, Inc. Exygen Research Page 2 of 29 Page 65 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 Title: EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM RED BLOOD CELLS FOR ANALYSIS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY 1. PURPOSE The purpose of this study is to analyze mallard and quail red blood cells samples for residues of perfluorooctanesulfonate (PFOS) using method entitled, "Extraction Of Potassium Perfluorooctanesulfonate Or Other Fluorochemical Compounds From Serum For Analysis -Using HPLC-Electrospray/Mass Spectrometry" (3M method number ETS-8-4.I) with the modifications listed in Section 9. The analytical phase will be conducted under EPA TSCA Good Laboratory Practice standards 40 CFR Part 792 (1). 2. REFERENCE MATERIAL The following analytical standard 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: Perfluorooctanesulfonate IUPAC Name: 1-Octanesulfonic acid,1,1,2,2,3,3,4,4,5,5,6 ,6 ,7,7,8 ,8 ,8 - heptadecafluoro-, potassium salt CAS Number: 2795-39-3 Molecular Weight: 499 (CsFnSOa-) O CsFiTjS O" o Note: The neutral molecule and standard from which the PFOS (anion) is obtained is perfluorooctanesulfonate potassium salt [C8F17SO3K], molecular weight 538. A record of test and reference substance receipt, storage conditions, and a record of use will be maintained at Centre Analytical Laboratories, Inc. Forms Centre Analytical Laboratories, Inc. Page 3 of 29 Exygen Research Page 66 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 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 Sponsor Representative: Rochelle Robideau TELEPHONE: 651-778-7065 FAX NUMBER: 651-778-6176 4. TESTING FACILITY Wildlife International, Ltd. 8598 Commerce Drive Easton, MD 21601 STUDY DIRECTOR: Sean Gallagher TELEPHONE: 410-822-8600 FAX NUMBER: 410-822-0632 5. PERFORMING LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 PRINCIPAL INVESTIGATOR: Emily R. Stauffer, Centre TELEPHONE: 814-231-8032 FAX NUMBER: 814-231-1580 6. PROPOSED EXPERIMENTAL TIME-FRAME Analytical Start Date Analytical Termination Date Report Issued April 23,2001 April 30,2001 May 31, 2001 1, 1 1 i j j" j j | i ! ; |. j i; 1 | j Centre Analytical Laboratories, Inc. Exygen Research Page 4 of 29 Page 67 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 7. JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM Mallaid and quail red blood cells samples collected by Wildlife International, Ltd. will be used as the analytical samples for this phase. Mallards and quails represent wild bird populations and they are EPA recommended species because they do well in a laboratory environment. Quail habitats include bushy pastures, grassy roadsides, farmlands and open woodlands, and they are considered land grazers. Mallard habitats include ponds, lakes and marshes, and they are considered aquatic grazers. The use of these matrices for the study was to determine if PFOS was deposited in the whole blood of mating adults that were fedPFOS. A complete description of the test system along with all of the in-life parameters is documented in Mallard Protocol No. 454/010600/MP/SUB454, Project No. 454-105 and Quail Protocol No. 454/010600/QP/SUB454, Project No. 454-104. This project is the analytical portion of those studies. 8. SAMPLE PROCESSING, STORAGE AND IDENTIFICATION No processing will be required for the any of the samples. 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 container or cross-referenced to the container. 9. ANALYTICAL METHOD All samples will be analyzed according to the analytical method titled "Extraction Of Potassium Perfluorooctanesulfonate Or Other Fluorochemical Compounds From Serum For Analysis Using HPLC-Electrospray/Mass Spectrometry" (3M method number ETS-8-4.1) with the following modifications: 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 Centre Analytical Laboratories, Inc. Exygen Research Page 5 of 29 Page 68 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 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. b. 0.1 ug/mL Fortification Solution - Pipette 10.0 mL of the 1.0 pg/ml fortification solution into a 100-mL volumetric flask and bring up to volume with methanol. c. 0.01 ue/mL Fortification Solution - Pipette 10.0 mL of the 0.1 gg/mL fortification solution into a 100-mL volumetric flask and bring up to volume with methanol Store all fortification standard solutions (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximum period of 6 months from the date of preparation. Calibration Standards Prepare six LC/MS/MS calibration standards in methanol via dilution of the 0.1 pg/mL and 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) 5 2 1 5 2 1 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 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. 3. The sample volume for the red blood cells samples will be 100 pL. The amount of 0.5 M TBA added to the sample will be 0.5 mL and the amount of 0.25 M sodium carbonate/sodium bicarbonate buffer added will be 1.0 mL. The samples will only be filtered with 0.2 pm nylon mesh filter if necessary. 4. The samples will be analyzed under the following conditions: | ! j I i ; Centre Analytical Laboratories, Inc. Exygen Research Page 6 of 29 Page 69 of 98 Exygen Study No.: 023-066 \ Centre Protocol No. 01P-023-066 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 Dell UltraScan P1110 Software: PE SciexAnalyst 1.1 Windows NT HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC ColumnrGenesis 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 Flow Rate: 0.3 mL/min. Time 0. 1.0 7.0 7.5 11.0 %A 60 0 0 60 60 %B 40 100 100 40 40 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 Cis etc.) can be used, provided equivalent chromatography is obtained. Ions monitored: Analvte PFOS Mode negative Transition Monitored 499 -> 99 Approximate Retention Time (min) 4.40 On a day-to-day basis, the retention times may vary slightly depending on the batch of mobile phase, etc. Centre Analytical Laboratories, Inc. Exygen Research Page 7 of 29 Page 70.of 98 Exygen Study No.: 023-066 CentreProtocol No. 01P-023-066 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. 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. 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 Hows Nebulizer Gas Curtain Gas Collision Gas TIS Temperature Set 12 13 4 350C Calibration Procedures a. Inject the same volume (between 10 to 20 p.L) 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, may be excluded from the calibration curve. However, the total number of calibration standards that may be excluded must not exceed 20% of the total number of standards injected. c. The correlation coefficient (r) for calibration curves generated must be >0.9925 (^>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. Centre Analytical Laboratories, Inc. Exygen Research Page 8 of 29 Page 71 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 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. Each set must also include at least one methanol blank and one ASTM Type IH2O blank. 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 (10 ng/mL) and 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 the response of the lowest standard (0.0001 pg/mL) will be reported as NQ (not quantifiable). 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. i $ p I [ | i ; j I j. | i ' j j | j S Centre Analytical Laboratories, Inc. Exygen Research Page 9 of 29 Page 72 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 10. EXPERIMENTAL DESIGN Samples obtained by Wildlife International Ltd. will be shipped to Centre Analytical Laboratories, Inc. for analysis. There are no known contaminants expected in the test system that would affect the results of this study. Only those samples designated by the Sponsor will be extracted and analyzed at Centre according to method, "Extraction Of Potassium Perfluorooctanesulfonate Or Other Fluorochemical Compounds From Serum For Analysis Using HPLCElectrospray/Mass Spectrometry" (3M method number ETS-8-4.1) with the modifications listed in Section 9. 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. Fortification will be made to the matrix prior to extraction at levels ranging from the LOQ to above the highest residue found. The fortification solution containing the test/reference substance will be applied to the designated samples via a Hamilton syringe, micropipette, pipet, or equivalent, to maintain consistency and accuracy. The average recovery and relative standard deviation of the fortified samples will be calculated. If necessary, apply a standard test for outliers. If an outlier exists, then it may be excluded from the statistical analysis. Also, the average residue found and standard deviation for each matrix will be calculated. The following equations will be used for the calculations. Equation 1: Analyte found (ng/mL) = (Teak 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, use Equation 3 to calculate the percent recovery. Centre Analytical Laboratories, Inc. Exygen Research Page 10 of 29 Page 73 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 Equation 3: Recovery (%) = ' ______ ((anal, found (pg/mL) - avg. anal, in Ctrl (pg/mL)) x (1000 ng/1 pg)) x 100 amount added (ng/mL) 11. PROTOCOL AMENDMENTS AND DEVIATIONS Any deviations from the protocol or from the analytical method as provided will be documented and reported promptly to the Study Director and Sponsor Representative. Planned changes to this protocol or to die analytical method will be made in writing as an amendment and approved by the Principal Investigator, the Study Director, and the Sponsor Representative. 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, Study Director, and Sponsor Representative. Copies of amendments will be sent to the quality assurance unit. 2. Protocol deviations: Protocol deviations, which are one time and unplanned deviations from the protocol, shall be documented in the study records, noting the nature of the deviation, potential effect or impact on the study, and corrective action if required. Protocol deviations are signed and dated by the Principal Investigator, Study Director, and Sponsor Representative and reviewed by QAU. 12. 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: ; p t;-; f. iv, \x j f j j j.v f j | | I j ; Centre Analytical Laboratories, Inc. Page 11 of 29 Exygen Research Page 74 of 98 . Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 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. 13. QUALITY ASSURANCE 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 GLP standards are met. 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. 14. 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 report will be issued by Centre, approved and signed by the Study Director and the Sponsor Representative 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. Reference materials identified by name, lot, purity, and other characteristics 4. Name of performing laboratory and analytical start and termination 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 and Sponsor Representative, who will return the report with comments. Centre Analytical will make revisions and finalize the report with the approval of the Study Director and Sponsor Representative. The Centre QA unit will conduct an audit of the final draft report and raw data package to assure accuracy and GLP compliance. The Centre Analytical Laboratories, Inc. Page 12 of 29 Exygen Research Page 75 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 report must be approved by the Study Director and Sponsor Representative 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, Study Director and Sponsor Representative. REPORT DISTRIBUTION: Original: Study Director Copy Sponsor Representative Copy: Centre Archives 15. 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. 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. 16. 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. Exygen Research Page 13 of 29 Page 76 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 17. PROTOCOL APPROVAL This protocol was audited by the Quality Assurance Unit of Centre Analytical Laboratories, Inc. Quality Assurance Reviewer, Centre Ang< a L. Morgan \ Qual y Assurance Auditor Principal Investigator, Centre Facility Management, Centre J b h n Flaherty * Onppe.rnattiinonnQs Manager Study Director, Wildlife International, Ltd. Date r Centre Analytical Laboratories, Inc. Exygen Research Page 14 of 29 Page 77 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 APPENDIX: ANALYTICAL METHOD A. "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-Electrospray/Mass ' Spectrometry" Centre Analytical Laboratories, Inc. Exygen Research Page 15 of 29 Page 78 of 98 Exygen Study No.: 023-066 Centre Protocol No. QlPr023-066. O 3M ENYIRONMF.NTAL LABORATORY Sf *7 M ethod `-S '^ ' E x t r a c t io n o f P o t a s s iu m P e r f l u o r o o c t a n e s u l f o n a t e o r O t h e r ^^XJOROCHEMICAL COMPOUNDS FROM SERUM FOR ANALYSIS USING HPLC- \ Electrospray/M ass Spectrom etry ' M ethod Number: ETS-S-4.1 '! iK : /J s A ^ <5^5 ./>-j JL-j ;** Author: l i s a Clemen, Glenn Langenbrg If _,4& \ . Adoption Date: 03/01/99 Revision Date: Approved By; , _____ i> f u ~ - ^ Laboratory Manager L I l ------- Group Leader rr->^ Techriical Reviewer v-P Date v- ..%Py/u/s3 ./Date .'.-jS s, >. u--' . 'l Date. 1.0 Scope and Application 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compoimds from serum. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. ^ 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report J Word 6/95 ETS-8-4.1 ; of PFOS ftom Serein Page 1 o f 14 Centre Analytical Laboratories, Inc. Page 16 of 29 Exygen Research Page 79 of 98 Exygen Study No.: 023-066 - Centre Protocol No. 01P-023-066 o . This method describes die procedure for extracting potassium perfluorooctanesulfonate (PFOS) or flier fluorochemical surfactants from serum, or other fluids, using an ion ' / / ' paring reagint and methyl-ferf-butyl ether (MtBE). In this method, seven 'V _ fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, f t y M 556, and surrogate standard (see 3 .0 Definitions). An ion pairing reagent is added to %. thesample and the analyte ion pair is partitioned into MtBE. The MtBE extract is Nsil^femoved and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 .'. ! '" llio f metfianol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filteriSo-Sassnutovials. H v S f t ,, *J v 2.2 These sample `extracts are analyzed following method ETS-8-5.1 or other appropriate m ethods.^p^'.A S 3.0 Definitions ^ 3.1 PFOS*. perfluorooctanesulfonate (anion of potassium salt) C,F,7S03" . . s . 3.2 PFOSA: pafluorooctarie sulfonylamide CtFt7S01NH1 3.3 PFOSA: perfluorooctansulj^ylaimdo (ethyl)acetate CjF ^ O jNCHjCHjJCHjCOj' 3.4 EtFOSE-OH: 2(N-ethylperfluoroctaiie sulfonamido>ethyl alcohol C,FnS02N(CH2CH3)CT2CH20 H \ ^ 3.3 PFOSEA pifluoroctane sulfonyl cthylamide CjF^SOjNCHjCH^H 3.6 M556: CtF,,SOJN(H)(CHJCOOH) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfomc acid ' 4.0 W arnings ano Cautions _________ ______ 4.1 Health and'safety warnings ' 4.1.1 U se universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. ^ 5.0 Interferences r* 5.1 There are no interferences known at this time. 6.0 E quipment - "K 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. _V 6.1.1 Vortex mixer, VWR, Vortex Genie 2 . `` 6.1.2 Centrifuge, Mistral 1000 or IEC o 6 .1 3 Shaker, Eberbach or VWR - ETS-S-4.1 Extraction of PFOS from Serum Page 2 of 14 Centre Analytical Laboratories, Inc. Exygen Research Page 17 of 29 Page 80 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 ' v-'- 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0 .100 g) ' f \ i. -3 7.0 Supplies and M aterials____________ __ 7-C :iS * " 7.2 'f- Eppendorf or disposable pipettes 7 3 ^ ^Nalgene bottles, e n a b le o f bolding 250 mL and 1 L 7.4 ^'WVolum`iestriAc.Nflasks, glass, type A 7.5 T-CHEM-vials; glass., 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 " 't" > v 7.8 Oxford DispeiKiT^jLOJo 10.0 mL 7.9 S_yringes, capa.ble oaEfsmi easuring \5 jiL to 50 pL 7.10 Graduated pipettes ' i 7.11 Syringes, disposable p la sb c ^ c c 7.12 Syringe filters, nylon, 0 2 pm, 25 jn m . _ 7.13 Timer '*' . tp*' 7.14 Crimp cap autovials and caps \ 7/.a1o5 cCrriimmppers a Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTM water. Rinse syringes a minimum o f 9.times with methanol, 3 rinses from 3 separate vials.- ` J 8.0 Reagents anp Standards .*'`<*.'*'*A*X 8.1 Type I reagent grade water, Milli:QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC PliisTM system 8.2 Sodium hydroxide (NaOH), 3.T Baker or equivalent % & - I8 2 Tetrabutylammonium hydrogen sulfhtefTBA), Kodak or equivalent Vi1 k 8.4 Sodium carbonate (NajCOj), J.T. Baker or equivalent 82 Sodium bicarbonate (NaHCOj), I.T. Baker or equivalent ": A W 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled orHPLC grade ,Vy 8.7 Methanol, Omnisolv, glass distilled orHPLC grade ' ,y 8.8 Seram or blood, frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight= 5 3 8 J 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ' ETS-8-4.1' Extraction ofPFOS from Senna Page 3 of 14 Centre Analytical Laboratories, Inc. Page 18 of 29 Exygen Research Page 81 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 D O V> : : . . . - . ; i r \ * ; "i-r .v - :- " ;- - ! 'T .'-1 ' .. ' '.'i';;: .. . . -V 'V * 8 .9 3 PFOSAA(3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8 .9 3 PFOSEA. (3M Specialty Chemical Division), molecular weight = 527 jy .8.9.6 M56 (3M Specialty Chemical Division), molecular weight = 557 ; 8 , 9 . 7 Surrogale standard: 4-H, perfluorooctane sulfonic acid (1-H .l-H , 2-H, 2-H i^i C,F,jSOjH) mole..c.u..l.a.r..w..e..i.g..h..t.=..4..2.8.. \ -.8.9.8 Other fluorochemicals, as appropriate 8.10 -'Reagentpreparation ' m NOTE: ,When preparing larger volumes than listed in reagent, standard, or surrogate I p * preparation, adjust accordingly. ' ' 8.10.1 104N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a i 1OmL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolvedyStore in a 1 L Nalgene bottle. 8.10.2 1 N sodiimfhydroxide (NaOH): Dilute 1 0 N NaOH 1:10. Measure 10 mL o f 10 ! .N NaOH solution into a'l 00 mL volumetric flask and dilute to volume using Milli-QTM watefS'Storejn a 125 mLNalgene bottle. 8 .1 0 3 0.5 M tetrabutylmiMmumlylrogen sulfate (TBA): W eigh approximately 169 g o f TBA into a 1 L vohrmeinc containing 500 mL Milli-QTM water. Adjust to pH ! Ip using approximately-114to 54 md^of 10 NNaO H (While adding the last mL o f NaOH, add slowly because th pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a l 'LNalgene bottle. 8.103.1 TBA requires a check prior to each u to ensurepH = 10. Adjiistas needed using I N NaOH solution^ / \ 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NajCO/NaHCOj): Weigh approximately 26.5 g o f sodium carbonate (j^ajCOj) said 21.0 g o f sodium bicarbonate (NaHCOJ into a 1 L volumetric flask.and bring to volume with M illi QTM water. Store in a 1 LNalgenebottle. 8.11 Standards preparation ' 8.11.1 Prepare PFOS standards for the standard curve. ^ 8 .1 1 3 Prepare other fluorochemrcal standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard^ solution containing 1.00 ppm PFOS, 1.02 ppmPFOSA, 0.987 ppm PFOSA+ and ! U O ppm EtFOSE-OH.) . - 8 .113 Weigh approximately 100 mg o f PFOS into a 100 mL volumetric flask'and r e c o r d V | the actual w eigh t ' 8.11.4 Bring to volume with methanol for a stock standard o f approximately 1000 p p m i^ ' (pg/mL). ', 8 .1 1 3 Dilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution o f approx; 5.0 ppm. ETS-S-4.I Extraction of PFOS from Scrum ri.-.''. Page 4 of 14 Centre Analytical Laboratories, Inc. Page 19 of 29 Exygen Research Page 82 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 r * ...if J?** ** .< \ O f JU 2 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution o f approx. 0.50 ppm.: Surrogate stock standard preparation r 8.12.1 Weigh approximately 50-60 mg o f surrogate standard 1-H.l-H, 2-H, 2-H, C,l*`|iOjH into a 50 mL volumetric flask and record the actual weight 8.12.2 Bring to volum e with methanol for a surrogate stock o f approximately 1000-1200 W i3j:Jt8.12J5prepare a surrogate woridng standard. Transfer approximately 1 mL o f surrogate " . f Pstock to a 10 m L volumetric flask and bring to volume with methanol for a \ afworkmg standard o f 100 ppm. Record the actual volume transferred. ' 9.0 Sample HANmrar: 9.1 A ll samples are received frozen and must be kept frozen until the extraction is performed. 9.2 A llow samples t^thiaw to room temperature prior to extraction. JF \ 10.0 Quality Control " fw a<_____________________________________ ___________ 10.1 Solvent Blanks, M ethod blanks and matrix blanks 10.1.1 An aliquot o f 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots o f Milli-QTM water following this procedure and use . as method blanks. 10.1.3 Extract two 1.0 mL aliquots o f the serum.fbllqwing this procedure and use as matrix blanks. See 11.1.4. ^ 10.2 M atrix spikes Sf V 0 w. 10.2.1 Prepare and analyze matrix spike and matrixspike duplicate samples to determine the accuracy o f the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample se t ^ 10.2.3 Expected concentrations will fall in the mid-range o f the initial calibration curve. Additional spikes may be included and may fall in the low-range q ftiie initial calibration curve. I |f tj` 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a b x minimum o f 2 matrix spikes per batch. " 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy o f the initial calibration curve. . V i? ' T. J* 10.3.2 Prepare, at a minimum, one continuing check per group o f 10 samples. For ' example, i f a sample set = 34, four checks are prepared and extracted. r 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. ETS-8-4.1 i Extraction ofPFOS fromSerum . W iV - Page 5 of 14 Centre Analytical Laboratories, Inc. Page 20 of 29 Exygen Research Page 83 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 _ ; vt-* -v- * .* '3S :* r 1 0 3 .4 The expected concentrations will fall within'the mid-range o f the initial. calibration curve. Additional spikes may be.included that fell in the low-range o f the initial calibration curve. This is necessary.if the analyst must quantitate using r % only the low end o f the calibration curve (for example, 5 ppb - 1 0 0 ppb, rather v - ' to" / | . ' 5 p p b - 1000 ppb). 11. C alibration <np Standardization -v 11.1^,.,'Preare matrix calibration standards "11.1.1 iTrahsfer 1 mL o f serum to a 15 mL centrifuge tube. St . 11.1.21I f mostsample volumes are less than 1.0 mil, extract standards with matrix ^volumesequal to the sample volumes. Do not extract less than i).50 mL o f matrix^Record each sample volume on the extraction sheet 11.13 ^ While prepariInKg' a total o f twenty aliquots in 15 mL centrifuge tubes, mix or shake between'<ayliquots.) ! 11.1.4 Two 1 mL aliquots,'or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f thissectiorC to spike, in duplicate, two standard curves, for a total o f eighteen standards,- two matrix blanks, and two method blanks. 1 1 .1 3 Refer to validation report ETS-8-4.() & ETS-8-5.0-V-1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 U se Attachment D as an aid in calculating the concentrations o f the working standards. See Section 13.0 to'calculate actual concentrations o f PFOS in calibration standards. Np*"' 11.2 . njkp'V r v. To each standard, blank, or continuing check,'add appropriate amount o f surrogate working standard for the concentration to fall within the calibration curve range 5 ppb - 1000 ppb. 'vgp ' Jf % 1 1 3 Extract spiked matrix standards following 12.6-12.16bf.this method. Use these standards to establish each initial curve on the mass spectrometer. r ? r-.i. '"'v7s5k-"' A? \ 3. .x S \.v *. T D HTS-8-4.1 i' Extraction of PFOS front Scrum .. Page 6 o f 14 Centre Analytical Laboratories, Inc. Page 21 of 29 Exygen Research Page 84 of 98 Exygen Study No.: 023-066 CentraProtocol No. 01P-023-066 stie Table 1 >..4/T . Approximate spiking amounts for standards and spikes . t? Using 1.0 iuL of matrix Working standard. pL . Approx, final cone, o f ' (approx, cone.) ' analyte in matrix i*f' a 4 A$ >. . 0.500 ppm . 10 . Blank ; 0.005 ppm & v 0.500 ppm ' 20 0.010 ppm x ' ^ 5 , 0 0 ppm - W 5.00, ppm . 5. 10 0.025 ppm " 0.050 ppm v.5,00 ppm : 20 0.100 ppm '^*50.0 ppmv . 5 0.250 ppm - ' 50,0/ppm 10 0-500 ppm 50.0'ppmSi' 15 . 0.750 ppm 50.0 ppm M r " v 20 1.00 ppm 12.0 P rocedure 'V ^ # X- 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 .M . Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge, tube. 12.3 Return unused sarnples to freezer after extrac'n'amounts have been removed. 12.4 Record the initial volume on the extraction worksheet, 12.5 Label th tube with the study number, sample ID, daiem d analyst initials. S ee attached worksheet for documenting the remaining steps. .Ss 12.6 Spike all samples, including blanks and standards, ready for exfraction with surrogate standard as described in 11.2. i f ir ....... yj * .. 12.7 Spike each matrix with the appropriate amount o f standard as described m l l . l , or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. , .^ 12.8 Vortex m ix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. ] ' V .-'. J, 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. I f not, adjust accordingly/' ' 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL o f 0.25M sodium caibonate/sodiuni^p - bicarbonate buffer. . ' . 12.11 Using an Oxford Dispenser, add 5 mL methyl-tert-butyl ether. .* 12.12 Cap each sample and put on the shaker at a setting o f 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting o f 3500 rpm, or until layers are Well separated. . ETS-8-4.I . . : Extraction of PFOS from Serum Page 7 of 14 Centre Analytical Laboratories, Inc. Page 22 of 29 Exygen Research Page 85 of 98 Exygen Study No.: 023-066 Centre Protocol No_01P.-023-0.66. 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL o f the organic layer to this clean 15 mL centrifuge tube. ^.12.16 Put' each sam pleon the analytical nitrogen evaporator until dry, approximately 1 to 2 \ ours. *' 12.17_.Add 1.0 mL ofmethanol to each centrifuge tube using a graduated pipette. ,*~ 1r2V;l8 -Vortex^mix for 30 se.con.ds.. . 12J9 ^Attach a 0.2 jim nylon mesh filter to a 3 cc syringe and transfer the sample to this . w^syri5fige$ Filter into a 1.5 t^T. glass antovial or low-volume autovial when necessary. 12.20 l i b elthe autovial with the study number, animal number and gender, sample timepoint, matrix^final solvent, extraction date, and analyses) performing the extraction. 12.21 Cap and store"extracts at room temperature or at approximately 4 C until analysis. 12.22 Completethe extractronworksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 'i f Sj&B' - & 13.0 Data Analysis and Calculations____________:______________ __________ ____ 13.1 Calculations \ \ 13.1.1 Calculate actual concentrations ofPFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL o f standard x concentration of standard (ug/mL^_________________ mL o f standard + mL o f surrogate standard s*initial matrix volume (mL) ,4 # '\ Final Concentration (pg/mL) o f PFOSin matrix v 14.0 Method P-e--r-f-o-r--m-a--n-c-e--;---- :-------- \ .\yji 14.1 The method detection limit (MDL) is analyte and matrixspectfic. Refer to M DL report for specific MDL and limit o f quantitation (LOQ) values (see Attachments B and C). 14.2 The following quality control samples are extracted with each hatch of samples to evaluate the quality o f the extraction and analysis: .- 14.2.1 Methodblanks and matrix blanks. g. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accurac/ind^v precision o f the extraction. '*K-iss> 14.2.3 Continuing calibration check samples to determine the continued accuracy'ofthe - initial calibration curve. ' 14.3 Refer to section 14 o f ETS-8-5.1 for method performance criteria. v. 15.0 Pollution Prevention and Waste Management-------------------------------:-- 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers locat1ed in the laboratory. ` ETS-8-4.1 Extraction of PFOS from Serum Page 8 of 14 Centre Analytical Laboratories, Inc. Page 23 of 29 Exygen Research Page 86 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 f: ' o 16.0 Records 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. ' f * ~ : . 17.0 'Attachments ** ____________________ tXif . \ ' 17.1 . Attachment A, Extraction worksheet \ >1 ? V 17.2^Attachm ent B , MDL/LOQ values and summary ~_________ ____________ .. 17.3 -Attachment C, Calibration standard concentration woricsheet 18.0 References .g-5 18.1 The validation rcporfassociated with this method is ETS-8-4.0 & 5.0-V -l. 18.2 FACT-M-3.1 ,"Analysia o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-ElectrospiayMass Spectrometry" 19.0 Affected PoaiMKNTSg ______________ ____________________ _____ 19.1 ETS-8-5.1, "Analysis o f Senun'or Otner Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R evisions : x . ..tx____________ . . Revision Number 1 \ Reason For Revisini Section 12.21 Changed to include sample storage'at room temperature. Section 12.13 Added th shaker speed.^y*'" p ' ' X . Section 12.17 Final volume is 1.0 mL; not adjustedforinitial volumes less than 1.0 mL. Revision Date 04/02/99 <&* % %. g, V, t ; ETS-34.1 Extraction ofPFOS from Setum Centre Analytical Laboratories, Inc. ; Exygen Research Page 9 of 14 Page 24 of 29 Page 87 of 98 Exygen Study No.: 023-066 CentreProtocol No. 01P-023-066 Attachment A ETS-M-1 Extraction of PFOS fiom S e n im - Centre Analytical Laboratories, Inc. Exygen Research Page 10 o f 14 Page 25 of 29 Page 88 of 98 Exygen Study No.: 023-066 Centre Protocol Na 01P-023-066 ,, -> - r.O.y'' O r Compound MDL (ppb) ^ N -, PpOS- I.Y4 PpOSAv, 1.51 ' POSAM 3.46 EtFOSErOH \ 11.4 M 556t^ i 6.03 PFOSEA' f i 5.71 LOQ (ppb) 5.55 4.79 20.5 3651 1951 18.2 Linear Calibration Range (LCR) . Approximate concentrations to be used for preparing the Standard Calibration Curve 5 ppb -1 0 0 0 ppb 5 ppb -1 0 0 0 ppb - 5 ppb -1 0 0 0 ppb 5 ppb-1 0 0 0 ppb : 5 ppb-1000 ppb 5 ppb -1 0 0 0 ppb detennined. Two cuiycs' ineach of these matrices were extracted and analyzed with the rabbit senmt curves to determine 'equivalence. Responses in the rat, bovine, monkey, and human were equivalent to the rabbit responses,`thereforej'their MDL and LOQ will be the same values as detennined in rabbit senun. I X $ * ",**. , Please see LOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-1 for further information. V? A?f \ V ^ r 'V' t ; V ' ,-$ P \ #$ % k^r V.9? /fiP N 1 f .1 '" t, v X i __ r*^;N I I o Attachment B: MDL/LOQ Summary ETS-8-4.1 ..;1. Extraction of PFOS from Senun . Centre Analytical Laboratories, Inc. Exygen Research Page 11 o f 14 I Page 26 of 29 Page 89 of 98 Exygen Study No..1023-066 Centre Protocol No. 01P-023-066 ... V'* -V::-7T:''.\ V` . , -75'v* V-. ; D Compound: PFOS ; Prepared range Rabbit Serum o f standards (ppb) (ngtaiL) \ 'V i Fuji Range J 0.99s"'-978 iy Low Curve/ * .vie;.-*tv**' \ 4.94.-248 . High c u. rv y e d | |7.8-978 1I X A / < / *rjr- A 0.995:-978 .$v LCR from curve . (PP*) (ng/mL) 24.8-978 % Recovery Range RSD Range. i- . 83-108 ` - 4.67-n.O 4.94 - 248' ; 85-104 5.34-12.0 97.8 - 978 85-106 . 4.84-9.80 4.94-978 94-111 4.60-10.5 Rabbit Scmm Full Range Low Curve High curve Prepared range , LCR from o f standards,^ ^ . curve (ppb) (ng/m)'a -*jt(ppb) $ ii(hg/m L) .0.993 - 976 . 4.93^976. `t-jA.X . .I'jnT 4.93-97-6 4.93-97.6' ' y.-rKv* 24.8 - 976 24.8?978 . % Recovery . Range 88-103 87-105 ,493-102 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 l/X .. 0.993-976 4.93 - 976''' ( m ok 5.10-14.5 . Compound: PFOSAA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1 0.991-974 . 4.92 - 247 4922- 974 0.991 -974 LCR from curve ' (Ppb) (ng/mL) 24.7-974 .9.74-247 97.4 - 974 * 9.74-974 %Recovery R S D Range y Range Vl*` N ' # ' 81-111 . 97-107 85-1Q8 95-115 4.18-1ti0ti./6** 638-21X.8 433-123 > 4P \ tf 4.11-23j Viti'' "f o Attachment B: MDL/LOQ Summary ETS-8-4.1 : , . '- ' Extraction of PFOS from Serum Centre Analytical Laboratories, Inc. Exygen Research Page 12 o f 14 Page 27 of 29 Page 90 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 O Componnd: EtFOSE-OH Prepared range ;Rabbit Serum o f standards 1. e v y a ' (ppb). (ng/mL) -s Full Range. i*' % 0993 - 976 tow Carre 4.93 - 97.6 LCR from curve (ppb) (ng/mL) 49.3-976 9.76-97.6 % Recovery Range 77-110 97-107 High curyb> : . ;]y 4 9 J -9 7 6 97.6-976 1/X . ' ( y v 0.993X493 : x _ V, Compound: PFOSEA . 9.76 - 976 Rabbit Serum Prepared range t LCR from of standmdsy- ; curve (ppb) (ng/tnL) ^ i / (ng/mL) Full Range 0993 - 976 . 24.8-976 90-109 86-111 94 Recovery Range 96-106 Low Curve High curve 493-248 ' 493-976 9.724S *w pi-no 49.3-976 ' 0 M O 6 l/X . 0993 - 976 9.76 - 976 \ 93-117',. RSD Range 113-255 14.1-213 11-5-19.6 11.1-213 RSD Range 10.1-163 11.8-195 103-183 10.1-19.1 Compound: M556 Rabbit Serum Prepared range o f standards . (ppb) (ng/mL) Full Range 0.993 - 976 LCR fromcurve (ppb) (ng/mL) 24.8-976 Low Curve 493-97.6 9.76-97.6 High curve 97.6 - 976 97.6-976 l/X 0993 - 976 . 9.76-976 94 Recovery y RSD Range `x " J&nge 88-106 K 4.82-17S* 100-105 . 595-183^, 81-111 97-110 5.11-9.74 4.77-195 \ %*!/ X J o Attachment B: MDL/LOQ Sununaiy ETS-8-4.1 Extraction of PBOS from Serum Centre Analytical Laboratories, Inc. Exygen Research Page 13 of 14 Page 28 of 29 Page 91 o f 98 Exygen Study No.: 023-066 .'* 1r Centre Protocol No. 01P-023-066 SSi. ; Ion Pair Standard Curves - Fluids Prep date(s): i; Standard number: ` Anaiyte(s): : \ Equipment number: feipv Sample matrix: : --`7i Final solvent and TN: Fv '!,*' : \ - M ethod/revisionf ^ ! . .Blank QuidAdentiQer: Targttanalyte(s): EC m h std approx. 0300 ppm: FC m urtd^approx. 5.00 ppm: FC mix std app ro x 50.0 ppm: Surrogate std approx. 100 ppm: PFOS Std cone ug/mL 0.500 0.500 5.00 5.00 5.00 50.0 50.0 50.0 50.0 PFOSA^iPFOSAAv EtFOSE PFOSEA M556 Stdconc^Y Std conclus Std cone Std cone . Std cone ug/mL uglinL* Jf^ug/mL - ug/mL ug/mL 0.507 0332.1? 0.501 0.521 0301 . 0.507- 0.532:1 .0301V 0.521 0301 5.07 532 H ?>.01 S 531 5.01 5.07 532 'H 5.01 s 3 3 1 5.01 5.07 532 >5.01 /.:i5.21 5.01 50.1 533 50.1 ? ?"52.1 50.1 50.1 533 50.1-7.7 52.1 ^ 50.1. 50.1 533 50.1 **_52.1 .iti ^ 50.1 50.1 533 50.1 .52:1 50.1 All Am't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 AU Final voi mL ' 1.015 1.025 1.010 1.015 . 1.025 1.010 1.015 1.020 1.025 Calculated concentrations of standards in thVe s' ample matrix PFOS Final cone ng/mL 4.93 PFOSA Final cone ng/mL 5.00 PFOSAA Final cone ng/mL 5.24 EtFOSE . PFOSEA -5>M556 ' Surrogate Final cone Final cone Final cone Sam c ng/mL ng/mL ng/mL i^ng/raL 4.94 5.01 5.13.S t 100. All Am't spiked mL 0.005 9.76 24.8 4M 97.6 248 493 9.89 25.1 SO.O 98.9 251 500 . 10.4 26.3 52.4 104 263 524 9.78 24.8 49.4 97.8 248 494 9.93 25.2 50.1 99.3 . 252 501 10.2 X .,, y 25.8 Surrogate 5L3 Final cone; P \ ' 102 258 "Ss^Af V 513 735 976 746 . 989 782 1038 737 1 - 7 4 9 978 1 993 766 1017 if Validated ranges - approximate conceutrations Senna PFOS- PFOSA PFOSAA Rabbit 5.00-1000 1 5.00-1000 1 5.00-1000 Bovine Estimatesonto. Use values for rabbit Rat Estimatesonly. Use values foerabbit. Monkey & Plasma Estimates only. Use values for rabbit Human Estimates only. Use values forrabbit EtFOSE-OH PFOSEA 1 5.00-1000 1 5.00-1000 \-vM556 ^ 1 5.00-1000 . " . i j f 3 Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extraction of PFOS from Scram j, Centre Analytical Laboratories, Inc. Page 14 of 14 Page 29 of 29 Research Page 92 of 98 Exygen Study No.: 023-066 Centre Analytical Laboratories, Inc. 3048 Research Drive, Stote College, PA 16801. Phone: (814)231-8032, Facsimile: (814)231-1253 PROTOCOL AMENDMENT Page 1 of 1 Centre Study Number: 023-066 Amendment Number: I Effective Date: 06/27/01 Centre Protocol Number: 01P-023-066 DESCRIPTION OF AMENDED SECTION 1. 7: Justification for the Selection of the Test System AMENDED TO Change the first sentence to read: Mallard and quail red blood cells and serum samples collected by Wildlife International, Ltd. will be used as the analytical samples for this phase. R A T IO N A L E 1. At the request of the sponsor, all serum samples are to be analyzed with this study. IMPACT ON THE STUDY 1. No negative impact on the study. Stud11y/ Dn tirrae/ 'cftrotrr SC iign nn an tfuu r e * \ Date i* Centre QAU Review tjh jii February 12,1998/1 Exygen Research Page 93 of 98 RESEARCH ^Precise Research. \ Proven Results. Exygen Study No.: 023-066 Exygen Research Page 94 of 98 Exygen Study No.: 023-066 Centre Analytical Laboratories, Inc. 3048 Research Drive, State College, PA 16801. Phone: (814) 231-8032, Facsimile: (814) 231-1253 PROTOCOL DEVIATION Deviation Number: 1 ' Date o f Occurrence: (1,2) 05/23/01, (3) 05/24/01 Centre Study Number: 023-066 Centre Protocol Number: 01P-023-066 DESCRIPTION OF DEVIATION 1. 9 Analytical Method, Sample Analysis d.: Quantified Centre sample 0107261 and its duplicate injection when the concentrations were greater than that o f the highest standard in Set 052201AD. 2. 9 Analytical Method Sample Analysis e.: Accepted recovery o f 76%.the duplicate injection of Centre sample 0107378 Spk A2 in Set 052201B. 3. 1. 9 Analytical Method, Sample Analysis d.: Quantified Centre samples 0107092 and 0107134 and their duplicate injections when the concentrations were greater than that o f the highest standard in Set 052201CD. ACTIONS TAKEN i.e.. amendment issued. SOP revision, etc.. 1-3. Protocol deviation issued. Recorded B y/D ate:___________ IMPACT ON THE STUDY 1,3. No negative impact because the areas were < 15% greater than the highest standard. 2. No negative impact because the overall average o f all of the fortifications were within the acceptable range. fPoBcipal Inv| stigator Signature P- dy Director Signature / Date A \ Date J k -> Sponsor Management Signature Date CAL QAU Review. tiCL q/y/oz js W y fta rree -;1to-ec) Vo S u s p e d c d February 12,1998/2 Exygen Research Page 95 of 98 Exygen Study No.: 023-066 Centre Analytical Laboratories, Inc. 3048 Research Drive, State College, PA 16801. Phone: (814) 231-8032, Facsimile: (814) 231-1253 PROTOCOL DEVIATION Deviation Number: 2 ' Date o f Occurrence: (1) 5/18/01 Centre Study Number: 023-066 Centre Protocol Number: 01P-023-066 DESCRIPTION OF DEVIATION 1. 9 Analytical Method, 3. Sample volumes used for Centre samples 0107080, 0107081,0107084, and 0107085 were 0.05 r L, 0.03 RL, 0.025 uL, and 0.025 rL, respectively, i ro(D t * 0 vO ACTIONS TAKEN i.e.. amendment issued. SOP revision, etc.. 1. Protocol deviation issued. Recorded B y/D ate:_________ IMPACT ON THE STUDY 1. No negative impact because the actual volume was used in the calculations. 4 ? ,1 nncipal Investigator Signature Date Study Director Signature ilb k Date Sponsor Management Signature / Date CAL QAU R eview . S /g /d S tJC U i t ' l l (TL- February 12,1998/2 'tf u 'i c W a -b o r i H \oe-\Na r e - i ^ u e d u L -4 o S u s p e c f'& f1 0 > * s ( r | h Ic2. Exygen Research Page 96 of 98 Exygen Study No.: 023-066 Centre Analytical Laboratories, Inc. 3048 Research Drive, State College, PA 16801. Phone: (814) 231-8032, Facsimile: (814) 231-1253 PROTOCOL DEVIATION Deviation Number: 3 ' Date of Occurrence: (1) 7/23/01 Centre Study Number: 023-066 Centre Protocol Number: 01P-023-066 DESCRIPTION OF DEVIATION 1. 9 Analytical Method, 3. Sample volumes used for Centre samples 0107159,0107167,0107168, and 0107209 were 0.05 mL. ACTIONS TAKEN i.e.. amendment issued. SOP revision, etc.. 1. Protocol deviation issued. Recorded By/D ate:_________ IMPACT ON THE STUDY 1. No negative impact because the actual volume was used in the calculations. stigator Signature StuIldHyv TD>iirrfec.f*ttonrr SSiiognnaattnurrAe Sponsor Management Signature Date f>\\Ao3 Date 1mlZ Date CAL QAU Review. February 12, 1998/2 iI Exygen Research Page 97 of 98 RESEARCH i Precise Research. Proven Results. Exygen Study No.: 023-066 Exygen Research Page 98-of 98