Document OJa7Vr2D1XV1qp5MOXgg20R6K

/)/ea~/g5f 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 001210 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. L Sean Gallagher Study Director Wildlife International, Ltd. Exygen Research Date Date Date 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 G ';l 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: f r / z - M __________ Date Exygen Facility Management: ic-oZ Date Study Director, Wildlife International: Sean Gallagher Wildlife International, Ltd. ' Date Exygen Research 2 Date 03 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 POBox 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 FACIL1TY/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 M ETHOD...........................................................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 1. LIST OF TABLES Page Summary of PFOS in Reagent Blanks............................................................ 21 Table n. Summary of PFOS in Mallard Red Blood CellBlanks...................................22 Table HI. Summary of PFOS in Mallard Serum Blanks.................................................22 Table IV. Summary of PFOS in Quail Red Blood Cell B lanks..................................... 23 Table V. Summary of PFOS in Quail Scrum 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 VIII. Summary of PFOS Recoveries in Quail Red Blood C ells............................. 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 XIH. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 1 0 ............................................................................................................... 30 Table XIV. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 1 0 ............................................................................................................... 31 Table XV. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 1 5 ............................................................................................................... 32 Table XVI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 1 5 ............................................................................................................... 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 XXH Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 10..........................................................................................................................37 Table XXIJ3. 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 TER M ............................................................................................................... 39 Table XXVI. Summary of PFOS in Quail Red Blood Cell Samples at 0, ppm a.i. for Week 5 ........................................................................................................40 Table XXVH 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 XXXVUI. 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 1 5 51 Table XLI. Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for T E R M ....... 51 Exygen Research Page 9 of 98 Exygen Study No.: 023-066 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFOS...................................................................53 Figure 2. Chromatogram Representing a 0.1 ng/mL standard for P F O S ........................ 54 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PF O S........................ 55 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Exygen ID Reagent Blank A, Set: 051801 A )................................................. 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-W k l5, 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 10 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 01 P-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-EIectrospray/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 Exygen ID 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 (CsFnSCL') O C8F17S -- cr O Note: The neutral molecule and standard form from which PFOS (anion) is derived, is potassium perfluorooctanesulfonate [CgFnSCLK], 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)] 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: Analyte PFOS Mode Transition Monitored negative 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 fmL)) 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 ED 0003803 Spk A (Set: 112100B), fortified at 10 vith PFOS, where: peak area = 6558 intercept = 515 slope dilution factor ng added (fort level) = = = 7740 1 1 ng avg. amt in controls final volume extraction volume aliquot volume = 0 (Not quantifiable) = 1 mL -- 5 mL = 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 fig (4 mL x 0.1 mL) 1000 ng = 0.00976 pg/mL From equation 3: % Recovery = ((0.00976 ug/mL x - 0 ug/mLl x 10001x 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 G 'il- 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 COlVi -4 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 05180IB 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 (ng/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 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 5/23/01 7/19-20/01 7/19-20/01 7/21/01 7/21/01 7/21-22/01 7/21-22/01 7/25/01 7/25/01 NQ ND ND ND ND NQ ND NQ NQ 7/24/01 7/24/01 7/24-25/01 7/24-25/01 AVERAGE: STANDARD DEVIATION: NQ NQ NQ NQ NQ NQ * Duplicate injection NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) 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 o f 98 O O lX .iO 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 05210IB 052201A 052201A 05220IB 05220IB 052201C 052201C Extraction Analysis Analyte Date Date Found (pg/mL) 5/21/01 5/22/01 5/21/01 5/22/01 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 0.00224 0.00184 NQ NQ 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 Date Analysis Analyte 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 05180IB 051801B 052101A 052101A 052201C 052201C Extraction Date Analysis Analyte Date Found (pg/mL) 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/21/01 5/22/01 5/21/01 5/22/01 5/22/01 5/23/01 5/22/01 5/23/01 AVERAGE: STANDARD DEVIATION: NQ NQ NQ NQ NQ NQ NQ NQ 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 07230IB 07230IB 072301C 072301C 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 AVERAGE: STANDARD DEVIATION: 0.00309 0.00272 NQ NQ 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 98 001 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 052201B 052201B 052201B 052201B 052201C 052201C Extraction Analysis Amt Date Date Added (ng/mL) 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 5/23/01 10 10 50000 50000 10 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 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 10 50000 50000 10 10 50000 50000 50000 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 |ig/mL for red blood cells and serum Exygen Research Page 24 of 98 Exygen Study No.: 023-066 Table VIL Summary of PFOS Recoveries in Mallard Serum Sponsor ID control control control control control control control control control control control control Centre Set ID Number 0107377 Spk A3 0107377 Spk A3* 0107377 Spk B3 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* 071901A 071901A 071901A 071901A 072001A 072001A 072001A 072001A 072001B 07200IB 07200IB 07200IB Extraction Analysis Amt Date Date Added (ng/mL) 7/19/01 7/19/01 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 7/19-20/01 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 10 10 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 |Jg/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 1D 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 052101A 0107376 Spk A* 052101A 0107376 Spk B 052101A 0107376 Spk B* 052101A 0107376 Spk A 052201C 0107376 Spk A* 052201C Extraction Analysis Amt Date Date Added (ng/mL) 5/18/01 5/18/01 5/18/01 5/21-22/01 5/21-22/01 5/21-22/01 10 10 50 5/18/01 5/18/01 5/21-22/01 5/21-22/01 50 10 5/18/01 5/18/01 5/18/01 5/21/01 5/21/01 5/21-22/01 5/21-22/01 5/21-22/01 5/22/01 5/22/01 10 50 50 10 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 (Jg/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 ID Set Number 0107375 Spk A 072301AR 0107375 Spk A* 072301AR 0107375 Spk B 072301AR 0107375 Spk B* 072301AR 0107375 Spk A2 07230ID 0107375 Spk A2* 072301D 0107375 Spk B2 07230IB 0107375 Spk B2* 07230IB 0107375 Spk A3 072301D 0107375 Spk A3* 072301D 0107375 Spk B3 072301C 0107375 Spk B3* 072301C Extraction Analysis Amt Date Date Added (ng/mL) 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 |Jg/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 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 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 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/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 Date Date 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 , 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 24.4 24.7 11.2 10.0 11.8 12.3 15.1 13.1 30.1 30.8 4.88 3.95 18.9 18.8 26.4 28.5 17.8 8.80 * Duplicate injection LOQ = 0.01 ng/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 052201 CD 052201CD Extraction Analysis Date Date 5/22/01 5/22/01 5/22/01 5/24/01 5/24/01 5/24/01 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ue/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-Wkl0 454-109-3250-WklO* 454-109-3251-Wk 10 454-109-3251 -Wk 10* 454-109-3252-Wkl0 454-109-3252-Wk 10* 454-109-3253-Wkl0 454-109-3253-Wkl0* 454-109-3254-Wk 10 454-109-3254-WklO* 454-109-3255-Wk 10 454-109-3255-WklO* 454-109-3256-WklO 454-109-3256-Wkl0* Centre ID 0107250 0107250 0107251 0107251 0107252 0107252 0107253 0107253 0107254 0107254 0107255 0107255 0107256 0107256 0107257 0107257 * Duplicate injection Set Number 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A Extraction Analysis Date Date 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 5/22/01 5/23/01 5/22/01 5/23/01 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 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 0.00245 0.000200 0.000600 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-WklO* 454-109-3290-WklO 454- 109-3290-WklO* 454-109-3291-Wkl0 454-109-3291 -WklO* 454-109-3292-WklO 454-109-3292-WklO* 454-109-3293-Wkl0 454-109-3293-Wkl0* 454-109-3294-Wk 10 454-109-3294-WklO* 454-109-3295-WklO 454-109-3295-WklO* 454-109-3296-WklO 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 5/22/01 5/23-24/01 5/23-24/01 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/pi 5/23-24/01 5/23-24/01 5/23-24/01 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 5/22/01 5/22/01 5/23-24/01 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 (ag/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-Wk 15 454-109-3249-Wk 15* 454-109-3250-Wk 15 454-109-3250-Wkl5* 454-109-3251-Wk 15 454-109-3251 -Wkl 5* 454-109-3252-Wkl5 454-109-3252-Wk 15* 454-109-3253-Wkl5 454-109-3253-Wkl5* 454-109-3254-Wk 15 454-109-3254-Wkl5* 454-109-3255-Wkl5 454-109-3255-Wkl 5* 454-109-3256-Wkl5 454-109-3256-Wkl5* 454-109-3236-Wk 15 454-109-3236-Wkl5* Centre ID 0107266 0107266 0107267 0107267 0107268 0107268 0107269 0107269 0107270 0107270 0107271 0107271 0107272 0107272 0107273 0107273 0107274 0107274 Set Number 052201B 052201B 052201B 05220IB 052201B 05220IB 05220IB > 052201B 05220IB 05220IB 05220 IB 052201B 05220IB 052201B 052201B 05220IB 05220IB 05220IB Extraction Date Analysis Date Analyte Found (ue/mL) 5/22/01 5/22/01 5/23/01 5/23/01 NQ NQ 5/22/01 5/22/01 5/23/01 5/23/01 NQ NQ 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 0.00179 0.00188 NQ NQ NQ NQ ND ND 5/22/01 5/23/01 NQ 5/22/01 5/23/01 NQ 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 AVERAGE: NQ NQ NQ NQ 0.000243 STANDARD DEVIATION: 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-Wkl5 454-109-3289-Wkl5* 454-109-3290-WklS 454-109-3290-Wk 15* 454-109-3291-Wk 15 454-109-3291-Wkl5* 454-109-3292-Wk 15 454-109-3292-Wk 15* 454-109-3293-Wkl5 454-109-3293-Wkl5* 454-109-3294-Wkl5 454-109-3294-W kl5* 454-109-3295-Wkl5 454-109-3295-Wk 15* 454-109-3296-Wk 15 454-109-3296-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 052201BDR 052201BDR 052201BDR 052201BDR 052201BDR 052201BDR 052201BD 052201BD Extraction Analysis Date Date 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: STANDARD DEVIATION: Analyte Found (pg/mL) 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 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 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) 275 272 406 418 343 80 LOQ = 0.01 |ig/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 Date Analysis Date 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 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 i 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 072001 AD 072001 AD 072001 AD 072001 AD 072001 AD 072001 AD 072001 AD 072001 AD 072001 AD 072001 AD Extraction Date Analysis 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 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 Centre ==mi__i=j r ^ ===s=_ =_= = j D =^ ^ 454-109-3329-Wk5 0107317 454-109-3329-Wk5* 0107317 454-109-3330-Wk5 0107318 454-109-3330-Wk5* 0107318 Set Extraction Analysis Analyte 072001AD 072001AD 072001AD 0720Q1AD 7/20/01 7/22/01 376 7/20/01 7/22/01 400 7/20/01 7/20/01 7/22/01 320 7/22/01__________ 290 AVERAGE: STANDARD DEVIATION: 347 50 * Duplicate injection LOQ = 0.01 |4g/mL for red blood cells and serum Exygen Research Page 35 of 98 COI 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-WklO* 454-109-3250-Wkl0 454-109-3250-WklO* 454-109-3251-Wkl0 454-109-3251-Wkl0* 454-109-3252-Wkl 0 454-109-3252-WklO* 454-109-3253-WklO 454-109-3253-Wk 10* 454-109-3254-Wkl0 454-109-3254-Wkl0* 454- 109-3255-Wkl0 454-109-3255-Wkl0* 454-109-3256-WklO 454-109-3256-WklO* Centre ID 0107322 0107322 0107323 0107323 0107324 0107324 0107325 0107325 0107326 0107326 0107327 0107327 0107328 0107328 0107329 0107329 * Duplicate injection Set Number 07200IB 07200IB 07200IB 072001B 07200IB 07200IB 07200IB 07200IB 07200IB 07200IB 072001B 072001B 07200IB 072001B 072001B 07200IB Extraction Analysis Date Date 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 , 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/20/01 7/20/01 7/21-22/01 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 (jug/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 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 |ig/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 COI 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-Wk 10* 454-109-3290-WklO 454-109-3290-WklO* 454-109-3291-Wk 10 454-109-3291-WklO* 454-109-3292-Wk 10 454-109-3292-WklO* 454-109-3293-Wkl0 454-109-3293-Wk 10* 454-109-3294-WklO 454-109-3294-WklO* 454-109-3295-WklO 454-109-3295-Wkl0* 454-109-3296-WklO 454-109-3296-WklO* Centre ID 0107330 0107330 0107331 0107331 0107332 0107332 0107333 0107333 0107334 0107334 0107335 0107335 0107336 0107336 0107337 0107337 Set Number 072001BD 072001 BD 072001 BD 072001BD 072001BD 072001BD 072001 BD 072001 BD 072001BD 072001 BD 072001 BD 072001BD 072001BD 072001 BD 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 7/22/01 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: Anaiyte Found (ug/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 |ig/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-Wkl5 454-109-3236-Wkl5* 454-109-3249-Wkl5 454-109-3249-Wk 15* 454-109-3250-Wkl5 454-109-3250-Wk 15* 454-109-3251-Wkl5 454-109-3251-Wkl5* 454-109-3252-Wk 15 454-109-3252-Wkl5* 454-109-3253-Wkl5 454-109-3253-Wkl5* 454-109-3254-Wkl 5 454-109-3254-W kl5* 454-109-3255-Wkl5 454-109-3255-Wkl5* 454- 109-3256-Wkl 5 454-109-3256-Wk 15* 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 7/19-20/01 7/19-20/01 7/19/01 7/19/01 7/19-20/01 7/19-20/01 7/19/01 1 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 7/19/01 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 7/19/01 7/19-20/01 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 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/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 c01 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-Wk 15 454-109-3289-Wkl5* 454-109-3290-Wk 15 454-109-3290-Wkl5* 454-109-3291-Wkl5 454-109-3291-Wkl5* 454-109-3292-Wkl5 454-109-3292-W kl5* 454-109-3293-WU5 454-109-3293-Wkl5* 454-109-3294-W k l5 454-109-3294-Wk 15* 454-! 09-3295-Wk 15 454-109-3295-Wkl5* 454-109-3296-W k l5 454-109-3296-Wk 15* Centre ID 0107347 0107347 0107348 0107348 0107349 0107349 0107350 0107350 0107351 0107351 0107352 0107352 0107353 0107353 0107354 0107354 Set Number 071901AD 071901 AD 071901 AD 071901 AD 071901AD 071901 AD 071901 AD 071901AD 071901AD 071901 AD 071901 AD 071901 AD 071901AD 071901 AD 071901 AD 071901 AD Extraction Date Analysis Date 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 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 (ug/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 072001AD 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/mL) 685 601 664 601 638 43 LOQ = 0.01 |Jg/mL 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 Date Analysis Date 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18-19/01 5/18-19/01 5/18-19/01 5/18-19/01 5/18-19/01 5/18-19/01 5/18-19/01 5/18-19/01 5/18-19/01 5/18-19/01 5/18-19/01 5/18-19/01 5/18-19/01 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 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/21-22/01 5/21-22/01 5/22/01 5/22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/21-22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ufi/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 |Jg/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 052201 CD 052201CD 052201CD Extraction Date Analysis Date 5/22/01 5/22/01 5/22/01 5/22/01 5/24/01 5/24/01 5/24/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/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-WklO 454-108-361 -Wkl 0* 454-108-362-Wkl0 454-108-362-Wkl0* 454-108-363 -Wk 10 454-108-363-Wkl0* 454-108-364-Wkl0 454-108-364-Wkl0* 454-108-365-Wk 10 454-108-365-Wkl0* 454-108-366-Wk 10 454-108-366-Wkl0* 454-108-367-Wk 10 454-108-367-Wkl0* 454-108-368-Wk 10 454-108-368-Wkl 0* 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 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B Extraction Analysis Analyte Date Date Found (ue/mL) 5/18/01 5/19/01 NQ 5/18/01 5/19/01 NQ 5/18/01 5/19/01 NQ 5/18/01 5/19/01 NQ 5/18/01 5/19/01 NQ 5/18/01 5/19/01 NQ 5/18/01 5/19/01 NQ 5/18/01 5/19/01 NQ 5/18/01 5/19/01 NQ 5/18/01 5/19/01 NQ 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/19/01 NQ 5/19/01 NQ 5/19/01 NQ 5/19/01 NQ 5/19/01 NQ 5/19/01 ____ NQ_____ AVERAGE: NQ STANDARD DEVIATION: 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-Wk 10* 454-108-402-WklO 454-108-402-Wk 10* 454-108-403-WklO 454-108-403-Wk 10* 454-108-404-Wkl0 454-108-404-Wk 10* 454-108-405-Wkl0 454-108-405-Wkl0* 454-108-406-WklO 454-108-406-Wk 10* 454-108-407-WklO 454-108-407-WklO* 454-108-408-Wk 10 454-108-408-Wk 10* 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 Analyte Date Date Found (pg/mL) 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 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 AVERAGE: STANDARD DEVIATION: 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 |jg/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-Wkl 5 454-108-361-Wkl5* 454- 108-362-Wk 15 454-108-362-Wkl5* 454-108-363-Wkl5 454-108-363-Wkl5* 454-108-364-W k l5 454-108-364-Wkl5* 454-108-365-Wkl5 454-108-365-Wkl5* 454-108-366-Wk 15 454-108-366-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 0107116 0107116 0107117 0107117 0107118 0107118 0107119 0107119 0107120 0107120 0107121 0107121 0107122 0107122 0107123 0107123 0107124 0107124 * Duplicate injection Set Number 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A Extraction Date Analysis Date 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 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 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 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 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 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-Wkl5* 454-108-402-Wk 15 454-108-402-Wk 15* 454-108-403-Wk 15 454-108-403-Wk 15* 454-108-404-Wk 15 454-108-404-W kl5* 454-108-405-Wk 15 454-108-405-Wkl5* 454-108-406-Wk 15 454-108-406-Wkl5* 454-108-407-Wk 15 454-108-407-Wk 15* 454-108-408-Wkl5 454-108-408-W kl5* 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 Extraction Analysis Number Date Date 052101AD 052101AD 052101AD 052101 AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101 AD 052101AD 052101AD 052201CD 052201CD 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/24/01 5/24/01 052101AD 052101AD 5/21/01 5/22-23/01 5/21/01 5/22-23/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 72.2 69.1 2.36 1.65 21.2 18.8 1.30 1.22 15.8 17.9 3.61 3.23 13.3 9.60 1.24 1.55 56.7 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 052201 CD 052201CD 052201 CD Extraction Date Analysis 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 (ug/mL) 361 356 106 114 234 144 * Duplicate injection LOQ = 0.01 |Jg/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 07230IAR 072301AR 07230 IAR 07230 IAR 072301AR 07230 IAR Extraction Date Analysis 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 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 , 7/23/01 7/25/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ufi/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 jig/inL for red blood cells and serum Exygen Research Page 46 of 98 0 1 "= 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-l08-407-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 072301 AD 072301 AD 072301AD 072301 AD 072301AD 072301 AD 072301 AD 072301 AD 072301 AD 072301 AD 072301 AD 072301AD 072301 AD 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 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 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 (mk/iuL) 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 7/23/01 7/23/01 7/23/01 7/26/01 7/26/01 7/26/01 7/26/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 551 562 529 520 541 19.4 * Duplicate injection LOQ = 0.01 [ag/mL for red blood cells and serum Exygen Research Page 47 of 98 oo: 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-Wk 10 454-108-361-Wkl0* 454-108-362-WklO 454-108-362-WklO* 454-108-363-Wkl0 454-108-363-Wkl0* 454-108-364-WklO 454-108-364-WklO* 454-108-365-Wkl0 454- 108-365-Wk 10* 454-108-366-WklO 454-108-366-WklO* 454-108-367-Wkl0 454-108-367-Wkl0* 454-108-368-WklO 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 072301B 072301B 07230IB 07230IB 072301B 07230IB 07230IB 072301B 07230IB 072301B 072301B 072301B 072301B 07230IB 072301B Extraction Analysis Date Date 7/23/01 7/24/01 7/23/01 7/24/01 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 7/23/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 , AVERAGE: STANDARD DEVIATION: Analyte Found (ug/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 |ig/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 -WklO* 454-108-402-Wk 10 454-108-402-WklO* 454-108-403-WklO 454-108-403-Wkl0* 454-108-404-WklO 454-108-404-WklO* 454-108-405-Wkl0 454-108-405-Wkl0* 454-108-407-Wk 10 454-108-407-WklO* 454-108-408-WklO 454-108-408-Wk 10* Centre ID 0107185 0107185 0107186 0107186 0107187 0107187 0107188 0107188 0107189 0107189 0107190 0107190 0107191 0107191 Set Number 072301DD 072301 DD 072301 DD 072301DD 072301 DD 072301DD 072301D 072301D 072301 DD 072301DD 072301 DD 072301DD 072301DD 072301DD Extraction Analysis Date Date 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 1 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 (us/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 |ig/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-Wk 15 454-108-362-Wkl5* 454-108-363-Wkl5 454-108-363-Wk 15* 454- 108-364-Wkl5 454-108-364-W kl5* 454- 108-365-Wk 15 454-108-365 -Wk 15* 454-108-366-Wkl5 454-108-366-Wkl5* 454- 108-367-Wkl5 454-108-367-Wkl5* 454-108-368-Wk 15 454- 108-368-Wk 15* 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 Date Analysis Date 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 , 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 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-Wk 15 454-108-402-Wk 15* 454-108-403-Wk 15 454-108-403-Wk 15* 454-108-404-Wk 15 454-108-404-Wk 15* 454-108-405-Wk 15 454-108-405-Wk 15* 454-108-406-W k l5 454-108-406-Wkl5* 454-108-407-Wkl5 454-108-407-Wkl5* 454- 108-408-Wk 15 454- 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 072301 DD 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 7/23/01 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 7/23/01 7/27-28/01 7/27-28/01 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 7/23/01 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 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/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 Date Analysis Date 7/23/01 7/23/01 7/27-28/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/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 I 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 Exygen Research Page 54 of 98 Exygen Study No.: 023-066 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PFOS Exygen Research Page 55 of 98 cn m Exygen Study No.: 023-066 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Exygen ID Reagent Blank A, Set: 051801A) Sample Name: *OfiiaoiA>ltO' Sample ID: 'Reagent Bfcnh" Pm * Name. 'PFOS' 4M.IVW.0 mu' Comment 'Reagent alerte A' Aimotabcn: * Sample Index i 10 Sample Type I Unknown ^ ncentrat ion i n/a le u la t e d Cone i * 0 Acq. D atei 05/18/01 Aeq. T inei D109 116 : File: 'quell blood.** M odified. nchlnq F ectoi i N oiee Thresholdi Threshold i Nu b . Smoothes No 1 E x p ected KT. Sep. W idth. Sep. H eight. Exp. Peak R a cio i Exp. Ad}. R a tio i Exp. V al. R a tio Uee R e l a t iv e RTi Vo In t. Type. Bear To Baae R e t e n t io n Time i 4 .4 2 tain A reas 1099.9 counte H eight! l.tle * 0 C 2 opa sta rt T iee: 4.19 in End T in e i 4 .5 1 min i 13591 |!. i In *l\'-5eo* -147 272 96/h,JV^ t v l4 A 7 W ^ y g ; / ' 2 0 2 6 3.0 3.5 46 60 SS 80 5 70 76 Exygen Research Page 56 of 98 O' 1 Exygen Study No.: 023-066 Figure 5. Chromatogram Representing Control Quail Red Blood Cells for PFOS (Exygen ID: 0107376 Blank A, Set: 051801A) I StnuJeName: *061#0tA-l12* Sample ID: 'Blank' 1 Peak Nam* -PFOS '4 9 9 0 * 9 .0 amu' Comment: '0107376 Blank A` AnnctaBon: " Stap le Index: 12 S ta p le Type: Unknown C oncentrationi tt/A C alculated Conei Acq. D atei 0 1 /1 8 /0 1 Acq. Time: 0 4 : 1 4 :3 s PH Fie M M odified > Bunch i ng P r t o n H oiee Thteahold: Are* T hreehold: Mu* Sm ooth: RT Window F.xpected IT . S ep . W idth: Sep. H eight: Exp. Peek ile t iu i Exp. A dj. B etioi Exp. V al. R eto: Uee R e l i t iv a RTi Nc 1 .7 6 .8 0 1 0 .0 < -tl 0 .2 0 0.01 1 .0 0 4 .0 0 3 .0 0 No epe epe aee 64 62 to n 76 I n t. Types B a to To Btae R e t e n t io n Timo > 4 .4 3 min Asee i 110.4 counts Height: i 6 . 2 1 # 00 1 epe S t e t t Ttmei 4 -12 min End Timei 4 .1 ? min 70 4 4295 666452 60- 64 S2 I a 40 36 36 34 32 X 24 2220II 16>4- Exygen Research Page 57 of 98 c f'H.A. ' Exygen Study No.: 023-066 Figure 6. Chromatogram Representing Control Quail Serum for PFOS, (Exygen ID: 0107375 Blank A, Set: 072301C) ISample Name. '07J30IC -312' Sample 10.'Blank' Peak Mama: 'P FO S' Maaa(ea): *499 (VM 0 arm.* Comment *0107375 Blank A3' Amrtaticrv ** Saatpl* In d em Sam ple Type i 12 Unknown C oa nl ccuelnattreadt i oCn : N/A File `quail arum ntlf 270 M odified: Bunching F a c to r) N oiae T hreaholdi Are Tftceaholdi Nun. Seoothei RT Window: E x p e c te d RTi 4.9806 1 )4 8 .B 2 .43e*002 1.28 200 196 190 166 160 17S 170 1S l0 1&S160 145 I 140 135 190 125 120 115 no 105 96 90 5 60 76 70 66 5606 i\!\ 1 1.4220 1 9002 :MvV ^ ^V \J 20 2 6426 3 296^ * *-"**-i 2 6 3.0 9 5 6.0 5 6 6.0 6 S 7 0 7 5 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) 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) I Sample Name: `072301D-S09* Sample IO: `Sph A' Peek Name' 'PFOS' M*M{eeJ '4 M 9 9 .0 aim / Comment *0107876 Spk A2 Cup InJ - An Buapl* Index > S( Saap lc Type : QC C o i c e T .t * ti o T > s a .01 rwj le i. n U li d Cone; 0.905 ng/mL Date i 07/26/01 T ie. 0 7 i 19>02 PH M o d i < iedi Bunching f e c te r . Naif Thiffheld. Ac* Thrfhold. Nue. SeosChs. RT in d o * i E x p ccted RT. sp ttidtl.. Sep. H eighci Enp. Pk R it i , txp. Adj. R atio. Exp . V a l. R a tio R a la t lo RTi In t. Type. 1 0 .0 1 5 0 .2 0 3 0 .0 4 .0 0 3 .0 0 Ho epa cp **c 45004400430042004100 4000- n O 0 .5 counl .7J'003 cp. 4 .3 7 min 4 .8 2 min 3000 3600- 3700- 3800- 3500 3400-1 3300- 3200- 3100 3000 .4296 28002700 2800 26OG | . l i 2300 2200 2100 1900 1800 1700- 1600- 1400- 1300- 1200 1100- 1000- 900 B00 700 DO 900 400 300 200 too oLiizsia006870 1.1326 LO 17096-' 2 01342 3029 7 60822 9043 3 4006 1.6 2 0 2.6 3 0 9.6 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) I S tm p tt Name "OSI601CR-417' SampM ID: 'S am ple' Fil*: 'q udl blood wilt* Peak Nm#: *PF06' `499 0/96.0 arou' C om m ent'0107064 (464-106*401-YVkS) DP * SOO* ArmoMMon.'* 5SaAmmple TInydpe.x . Unk)6nown C oncentretion: C a lc u lated Cone/ N/A 4.54* rij/rnL 1.06*4- Acq. D ate. Aeq. Time. 0 5 /2 1 /0 1 0 7 ,0 9 .5 3 PK 1.04*4 Nod i f i ad i Bunching f a c t o r . Noi.ee T h resh o ld . Area T tiraeh old. nub. sm oothsi RT Window. E x p ec ted RT. Sep. W idth. Sep. H eight i Exp. Peak R a tio : Exp. A dj. R atio i Exp. V*1 . R a tio . U se R e l a t iv e RT. Yes 1 10.04 SO. 20 2 3 0 .D 1 .3 * 0 .2 0 0 .0 1 5 .0 0 4 .0 0 3 .CO No m e . Type. Hernial R eten tio n Tima: * .4 1 min Area: 76457.1 count. H e.flht: 1 .0 * a * 0 0 l epa S ta r t Tima. 4 .2 6 min End Timei 4 .8 3 min 1.02*41 00*4saw oo M X 0094X QC 9200 009003 006600 0066X .X B4C0.0Q- 6200 00- ooo.oo- 7600.X - 74M .X 7300 00 7000.00BBOC.OO 6600 00 6*00 00 6200.00 5600.00 5600 00 I 5*00.00 5200.005000.00 4600.00 4600.00 4400.00 4200.00 4000 00 36X00 00 X00.00 3200 00- I 4043 Exygen Research OS 1 0 1 6 2 0 2.6 3.0 3.6 4 0 4 5 ' s 'o 6 5 fl.O 6 5 7C 7.5 _____________________________________ ___ ________________________Tima, mr._______ _______________________________________________________ [ I Page 61 of 98 Exygen Study No.: 023-066 Figure 10. Chromatogram Representing Quail Serum Sample (Exygen ID: 0107207, Sponsor ID: 454-108-407-Wkl5, Set: 072301DD) 0107207 (454-108-407-Wk15) DF = 5000 072301 DO-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 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 _____________________________________________________________________ Page TABLE OF CONTENTS...................................................................................................... 2 1. PURPOSE.............................................. 1................................................................. 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.......................................................................................12 14. DATA AND REPORT............................................................................................ 12 15. ARCHIVE STATEMENT...................................................................................... 13 16. REFERENCES.........................................................................................................13 17. PROTOCOL APPROVAL......................................................................................14 APPENDIX: ANALYTICAL METHOD........................................................................... 15 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 lilSl 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 pcrfluorooctanesulfonate (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-8-4.1) 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 08731/01 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 (CsFnSOs" ) O II C$F17S O O Note: The neutral molecule and standard from which the PFOS (anion) is obtained is perfluorooctanesulfonate potassium salt [CsFnSOjK], 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 r 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-Q9 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 | j , Ii . | ; | [ j ; i; | j i i k j Centre Analytical Laboratories, Inc. Exygen Research Page 4 of 29 Page 67 of 98 Exygen Study No.: 023-066 CentreProtocol No. 01P-023-066 7. JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM Mallard 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 fed PFOS. 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 Seram 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 ue/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 ue/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 pg/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. (ug/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. (ug/mL) 0.005 0.002 0.001 0.0005 0.0002 0.0001 Store all calibration standard solutions (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximum period of 6 months from the date of preparation. 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: I: IP- I I g I Centre Analytical Laboratories, Inc. Exygen Research Page 6 of 29 Page 69 of 98 Exygen Study No.: 023-066 s 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 Column:Genesis C* (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 How Rate: 0.3 ibL/min. Time %B 0 60 40 1.0 0 100 7.0 0 100 7.5 60 40 11.0 60 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 C|g etc.) can be used, provided equivalent chromatography is obtained. Ions monitored: Analvte PFOS Mode negative Transition Monitored 499 -> 99 Approximate Retention Time ('mini 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 98 001 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 -4200.0 -51.0 -230.0 10.0 -70 -5 300.0 2800.0 Gas Flows Nebulizer Gas Curtain Gas Collision Gas TIS Temperature Sei 12 13 4 350C Calibration Procedures a. Inject the same volume (between 10 to 20 pL) of each calibration standard (prepared in MeOH) into the LC/MS/MS. b. Use linear, 1/x weighted standard curves for quantification. Linear standard curves are generated for each set by linear regression using the appropriate software system. Any calibration standards falling outside 30%, based on its calculated concentration, 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 20.9925 (^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 Cent 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 I HjO 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. j i jv $ p i \ | ! ! 1* j ; i Centre Analytical Laboratories, Inc. Exygen Research Page 9 of 29 Page 72 of 98 G O l' 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/ieference 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) = (Peak area - intercept! slope Equation 2: Analyte found (jrg/mL) = ((anal, found (ng/mL) x FV (mL) x DF x EV (mL)) x 1 ug (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 (ug/mL) - avg, anal, in Ctrl (ug/mL)) x (1000 ngfl ug)) x 100 amount added (ng/mL) 11. PROTOCOL AMENDMENTS AND DEVIATIONS I 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 the 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: Centre Analytical Laboratories, Inc. Page 11 of 29 Exygen Research Page 74 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-Q23-O66 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 l ' I j 1 ! i I j [. ; j. | jt : , j 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 i Page 76 of 98 Q -0X 2 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 j l 3 Lv jp s^ ' aL. Morgan y y Assurance Auditor Principal Investigator, Centre j U A >1 Date C Z L /r? JoohhnFnFIhlarhte r t Y 7 ' Operations Manager Study Director, Wildlife International, Ltd. Date 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 BEST COPY AVAILABLE Exygen Study No.: 023-066 Centre Protocol No. QlPr023-Q66. O 3.M E n v ir o n m en t AL LABORATORY V.. ' M ethod i; - ' Extraction of Potassium Perfluorooctanesulfonate or Other \ ^ uorochem3cal compounds from Serum for A nalysis Using HPLC- . Electrospray/M assSpectrometry S< y\ Method Number: ET^-8-4.1 ^ 'x 'X t s i. i Author; LisaClemen,^Gle&nn4 Langeabing Adoption Date: 03/01/99 : Revision Date: 1/3 W9 ApprovedBy: libi l i ~ LaboratoryManager U i-- Group Leader X !V ' fi NSy V , ' , :v-v \ Date W 'x V ' lh u /u /1 3 'M T e . , d * A CL ^ TechnicalReviewer o i/u /n -. & 1.0 Sco pe anp Application : M l* h; 1.1 Scope: This methodis for theextraction ofpotassiumperfluorooctanesulfonate (PFOS);-- or other fluorochemical compounds from serum. v* 1.2 Applicable compounds: Fluorochemical surfactants or other Quorinatedcompounds. ^ 13 Matrices: Rabbit, rat, bovine, monkey, andhuman serumor otherfluids asdesignated in the validation report J Word 6/95 J ETS4^.1 Ltaacdoo of PFOS from Serum Page 1of 14 Centre Analytical Laboratories, Inc. Page 16 of 29 Exygen Research Page 79 of 98 BEST COPY AVAILABLE Exygen Study No.: 023-066 .... . Centre Protocol No. 01P-023-066. .'W. JfIflvfP S - O' &<>_SVMMary o?M ethod i'-p'l-l .This method describesthe procedure for extractingpotassiumperfluorooctanesulfonate (PFOS)or otjjti fluorochemical surfactantsfrom serum,or otherfluids, using anion '( ' ^'pairing reaginUnd methyl-tert-butyl ether(MtBE). In thismethod, seven 'V, ^f^fluprochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH,PFOSEA, r tf ': M556, andsurrogatestandard (see3.0Definitions). Anionpairing reagentis addedto .the"sampleandthe analyteion pairis partitioned intoMtBE. TheMtBE extractis 'miremoVed andpot onto anitrogenevaporatoruntil dry. Eachextract is reconstitutedin 1.0 ' .nlf'ofjMtfianol, thenfiltered througha3ccplasticsyringeattachedto a02 jun nylon filterffito^ashautovials. Z2 Theseaampletracts'areanalyzedfollowingmethodETS-8-5.1 or otherappropriate methods.'^^' / \ 3,0 Definitions- 3-1 PFOS;petfluorooctraesurmde (anion ofpotassiumsalt) C,F17SOj' 3.2 PFOSA: perfhtorooclanfmlfonylamideC^17SO,NH, 3.3 PFOSAA: perfIuorooctaire"s1filiylamido (ethyl)acetateCtF11S01N(CH1CH1)CK,C0,' jw , 3.4 EtFOSE-OH: 2 (N-ethylperfluorooctanesulfonamido)-ethyl alcohol o C1F,,SOJN(CHJCHJ)CH1CH1O H \5^ 3.5 PFOSEA: prffuoroctanesulfonyl ethyUimde CJ|,SO,N(CH,CH^H 3.6 3.7 MSu5.r5.r.o.6.g.:.a.C.t.e..^.s..,t.a^..n.0.d..1a.N.r.d.C.:.H.1..).H(.C..-.1H..H.I.C.-.2..0H..0.-..2H..H.]...p..e.rfluor_oS_o_Qct?anfyesurilfoonxvic aj,cci<id 4,0 W arnings and C autions 4.1 Health and'sufetywarnings jfi'.'JZL, 4.1.1 Usduniversal precautions, especially laboratorycoats, goggles, andgloves when handling animal tissue, whichmay containpathogens. 5,0 iNTEUFERKwrys__________________ ________________ ^ - S.1 There areno interferencesknown at this time. 6.0 Equipment 'v* -v*. : V 6.1 The following equipment is usedwhile perfotmmg thismethod. Equivalent equipmentjs ^ acceptable. V 6.1.1 Vortex mixer, VWR, Vortex Genie2 . 6.1.2 Centrifuge, Mistral 1Q00orIEC o 6.13 Shaker, Eberbachor VWR .- ETS-W.1 Extraction ofPFOS fiom Serum Pige 2 of 14 Centre Analytical Laboratories, Inc. Page 17 of 29 Exygen Research Page 80 of 98 BEST COPY AVAILABLE Exygen Study No.: 023-066 . Centre Protocol No. 01P-023-066 nr. ry . 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance( 0.100 g) ' ' d p * 7 J u?>LIES AWC MATERIALS------------------------ \ 7'^ S JV" S * * ' : Eppendorfor disposable pipettes ''yv H Q ' 7-3 ,Nilgcne bottles, capableofholding250 mLand 1L n7.4A ^VolisjMtricflasks, glass,typeA 7.5 Tf-Cn HtmEM/.ivti.iatdli.;g_lia_ss,4in0_mrL. glass 7.6 Centrifuge tubesTpolypropytene, 15mL 7.7 Labels 7.8 OxfordDispenser-T-'3.0 to 10.0mL 7.9 Syringes, capableofmeasuring5 pL to 50 pL 7.10 Graduatedpipettes 1 ptif ' & 7.11 Syr.inges,disposableplaVstic,^3-cc? \ 7.12 Syringe filters, nylon, 0.2 pm,25 Sun . _ 7.13 Timer \ 7.14 Crimpcap autovialsandcaps 7.15 Crimpem . J/ ^ \' ' >- . Note: Pruriuoir trou ussiingg gglaasssswwaarree aannda bootutliecss,, riuinuske*j3tuimiueswitahi muiebtuhmaunwol uanwd-3j thimukess winthn MiUi-QTMwater. Rinsesyringes4 minimumofprimes methanol, 3rinses from 3 separatevials. M 8-0 R eagents ano Standards 8.1 Type1reagentgradewater,Milli:QTM or equivalent; all water usedin this method should beMilli-QTM waterandmaybeprovidedby aMilli-QTOC PlusTM systenif- 8.2 Sodiumhydroxide (NaOH), J.TBakeror equivalent V. 83 TefrabutylarornoniumhydrogensuifoteTBA),Kodakorequivalent 8.4 Sodium carbonate(Na,COj), J.T. Bakeror equivalent 83 Sodiumbicarbonate (NaHCOJ, J.T.Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled orHPLCgrade 8.7 Methanol, Omnisolv, glassdistilled or HPLC grade 8.8 Serumor blood, frozen from supplier 8.9 Fluorocbemicnlstandards ' 8.9.1 PFOS(3M Specialty Chemical Division),molecularweight- 538 j 8.9.2 PFOSA (3M SpecialtyChemical Division),molecular weight 499 ' ETS-J-4.1' Extraction ofPFOS fromS en a j Page 3 o f 14 Centre Analytical Laboratories, Inc. Page 18 of 29 Exygen Research Page 81 of 98 Ai BEST COPY AVAILABLE Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 I 0 o ' *:*--*,'.. -i j-i r\ \ .* .yr v . -A:C :. VXiV.ft :v- 8.9.3 PFOSAA(3MSpecialty ChemicalDivision), molecularweight585 9.4 EtFOSE-OH(3M Specialty ChemicalDivision), molecularweight 570 \ 8.9.5 PFOSEA.(3M Specialty Chemical Division), molecularweight 527 .8.9.6 M556 (3M Specialty Chemical Division), molecularweight - 557 8.9.7 Surrogale standard: 4-H, perfluorooctane sulfonic acid (1-H.l-H, 2-H, 2-H '"' C,F,,SO,H) molecularweight **428 ...8.9.81Other Ouorocbemicals, asappropriate 8 .1 0 -Reagentpreparation NOTE?/Whenpreparinglargervolumes thanlisted in reagent; standard, orsurrogate 'I y preparation, adjustaccordingly. ' 8.70.1 104N sodium'hydroxide(NaOH): Weighapproximately200gNaOH.Pourinto a . 1000mLb^cffjContauiing 500mLMilli-Q1*4water, mixuntil allsolids are i dissolved.yStore in f&a? a 1L Na. lgenebottle. 8.10.2 1 Nsodium'hydrpxide (NaOH): Dilute lONNaOH 1:10. Measure 10mL of 10 i .N NaOHsolutioninto a'lOOiriL volumetric Sasicanddilute to volume using ? Milli-QTM waterTVfStore H?a125mLNalgenebottle. 8.10J 0.5 M tetrabutylammoiuum'hydrogen sulfate(TBA): Weigh approximately 169 g ofTBA into a 1L volumetric containing500mL Milli-QTM water. Adjust to pH \ 10using approximately^ to 54mt^pf10NNaOH (While addingthe lastmL of NaOH, addslowlybecausethepH'changes abruptly). Dilute to volumewith Milli-Q1*4water. Storein aHINalgene bottle. ' 8.10.3.1'TBA requires s checkpriorto eachuseto ensurepH-10. Adjust as neededusing 1NNaOHsohfio&v' Jr 8.1 0.4 0.25 M sodiumcarbonate/sodiumbicarbonatebuffer(Na,COj/NaHCOj): Weigh approximately26J gofsodiumcarbonateQ>j&jCO,)md21,0g ofsodium bicarbonate(NaHCOj) into a 1L volumetric flaskawlbring to volumewith M illi- . QTM water. Store in a 1L Nalgenebottle. . flgsv 8.11 Standards preparation \ `. 8.11.1 PreparePFOSstandardsfor the standardcurve. ^ 8.1 1J Prepareotherfluorochemical standards, asappropriate. Multicomponent fluorochemicalstandardsareacceptable(for example, oneworkingstariSard solutioncontaining I.OOppmPFOS, L02 ppmPFOSA, 0.987ppmPjjdSAA}and 1.10ppmEtFOSE-OH.) V 8.1lJ Weighapproximately 1Q0mgofPFOSinto a700mL volumetric flasic`andrecord"v | the actual weight 8.11.4 Bring to volumewith methanol for astock standardofapproximately 1000p pm ^" (jig/mL). . r% 8.11.5 Dilute thestock solutionwith methanolfor aworkingstandard I soludonof approximately 50ppm. 8.11.6 Dilute working standard l with methanol for aworking standard2 solution of approx; 5.0ppm. ETS-S-4.1 ( ` Extraction a f PFOS from Scrura Page 4 of 14 Centre Analytical Laboratories, Inc. Page 19 of 29 Exygen Research Page 82 of 98 Exygen Study No.: 023-066 SEST COPY AVAILABLE Centre Protocol No. 01P-023-066 y?**.* "'< r' ; ) 8.11.7 Dilute working standard 1 with methanol for aworking standard3 solution of approx. 6JO ppm.: ,.- 8 .1 2 Surrogate stock standard preparation :l r " V ' V Weigh ipproximately 50-60mg ofsurrogatestandard 1-H.l-H, 2-H, 2-H, ' ' "V C^jSOjH into a50mL volumetricflask andrecordthe actualweight. 8.123 Bring to volumewith methanol for asurrogatestockofapproximately 1000-1200 ' jM. vppm-. tj![t8.123-!|Wepare asurrogateworking standard. Transferapproximately 1mLofsurrogate ' y " stockto a 10mL volumetric flaskandbring tovolumewith methanol for a working standardof 100ppm. Recordthe actualvolumetransferred. 9.0 Sample HArtpflrfc P - . ______ ^ ------------------------ :------!___ ' _ 9.1 All samples arereceivedfrozen andmust bekept frozen until the extraction isperformed. 9.2 _Allow samplestoNSkw^o roomtemperatureprior to extraction. 10.0 Q u a l it y C o n t r o l W a ________________ !------------------------- c_------------ 10.1 Solvent Blanks, MethoMdflan-AkYs?anVd matrix blanks 10.1.1 An aliquot of 1.0 mL me^mol is usedasasolventblank. 10.1.2 Extract two 1.0 mL ahqubtsofMillirQTM water following thisprocedure anduse . asmethodblanks. v 10.13 Extract two 1.0mL aliquots oftheserumlollqwing this procedureanduseas matr`ixblanks. See11.1.4. ^ 10.2 Matrix spikes ^ e y >x 1 0 .il Prepareandanalyzematrix spike andmatrixspike duplicate samplesto determine the accuracyoftheextraction .S ff 10.2.2 Prepare eachspike using asamplechosenbythe analyst,usuallythe control matrix receivedwith eachsampleset "V^ 10.23 Expected concentrationswill fall in the mid-rangeof theinitial calibration olive. Additional spikesmaybeincluded andmay fall in thelow-range'ofdie initial calibration curve. |p '' j5j 10.2.4 Prepareone matrix spike andmatrixspikeduplicateper40 samples,with a ij, minimum of2 matrix spikesperbatch. * ,^ jr' .< 103 Continuing calibration checks ^ r<"'*' > 103.1 Preparecontinuingcalibration checksamplesto ensurethe accuracyof theinitial " calibration curve. . *. * 103.2 Prepare, at aminimum, onecontinuing checkper group of 10samples. Fpr v example, if asampleset " 34, four checksarepreparedandextracted. ' 1033 Prepare eachcontinuingcalibration check from thesamematrix usedto prepare the initial curve. - ETS-8-4.1 ;j ^Extraction o f PFOS from Senna . Pate 5 of 14 Centre Analytical Laboratories, Inc. Page 20 of 29 Exygen Research Page 83 of 98 0012 BEST COPY AVAILABLE . Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 _ 103.4 The expectedconcentrationswill fall withirithe mid-rangeoftheinitial. calibrationcurve. Additional spikes maybe.includedthatfill in thelow-range of theinitial calibrationcurve. This is necessary.iftheanalystmustquantitateusing f- >- onlythelow endofthecalibrationcurve (forexample, 5ppb-100 ppb, rathen than * 5ppb-1000 ppb). '\ ``a'`w Oav'y'.KVl\ -551,I W C ALIBRATION AND STANDARDIZATION .* I 11-1 ^.^re^JUje matrii calibration standards ,. '31.1.1 ijrahsfer 1mL ofserumto a 15diL centrifugetube. 11.1 .2 Tfmostsample volumesarelessthan 1.0 ml;, extractstandardswith matrix 9 volumes'equal to thesamplevolumes. Do not extractlessthan0.50mL of m'eaytri'^TRecordeachsamplevolumeon theextractionsheet 11.13 Whilepreparing atotal'oftwenty aliquots m l 5mLcentnfugetubes, mix orshake bctweeVn'faliqpiu.'*o'*ts''! ;, 11.1.4 Two 1mL aliquots,'orotherappropriate volume, serveasmatrix blanks. Typicallyusethestandardconcentrationsandspiking amountslisted in Table 1, at die endofthissectiorCto spike, mduplicate, twostandardcurves, for atotal of eighteenstandardvtvro matrixblanks, andtwomethod blanks. 11.13 Referto validationreportjHEHTS' -S-4.0 & ETS-8-5.D-V-1, which lists theworking rangesandthe Linear Calibration Range(LCR) for calibrationcurves. 11.1.5 UseAttachmentD osanaidPrncalculatingtheconcentrations oftheworking standards. SeeSection13.0to'calculateactual concentrationsofPFOS in calibration standards. ' 'O. J*" t r V / v. 11.2 To each standard,blank,or continuing check,'addappropriateamountofsurrogate working standardfor theconcentration to fell within thecalibrationcurve: range55 pipb - 1000 ppb. v5#' W J 113 Extract spikedmatrix standardsfollowing 12.6-12.16 of.this method. Usethesestandards ' to establish eachinitial curveonthemassspectrometer. J ETS-8-4.1 i' Extractioa of PFOS from Seram ; : Yi.s Centre Analytical Laboratories, Inc. f'- J , X ?t*' ,'b'. v X Page ( o f 14 Page 21 of 29 Exygen Research Page 84 of 98 BESTCOPYAVAILABLb Exygen Study No.: 023-066 Cento. Protocol No. 01P-0234)6$ .. . -f M. o i is fe??- v" ' |, , >v*' ` - ' /-"'./p'v..? a- Table 1 . . ; Approximate spiking amounts for standards andspikes - .S ' : . Using 1.0 mL ofmatrix " r Working standard.. pL . Approx, final cone, of AT- A (approx, cone.) ' - .* . analytein matrix - Blank vVrnp- - K- . 0.500ppm . v0.500 ppm ' t ,0 , 0 0 ppm : 10 20 ' 5. 0.005 ppm 0.010 ppm ' 0.025ppm W .5,00.ppm . 10 0.050ppm .v5,00jtpm - 20 0.100 ppm 'x$ii`50.0ppmv 5 0.250ppm - ' 50.0ppin & 10 0.500ppm 50.0'ppmBP' 15 0.750 ppm 50.0ppm v 20 ' 1.00 ppm' 0 12.0 P rocbim ib ......... ^ .^ s g jr ,, 12.1 Obtain frozen samplesandallow to thawatroom temperatureorin alukewarm waterbath. . 12.2 Vortex mix for 15seconds, thentransfer i.OmL orother appropriatevolume to a15mL polypropylenecenriifugetube. 12J Returnunusedsamplesto freezer afterextract]on"amountshavebeenremoved. 12.4 Record the initial volume onthe extractionworksheet. 12.5 Label the tubewith thestudy number, sampleID, daleandanalystinitials. Seeattached worksheet for documentingtheremaining step's. N sS ^ 12.6 Spike ali samples, includingblanks and standards,readyfor extra&onwith surrogate standard asdescribedin 11J. W' ^ 12.7 . ' . id . ' Spikeeachmatrixwith theappropriateamountofstandardasdescribedin ll.l. orTable 1in thatsection, for thecalibrationcurvestandards. Also preparemarinespikesand continuingcalibration standards. ,. \ 12.8 Vortex mix the standardcurve samples, matrix spike samples, andcontinuingcalibration samplesfor 15seconds. j ^ 12.9 Checkto ensurethe 0.5 M TBAreagent is atpH 10. If not, adjust accordingly?- ' 12.10 To eachsample, add 1mL 0.5 M TBA and2mL of0.25M sodiumcarbonate/sodiura.,-riibicarbonate buffer. 12.11 Using anOxford Dispenser, add5mL methyl-tert-butyl ether. .* 12.12 Capeach sample andput on the shaker at asetting of300rpm, for20minutes. 12J3 Centrifugefor 2 0 to 25 minutesatasettingof3500rpm, or until layersarewell separated. EIS4-4.1 . . Extraction of PFOS from Semm Page 7 of 14 Centre Analytical Laboratories, Inc. Page 22 of 29 Exygen Research Page 85 of 98 O O 2X 1. Exygen Study No.: 023-066 b e s t c o p y a v a il a b l e ....... Centre Protocol No-01E.-023-0.66i. 12.14 Label afresh 15mLcentrifagetubewith thesameinformationasin 12.5. 1X15 Remove4.0 mL ofthe organiclayerto this clean 15mL centrifiige tube. [hvIXIS Put eachsample,on the analytical nitrogen evaporatoruntil dry, approximately 1to 2 V-,li*o'urs. ,I** * t'' ,% 12.1XjAdd 1.0mL of.methanolto eachcentrifugetubeusingagraduatedpipette. l,X'l 8 -V^ortex. mix fcr 30 se.co.nds. . 12J9 ^Attacha02 jun nylonmeshfilter to a3ccsyringe andtransferthe sampleto this " ^'3yifiige|Filter into a1.5mLglassautovial or low-volumeautovial whennecessary. 12.20 'Labelthe autovial with thestudynumber, animal numberandgender, sampletimepoint, matrixjfinalsolvent, extractiondale, andanalyst(s)performingthe extraction. 12-21 Cap andstore"pttra*cAts atroomtemperatureor atapproximately4C until analysis. 12.22 Completetheextraction.worksheet, attachedto this document, andtapein thestudy notebookor include'instudybinder, asappropriate. 13-0 D ata Analysis awp C alculatiows-------------------- :---------------------------------------------- 13.1 Calculations t V 13.1.1 Calculate actual concentrationsofPFOS,orotherapplicablefluorochemicsl, in calibration standards tmfthe following equation: mLofstandard x concentration-o/f'iswta,,ndard(ug/mLi ______ - mLofstandard+mL ofsurrogate istandard.+initial matrix volume(mL) Final Concentration(pg/mL) ofPFOSinmalnx ,, X y ,&> L4.D-METHOD PERFORMANCE '__________ 14.1 The methoddetection limit (MDL) is analyteandmatri^specific. RefertoMDL report for specificMDL and limit ofquantitation (LOQ)values(seeAttachments B and C). 142 The following quality control samplesareextractedwith eachbatch ofsamplesto evaluate thequality ofthe extraction andanalysis; *' 142.1 Methodblanks andmatrixblanks. t# 14.22 Matrix spikeandmatrix spikeduplicatesamplesto determine accuracyshd^ precisionofthe extraction. ' 1422 Continuing calibrationchecksamplesto determinethecontinuedaccuracy'ofthei . initial calibrationcurve. 142 Refer to section.14ofETS-8-5.1 formethodperformancecriteria. 15.0 P ollutio n P revention and W aste M anagement_____________________ 3 15.1 Samplewasteis disposed in biohazard containers, flammablesolventwasteis disposed in high BTU containers, andusedglasspipettewasteis disposedin brokenglasscontainers located in the laboratory. ETS-W.l Extraction of PFOS from Scrum Poge 8 o f 14 i-: I Centre Analytical Laboratories, Inc. Page 23 of 29 Exygen Research Page 86 of 98 Exygen Study No.: 023-066 BEST COPY AVAILABLE Centre Protocol No. 01P-023-066 V*-*:'/ -' \ ., ^.'* ' .? ' *t; * '.a. jV ')' -. -. ' 1 vt'iwt..j.`. ' o 16.0 R ecords ^.,.16.1 Completetheextractionworksheetattachedto this method, andtapein the study *'notebookorinclude in the3-ring studybinder, asappropriate. fy -j*' \ 17.0 A ttachments * '________ J_____ _________________ :-------------------------------- . 1'47.1 -.itA'/tta\chmentA, E:xtractionworicsheet 17.2^gVttachmentB, MDL/LOQvalues andsummary 17.3 -AttachmentC, Calibrationstandardconcentrationworksheet 13-fi BBTONK 18.1 Thevalidationrcportassociatedwith thismethodisETS-8-4.0 & 5.0-V-l. 182 FACT-M-3.1,"Asiysi^of SerumorOtherFluid Extractsfor Fluorocfaemicalsusing HPIXli-Electroaspriravy'MMasas Snpectrometry" :f -- IS A Affected Documents 19.1 ETS-8-5.1, "Analysis ofSerna or pifeerFluidExtracts lorFluorochemicalsusing HPLC-ElectrosprayMass Spectrometry" 20.0 R evisions Revision Number 1 ReaswonF.or Rev;isioxn1 Section 1221 Changedto include samplestorage'atroomtemperature. Section 12.13 Addedtheshakerspeedier j? ' Section 12.17Final volume is 1.0mL;notadjutf^for initial volumes lessthan 1.0mL. >r Revision Date 04/02/99 y- ^ */ 4 ,SVix ,w \ ;>V v i# " O / ETS-S-4-! Extraction ofPFOS firm Satan Paie 9 of 14 Centre Analytical Laboratories, Inc. Exygen Research Page 24 of 29 Page 87 of 98 BEST c o p y a v a il a b l e Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 i I kI & r- 1 Attachment A ETS-W.1 ; Centre Analytical Laboratories, Inc. Exygen Research Pige IQ of 14 Page 25 of 29 Page 88 of 98 G O jU best cori kjwiabi Exygen Study No.: 023-066 Centre Protocol N a 01P-023-066 ` 't 'tfv. \;w;; O Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be usedfor preparing the N : Standard Calibration Curve PEOS-" IA4 5.55 5ppb-1000ppb 1 .! PEOSV 1.51 ' 4.79 5 ppb-1000 ppb PfOSAA-'j 3.46 20.5 5 ppb-1000 ppb EtFOSOH s 11.4 362 5ppb-1000 ppb : MJ36^ 7 . i 6.03 192 5ppb-1000 ppb PFOSEA' f|i ,5:71 182 5ppb- 1000ppb detennined. Two cu rv w ^ each of these matrices were extracted and analyzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, mooliey, and human were equivalent to the rabbit responsei'therefor^their MDL and T,,OQwill be the same values as determined in rabbit smm- j Please see LOQ Summary andMDLsSSy m ETS-8-4.0 iJ.O-V-1 for further information. - i" 1 * V ! \ ~ * W -0S - w, w : '* V ! & V X A. I \\ o Attachment B: MDL/LOQ Summary BTS-8-4.1 Extraction of PFOS from Scrum /*., Centre Analytical Laboratories, Inc. Exygen Research Page It of 14 t Page 26 of 29 Page 89 of 98 :U Exygen Study No.: 023-066 best copy available CentreProtocol No. 01P-023-066 ........ .'yV,.: ' '. 3Y. 3 riv" .'.VtX-' ; i ' tr.isfvivS.'. v-f ***>. 3 Compound! PPOS Preparedrange LCRfrom %Recover/ RSD RabbitSerum ofstandard] curse Range Range. (ppb) (ng/raL) .(pp<>) (ng/mL) t F*uXllRV'ige+. w'o.99i'- 978 24.8- 978 89-108 ` - 4.67-11.0 S 454.-248 . Highcu.rvXed . 1 I ? - 78 4.94-248' ; 97.8-978 85-104 89-10 534-12.0 . 4.84-9.80 l/o M ^ rn 4.94- 978 94-111 4.60-10.5 R a b b it S e r u m rx P repared rango"- , L C R fro m - o f sta n d a r d s.^ j . cu rv e (p p b ) C V m L ) ^ -r (P P b ) r i ' t f g / m L ) % R ecovery . R ange R SD R ange F u ll R a n g e L ow C urve . 0 .9 9 3 - 9 7 t 4 .9 3 -9 7 . . ' 4 . 9 3 `- 9 7 6 , ,rtn > i \ . .SJ 4 8 8 -1 0 3 87-105 5 ,1 0 -1 4 .7 9 .8 5 -1 4 .7 ,1 - H ig h cu rv e 2 4 ,8 -9 7 6 2 4 .8 ? ^ A h -iv z *A iS 5 .0 8 -1 3 .9 1* . 0 .9 9 3 - 9 7 f * - m X4 5 3 - 9 7 6 ^ 1 5 .1 0 -1 4 5 . Compound: PFOSAA R a b b it S e r u m P repared ran ge o f stan d ard s . (p p b ) (n g /m L ) F u ll R a n g e L o w C urve H ig h cu rv e l/X 0 9 9 1 -9 7 4 . 4 .9 2 -2 4 7 4 9 ,2 -9 7 4 0 5 9 1 -9 7 4 L C R fro m curve (p p b ) (n g /m L ) 2 4 .7 -9 7 4 9 .7 4 -2 4 7 9 7 .4 -9 7 4 9 .7 4 - 9 7 4 Hi % R ecovery jsS R S D R a n g e v- I , R a n g e \ . 81-111 . 97-107 8 5 -1 0 8 9 5 -1 1 5 4 .1 0 -1 0 .6 5 tk S *a 6 J 8 -2 1 J *1, > 4 3 3 -1 2 5 4 .1 1 -2 3 2 f i' X 'y \ v13^.>VL'K"y-Z'-,,*/'. * O A tta ch m en t B : M D L /L O Q Su m m a ry / - E T S -8 -4 .1 , - ^ , . E x tra ctio n o f P F O S fro m S n n m .! Centre Analytical Laboratories, Inc. Exygen Research Page 12 of 14 Page 27 of 29 Page 90 of 98 i best COPVavailable Exygen Study No.: 023-066 Centre Protocol No, 01P-23-066 o//'rVVr.-V..V Preparedtinge riJP RabbitScrum y -' \ m Ringe^ ofstandards (ppb). fng/mL) J 0593-976 ' towCmve1 453-97. LCR from curve (b) (ng/mL) 493-976 9.76-97. y. Recovery Range 77-110 97-107 Highcur\ve : !b n - TM 97.6-97 l/X ( r . 9.7-97 ,.r*r JN Prcpared nnge . LCR from Rabbit Serum ofstiusutb/^ ' curve (ppb) (ngtL) .- jF f r p y ^ / (ng/mL) Full Range 0593 - 97 . 90-109 85-111 % Recovery Range 96-106 Low Curve High curve 1 . 453-148 ` 493-976 0593 - 97 9.76v^*248 493 - 976 ^ 9.76 - 976 91-110 ,h> W 06 w v 95-117#>t RSD Range 113-15J 14.1-113 115-19.6 11.1-313 RSD Range 10.1-163 113-195 103-183 10.1-19.1 Rabbit Serum Full Range Prepared range of standards . (ppb) (ng/mL) 0.993 - 976 LCR fromcurve (ppb) (ng/mL) 34.8-97 Low Curve 4.93 - 97. 9.76- 975 High curve 97.6 - 976 97.6-976 l/X 0593-976 . 9.76-97 % Recovery - / RSD ' Range Range 88-106 wJS. 4.82-175 \ 100-103 . 355-183^. 81-111 97-110 3.11-9.74 i 4 \ - 4.77-195 W . 4 K o Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum Centre Analytical Laboratories, Inc. Exygen Research Page13of14 I Page 28 of 29 Page 91 of 98 G O tU O O CO P"' Q Sff 'iernt*9* Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 . r. : ** i ' : . ; ' ;; [ O ! Ion Pair Standard Curves-Fluids Prep date(i): ' 'j ' Standard amber: l__Aaalyte{j): \ Equipment number: ^ Jj^ S iu n p le matrix: : #'; Final lotvent and TN: W * \ ,y i BlankQuld/ideatifien 'V - Method/revbionr'' I 's " ' Tu^Tiolyte(s): ,FjC m lx itd p jp ro x .0 .5 0 0 p p m : : j''j' . ' FC mix jtd approx. 50.0 ppm: . S u iro p te Jtd a ^ ro x . 1Q0 ppm: Actual concentration of standards tn the FC mix rFOS FO SM rO SA A . EtFOSB PFOSEA MS56 Stdcouc Sricotur^ SriconN Sricone Sri cone Sricooc ugAuL ug/mL` v i f S ? jiue/mL ugAnL ug/mL 0500 0500 05070507- 053Z.iT 0.501 0521 0532ii ,;0501V 0521 0501 0501 5.00 5.07 532 X S75.01 ! 331 5.01 5.00 5.07 5.00 5.07 532 'S 5.01 s <531 532 V5.0UT .531 531 5.01 200 50.1 333 50.1 * W s u sai 50.0 50.1 533 50.0 50.1 533 50.0 50.1 533 50.1X7 531 . 50.1. 30.1 ' .__52.1 ./Ti ^ 50.1 30.1 ' .52.1 Y 50.1 AU Ain'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 ofstandards toithe samplctnatrii PFQS Final cone PFOSA Final ease FFQ5AA Findeooe EtFOSE Finalcooc FFOSEA >j-M556 ' SarrofaU Fmalcone rindcone A^Sjcone 431 9.74 ' 24.1 49J 97.4 241 493 735 976 S.00 9.89 25.1 $0.0 98.9 251 500 . 744 . 989 5-24 10.4 265 514 \04 243 524 782 1038 4.94 9.78 24.8 49.4 97. 248 494 737 978 3X1 9.93 253 50.1 99.3 252 SOI 749 993 .I3: ioa 103 X. 25.8 Surrogale SU fimiMMT 102 itg/nL^ 258 500" 513 746 ion Senta Rabbit Bovina Rat Monkey 8c.Plasma Human ffe PFOSA PFOSAA 1.00-1000 1 5.00-100 1 3.00-1000 Estimatesonly. Usavalues forrabbit Estimate*only. Usevalues forrabbit Esrimatts only. Use valuesforrabbit Estimatesonly. Usevalues forrabbit LlFOSE-OH PFOSEA 1 5.00-1000 1 5.00-1000 'w M55i -i1 5.00-1000 .. ,v* 'ijf .* o i 14 o f 14 Centre Analytical Laboratories, Inc. Exygen Research Page 29 of 29 Page 92 of 98 001301 Exygen Study No.: 023-066 3048ResearchDrive, State College, PA 16801. Phone: (814)231-8032, Facsimile: (814)231-1253 PROTOCOL AMENDMENT Page 1 of 1 Centre Study Number: 023-066 Amendment Number: 1 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. RATIONALE 1. At the request of the sponsor, all serum samples are to be analyzed with this study. IMPACT ONTEE STUDY 1. No negative impact on the study. Study Director Signature iVvV\ Date Centre QAU Review ijh h * February 12,1998/1 Exygen Research Page 93 of 98 '\*fl < en RESEARCH Precise Research. \ Proven Results. Exygen Study No.: 023-066 Exygen Research Page 94 of 98 0; Exygen Study No.: 023-066 Centre Analytical Laboratories, Inc. 3048ResearchDrive, State College,FA 16801. Phone: (814) 231-8032, Facsimile: (814) 231-1253 PROTOCOL DEVIATION Deviation Number: 1 ' Date of Occurrence: (1,2) 05/23/01, (3) 05/24/01 Centre Study Number: 023-066 1Centre 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 of the highest standard in Set 052201AD. {or `iH /0 2 - 2. 9 Analytical Method Sample Analysis e.: Accepted recovery of 76%.the duplicate injection of Centre sample 0107378 Spk A2 in Set 052201B. A 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 of the highest standard in Set 052201CD. ACTIONS TAKEN i.c.. amendment issued. SOP revision, etc.. 1-3. Protocol deviation issued. Recorded By/Date:_________ 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 of all of the fortifications were within the acceptable range. Pnncipl Investigator Signature Study Director Signature / Date s\vAoi Date Spcrasor Management Signature S lK j -i Date CAL QAU Review. u c l q/y/d? ________ ______________________________________________________Freepbruaarryy 1122,1998/2 % i (W'&Aool i Wi f(a ce7 cW^- fa iaspeok b>&> of-'V^ve. 0( Exygen Research Page 95 of 98 Exygen Study No.: 023-066 Centre Analytical Laboratories, Inc. 3048ResearchDrive, State College,PA 16801. Phone: (814)131-8032,Facsimile: (814) 231-1253 PROTOCOL DEVIATION Deviation Number: 2 ' Date of 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 0.03 gtL, 0.025 pi*, and 0.025 j/L, respectively. If\u) W> MG? ffi ACTIONS TAKEN i.e,, amendmentissued. SQPrevision, etc... 1. Protocol deviation issued. Recorded By/Date:______ IMPACT ON THE STUDY 1. No negative impact because the actual volume was used in the calculations. incipal Irttestigator Signature Date Study Director Signature 3 Date s/g /a s Date CAL QAU Review. ____ tJC<^ N I vlFebruary 12,1998/2 cW stcH H W'.Na re-issued ue^-fo uspect-eti) Co*s ofHfte. q |n k Exygen Research Page 96 of 98 Exygen Study No.: 023-066 Centre Analytical Laboratories, Inc. 3048ResearchDrive, Slate 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-Q23-066 DESCRIPTION OFDEVIATION 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/Date:_______ IMPACT ON THE STUDY 1. No negative impact because the actual volume was used in the calculations. icrjal Investigator Signature Study Director SignianttuurreA Date sW\<>3 Date ^ Sponsor Management Signature Z~ Date CAL QAU Review________b t C February 12,1998/2 I Exygen Research I Page 97 of 98 en RESEARCH Precise Research. Proven Results. Exygen Study No.: 023-066 Exygen Research Page 98 of 98