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AR226-1739 STUDY TITLE Extraction of Potassium Perfluorooctanesulfonate from Mallard Serum and Mallard Liver for Analysis Using HPLC-ElectrosprayiMassSpectrometry DATA REQUIREMENTS Analytical Method Requirements STUDY DIRECTOR Sean Gallagher STUDY COMPLETED ON September 06,2001 -PERFORMING LABORATORY / TESTING FACILITY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 Phone: 814-231-8032 STUDY SPONSOR 3M EnvironmentalTechnology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 PROJECT Centre Protocol Number: 0032-023-042 Centre Study Number: 023-042 Total Pages: 104 centre Study No.!023-042 GOOD LA,BORATORY PX(ACTICECOMPLlANCESTATE MENT Centre Study Number 023-042, entitled "Extraction of Potadurn Perfluomoccanesulfmate from Mallard S e m and Mallard Ever far Analysis Ueing HPLC-Elsctmspray/Mass Spc~tromelry,"conducted for 3M Envimmxntal Tcchnoloay and Safety Services, was parformedin compliance with US EPA TSCA OoodLaboracory Practice Standards (40CFR Part 792)by Centre Analytical taboratodes,Inc, Date Principa JDvcatigator Centre Analytiaal Laboratorioti, Inc. Sean Gallagher J Data Study Director Wildlife Znternatianal, Ltd. and Safety Servlce~ Csatre Analytical Laboratories, hc. Page 2 of 104 Centre Study No.: 023-042 QUALITY ASSURANCE STATEMENT Centre Analytica Laboratories' Quality Assurance Unit reviewed Centre StuGj Num ier 023-042, entitled, "Extraction of Potassium Perfluorooctanesulfonate from Mallard Serum and Mallard Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry". All reviewed phases were inspected for conduct according to Centre Analytical Laboratories' Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Study Director and to management. Phase 1. Protocol Review 2. Extraction, Fortification 3. Raw Data Review 4. Raw Data anti Draft Report Review 5. Final Report Review Date Inspected 11/20/00 11/20/00 Date Reported to Date Reported to Centre Study Director and Management Sponsor Management 11/30/00 12/28/00 11/30/00 12/28/00 12112/00 3118-21/00 1212 1/00 312910 1 12/28/00 51710 1 91610 1 91610 1 91610 1 L C u - - ; &v&v- Naomi Lovallo Sr. Quality Assurance Auditor W6(0 I Date Centre Analytical Laboratories, Inc. Page 3 of 104 Centre Study No.:023442 CERTnICATIOB-FO This repon, for Centre Study Number 023-042,Is a true and complete repmentation of the raw data foi' the study. Submitted by: CmtR Anal$cd Laboratdes, Inc. 3048 Research Drive State College, PA 16801 (814) 231-8032 Centre Analytical Laboralories,Inc. Facility Management: President '\ Centre Analytiical Laboratories,Xnc. Study Director, Wildlife International: Sam Gallagher U Wildlife Inten3aeiona1, Ltd, Sponsor Ropceenta Envltonrrmtal T&hnology and Safety Services Centre Analytical Labaraturiea,hc. Date Date #- Page 4 of 104 Centre Study No.: 023-042 STUDY IDENTIFICATION Extraction of Potassium Perfluorooctanesulfonate from Mallard Serum and Mallard Liver for Analysis Using HPLC-ElectrosprayMassSpectrometry CENTRE PROTOCOL NUMBER: OOP-023-042 CENTRE STUDY NUMBER: TYPE OF STUDY: SAMPLE MATRIX: 023-042 Analytical Mallard Serum and Mallard Liver TEST SUBSTANCE: SPONSOR: Peffluorooctanesulfonate (PFOS) 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 STUDY DIRECTOR: Sean Gallagher Wildlife International, Ltd. 8598 Commerce Drive Easton, MD 21601 SPONSOR REPRESENTATIVE: Rochelle Robideau 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 TESTING FACILITY: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: Experimental Start Date: Experimental Termination Date: Study Completion Date: 11/10/00 11/17/00 03/13/01 09/06/0 1 Centre Analytical Laboratories, Inc. Page 5 of 104 Centre Study No.: 023-042 PROJECT PERSONNEL The Study Director for this project was Sean Gallagher at Wildlife International, Ltd. The folowing personnel from Centre Analytical Laboratories, Inc., were associated with various phases of the study: Name Emily Stauffer Dave Hell Melissa Kennedy Whitsel Tiffany Proctor Angela Morgan Michelle Arjmand Lawrence Ord Rickey Keller Karen Risha Title Scientist Scientist Groupmeam Leader Technician Technician Technician Sample Custodian Sample Custodian Scientist Centre Analyticail Laboratories, Inc. Page 6 of 104 Centre Study .No. 023-042 TABLE OF CONTENTS TITLE PAGE ....................................................................................................................... 1 GOOD LABOIUTORY PRACTICE COMPLIANCESTATEMENT.............................. 2 QUALITY ASlSURANCE STATEMENT .......................................................................... 3 CERTIFICATIONOF AUTHENTICITY........................................................................... 4 STUDYIDENTIFICATION................................................................................................ 5 PROJECT PEFSONNEL..................................................................................................... 6 TABLE OF CONTENTS..................................................................................................... 7 LIST OF TABLES ............................................................................................................... 8 LIST OF FIGURES............................................................................................................ 10 LIST OF APPE:NDICES .................................................................................................... 11 1.0 SUMMAR.Y................................................................................................................ 12 2.0 OBJECTIVE ............................................................................................................... 12 3.0 INTRODUCTION....................................................................................................... 12 4.0 TEST SYSTEM .......................................................................................................... 13 5.0 REFERENCE MATERIAL........................................................................................ 13 6.0 DESCRIPTION OF ANALYTICAL METHOD ........................................................ 14 6.1 Extraction Procedure ................................................................................................ 14 6.2 Preparationof Standardsand Fortification Solutions............................................... 14 6.3 Chromatography ....................................................................................................... 15 6.4 Instrumenit Sensitivity............................................................................................... 15 6.5 Description of Instrument and Operating Conditions............................................... 15 6.6 Quantitation and Example Calculation..................................................................... 16 7.0 EXPERIMIENTAL DESIGN ...................................................................................... 18 8.0 RESULTS ................................................................................................................... 18 9.0 CONCLUSIONS......................................................................................................... 18 10.0 RETENTION OF DATA AND SAMPLES .............................................................. 19 Centre Analytical. Laboratories. Inc. Page 7 of 104 Centre Study No .. 023-042 LIST OF TABLES Table I. Sulmmary of PFOS in Reagent Blanks............................................................. 21 Table I1. Sulmmary of PFOS in Mallard Liver Blanks................................................... 22 Table I11. Sulmmary of PFOS in Mallard Serum Blanks................................................. 22 Table IV. Sulmary of PFOS Recoveries in Mallard Liver ............................................ 23 Table V. Sulnmary of PFOS Recoveries in Mallard Serum .......................................... 24 Table VI. Summary of PFOS in Adult Liver Samples at 0 ppm a.i. (Test Termination 19 Weeks) .................................................................................. 25 Table VI1. Summary of PFOS in Adult Liver Samples at 1.8 ppm a.i. (Test Termination 6 Weeks) .................................................................................... 26 Table VI11. Summary of PFOS in Adult Liver Samples at 6.2 ppm a.i. (Test Termination 6 Weeks) .................................................................................... 27 Table IX. Sunmary of PFOS in Adult Liver Samples at 17.6 ppm a.i. (Test Termination 19 Weeks) .................................................................................. 28 Table X. Swnmary of PFOS in Juvenile Liver Samples at 0 ppm a.i. (Test Temination 12 Weeks) .................................................................................. 29 Table XI. Surnmary of PFOS in Juvenile Liver Samples at 1.8 ppm a.i. (Test Termination 12 Weeks) .................................................................................. 30 Table XI1. Surnmary of PFOS in Juvenile Liver Samples at 6.2 ppm a.i. (Test Termination 12 Weeks) .................................................................................. 31 Table XI11. Surnmary of PFOS in Juvenile Liver Samples at 17.6 ppm a.i. (Test Termination 12 Weeks) .................................................................................. 32 Table XIV . Surnmary of PFOS in Control Adult Serum Samples..................................... 33 Table XV . Surnmary of PFOS in Adult Serum Samples at 0 ppm a.i. (Test Termination 19 Weeks) .................................................................................. 34 Table XVI. Surnmary of PFOS in Adult Serum Samples at 1.8 ppm a.i. (Text Termination 6 Weeks) .................................................................................... 35 Table XVII. Summary of PFOS in Adult Serum Samples at 6.2 ppm a.i.(Test Termination 6 Weeks) .................................................................................... 36 Table XVIII. Summary of PFOS in Adult Serum Samples at 17.6pprn a.i. (Test Termination 19 Weeks) .................................................................................. 37 Centre Analytical Laboratories. Inc. Page 8 of 104 Centre Study No.: 023-042 LIST OF TABLES (Continued) Paae Table XIX. Suunmary of PFOS in Adult Serum Samples at 17.6 ppm a.i. (6 Week Samples) ....................................................................................................... ..38 Table XX. Summary of PFOS in Juvenile Serum Samples at 0 ppm a i . (Test Teimination 12 Weeks) .................................................................................. 39 Table XXI. Sulnmary of PFOS in Juvenile Serum Samples at 1.8 ppm a.i. (Test Termination 12 Weeks) ................................................................................. .40 Table XXII. Silmmary of PFOS in Juvenile Serum Samples at 6.2 ppm ai. (Test Termination 12 Weeks) ................................................................................. .41 Table XXIII. Slummary of PFOS in Juvenile Serum Samples at 17.6 ppm a.i. (Test Termination 12 Weeks) ................................................................................. .42 Table XXIV. hmmary of Percent Moistures for Mallard Liver Samples ....................... 43 Centre Analytical Laboratories, Inc. Page 9 of 104 Centre Study No.: 023-042 Figure 1. Figure 2. Figure 3. LIST OF FIGURES Pag;e Typical Calibration Curve for PFOS .............................................................. 46 ChromatogramRepresenting a 0.1 ng/mL standard for PFOS ....................... 47 Chromatogram Representing a 1.0 ng/mL standard for PFOS ....................... 48 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Centre ID Reiagent Blank A, Set: 112000A) ................................................................... 49 Figure 5. Chromatogram Representing Control Mallard Serum for PFOS (Centre ID: 0003803 Blank A, Set: 112lOOB)................................................................... 50 Figure 6. Chromatogram Representing Control Mallard Liver for PFOS, (Centre ID: OOO3804 Blank A, Set: 112000A).................................................................. 51 Figure 7. ChromatogramRepresenting Control Mallard Senun fortified with 10 ppb of PFOS (Centre ID: 0003803 Spk A, Set: 112100B)............................. 52 Figure 8. Chromatogram Representing Control Mallard Liver Fortified at 1000ppb with PFOS (Centre ID: 0003804 Spk A, Set: 112100A)................................ 53 Figure 9. Chromatogram Representing Juvenile Mallard Serum Sample (Centre ID: 0008143, SponsorID: 2332, Set: 112700A)................................................... 54 Figure 10. Chromatogram Representing Juvenile Mallard Liver Sample (Centre ID: 0008108, Sponsor ID: 2337, Set: 1121OOA)................................................... 55 Centre Analytical Laboratories, Inc. Page 10 of 104 Centre Study No.: 023-042 LIST OF APPENDICES Appendix A AStmudenydPmroetnotcsoal nOdODPe-v0i.2a3t-i.0o4n2..(.C...e.n..t..r..e..S..t.u...d..y..N...o.....0..2..3..-..0..4..2..)..a..n..d...................56 Centre Analytical Laboratories, Inc. Page 1 1 of 104 Centre Study No.: 023-042 1.0 SUMMARY Centre Analytical Laboratories, Inc. (Centre) extracted mallard liver samples and mallard serum samples for the determination of perfluorooctanesulfonate (PFOS) in mallard serum and mallard liver according to protocol OOP-023-042 (Appendix A). The limit of qiiantitation for mallard liver was 10 ppb and 10 ng/mL for mallard serum. The LOQ for leach matrix was determined in a method verification study performed at Centre (Centre Study: 023-044). Residues of PFOS in the mallard liver samples ranged from non-quantifiable levels (a peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/rnL,)) to 22.8 pg/g. These results are reported on a wet weight basis. PFOS in the mallard serum samples ranged from non-quantifiable levels to 218 pg/g. The average percent recovery k standard deviation for PFOS in liver samples was 95% f 12%. The average percent recovery i-standard deviation for PFOS in serum samples was 93% & 10%. 2.0 OBJECTIVE The objective of this study was to determine levels of perfluorooctanesulfonate (PFOS) in specimens of mallard liver and mallard serum using the analytical methods described in protocol OOP-O:23-042. 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFOS in mallard liver and mallard serum, using the analytical methods entitled, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC,-Electrosprayhlass Spectrometry" and "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-Electrosprayhlass Spectrometry". The study was initiated on November 10, 2000, when the study director signed Centre protocol number OOP-023-042. The experimental start date was November 17,2000, and the experimental termination date was March 13,2001. Centre Analyticad Laboratories, Inc. Page 12 of 104 Centre Study No.: 023-042 4.0 TEST SYSTEM The control mdlard liver and serum used for the matrix blanks and matrix fortifications was received on May 18,2000from Wildlife International Ltd., Easton, MD. The control mallard liver was assigned the Centre ID of 0003804 and the control mallard serum was assigned the Centre ID of 0003803. Sixty mallard liver samples and sixty mallard serum samples were received at Centre on August 10, 2000. The samples were stored frozen until they were logged in by Centre personnel on August 16, 2000 and stored frozen. An additional twenty mallard liver samples and forty mallard serum samples were received on February 24,2001 and stored frozen until they were logged in by Centre personnel on March 1,2001. Sample log-in and chain of custody information can be found in the raw data package associated with1 this study. Storage records will be kept at Centre Analytical Laboratories, Inc. and a true copy of the storage records can be found in the raw data package associated with.this study. 5.0 REFERENCE MATERIAL The analytical standard PFOS was received at Centre on June 3, 2000 from 3M Environmental Technology and Services. The available information for the reference material is listed below. The reference material was stored frozen. Compound PFOS Centre Control No.. TCR No. Purim (%I Expiration Date 00-023-042 TCR 00017-46 97.9 08/31/O 1 The molecular structure of PFOS is given below. PFOS Chemical Name - Molecular weight = U Peffluorooctanesulfonate 499 (CsF17SO3-) Note: The neutral molecule and standard form from which PFOS (anion) is derived,,is potassium periluorooctanesulfonate [ C S F ~ ~ S O ~mKol]e,cular weight 538. Centre Analyticad Laboratories, Inc. Page 13 of 104 Centre Study No.: 023-042 6.0 DESCRIPTION OF ANALYTICAL METHOD Analytical methods entitled "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC-ElectrosprayMass Spectrometry" and "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-ElectrosprayMass Spectrometry" were used for this study. 6.1 Extraction Procedure A - 0.50 g procedure. sample of liver and a 100 pL aliquot of the serum was After fortification of appropriate samples, the samples used were - for the extraction vortexed for 15 seconds. One milliliter of 0.5 M TBA was added to the liver samples and 0.5 milliliter was added to the serum samples. Two milliliters of 0.25 M sodium carbonatehodium bicarbonate was added to the liver samples and 1 milliliter was added to the serum samples. Five milliliters of MTBE sample was placed on wrist-action - was added to shaker for both the 20 min. liver and and serum samples. then centrifuged for Each - 15 min. Four milliliters of the organic layer was taken and dried on a nitrogen evaporator and then reconstituted with 1 milliliter of methanol. Each sample was analyzed by LCMSMS electrospray. 6.2 Preparation of Standards and Fortification Solutions Standard solutions were prepared on September 18, 2000 as specified in Centre Analytical Laboratories' protocol OOP-023-042. An individual stock standard solution of PFOS was prepared at a concentration of 100 pg/mL by dissolving 10 mg of the standard (corrected for purity and salt content) in methanol. From this solution, a 1.0 pg/mL fortification standard solution was prepared by taking 1 mL of the stock and bringing the volume up to 100 mL with methanol. A 0.1 pg/mL fortification standard was prepared by taking 10 mL of the 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol. A 0.01 pg/mL standard was prepared by taking 10 mL of the 0.1 pg/mL standard and bringing to 100 mL with methanol. A set of standards containing PFOS was prepared by serial dilution of the 0.1 pg/mL and 0.01 pg/mL solutions in the following manner: Initial Conc. (pg/mL)' 0.1 0.1 0.1 0.01 0.01 ' of 0.01 PFOS Volume (mL) 5 2 1 5 2 1 Diluted to (mL) 100 100 100 100 100 100 Final Conc. (pg/mL) 0.005 0.002 0.001 0.0005 0.0002 0.0001 Centre Analytical Laboratories, Inc. Page 14 of 104 Centre Study No.: 023-042 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4" f 2OC) 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 time of PFOS 'was 4.4 was min. accomplished by LC/MS/MS electrospray. Peaks were detected in the control matrices The retention 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 Descriptioin of Instrument and Operating Conditions Instrument: PE SCIEX API 3000 Biomolecular Mass Analyzer Interface: SCIEX TurboIon Spray Liquid Introduction Interface Computer: Dell Ultrascan P1110 ,Software: "PLC: PE SciexAnalyst version 1.1 Windows NT, version 4 Hewlett Packard (HP) Series 1 100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven :HPLCCo1umn:Genesis CS(Jones Chromatography), 2.1 mm x 50 mm, 4p Column Temp.: 35" C :InjectionVol.: 10 pL :MobilePhase (A): 2 mM Ammonium Acetate in ASTM type I water :Mobile Phase (B): Methanol :Flow Rate: 0.3 mL/min. Centre Analytical Laboratories, Inc. Page 15 of 104 Time %A - % B 0 60 40 1.o 0 100 7.0 0 100 7.5 40 60 11.0 40 60 Centre Study No.: 023-042 Ions monitored: Analyte PFOS Mode negative Transition Monitored 499 3 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 -5 1.O -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 l/x fit weighted linear regression) by Analyst software using six concentrations of standards. The concentration for mallard liver (pg/g) and mallard serum (pg/mL) was determined from the equations below. Equation 1 calculated the amount of analyte found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Then Equation 2 calculated the amount of analyte found in pg/mL (the equations for serum are shown). Centre Analytical Laboratories, Inc. Page 16 of 104 Centre Study No.: 023-042 Equation 1: Analyte found (ng/mL) = (Peak area - intercept) slope Equation 2: Analyte found (pg/mL) = ((anal. found (ndmL) x FV (mL) x DF x EV (d)x ) & (AV (mL) x sample vol. (mL)) 1000 ng Where: FV = Final Volume DF = Dilution Factor. EV = Extraction Volume AV = Aliquot Volume For samples hrtified 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/l pg)) x 100 - amount added (ng/mL) An example of a calculation using an actual sample follows: Mallard seinun sample Centre ID 0003803 Spk A (Set: 112100B), fortified at 10 ng/mL with. PFOS, where: peak area intercept slope dilution factor -- 6558 - 515 -- 7740 - 1 ng added (fort level) = 1 ng avg. amt in controls - 0 (Not quantifiable) find volume -- 1 mL extraction volume -- 5 mL aliquot volume -- 4 mL sample weight (volume) = 0.1 mL From equation 1: Analyte found (ng/mL) = 16558-5151 7740 = From equation 2: Analyte found (pg/mL) 0.8ng/mL = (0.8 ng-/mL x 1 mL x 1 x 5 mL) x & (4 mL x 0.1 mL) 1000 ng = 0.00976 pg/mL Centre Analyticail Laboratories, Inc. Page 17 of 104 Centre Study No.: 023-042 From equation 3: % Recovery = ((0.009'76 UdmLx - 0 n d d ) x 1000)x 100 10 ng/mL, = 98% 7.0 EXPERIMENTAL DESIGN Each set of samples (liver two matrix blanks fortified - or serum) consisted of one reagent blank, at known concentrations, and 20 samples. one matrix blank, Each sample was extracted using the appropriate method and then analyzed in duplicate. 8.0 RESULTS The PFOS found in the reagent blanks are listed in Table I. The PFOS in the mallard liver blanks and mallard serum blanks are given in Tables I1 & 111. Individual recoveries for PFOS in the liver samples are detailed in Table IV. The average percent recovery k standard deviation for PFOS in liver samples was 95% k 12%. Individual recoveries for PFOS in the serum samples are detailed in Table V. The average percent recovery k relative standard deviation for PFOS in serum samples was 93%k 10%. PFOS in the mallard liver samples ranged from non-quantifiable levels (a peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) to 22.8 pg/g. Individual results are liste'din Tables VI-XIII. The results are reported on a wet weight basis. PFOS in the mallard serum samples ranged from non-quantifiable levels (a peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001pg/mL)) to 218 pg/g. Individual results are listed in Tables XIV-XXIII. The percent moistures for the mallard liver samples are given in Table XXIV 9.0 CONCLUSIONS The mallard liver and serum samples were successfully extracted and analyzed according to protocol OOP-023-042. Centre Analytical Laboratories, Inc. Page 18 of 104 Centre Study No.: 023-042 10.0 RETENTION OF DATA AND SAMPLES When the final report is complete, all original paper data generated by Centre Analytical Laboratories, Inc. will be shipped to the sponsor. This does not include facility-specific raw data such as instrument logs, however exact copies of temperature logs will be submitted. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Centre Analytical Laboratories, hic. archives for the period of time specified in 40 CFR Part 792. Retained samples of reference substances are archived by the sponsor. Centre Analytical Laboratories, Inc. Page 19 of 104 Centre Study No.: 023-042 TABLES Centre Analytical Laboratories, Inc. Page 20 of 104 Centre Study No.: 023-042 Table I. Summary of PFOS in Reagent Blanks Sponsor ID na na na na na na na na na na na na na na na na na na Centre ID Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Realgent Blank A Reagent Blank A* Realgent Blank A Reaaent Blank A* * Duplicate injection Set Number 11 1700A 111700A 112000A 112000A 112100A 112100A 112100B 112100B 112700A 112700A 112700B 112700B 030701A 030701A 030701B 030701B 030801A 030801A Extraction Date 11/17/00 11/17/00 11/20/00 11/20/00 11/21/00 11/21/00 11/21/00 11/21/00 11/27/00 11/27/00 11/27/00 11/27/00 31710 1 31710 1 31710 1 31710 1 3/8/0 1 31810 1 Analysis Date 11118-19/00 11118-19/00 11120-21/00 11120-21/00 11122-23/00 11122-23/00 11/23/00 11/23/00 11128-29/00 11128-29/00 11/29/00 11/29/00 3 1910 1 31910 1 319- 10101 319- 10101 311 1/01 311 1/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) NQ NQ NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) For values recorded as NQ, 0.00005 pg/g (half the value o f the lowest calibration standard, 0.000~1pg/mL) was used to calculate the average and standard deviation. LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 21 of 104 Centre Study No.: 023-042 Table 11. Summary of PFOS in Mallard Liver Blanks Sponsor ID na na na na na na na na Centre ID 0003804 Blank A 0003804 Blank A* 0003804 Blank A 01003804 ~ i a n Ak * 0003804 Blank A 0,003804Blank A* 0003804 Blank A 0003804 Blank A* Set Number 111700A 111700A 1 12000A 112000A 112100A 112100A 030801A 030801A Extraction Analysis Date Date 11/17/00 11118-19/00 11/17/00 11/18-19/00 11/20/00 11/20/00 11/21/00 11/21/00 11/20-21/00 11120-21/00 11122-23/00 11122-23/00 3/8/0 1 3/8/0 1 3/11/01 311 1/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 0.000294 0.000300 NQ NQ NQ NQ NQ NQ NQ NQ Table 111. ,Summaryof PFOS in Mallard Serum Blanks Sponsor ID na na na na na na na na na na Centre 0003803 Blank A 0003803 Blank A* 0003803 Blank A 0003803 Blank A* (11003803Blank A 0003803 Blank A* (11003803Blank A 0003803 Blank A* 0003803 Blank A 0003803 Blank A* Set Extraction Analysis 112100B 112100B 112700A 112700A 112700B 1 12700B 030701A 030701A 030701B 030701B 11/21/00 11/23/00 11/21/00 11/23/00 11/27/00 11/28-29/00 11/27/00 11128-29/00 11/27/00 11/29/00 11/27/00 11/29/00 31710 1 3/9/0 1 3/7/0 1 3/9/0 1 3/7/0 1 3/9- 10/01 31710 1 319- 1010 1 AVERAGE: STANDARD DEVIATION: Analyte NQ NQ NQ NQ NQ 0.00159 0.00826 0.00825 0.00159 0.00397 0.00239 0.00333 * Duplicate injection NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.000 1 pg/mL) was used to calculate the average and standard deviation. Centre Analytical Laboratories, Inc. Page 22 of 104 Centre Study No.: 023-042 Table IV. Summary of PFOS Recoveries in Mallard Liver Sponsor ID na na na na na na na na na na na na na na na na Centre ID 0003804 Spk A 0003804 Spk A* 0003804 Spk B 0003804 Spk B* 0003804 Spk A 0003804 Spk A* 0003804 Spk B 0003804 Spk B* 0003804 Spk A 0003804 Spk A* 0003804 Spk B 0003804 Spk B* 0003804 Spk A 0003804 Spk A* 0003804 Spk B 0003804 Spk B* Set Number 111700AD 111700AD 11 1700A 111700A 112000AR 112000AR 112000A 112000A 112100A 112100A 112100AD 112100AD 030801A 030801A 030801AD 030801AD Extraction Date Analysis Date Amt Added (ng/g) 11/17/00 11/19/00 10 11/17/00 11/19/00 10 11/17/00 11118-19/00 50 11/17/00 11118-19/00 50 11/20/00 11/22/00 100 11/20/00 11/22/00 100 11/20/00 11120-21/00 4000 11/20/00 11120-21/00 4000 11/21/00 11122-23/00 1000 11/21/00 11122-23/00 1000 11/21/00 11/27/00 20000 11/21/00 31810 1 11/27/00 311 1/01 20000 20 31810 1 311 1/01 20 318101 3111-12/01 200 3/8/01 3111-12/01 200 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: YO Recovery 79 77 85 84 114 115 106 107 89 88 90 89 92 92 107 101 95 11 12 * Duplicate injectison LOQ = 0.01 pglg for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 23 of 104 Centre Study No.: 023-042 Table V. Summary of PFOS Recoveries in Mallard Serum Sponsor Centre Set Extraction Analysis Amt YO ID na 0003803 Spk A 112100B 11/21/00 11/23/00 10 98 na 0003803 Spk A* 112100B 11/21/00 11/23/00 10 98 na 0003803 Spk B 112100B 11/21/00 11/23/00 500 84 na 0003803 Spk B* 112100B /21/00 11/23/00 500 86 na 0003803 Spk A 112700A /27/00 11128-29/00 10 81 na 0003803 Spk A* 112700A /27/00 11128-29/00 10 81 na 0003803 Spk B 112700A l27/00 11128-29/00 10000 91 na 0003803 Spk B* 112700A l27/00 11128-29/00 10000 91 na 0003803 Spk A 112700B 11/27/00 11/29/00 500 97 na 0003803 Spk A* 112700B 11/27/00 11/29/00 500 97 na 0003803 Spk B 112700B 11/27/00 11/29/00 100000 89 na 0003803 Spk B* 112700B 11/27/00 11/29/00 100000 89 na 0003803 Spk A 030701A 317101 31910 1 100 79 na 0003803 Spk A* 030701A 317101 31910 1 100 77 na 0003803 Spk B 030701A 317101 31910 1 1000 89 na 0003803 Spk B* 030701A 317101 31910 1 1000 93 na 0003803 Spk A2 030701B 317101 319-10101 100 90 na 0003803 Spk A2* 030701B 317101 319-10101 100 90 na 0003803 Spk B2 030701B 317101 319-10101 1000 118 na 0003803 Spk B2* 030701B 3/7/01 319-10101 1000 119 * Duplicate injectilon AVERAGE: 92 STANDARD DEVIATION: 11 RELATIVE STANDARD DEVIATION: 12 LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 24 of 104 Centre Study No.: 023-042 Table VI. Summary of PFOS in Adult Liver Samples at 0 ppm a.i. (Test Termination 19 Weeks) Sponsor ID 1961 1961* 1962 1' 1962* 1963 v, 1963* 1964 1964* 1965 1965* 1966 f 1966* 1967 j'.' 1967* 1968 ? 1968* 1969f l 1969* 1970 :1 1970* Centre ID 0008006 0008006 0008007 0008007 0008008 0008008 0008009 0008009 00080 10 00080 10 000801 1 0008011 00080 12 0008012 00080 13 00080 13 0008014 0008014 0008015 00080 15 * Duplicate injection Set Number 1 1 1700A 1 1700A 1 1700A 11700A 11700A 11700A 111700A 11 1700A 1 1 1700A 11 1700A 111700A 1 1 1700A 111700ADD 111700ADD 111700AD 11 1700AD 1 1 1700AD 111700AD 111700AD 1 1 1700AD Extraction Analysis Date Date 11/17/00 11/18-19/00 11/17/00 11/17/00 1/18-19/00 1/18-19/00 11/17/00 11/17/00 1/18- 19/00 1/18-19/00 11/17/00 11/17/00 1/18-19/00 11118-19/00 11/17/00 11118-19/00 11/17/00 11/17/00 11/18-19/00 11118-19/00 11/17/00 11/17/00 11/17/00 11/17/00 11/18-19/00 11/18-19/00 11/20/00 11/20/00 11/17/00 11/19/00 11/17/00 11/19/00 11/17/00 11/19/00 11/17/00 11/19/00 11/17/00 11/19/00 11/17/00 11/19/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 0.0008 13 0.000763 0.000509 0.00063 1 0.000937 0.000887 0.00155 0.00157 0.001 10 0.001 11 0.00446 0.00432 0.662 0.684 0.238 0.246 0.115 0.106 0.355 0.343 0.138 0.219 LOQ = 0.01 pgJg for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 25 of 104 Centre Study No.: 023-042 Table VII. Summary of PFOS in Adult Liver Samples at 1.8 ppm a i . (Test Termination 6 Weeks) Sponsor ID EO 1-0263-21065 f l EO 1-0263-2 1065* E01-0263-21066 /'h EO 1-0263-21066* E01-0263-21067 & EO 1-0263-21067* EO1-0263-210681 A. E01-0263-21068* EO1-0263-21069 P EO 1-0263-21069" EO1-0263-21075 I' EO 1-0263-21075'' E01-0263-21076 E01-0263-21076" E01-0263-21077 E01-0263-2 1077' EO1-0263-21078 6 EO 1-0263-2 1078" EO1-0263-21079 ?' E01-0263-21079" Centre ID 0103870 0 103870 010387 1 0103871 0103872 0103872 01 03873 0103873 0 103874 0103874 0103880 0 103880 0103881 010388 1 0103882 0 103882 0 103883 0103883 0 103884 0103884 * Duplicate injection Set Number 030801AD 030801AD 030801AD 030801AD 030801AD 030801AD 03080 1AD 030801AD 03080 1AD 030801AD 030801AD 030801AD 030801AD 03080 1AD 030801AD 03080 1AD 030801AD 030801AD 030801AD 030801AD Extraction Analysis Date Date 31810 1 31810 1 3/8/0 1 31810 1 311 1-12/01 311 1-12/01 311 1-12/01 311 1-12/01 3/8/0 1 311 1-12/01 3/8/0 1 31810 1 311 1-12/01 311 1-12/01 3/8/0 1 31810 1 31810 1 3/11-12/01 311 1-12/01 311 1-12/01 31810 1 3/8/0 1 3/8/0 1 311 1-12/01 311 1-12/01 3/11-12/01 31810 1 31810 1 311 1-12/01 311 1-12/01 31810 1 3111-12/01 31810 1 3/8/01 311 1-12/01 3/11-12/01 31810 1 311 1-12/01 3/8/01 3/11-12/01 AVERAGE: STANDARD DEVIATION: Analyte Found (p.g/g) 0.359 0.350 0.647 0.63 1 0.886 0.880 2.1 1 2.04 0.222 0.232 0.3 17 0.326 0.219 0.230 0.194 0.197 0.346 0.342 0.0893 0.0888 0.535 0.574 LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 26 of 104 Centre Study No.: 023-042 Table VIII. Summary of PFOS in Adult Liver Samples at 6.2 ppm a.i. (Test Termination 6 Weeks) Sponsor Centre ID ID EO1-0263-21070 ,L E01-0263-21070* EO 1-0263-2107I EO 1-0263-21071* E01-0263-21072 E01-0263-21072' E01-0263-21073 E01-0263-21073 * ' EO 1-0263-21074 E01-0263-2 1074" E01-0263-21080 + E01-0263-21080* EO 1-0263-21081 0103875 0103875 0103876 0103876 0103877 0103877 01 03878 0103878 0103879 0103879 0103885 0 103885 0103886 E01-0263-21081" 0103886 E01-0263-2 1082 0 103887 EO 1-0263-21082' 0103887 E01-0263-21083 E01-0263-21083' , 0103888 0103888 ' E01-0263-21084 ,~0103889 E01-0263-2 1084' 0 103889 * Duplicate injection Set Number 030801AD 030801AD 03080 1AD 030801AD 03080 1AD 030801AD 030801AD 030801AD 030801AD 03080 1AD 030801AD 030801AD 030801AD 030801AD 030801AD 03080 1AD 030801AD 03080 IAD 030801AD 030801AD Extraction Analysis Date Date 31810 1 31810 1 311 1-12/01 311 1-12/01 31810 1 311 1-12/01 31810 1 311 1-12/01 31810 1 31810 1 311 1-12/01 311 1-12/01 31810 1 311 1-12/01 31810 1 31810 1 3/8/01 31810 1 311 1-12/01 311 1-12/01 311 1-12/01 341-12/01 31810 1 31810 1 31810 1 31810 1 31810 1 31810 1 3/8/0 1 31810 1 311 1-12/01 311 1-12/01 311 1-12/01 3/11-12/01 311 1-12/01 311 1-12/01 311 1-12/01 311 1-12/01 31810 1 311 1-12/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 1.04 1.01 0.963 0.907 1.92 1.81 0.791 0.827 1.28 1.34 1.01 1.03 0.0614 0.0578 0.473 0.517 1.66 1.52 0.0862 0.0878 0.920 0.576 LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 27 of 104 Centre Study No.: 023-042 Table IX. Summary of PFOS in Adult Liver Samples at 17.6 ppm a.i. (Test Termination 19 Weeks) Sponsor ID 1991 j.1 1991* 1992 F 1992* 1993 *' 1993* 1994' G 1994* 1995 4 ' 1995* 1996 i 1996* 1997 IC' 1997* 1998 1998* 1999 .U 1999* 2000 2000* Centre ID 00080 16 00080 16 00080 17 00080 17 00080 18 0008018 00080 19 0008019 0008020 0008020 0008021 000802 1 0008022 0008022 0008023 0008023 0008024 0008024 0008025 0008025 * Duplicate injection Set Number 1 1 1700ADD 111700ADD 1 1 1700ADD 111700ADD 1 1 1700ADD 111700ADD 11 1700AD 111700AD 111700AD 111700AD 111700ADD 1 1 1700ADD 111700AD 1 1 1700AD 111700AD 11 1700AD 111700ADD 111700ADD 1 1 1700AD 111700AD Extraction Analysis Date Date 11/17/00 11/17/00 11/20/00 11/20/00 11/17/00 11/20/00 11/17/00 1 1/20/00 11/17/00 11/17/00 11/17/00 11/20/00 11/20/00 11/19/00 11/17/00 11/17/00 11/17/00 11/17/00 11/17/00 11/19/00 11/19/00 11/19/00 11/20/00 11/20/00 11/17/00 11/19/00 11/17/00 11/19/00 11/17/00 11/17/00 11/19/00 11/19/00 11/17/00 11/20/00 11/17/00 11/17/00 11/20/00 11/19/00 11/17/00 11/19/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 15.9 16.4 22.8 21.9 7.02 6.69 2.82 2.93 3.53 3.60 13.2 13.5 5.62 5.58 3.72 3.86 15.5 16.1 5.97 6.10 9.64 6.57 LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 28 of 104 Centre Study No.: 023-042 Table X. Summary of PFOS in Juvenile Liver Samples at 0 ppm a i . (Test Termination 12 Weeks) Sponsor ID 2301' b 2301* 2302 C 2302* c 2303 '2303* 2305 ''i 2305* 2307 Ni 2307* 2308 m 2308* 2314 '"v 23 14* 2316 f l 2316* 2317 " 2317* 2320 <' 2320* Centre ID 0008086 0008086 0008087 0008087 0008088 0008088 0008089 0008089 0008090 0008090 0008091 000809 1 0008092 0008092 0008093 0008093 0008094 0008094 0008095 0008095 * Duplicate injection Set Number 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 112000A 1 12000A 112000A 112000A 112000A 112000A 112000A Extraction Analysis Date Date 11/20/00 11/20-21/00 11/20/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20-21/00 11/20-21/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ N Q = N o t Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. Centre Analytical Laboratories, Inc. Page 29 of 104 Centre Study No.: 023-042 Table XI. Summary of PFOS in Juvenile Liver Samples at 1.8 ppm a i . (Test Termination 12 Weeks) Sponsor ID 2322 2322* 2323 Q 2323* 2325 ~5 2325* 2326 2326* 2328 I\' 2328* 2330 (.= 2330* 2331 ri\ 2331* 2332 ? 2332* 2333 IC 2333* 2334 P''< 2334* Centre ID 0008096 0008096 0008097 0008097 0008098 0008098 0008099 0008099 0008 100 0008100 0008 101 0008 10 1 0008102 0008 102 0008 103 0008 103 0008 104 0008 104 0008 105 0008105 Set Number 112000A 112000A 1 12000A 112000A 1 12000A 112000A 1 12000A 1 12000A 112000A 1 12000A 112000A 112000A 1 12000A 112000A 112000A 1 12000A 112000A 1 12000A 112000A 112000A Extraction Analysis Date Date 11/20/00 11120-21/00 11/20/00 11/20-21/00 11/20/00 11120-21/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20/00 11/20/00 11/20/00 11/20-21/00 11120-21/00 11/20-21/00 11/20/00 11/20/00 11/20-21/00 11/20-21/00 11/20/00 11/20/00 11/20-21/00 11120-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 11/20/00 11/20-21/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 0.00454 0.00458 0.00124 0.00129 0.00 150 0.00 154 0.00236 0.00245 0.00 167 0.00165 0.00185 0.00 182 0.000719 0.000763 0.000425 0.000430 0.000969 0.000966 0.00356 0.00355 0.00189 0.00126 , * Duplicate injection LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 30 of 104 Centre Study No.: 023-042 Table XII. Summary of PFOS in Juvenile Liver Samples at 6.2 ppm a i . (Test Termination 12 Weeks) Sponsor ID 2335 17 2335* 2336 1' 2336* 2337 ? 2337* 2338 f l 2338* 2339 1' 2339* 2341 IV' 2341* 2342 2342* . 2343 $'\ 2343* 2344 $ 2344* 2346 '1 2346* Centre ID 0008106 0008 106 0008 107 0008 107 0008 108 0008 108 0008 109 0008 109 00081 10 00081 10 00081 11 00081 11 00081 12 00081 12 00081 13 00081 13 0008114 00081 14 00081 15 00081 15 Set Number 112100A 112100A 112100A 112100A 112100A 1 2100A 1 2 1OOA 1 2 1OOA 1 2 1OOA 1 2 1OOA 1 2100A 1 2 1OOA 1 2 1OOA 1 2100A 112100A 112100A 112100A 112100A 112100AD 112100AD Extraction Analysis Date Date 11/21/00 11/21/00 11/21/00 11122-23/00 11122-23/00 11122-23/00 11/21/00 11122-23/00 11/21/00 11/21/00 11122-23/00 11122-23/00 11/21/00 11/21/00 11122-23/00 11122-23/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11/21/00 11122-23/00 11122-23/00 11122-23/00 11122-23/00 11122-23/00 11122-23/00 11122-23/00 11/21/00 11/21/00 11/21/00 11122-23/00 11122-23/00 11122-23/00 11/21/00 11/21/00 11/27/00 11/27/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pglg) 0.00197 0.00200 0.00377 0.00378 0.00380 0.00363 0.00555 0.00535 0.00540 0.005 18 0.00306 0.00313 0.003 12 0.00320 0.00329 0.00332 0.00402 0.00405 0.0141 0.0146 0.00482 0.00341 * Duplicate injection LOQ = 0.01 pglg for liver and 0.01 pg/mL for serum Centre Analytical Laboratories,Inc. Page 31 of 104 Centre Study No.: 023-042 Table XIII.Summary of PFOS in Juvenile Liver Samples at 17.6 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 2347 P 2347* 2348 F 2348* 2353 F 2353* 2354 1/) 2354* 2355 f i 2355*ff 2357 f i 2357* 2358 C 2358* 2359 F 2359* 2360 fl 2360* 2363 f i 2363* Centre ID 00081 16 0008116 00081 17 0008117 0008118 0008118 00081 19 0008119 0008 120 0008120 0008121 0008121 0008 122 0008122 0008 123 0008 123 0008124 0008 124 0008125 0008125 Set Number 112100AD 112100AD 112100AD 112100AD 112100AD 112100AD 112100AD 112100AD 112100A 112100A 112100AD 112100AD 112100AD 112100AD 112100AD 112100AD 112100A 112100A 112100AD 112100AD Extraction Analysis Date Date 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 11/27/00 11/21/00 1 iniioo 11/27/00 11/27/00 11/21/00 11/27/00 11/21/00 11/22-23/00 11/21/00 11/22-23/00 11/21/00 11/27/00 11/21/00 11/27/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pug) 0.0383 0.0370 0.0456 0.0465 0.0234 0.0227 0.0 1 84 0.0189 0.01 10 0.0105 0.0616 0.0629 0.0175 0.0189 0.021 1 0.021 1 0.00623 0.00628 0.0172 0.0191 0.0262 0.0168 * Duplicate injection LOQ = 0.01 pg/g for liver and 0.01 p g / d for serum Centre Analytical Laboratories, Inc. Page 32 of 104 Centre Study No.: 023-042 Table XIV. Summary of PFOS in Control Adult Serum Samples Sponsor ID EO1-0263-21005 4 EO 1-0263-21005* EO1-0263-21006 4 EO 1-0263-21006* EOl-0263-210074 EO1-0263-21007* E01-0263-21008M EO 1-0263-21008* EO1-0263-21009J\ EO1-0263-21009* E01-0263-21025 EO1-0263-21025* E01-0263-21026 E01-0263-21026* EO1-0263-210276 EO 1-0263-21027* EO1-0263-210286 E01-0263-21028* E01-0263-21029r'/ EO1-0263-21029* Centre ID 0 1038 10 01038 10 0103811 010381 1 01038 12 0103812 01038 13 0 1038 13 0 103 8 14 01 038 14 0 103830 0103830 0103831 0103831 0 103832 0103832 0103833 0 103833 0 103 834 0103834 * Duplicate injection Set Number 030701A 030701A 030701A 030701A 030701A 030701A 030701A 030701A 030701A 030701A 030701B 03070 1 B 030701B 030701B 03070 1 B 030701B 030701B 030701B 030701B 030701B Extraction Analysis Date Date 317101 ' 319101 31710 1 31910 1 31710 1 31910 1 31710 1 31910 1 31710 1 31910 1 31710 1 31910 1 3/7/01 31910 1 31710 1 31910 1 3/7/01 31910 1 31710 1 31910 1 3/7/01 319-10101 31710 1 31710 1 319-10101 319-10101 31710 1 319-10101 31710 1 319-10101 31710 1 319-10101 317101 31710 1 319-10101 319-10101 31710 1 319-10101 31710 1 319-10101 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.0463 0.0455 0.00828 0.00928-0.00954 0.0103 r 0.-0000435219> 0.00423\ 0.00616 0.00285 0.00207 NQ NQ NQ NQ 0.00428 0.00265 NQ NQ 0.00797 0.0134 NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards(0.0001 pg/mL,)) For values recorded as NQ, 0.00005 pg/rnL (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/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 33 of 104 Centre Study No.: 023-042 Table XV. Summary of PFOS in Adult Serum Samples at 0 ppm ami. (Test Termination 19 Weeks) Sponsor ID 1961 m 1961* 1962 1962* ,F ,, 1963 1963* 1964 F 1964* 1965 f i 1965* 1966 6 1966* 1967 1967* 1968 i' 1968* 1969 4 1969* 1970 1970* Centre ID 0008026 0008026 OW8027 0008027 0008028 0008028 0008029 0008029 0008030 0008030 000803 1 0008031 0008032 0008032 0008033 0008033 0008034 0008034 0008035 0008035 set Number 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100B 112100BD 112100BD 112100BD 112100BD 112100BD 112100BD 1121OOBD 112100BD Extraction Date Analysis Date Analyte Found (pg/mL) 11/21/00 11/21/00 11/21/00 11/21/00 11/23/00 11/23/00 11/23/00 11/23/00 0.00605 0.00594 0.00494 0.00501 11/21/00 11/23/00 11/21/00 11/23/00 11/21/00 11/23/00 11/21/00 11/23/00 11/21/00 11/23/00 11/21/00 11/23/00 11/21/00 11/23/00 11/21/00 11/23/00 11/21/00 11/27-28/00 11/21/00 11/27-28/00 11/21/00 11/27-28/00 11/21/00 11/27-28/00 11/21/00 11/27-28/00 11/21/00 11/27-28/00 11/21/00 11/27-28/00 11/21/00 11/27-28/00 AVERAGE: STANDARD DEVIATION: 0.00844 0.00825 0.00798 0.00761 0.0104 0.0103 0.0358 0.0357 6.1 1 6.02 3.80 3.81 0.843 0.849 1.41 1.38 1.22 2.03 * Duplicate injection LOQ = 0.01 pg/g for liver and 0.01 pg/mL for senun Centre Analytical Laboratories, Inc. Page 34 of 104 Centre Study No.: 023-042 Table XVI. Summary of PFOS in Adult Serum Samples at 1.8 ppm a.i. (Test Termination 6 Weeks) Sponsor ID E01-0263-21010 N) EO1-0263-2 1010* E01-0263-210114 EO1-0263-21011* E0l-0263-21012 f l EO 1-0263-2 1012* E01-0263-21013 4 EO 1-0263-2 1013* EO1-0263-21014 r" EO 1-0263-21014* E01-0263-21030 C EO 1-0263-21030* E01-0263-21031 C EO1-0263-21031* EO1-0263-21032 6 EO1-0263-21032* EO1-0263-21033 EO1-0263-21033' EO1-0263-21034 l9 E01-0263-2 1034* Centre ID 0103815 0103815 0103816 0103816 0103817 0103817 0103818 01038 18 0103819 0103819 0103835 0103835 0103836 0 103836 0103837 0103837 0103838 0103838 0 103839 0 103839 Set Number 030701AD 030701AD 030701AD 030701AD 03070 1AD 030701AD 030701AD 030701AD 030701AD 030701AD 03070 1BD 03070 1BD 030701BD 030701BD 030701BD 030701BD 030701BD 030701BD 03070 1BD 03070 1BD Extraction Analysis Date Date 31710 I 311 010 1 31710 1 3110101 31710 1 311 010 1 31710 1 311 010 1 31710 1 31710 1 311 010 1 311 010 1 3n10 I 3110101 31710 1 3/10/01 31710 1 31710 1 311 010 1 311 010 1 31710 1 3110-1 1101 31710 1 3/10-11/01 31710 1 3110-1 1101 31710 1 3110-1 1/01 3nIO 1 3110-1 1101 31710 1 3110-1 1101 31710 1 3110-1 1101 31710 1 3110-11101 31710 1 3110-11101 31710 1 3110-1 1/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 12.8 12.1 18.2 20.1 22.5 24.0 20.4 20.4 10.6 10.6 0.870 0.854 6.70 6.43 0.443 0.468 8.88 8.38 1.68 1.74 10.4 8.15 * Duplicate injection LOQ = 0.01 vg/g for liver and 0.01 p g / d for serum Centre Analytical Laboratories, Inc. Page 35 of 104 Centre Study No.:023-042 Table XVII. Summary of PFOS in Adult Serum Samples at 6.2 ppm a i . (Test Termination 6 Weeks) Sponsor ID E01-0263-21015 rJI E01-0263-21015* E01-0263-21016 NI EO 1-0263-2 1016* E01-0263-21017 4 EO1-0263-21017* E01-0263-21018 4 EO1-0263-21018* EO1-0263-21019 4 EO1-0263-2 1019* E01-0263-21035 F EO 1-0263-21035* EO1-0263-21036 E01-0263-2 1036* E01-0263-21037 F EO1-0263-2 1037* E01-0263-21038 F EO 1-0263-2 1038* EO1-0263-21039 f' E01-0263-2 1039* Centre ID 0103820 0103820 0103821 010382 1 0103822 0103822 0103823 0 103823 0103824 0103824 0103840 0103840 0103841 0103841 0103842 0 103842 0103843 0103843 0103844 0103844 Set Number 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701AD 030701BD 03070 1BD 030701BD 030701BD 030701BD 03070 1BD 030701BD 030701BD 030701BD 030701BD Extraction Analysis Date Date 31710 1 3110101 31710 1 311 010 1 31710 1 3110101 31710 1 311 010 1 31710 1 31710 1 311 010 1 3110101 31710 1 311 010 1 31710 1 311 010 1 31710 1 3110101 31710 1 3110101 3mO 1 3110-1 1101 31710 1 3110-1 1101 31710 1 3110-1 1101 31710 1 3110-1 1101 3mO 1 3110-1 1101 31710 1 3110-1 1101 31710 1 3110-11101 31710 1 3110-1 1101 31710 1 3110-1 1101 31710 1 3110-1 1101 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 47.1 44.7 40.4 35.5 45.5 44.4 23.5 24.7 20.3 20.8 17.8 17.3 2.83 2.87 3.48 4.12 61.9 60.3 3.69 2.79 26.2 20.0 * Duplicate injection LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 36 of 104 Centre Study No.: 023-042 Table XVIII. Summary of PFOS in Adult Serum Samples at 17.6 ppm a.i. (Test Termination 19 Weeks) Sponsor ID 1991 M 1991* 1992 F 1992* 1993 M 1993* 1994 r-' 1994* 1995 4 1995* 1996 ;1 1996* 1997 PI 1997* 1998 F 1998* 1999 /tz 1999* v 2000 2000* Centre ID 0008036 0008036 0008037 0008037 0008038 0008038 0008039 0008039 0008040 0008040 0008041 0008041 0008042 0008042 0008043 0008043 0008044 0008044 0008045 0008045 * Duplicate injection Set Number 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 1121OOBDD 121OOBDD 121OOBDD 121OOBDD 12100BDD 121OOBDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 112100BDD 1121OOBDD 112100BDD Extraction Analysis Analyte Date Date Found (pg/mL) 11/21/00 11/28/00 41.0 11/21/00 11/28/00 41.1 11/21/00 11/28/00 47.0 11/21/00 11/28/00 49.5 11/21/00 11/28/00 199 11/21/00 11/28/00 198 11/21/00 1/28/00 93.9 11/21/00 1/28/00 93.0 11/21/00 1/28/00 182 11/21/00 1/28/00 180 11/21/00 1/28/00 218 11/21/00 11/28/00 217 11/21/00 11/28/00 214 11/21/00 11/28/00 212 11/21/00 11/28/00 156 11/21/00 11/28/00 156 11/21/00 11/28/00 156 11/21/00 11/28/00 161 11/21/00 11/28/00 200 11/21/00 11/28/00 205 AVERAGE: 151 STANDARD DEVIATION: 65.0 LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 37 of 104 Centre Study No.:023-042 Table XM. Summary of PFOS in Adult Serum Samples at 17.6 ppm a i . (6 Week Samples) Sponsor ID E01-0263-21020 E01-0263-21020* EO 1-0263-21021 EO 1-0263-2 1021* EO1-0263-21022 ry\ E01-0263-21022* EO 1-0263-2 1023 4 EOl-0263-21023* E01-0263-21024 /' E01-0263-21024* EO1-0263-21040 E E01-0263-21040* EO1-0263-21041 F EO1-0263-2 1041* EO1-0263-21042 F EO 1-0263-21042* E01-0263-21043 C J E01-0263-21043* EO 1-0263-2 1044 Eo 1-0263-2 1044* Centre ID 0 103825 0103825 0 103826 0103826 0103827 0103827 0103828 0103828 0 103829 0103829 0103845 0103845 0 103846 0 103846 0103847 0 103847 0 103848 0103848 0 103849 0 103849 Set Number 030801AD 030801AD 030701AD 03070 1AD 030701AD 030701AD 030701AD 030701AD 030801AD 030801AD 03070 1BD 030701BD 03070 1BD 03070 1BD 03070 1BD 03070 IBD 03070 1BD 030701BD 030701BD 030701BD * Duplicate injection Extraction Analysis Date Date 31710 1 311 1-12/01 31710 1 311 1-12101 31710 1 311 010 1 3mO 1 3110101 31710 1 311 010 1 31710 1 3110101 31710 1 311 010 1 31710 1 311 010 1 31710 1 311 1-12/01 31710 1 311 1-12101 31710 1 3110-1 1101 31710 1 3110-1 1/01 31710 1 3110-1 1101 31710 1 3110-1 1101 31710 1 3110-1 1101 31710 1 3110-1 1101 31710 1 3110-1 1101 31710 1 3110-11101 31710 1 3110-1 1101 31710 1 3110-11101 AVERAGE: STANDARD DEVIATION Analyte Found (pg/mL) 18.6 18.1 152 151 156 157 137 138 42.2 39.1 55.9 62.1 49.8 43.2 15.8 17.7 18.5 17.3 14.1 14.4 65.9 57.5 LOQ = 0.01 pg/g for liver and 0.01 p g / d for serum i Centre Analytical Laboratories,Inc. Page 38 of 104 Centre Study No.: 023-042 Table XX. Summary of PFOS in Juvenile Serum Samples at 0 ppm a i . (Test Termination 12 Weeks) Sponsor ID 2301 C 2301* 2302 f c 2302* I 2303 2303* 2305 f i 2305* 2307 4 2307* 2308 /vi 2308* 2314 4 23 14* 2316 /v\ 23 16* 2317 I2317' 2320 y 2320* Centre ID 0008 126 0008 126 0008 127 0008127 0008128 0008128 0008 129 0008 129 0008130 0008130 0008131 0008 131 0008 132 0008 132 0008133 0008133 0008134 0008134 0008135 0008135 * Duplicate injection Set Number 1 12700A 112700A 112700A 112700A 112700A 1 12700A 112700A 112700A 112700A 112700A 1 12700A 1 12700A 1 12700A 112700A 112700A 112700A 112700A 112700A 112700A 112700A Extraction Date Analysis Date Analyte Found (pg/mL) 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11128-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 11/27/00 11/28-29/00 AVERAGE: STANDARD DEVIATION: NQ NQ NQ NQ 0.00458 0.00453 NQ NQ 0.00278 0.00272 NQ NQ NQ NQ 0.00381 0.00379 0.00626 0.00601 0.00141 0.00153 0.00190 0.00222 N Q =Not Quantifiable (A peak was detected at the corresponding d y t e retention time but was less thanthe lowest concentration of the calibration standards (0.0001 pg/mL)) 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/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 39 of 104 Centre Study No.: 023-042 Table XXI. Summary of PFOS in Juvenile Serum Samples at 1.8 ppm ami(.Test Termination 12 Weeks) Sponsor ID 2322 2322* 2323 f`V 2323* 2325 F 2325* 2326 1"2326* 2328f l 2328* 2330 P 2330* 2331 r/\ 2331* 2332 F 2332* 2333 2333* 2334 2334* Centre ID 0008136 0008136 0008137 0008137 0008138 0008138 0008139 0008139 0008140 0008 140 0008141 0008141 0008 142 0008142 0008143 0008143 0008 144 0008144 0008145 0008145 Set Number 1 12700A 112700A 112700A 1 12700A 1 12700A 112700A 112700A 112700A 112700D 112700D 112700A 112700A 1 12700A 1 12700A 112700A 112700A 1 12700A 112700A 112700A 112700A Extraction Analysis Analyte Date Date Found (pg/mL) 11/27/00 11/28-29/00 0.0464 11/27/00 11/27/00 11/28-29/00 11/28-29/00 0.0472 0.0274 11/27/00 11/28-29/00 0.0281 11/27/00 11/27/00 11/28-29/00 11/28-29/00 0.0497 0.0487 11/27/00 11/27/00 11/27/00 11/28-29/00 11/28-29/00 11/29-30100 0.0160 0.0156 0.249 11/27/00 11/29-30/00 0.250 11/27/00 11/28-29/00 0.00788 11/27/00 11/28-29/00 0.00747 11/27/00 11/28-29/00 0.0101 11/27/00 11/28-29/00 0.00998 11/27/00 11/28-29/00 0.0113 11/27/00 11/28-29/00 0.01 10 11/27/00 11/28-29/00 0.0339 11/27/00 11/28-29/00 0.0339 11/27/00 11/28-29/00 11/27/00 11/28-29/00 AVERAGE: STANDARD DEVIATION: 0.0554 0.0555 0.0507 0.0701 * Duplicate injection LOQ = 0.01 pg/g for liver and 0.01 p g / d for serum Centre Analytical Laboratories, Inc. Page 40 of 104 Centre Study No.:023-042 Table XXII. Summary of PFOS in Juvenile Serum Samples at 6.2 ppm a i . (Test Termination 12 Weeks) Sponsor ID 2335 F 2335* 2336 f 2336* 2337 F 2337* 2338 M 2338* I 2339 4 2339* 2341 A 2341* 2342 2342* 2343 Ir\ 2343* 2344 F 2344* 2346 2346* Centre ID 0008 146 0008 146 0008147 0008147 0008148 0008 148 0008 149 0008149 0008150 0008 150 0008151 0008151 0008152 0008 152 0008 153 0008 153 0008154 0008154 0008 155 0008155 Set Number 1 12700B 1 12700B 112700D 112700D 112700D 1 12700D 112700D 1 12700D 112700D 112700D 1 12700D 1 12700D 112700B 112700B 112700D 1 127OOD 112700D 112700D 112700B 112700B Extraction Analysis Date .Date 11/27/00 11/27/00 11/27/00 11/27/00 11/29/00 11/29/00 11/29-30100 11/29-30/00 11/27/00 11/27/00 11R9-30100 11/29-30100 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/29-30100 11/29-30100 11/29-30100 11/29-30100 11/29-30100 11/27/00 11/27/00 11/29-30/00 11/29/00 11/27/00 11/27/00 11/29/00 11/29-30100 11/27/00 11/29-30100 11/27/00 11/29-30100 11/27/00 11/29-30100 11/27/00 11/29/00 11/27/00 11/29/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.0159 0.0162 0.1 18 0.124 0.181 0.183 0.0584 0.0592 0.0889 0.0840 0.0556 0.0558 0.0380 0.0383 0.0700 0.07 17 0.0676 0.0688 0.0371 0.0373 0.0734 0.0466 * Duplicate injection LOQ = 0.01 vg/g for liver and 0.01 p g / d for serum Centre Analytical Laboratories, Inc. Page 41 of 104 Centre Study No.: 023-042 Table XXIII. Summary of PFOS in Juvenile Serum Samples at 17.6 ppm a i . (Test Termination 12 Weeks) Sponsor ID 2347 F 2347* 2348 2348* 2353 F 2353* 2354 h 2354* 2355 y 235S 2357 N? 2357* 2358 2358* 2359 1s 2359* 2360 2360* 2363 A 2363* Centre ID 0008 156 0008156 0008157 0008157 0008158 0008 158 0008 159 0008159 0008160 0008160 0008161 0008161 0008162 0008 162 0008 163 0008163 0008164 0008164 0008 165 0008 165 Set Number 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D 112700D Extraction Analysis Date Date 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/00 11/27/OO 11/27/00 11/27/00 11/27/00 11/27/00 11/29-30/00 AVERAGE: STANDARD DEVIATION Analyte Found (pg/mL) 0.490 0.496 0.150 0.156 0.894 0.908 0.591 0.62 1 0.399 0.384 0.547 0.547 0.5 12 0.517 0.402 0.404 0.52 1 0.514 0.439 0.434 0.496 0.184 * Duplicate injection LOQ = 0.01 &g for liver and 0.01 p g / d for serum Centre Analytical Laboratories, Inc. Page 42 of 104 Centre Study No.: 023-042 Table XXIV. Summary of Percent Moistures for Mallard Liver Samples Sponsor Centre Average ID ID YOMoisture 1961 0008006 69.7 1962 0008007 73.4 1963 0008008 75.7 1964 0008009 69.2 1965 0008010 71.9 1966 1967 1968 1969 1970 0008011 75.0 0008012 72.8 00080 13 70.4 0008014 69.5 0008015 72.9 1991 00080 16 75.3 1992 0008017 71.8 1993 0008018 71.9 1994 00080 19 71.8 1995 0008020 71.5 1996 000802 1 72.4 1997 0008022 70.5 1998 1999 2000 2301 2302 2303 2305 2307 2308 23 14 2316 2317 2320 2322 2323 2325 2326 2328 2330 233 1 2332 2333 2334 2335 0008023 71.3 0008024 69.8 0008025 72.0 0008086 76.6 0008087 77.3 0008088 72.7 0008089 72.5 0008090 72.3 000809 1 72.0 0008092 75.0 0008093 69.6 0008094 71.8 0008095 71.6 0008096 71.6 0008097 72.5 0008098 73.2 0008099 72.6 0008100 72.0 0008 10 1 68.9 0008 102 71.4 0008103 71.7 0008 104 69.8 0008 105 68.1 0008106 71.7 Centre Analytical Laboratories, Inc. Page 43 of 104 Centre Study No.: 023-042 Table XXTV (cont'). Summary of Percent Moistures for Mallard Liver Samples Sponsor Centre Average ID ID YOMoisture 2336 0008107 69.7 2337 0008108 69.7 2338 0008 109 69.8 2339 00081 10 70.6 2341 000811 1 72.6 2342 00081 12 78.2 2343 00081 13 70.9 2344 0008114 69.2 2346 0008 1 15 70.9 2347 00081 16 69.7 2348 0008117 70.3 2353 OO08118 73.2 2354 0008119 68.5 2355 0008 120 67.8 2357 0008 12 1 74.5 2358 0008122 72.0 2359 0008 123 73.6 2360 0008 124 69.0 2363 0008 125 74.1 EO1-0263-21065 0103870 73.8 EO1-0263-21066 0103871 81.0 E01-0263-21067 0103872 82.4 EO1-0263-21068 0103873 75.8 EO1-0263-21069 0103874 78.2 E01-0263-21070 0103875 77.4 E01-0263-21071 0103876 77.5 E01-0263-21072 0103877 74.8 EO1-0263-21073 0103878 77.5 EO1-0263-21074 0103879 82.7 EO1-0263-21075 0103880 71.7 EO1-0263-21076 0103881 77.9 EO1-0263-21077 0103882 77.5 EO1-0263-21078 0103883 76.0 E01-0263-21079 0103884 63.8 E01-0263-21080 0103885 78.2 EO1-0263-21081 0103886 73.6 E01-0263-21082 0103887 76.7 E01-0263-21083 0103888 81.4 EO1-0263-21084 0103889 72.3 Centre Analytical Laboratories, Inc. Page 44 of 104 Centre Study No.: 023-042 FIGURES Centre Analytical Laboratories, Inc. Page 45 of 104 3.5e4 3.0e4 2.5e4 2.0e4 * I 1S e 4 I.Oe4 500p.O 0.0 ..,:. . 0 I .. I I . I . ?<,,..,' / /'. . .. I' .' , ,,--' 0 ,.- ,/ . . . C:nncet;trntinn n g l m L Centre Study No.: 023-042 Figure 2. Chromatogram Representing a 0.1 ng/mL standard for PFOS Sample Index: Sample Type: Standard Concentration: 0.100 450 Acq. Date: 11/22/00 Acq. Time: 12:11:52 PH Modified: Bunching Factor: Noise Threshold: Yes 1 2.82 Area Threshold: 1 4 . 0 8 8 Num. Smooths: 1 2 RT Window: 30.0 sec e Expecte'd RT: 4.52 mn - Sep. Width: 0.20 - Sep. Height: 0.01 E Exp. Peak Ratio: 5.00 Exp. Add. Ratio: Exp. Val. Ratio: 4.00 3.00 U s e R e l a t i v e RT: NO I n t . Type: Retention T i m : Area: Manual . 4 . 5 5 rain 1540.4 counts Centre Analytical Laboratories, Inc. Page 47 of 104 Centre Study No.: 023-042 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PFOS j Sample Type: 'I Concentration: Acq. Date: ' Acq. Time: Standard 1.000 11/22/00 12:49:51 PM Modified: i Bunching Factor: Y. e. s I I Noise Threshold: 2.82 I Area Threshold: 14.08 II i Num. Smooths: 1 RT Window: 1 Expected RT: j Sep. Width: i, Sep. Height: Exp. Peak Ratio: Exp. Adj. Ratio: Exp. Val. Ratio: - 4 2 2 min 0.20 L 0:Ol 5.00 4.00 3.00 Use R e l a t i v e RT: YO j'Int. Type: Retention Time: Area: Manual 4.54 8355.5 nin counts 2695 2500 2000 1500 1000 500 0 4 - 1 2. 3 4 5 6 7 Time, min Centre Analytical Laboratories, Inc. Page 48 of 104 Centre Study No.: 023-042 Figure 4. ChromatogramRepresenting a ReagentBlank for PFOS (Centre ID Reagent Blank A, Set: 112000A) Sample Type: Blank Concentration: 0.000 Acq. Date: 11/20/00 j1 Acq. Time: 05:21:16 pF.I Modified: Yes Bunching Factor: 1 Noise Threshold: 2.82 - Area Threshold: 14.08 ! Num. Smooths: 1- 2 I RT Window: 30.0 sec i Expected RT: 4.52 min * - ! Sep. Width: 0.20 5 - _ ' Sep. Height: 0.01 c Exp. Peak Ratio: 5 . 9 0 1 Exp. Adj. Ratio: 4.00 1 Exp. Val. Ratio: ~ U s e R e l a t i v e RT: 3.00 NO 1331 120 i loo/ 80: I 60: i4 0 1 I .I . - .. ; I n t . Type: I Retention Time: 4 Area: Uanwl 4.57 432.0 min counts 1 . . .. . ..:. ., . . - . . . .. -. 1 2 3 4 5 Time, min 6 7 ~- Centre Analytical Laboratories, Inc. Page 49 of 104 Centre Study No.: 023-042 Figure 5. Chromatogram Representing Control Mallard Serum for PFOS (Centre ID: 0003803 Blank A, Set: 112100B) Sample Type: Blank Concentration: 0.000 200 Acq. Date: 11/23/00 Acq. Time: 03:38:52 AM ModiZied: Bunching Factor: Noise Threshold: Area Threshold: Num. Smooths: Yes A 2.82 14.08 1 150 m 8 2 RT Window: Expected RT: Sep. Width: - Sep. Height: 30.0 sec 100 4 . 5 2 min e 0.20 2 0.01 Exp. Peak Ratio: 5 . 0 0 Exp. Adj. Ratio: 4.00 50 Exp. Val. Ratio: Use Relative RT: 3.00 No I n t . Type: Retention Time: -ea: 4 I3 1 1 .4.66 4.45; Centre Analytical Laboratories, Inc. Page 50 of 104 _ _ ~ ~ Centre Study No.:023-042 Figure 6. Chromatogram Representing Control Mallard Liver for PFOS,(Centre ID: 0003804 Blank A, Set: 112000A) Sample Type: Blank , Concentration: 0.000 227 Acq. Date: 11/20/00 Acq. Time: 05:46:39 m' 200 Modified: Yes Bunching Factor: 1 Noise Threshold: Area Threshold: 2.82 14.08 . 150 Num. Smooths: RT Window: Expected RT: Sep. Width: Scp. Height: 1 1 8 2 30.0 sec 4.52 min E 100 - 0 . 2 0 - 0.01 c Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: Exp. Val. Ratio: 4.00 3.00 . SO U s e R e l a t i v e RT: No I n t . Type: Manual Retention Time: 4.56 a n . 0 Area: 829.8 counts I Time. min _ .-.-__.. Centre Analytical Laboratories, Inc. Page 51 of 104 Centre Study No.: 023-042 Figure 7. Chromatogram Representing Control Mallard Serum fortified with 10 ppb of PFOS (Centre ID: 0003803 Spk A, Set: 112100B) Sample Type: Concentration: Acq. Date: Acq. Time: Qc. 0.800 11/23/00 04:04:15 An 2150 2000 Hodifi e d: Bunching Factor: Noise Threshold: Area Threshold: . Nun. Smooths: Yes 1 2.82 14.08 1 1500 RT Window: Expected RT: . Sep. Width: Sep. Height: 30.0 sec 4.52 min 0.20 0.01 1ooo Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 500 Exp. Val. Ratio: 3 . 0 0 Use R e l a t i v e RT: No . I n t . Type: Manual Retention Time: 4...5-3- min.. 0 .- 1 2 3 4- Area: . 6557.7 I_ counts - 5 6 7 Time, E$__- Centre Analytical Laboratories, Inc. . .. . . .. ~.____.......I . ... .... Page 52 of 104 . . .. - .. . . .- , -_.. .- -. Centre Study No.: 023-042 Figure 8. Chromatogram Representing Control Mallard Liver Fortified at 1000 ppb with PFOS (Centre ID: 0003804 Spk A, Set: 112100A) ,....-, ...... 1 Sample Type: pc 4 3 I i Concentration: 1.600 3251 Acq. Date: 11/22/00 3000, I Acq. Time: 02:31:00 OM I i I Modified: YO3 ! Bunching Factor: j Noise Threshold: , Area Threshold: 8 Nwn. Smooths: 1 2.82 .. 14.08 1 2000 i 1 ' RT Window: 30.0 . sec Expected RT: . 4.52 min E 1500 Sep. Width: 0.20 9 - . Sep. Height: 0.01 ; 1 Exp. Peak Ratio: 5.00 1000 I Exp. Ad]. RatLo: 4.00 i Exp. Val. Ratio: 3.00 i ' U s e R e l a t i v e RT: No 500 ' I n t . Typ.e: Manual I I Retention Time: 4;54 min 0' 1 2 3 4 5 6 7 Area: . 1056310 counts Time, min _. - Centre Analytical Laboratories, Inc. Page 53 of 104 Centre Study No.: 023-042 Figure 9. Chromatogram Representing Juvenile Mallard Serum Sample (Centre ID: 0008143, Sponsor ID: 2332, Set: 112700A) Acq. T i m : ._ _ .. 04:07:58 UI i "O0 1Mo 3 Hodifi.d: BunchLnq Factor: Noire Threrhold: AT-. ThI.ahold: Y*8 1 2.12 14.08 1200. I 1100 2 NW. S-tha: 1 . RT Window: 30.0 I:' Expascad RT: 4.52 mi0 ~ I SeP. Width: 0~ . 2.0.. iI S-p. Height: 0.01 800- Cxp. Peak Patio: 5.00 I Lap. MY Patio: 4.00 s ulp. Val! Patio: 3.00 700 4 I (Ira h l a t i w RT: So 1 Inc. Type: nanu.1 i R..t.ntion Th: 4.50 min At..: : 4677.3 H.iqht: 1. 41.+003 2 i Start T h : 4.40 End T i m : 65o0o0 400. MO 200 ! 100.I 0' 05 10 15 20 25 30 35 40 Tim.. mlll Centre AnalyticalLaboratories,Inc. Page 54 of 104 Centre Study No.: 023-042 Figure 10. Chromatogram Representing Juvenile Mallard Liver Sample (Centre ID: 0008108, Sponsor ID: 2337, Set: 112100A) 1 Sample Type: Concentration: Acq. Date: 1 . Acq. Time: i LL Unknown N/A 11/22/00 04:25:14 Pn 3409080 : f I Modified: { i Bunching Factor: 1 Noise Threshold: Area Threshold: 3 Num. Smooths: Ycs 1 2.82 14.08 1 2500 I 1 RT Window: 1 Expected RT: j Sep. Width: 30.0 sec 4 . 5 2 min 5 0.20 e ' - Sep. Height: 0.01 ~ Exp. Peak Ratio: i Exp. Adj. Ratio: 5.00 4.00 ! Exp. Val. Ratio: Use Relative RT: i 1 Int. Type: : Retention Time: i Area: 3.00 NO Manual 4.54 11166.9 min counts - I4.54' Time. min ~ --- Centre Analytical Laboratories,Inc. Page 55 of 104 Centre Study No.:023-042 APPENDIX A Study Protocol OOP-023-042 (Centre Study No. 023-042) and Amendments and Deviation Centre Analytical Laboratories, Inc. Page 56 of 104 Centre Study No.: 023-042 Centre ProtocolNo.OOP-023-042 . STUDY PROTOCOL STUDY TITLE EXTRACTIONOF POTASSIUMPERFLuoROOCTANESULFONATEI%OM MALLARD SERUM AND MALLARD LDER FOR ANALYSIS USING HPLC- ELECTROSPRAYMSS SPECTROMETRY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St.-Ad,M N 55133-3331 DATA REQUIREBEN?S Analytical Method Requirements TESTING LABORATORY Centre Analytical Laboratories,Inc. (Centre) 3048 Research Drive State College. PA 16801 Phone 8 14-23 1-8032 PROTOCOL IDENTIFICATION NUMBER OOP-023-042 Centre Analytical Laboratories. hc. ' Centre Analytical Laboratories, Inc. __ - . . ,. . - . _. .. .. - Page 1 of 43 Page 57 of 104 . .. . .. .__I_ .. . . - -......c- -. - ----.---------- Centre.StudyNO.:023-042 - _ _ _ _ _ _ .._______._ Centre Protocol No. OOP-023-042 TABLE OF CCNI"TNTS Page TABLEOFCONTENTS.................................................... -............................................... 2 1. PURPOSE.................................................................. ............ .....................3 2. MATERIALS .................................................................. ............................. 3 3. SPONSOR............ ............................................................... .........t..............4 4. TESTING FACILITY AND PERFORMING LABORATORY............................ 4 5. PROPOSEDEXPERMENTALTIME-FRAME................. .................................. 4 6. JUSTIFICATIONFOR THE SELECTIONOF THE 'ESTSYSTEM.................4 7. SAMPLEPROCESSING,STORAGEAND IDENTIFICATIONa............ 5 8. M a y T I C A LMETHOD..................................................................................... 5 9. ExpERIMENTALDESIGN................................................................................. 10. 10. PROTOCOLAMENDMENTS AND DEVIATIONS.......................................... io 11. RECORDS ............................................................................................................ 11 12. Q U M ASSURANCE................................................... -................................ 11 13. DATA W O R T................................................... -...................................... 12 14. A R C m STATEMENT.................................................................................... 12 15. REFERENCES........................................................................................ .........,...13 16. PROTOCOLAPPROVAL.............................................. ...................................... 14 APPENDLX:ANALYTICALMETHODS........... .................................................. 15 __ - .-. . Centre Analytical Labomtories. Inc. Centre Analytical Laboratories, IIIC. ... -. .- ._ Page 2 of 43 Page 58 of 104' Centre Study NO.:023-042 . .. ..-- Centre Protocol NO. 00~-023042 Studv Title: EXTRACTION OF POTASS& PERFLuoRoO~ANESULFONATE FROM MALLARD SERUMAND MALLARDLIVER FOR ANALYSIS USING HPLC- ELECTROSPRAYMASS SPECTROMETRY 1. PURPOSE The purpose of this study is to analyze mallard serum samples and mallard liver . samples for residues of perfluorooctanesulfonate(PFOS) using methods entitled, "'Extraction Of Potassium Perfluorooctanesulfonate Or Other Fluorochemical Compounds From Serum For Analysis Using HPLC-ElectrosprayMass Spectromeny" (3M method number ETS-8-4.1) and 'Extraction Of Potassium PeffluorooctanesulfonateOr Other Fluorodwnical Compounds From Liver For Analysis Using HPLC-ElectrosprayMass SpeCW~metry"(3M method number FTS-8-6.0)with'the modifications listed in Section 8. The Quality Assumnde Unit of Centre Analytical Laboratories, Inc., will audit the study for compliance with EPA TSCA Good Laboratory Practice stan&& 40 CFR Part 792(1). 2. TESTMATERIALS The following analytical standards will be used - I Test Material I PFOS I TCRNumber I Purity(%) I ExpirationDate I I TCROOOl7-46 I - 97.9 I 0 8 / 3 1 / 7 1 Chemical name and structure of the compound is presented below. PFOS . Chemical Name: W A C Name: CAS Number: Molecular Weight: Perfluorooctanesulfonate 1-Octanesulfonicacid,l, 1,2~!3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-,potassiumS d t 2795-39-3' 499 (CBFI~S@T 0 0 - Note: The neutral molecule and standard form that PFoS (anion) is derived from is perfluorocctanesulfonatepotassiumsalt [CBFI~SO~mKoI,lecularweight 538. Centre Analytical Laboratories. Inc. I Page 3 of 43 Centre Analytical Laboratories, Inc. Page 59 of 104 . -. Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 A record of test and reference substance receipt. storage conditions, and a record of use will be maintained at Centre Analytical Laboratories, Inc. F O ~ S documenting chain-of-custody and shipping records for tracking of fie test substanceswill be included as part of the raw data package. All standards/test substancesand any prepared solutionsmust be identified with a unique label or number on the containeror cross-referenced to the container. , HAZARD INFORMATION A current MSDS for the chemical(s).usedin this study will be maintained at the testing facility. 3. SPONSOR 3M EnvironmentalTechnolosy and SafetyServices . Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 STUDY DIRECTOR Rochelle Robideau TELEPHONE: 651-778-7065 . FAX NUMBER: 651-778-6176 4. ..TESTING FACILITYAND P E R F O W G LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 PRINCIPAL INVESTIGATOR Emily R. Stauffer,Centre TELEPHONE: 814-231-8032 F A X " B E R 814-231-1580 5. PROPOSEDEXPERIMENTALTIME-FRAME Exp-mental Start Date ExperimentalTermination Date Report Issued Nov. 6,2000 Nov. 24.2000 Dec. 30,2000 6. JUSTIFICATIONFOR THESELECTIONOF THE TESTSYSTEM Mallard serum sllmples and liver samples collected by Wildlife International, Ltd. will be used as the test systems for this study. Mallards represent wild bird populationsand they are an EPA recommendedspecies because they do well in a laboratory environment. Mallard habitats include ponds, lakes and marshes, and I Centre Analytical Laboratories, Inc. Centre Analytical Laboratories,Inca. Page 4 of 43 Page 60 of 104 ._.-. _ _ . -._-. Centre Study No.: 023-042 . ... .. .-.. . --------._______I_-__.- . . - . - Centre protoC0lNo.OOP-023-042 I 7. 8. - they are considered aquatic grazers. The use of these matrices for the study was to if PFOS was deposited in the liver and serum of mating adults that were fed PFOS. SAMPLE PROCESSINGS,TORQGEAND IDENTIFICATION No processing will be required for the serum samples. Due to the small mount of sample available, the liver samples will not be homogenized prior to extraction; however, they will be homogenized by lying the Samples on a flat surface and mashing them by hand as best as possible.. Each sample will be assigned a unique sample identification number at Centre, which will be used for tracking 3nd identification Of the samples. The samples will be stored in a temperature-monitored freezer, maintained at c -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. ANALYTICAL METHOD - The serum samples wii be analyzed according to the analytical method titled "Extraction Of Potassium PerflUOrOoctanesUlfOnate Or Other Fluorochemical Compounds From Serum For Analysis Using HPLC-Electrospray/Iviass Spectrometry"(3Mmethod number ETS-8-41) and the control liver samples will be analyzed according to the method titled "Extraction Of Potassium Perfluorooctanesulfonate Or Other Fluorochemical Compounds From Liver For Analysis Using HPLC-Electrosprayh'lass Spectrometry" (3M method number ETS-8-6.0). The following modificationswill be applied to each method 1. The=- will be no surrogate standard used in the extraction procedure. The stock, fortification,and calibration standards will be prepared as follok: Stock Solution Prepan a stock solution of PFOS at 100 pg/mc by weighing out 10.0 mg of analyticalstandard (comcted for percent salt and percent purity). Adjust final volume to 100 mL with methanol in a 100-mL volumetric flask. Store this stock solution (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maXimum period of 6 months from the date.of preparation. Fortification Solutions a. 1.0 udmLFortification Solution -Pipette 1.0 rnL of the 100 p g l d stock solution into a 100 mL volumetric flask. Bring up to volume with methanol. Centre Analytical Labontoria, Inc. Centre Analytical Laboratories, Inc. Page 5 of 43 Page 61 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP423-042 I I. 0.1 ue,.lL Fortification Solution - Pipette 10.0 mL of the 1.0 pg/& fortification solution into a 100-mL volumetric flask and bring up to volume with methanol. c. 0.01 ug/mL Fortification Solution - Pipette 10.0 mL of the 0.1 pglmL fortification solution into a 100-mL VOhmetriC flask and bring up to volume with methanol Store all fortification standard solutions (in 125-mL LDPE bottles) in a refrigerator at 2C to 6pC for a maximum period of 6 months from the date of preparation. Calibration Standards prepare six LC/IvfS/MS calibration standards in methanol via 'dilution of the 0.1 pg/mL and the 0.01 p g / d fortificationsolutions. This is a typical example; additional concentrations may be prepad as needed. Initial Conc. (pg/mL) 0.10.1 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 Conc. ( p g / d ) 0.005 0.002 0.001 0.0005 0.0002 0.0001 Store all calibration standard solutions (in 125-mL LDPE bottles) in a nfrigerator at 2C to 6C for a maximum period of 6 months from the date of preparation. 2. The liver samples will not be processed to make the homogenate described in the method Section 12.2 to.12.12. Due to limited sample available, samples will be mashed on a flat surfaceby hand prior to extraction. 3. The sample weight for liver'will be 0.50 g (k 0.05) and the sample volume for semm will be 100pL. The amount of 0.5 M TBA added to the s e m sample will be 0.5 mL and the amount of 0.25 M sodium carbonatdsodium - bicarbonate buffer added will be 1.0 mL. Samples will be placed on a wrist- action shaker for @ 20 min. at level 10 and then cenaifuged for 15 mjn @ -3oo(J rpm. The samples will only be filtered with 0.2 pm nylon mesh filter if necessary. Samples will be reconstituted in a final volume of. 1 mL of methanol for both matrices. Centre AnalyticalLabontdes.Inc. Centre Analytical Laboratories, Inc. .... - .... - .~.. Page 6 of 43 Page 62 of 104 Centre Study No.: 023-042 . . . ._____ -.. Centre Protocol No. OOP-023-042 4. The samples will be analyzedunder the followingconditions: ' LC/Ms/Ms System and Operating Conditions (TurboIonspray) Mass Spec: . PE SCIEX API 3000 Biomolecular Mass Analyzer Interface:. SCIEX TurboIon Spray Liquid IntroductionInterface Harvard infusion pump Computer: Power Macintosh G3 Software: PE SciexAnalyst 1.1 Windows NT HPLC: Hewlett Packard (HF') Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Co1umn:Genesis CS(Jones Chromatography),2.1 mm x 50 -,'4p Column Temp.: 35' C Injection Vol.: 10 pL - Mobile Phase (A): 2 mM Ammonium Acetate in ASTM type I water Mobile Phase (B): Methanol Flow Rate: 0.3 d m i n . - Time %A 9bB 0 - 60 40 1 .o 0 100 7.0 0 100 7.5 40 60 11.0 40 60 It may be necessary to adjust the HPLC gradient in order to optimize instrument performance. Columns with different dimensions (e.g., 2.1 x 30 mm) and columns from different manufacturers (Keystone Bemil Cis etc.) can be used, provided equivalent chromatography is obtained. Ions monitored - Analvte PFOS - Mode negative + Transition Monitored 499 99 Approximate Retention Time (mjn) 4.20 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 7 of 43 Page 63 of 104 .. Centre Study No.:023-042 Centre Protocol No. OOP-023-042 On a day-to-day basis, the retention times may vary slightly depending on the batch of mobile phase. etc. Example Tune File Parameters The following values are provided as an example. Actual values may vary from instrument to instrument. Also, these values may be changed from time to time in order to optimize for greatest sensitivity. The mass spectrometer is tuned using a 0.5ps/mL PFOS solution, prepared via dilution of the stock solution in methanol. The solution is infused (wing a connector) at 10 U m i n into a 0.2 mUmin stream of mobile phase consisting of 40% methanol and 60% 2 mM ammonium acetate. The analytes are initidly tuned for the parent ion and then tuned for the product ion. Once the instrument is tuned, the optimized parameters are saved Bs a "tune file". This tune fileis then used during routine analysis. The tuning procedure may be repeated as necessary to ensurioptimal sensitivity. Controls * IS-Iospray OR-Orifice RNG-Focus Ring . QO-Quad 0 Rod Offset IQ1- Inter quad 1 lens ST-Stubbies ROl-Quad1 Rod Offset . IQ2-Inter quad 2 lens RO2-Quad 2 rod offset ST3-Stubbies R03- Quad 3 rod offset DF-CEM Deflection Plate CEM-Channel Electron Multiplier . Gas Flows Nebulizer Gas Curtain Gas Collision Gas TIS Temperature Set -4000.0 - -61.0 -270.0 10.0 - 9.3 15.0 9.3 20.0 - 84.0 100.0 86.0 300.0 2800.0 -Set 12 13 4 350C a) Calibration Procedures a. Inject the same volume (between 10 to 20 of each calibration standard (preparedin Meow into the LCIMSIMS. b. Use linear, l/x weighted standard curves for quantification. finear standard curves are generated for each set by linear regression using the appropriate software system. Any calibration standards falling outside f 30%, based on its calculated concentration, must Centre Analytical Labontdes. hc. Centre Analytical Laboratories, Inc. -. . ._ _ ._.. ._.. . . - .- _ _ ._ ._ I Page 8 of 43 Page 64 of 104, . . . . . . . .. - . . . ., .- - , _ ... . _ _ ,. ..._-~~ Centre Study No.: 023-042 Centre Protocol No.OoP-023-042 be excluded from the cdibration curve. However, the total number of calibration standards that may be excluded must not exceed 30% of the total number of standards injected. c. n e cornlation coefficient (r) for calibration curves generated must be 20.9925 (r220.9Ss). If calibrationresults fall outside these limits, then appropriate steps should be taken to adjust i n s w e n t opention, and the relevantset of samplesmust be reanalyzed. I Sample Analysis Inject the same volume used for the calibration Standards (between 10 to 20 @). of each sample, fortification,control, etc. into the I-c/Ms/Ms. Each sample must be analyzed in duplicate. Standards corresponding to at least six concentrations must be included in an analytical set. Inject an entire set of standards (Six) at the beginning of the run and inject stand;lrds interspersed about every 5-10 samples. 41 sample injections must be bracketed by standard injections. * Each set of samples analyzed (not to exceed 25) must include at least one reagent control (extraction solvents only), at least one m a ~ x control, and two matrix control Samples fortified at known concentrations and carried through the procedure to verify recovery. 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. Fortification recoveries within 80 to 120% are acceptable for fortifications at the LOQ level. Recoveries between 80 to 120% are acceptable for fortifications at levels greater than the LOQ. Failure to meet these criteria requires w investigation of cause and a fuli reanalysis of the affected samples. f. Shples in which no peaks are detected at the corresponding analyte retention times will be reported ;LS ND (not detected). Samples in which peaks are detected at the correspondinganalyte retention times but are less than 3:l signaknoise Will be reported as NQ (not quantifiable). I Centre AnalyticalLabontories. Inc. Centre Analytical Laboratories,I~c. - _. .... Page 9 of 43 Page 65 of 104 ... -.-.. .-. .. - . . Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 g. Background levels of analyte found in cont.ro,Ublank samples that correspondto values below the LOQ, but ;Ire 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 analyzedpreferablywithin a week. 3. EXPERIMENTAL DESIGN Samples obtained by Wildlife International Ltd. have been shipped to Centre AnalyticalLaboratories,Inc. for analysis. All samples will be analyzed according to methods, "Ektraction of Potassium Peffluorooctanesulfonateor Other Fluorochemicd Compounds From Serum for Analysis Using HPLC-Elecuospray/Mass SpectrometrJ"' and 'Extraction of Potassium Peffluorooctanesulfonateor Other Fluorochemical Compounds From Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" with the modificationsdetailed in Section 8. Methods to control bias will include assay of untreated control samples, fortification of untreated control samples to obtain recovery data, and replicate analysis of fortifiedsamplest9 provide an indication of reproducibility. The average recovery and relative standard deviation of the fortified samples will be calculated. Also, the average residue found and Standard deviation for each matrix will be calculated 10. PROTOCOL AMENDMENTS A N D DEVIATIONS Any deviations from the protocol or from the method as provided will be documented and reported promptly to the Study Director. Planned changes to this protocol or to the analytical method will be made in Writing as an amendment and approved by the Principal Investigator and the Study Director. Any amendments or deviations will be appended to this protocol and included in the final report. 1. Protocol amendments; Planned changes to the. approved protocol shall be documented by amendments that clearly describe the change,justification for the change, and impact on the study. Amendments will be signed and dated by the Principal Investisator and Study Director. Copies of amendments will be sent to the quality assunnce unit. Centre Analytical b h t o r i e s , Inc. Centre Analytical Laboratories, Inc. Page 10 of 43 Page 66 of 104 .. . . Centre StudyNo.:023-042 - . _--.____ Centre Protocol No.OOP-023-042 2. Protocol deviations: Protocol deviations. which im one time and unplanned deviationsfrom the protocol, shallbe documentedin the studyrecords, noting the natun of the deviation, potential effect or impact on the study, and corrective action if required. Protocol deviations are signed and dated by the Principal Investigator and Study Director and reviewed by QAU. 11. RECORDS Records to be maintained inciude, but are not limited the following (8 appropriate): 1. Sample tracking sheet@) 2. Samplereceipt records, storagehistory, and chainsof custody 3. History and preparationof standards (stock, fortification,calibration) 4. Description of any modificationsto the method 5. Instrument run sheets, bench-sheets or logs 6. Analytical data tables 7. All chromatographicand inswmental conditions 8. Sampleextractionand analysisdates 9. A completelisting of study personnel, signaturesand initials 10. Chronologicalpresentationof all study correspondence 11. Any otherdata ne-cessary for the reconst?uction of the study All chromatogramswill containthe following: a. Sample identification, date, arrow m'other indication of the m a 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+, pg/ml, ng/mL. ppb, ppt. etc.). Each data set will contain information on temperatures, flow rates, column parameters, gases, instrument parameters, and instrument type. etc. The Centre study number will also be recorded on the first chromatogram of each daily analytical run. 12. QUALITY ASSURANCE Centre QA Unit will review the protocol, audit the study conduct &d the study documentation(includingraw data and final report) to ascertainthat all QMGLP procedures art adhered to. Centre QA Unit will inspect the study at intervals adequate to assure compliance to GLP's, and will report the findingsof the'audits to the Principal Investigator, Centre Management. and the Study Director. kc. Centre Analyticai Laboratories. Centre Analytical Laboratories, Inc. Page 11 of 43 Page 67 of 104 _.- .- .. -.... .. .. . Centre Study No.: 023-042 .............. ... - ...._______ Centre Protocol No. OOP-023-042 13. DATA AND REPORT 1. All raw data and the original signed prOtOcO1 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 C U ~ V ~ S , 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 find report will be issued by Centre, approved and signed by the Study Dimtor and sufficient for submission to EPA. The report contents should include, but not limited to: 1. Objectives and procedures stated in the protocol 2. Analytical and statistical methods used 3. Test materials identifiedby name, lot, punty, and other characteristics 4. Name of testing facility and studyinitiation and completiondates 5. Statement prepared and signedby the quality assuranceunit 6. Tables containingall applicabledata . 7. All chromatographic and instrumental conditions 8. A complete listing of Centre study personnel Centre will sezd a copy of @edraft report to the Study Director, who will r e m the report with comments. Centre Analytical will make revisions and finalize the report with the approval of the Study Director: The Centre QA unit will conduct .an audit of the final draft report and data files to assure accuracy and GLp compliance. The final report must be approved by the Study Director priorto signature. A statement of QAU inspections and a statement of GLP compliance will be included in the final nport. . . Any corrections or additions to the final report shall be in the form of an amenbent by the Principal Investigator. The amemhent shall clearly identify the part of the final report that is being corrected and the justification for correction. The amendment shall be signed and dated by the Principal Investigator and the Study Director. REPORT DISTRIBU?'ION: original: Study Director Copy: Centre Archives 14. ARCHIVE STATEMENT Study records to be maintained Records to be maintained for the study include all raw data, observations recorded during the conduct Of the study, documentation, c h r o m a t o m , specimen tracking information, and study related correspondence. Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. ___ - .... . . .______ -- - . . - . . . . . . . . . . . . . , . . .. Page 12 of 43 Page 68 of 104 .. ....... Centre Study NO.:023-042 .---- ._.. .______-- Centre Protocol No.OOP-023-042 This includes a description of equipment used during the conduct of the study. ~ 1 1 characterization data and any shipping records shall be retained. Document archives: Upon completion of the study, the study records, protocol md 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 Etain facility-related originals and a COPY of the final report and raw data package. 15. REFERENCES 1 U.S.Environmental Protection Agency. Toxic Substances Control Act (TSCA). Good LaboratoryPractice Standards. Final Rule, 40 CFR Part 792. Centre Analytical Laboratories. Inc. Centre Analytical Laboratories, Inc. , . . . -. . - . . . . .. . . . . -. .. - .. .... . - I Page 13 Of43 Page 69 of 104 - ....- .- . .. . . . .. Centre.StudyNo.: 023-042 .._ .. . . . . ~ Centre Protocol No. OOP-023-042 16. PROTOCOL APPROVAL This protocol was audited by the Quality Assurance Unit of Centre Analytical Laboratories, Inc. Quality Assurance Reviewer, Centre ub-2--2 Naomi Lovallo, Ph.D. Quality Assurance Auditor Principal Investigator, Centre DateI I' / s p C r c :Facility Management, Centre President Study Director, 3M ~ m e n t a l T e c h n o fl o* t CentreAnalytical Labontoria,Inc. .... .- _'' . Centre'AnalyticalLaboratories,Inc. .. . IS- twv - 00 Date Page 14 of 43 Page 70 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 APPENDIX: ANALYTICAL METHODS A. "Extractionof PotassiumPeffluorooctanesulfonateor Other Fluorochemicd Compounds From Serum for Analysis Using HPLC-Eiectrospray/Mas~ Spectrometry" B. "Extraction ofPotassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" Centre Analytical Laboratories. Inc. Centre AnalyticalZaboratories, Inc. . " ' . Page I5 of 43 . Page 71 of 104 Centre Study No.: 023-042 - ..... .--. .----_-_. - . i_.. .. . . . .__.__. _. . _ _..... Centre ProtocolNo.OOP-OU-042 Centre Analytical Laboratories, Inc. . . . . . . . . . .--. . . . . . . . - -- . ........... - .: --------.__ Page 16 of 43 Page 72 of 104 Centre Study No.: 023-042 Centre Analytical Laboratories, Inc. .- . .-. . Page 73 of 104 Centre Study No.: 023-042 .. Centre Protocol No. OOP-023-042 Centre Analytical Laboratories, Inc. ... - ._--__' __Page l8Of 43 Page 74 of 104 , -.-.. ...- Centre Study No.: 023-042 Centre Protocol No.OOP-02:3-042 Centre Analytical Laboratories, Inc. Page 75 of 104 Centre Study No.: 023-042 ..-. .-. . ....__ - -. - .. . ._. . _ __.-- Centre Protocol No.oOP-023-042 Centre Analytical Laboratories, Inc. Page 76 of 104 Centre Study No.: 023-042 .... ........ ....... --.- ......... . .. ........ -- .... .... - .... .-._ ...... .............. -. .- Centre Protocol No. OOP-023-042 I-- - ..... ..entre C. inalytic_al.-L--aboratorick: In!. ...... ..... Centre Analytical Laboratories, Inc. ... .-_- --.-- Page 21 O.f 43 Page 77 of 104 Centre Study No.: 023-042 . -. .- --..-. _ _ .. - Centre Protocol No.OOP-023-042 - . Centre ____ Analytical-La.b.oratories,Inc. Centre Analytical Laboratories,Inc. _- Page 78 of 104 ....... ......... . Centre Study NO.:023-042 ... ................... . . ._- ..-- -.. _ _ .. ................... .. -- --------. ....... - __I_ -. . ..... Centre Protocol No. oOP-023-042 ... ...... .- Centre Analytical Laboratories, Inc. - -- -- - - - - Page 79 of 104 ....... ......... .............. ...... ~ ~~ Centre Study No.: 023-042 ...... ...... .... - . . . Centre Analytical Laboratories, Inc. .............. .- . . .-..... Page SO of 104 _ _ ~ ~ ~ ......... Centre Study No.: 023-042 - - ......... - ........... . ........... Centre Protocol No.OOP-023-042 - __--...._P_ag_e 2_5 of 43 Page 81 of 104 ..... ._ . . . . Centre Study No.: 023-042 ..- . . . . . - - .._-..- . . -. ..... - I _. .. ------ Centre Analytical Labontdries; Inc. - - ....... .... ... Centre Analyticail Laboratories, Inc. .. .. ... -. .... -.- - P..a-g.e..2..6.-o.-f 43 Page 82 of 104 .. .. _ .. - . . . . . - - __ . . . . ~~ Centre Study No.: 023-042 ------ . . . . . . . . . . . . . . . ,. . . . . . . . . . . . . - ....... ... -.. . . . Centre Protocol No. oOP-02.3-042 __':_ Centre Ar,alyticai.Labora.tor.ies~, n c . :entre Analytkal Laboratories, Inc. I ...--...--.... . . ----- -..___P_,ag..e 27 of 43 Page 83 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-0:~-042 --- --._- Anat lytical Laboratories_, Inc_ . --- - - Centre Analytical Laboratories, Inc. I I Page - 28- * _ o_f _43 Page 84 of 104 ....... ....... . . ................................ Centre Study No.: 023-042 .. . .......... -. . -- Centre Protocol No. OoP-0:!3-042 ' ---. ... . CentreAn_alyticalLabontories,inc, !. Centre Analytical Laboratories,Inc. ............. .... .. - , Pag.e_2_9_of. 43 Page 85 of 104 Centre Study No.: 023-042 Centre Protocol No. OOP-023-042 -------_-__C_en_t& Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. . ------__ _ _ Page 30 of 43 Page 86 of 104 Centre Study No.: 023-042 . . Centre Pratccol No. oOP423-042 _ _ ~ - -Centree\nalyticalLaboratories-, hc. ----__ Page 3-1--o-f 4.3 Centre Analytical Laboratories, Inc. Page 87 of 104 _- .. ... _.......... . . . __-- .......... Centre Study No.: 023-042 ........... .... . . - . --- ---_-____- Centre Protocol No.oOP-o2:3-&~ -.---' Centre Analytical La.b.o.ra..t-o.-r.ie.s,-b..c... . ! ....... - .- .,.I --.-_. Centre Analytical Laboratories, Inc. _. . Page 32 of 43 . Page 88 of 104 .... Centre Study No.: 023-042 - -- -- . . .- - .-._ ---.- --.-__---I - . . . . . ........... ...... ..- . .... Centre Protocol No. OOP-023-042 , Cepg-e-Analytical Laboratories. Inc. ----- -_._. _---- I -- Page 33 of '43 -..-.__ Centre Analytical Laboratories, Inc. Page 89 of 104 ... ................ - ........ . .. . - . -. Centre Study No.: 023-042 . - . _.,".. . . . ,, . _ . ..- ____ - .- . . -_----I- _ _ .--- . .. . . -. .. -. . . --- .-. Centre Protocol No.DoP-023-042 -- ... - Centre Anal.yt.i.c.a.-l-.Laboratories. Inc. Centre Analytical Laboratories, Inc. . ..-_____, Page 34 of 43 ..... Page 90 of 104 Centre Study No.: 023-042 .. Centre ProtocolNo.OOP-023-042 ...--_ 7C- entre kia.ly.t- i.cH..l Laboratories,I&. _.-.- +-.- Centre Analytical Laboratories, Inc. .. ------ - -- -Page 3.5-o._f_..43 Page 91 of 104 Centre Study No.: 023-042 CentreAmhytical Laboratories, ~ n c . --________~-_---. Centre Analytical Laboratories, Inc. . .: - -_----- .. .. .. .- Page 3.6 of.43 Page 92 of 104 .. . Centre Study No.: 023-042 I Centre Protocol No. OOP-023-042 --... --- Centre Ana-ly- tic-al Labntories-.-I-nc. . 1 Centre Analytical Laboratories, Inc. --- . - . _ I .--._. _.. Page 37 of 43 Page 93 of 104 Centre Study No.: 023-042 -. . -C-en.-tre..A. nalytical Laboratories, .. - Inc. I . ~ Centre Analytical Laboratories, Inc. -- _----~ _ _ - __ ~ -. - ._. Page38 of 43 ______ Page 94 of 104 -. Centre Study No.: 023-042 Centre Protocol No. oOP-023-042 - - Centre ---_--- Arialytical Laboratories, Inc. -- - Centre Analytical Laboratories, Inc. ------- .- _ _ _ Page 39 o- f_4.3- Page 95 of 104 ...... . . -. . . ........ Centre Study No.: 023-042 Centre Protocol No. oOP-023-042 -- - - - ____ . --C-entre AnalyticalLaboratori_es,_In_c. ____-- -- - Centre Analytical Laboratories,Inc. . . . . . _..._ ....... .... .. --__ P_a_ge 40 Of43 Page 96 of 104 - - Centre Study No.: 023-042 Centre ProtocolNo.OOP-023-042 I . . - _---.-.Centre Arial-yt-ical Laboratories, -. Inc. ' Centre Analyticad Laboratories, Inc. _.... . - -. .. . . _ .- . . Page 41 of 43 Page 97 of 104; -. . . . . . . . . ........... .... Centre Study No.: 023-042 - .... _-. --- ---_ ___ . ................ Centre Protocol NO. 00P-023&2 ........ ..... , .C.entre.A..n..a..l.y.t.i.cal Laboratories, Inc. ..... Centre Analytical Laboratories, Inc. ..... . I ? . ....... .- Page 42 of 43 .. Page 98 of 104 Centre Study No.: 023-042 .. - .. ...... ... ...... ..... -. --. .-___.-.-._.-.__I. ... Centre Protocol No. oOP-023-042 .-. . .._..___ ........................... ........ . . . .,-. ... .. .:.. . . . .. . .. .. . . . . .. .. I. . . . . . . .. .. Ion Pair Standard Curves -Tissue --- . . . . : .. :: .. aa ... . .' .... .. .. . I .. C.e.n.tre Analytic_a.l.. L.aboratories, Inc. . - --- - . ... ..__ Page 43 of 43 Centre Analytical Laboratories, Inc. Page 99 of 104 Centre StudyNO.:023-042 - CEntrE Analytical Laboratorks, Inc, -30448 ResearchDrive,State College, PA 16801. Phone: (814)231-8032, Famimile: (814)231-1253 PROTOCOL AMEh'DMENT Page 1 of 1 Centre Study Number 023-042 Amendment Number: 1 Effective Date: 11/21/00 Centre Protocol Number 00P-023-042 DESCRIPTIONOF AMENDEDSECTION 1. Q 6: Justification for the Selection of the Test System AMENDED TO 1. Add the following: -.Acompletedescription of the test system along with all of the in-life paramett:rs is jdocemented in the sponsor's protocol no. 454/010600/Mp/SUB454, Project No.454105 that is associated with this analytical protocol. RATIONALE 1. At the request of the sponsor, a description of the in-iife parameters for the test system was not to be includedin this protocol that covers the analyticalphase for the analysis -of mallard liver and serum. IMPACT ON THESTUDY 1. No negative impact on the study. c - - Centre Analytical Laboratories, Inc. -. Centre QAU Review lvcL ///3.'/ob February 12.1998/1 Page 100 of 104 ~~ Centre Study No.: 023-042 @0I0 3 PROTOCOL Ab- Page 1 of n ECEIMD TIME SEP. 6. 4:06PM Centre Analytical Laboratories,Inc. Page 101 of 104 Centre Study No.: 023-042 .- ... . .. -Xu58.2681 3:EPH EXYEN N0.555 P.4 I @mhC ~ Lab n t Ar n~alytical 0ratories, Inc. pngc 1 of I , 1 i i I I ! ! I I I I 4 ECEIVED TIME SEp. 6. 4:06pfl Centre Analytical Laboratories, Inc. I I / I PRINT TIM SEP. 6. 4:88p~ Page 102 of 104 ~ ~~~~ Centre Study No.: 023-042 Centrtz AnaIyticai m4l!\ Laboratories, Inc, -3048 Research Drive, State College, PA 16801. Phone: (814)231-8032, Facsimile: (814)231-1253 Page 1 of PROTOCOL A&ENDMENT Amendment Number: 4 Date of Occurrence: 08/24/01 Centre: Study Number: 023-042 Centre Protocol Number: OOP-023-042 DESCRIPTIONOF AMENDED SECTION It- AMENDED TO 1. Change the study director horn Rochelle Robideau at 3 M Environmental Tecfmoiogy and Safety Services to Sean Gallagher at Wildlife International, LTD, 8598 Commerce Drive, Easton, MD 21601, phone: 410-822-8600, fax: 410-822-0632. 2. Change Rochelle Robideau to the Sponsor Representative. RATIONALE 1-2. By request of Rochelle Robideau (fax dated 08/24/01 included in correspondencesection of the raw data associated with this study) IMPACT ON THE STUDY 1-2. No negative impact on the study. Date epd,L Incoming Study Director Signature sI3d 01 Date ' Centre QAU Review !I31L. I 7 13010 February 12. 1998/1 Centre Analytical Laboratories, h c . Page 103 of 104 Centre Study No.: 023-042 411002 I I i i I ! I I I j 1 I i I I I ! I I ! 2 EGI- TIME SEP. 6. 4:06R1 Centre Analytica 1 Laboratories, Inc. 1 1 PRINT TIME SEP. 6. 4 : a m Page 10-4 of -104