Document G5wEyaVZ6XVpX0O0ZwzonLOV4

BACK TO MAIN SUMMARY OF ANALYTICAL RESULTS: MULTI-CITY FOOD STUDY The Multi-City Study was originally designed by Battelle Memorial Institute (Columbus, OH), to obtain preliminary data about the presence of fluorochemicals in foods and in drinking water to understand the potential sources of human exposure. The Multi-City Study paired each of three cities having manufacturing or commercial use of fluorochemical products (test cities) with three cities that do not (control cities). Information on residues in selected foods was obtained from analyses of food samples collected in a market basket study. The market basket sampling of the original Multi City Study design was implemented by Pace Analytical Services, Inc., Minneapolis, MN. The samples were analyzed for PFOS, PFOA, and FOSA by Centre Analytical Laboratories, Inc., State College, PA. The distributions of the PFOS, PFOA, and FOSA residue data by food and city category reveal similar patterns of residue concentrations in the control and test cities for each type of food. A total of 12 samples were found to contain levels of fluorochemical residues above the limit of quantification. Of the 12 samples with measurable fluorochemical residue levels, eight were samples collected in test cities. Measurable quantities of PFOS were found in five samples: four whole milk samples (three from test cities) and a ground beef sample (test city). PFOS residues found in the foods ranged from non-quantifiable levels to 0.852 ng/g. Measurable quantities of PFOA were found in seven samples: two ground beef samples (neither from test cities); two bread samples (one from a test city); two apple samples (both from test cities); and one green bean sample (from a test city). PFOA residue levels ranged from non-quantifiable levels to 2.35 ng/g. A value of 14.7 ng/g was found for PFOA in a bread sample from a control city, but was considered "suspect" by Centre Analytical. BACK TO MAIN ANALYTICAL REPORT STUDY TITLE Analysis of PFOS, FOSA and PFOA From Various Food Matrices Using HPLC Electrospray/Mass Spectrometry DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792 STUDY DIRECTOR Karen Smith, Centre SPONSOR REPRESENTATIVE Susan A. Beach, 3M ANALYTICAL REPORT COMPLETION DATE June 21, 2001 PERFORMING LABORATORY / TESTING FACILITY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 Phone: 814-2.31-8032 STUDY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 5:5133-3331 PROTECT IDENTIFICATION Centre Protocol Number: 00P-023-057 Centre Study Number: 023-057 Total Pages: 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Centre Study Number 023-057, entitled "Analysis of PFOS, FOSA and PFOA From Various Food Matrices Using HPLC Electrospray/Mass Spectrometry," conducted for 3M Environmental Technology and Safety Services, was performed in compliance with EPA TSCA Good Laboratory Practice Standards (40 CFR, Part 792) by Centre Analytical Laboratories, Inc. with the following exceptions: 1. The FOSA reference substance (analytical standard) used in this study has not been characterized under GLP's. 792.105(a). 2. Specimen (egg sample) identification for three samples was not according to 792.130(c) because the labels became removed from the containers. Study Director Centre Analytical Laboratories, Inc. Date Susan A. Beach Sponsor Representative 3M Environmental Technology and Safety Services Centre Analytical Laboratories, Inc. Page 2 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 QUALITY ASSURANCE STATEMENT Centre Analytical Laboratories' Quality Assurance Unit reviewed Centre Study Number 023-057, entitled, "Analysis of PFOS, FOSA and PFOA From Various Food Matrices Using HPLC Electrospray/Mass Spectrometry". All phases were reviewed 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 Date Inspected 02/14/01 2. Extraction and Fortification 02/14/01 Date Reported to Study Director* 03/23/01 03/23/01 Date Reported to Centre Management 02/26/01 02/26/01 Date Reported to Sponsor Management 03/23/01 03/23/01 3. Raw Data and Draft Report Review 04/10-13/01 05/21/01 06/04/01 06/15/01 4. Raw Data and Draft Report Review 05/30/01 06/01/01 06/04/01 06/15/01 5. Final Report Review 06/19/01 06/20/01 06/21/01 06/21/01 *The Study Director resided at the sponsor until 05/01/01 at which time a protocol amendment transferred Study Director duties to Centre Analytical Laboratories. Naomi Lovallo Senior Quality Assurance Auditor Date Centre Analytical Laboratories, Inc. Page 3 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 CERTIFICATION OF AUTHENTICITY This report, for Centre Study Number 023-057, is a true and complete representation of the raw data for the study. Submitted by: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 (814) 231-8032 Centre Analytical Laboratories, Inc. zitelloI Date Centre Analytical Laboratories, Inc. Facility Management: m Dhn Flaherty Laboratory Manager Centre Analytical Laboratories, Inc. Date Sponsor Representative, 3M Q_- Susan A. Beach 3M Environmental Technology and Safety Services &U ^ l \ Date Centre Analytical Laboratories, Inc. Page 4 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 STUDY IDENTIFICATION Analysis of PFOS, FOSA and PFOA From Various Food Matrices Using HPLC Electrospray/Mass Spectrometry CENTRE PROTOCOL NUMBER: 00P-023-057 TYPE OF STUDY: Residue TEST SYSTEM: Green beans, apples, pork muscle, cow milk, chicken muscle, chicken eggs, bread, hot dogs, catfish and ground beef TEST MATERIAL: PFOS, FOSA and PFOA SPONSOR: 3M Environmental Technology and Safety Services Building 2-3E-09 POBox 33331 St. Paul, MN 55133-3331 STUDY DIRECTOR: Karen Smith Centre Analytical Laboratories, Inc. Phone: (814) 231-8032 TESTING FACILITY: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: Analytical Start Date: Analytical Termination Date: 01/09/01 01/11/01 05/22/01 Centre Analytical Laboratories, Inc. Page 5 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 PROJECT PERSONNEL The Study Director for this project at Centre Analytical Laboratories, Inc. was Karen Smith. The following personnel from Centre Analytical Laboratories, Inc., were associated with various phases of the study: Name Karen Smith Emily Stauffer Angela Morgan Tiffany Proctor Rickey Keller Lawrence Ord Dave Bell Sharareh Zolghadr Jason Farabaugh Mitra Arjmand Yu Qiong Tan Ling Ling Liu Xiaoming Zhu Title Scientist Scientist Technician Technician Sample Custodian Sample Custodian Scientist Technician Scientist Technician Technician Technician Technician Centre Analytical Laboratories, Inc. Page 6 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 TABLE OF CONTENTS Page TITLE PAGE...................................................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................. 2 QUALITY ASSURANCE STATEMENT.......................................................................... 3 CERTIFICATION OF AUTHENTICITY........................................................................... 4 STUDY IDENTIFICATION............................................................................................... 5 PROJECT PERSONNEL.................................................................................................... 6 TABLE OF CONTENTS.................................................................................................... 7 LIST OF TABLES.............................................................................................................. 8 LIST OF FIGURES............................................................................................................. 9 LIST OF APPENDICES................................................................................................... 11 1.0 SUMMARY............................................................................................................... 12 2.0 OBJECTIVE............................................................................................................... 12 3.0 INTRODUCTION.......................................................................................................12 4.0 TEST SYSTEM......................................................................................................... 13 5.0 REFERENCE MATERIAL........................................................................................14 6.0 EXPERIMENTAL DESIGN...................................................................................... 15 7.0 DESCRIPTION OF ANALYTICAL METHOD........................................................15 7.1 Extraction Procedure........................................................................................... 15 7.2 Preparation of Standards and Fortification Solutions...........................................15 7.3 Chromatography.................................................................................................. 16 7.4 Instrument Sensitivity.......................................................................................... 17 7.5 Description of Instrument and Operating Conditions.......................................... 17 7.6 Quantitation and Example Calculation................................................................ 18 8.0 RESULTS AND DISCUSSION.............................................................................. 20 9.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA.................... 21 10.0 RETENTION OF DATA AND SAMPLES............................................................. 21 11.0 TABLES................................................................................................................... 22 12.0 FIGURES.................................................................................................................41 13.0 APPENDICES.......................................................................................................... 65 Centre Analytical Laboratories, Inc. Page 7 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 LIST OF TABLES Page Table I. Summary of PFOS, FOSA and PFOA Fortification Recoveries....................... 23 Table II. Summary of PFOS, FOSA and PFOA Residues Found in Samples................ 26 Centre Analytical Laboratories, Inc. Page 8 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 LIST OF FIGURES Page Figure 1. Typical Calibration Curve for PFOS................................................................42 Figure 2. Typical Calibration Curve for FOSA...............................................................43 Figure 3. Typical Calibration Curve for PFOA...............................................................44 Figure 4. Chromatogram Representing a 0.5 ng/mL mixed calibration standard of PFOS, FOSA and PFOA...............................................................................45 Figure 5. Chromatogram Representing a 2.5 ng/mL mixed calibration standard of PFOS, FOSA and PFOA...............................................................................46 Figure 6. Chromatogram Representing a Control Apple Sample (Centre ID: 0009347 Blank A l, Data Set: 020901C Apple)........................................................... 47 Figure 7. Chromatogram Representing a Control Apple Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009347 Spk Al, Data Set: 020901C Apple)............................................................................ 48 Figure 8. Chromatogram Representing an Apple Sample (Centre ID: 0012927, Sponsor ID: Publix, 18257, Data Set: 020901C Apple)............................... 49 Figure 9. Chromatogram Representing a Control Green Bean Sample (Centre ID: 0009346 Blank A2, Data Set: 020601C Bean)............................................. 50 Figure 10. Chromatogram Representing a Control Green Bean Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009346 Spk A2, Data Set: 020601C Bean).............................................................................. 51 Figure 11. Chromatogram Representing a Green Bean Sample (Centre ID: 0012910, Sponsor ID: Bruno's, 18341, Data Set: 020601C Bean)............................... 52 Figure 12. C hrom atogram Representing a C ontrol P ork M uscle Sam ple (C entre ID: 0009348 Blank Al, Data Set: 011501A Pork).............................................. 53 Figure 13. Chromatogram Representing a Control Pork Muscle Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009348 Spk Al, Data Set: 011501A Pork).............................................................................. 54 Figure 14. Chromatogram Representing a Pork Muscle Sample (Centre ID: 0012795, Sponsor ID: Winn-Dixie, 18227, Data Set: 011501A Pork)......................... 55 Figure 15. Chromatogram Representing a Control Milk Sample (Centre ID: 0009349 Blank A l, Data Set: 020701A M ilk)............................................................ 56 Figure 16. Chromatogram Representing a Control Milk Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009349 Spk Al, Data Set: 020701A Milk).............................................................................. 57 Centre Analytical Laboratories, Inc. Page 9 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 LIST OF FIGURES (CONTINUED) Figure 17. Chromatogram Representing a Milk Sample (Centre ID: 0012871, Sponsor ID: Winn-Dixie, 18202, Data Set: 020701A M ilk)........................ 58 Figure 18. Chromatogram Representing a Control Chicken Egg Sample (Centre ID: 0009342 Blank A3, Data Sets: 050901A Egg for PFOS, 012401A Egg for FOSA, and 052101A Egg for PFOA)...................................................... 59 Figure 19. Chromatogram Representing a Control Chicken Egg Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009342 Spk A3, Data Sets: 050901A Egg for PFOS, 012401A Egg for FOSA, and 052101A Egg for PFOA).............................................................................. 60 Figure 20. Chromatogram Representing a Chicken Egg Sample (Centre ID: 0012861, Sponsor ID: Save, 18167 and 21226, Data Sets: 050901A Egg for PFOS, 012401A Egg for FOSA, and 052101A Egg for PFOA).............................. 61 Figure 21. Chromatogram Representing a Control Bread Sample (Centre ID: 0009343 Blank Al, Data Set: 020901A Bread)........................................................... 62 Figure 22. Chromatogram Representing a Control Bread Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009343 Spk Al, Data Set: 020901A Bread)............................................................................ 63 Figure 23. Chromatogram Representing a Bread Sample (Centre ID: 0012887, Sponsor ID: Albertson's, 18244, Data Set: 020901A Bread)....................... 64 Centre Analytical Laboratories, Inc. Page 10 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 LIST OF APPENDICES Page Appendix A Study Protocol 00P-023-057 (Centre Study No. 023-057), Amendments and Deviation............................................... 65 Centre Analytical Laboratories, Inc Page 11 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 1.0 SUMMARY The purpose of this study was to analyze residues of PFOS, FOSA and PFOA in various food matrices as specified in Centre Protocol 00P-023-057. The analytical method used for this study was entitled, "Method of Analysis for the Determination of Fluorochemicals in Green Beans, Apples, Pork Muscle, Cow Milk, Chicken Muscle, Chicken Eggs, Bread, Hot Dogs, and Catfish by LC/MS/MS." The limit of quantification for PFOS, FOSA and PFOA in food was 0.5 ppb (ng/g). Average fortification recoveries and relative standard deviations were 101% 9.0% for PFOS, 96% 13% for FOSA and 104% 14% for PFOA. Residues ranging from non-quantifiable levels to 0.852 ppb (ng/g) for PFOS, non quantifiable levels for FOSA, and non-quantifiable to 2.35 ng/g for PFOA (one value of 14.7 ppb (ng/g) was found for PFOA but was considered suspect) were found in the food samples. 2.0 OBJECTIVE The objective of this study was to determine levels of PFOS, FOSA and PFOA in food samples collected from local grocery stores from six test cities outlined in 3M Quality Assurance Project Plan for Empirical Human Exposure Assessment Multi-City Study Sampling Task using the analytical method entitled "Method of Analysis for the Determination of Fluorochemicals in Green Beans, Apples, Pork Muscle, Cow Milk, Chicken Muscle, Chicken Eggs, Bread, Hot Dogs, and Catfish by LC/MS/MS." 3.0 INTRODUCTION This report details the results of the residues of PFOS, FOSA and PFOA detected in green beans, apples, pork muscle, cow milk, chicken muscle, chicken eggs, bread, hot dogs, catfish and ground beef, using the analytical method entitled, "Method of Analysis for the Determination of Fluorochemicals in Green Beans, Apples, Pork Muscle, Cow Milk, Chicken Muscle, Chicken Eggs, Bread, Hot Dogs, and Catfish by LC/MS/MS." Complete details of the analytical methodology can be found in Appendix A. The study was initiated on January 09, 2001, when the study director signed the protocol 00P-023-057. The complete protocol and amendments can be found in Appendix A. The analytical start date was January 11, 2001, and the analytical termination date was May 22, 2001. Centre Analytical Laboratories, Inc. Page 12 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 4.0 TEST SYSTEM The food samples analyzed in this study were received frozen from Pace Analytical on December 09, 2000 and stored frozen (<-10C) upon receipt. Pace Analytical collected the samples for 3M Environmental Laboratory. The samples were then logged in on December 18, 2000 by Centre personnel and transferred to different frozen storage locale. All of the samples were processed according to the method, "Method of Analysis for the Determination of Fluorochemicals in Green Beans, Apples, Pork Muscle, Cow Milk, Chicken Muscle, Chicken Eggs, Bread, Hot Dogs, and Catfish by LC/MS/MS." Exact details of processing can be found on page 91 of this report. After processing, the samples were stored in a freezer at a temperature of -10C or below until used and placed back into a freezer immediately after use. At the request of the sponsor, the egg samples were returned frozen to 3M Environmental Laboratory on February 26, 2001 for possible reanalysis. The egg samples were returned unanalyzed from 3M Environmental Laboratory to Centre Analytical Laboratories, Inc. These samples were received frozen from 3M Environmental Laboratory on May 03, 2001 for PFOS and PFOA re-extraction and analysis. They were assigned different 3M and Centre ID's. The food samples used for the controls and matrix spikes were the same samples used in the method validation study (Centre Study No. 023-043). They were purchased from Giant, Store #111 in State College, PA and assigned the following sample IDs upon log in at Centre: Centre Sample ID 0009341 0009342 0009343 0009344 0009345 0009346 0009347 0009348 0009349 0100006 Sample Description Chicken Muscle Chicken Eggs White Bread Hot Dogs Catfish Green Beans Apples Pork Muscle Whole Milk Ground Beef All of the control samples were purchased on September 27, 2000 except for the ground beef, which was purchased on January 03, 2001. The samples were stored in a refrigerator (4 2C) until processed. All of these control samples were processed with dry ice in a Hobart food chopper on October 02, 2000 except for the ground beef, which was processed on January 03, 2001. After processing, the samples were stored in a freezer at a temperature of -10C or below until used and placed back into a freezer immediately after use. Centre Analytical Laboratories, Inc. Page 13 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Sample login and chain of custody information can be found in the raw data package associated with this study. Storage records will be kept at Centre Analytical Laboratories, Inc. and a true copy of the storage records can be found in the raw data package associated with this study. 5.0 REFERENCE MATERIAL PFOS was received at Centre on June 05, 2000, FOSA was received on February 15, 2000 and PFOA was received on July 06, 2000 from 3M Environmental Technology and Services. Characterization of the reference materials PFOS and PFOA was performed at Centre on August 31, 2000 and November 06, 2000, respectively, and documentation can be found in raw data package associated with this report. The characterization of FOSA is in progress and the necessary information will be added upon complete characterization of the reference materials. All analyses were completed prior to standard expiration dates. The available information for the reference material is listed below. FOSA was stored at room temperature and PFOS and PFOA were stored frozen at < -10C. Compound PFOS FOSA PFOA TCR Substance No. TCR00017-46 SD-029 TCR99030-30 Batch No. NA L-15709 332 Purity (%) 97.9 95.1 95.0 Expiration Date 08/31/01 02/15/01 10/31/01 Molecular structures of PFOS, FOSA and PFOA are given below. PFOS Chemical Name Molecular weight o = = Perfluorooctane sulfonate 499 (CgFnSOa-) C8F17S -- O" 0 Note: The neutral molecule and standard form which PFOS (anion) is derived from is potassium perfluorooctane sulfonate [C8F17SO3K], molecular weight 538. FOSA Chemical Name Molecular weight O C 8Fi 7S -- NH2 o = = Perfluorooctane sulfonylamide 499 Centre Analytical Laboratories, Inc. Page 14 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 PFOA Chemical Name Molecular weight Perfluorooctanoate 413 C7F-I5COO' Note: The neutral molecule and standard form which PFOA (anion) is derived from is ammonium perfluoroctanoate [C7F15COONH4], molecular weight 431. 6.0 EXPERIMENTAL DESIGN Each sample was extracted in duplicate. A set contained one matrix blank and two matrix blanks spiked with known amounts of PFOS, FOSA and PFOA and 6 - 1 0 samples. The re-extracted egg sets also included one reagent blank. The extracts were analyzed by LC/MS/MS. Samples with residues below the linear range of the calibration curve were not quantified; however, residues in the control samples were calculated up to 20% of the LOQ by extrapolation of the calibration curve, in order to correct fortification recoveries. 7.0 DESCRIPTION OF ANALYTICAL METHOD Analytical method entitled "Method of Analysis for the Determination of Fluorochemicals in Green Beans, Apples, Pork Muscle, Cow Milk, Chicken Muscle, Chicken Eggs, Bread, Hot Dogs, and Catfish by LC/MS/MS" was used for this study. 7.1 Extraction Procedure Five grams of processed sample were weighed into 50 mL polypropylene centrifuge tubes and fortified (if necessary). The samples were extracted with ACN followed by a SPE column (florisil, silica gel, carbon and LC-NH2) clean up. The column eluate was collected and concentrated to near dryness. Final volume was adjusted to 2 mL with 2% ascorbic acid in methanol. The samples were analyzed using electrospray LC/MS/MS. 7.2 Preparation of Standards and Fortification Solutions Standard stock solutions of PFOS, PFOA and FOSA were prepared on September 18, 2000, September 26, 2000 and October 03, 2000, respectively. Fortification and calibration standard solutions were prepared on October 17, 2000 as specified in Centre Analytical Laboratories' analytical method entitled, "Method of Analysis for the Determination of Fluorochemicals in Green Beans, Apples, Pork Muscle, Cow Milk, Centre Analytical Laboratories, Inc. Page 15 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Chicken Muscle, Chicken Eggs, Bread, Hotdogs, and Catfish by LC/MS/MS." Individual stock standard solutions of PFOS, FOSA and PFOA were prepared at concentrations of 100 pg/mL by taking 10 mg of the standard (corrected for purity and salt content where applicable) and bringing it up to 100 mL with methanol. From these solutions, a 1 pg/mL mixed fortification standard solution was prepared by taking 1 mL of each stock and bringing the volume up to 100 mL with methanol. The 1 pg/mL mixed fortification standard was diluted ten-fold with methanol to make the 0.1 pg/mL mixed fortification standard. Then, the 0.1 pg/mL mixed standard was diluted ten-fold with methanol to make the 0.01 pg/mL fortification standard. LC/MS/MS calibration standards were prepared in methanol at the following concentrations 50,25,10, 5.0,2.5,1.0, and 0.5 ng/mL according to the following table: Initial Concentration (ng/mL) 1000 1000 1000 50 25 10 5.0 Volume (mL) 5.0 2.5 1.0 10.0 10.0 10.0 10.0 oo Diluted to (mL) 100 100 100 100 100 100 Final concentration (ng/mL) 50 25 10 5.0 2.5 1.0 0.5 New standard stock, fortification and calibration solutions were made for PFOS and PFOA in order to perform the egg re-extraction and analysis. The PFOS standard stock solution was prepared on March 14, 2001 and the PFOA standard stock solution was prepared on April 11, 2001. Mixed fortification and calibration solutions of PFOS and PFOA were prepared from the stock solutions on May 03, 2001 as described above. The only modification from the above procedure is that FO S A w as om itted from the m ix since the re-extraction and analysis was for PFOS and PFOA only. The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation can be found in the raw data associated with this report. 7.3 Chromatography Quantification of PFOS, FOSA and PFOA was accomplished by LC/MS/MS analysis using electrospray LC/MS/MS. The retention times of PFOS, FOSA and PFOA were ~ 5.4 min, ~ 6.0 min and ~ 5.3 min., respectively. The matrices did not contribute any interfering peaks corresponding to the Centre Analytical Laboratories, Inc. Page 16 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 analyte retention times; however, there were some occasions where the control matrices appeared to contain analyte. 7.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run was 0.5 ng/mL which is equivalent to 0.2 ng/g of PFOS, FOSA and PFOA in matrix. 7.5 Description of Instrument and Operating Conditions A Micromass Quattro Ultima LC/MS/MS coupled to a Hewlet Packard HPLC system was used. Data acquisition and processing were performed using Masslynx 3.4 software. Detailed operating conditions are listed below: Instrument: Micromass Quattro Ultima ELECTROSPRAY ION SOURCE: Capillary: 2.0 kV Hexapole 1:0 V Aperture 1: 0 V Hexapole 2: 0 V Source Block Temp.: 100C Desolvation Temp.: 400C ANALYZER: LM Res 1: 10.5 V H M R es 1: 10.5 V IEnergy 1: 1.0 V Entrance: -2 V Exit: 2 V LM Res 2: 12.0 V H M R es 2: 12.0 V IEnergy 2: 2.0 V Multiplier: 650 V GAS FLOWS AND PRESSURE: Desolvation N2Flow Rate: -700 L/hr Nebuliser N2Flow Rate: -150 L/hr Gas Cell Pressure: -0.0031 mbar Computer: Software: COMPAQ Professional Workstation AP200 Microsoft Windows NT: Version 4 Build 1381: Service Pack 5 Micromass Limited: Masslynx 3.4 Build 004 HPLC Equipment: Hewlett Packard (HP) Series 1100 HP Binary Pump HP Autosampler HP Vacuum Degasser HP Column Oven HPLC Column: Genesis C-8, 5 cm x 2.1 mm i.d. x 4 p Column Temperature: 35C Mobile Phase (A) : 2 mM Ammonium Acetate in Type I Water Centre Analytical Laboratories, Inc. Page 17 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Mobile Phase (B) : Methanol Time (min) %A 0.0 60.0 0.4 60.0 1.0 10.0 7.0 10.0 7.5 0.0 9.0 0.0 9.5 60.0 13.5 60.0 14.0 60.0 Total run time = 1 4 min %B 40.0 40.0 90.0 90.0 100.0 100.0 40.0 40.0 40.0 Flow Rate (mL/min) 0.3 0.3 0.3 0.3 0.3 0.4 0.4 0.4 0.3 Injected Volume: 15 pL Ions monitored : Analvte PFOA FOSA PFOS Parent ion 413 498 499 Daughter ion 369 78 99 Dwell (secs') 0.2 0.2 0.2 Coll Enerev (eVi 10 40 50 Cone(VI 30 30 30 7.6 Quantitation and Example Calculation Fifteen microliters of sample or calibration standard was injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x weighted linear regression) by Masslynx software using seven concentrations of standards. The residue concentration in food samples was determined using the following equations: Equation 1 was used to calculate the amount of analyte found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Masslynx software program. Equation 1: Analyte found (ng/mL) = (Peak area - intercept! slope The component residue concentration (in ng/g) in matrix was determined by using Equation 2. Centre Analytical Laboratories, Inc. Page 18 of 151 BACK TO MAIN Equation 2: Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Analyte found (ng/g) = [analyte found (ng/mL) - average analyte found in blanks (ng/mL)] x DF x FV (mL) sample weight (g) where DF = dilution factor and FV = final volume Note: the process of subtracting the average analyte found in the blanks was done only for control matrix spikes or if a reagent blank contained residue. For samples fortified with known amounts of analytes prior to extraction, Equation 3 was used to calculate the percent recovery. Equation 3: Recovery (%) = " " ly<efoi.d(ngfe) xlOO analyte added (ng/g) An example of a calculation using an actual sample follows: Pork sample Centre ID 0009348 Spk A1 (Set No.: 011501A Pork), fortified at 2.5 ng/g of PFOS, FOSA and PFOA: (Calculation is using values from FOSA) Where: peak area intercept slope final volume dilution factor sample weight ng/g added (fort level) amt found in control = = = = = = = = 149454 2524.48 25194.9 2 mL 1 5g 2.5 ng/g 0 (NQ = Not quantifiable; assumed to be 0) From equation 1: Analyte found (ng/mL) = f149484 - 2524.481 25194.9 = 5.83 ng/mL Centre Analytical Laboratories, Inc. Page 19 of 151 From equation 2: Analyte found (ng/g) BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 = (5.832- 0 ) x 1 x 2 5.0 = 2.33 ng/g From equation 3: % Recovery = 2.33 x 100 2.5 = 93% Note: This example calculation was done using rounded numbers, and therefore may be slightly different from the values shown in the RAW DATA. Any residues found for PFOA were adjusted for PFOA purity (95%). This was done by taking the residue found (ng/g) for PFOA and multiplying by the purity (0.95). Other statistical methods used in analyzing this data were: (*,. - x f Standard Deviation = 1 n- 1 Mean = x = Relative Standard Deviation (RSD or Coefficient o f Variation (CV)) = Standard Deviation x 100% Mean 8.0 RESULTS AND DISCUSSION Average fortification recoveries and relative standard deviations were 101% 9.0% for PFOS, 96% 13% for FOSA and 104% 14% for PFOA. A summary of all of the fortification recoveries can be found in Table I. Residues ranging from non-quantifiable levels to 0.852 ppb (ng/g) for PFOS, non quantifiable levels for FOSA, and non-quantifiable to 2.35 ng/g for PFOA (one value of 14.7 ppb (ng/g) was found for PFOA but was considered suspect) were found in the food samples. The residues found in all of the samples are detailed in Table II. Centre Analytical Laboratories, Inc. Page 20 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Typical calibration curves and chromatograms representing standards, controls, fortifications, and samples are depicted in Figures 1-23. In this study, analytes were not quantified below 0.5 ng/g. If lower detection levels are required, a more definitive study, including extraction blanks in addition to matrix blanks, could be done. Initially the PFOA standard and fortification solutions were not corrected for purity, as purity information had not been received at the time. Purity data was received after residue analyses were completed and the residues found were then corrected for PFOA purity. All ng/g residues of PFOA in this report have been adjusted to reflect the purity of the PFOA used in this study. There was some PFOA contamination from the lab while performing the extraction for eggs. It was believed to be coming from the extraction solvent itself. Using a different brand and lot number of ACN alleviated the problem because upon reanalysis, there were no quantifiable residues of PFOA in the egg samples. 9.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA There were no circumstances that may have affected the quality or integrity of the data. 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 subm itted. E xact copies o f all raw data, as w ell as a signed copy o f the final analytical report and all original facility-specific raw data, will be retained in the Centre Analytical Laboratories, Inc. archives for the period of time specified in 40 CFR 792.195 (b). Retained samples of reference substances are archived by the sponsor. Centre Analytical Laboratories, Inc. Page 21 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 11.0 TABLES Centre Analytical Laboratories, Inc. Page 22 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table I. Summary of PFOS, FOSA and PFOA Fortification Recoveries Extraction Analysis Fort Level Recovery (%) Centre ID Sample Description Date Date (ng/g) PFOS FOSA PFOA 0009341 Spk Al 0009341 SpkBl 0009341 Spk A2 0009341 SpkB2 0009341 Spk A3 0009341 Spk B3 0009341 Spk A4 0009341 Spk B4 0009348 Spk Al 0009348 SpkBl 0009348 Spk A2 0009348 Spk B2 0009348 Spk A3 0009348 SpkB3 0009348 Spk A4 0009348 Spk B4 0009344 Spk Al 0009344 SpkBl 0009344 Spk Al 0009344 SpkBl 0009344 Spk A2 0009344 Spk B2 0009344 Spk A3 0009344 Spk B3 0009344 Spk A4 0009344 Spk B4 0009345 Spk Al 0009345 SpkBl 0009345 Spk A2 0009345 Spk B2 0009345 Spk A2 0009345 Spk B2 0009345 Spk A3 0009345 SpkB3 0009345 Spk A4 0009345 SpkB4 Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Pork Pork Pork Pork Pork Pork Pork Pork Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish 01/11/01 01/11/01 01/11/01 01/11/01 01/12/01 01/12/01 01/12/01 01/12/01 01/15/01 01/15/01 01/15/01 01/15/01 01/16/01 01/16/01 01/16/01 01/16/01 01/17/01 01/17/01 01/17/01 01/17/01 01/17/01 01/17/01 01/18/01 01/18/01 01/18/01 01/18/01 01/19/01 01/19/01 01/19/01 01/19/01 01/30/01 01/30/01 01/22/01 01/22/01 01/22/01 01/22/01 01/11-12/01 01/11-12/01 01/12/01 01/12/01 01/15/01 01/15/01 01/15/01 01/15/01 01/15-16/01 01/15-16/01 01/16/01 01/16/01 01/16/01 01/16/01 01/16-17/01 01/16-17/01 01/17-18/01 01/17-18/01 01/18/01 01/18/01 01/18/01 01/18/01 01/18-19/01 01/18-19/01 01/19/01 01/19/01 01/19-20/01 01/19-20/01 01/20/01 01/20/01 01/31/01 01/31/01 01/22-23/01 01/22-23/01 01/23/01 01/23/01 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 104 93 94 89 84 87 110 94 98 91 89 91 108 107 106 106 105 111 106 105 107 97 90 98 105 93 116 99 86 110 98 87 105 84 76 95 98 93 106 81 78 91 90 86 93 82 79 102 100 95 92 92 * * - - 97 - - 95 113 106 114 95 91 87 99 94 102 89 77 89 104 95 96 91 84 84 100 68 105 87 56 87 86 * 85 86 * 86 - 126 - - 110 - I l l 76 I l l 95 65 101 106 73 103 89 62 89 * = Rejected due to an unacceptable recovery. Replaced with re-extracted data. - = Not Analyzed For Centre Analytical Laboratories, Inc. Page 23 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table I continued: Summary of PFOS, FOSA and PFOA Fortification Recoveries Extraction Analysis Fort Level Recovery (%) Centre ID Sample Description Date Date (ng/g) PFOS FOSA PFOA 0009342 Spk Al 0009342 SpkBl 0009342 Spk A2 0009342 Spk B2 0009342 Spk A3 0009342 SpkB3 0009342 Spk A4 0009342 Spk B4 0009346 Spk Al Egg Egg Egg Egg Egg Egg Egg Egg Green Bean 01/23/01 01/23/01 01/23/01 01/23/01 01/24/01 01/24/01 01/24/01 01/24/01 02/05/01 01/23-24/01 01/23-24/01 01/24/01 01/24/01 01/24-25/01 01/24-25/01 01/25/01 01/25/01 02/05/01 2.5 10 2.5 10 2.5 10 2.5 10 2.5 104 96 84 98 80 67 117 106 78 101 98 77 115 109 105 92 90 85 117 111 90 102 97 82 98 114 129 0009346 SpkBl Green Bean 02/05/01 02/05/01 10 106 106 128 0009346 Spk A2 Green Bean 02/06/01 02/07/01 2.5 92 99 124 0009346 Spk B2 Green Bean 02/06/01 02/07/01 10 111 103 130 0009346 Spk A3 Green Bean 02/08/01 02/09/01 2.5 99 100 125 0009346 Spk B3 Green Bean 02/08/01 02/09/01 10 94 97 125 0009346 Spk A4 0009346 Spk B4 0009349 Spk Al 0009349 SpkBl Green Bean Green Bean Milk Milk 02/08/01 02/08/01 02/07/01 02/07/01 02/12/01 02/12/01 02/09-10/01 02/09-10/01 2.5 10 2.5 10 83 85 103 96 95 112 99 103 129 91 90 128 0009349 Spk A2 Milk 02/07/01 02/10/01 2.5 114 101 123 0009349 Spk B2 Milk 02/07/01 02/10/01 10 90 91 123 0009349 Spk A3 Milk 02/08/01 02/10/01 2.5 101 103 126 0009349 SpkB3 Milk 02/08/01 02/10/01 10 89 91 129 0009349 Spk A4 Milk 02/08/01 02/10-11/01 2.5 113 103 124 0009349 Spk B4 0009347 Spk A l Milk Apple 02/08/01 02/10-11/01 02/09/01 02/12/01 10 2.5 93 94 123 113 110 122 0009347 SpkBl 0009347 Spk A2 Apple Apple 02/09/01 02/12/01 10 105 104 119 02/12/01 02/13/01 2.5 111 107 108 0009347 SpkB2 Apple 02/12/01 02/13/01 10 100 102 101 0009347 Spk A3 Apple 02/12/01 02/13-14/01 2.5 109 105 110 0009347 Spk B3 Apple 02/12/01 02/13-14/01 10 98 96 100 0009347 Spk A4 Apple 02/12/01 02/14/01 2.5 108 101 102 0009347 Spk B4 Apple 02/12/01 02/14/01 10 99 96 99 Centre Analytical Laboratories, Inc. Page 24 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table I continued: Summary of PFOS, FOSA and PFOA Fortification Recoveries Extraction Analysis Fort Level Recovery (%) Centre ID Sample Description Date Date (ng/g) PFOS FOSA PFOA 0009343 Spk Al Bread 02/08-09/01 02/11/01 2.5 87 106 118 0009343 SpkBl Bread 02/08-09/01 02/11/01 10 96 96 109 0009343 Spk A2 Bread 02/08-09/01 02/11/01 2.5 106 101 119 0009343 SpkB2 Bread 02/08-09/01 02/11/01 10 94 93 106 0009343 Spk A3 Bread 02/12-13/01 02/14/01 2.5 106 103 103 0009343 SpkB3 Bread 02/12-13/01 02/14/01 10 99 92 97 0009343 Spk A4 Bread 02/12-13/01 02/14/01 2.5 111 109 106 0009343 SpkB4 Bread 02/12-13/01 02/14/01 10 102 100 100 0100006 Spk Al Ground Beef 02/13/01 02/14-15/01 2.5 104 104 100 0100006 SpkBl Ground Beef 02/13/01 02/14-15/01 10 103 99 95 0100006 Spk A2 Ground Beef 02/13/01 02/15/01 2.5 111 102 103 0100006 Spk B2 Ground Beef 02/13/01 02/15/01 10 103 98 98 0100006 Spk A3 Ground Beef 02/14/01 02/15/01 2.5 121 123 120 0100006 Spk B3 Ground Beef 02/14/01 02/15/01 10 106 116 114 0100006 Spk A4 Ground Beef 02/14/01 02/15-16/01 2.5 112 108 105 0100006 Spk B4 Ground Beef 02/14/01 02/15-16/01 10 102 101 102 0009342 Spk Al Egg 05/07-08/01 05/08/01 2.5 111 - - 0009342 SpkBl Egg 05/07-08/01 05/08/01 10 99 - - 0009342 Spk A2 Egg 05/07-08/01 05/08-09/01 2.5 111 - - 0009342 SpkB2 Egg 05/07-08/01 05/08-09/01 10 102 - - 0009342 Spk A3 Egg 05/08-09/01 05/09/01 2.5 106 - - 0009342 SpkB3 Egg 05/08-09/01 05/09/01 10 94 - - 0009342 Spk A4 Egg 05/08-09/01 05/09-10/01 2.5 109 - - 0009342 Spk B4 Egg 05/08-09/01 05/09-10/01 10 102 - - 0009342 Spk At Egg 05/18/01 05/18/01 2.5 - - 110 0009342 SpkBl Egg 05/18/01 05/18/01 10 - - 99 0009342 Spk A2 Egg 05/18/01 05/18-19/01 2.5 - - 102 0009342 SpkB2 Egg 05/18/01 05/18-19/01 10 - - 108 0009342 Spk A3 Egg 05/20-21/01 05/21/01 2.5 - - 96 0009342 SpkB3 Egg 05/20-21/01 05/21/01 10 - - 96 0009342 Spk A4 Egg 05/20-21/01 05/21-22/01 2.5 - - 95 0009342 Spk B4 Egg 05/20-21/01 05/21-22/01 10 - - 96 AVERAGE: 101 96 104 STD DEV: 9.0 13 14 RELATIVE STD DEV: 9.0 13 14 n: 88 80 88 - = Not Analyzed For Centre Analytical Laboratories, Inc. Page 25 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II. Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID Data Set No. Sponsor ID Store Name Location 0012776 011101A Chicken 0012776 Dup 011101A Chicken 0012777 011101A Chicken 0012777 Dup 011101A Chicken 0012778 011101A Chicken 0012778 Dup 011101A Chicken 0012779 011101A Chicken 0012779 Dup 011101A Chicken 0012780 011101A Chicken 0012780 Dup 011101A Chicken 0012781 011101B Chicken 0012781Dup 011101B Chicken 0012782 011101B Chicken 0012782 Dup 011101B Chicken 0012783 011101B Chicken 0012783 Dup 011101B Chicken 0012784 011101B Chicken 0012784 Dup 011101B Chicken 0012785 011101B Chicken 0012785 Dup 011101B Chicken 0012786 011201A Chicken 0012786 Dup 011201A Chicken 0012787 011201A Chicken 0012787 Dup 011201A Chicken 0012788 011201A Chicken 0012788 Dup 011201A Chicken 0012789 011201A Chicken 0012789 Dup 011201A Chicken 0012790 011201A Chicken 0012790 Dup 011201A Chicken 0012791 011201B Chicken 0012791Dup 011201B Chicken 18249 18249 18226 18226 18239 18239 18356 18356 18197 18197 18345 18345 18209 18209 18335 18335 18323 18323 18312 18312 18301 18301 18187 18187 18174 18174 18163 18163 18283 18283 18260 18260 Publix Port St. Lucie, FL Publix Port St. Lucie, FL Winn-Dixie Port St. Lucie, FL Winn-Dixie Port St. Lucie, FL Albertson's Port St. Lucie, FL Albertson's Port St. Lucie, FL Delchamps II Mobile, AL Delchamps II Mobile, AL Winn-Dixie Columbus, GA Winn-Dixie Columbus, GA Delchamps I Mobile, AL Delchamps I Mobile, AL Piggly Wiggly Columbus, GA Piggly Wiggly Columbus, GA Bruno's Mobile, AL Bruno's Mobile, AL Kroger's Decatur, AL Kroger's Decatur, AL Winn-Dixie Decatur, AL Winn-Dixie Decatur, AL Haloway Decatur, AL Haloway Decatur, AL Cub Columbus, GA Cub Columbus, GA Bi-Lo Cleveland, TN Bi-Lo Cleveland, TN Save-a-Lot Cleveland, TN Save-a-Lot Cleveland, TN Albertson's Pensacola, FL Albertson's Pensacola, FL Food World Pensacola, FL Food World Pensacola, FL M atrix Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Residue Found (ng/g)* PFOS FOSA PFOA NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. *Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for Centre Analytical Laboratories, Inc. Page 26 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID Data Set No. Sponsor ID Store Name Location 0012792 011201B Chicken 0012792 Dup 011201B Chicken 0012793 011201B Chicken 0012793 Dup 011201B Chicken 0012794 011501A Pork 0012794 Dup 011501A Pork 0012795 011501A Pork 0012795 Dup 011501A Pork 0012796 011501APork 0012796 Dup 011501APork 0012797 011501A Pork 0012797 Dup 011501A Pork 0012798 011501A Pork 0012798 Dup 011501A Pork 0012799 011501B Pork 0012799 Dup 011501B Pork 0012800 011501B Pork 0012800 Dup 011501B Pork 0012801 011501B Pork 0012801Dup 011501B Pork 0012802 011501B Pork 0012802 Dup 011501B Pork 0012803 011501B Pork 0012803 Dup 011501B Pork 0012804 011601A Pork 0012804 Dup 0 1 1601A Pork 0012805 011601A Pork 0012805 Dup 011601APork 0012806 011601A Pork 0012806 Dup 011601A Pork 0012807 011601A Pork 0012807 Dup 011601A Pork 18151 18151 18272 18272 18250 18250 18227 18227 18238 18238 18357 18357 18198 18198 18346 18346 18210 18210 18334 18334 18322 18322 18311 18311 18299 18299 18186 18186 18175 18175 18164 18164 Watson's Cleveland, TN Watson's Cleveland, TN Food World II Pensacola, FL Food World II Pensacola, FL Publix Port St. Lucie, FL Publix Port St. Lucie, FL Winn-Dixie Port St. Lucie, FL Winn-Dixie Port St. Lucie, FL Albertson's Port St. Lucie, FL Albertson's Port St. Lucie, FL Delchamps II Mobile, AL Delchamps II Mobile, AL Winn-Dixie Columbus, GA Winn-Dixie Columbus, GA Delchamps I Mobile, AL Delchamps I Mobile, AL Piggly Wiggly Columbus, GA Piggly Wiggly Columbus, GA Bruno's Mobile, AL Bruno's Mobile, AL Kroger's Decatur, AL Kroger's Decatur, AL Winn-Dixie Decatur, AL Winn-Dixie Decatur, AL Haloway Decatur, AL Haloway Decatur, AL Cub Columbus, GA Cub Columbus, GA Bi-Lo Cleveland, TN Bi-Lo Cleveland, TN Save-a-Lot Cleveland, TN Save-a-Lot Cleveland, TN M atrix Chicken Chicken Chicken Chicken Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Pork Residue Found (ng/g)* PFOS FOSA PFOA NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for Centre Analytical Laboratories, Inc. Page 27 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID Data Set No. !Sponsor ID Store Name Location Matrix 0012808 011601A Pork 18282 Albertson's Pensacola, FL Pork 0012808 Dup 0012809 0012809 Dup 0012810 0012810Dup 0012811 011601A Pork 011601B Pork 011601B Pork 011601B Pork 011601B Pork 011601B Pork 18282 18261 18261 18152 18152 18271 Albertson's Food World Food World Watson's Watson's Food World II Pensacola, FL Pensacola, FL Pensacola, FL Cleveland, TN Cleveland, TN Pensacola, FL Pork Pork Pork Pork Pork Pork 0012811Dup 011601B Pork 18271 Food World II Pensacola, FL Pork 0012812 011701AHotDog 18251 0012812Dup 011701A Hot Dog 18251 Publix Port St. Lucie, FL Hot Dog Publix Port St. Lucie, FL Hot Dog 0012813 O inO lA H otD og 0012813Dup 011701A Hot Dog 0012814 011701A Hot Dog 18228 18228 18240 Winn-Dixie Port St. Lucie, FL Hot Dog Winn-Dixie Port St. Lucie, FL Hot Dog Albertson's Port St. Lucie, FL Hot Dog 0012814Dup 011701A Hot Dog 18240 Albertson's Port St. Lucie, FL Hot Dog 0012815 011701A Hot Dog 0012815Dup 011701A Hot Dog 0012816 011701AHotDog 0012816Dup O inO lA H otD og 0012812 011701AR Hot Dog 0012812 Dup 011701AR Hot Dog 0012813 011701AR Hot Dog 0012813 Dup 011701AR Hot Dog 18358 18358 18199 18199 18251 18251 18228 18228 Delchamps II Mobile, AL Delchamps II Mobile, AL Winn-Dixie Columbus, GA Winn-Dixie Columbus, GA Publix Port St. Lucie, FL Publix Port St. Lucie, FL Winn-Dixie Port St. Lucie, FL Winn-Dixie Port St. Lucie, FL Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog 0012814 011701AR Hot Dog 18240 Albertson's Port St. Lucie, FL Hot Dog 0012814 Dup 011701AR Hot Dog 18240 Albertson's Port St. Lucie, FL Hot Dog 0012815 O inO lA RH otD og 0012815 Dup 011701AR Hot D og 0012816 011701AR Hot Dog 0012816 Dup 011701AR Hot Dog 0012817 011701B Hot Dog 0012817Dup 011701B Hot Dog 18358 18358 18199 18199 18347 18347 Delchamps II Delchamps II Winn-Dixie Winn-Dixie Delchamps I Delchamps I Mobile, AL Mobile, AL Columbus, GA Columbus, GA Mobile, AL Mobile, AL Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog 0012818 011701B Hot Dog 18211 Piggly Wiggly Columbus, GA Hot Dog 0012818 Dup 011701B Hot Dog 18211 Piggly Wiggly Columbus, GA Hot Dog Residue Found (ng/g)* PFOS FOSA PFOA NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ ** NQ NQ ** NQ NQ ** NQ NQ ** NQ NQ ** NQ NQ ** NQ NQ 5k* NQ NQ ** NQ NQ ** NQ NQ ** - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for Centre Analytical Laboratories, Inc. Page 28 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID Data Set No. Sponsor ID Store Name Location Matrix 0012819 011701B Hot Dog 0012819Dup 011701B Hot Dog 0012820 011701B Hot Dog 0012820 Dup 011701B Hot Dog 0012821 011701B Hot Dog 0012821Dup 011701B Hot Dog 0012822 011801A Hot Dog 0012822 Dup 011801AHotDog 0012823 011801A Hot Dog 0012823 Dup 011801A Hot Dog 0012824 011801AHotDog 0012824Dup 011801AHotDog 0012825 011801A Hot Dog 0012825 Dup 011801A Hot Dog 0012826 011801AHotDog 0012826 Dup 011801A Hot Dog 0012827 011801B Hot Dog 0012827 Dup 011801B Hot Dog 0012828 011801B Hot Dog 0012828 Dup 011801B Hot Dog 0012829 011801B Hot Dog 0012829 Dup 011801B Hot Dog 0012830 011901A Fish 0012830Dup 011901A Fish 0012831 011901A Fish 0 0 1 2 8 3 1 Dup 011901A Fish 0012832 011901A Fish 0012832 Dup 011901A Fish 0012833 011901A Fish 0012833 Dup 011901A Fish 0012834 011901A Fish 0012834Dup 011901A Fish 18336 18336 18324 18324 18313 18313 18302 18302 18188 18188 18176 18176 18165 18165 18285 18285 18263 18263 18153 18153 18273 18273 18252 18252 18229 18229 18241 18241 18359 18359 18200 18200 Bruno's Mobile, AL Bruno's Mobile, AL Kroger's Decatur, AL Kroger's Decatur, AL Winn-Dixie Decatur, AL Winn-Dixie Decatur, AL Haloway Decatur, AL Haloway Decatur, AL Cub Columbus, GA Cub Columbus, GA Bi-Lo Cleveland, TN Bi-Lo Cleveland, TN Save-a-Lot Cleveland, TN Save-a-Lot Cleveland, TN Albertson's Pensacola, FL Albertson's Pensacola, FL Food World Pensacola, FL Food World Pensacola, FL Watson's Cleveland, TN Watson's Cleveland, TN Food World II Pensacola, FL Food World II Pensacola, FL Publix Port St. Lucie, FL Publix Port St. Lucie, FL Winn-Dixie Port St. Lucie, FL W inn-Dixie Port St. Lucie, FL Albertson's Port St. Lucie, FL Albertson's Port St. Lucie, FL Delchamps II Mobile, AL Delchamps II Mobile, AL Winn-Dixie Columbus, GA Winn-Dixie Columbus, GA Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Hot Dog Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Residue Found (ng/g)* PFOS FOSA PFOA NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. *Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for Centre Analytical Laboratories, Inc. Page 29 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID 0012835 0012835 Dup 0012836 0012836 Dup 0012837 0012837 Dup 0012838 0012838 Dup 0012839 0012839 Dup 0012835 0012835 Dup 0012836 0012836 Dup 0012837 0012837 Dup 0012838 0012838 Dup 0012839 0012839 Dup 0012840 0012840Dup 0012841 0012841Dup 0012842 0012842 Dup 0012843 0012843 Dup 0012844 0012844 Dup 0012845 0012845 Dup Data Set No. 011901B Fish 011901B Fish 011901B Fish 011901B Fish 011901B Fish 011901B Fish 011901B Fish 011901B Fish 011901B Fish 011901B Fish 013001A Fish 013001A Fish 013001A Fish 013001A Fish 013001A Fish 013001AFish 013001AFish 013001A Fish 013001A Fish 013001A Fish 012201A Fish 012201A Fish 012201A Fish 012201A Fish 012201A Fish 012201A Fish 012201A Fish 012201A Fish 012201A Fish 012201A Fish 012201B Fish 012201B Fish Sponsor ID Store Name Location 18348 18348 18212 18212 18337 18337 18325 18325 18314 18314 18348 18348 18212 18212 18337 18337 18325 18325 18314 18314 18303 18303 18177 18177 18166 18166 18284 18284 18262 18262 18154 18154 Delchamps Delchamps Piggly Wiggly Piggly Wiggly Bruno's Bruno's Kroger's Kroger's Winn-Dixie Winn-Dixie Delchamps Delchamps Piggly Wiggly Piggly Wiggly Bruno's Bruno's Kroger's Kroger's Winn-Dixie Winn-Dixie Haloway Haloway Bi-Lo Bi-Lo Save-a-Lot Save-a-Lot Albertson's Albertson's Food World Food World Watson's Watson's Mobile, AL Mobile, AL Columbus, GA Columbus, GA Mobile, AL Mobile, AL Decatur, AL Decatur, AL Decatur, AL Decatur, AL Mobile, AL Mobile, AL Columbus, GA Columbus, GA Mobile, AL Mobile, AL Decatur, AL Decatur, AL Decatur, AL Decatur, AL Decatur, AL Decatur, AL Cleveland, TN Cleveland, TN Cleveland, TN Cleveland, TN Pensacola, FL Pensacola, FL Pensacola, FL Pensacola, FL Cleveland, TN Cleveland, TN Matrix Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Residue Found (ng/g)* PFOS FOSA PFOA NQ ** NQ NQ ** NQ NQ ** NQ NQ ** NQ NQ ** NQ NQ ** NQ NQ ** NQ NQ ** NQ NQ ** NQ NQ ** NQ - NQ - NQ - NQ - NQ - NQ - NQ - NQ - NQ - NQ - NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for Centre Analytical Laboratories, Inc. Page 30 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID 0012846 0012846 Dup 0012903 0012903 Dup 0012904 0012904 Dup 0012905 0012905 Dup 0012906 0012906 Dup 0012907 0012907 Dup 0012908 0012908 Dup 0012909 0012909 Dup 0012910 0012910Dup 0012911 0012911Dup 0012912 0012912 Dup 0012913 0012913Dup 0012914 0012914Dup 0012915 0012915 Dup 0012916 0012916Dup 0012917 0012917Dup Data Set No. 012201B Fish 012201B Fish 020501A Bean 020501A Bean 020501A Bean 020501A Bean 020501A Bean 020501A Bean 020501A Bean 020501A Bean 020501A Bean 020501A Bean 020601C Bean 020601C Bean 020601C Bean 020601C Bean 020601C Bean 020601C Bean 020601C Bean 020601C Bean 020601C Bean 020601C Bean 020801C Bean 020801C Bean 020801C Bean 020801C Bean 020801C Bean 020801C Bean 020801C Bean 020801C Bean 020801C Bean 020801C Bean Residue Found (ng/g)* Sponsor ID Store Name Location M atrix PFOS FOSA PFOA 18274 Food World II Pensacola, FL Fish NQ NQ NQ 18274 Food World II Pensacola, FL Fish NQ NQ NQ 18256 Publix Port St. Lucie, FL Green Bean NQ NQ NQ 18256 Publix Port St. Lucie, FL Green Bean NQ NQ NQ 18234 Winn-Dixie Port St. Lucie, FL Green Bean NQ NQ NQ 18234 Winn-Dixie Port St. Lucie, FL Green Bean NQ NQ NQ 18245 Albertson's Port St. Lucie, FL Green Bean NQ NQ NQ 18245 Albertson's Port St. Lucie, FL Green Bean NQ 18363 Delchamps II Mobile, AL Green Bean NQ NQ NQ NQ 0.543 18363 Delchamps II Mobile, AL Green Bean NQ NQ NQ 18204 Winn-Dixie Columbus, GA Green Bean NQ NQ NQ 18204 Winn-Dixie Columbus, GA Green Bean NQ NQ NQ 18352 Delchamps I Mobile, AL Green Bean NQ NQ NQ 18352 Delchamps I Mobile, AL Green Bean NQ NQ NQ 18216 Piggly Wiggly Columbus, GA Green Bean NQ NQ NQ 18216 Piggly Wiggly Columbus, GA Green Bean NQ NQ NQ 18341 Bruno's Mobile, AL Green Bean NQ NQ NQ 18341 Bruno's Mobile, AL Green Bean NQ NQ NQ 18329 18329 Kroger's Kroger's Decatur, AL Green Bean NQ NQ NQ Decatur, AL Green Bean NQ NQ NQ 18318 Winn-Dixie Decatur, AL Green Bean NQ NQ NQ 18318 Winn-Dixie Decatur, AL Green Bean NQ NQ NQ 18307 Haloway Decatur, AL Green Bean NQ NQ NQ 18307 Haloway Decatur, AL Green Bean NQ NQ NQ 18193 Cub Columbus, GA Green Bean NQ NQ NQ 18193 Cub Columbus, GA Green Bean NQ NQ NQ 18182 Bi-Lo Cleveland, TN Green Bean NQ NQ NQ 18182 Bi-Lo Cleveland, TN Green Bean NQ NQ NQ 18289 Albertson's Pensacola, FL Green Bean NQ NQ NQ 18289 Albertson's Pensacola, FL Green Bean NQ NQ NQ 18267 Food World Pensacola, FL Green Bean NQ NQ NQ 18267 Food World Pensacola, FL Green Bean NQ NQ NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. *Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for Centre Analytical Laboratories, Inc. Page 31 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID 0012918 0012918 Dup 0012919 0012919 Dup 0012867 0012867 Dup 0012868 0012868 Dup 0012869 0012869 Dup 0012870 0012870Dup 0012871 0012871Dup 0012872 0012872 Dup 0012873 0012873 Dup 0012874 0012874 Dup 0012875 0012875 Dup 0012876 0012876 Dup 0012877 0012877 Dup 0012878 0012878 Dup 0012879 0012879 Dup 0012880 0012880 Dup Data Set No. 020801D Bean 020801D Bean 020801D Bean 02080ID Bean 020701A Milk 020701A Milk 020701A Milk 020701A Milk 020701A Milk 020701A Milk 020701A Milk 020701A Milk 020701A Milk 020701A Milk 020701B Milk 020701B Milk 020701B Milk 020701B Milk 020701B Milk 020701B Milk 020701B Milk 020701B Milk 020701B Milk 020701B Milk 020801A Milk 020801A Milk 020801A Milk 020801A Milk 020801A Milk 020801A Milk 020801A Milk 020801A Milk Residue Found (ng/g)* Sponsor ID Store Name Location Matrix PFOS FOSA PFOA 36868 Watson's Cleveland, TN Green Bean NQ NQ NQ 36868 Watson's Cleveland, TN Green Bean NQ NQ NQ 18278 Food World II Pensacola, FL Green Bean NQ NQ NQ 18278 Food World II Pensacola, FL Green Bean NQ NQ NQ 18254 Publix Port St. Lucie, FL Milk NQ NQ NQ 18254 Publix Port St. Lucie, FL Milk NQ NQ NQ 18231 Winn-Dixie Port St. Lucie, FL Milk NQ NQ NQ 18231 Winn-Dixie Port St. Lucie, FL Milk NQ NQ NQ 18243 18243 Albertson's Port St. Lucie, FL Milk Albertson's Port St. Lucie, FL Milk NQ NQ NQ NQ NQ NQ 18361 Delchamps II Mobile, AL Milk NQ NQ NQ 18361 Delchamps II Mobile, AL Milk NQ NQ NQ 18202 Winn-Dixie Columbus, GA Milk NQ NQ NQ 18202 Winn-Dixie Columbus, GA Milk 0.693 NQ NQ 18350 Delchamps I Mobile, AL Milk NQ NQ NQ 18350 18214 18214 18339 18339 Delchamps I Piggly Wiggly Piggly Wiggly Bruno's Bruno's Mobile, AL Columbus, GA Columbus, GA Mobile, AL Mobile, AL Milk Milk Milk Milk Milk NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 18327 18327 18316 18316 Kroger's Kroger's Winn-Dixie Winn-Dixie Decatur, AL Decatur, AL Decatur, AL Decatur, AL Milk NQ NQ NQ Milk NQ NQ NQ Milk NQ NQ NQ Milk NQ NQ NQ 18305 18305 18190 18190 18179 18179 18168 Haloway Haloway Cub Cub Bi-Lo Bi-Lo Save-a-Lot Decatur, AL Decatur, AL Columbus, GA Columbus, GA Cleveland, TN Cleveland, TN Cleveland, TN Milk M ilk Milk Milk Milk Milk Milk NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 18168 Save-a-Lot Cleveland, TN Milk 0.573 NQ NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for Centre Analytical Laboratories, Inc. Page 32 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID 0012881 0012881Dup 0012882 0012882 Dup 0012883 0012883 Dup 0012884 0012884 Dup 0012926 0012926 Dup 0012927 0012927 Dup 0012928 0012928 Dup 0012929 0012929 Dup 0012930 0012930 Dup 0012931 0012931Dup 0012932 0012932Dup 0012933 0012933 Dup 0012934 0012934 Dup 0012935 0012935 Dup 0012936 0012936 Dup 0012937 0012937 Dup Data Set No. 020801A Milk 020801A Milk 02080IB Milk 02080 IB Milk 02080IB Milk 020801B Milk 02080 IB Milk 02080IB Milk 020901C Apple 020901C Apple 020901C Apple 020901C Apple 020901C Apple 020901C Apple 020901C Apple 020901C Apple 020901C Apple 020901C Apple 021201A Apple 021201A Apple 021201A Apple 021201A Apple 021201A Apple 021201A Apple 021201A Apple 021201A Apple 021201A Apple 021201A Apple 021201B Apple 021201B Apple 021201B Apple 021201B Apple Sponsor ID Store Name Location 18287 Albertson's Pensacola, FL 18287 Albertson's Pensacola, FL 18265 18265 Food World Pensacola, FL Food World Pensacola, FL 18156 Watson's Cleveland, TN 18156 Watson's Cleveland, TN 18276 Food World II Pensacola, FL 18276 Food World II Pensacola, FL 18205 18205 18257 18257 Winn-Dixie Columbus, GA Winn-Dixie Columbus, GA Publix Port St. Lucie, FL Publix Port St. Lucie, FL 18235 Winn-Dixie Port St. Lucie, FL 18235 Winn-Dixie Port St. Lucie, FL 18246 Albertson's Port St. Lucie, FL 18246 Albertson's Port St. Lucie, FL 18364 Delchamps II Mobile, AL 18364 Delchamps II Mobile, AL 18353 Delchamps I Mobile, AL 18353 Delchamps I Mobile, AL 18217 18217 18342 18342 Piggly Wiggly Piggly Wiggly Bruno's Bruno's Columbus, GA Columbus, GA Mobile, AL Mobile, AL 18330 Kroger's Decatur, AL 18330 18319 Kroger's Winn-Dixie Decatur, AL Decatur, AL 18319 Winn-Dixie Decatur, AL 18308 18308 Haloway Haloway Decatur, AL Decatur, AL 18194 18194 Cub Columbus, GA Cub Columbus, GA M atrix Milk Milk Milk Milk Milk Milk Milk Milk Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Residue Found (ng/g)* PFOS FOSA PFOA NQ NQ NQ NQ 0.852 NQ NQ NQ NQ 0.605 NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 2.35 NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for Centre Analytical Laboratories, Inc. Page 33 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS,, FOSA and PFOA Residues Found in Samples Centre ID Data Set No. Sponsor ID Store Name Location 0012938 021201B Apple 0012938 Dup 021201B Apple 18183 18183 Bi-Lo Bi-Lo Cleveland, TN Cleveland, TN 0012939 021201B Apple 0012939 Dup 021201B Apple 18170 18170 Save-a-Lot Cleveland, TN Save-a-Lot Cleveland, TN 0012940 0012940 Dup 0012941 0012941Dup 0012942 0012942 Dup 0012943 0012943 Dup 0012885 0012885 Dup 0012886 0012886 Dup 0012887 0012887 Dup 0012888 0012888 Dup 0012889 0012889 Dup 0012890 0012890 Dup 0012891 0 0 1 2 8 9 1 Dup 0012892 0012892 Dup 0012893 0012893 Dup 0012894 0012894 Dup 021201B Apple 021201B Apple 021201C Apple 021201C Apple 021201C Apple 021201C Apple 021201C Apple 021201C Apple 020901A Bread 020901A Bread 020901A Bread 020901A Bread 020901A Bread 020901A Bread 020901A Bread 020901A Bread 020901A Bread 020901A Bread 020901B Bread 02090IB Bread 02090 IB Bread 02090IB Bread 02090 IB Bread 02090IB Bread 02090 IB Bread 02090IB Bread 02090 IB Bread 02090IB Bread 18290 18290 18268 18268 18159 18159 18279 18279 18255 18255 18233 18233 18244 18244 18362 18362 18203 18203 18351 18351 18215 18215 18340 18340 18328 18328 18317 18317 Albertson's Pensacola, FL Albertson's Pensacola, FL Food World Pensacola, FL Food World Pensacola, FL Watson's Cleveland, TN Watson's Cleveland, TN Food World II Pensacola, FL Food World II Pensacola, FL Publix Port St. Lucie, FL Publix Port St. Lucie, FL Winn-Dixie Port St. Lucie, FL Winn-Dixie Port St. Lucie, FL Albertson's Port St. Lucie, FL Albertson's Port St. Lucie, FL Delchamps II Mobile, AL Delchamps II Mobile, AL Winn-Dixie Columbus, GA Winn-Dixie Columbus, GA Delchamps I Mobile, AL Delchamps I Mobile, AL Piggly Wiggly Columbus, GA Piggly W iggly Columbus, GA Bruno's Mobile, AL Bruno's Mobile, AL Kroger's Decatur, AL Kroger's Decatur, AL Winn-Dixie Decatur, AL Winn-Dixie Decatur, AL M atrix Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Apple Bread Bread Bread Bread Bread Bread Bread Bread Bread Bread Bread Bread Bread Bread Bread Bread Bread Bread Bread Bread Residue Found (ng/g)* PFOS FOSA PFOA NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 1.13 NQ NQ NQ NQ NQ NQ NQ NQ NQA NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. *Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for A= Sample result fell below 0.5 ng/g when adjusting for purity of PFOA. Centre Analytical Laboratories, Inc. Page 34 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Residue Found (ng/g)* Centre ID Data Set No. Sponsor ID Store Name Location M atrix PFOS FOSA PFOA 0012895 0012895 Dup 0012896 0012896 Dup 021301A Bread 021301A Bread 021301A Bread 021301A Bread 18306 18306 18192 18192 Haloway Haloway Cub Cub Decatur, AL Decatur, AL Columbus, GA Columbus, GA Bread Bread Bread Bread NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 0012897 0012897 Dup 0012898 0012898 Dup 0012899 0012899 Dup 0012900 0012900 Dup 0012901 021301A Bread 021301A Bread 021301A Bread 021301A Bread 021301A Bread 021301A Bread 021301B Bread 021301B Bread 021301B Bread 18181 18181 18172 18172 18288 18288 18266 18266 18158 Bi-Lo Bi-Lo Save-a-Lot Save-a-Lot Albertson's Albertson's Food World Food World Watson's Cleveland, TN Cleveland, TN Cleveland, TN Cleveland, TN Pensacola, FL Pensacola, FL Pensacola, FL Pensacola, FL Cleveland, TN Bread Bread Bread Bread Bread Bread Bread Bread Bread NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 14.7A NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 0012901Dup 021301B Bread 18158 Watson's Cleveland, TN Bread NQ NQ NQ 0012902 021301B Bread 18277 Food World II Pensacola, FL Bread NQ NQ NQ 0012902 Dup 0012758 0012758 Dup 0012759 0012759 Dup 0012760 0012760Dup 0012761 0012761Dup 021301B Bread 021301C Beef 021301C Beef 021301C Beef 021301C Beef 021301C Beef 021301C Beef 021301C Beef 021301C Beef 18277 18248 18248 18225 18225 18237 18237 18355 18355 Food World II Pensacola, FL Bread Publix Port St. Lucie, FL Ground Beef Publix Port St. Lucie, FL Ground Beef Winn-Dixie Port St. Lucie, FL Ground Beef Winn-Dixie Port St. Lucie, FL Ground Beef Albertson's Port St. Lucie, FL Ground Beef Albertson's Port St. Lucie, FL Ground Beef Delchamps II Mobile, AL Ground Beef Delchamps II Mobile, AL Ground Beef NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 0.524 NQ NQ NQ 0.504 NQ NQ NQ NQ NQ 1.09 NQ NQ NQ NQ NQ NQ 0012762 0012762 Dup 0012763 0012763 Dup 0012764 0012764 Dup 0012765 0012765Dup 021301C Beef 021301C Beef 021301DBeef 021301D Beef 021301D Beef 021301D Beef 021301D Beef 021301D Beef 18196 18196 18344 18344 18208 18208 18333 18333 Winn-Dixie W inn-D ixie Delchamps I Delchamps I Piggly Wiggly Piggly Wiggly Bruno's Bruno's Columbus, GA Columbus, GA Mobile, AL Mobile, AL Columbus, GA Columbus, GA Mobile, AL Mobile, AL Ground Beef NQ Ground Beef NQ Ground Beef 0.570 Ground Beef 0.587 Ground Beef NQ Ground Beef NQ Ground Beef NQ Ground Beef NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. A= Result is suspect. Apparent contamination. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for Centre Analytical Laboratories, Inc. Page 35 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID 0012766 0012766 Dup 0012767 0012767 Dup 0012768 0012768 Dup 0012769 0012769 Dup 0012770 0012770 Dup 0012771 0012771Dup 0012772 0012772 Dup 0012773 0012773 Dup 0012774 0012774 Dup 0012775 0012775 Dup 0012847 0012847 Dup 0012848 0012848 Dup 0012849 0012849 Dup 0012850 0012850 Dup 0012851 0012851Dup 0012852 0012852 Dup Data Set No. 021301D Beef 021301D Beef 02130ID Beef 021301DBeef 021401A Beef 021401ABeef 021401A Beef 021401A Beef 021401A Beef 021401A Beef 021401A Beef 021401A Beef 021401A Beef 021401A Beef 021401B Beef 021401B Beef 02140 IB Beef 021401B Beef 021401B Beef 021401B Beef 012301A Egg 012301A Egg 012301A Egg 012301AEgg 012301A Egg 012301A Egg 012301A Egg 012301AEgg 012301A Egg 012301AEgg 012301B Egg 012301B Egg Residue Found (ng/g)* Sponsor ID Store Name Location M atrix PFOS FOSA PFOA 18321 Kroger's Decatur, AL Ground Beef NQ NQ NQ 18321 Kroger's Decatur, AL Ground Beef NQ NQ NQ 18310 Winn-Dixie Decatur, AL Ground Beef NQ NQ NQ 18310 Winn-Dixie Decatur, AL Ground Beef NQ NQ NQ 18300 Haloway Decatur, AL Ground Beef NQ NQ NQ 18300 Haloway Decatur, AL Ground Beef NQ NQ NQ 18185 Cub Columbus, GA Ground Beef NQ NQ NQ 18185 Cub Columbus, GA Ground Beef NQ NQ NQ 18173 Bi-Lo Cleveland, TN Ground Beef NQ NQ NQ 18173 Bi-Lo Cleveland, TN Ground Beef NQ NQ NQ 18162 Save-a-Lot Cleveland, TN Ground Beef NQ NQ NQ 18162 Save-a-Lot Cleveland, TN Ground Beef NQ NQ NQ 18281 Albertson's Pensacola, FL Ground Beef NQ NQ NQ 18281 Albertson's Pensacola, FL Ground Beef NQ NQ NQ 18259 Food World Pensacola, FL Ground Beef NQ NQ NQ 18259 Food World Pensacola, FL Ground Beef NQ NQ NQ 18161 Watson's Cleveland, TN Ground Beef NQ NQ NQ 18161 Watson's Cleveland, TN Ground Beef NQ NQ NQ 18270 Food World II Pensacola, FL Ground Beef NQ NQ NQ 18270 Food World II Pensacola, FL Ground Beef NQ NQ NQ 18253 Publix Port St. Lucie, FL Egg A A NQ A A 18253 Publix Port St. Lucie, FL Egg A A NQ A A 18230 Winn-Dixie Port St. Lucie, FL Egg A A NQ A A 18230 Winn-Dixie Port St. Lucie, FL Egg A A NQ A A 18242 18242 18360 Albertson's Port St. Lucie, FL Albertson's Port St. Lucie, FL Delchamps II Mobile, AL Egg Egg Egg A A NQ A A AA N Q AA A A NQ A A 18360 Delchamps II Mobile, AL Egg AA NQ AA 18201 Winn-Dixie Columbus, GA Egg A A NQ A A 18201 Winn-Dixie Columbus, GA Egg A A NQ A A 18349 Delchamps I Mobile, AL Egg AA NQ AA 18349 Delchamps I Mobile, AL Egg AA NQ AA NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. *Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for Centre Analytical Laboratories, Inc. Page 36 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID 0012853 0012853 Dup 0012854 0012854 Dup 0012855 0012855 Dup 0012856 0012856 Dup 0012857 0012857 Dup 0012858 0012858 Dup 0012859 0012859 Dup 0012860 0012860Dup 0012861 0012861Dup 0012862 0012862 Dup 0012863 0012863 Dup 0012864 0012864 Dup 0012865 0012865 Dup 0012866 0012866Dup 0106429 0106429 Dup 0106430 0106430 Dup Data Set No. 012301B Egg 012301B Egg 012301B Egg 012301B Egg 012301B Egg 012301B Egg 012301B Egg 012301B Egg 012401A Egg 012401AEgg 012401A Egg 012401AEgg 012401A Egg 012401AEgg 012401A Egg 012401A Egg 012401A Egg 012401A Egg 012401B Egg 012401B Egg 012401B Egg 012401B Egg 012401B Egg 012401B Egg 01240 IB Egg 012401B Egg 01240 IB Egg 012401B Egg 050801A Egg 050801A Egg 050801A Egg 050801A Egg Sponsor ID Store Name Location 18219 18219 18213 18213 18338 18338 18326 18326 18315 18315 18304 18304 18189 18189 18178 18178 18167 18167 18286 18286 18264 18264 18296 18296 18155 18155 18275 18275 21213 21213 21214 21214 Peggy's Port St. Lucie, FL Peggy's Port St. Lucie, FL Piggly Wiggly Columbus, GA Piggly Wiggly Columbus, GA Bruno's Mobile, AL Bruno's Mobile, AL Kroger's Decatur, AL Kroger's Decatur, AL Winn-Dixie Decatur, AL Winn-Dixie Decatur, AL Haloway Decatur, AL Haloway Decatur, AL Cub Columbus, GA Cub Columbus, GA Bi-Lo Cleveland, TN Bi-Lo Cleveland, TN Save-a-Lot Cleveland, TN Save-a-Lot Cleveland, TN Albertson's Pensacola, FL Albertson's Pensacola, FL Food World Pensacola, FL Food World Pensacola, FL Farmer's Market Mobile, AL Farmer's Market Mobile, AL Watson's Cleveland, TN W atson's Cleveland, TN Food World II Pensacola, FL Food World II Pensacola, FL Winn-Dixie Port St. Lucie, FL Winn-Dixie Port St. Lucie, FL Albertson's Port St. Lucie, FL Albertson's Port St. Lucie, FL M atrix Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Residue Found (ng/g)* PFOS FOSA PFOA AA NQ A A NQ A A NQ A A NQ A A NQ AA NQ A A NQ A A NQ A A NQ A A NQ A A NQ AA NQ A A NQ A A NQ AA NQ A A NQ AA NQ A A NQ AA NQ A A NQ A A NQ A A NQ A A NQ A A NQ A A NQ AA N Q A A NQ AA NQ NQ NQ NQ NQ - AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. *Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for Centre Analytical Laboratories, Inc. Page 37 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID 0106431 0106431Dup 0106432 0106432 Dup 0106433 0106433 Dup 0106434 0106434 Dup 0106435 0106435 Dup 0106436 0106436 Dup 0106437 0106437 Dup 0106438 0106438 Dup 0106439 0106439 Dup 0106440 0106440 Dup 0106441 0106441Dup 0106442 0106442 Dup 0106443 0106443 Dup 0106444 0106444 Dup 0106445 0106445 Dup 0106446 0106446 Dup Data Set No. 050801A Egg 050801A Egg 050801A Egg 050801A Egg 050801A Egg 050801A Egg 050801B Egg 050801B Egg 05080IB Egg 050801B Egg 050801B Egg 05080IB Egg 050801B Egg 050801B Egg 050801B Egg 05080 IB Egg 050901A Egg 050901A Egg 050901A Egg 050901A Egg 050901A Egg 050901A Egg 050901A Egg 050901A Egg 050901A Egg 050901A Egg 05090IB Egg 050901B Egg 050901B Egg 05090IB Egg 05090IB Egg 050901B Egg Sponsor ID Store Name Location 21216 21216 21217 21217 21218 21218 21219 21219 21221 21221 21222 21222 21223 21223 21224 21224 21225 21225 21226 21226 21227 21227 21228 21228 21229 21229 21230 21230 21231 21231 na na Winn-Dixie Columbus, GA Winn-Dixie Columbus, GA Delchamps I Mobile, AL Delchamps I Mobile, AL Peggy's Port St. Lucie, FL Peggy's Port St. Lucie, FL Piggly Wiggly Columbus, GA Piggly Wiggly Columbus, GA Kroger's Decatur, AL Kroger's Decatur, AL Winn-Dixie Decatur, AL Winn-Dixie Decatur, AL Haloway Decatur, AL Haloway Decatur, AL Cub Columbus, GA Cub Columbus, GA Bi-Lo Cleveland, TN Bi-Lo Cleveland, TN Save-a-Lot Cleveland, TN Save-a-Lot Cleveland, TN Albertson's Pensacola, FL Albertson's Pensacola, FL Food World Pensacola, FL Food World Pensacola, FL Farmer's Market Mobile, AL Farmer's Market M obile, AL Watson's Cleveland, TN Watson's Cleveland, TN Food World II Pensacola, FL Food World II Pensacola, FL $$ $$ M atrix Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Residue Found (ng/g)* PFOS FOSA PFOA NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ - AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. *Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for $ = Sample labels fell off during second shipping and there was no way of telling samples apart. Centre Analytical Laboratories, Inc. Page 38 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID 0106447 0106447 Dup 0106448 0106448 Dup 0106429 0106429 Dup 0106430 0106430 Dup 0106431 0106431Dup 0106432 0106432 Dup 0106433 0106433 Dup 0106434 0106434 Dup 0106435 0106435 Dup 0106436 0106436 Dup 0106437 0106437 Dup 0106438 0106438 Dup 0106439 0106439 Dup 0106440 0106440 Dup 0106441 0106441Dup 0106442 0106442 Dup Data Set No. 050901B Egg 050901B Egg 050901B Egg 050901B Egg 051801A Egg 051801A Egg 051801A Egg 051801A Egg 051801A Egg 051801A Egg 051801A Egg 051801A Egg 051801A Egg 051801A Egg 051801B Egg 051801B Egg 051801B Egg 051801B Egg 051801B Egg 051801B Egg 051801B Egg 05180 IB Egg 051801B Egg 051801B Egg 052101AEgg 052101A Egg 052101AEgg 052101A Egg 052101AEgg 052101A Egg 052101AEgg 052101A Egg Sponsor ID Store Name Location na na na na 21213 21213 21214 21214 21216 21216 21217 21217 21218 21218 21219 21219 21221 21221 21222 21222 21223 21223 21224 21224 21225 21225 21226 21226 21227 21227 21228 21228 $$ $$ $$ $$ Winn-Dixie Port St. Lucie, EL Winn-Dixie Port St. Lucie, FL Albertson's Port St. Lucie, FL Albertson's Port St. Lucie, FL Winn-Dixie Columbus, GA Winn-Dixie Columbus, GA Delchamps I Mobile, AL Delchamps I Mobile, AL Peggy's Port St. Lucie, FL Peggy's Port St. Lucie, FL Piggly Wiggly Columbus, GA Piggly Wiggly Columbus, GA Kroger's Decatur, AL Kroger's Decatur, AL Winn-Dixie Decatur, AL Winn-Dixie Decatur, AL Haloway Decatur, AL Haloway Decatur, AL Cub Columbus, GA Cub Columbus, GA Bi-Lo Cleveland, TN Bi-Lo Cleveland, TN Save-a-Lot Cleveland, TN Save-a-Lot Cleveland, TN Albertson's Pensacola, FL Albertson's Pensacola, FL Food World Pensacola, FL Food World Pensacola, FL Matrix Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Residue Found (ng/g)* PFOS FOSA PFOA NQ - AA NQ - AA NQ - AA NQ - AA - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for $ = Sample labels fell off during second shipping and there was no way of telling samples apart. Centre Analytical Laboratories, Inc. Page 39 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Table II continued: Summary of PFOS, FOSA and PFOA Residues Found in Samples Centre ID 0106443 0106443 Dup 0106444 0106444 Dup 0106445 0106445 Dup 0106446 0106446 Dup 0106447 0106447 Dup 0106448 0106448 Dup Data Set No. 052101AEgg 052101AEgg 052101B Egg 052101B Egg 052101B Egg 052101B Egg 052101B Egg 052101B Egg 052101B Egg 05210IB Egg 052101B Egg 052101B Egg Sponsor ID Store Name Location 21229 21229 21230 21230 21231 21231 na na na na na na Farmer's Market Mobile, AL Farmer's Market Mobile, AL Watson's Cleveland, TN Watson's Cleveland, TN Food World II Pensacola, FL Food World II Pensacola, FL $$ $$ $$ $$ $$ $$ Matrix Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Residue Found (ng/g)* PFOS FOSA PFOA - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ - - NQ NQ = Not Quantifiable. The residue detected, if any, was below the limit of the study which is 0.5 ng/g. *Value is based on wet weight (5.0 g sample processed as received) ** Values rejected based on unacceptable recoveries. Data has been replaced with either reanalysis or re-extraction values. AA= Results rejected based on apparent contamination. Data has been replaced with re-extracted values. - = Not analyzed for $ = Sample labels fell off during second shipping and there was no way of telling samples apart. Centre Analytical Laboratories, Inc. Page 40 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 12.0 FIGURES Centre Analytical Laboratories, Inc. Page 41 of 151 BACK TO MAIN Figure 1. Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Typical Calibration Curve for PFOS Cantra Analytical Laboratoriaa, Inc. Centre Analytical Laboratories, Inc. Page 42 of 151 BACK TO MAIN Figure 2. Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Typical Calibration Curve for FOSA Centra Analytical Laboratories, Zno. Centre Analytical Laboratories, Inc. Page 43 of 151 BACK TO MAIN Figure 3. Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Typical Calibration Curve for PFOA Cantra Analytical Laboratori, Inc. Centre Analytical Laboratories, Inc. Page 44 of 151 BACK TO MAIN Figure 4. Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Chromatogram Representing a 0.5 ng/mL mixed calibration standard of PFOS, FOSA and PFOA Hama: 021201A-1001 Taxt: CIO1700-7, 0.5 ng/mL Standard 1 : PFOS 2 : FOSA 3 : PFOA Cantra Analytical Laboratori#, Inc. Centre Analytical Laboratories, Inc. Page 45 of 151 BACK TO MAIN Figure 5. Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Chromatogram Representing a 2.5 ng/mL mixed calibration standard of PFOS, FOSA and PFOA Nanai 021201A-1003 Taxts C101700-5, 2.5 ng/aL Standard 1 : PFOS C101700-5,2.5 ng/mL Standard 13-Feb-2001 14:42:56 LC/MS/MS #6 021201A-1003 Sm (Mn, 2x3) 100n 5.22 23032 iI MRM of 3 Channels ES499 > 99 1.26e5 I Area %- (P i { i i l i t r i t i i i i i t { n r i i i i u p i 1' I' ' ' j ' 1i i '' ' i r ' ' i 111111 i ' i >Timo 1.00 2.00 3.00 4.00 5.00 6.00 7.00 2 : FOSA C101700-5,2.5 ng/mL Standard 021201A-1003 Sm (Mn, 2x3) 100-1 % 5.85 81735 13-Feb-2001 14:42:56 LC/MS/MS #6 MRM of 3 Channels ES498 > 78 4.07e5 Area T 11r ~r r~r 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Time Cantra Analytical Laboratorias, Inc. Centre Analytical Laboratories, Inc. Page 46 of 151 BACK TO MAIN Figure 6. Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Chromatogram Representing a Control Apple Sample (Centre ID: 0009347 Blank A l, Data Set: 020901C Apple) Nanai 020901C-3009 Text: 0009347 Blank XI 1 : PFOS 2 : FOSA 3 : PFOA Cantra Analytical Laboratories, Xne. Centre Analytical Laboratories, Inc. Page 47 of 151 BACK TO MAIN Figure 7. Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Chromatogram Representing a Control Apple Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009347 Spk A l, Data Set: 020901C Apple) Nu m i 020901C-3010 Text 0009347 8pk Al, 2.5 ppb 1 : PFOS 0009347 Spk A 1.2.5 ppb 0209131C-3010Sm (Mn, 2x3) 100n %: 5.21 54931 iI I 12-Feb-2001 18:43:40 LC/MS/MS *6 MRM of 3 Channels ES499 >99 2.90e5 Area o- ..........................i 1............. '"i i 11 11P 111 i 1.00 2.00 3.00 4.00 ITT i i ' i j i i >i i > >i > i inl 5.00 6.00 7.00 8.00 2 : FOSA 0009347 Spk A1, 2.5 ppb 020901C-3010 Sm (Mn. 2x3) 100 5.87 206244, I i 12-Feb-2001 18:43:40 LC/MS/MS #6 MRM of 3 Channels ES498 > 78 1.02e6 Area %: ____ Jl Cr`i i I i i i i I i Ti j i i i i I r i i I <i i I 111' I 11' 11111I 1' 1' 11 1* I T' 1' ! ' ' ' * 1' ' ' ' 1' ' ' ' 1' ' ' ' 1 i Hiw 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3 : PFOA 0009347 Spk A1. 2.5 ppb 12-Feb-2001 18:43:40 LC/MS/MS #6 020901 C-3010 Sm (Mn, 2x3) 100i 5.02 812689 ji I MRM of 3 Channels ES- 413 >369 4.47e6 Area i%: o-S-rri iii i ; i i i i j i i i i i i i { i i <H 11' I * 111I 1111I 11111111_1_I_1_1_1_1_I _' _11_1_I_1_T_*_1_I 1_1_1_1_I_1_1_1_' I__M_IIW_ 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Cantra Analytical Laboratoria, Inc. Centre Analytical Laboratories, Inc. Page 48 of 151 BACK TO MAIN Figure 8. Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Chromatogram Representing an Apple Sample (Centre ID: 0012927, Sponsor ID: Publix, 18257, Data Set: 020901C Apple) I m i 020901C-3016 Tuet I 0012927 1 : PFOS 2 : FOSA 3 : PFOA Cantra Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 49 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 9. Chromatogram Representing a Control Green Bean Sample (Centre ID: 0009346 Blank A2, Data Set: 020601C Bean) Kaaas 020601C-5009 Texts 000934 Blank *2 I s PFOS 2 : FOSA 3 : PFOA Cantra Analytical Laboratories, Xnc. Centre Analytical Laboratories, Inc. Page 50 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 10. Chromatogram Representing a Control Green Bean Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009346 Spk A2, Data Set: 020601C Bean) Nu m i 020601C-5010 Text 0009346 Spk 2, 2.5 ppb 1 : PFOS 0009346 Spk A 2 ,2.5 ppb 0206131C-5010 Sm (Mn, 2x3) 100i %- 5.29 45308 iI I 07-Feb-2001 09:15:32 LC/MS/MS #6 MRM of 3 Channels ES* 499 >99 3.01 e5 Area Q-1 1.00 2.00 2 : FOSA 0009346 Spk A 2 ,2.5 ppb 020601C-5010 Sm (Mn, 2x3) lOO- %- wr 1.00 2.00 3 : PFOA 0009346 Spk A2, 2.5 ppb 0206131C-5010 Sm (Mn, 2x3) 100i %: 111! I 1111 1' 1' 1' 1 j it i*| i >i 3.00 4.00 5.00 6.00 7.00 8.00 5.90 182368 iI 1 07-Feb-2001 09:15:32 LC/MS/MS #6 MRM of 3 Channels ES498 > 78 8.84e5 Area _____________ 3.00 4.00 5.00 6.00 7.00 8.00 5.12 606443 07-Feb-2001 09:15:32 LC/MS/MS #6 MRM of 3 Channels ES- 413 >369 3.39e6 Area o- ' 'I i 1i i 1i i i i i i i i i i rr i j ri i Timo 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Centra Analytical Laboratories, Xnc. Centre Analytical Laboratories, Inc. Page 51 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 11. Chromatogram Representing a Green Bean Sample (Centre ID: 0012910, Sponsor ID: Bruno's, 18341, Data Set: 020601C Bean) U H I 020601C-5020 Tcxti 00129X0 I s PFOS 2 : FOSA 3 : PFOA Cantra Analytical Laboratori#, Inc. Centre Analytical Laboratories, Inc. Page 52 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 12. Chromatogram Representing a Control Pork Muscle Sample (Centre ID: 0009348 Blank A l, Data Set: 011501A Pork) H U w i 011501A-1009 Text i 0009348 Blank Al 1: PFOS 2: FOSA 3: PFOA Centra Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 53 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 13. Chromatogram Representing a Control Pork Muscle Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009348 Spk; A l, Data Set: 011501A Pork) Hi m i 011501A-1010 Tanti 0009348 Spk Al, 2.5 ppb Centra Analytic! Laboratoriaa, Inc. Centre Analytical Laboratories, Inc. Page 54 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 14. Chromatogram Representing a Pork Muscle Sample (Centre ID: 0012795, Sponsor ID: Winn-Dixie, 18227, Data Set: 011501A Pork) Nu m i 011501A-1016 Taxt: 0012795 1 : PFOS 2 : FOSA 3 : PFOA Cantra Analytical Laboratori#, Inc. Centre Analytical Laboratories, Inc. Page 55 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 15. Chromatogram Representing a Control Milk Sample (Centre ID: 0009349 Blank A l, Data Set: 020701A Milk) Nu m i 020701A-3009 T u t I 0009349 Blank *1 1 : PPOS 2 : FOSA 3 : PFOA Caatra Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 56 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 16. Chromatogram Representing a Control Milk Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009349 Spk A l, Data Set: 020701A Milk) H i M t 020701A-3010 Tenti 0009349 Spk Al, 2.5 ppb 1 : PFOS 0009349 Spk A 1 ,2.5 ppb 10-Feb-2001 00:01:51 LC/MS/MS 6 020701A-3010 Sm (Mn, 2x3) 100-1 5.19 57181 II I MRM of 3 Channels ES499 > 99 2.93e5 Area %o: ^ i i I....... I i i i i I i ' 1 1 ' 1 ' 1 1 1 ' ' 1 ' rr i i i i i i i i i i i i i i i i ^ L ................................................. 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 2 : FOSA 3 : PFOA 0009349 Spk A 1 ,2.5 ppb 020701A-3010Sm (Mn,2x3) 100-1 %- 5.02 754985 10-Feb-2001 00:01:51 LC/MS/MS #6 MRM of 3 Channels ES413 >369 4.46e6 Area i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Centre Analytical Laboratories, Xnc. Centre Analytical Laboratories, Inc. Page 57 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 17. Chromatogram Representing a Milk Sample (Centre ID: 0012871, Sponsor ID: Winn-Dixie, 18202, Data Set: 020701A Milk) Nu m i 020701A-3024 Twit i 0012871 1: PFOS 2 : FOSA 3 : PFOA Cantra Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 58 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 18. Chromatogram Representing a Control Chicken Egg Sample (Centre ID: 0009342 Blank A3, Data Sets: 050901A Egg for PFOS, 012401A Egg for FOSA, and 052101A Egg for PFOA) Name: 050901A-3010 Text: 0009342 Blank A3 1: PFOS Centre Analytical Laboratories, Inc. Page 59 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 19. Chromatogram Representing a Control Chicken Egg Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009342 Spk A3, Data Sets: 050901A Egg for PFOS, 012401A Egg for FOSA, and 052101A Egg for PFOA) Hama: 050901A-3011 Text: 0009342 Spk A3, 2.5 ppb 1: PFOS 0009342 Spk A3, 2.5 ppb 050901A-3011 Sm (Mn, 2x3) 100^ %~ ry i i 11111 ' i ' l I I ` I ` ' 11 1.00 2.00 3.00 09-May-2001 19:32:10 LC/MS/MS #6 5.01 33412 I MRM of 3 Channels ES- 499 > 99 2.51e5 Area L i i - , >i ~ n Time 4.00 ` 5.00 6.00 7.00 son 2 : FOSA 0009342 Spk A3,2.5 ppb 012401A-5010 Sm (Mn, 2x3) 100n %- rvu - i - - i - i * - i 1.00 2.00 5.86 224346 I 1 24-Jan-2001 19:43:27 LC/MS/MS #6 MRM of 3 Channels ES- 498 > 78 1.08e6 Area ......... .. . . . . j . . . . j - . ._. i . . . . i 3.00 4.00 5.00 Li | w| r-i i ij i , i , | i i i .i ime 6.00 7.00 Centre Analytical Laboratories, Inc. Page 60 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 20. Chromatogram Representing a Chicken Egg Sample (Centre ID: 0012861, Sponsor ID: Save, 18167 and 21226, Data Sets: 050901A Egg for PFOS, 012401A Egg for FOSA, and 052101A Egg for PFOA) Name: 050901A-3017 Text: 0106440 1: PFOS 2 : FOSA 0012861 24-Jan-2001 23:24:17 012401A-5024 Sm (Mn, 2x3) \ LC/MS/MS #6 MRM of 3 Channels ES- 10Oj %-' -?10.04 5.76 .5.01 6.74 6.99 498 > 78 A / 533 \6.50 A n 7-36 5.58 I 1 \J \ J\J\l3.39 4.094 70l4.83 7.587.93 1.181.54 2^49 i ri i i i i i i i i i i i i i i i 1.00 2.00 3.00 4.00 5.001i 11 i ' ^ 1111i 'T' ' i ' ' Time 6.00 7.00 1: PFOA 0106 440 21-M ay-2001 19:26:45 0521 01A-3017 Sm (Mn, 2x3) 100: 5.53 %- Ch 2.00 L1.bo 1 _vn "1' 11M ` 3.00 ' 4.00 ' 5.00 6.00 LC/M S/M S #7 MRM of 3 Channels ES- 413 > 369 / v3.14e4 1111i 111i 1' ' ' T im e 7.00 Centre Analytical Laboratories, Inc. Page 61 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 21. Chromatogram Representing a Control Bread Sample (Centre ID: 0009343 Blank A l, Data Set: 020901A Bread) h u m i 020901A-1009 Text i 0009343 Blank *1 1: PFOS 0009343 Blank A1 020901A-1009 Sm (Mn, 2x3) 100i 5.02., 10497 A %- J i0-* '-i ' - 1 1 ' i 1' i 1 111 . 1' i i*i~i 11 11 ' ' ' T 1rT^ i ........ .... 1.00 2.00 3.00 4.00 5.00 11-Feb-2001 03:36:14 LC/MS/MS #6 MRM of 3 Channels ES499 >99 5.29e4 Area ____ i ' i ' 1' i i ' i ' lime 6.00 7.00 2: FOSA 3: PFOA Cantra Analytical Laboratoriaa, Xnc. Centre Analytical Laboratories, Inc. Page 62 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 22. Chromatogram Representing a Control Bread Sample Fortified with 2.5 ng/g of PFOS, FOSA and PFOA (Centre ID: 0009343 Spk A l, Data Set: 020901A Bread) H u m s 020901A-1010 Texts 0009343 Spk Al, 2.5 ppb 1 : PFOS 2 : FOSA 0009343 Spk A 1 ,2.5 ppb 020901A-1010 Sm (Mn, 2x3) 100-1 %- 5.88 250287 J 11-Feb-2001 03:52:01 LC/MS/MS #6| MRM of 3 Channels ES498 > 78 1.23e6 Area "I i i i i i i r rr p Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 3 : PFOA______________ ________________________________________________________________________________________ 0009343 Spk A1. 2.5 ppb 11-Feb-2001 03:52:01 LC/MS/MS #6 020901 A-1010 Sm (Mn, 2x3) MRM of 3 Channels ES- 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Centre Analytical Laboratories, Xnc. Centre Analytical Laboratories, Inc. Page 63 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Figure 23. Chromatogram Representing a Bread Sample (Centre ID: 0012887, Sponsor ID: Albertson's, 18244, Data Set: 020901A Bread) Nu m i 020901A-1020 Tant 0012887 1 : PFOS 2 : FOSA 3 : PFOA Cantra Analytical Laboratoriaa, Xnc. Centre Analytical Laboratories, Inc. Page 64 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 13.0 APPENDICES Centre Analytical Laboratories, Inc Page 65 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 APPENDIX A Study Protocol 00P-023-057 (Centre Study No. 023-057), Amendments and Deviation Centre Analytical Laboratories, Inc. Page 66 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 STUDY PROTOCOL i STUDY TITLE EXTRACTION OF PFOS, FOSA AND PFOA FROM VARIOUS FOOD MATRICES FOR ANALYSIS USING HPLC ELECTROSPRAY/MASS SPECTROMETRY SPONSOR 3M Environmental Technology and Safety Services . Building 2-3E-09 - PO Box 33331 St. Paul, MN 55133-3331 . DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792 TESTING LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 Phone 814-231-8032 PROTOCOL IDENTIFICATION NUMBER 00P-023-057 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 1 of 81 Page 67 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 Title: EXTRACTION OF PFOS, FOSA AND PFOA FROM VARIOUS FOOD MATRICES FOR ANALYSIS USING HPLC ELECTROSPRAY/MASS SPECTROMETRY TABLE OF CONTENTS ________ .________________________ __________________ '_____________ Page STUDY TITLE.......................................................................................................... 1 1. PURPOSE......................................................................................................... 2. ' TEST MATERIALS........................................................................................ .. 3. SPONSOR............. ......................................................................................... .. 4. TESTING FACILITY AND PERFORMING LABORATORY.......................... 5 5. PROPOSED EXPERIMENTAL TIME-FRAME.......'.........................................5 6 . TEST SYSTEM............................................................ 5 7. JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM................ 6 . 8. SAMPLE PROCESSING. STORAGE AND IDENTIFICATION........ ..... ;......6 9. ANALYTICAL METHOD..................................................._..........................6 10. EXPERIMENTAL DESIGN............................. 7 11. ' STATISTICAL METHODS TO BE USED........... ........................................... 7 ' 12. PROTOCOL AMENDMENTS AND DEVIATIONS......................................... 7 13. RECORDS AND DATA REPORTING....................................... '...................g 14. QUALITY ASSURANCE................................................................ ................g 15. DATA AND REPORT....................................................................................... 16. ARCHIVE STATEMENT.................................................. 10 17. REFERENCES................................................................ :................. ...........10 18. PROTOCOL APPROVAL............................................................................. ... APPENDIX: ANALYTICAL METHOD.......................... ........ ................................ 12 ; Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. I' Page 2 of 81 Page 68 of 151 BACK Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 Study title: EXTRACTION OF PFOS, FOSA AND PFOA FROM VARIOUS FOOD MATRICES FOR ANALYSIS USING HPLC ELECTROSPRAY/MASS SPECTROMETRY 1. PURPOSE The purpose of this study is to analyze PFOS, FOSA and PFOA in various food matrices using the analytical method entitled "Method of Analysis for the Determination of Fluorochemicals in Green; Beans, Apples, Pork Muscle, Cow Milk, Chicken Muscle, Chicken Eggs, Bread, Hotdogs, and Catfish by LC/MS/MS". A copy of the method is appended to this protocol (Appendix). The Quality Assurance Unit of Centre Analytical Laboratories, Inc. will audit the study for compliance with EPA TSCA Good Laboratory Practice standards 40 CFR 792 (1). 2. TEST MATERIALS The following analytical standards will be used: Test Material Lot or TCR Number Purity (%) Expiration Date PFOS PFOA TCR00017-46 332 97.9 . TBD 08731/01 07/06/01 FOSA L-15709 TBD= To be determined 95.1 02/15/01 Note: GLP characterization, solubility and stability data for PFOA and FOSA are unavailable at this time. However, available characterization data for the test materials will be supplied to the testing facility (Centre) by the sponsor (3M). Therefore, do not correct for percent purity when preparing standard solutions for these two fluorochemicals. I Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 3 of 81 Page 69 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 Chemical names and structures of the compounds are presented below. PFOS Chemical Name: Molecular Weight: CAS Number: 1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6 ,6 ,7,7,8,8,8- heptadecafluoro', potassium salt. 499 (CsFnSCV) 2795-39-3 . 0 c 8f )7s -- o 0 Note: The neutral molecule and standard form which PFOS (anion) is derived from is perfluorooctanesulfonate potassium salt [C8F17SO3K], molecular weight 538. PFOA Chemical Name: Molecular Weight: CAS Number: Octanoic acid, pentadecafluoro', ammonium salt 413 3825-26-1 (FC-143) C7f 15C 00- - Note: The neutral molecule and standard form which PFOA (anion) is derived from is perfluoroctanoate ammonium salt [C7F15COONH4], molecular weight 431. .' ' FOSA Chemical Name: Molecular Weight: CAS Number: 1-Octanesulfonamide, 1,1,2,2,3,3,4,4,5,5,6 ,6 ,7,7,8,8,8heptadecafluoro' - 499 754-91-6 0 , II C8F17S -- nh2 0 A record of test and reference substance receipt, storage conditions, and a record of use will be maintained at Centre Analytical Laboratories, Inc. Chain-ofcustody forms and shipping documents for tracking of the test substances will be included as part of the raw data package. !- Centre Analytical Laboratories, Inc. Page 4 of 81 Centre Analytical Laboratories, Inc. Page 70 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 All standards/test substances and any prepared solutions must be identified with a unique label or number on the container or cross-referenced to the container. HAZARD INFORMATION A current MSDS for the chemical(s) used in this study will be maintained at the testing facility. . 3. SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 POBox 33331 St. Paul, MN 55133-3331 STUDY DIRECTOR: ' Susan A. Beach, 3M TELEPHONE: 651-778-7452 . FAX NUMBER: 651-778-6176 - 4. TESTING FACILITY AND PERFORMING LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive . State College, PA 16801 PRINCIPAL INVESTIGATOR: Karen Smith, Centre TELEPHONE: 814-231-8032 FAX NUMBER: 814-231-1580 . ' 5. PROPOSED EXPERIMENTAL TIME-FRAME Experimental Start Date Experimental Termination Date Report Issued January 8, 2001 February 8, 2001 March 8, 2001 6. TEST SYSTEM Food samples obtained by Centre Analytical Laboratories, Inc. and used in the validation study (Centre Study No. 023-043) will be used as the control samples. Samples will be collected from local grocery stores from six test cities outlined in Centre Analytical Laboratories, Inc. Page 5 of 81 Centre Analytical Laboratories, Inc. Page 71 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 3M Quality Assurance Project Plan for Empirical ,Human Exposure Assessment Multi-City Study Sampling Task. All food samples obtained from grocery stores in the six test cities by the sponsor will be shipped to Centre Analytical Laboratories, Inc. for analysis. 7. JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM Representative samples such as, honey, vegetable (green beans, carrot, radish, lettuce, potato), fruit (tipple), livestock (pork muscle, cow milk), poultry (chicken muscle, egg), fish and processed foods (bread, hot dogs), will be collected by the sponsor and shipped to Centre for trace-level analysis of fluorochemical residues. The cities chosen as the collection sites either have major fluorochemical production facilities, major fluorochemical users, or no major fluorochemical production or usage facilities. The matrices were chosen to determine if fluorochemicals are being transferred from the environment to food sources. 8. SAMPLE PROCESSING, STORAGE AND IDENTIFICATION All food samples will be processed according to the method entitled, "Method of Analysis for the Determination of Fluorochemicals In Green Beans, Apples, Pork Muscle, Cow Milk, Chicken Muscle, Chicken Eggs, Bread, Hotdogs, and Catfish byLC/MS/MS." - . Upon receipt at Centre, the food samples wiH "be assigned unique sample identification numbers, which will be used for tracking and identification of the samples. The samples will be stored in a temperature monitored freezer, maintained at < -10 C, except when removed for 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 must be documented. All samples and any resulting sample extracts must be identified with a unique label or sample number. Such identification will be either, on the container or cross-referenced to the container. . . 9. ANALYTICAL METHOD The samples will be analyzed according to the analytical method entitled "Method of Analysis for the Determination of Fluorochemicals in Green Beans, Apples, Pork Muscle, Cow Milk, Chicken Muscle, Chicken Eggs, Bread, Hotdogs, and Catfish by LC/MS/MS" (see Appendix). ' Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 6 of 81 Page 72 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 10. EXPERIMENTAL DESIGN For each matrix, a set of samples will be extracted in duplicate and analyzed. Each set of samples analyzed will contain at least one matrix control and two matrix control samples fortified at known concentrations and carried through the extraction procedure to verify recovery. Methods to control bias will include assay of untreated control samples, fortification of untreated control samples to obtain recovery data, and replicate analysis of fortified samples to provide an indication of reproducibility. 11. STATISTICAL METHODS TO BE USED Standard (linear, 1/x weighted) curves used to determine the concentration of the compound being analyzed will be constructed from a minimum of six concentrations. Quantification of residues will only occur within the range of the - standard curve (residues in control samples may be calculated up to 2 0 % of- the LOQ [20% of LOQ = 0.1 ppb] by extrapolation of the calibration curve, in order to correct fortification recoveries). The correlation coefficient (r) for calibration curves generated must be greater than or equal to 0.9925 (r2> 0.985). ' Overall averages, standard deviations and relative standard deviations of fortification recoveries will be calculated. .- Other than these descriptive statistics, no other statistical methods will be used during the course of this study. 12. PROTOCOL AMENDMENTS AND DEVIATIONS Any deviations from the protocol or from the analytical method as provided will be documented and reported promptly to the Study Director. Planned changes to this protocol or to the analytical method will be made in writing as an amendment or modification and approved by the Principal Investigator and the Study Director. Any amendments will be appended to this protocol and included in the final report. . 1. Protocol amendments: Planned changes to the approved protocol shall be documented by amendments that clearly describe the change, justification for the change, and impact on the study. Amendments will be signed and dated by the Principal Investigator and Study Director. Copies of amendments will be sent to the quality assurance unit. - 2. Protocol deviations: Protocol deviations, which are one time and unplanned . deviations from the protocol shall be documented in the study records, noting the nature of the deviation, potential effect or impact on the study, and corrective action if required. Protocol deviations are signed by the Principal Investigator and Study Director and reviewed by QAU. Centre Analytical Laboratories, Inc. , Centre Analytical Laboratories, Inc. Page 7 of 81 Page 73 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 13. RECORDS AND DATA REPORTING Records to be maintained include, but are not limited to the following (as appropriate): 1. Sample tracking sheet(s) . 2. Sample receipt records, storage history, and chains of custody 3. History and preparation of standards (stock, fortification, calibration) 4. Description of any modifications to the method 5. Instrument run sheets, bench-sheets or logs 6 . Analytical data tables " 7. All chromatographic and instrumental conditions 8. Sample extraction and analysis dates 9 . A complete listing of study personnel, signatures and initials 10. Chronological presentation of all study correspondence ' 11. Any other data necessary for the reconstruction of the study All chromatograms will contain the following: a. Sample identification, date, arrow or other indication of the area of interest, and injection number corresponding to the run. b. Additionally, fortifications will include the fortification level of each analyte. ' c. Analytical standard chromatograms will additionally include the concentration (e.g., pg/ml, ng/mL, ppb, ppt, etc.). Each data set will contain information on temperatures, flow rates, column parameters, gases, instrument parameters, and instrument type, etc. if any of these differ from the method. The Centre study number will also be recorded on the first chromatogram of each daily analytical run. . 14. QUALITY ASSURANCE . Centre QA Unit will review the protocol and audit the study conduct and the study documentation (including raw data and final report) to ascertain that all QA/GLP procedures are adhered to. Centre QA Unit will inspect the study at . intervals adequate to assure compliance to GLP's, and will report the findings of the audits to the Principal Investigator, Centre Management, and the Study . Director. . 15. DATA AND REPORT i 1. All raw data and the original signed protocol will be maintained in the study file. This data includes the protocol amendments, protocol deviations, laboratory notebooks, analytical standard solution preparation, sample chain of custody sheets, sample work sheets, chromatograms, calibration curves, and any other appropriate data generated. Raw data not used will be stored in the study file. The reason for exclusion will be documented. Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 8 of 81 Page 74 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 2. A final report will be issued by Centre, approved and signed by the Study Director and sufficient for submission to EPA. The report contents should include, but are not limited to: 1. Objectives and procedures stated in the protocol 2. Analytical and statistical methods used 3. Test materials identified by name, lot, purity and other characteristics 4. Name of testing facility and study initiation and completion dates 5. Statement prepared andsigned by the quality assurance unit 6 . Tables containing all applicable data 7. All chromatographic and instrumental conditions . 8. A complete listing of Centre study personnel . Centre will send a copy of the draft report to the Study Director, who will return the report with comments. Centre Analytical will make revisions and finalize the - report with the approval of the Study Director. The Centre QA unit will conduct an audit of the final draft report and data files to assure accuracy and GLP compliance. The final report and copies of the raw data must be approved by the " Study Director prior to signature. A statement of inspections and a statement of compliance will be included in the final report . Any corrections or additions to the final report shall be in the form of ah amendment by the Principal Investigator. The amendment shall clearly identify the part of the final report that is being corrected and the justification for correction. The amendment shall be signed -and dated by the Principal Investigator and the Study Director. - REPORT DISTRIBUTION: Original: Study Director Copy: Centre Archives . Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 9 of 81 Page 75 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol. No: 00P-023-057 Centre Protocol No. 00P-023-057 16. ARCHIVE STATEMENT Study records to be maintained: Records to be maintained for the study include all raw data, observations recorded during the conduct of the study, documentation, chromatograms, specimens, and study related correspondence. This includes a description of equipment used during the conduct of the study. All characterization data and any shipping records shall be retained. Document archives: Upon completion of the study, the study records, protocol and ' amendments, and the final report and amendments shall be retained in the 3M document archives. If it is necessary to substitute a copy for an original record, it will be certified as an exact copy. Centre will retain facility-related originals and a copy of the final report and raw data package. 17. REFERENCES . 1. U.S. Environmental Protection Agency, Toxic Substances Control Act (TSCA). Good Laboratory Practice Standards. Final Rule, 40 CFR Part 792. Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 10 of 81 Page 76 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 18. PROTOCOL APPROVAL This protocol was audited by the Quality Assurance Unit of Centre Analytical Laboratories, Inc. ' Quality Assurance Reviewer, Centre Quality Assurance Auditor Principal Investigator, Centre Scientist Facility Management, Centre CpL,ft)fj/JeCi, John Flaherty / Laboratory Manager Date 3 -gftv-d; Date . Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 11 of 81 Page 77 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 APPENDIX: ANALYTICAL METHOD METHOD OF ANALYSIS FOR THE DETERMINATION OF FLUOROCHEMICALS IN GREEN BEANS, APPLES, PORK MUSCLE, COW MILK; CHICKEN MUSCLE, CHICKEN EGGS, BREAD, ' HOTDOGS, AND CATFISH BY LC/MS/MS. Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 12 of 81 Page 78 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057- ' TITLE . yMethod of Analysis for the Determination of Euorochemicals in Green Beans, Apples Pork Muscle, Cow Milk, Chicken Muscle, Chicken Eggs, Bread, Hotdo'gs and Catfish bv LC/MS/MS '. AUTHORS . Enaksha Wickremesinhe, Shaozhi Zheng and John Eaherty ' DATE ISSUED September 13,'2000 SPONSOR ' 3M Environmental Technology and Safety Services Building 2-3E-09 - PO Box 33331 . - St. Paul, MN 55133-3331 PERFORMING LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 CENTRE STUDY NUMBER 023-001 TOTAL NUMBER OFPAGES . 69 Lenite A....!; I is, Inc. Centre Analytical Laboratories, Inc. Page 13 of 81 Page 79 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 . ' MANAGEMENT APPROVAL As per 40 CFR 160.3, method development is not required to be conducted in compliance with GLP's. However, the work was in general conformance with applicable standard operating procedures and general GLP.regulations. Enaksha Wickremesinhe, PhD. Author, Principal Investigator Centre Analytical Laboratories, Etc Date Shaozhi Zheng J" Cj-lS-P Date ' Author, Principal Investigator . Centre Analytical Laboratories, Inc. tf-L.m y/iohn Flaherty / " Date Laboratory Manager 1 Centre Analytical Laboratories, Inc.: C=>{ v ct Richard A. Grazzini, PhD. President' . Centre Analytical Laboratories, Inc. ^ / 1 /16 Q Date L iz -------- Kris Hansen, PhD. Sponsor Representative 3M Environmental Laboratory Date 2oo<=> Centre Analytical Laboratories, Inc. Study 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 2 of 69 Page 14 of 81 Page 80 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 . TABLE OF CONTENTS - ' TITLE PAGE.....................- ....................... ..................................................................l m a nag em ent approval............................ ..........................................................2 t a ble of contents'-- :................... ....... .......... ........................ .......................3 list of t a b l e s ............- .......:............ ....................... - ..................... . !.............. . 4 LIST OF FIGURES________ :............................................................. ....... ................ 5 1. SUMMARY...........................................~........... .................................................. 7 2. EXPERIMENTAL C O M P O U N D S ........................................................;....... g 3. CHEMICALS AND SUPPLIES.......................... ........................... ....... ;.............. 9 3.1: Chemicals.......... -- ................- .................................................................................. 3.2. Standards.............................................. ........... -................................;................... 9 3.3. Equipment and Su pp l e s ................................ ........................................................ 3.4. S olutions........................................... -- .............................................................. .. 1 ' 3.5. Preparation of Stock, F ortification, and Calibration Solutions.......11 3.5.1. Stock Solutions.................................... :....... - .............................. u . 3.5.2. Fortification Solutions....................................................................u . 3.5.3. Calibration Standards-- .....................................:..... .....................j2 4. METHOD.........................L................................................ ...........................12 4.1. R ow Diagram................................................... .............. .......................... . 4.2. Sample Processing................................ -....... ......................... ............................ 13 . 4.3. Extraction.............................- ............................ .................................... ........... 13 ' 4.3.1. Apple, Green Beans, Bread, Hot Dog, and P o r k ....................1 3 . 4.3.2. Chicken and Milk.......................... .............................................. 4 ' 4.3;3. Egg and Catfish............. ........................--....................................... 4.3.4. SPE Column Preparation................................................................jg 43,5. Silanization of Pear-Shaped Flasks...................... ..........................g 4 .3 .6 . Standardization of SPE Columns.................................... ............... jg 4.4'. Analysis by LC/MS/MS.....-- .................................................................. .. . 4.4.1. LC/MS/MS System and Operating Conditions (Electrospray)........ 16 ; 4.4.2. Example Tune Hie Parameters ......................................................... 4.4.3. Calibration Procedures................... ......... -.................................... 9 4.4.4. Sample AnalysisTM........................................................ 19 4.5. Performance CrtierIa ...................................................................... 20 ' 4.6. T ime Required for Analysis.................................................. ............................ 21 5. CALCULATIONS....................... - ..... ....................... '..................... - ...............21 5.1. Component Residue Concentration.............. ............................................. 21 5.2. Fortification Recovery................................................................ 22 6 - SAFETY............................................................................................................... 22 Centre Analytical Laboratories, Inc. Study # 023-001 Centre Analytical Laboratories, Inc. Page 3 of 69 ' Page 15 of 81 Centre Analytical Laboratories, Inc. Page 81 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 LIST OFTABLES Table 1: Summary of Recoveries forEt-FOSE-OH, Me-FOSE-OHJ'FOS, POAA and PFOSA in Whole Apple............ .................................................24 Table 2: Summary of Recoveries forEt-FOSE-OH, Me-FOSE-OH, PFQS, POAA and PFOSA in GreenBeans'.............................................................. 25 Table 3: Summary of Recoveries for Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA and PFOSA in Chicken Eggs.................. :....................:...... ............. 26 Table 4: Summary of Recoveries forEt-FOSE-OH, Me-FOSE-OH, PFOS, - POAA and PFOSA in Chicken Muscle........................................................... 27 Table 5: Summary of-Recoveries forEt-FOSE-OH, Me-FOSE-OH, PFOS, ' . POAA and PFOSA in Cow Milk......... ...........................................................28 Table 6: Summary of Recoveries for Et-FOSE-OH, Me-FOSE-OH, PFO S,. . POAA and PFOSA in Bread....... 1.............................................................. 29 " Table 7: Summary of Recoveries for Et-FOSE-OH, Me-FOSE-OH, PFOS, ' POAA and PFOSA in Hot Dog:.................... ........................... !................... 30 Table 8: Summary of Recoveries for PFOS, POAA and PFOSA in Catfish................ 3 1 Table 9: Summary of Recoveries forEt-FOSE-OH, Ms-FOSE-OH,PFOS, .. POAA and PFOSA in Pork'Muscle........ .............................................. 32 . Centre Analytical-Laboratories, Inc. Study 4 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 4 o f 69 Page 16 of 81 Page 82 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 i Centre Protocol No. OOP-023-057----- I '. LIST OFFIGURES Figure 1 : Representative Chromatograms of a 0.5 ng/mL Mixed Calibration ' Standard of Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA and PFOSA-...... 34 Figure 2: Representative Chromatograms of a Control Whole Apple Sample ' , (Set 111799 Apple, Centre ID 9S3732 Apple Blank A ).......... .................35 Figure 3: Representadve Chromatograms from the Injection of Control Whole ' Apple Fortified at 500 ppt each of Et-l'OSE-OH, Me-FOSE-OH, PFOS, POAA, and PFOSA (Set 111799 Apple, Centre ID 983732 Spk A) 38 Figure 4: Representative Chromatograms of a Control Green Bean Sample (Set 111299 Beans, Centre ID 9908032 GreenBeans Blank A)................. 40 Figure 5: Representative Chromatograms from the Injection of Control GreenBeans 42' Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA', and PFOSA (Set 111299 Beans, Centre ID 9908032 Spk B).............. , ' Figure 6: Representative Chromatograms of a Control Chicken Egg Sample (Set 122999 Egg, Centre ID 99i2031 Egg Blank A)................................... Figure 7:. Representative Chromatograms from the Injection of Control Chicken Egg Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA, and PFOSA (Set 122999 Egg, Centre ID 9912031 SpkB )..J....... 46 Figure 8: Representative Chromatograms of a Control Chicken Muscle Sample -(Set 111999 Chicken, Centre ID 9910292 Chicken Blank B)....... .............43 - ' Figure 9: Representative Chromatograms from the Injection of Control Chicken Muscle Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA, and PFOSA (Set 111999 Chicken, Centre ID 9910292 Spk A)....'... 50 Figure 10: Representative Chromatograms of a Control Cow Milk Sample (Set 110999 Milk, Centre ID 972627 Milk Blank B)................................. 52 Figure 11 : Representative Chromatograms from the Injection of Control Cow Milk Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH, PFOS, . . POAA, and PFOSA (Set 110999 Milk, Centre ID 972627 Spk A).....;....... 54 Figure 12: Representative Chromatograms of a Control Bread Sample (Set 111999 Bread, CentreID 990S031 Bread Blank A)............................ gg Figure 13: Representative Chromatograms from the Injection of Control Bread Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH, PFOS, ' . POAA, and PFOSA (Set 111999 Bread, CentreID 9908031 Spk A).......... 58 Figure 14: Representative Chromatograms of a Control Hot Dog Sample (Set 110899 Hot Dog, Centre ID 9908035 Hot Dog Blank B )............... . go Figure 15: Representative Chromatograms from the Injection of Control Hot Dog .. - Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA, and PFOSA (Set 110899 Hot Dog, Centre ID 9908035 Spk B)..................go Figure 16: Representative Chromatograms of a Control Catfish Sample _ (Set 012400 Catfish, Centre ID 9912032 Catfish Blank A)........................ g4 - Centre Analytical Laboratories, Inc. Study # 023-001 Centre Analytical Laboratories, Inc. ' Centre Analytical Laboratories, Inc. . Page 5 of 69 Page 17 of 81 Page 83 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No: 00P-023-057 LIST OFFIGURES (CONTINUED) figure 17: Representative Chromatograms from the Injection of Control Catfish Fortified at 500 ppt each of PFOS, POAA, and PFOSA (Set 012400 Catfish, Centre ID 9912032 Spk A).....i.............................................. ...... figure 18: Representative Chromatograms of a Control Pork Muscle Sample (Set 122199 Pork, Centre ID 9911783 Pork Blank B )................................ figure 19: Representative Chromatograms from the Injection of Control Pork Muscle Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA and PFOSA (Set 122199 Pork, Centre ID 9911783 SpkB)....;...... ......... ' Centre Analytical Laboratories, Inc. Study # 023-001 Centre Analytical Laboratories, Inc: Centre Analytical Laboratories, Inc. Page 6 of 69 Page 18 of 81 Page 84 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 .. Centre Protocol.No. 0QP-023-057 1. SUMMARY - This document details a method of analysis for residues of 2(N-ethylperfiuorooctane- sulfonamido)-ethyl alcohol (Et-FOSE-OH), 2(N-methylperfluorooctanesulfonamido) ethyl alcohol (Me-FOSE-OH), perfluorooctaie sulfonate (PFOS), perfluorooctanoate (POAA), and periluorooctane sulfonylamide (PFOSA), in green beans, apples, pork muscle, cow milk, chicken muscle, chicken eggs, bread, and hotdogs, and PFOS, POAA and PFOSA in catfish. -The chemical formula;! of the analytes are given in Section 2 of this method.. . . '" Residues of these fivefluorochemicals are extracted from each matrix with acetonitrile (ACN). The ACN extract is passed through a multi-layered (florisil, silica gel" carbon and amino) solid-phase extraction (SPE) column. Approximately 2-3 drops of l'-octanol are added to the extract and evaporated to near dryness using rotary evaporation. Each extract is then reconstituted with methanol. Quantification of Et-FOSE-OH Me-FOSE-OH PFOS, POAA, and PFOSA is accomplished by liquid' chromatography/tandem mass spectrometry (LC'/MS/MS) analysis using selected reaction monitoring (SRM). . .. The proposed limit of quantitation, LOQ; is the lowest fortification specified by the method which gives adequate recovery according to EPA guidelines [OPPTS 860.1340 (c) (2)j and.for this method is 0.5 parts-per-billion (ppb) each for Et-FOSE-OH, MeFOSE-OH, PFOS, POAA, and PFOSA. This is based on studies conducted with whole apple, green bean, bread, chicken egg, chicken muscle, pork muscle, cow milk, hot do, and catfish control samples during the development of this method. ' The theoretical limit of detection (LOD) will be based on the signal to noise ratio and will be at least greater than 3 times the level of noise, based on the' instrumentation system used. For all five analytes (Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA, and PFOSA), the lowest analytical standard corresponds to 0.5 ng/mL, which is equivalent to 0.2 ns/ (0 .2 ppb) of matrix. Recoveries for ail matrices can be found in Tables 1-9. Lower recoveries for Me-FOSE-OH and broad peak shapes for Et-FOSE-OH have been observed in some animal matrices (chicken, pork and chicken egg). ' . NOTE: Perfluorononane sulfonate (PFNS) may be used as a surrogate standard, to verify extraction efficiency. - i - Centre Analytical Laboratories. Inc. Study # 023-001 'i Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 7 of 69 ' Page 19 of 81 Page 85 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No..00P-Q23-057. 2. EXPERIMENTALCOMPOUNDS Molecular structures ofEt-FOSE-OH, Me-FOSE-OH, PFOS, POAA, and PFOSA are given below. ' Et-FOSE-OH ' . molecular weight: 571 ' - .| ^ C H jCHjCH C8F,7S-- N II NCHH3 ! ' . Chemical Name = 2(N-ethyIpetfluorooctanesulfonamido)ethyl alcohol Me-FOSE-OH molecular weight: 557 0 / CHfitifiH Chemical Name = 2(N-meshylpt:rfluorooctanesulfonamido)ethyl alcohol 99PFOS molecular weight: 4 (CgFnSOs- ) ' . 0- CgJrnlS--0 . II - 0 .' ' Chemical Name = Perfluorooctaoe sulfonate Note: The neutral molecule and standard form which PFOS (anion) is derived from is potassiumperflucrooctane sulfonate [C8Fi7S03K], molecular weight 538. . ' POAA molecular weight: 413 ' . . C7l5COO" Chemical Name = Perfluorooctanoate ' . ' Note: The neutral.molecule and standard form which POAA (anion) is derived from is ammonium perfluoroctanoate [C7F15COONH4], molecular weiaht 431. . `. . ' '= i Centre Analytical Laboratories, Inc. Study # 023-001 ` , j p ag: 3 0f g 'i ' ' ' Centre Analytical Laboratories, Inc. ; Page 20 of 81 Centre Analytical Laboratories, Inc. Page 86 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 PFOSA molecular weight: 499 0 II c8f,7| -- nh2 . ' ' . ^ ' Chemical Name = Perfluorooccme sulfonylamide 3. CHEMICALSAND SUPPLIES 3.1. Chemicals ' Chemical_________ Grade_______ S o u r c e _______Catalog Nr Acetonitrile (ACN) Carbon ` Methanol (MeOH) . Silica gel (60-200 mesh) Florisil (60-100 mesh) Superclean LC-NHi 1 -O.ctanol Ammonium Acetate. L-Ascorbic acid Dimetyldicnlorosilane Toluene " . Water . HPLC 120/400 HPLC Reagent Reagent Reagent Reagent Reagent Reagent Reagent Reagent Type I - (VWR)J.T. Baker Supelco (VWKJJ.T. Baker (VvVR) J. T. Baker (VWR)J,T. Baker Supelco ' Aldrich Chemical . Sigma-Aldrich " . Sigma-Aldrich Supelco (VWR)J.T. Baker in house JT9017-3 57210-U JT9093-2 3405-05 3372-08 5-7205 111-87-5 A-7330 L-5960 3-3009 JT9460-3 - (Type I water = electrical resistivity, minimum of 16.67 Mi2/cm at 25C, from a Labconco waterpro workstation) ` 3.2. Standards S(andard Et-FOSE-OH Me-FOSE-QH PFOS ' POAA PFOSA i Grade - Analytical Analytical . Analytical Analytical Analytical Source 3M 3M 3M 3M 3M . Centre Analytical Laboratories, Inc. Study # 023-001 I ;_ Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page9 of 69 Page 21 of 81 Page 87 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Cntre Protocol No, OOP-023-057 3.3. Equipment and Supplies EQUIPMENT Balance, analytical ' Balance, top-loading Rotary evaporator Rotary evaporator trap ' Tissumizer . Sorvall RC 5C plus Centrifuge . IEC HN SB centrifuge Pear-shaped flasks - ` SPE tubes (20 mL) cat no. N057177 Vacuum pump . Visiprep vacuum manifold Wrist-action shaker. ' Bench-top centrifuge 50 mL disposable polypropylene.centrifuge tubes Disposable pipets, test tubes etc. 125-mL LDPE narrow-mouth bottles 2-mL clear HPLC vial kit (cat # 5181-3400) - Standard lab equipment (class A pipets and - volumetric flasks, graduated cylinders, etc.) LC/MS/MS and HPLC systems are SOURCE ' Mettler' Mettler ' Buchi ; Buchi Tekmar Dupont OEC Pyrex Supelco . Buchi Supelco Burrell IEC VWR , VWR Nalgene ' HP --" As described in Section 4 .4 .1 ` ` " Notes: . 1. In order to avoid contamination, the use of disposable labware is highly recommended (containers, tubes, pipets etc.). . ' ' 2. Teflon or teflon-lined containers or equipment, including teflon-lined - HPLC vial caps should not be used. ' 3. Silanize pear-shaped flasks before use. 4. It is necessary to check the solvents (methanol) for the presence of ; contaminants (especially POAA) by LC/MS/MS before use. Certain lot numbers have been found to be unsuitable for use. 5. Use disposable micropipets or pipets to aliquot standard solutions, 6.when preparing standards and samples for extraction. Equivalent materials may be substituted for those, specified 'in this ' method. However, the use of carbon from Supelco is strongly. recommended. ' " Centre Analytical Laboratories, Inc. Study # 023-001 , Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. . p^gj [q 0 f gg "- Page 22 of 81 Page 88 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 3.4. Solutions . 1. 50 mM ammonium acetate: Dissolve 3.85 g of ammonium acetate in 1 L of type I water. 2. 2 mM ammonium acetate: Dilute 40 mL of the 50 mM ammonium . acetate solution in a liter of type I water, for mobile phase A. 3. 2 % ascorbic acid in methanol: Dissolve 2 ,g ascorbic acid in 100 mL ' methanol. `' Note: The volumes shown are provided for guidance; alternative volumes may be prepared. 3.5. Preparation .of Stock, F ortification, and Calibration Solutions ' '' ' Analytical standards are prepared for two purposes. They are used to fortify untreated samples to determine analytical recovery, and to calibrate the response of the detector used in the analysis. The analyst may vary die absolute volumes of the standards as long as the correct proportions of solute to solvent are maintained. The solutions cited below-are 3.5.1.StockSolutionsgiven as an example; alternative concentrations may be preparedif needed. . .' Prepare individual stock solutions of Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA, andPFOSA at.100 pg/mL each, by weighing out 10.0 mg of analytical standard (corrected for percent salt). Adjust final volume to 100 mL with methanol in a 100-mL volumetric flask. Prepare a separate solution for each analyte. Store these stock solutions (in 125-mL LDPE bottles) in a refrigerator at 2C to 6 C for a maximum period of 6 months from the date of 3p.r5ep.2a.raFtioonr.tificationSolutions. . * _ a. 1,0 ug/mL Combined Fortification Solution - Pipet 1.0 mL each of Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA, and PFOSA 100 pg/mL ' stock solution in to a 100 mL volumetric flask. Bring up to volume with methanol. . - - b. 0.1 ua/mL CnmHined Fortification Solution - Pipet 10.0 mL of the -1.0 pg/mL combined fortification to a 100-mL volumetric flask and bring, up to volume with methanol. ' . | Cemre Analytical Laboratories, Inc. Study # 023-001 i` CeVitre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page II of 69 Page 23 of 81 Page 89 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 c. 0-01 us/mL Combined Fortification Solution - Pipet 10.0 mL of the 0 1 pg/mL combined fortification to a 100-mL volumetric flask and bring up to volume with methanol. Store all fortification standard solutions (in 125-mL LDP bottles) in a refrigerator at 2C to 6C for a maximum period of 6 months from the date 3.5.3of.prCepaalribatriaotni.on.Standards ' Prepare seven LOMS/MS mixed calibration standards in methanol via dilution of the 1.0 pg/mL fortification solution. This is a'typical example; additional concentrations may be prepared as : needed. ' ' Initial Cone. (pg/mL)` Volume (mL) Diluted to (mL) Final Cone, (ua/mLl . : 1.0 5.0 ` 100 0.05 : 1.0 ' 2.5 . 100 0.025 1.0 1.0 100 0.01 0.05 10.0 100 . 0.005 0.025 10.0 100 ' 0.0025 0.01 . 10.0 - 100 0.001 ' 0.005 10.0' 100 . 0.0005 'eachofEt-FOSE-OH, Me-FOSE-OH, PFOS, POAA, and PFOSA Store all fortification standard solutions (in 125-mL LDPE bottles) in a refrigerator at 2 C to 6C for a maximum period of 6 months from the date of preparation. ' 4. METHOD 4.1. FLOWDlAGRAiM The flow diagram of the method is given below, followed by a detailed description of each step. . . . . . . 5 g of matrix. Fortify :ifneeded i ' Extract with ACN ' i _ . SPE column clean up :i . . Concentrate to almost dryness .. ` 4- ' . i. '. 1Adjust final volume LC/MS/MS analysis ' ItCentre Analytical Laboratories. Inc. Study 023-001 Page 12 o f 69 Centre Analytical Laboratories, Inc. ' Page 24 of 81 Centre Analytical Laboratories, Inc. Page 90 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OQP-023-057 4.2. Sample Processing For apple, green beans, bread, hot dog, pork, chicken and catfish, place frozen samples in a Hobart food processor and homogenize with dry ice. Then place samples in polyethylene containers and leave open in frozen storage overnight to allow for CO: sublimation. Seal and place the samples in frozen storage below -10C unul time of analysis. For milk, shake sample container, transfer' to a polyethylene container, seal and place in frozen storage below -10C until time of analysis. For egg, place samples in a blender and homogenize. Transfer samoles to polyethylene containers,-seal and place in frozen storage below -10C until time of analysis. . . 4.3. Extraction - Note: A. Preparation/conditioning of SPE columns is described in Section 4.3.4. B. Silanization of pear-shaped flasks is described in Section 4.3.5. ' . C. Evaluation/standardization of SPEcolumns (if poor recoveries are obtained orwhen a different supplier is used etc.) is described in . 4.3.1.SeAcptipolne4,3G.6re.enBeans,B-read,HotDog,andPork ' a. Weigh 5 g of frozen sample into 50 mL disposable polypropylene centrifuge tube .(untreated control samples must be fortified at this point for determination of method recovery). .. - b. Add 30 mLof ACN, replace lid tightly, and shake on a wrist-action shaker for 15 minutes. Let the tubes sit for - 5 minutes, carefully decant, and transfer the extract on to a conditioned SPE column (see Section 4.3.4.). Collect the eluate in a 125 mL silanized pear-shaped flask (see Section 4.3.5.). ' ' Note: The 20-mL SPE column fits well inside the mouth (sleeve) of the . pear-shaped flask. Make sure that the pear-shaped flask is well supported and will not topple. No vacuum is needed for this step. ' c. Add 20 mL of ACN to the sample, replace lid tightly, and shake for 10 . minutes on a wrist-action shaker. Let the tubes sit for - 5 minutes, carefully decant and transfer the extract to the same SPE column. Collect the eluate into the same pear shaped flask and combine. d. Repeat Step c one more time. This time, centrifuge the tubes at - 2000 rpm for - 5 minutes before decanting the extract. Combine the eluate in the same pear-shaped flask as the original extract. Centre Analytical Laboratories. Inc. Study 023-001 Centre Analytical Laboratories, Incr Page 13 of 69 I ,_ Page 25 of 81 Centre Analytical Laboratories, Inc. Page 91 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 3-4e. Add drops of 1-octanol to the' extract in the pear-shaped flask and evaporate at reduced pressure using a rotaryevaporator (at <40C). f. Make the final volume, by adding 2 mL of 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. Transfer the extracts to ' . HPLC vials using disposable pipets. . . : 4.3.2.ChickenandMilk a. Weigh 5 g of frozen sample (allow milk to thaw and shake well before weighing) into 50 mL disposable polypropylene centrifuge tube (untreated control samples must be fortified at this point for determination of method recovery). . b. Add 30 mL of ACN, replace lid tightly mid shake on a wrist-action shaker ` for 15 .minutes. Place tubes in freezer (< ,10C) for -2 hours. If a stopping point is needed, samples; may be stored overnight in the freezer. Centrifuge the tubes at - 10,000 ipm for - 10 min. Carefully decant,"and transfer the extract onto a conditioned SPE column (see Section 4.3.4.) Collect the eluate in a 125 mL silanized pear-shaped flask (see Section 4.3.5.). ;" Note: The 20-mL SPE column-fits well inside th mouth (sleeve) of the pear-shaped flask. Make sure that the pear-shapd flask is well supported and will not topple. No vacuum is needed for this step. c. Add 20 mL of ACN to the sample, replace lid tightly, and shake the sample for 10 minutes on a wrist-action shaker. Place tubes in freezer (< -10C) for - 1 hour. Centrifuge tubes at - 10,000 rpm for - 10 minutes and carefully decant and transfer the extract to the same SPE column. Collect the eluate into the same pear shaped flask and com bine. d. Pass 20 mL ACN through the .SPE column and combine eluate in the same pear-shaped flask. . . -= - e. `Add 3-4 drops of 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotao' evaporator (at <40C). 2%f. Make the final volume, by adding 2 mL of ascdrbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. Transfer the extracts to HPLC vials using disposable pipets. . Centre Analytical Laboratories, Inc. Study 023-001- J Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 14 of 69 Page 26 of 81 Page 92 of 151 BACK TO MAIN Centre Study No.: 023-057 .Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 4.3.3.EggandCatfish ' a. Weigh 5 g of frozen sample (allow egg to thaw and shake well before weighing) into 50 mL disposable; polypropylene centrifuge tube (untreated control samples must be fortified at this point for determination of method recovery). . ' ' . b. Add 30 mL of ACN, replace lid tightly and shake on a wrist-action shaker for 15 minutes. Place tubes in freezer (< -10C) for - 1 hour. Centrifuge 43.4.).tubes at - 2,000 ipm for - 10 minutes. Carefully decant, and transfer the extract on to a conditioned SPE column (see Section Collect the eluate in a 125 mLsilanized pear-shaped flask (see Section 4 3 .5 .). Note: The 20-mL SPE column fits well inside the mouth (sleeve) of the ' - pear-shaped flask. Make sure that the pear-shaped flask is well supported and will not topple. No vacuum is needed for this step. c. Add 10 mL of ACN to the sample, homogenize the frozen fat phase usin' a Tissumizer for - 30 seconds and rinse the Tissumizer with 10 mL ACN into the tube. Replace lid tightly, and shake the sample for 10 minutes on a wrist-action shaker. Place tubes in freezer (< -1QC) for - l hour. ' Centrifuge tubes at - 2,000 rpm for - 10 minutes and carefully decant, extract to the same SPE column. Collect the eluate into the same pear shaped flask and combine with eluent from initial food extraction.. d. 'Pass 20 mL ACN through the SPE column and combine eluate in the same pear-shaped flask. e. Add 34 drops of 1-octanol to the extract in the pear-shaped flask and evaporate at reducedpressure using arotary evaporator (at < 40C). - f. Make the final volume, by adding 2 mL of 2% ascorbic acid,in methanol to the pear-shaped flask and swirl to mix/dissolve. Transfer the extracts to HPLC vials using disposable pipets. . /. Note; The extraction procedures described in Sections 4.3.1, 43.2. and 4.3.3. are given as a general guideline for each matrix. The procedures in Sections 4.3.2. and 4.3.3. were developed as a result of poor recoveries (for Et-FOSE-OH and Me-FOSE-OH) and ' broad peak shapes (for Et-FOSE-OH) when using the procedure described in Section 4.3.1. If problems are encountered with any ' matrix, it is recommended to try the other two procedures. Centre Analytical Laboratories, Inc. Study 023-001 Centre Analytical Laboratories, Inc. ' Centre Analytical Laboratories, Inc. Page 15 of 69 . Page 27 of 81 \ i i Page 93 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No, 00P-023-057 .4.3.4.SPEColumnPreparation, ' .a. Pack 20-mL SPE tubes with (in sequence) 2 g florisil, 2 g silica gel, 2 <* carbon, and 1 g LC-NH2 (Note: the florisil should be activated at - 160C . for at least 24 hours before use). b. Wash the columns with 20 mL MeOH, then 20 mL ACN. Discard all . washes. Do not allow the column to dry. ` ' Note: The SPE columns may be packed and conditioned at any point. Use the vacuummanifold to condition the columns. 4.3.5.SilanizationofPear-ShapedFlasks- . DonotlettheSPEcolumnsjundryatanytime. . Pear-shaped flasks are silanized before use, by rinsing with a 30% diraethyldichlorosilane in toluene solution. Then rinse with toluene (lx) followed by methanol (3x). The flasks are driedcompletely before use, either by allowin'* to air dryor with a streamof nitrogen. 4.3.6.StandardizationofSPEColumns - Standardize the SPE columns before analyzing samples in the followin'* manner, especially when using carbon from a- different supplier than recommended:- ' ` a. Using a mixed standard with a concentration - 50 ng/mL (each' of the five analytes) follow the elution schemes as outlined in Steps b to d in Section 4.3.1. Collect all eluting fractions. ;. b. Collect a post-elution fraction, after Step d, eluting with 20 mL of 50/50 MeOH/ACN. '. c. Adjust the final volume of all the fractions to 2 mLw'ith methanol. ' d. Analyze all the fractions by LC/MS/MS.. _ e. If the target fraction contains a minimum of 85% of the respective analytes, it may beconsidered acceptable. f. If the "post-elution" fraction contains a significant amount (> 10%) of analytes, the target elution volume may be increased. . 4.4. Analysis by LC/MS/MS 4.4.1. LCMS/MSSystemandOperatingConditions(Electrospray) Mass Spec': Interface: Computer Software: Micromass Quattro U1tima (Micromass) Electrosptay (Micromass) Harvard infusion pump COMPAQProfessional Workstation AP200 Windows NT, MassLynx 3.3 ; Centre Analytical Laboratories, Inc. Study # 023-001' Page 16 of 69 Centre Analytical Laboratories, Inc. Page 28 of 81 Centre Analytical Laboratories, Inc. Page 94 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 HPLC: : Hewlett Packard (HP) Series 1100 HPQuatPump' HP VacuumDegasser HP Autosampler : HP Column Oven . . Note: A 4 x 10 mm hypercarb drop-in -guard cartridge (Keystone, part yif 844017-400) is attached on-line after the purge valve"and before the sample injector port to trap any residue contaminants that may be in the mobile phase and/orHPLC system. HPLC Column: Genesis Cs (Jones Chromatography), 2.1 mm x 50 mm 4li Column Temp.: 35 C . '- Injection Vol.: 10 pL Mobile Phase (A): 2 mM Ammonium Acetate in Type I water Mobile Phase (B): Methanol - Time 0 0.4 1.0 7.0 73 9.0 93 133 14.0 - %A 60 60 10 10 0 0 60 60 60 ' %J3 F lo w Ram fmT 40 40 ' 90 90 - .100 ' 100 0.300 0.300 0300 0300 0.300 0.400 ' - ' 40 . 40 . 40 ' 0.400 0.400 0.300 It may be necessary to adjust the HPLC gradient in order to optimize instrument performance. Columns with different dimensions (e.g. 2.1 mm x 30 mm) and also columns from different manufacturers (Keystone Betasil C etc.) can be used provided equivalent chromatography is obtained. ' . Ions monitored: Analvte Et-FOSE-OH Me-FOSE-OH PFOS POAA PFOSA Mode negative negative negative negative negative Transition Monitored 6c0 - 59 616 - 59 499.- 99 413 - 369 - 498 -> 78 Approximate Retention Time, (mini 6.7 6.7 5.3 5.1 : 6.0 On'a day-to-day basis, the retention times may vary slightly depending on the batch of mobile phase, etc. | - ~ Centre Analytical Laboratories. Inc. Study #023-001 '' Centre Analytical Laboratories, Inc. . j . ' i- . Paeel7of69 Page 29 of 81 . . Centre Analytical Laboratories, Inc. . PaSe 95 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 4.4.2. ExampleTunerileParameters Tne 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 forgreatest sensitivity. The mass spectrometer is tuned using a solution of each analyte at - 0.5 pg/mL, prepared via dilution of the stock solution in methanol. Tne solution ' is infused (using a T ' connector) at 10 pIVmin into a 0.2 raL/min stream of. mobile phase consisting of 40% methanol and 60% 2 mM ammonium acetate. The analytes are initially tuned for the parent ion and then tuned for the product ion. Once the instrument is tuned, the optimized parameters are saved as a "tune file". This tune file is then used during routine analysis. Arialvte Et-FOSE-OH : Me-FOSE-OH PFOS . POAA . ; PFOSA Dwell (si 0.2 - 0.2 0.2 0.2 0.2 Collision Energy (eVl 31 31 43. 11 28 Cone fVi "IT " 30 76 05 34 ' . , .' ' Source . . Capillary Hexapole 1 Aperture 1 Hexapole 2 Source Block Temp. Desolvation Temp. ' Set' 2.56 kV. 0.5V _ 0.2 V ` 0.8 V 100C 400C ` . . -. -' ` Analvzer LMResl HMResl Energy 1 Entrance Exit LMRes2 HMRes 2 TEnergy2 .Multiplier . : 7 Set 13.0 V 13.0V 0.7 V . -2 V IV 11.0 V 11.0 V 1.0 V 650 V ` . . ' . . Gas Flows 'Cone Gas D. eso|lvadon Pressures Gas Cell Set -150I7hr ~700L/hr Read back - 3.0e-3 mbar Centre Analytical Laboratories. Inc. Study 023-001 I Centre Analytical Laboratories, Inc. . Page 18of 69 ' Page 30 of 81 Centre Analytical Laboratories, Inc. Page 96 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 Note: An alternative LC/MS/MS system may be used once demonstrated to 4.4.3.CbaeleibqruaivtiaolenntP.rocedures ' ' a. Inject the same aliquot (between 10 to 25 jxL) of.each calibration standard into the LC/MS/MS. ' . b. Use linear standard curves for quantification. Linear standard curves are generated for each analyte by linear regression using i/x fit weightin'* of peak area versus calibration standard concentration using the Windows NT, MassLynx 3.4 (or equivalent) software system. Any calibration standards falling outside 30%, based on .of its calculated concentration must be excluded from the calibration curve; However, the total number . of calibration standards that could be excluded must not exceed 30% of the total number of standards injected >0.9925(R2>0.9S5).c. The correlation coefficient (R) for calibration curves generated must be If calibration results fall outside these limits, then appropriate steps should.be taken to-adjust instrument operation, and the relevant set of samples must be remalyzed. - . . ' 4.4.4.SampleAnalysis a. Inject the same aliquot (between 10 to 25 jiL) of each sample, fortification control etc. into the LC/MS/MS. b. Standards corresponding to at least six concentrations must be included in an analytical set. . c. Include an entire set o f extracted calibration standards at the bezinnm o f a sample set. Continuing calibration verification standards (CCVs) can be interspersed between at least every 8-10 samples. As an alternative, an entire set of standards interspersed every 3-4 samples can be included to account for a second set of standards. In either case, a standard or CCV should be the last injection in a sample set (see Section 4 .5 ). : . d. Each set of samples analyzed for investigation purposes should include at" least one reagent control (method blank) at least one matrix control and two matrix control samples fortified at known concentrations and carried through the procedure to verify recovery. , '. . Note: The analysis performed during method development included fortifications at 0.5,1.0,2.0,5.0 and 10 ng/g (ppb) for each analyte. iCentre Analytical Laboratories. Inc. Study 023-001 Centre Analytical Laboratories, Inc. Page 19 o f 69 Page 31 of 81 Centre Analytical Laboratories, Inc. Page 97 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 e. The concentration of each sample, fortification, control etc. is determined from the standard curve, based on the peak area of each analyte. The standard responses should bracket responses of the residue found in each sample set. If necessary, dilute the samples and re-analyze to give a response within the standard curve range. .` f. Fortifications that bracket the highest residue expected in each treated matrix will be included with each sample set If residues are found outside these limits, additional fortifications will be included in a subsequent sample set to establish that method recoveries are available for all analytes of interest at concentrations exceeding those in treated samples. g. Fortification recoveries falling within 50 to 140% are considered ' acceptable for fortifications at the LOQ level and 60 to 130% for fortifications at levels greater than the LOQ. - h. Samples or controls in which no peaks are detected (i.e. signal : noise ratio < 3 :1) at the corresponding analyte retention times will be reported as ND (not detected). Samples or controls in which peaks are detected at the: corresponding analyte retention times -that have a quantification ' concentration of zero or a negative number will be reported as NQ (not quantifiable). Otherwise, all positive numbers will be reported. _ ' i. If samples are not loaded on the instrument to be analyzed the day thev are extracted, samples will be stored refrigerated at approximately 4C until analysis, and analyzed preferably within a week. v Recoveries from method development for all matrices can be found in Tables - 1-9. Lower recoveries for Me-FOSE-OH and broad peak shapes for . Et-FOSE-OH have been observed in some animal matrices (chicken, pork and chicken egg). - . 4.5. P erformance Criteria. ... The following two criteria should be met before the initial analysis of samples,' especially when using different instrumentation set-ups than those cited in this method. ' First Criterion - Inject a standard solution on LC/MS/MS corresponding to the estimated LOQ (1.25 ng/mL) and obtain a signal to noise ratio of at least 9:1 relative to the reagent blank. If this criterion cannot be met, optimize and change , 1" * instrument operating parameters. '# ' $Centre Analytical Laboratories, Inc. Study 023-001 ? I; Centre Analytical Laboratories, Inc. Page 20 of 69. Page 32 of 81 Centre Analytical Laboratories, Inc. Page 98 of 151 BACK TO MAIN Centre Study N.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 Second Criterion - Inject a set of standards of six or more concentration levels ranging from at or below the LOQ, up to the highest concentration level to be included in the analysis. Generate a calibration curve for each analyte and obtain a 1/x weighted linear regression with a coefficient of determination of at least - 0.985 for each analyte. ' 4.6. T im e R equired fo r A nalysis . . A set of 14 samples can be taken through the extraction procedure in approximately six hours by one person. The LC/MS/MS analysis (8 -10 standards ' and 14 samples) will take approximately 6 hours. 5. CALCULATIONS . Calculate the amount of analyte found (in ng/mL, based on peak area) using the standard curve generated by the Mass Lynx software program using Equation 1. Equation 1 *. - '. Analyte found (ng/mL) = (beak area - intercept) ; slope 5.1. C o m po n en t.Resid u e C oncentration . . Determine the component residue concentration using Equation 2 . Equation 2 : . Residue found (ng/g) (analyte found (ng/m L) X D F x FV (m L)) sample weight (g) Where DF = dilution factor andFV = final volume ; ' Note: Residues found in the samples should be corrected to reflect the average amount of residue Of any) found in the matrix blank. If the amount of residue found in the reagent blanks is similar to the amount ' 'found in the matrix blanks then the average of the reagent and matrix blanks will be taken to achieve a more accurate number. If however, the matrix blanks have residues higher than the reagent blanks then . correct only for the matrix blank.1:. Also, when taking averages, consider anything reported as not. quantinable (NQ) as equal to zero. Centre Analytical Laboratories, Inc. Study #0234X11 r "tre Analytical Laboratories, Inc. Page 21 of 69 ' Page 33 of 81 Centre Analytical Laboratories, Inc. Page 99 of 151 '1 ' . ' BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 5.2. F o r t i f i c a t i o n R e c o v e r y . For samples fortified with known amounts of analytes prior to extraction -calculate the percent recovery from Equation 3. . .' Equation 3: Recovery (%)= '_ (na/g found- (averageng/g found in control)) ng/g added or (analyte found (ng/mL)-(average analytefound in control (ng/mLI/DF! x DFx FV ' sample weight (g)x amount analyteadded (ng/g) SAFETY . There are no unusual hazards associated with this method. The. analyst should . read the material safety data sheets for all reagents before performin'' this method . Normal laboratory precautions should be taken. ' Centre Analytical Laboratories, Inc. Study # 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 22 o f 69 Page 34 of 81 Page 100 of 151 BACK TO Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 ' TABLES Centre Analytical Laboratories. Inc. Study # 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 23 of 69 Page 35 of 81 Page 101 of 151 Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 BACK TO MAIN O Crr--sI Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc, Page 36 of 81 Page 102 of 151 Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 BACK TO MAIN Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 37 of 81 Page 103 of 151 Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 BACK TO MAIN Centre Analytical Laboratories, Inc. Study II 023-001 I ; ' Centre Analytical Laboratories, Inc. ! Centre Analytical Laboratories, Inc Page 38 of 81 Page 104 of 151 Centre Study No.: 023-057 .Centre Protocol No: 00P-023-057 . Centre Protocol No. OOP-023-057 BACK TO MAIN Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 39 of 81. Page 105 of 151 Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 BACK TO MAIN Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 40 of 81 Page 106 of 151 Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No.:OOP-023-057 t' cCON\ BACK TO MAIN Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc Page 41 of 81 Page 107 of 151 Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 BACK TO MAIN Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. I Page 4 2 of 81 Page 108 of 151 Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 BACK TO MAIN , Centre Analytical Laboratories, Ihc. Centre Analytical Laboratories, Inc. I Page 43 of 81 Page 109 of 151 acuire rrotocol No: 00P-023-057 Centre Protocol No. OOP-023-057___ BACK TO MAIN Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 44 of 81 Page 110 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 FIGURES Centre Analytical Laboratories, Inc. Study # 023-001 i' Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. -Page 33 o f 69 . Page 45 of 81 Page 111 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 i Figure 1 : Representative Chromatograms of a 0.5 ng/mli Mixed Calibration Standard of Et-FOSE-OH, Me-FOSE-OH, PFOS, P0.4A and PFOSA N a = a : 111133-5001 - t x z : o.s s g / s L STD 1C1108S9-7) Centre Analytical Laboratories, Inc. Study # 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. I I. 1 Page 34 of 69 ; Page 46 of 81 Page 112 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 ___ ___;Centre Protocol No. OOP-023-057 I' . Figure 1 (continued): Representative Chromatograms of a 0.5 ng'mL Mixed Calibration Standard of Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA and PFOSA Sana: 1'1199-Eooi ' 0.5 =s/al, S3) (C110899-7) 4: PCAA 0.5 nc/mL STD (C110S99-7) 111199-5001 Sm (SG, 2x2) 100-1 . 5.13 - 10120^ S12-Nov-1999 00:3 5 :5 3 ' ' LC/MS7MS unit MRM of 5 C hannels S- -413 >363 1.19e5 Area 1.00 2.00 ' 3.00 4.00 5.00 6.00 7.00 8.00 - Time 5 : ??CSA ' 0.5 ng/ml STD (C1108S9-7) 111199-5001 Sm (SG, 2x2] lOOi : ' 6.08 3404^ 12-N ov-1999 00:35:5ff LC/MS(MS unit 6I MRM of 5 C hannels E3 - 498 > 78 .. 3.2294 Area 1.00 2.00 3.C0 4.00 5.00 6.00 7.00 8.00 T im e Centre Analytical Laboratories, Inc. Study * 023-001 'i " Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 35 of 69 Page 47 of 81 Page 113 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 Figure 2: Representative Chromatogram; of a Control Whole AddIcSim 1 111799 Apple, Centre ID 983732 Apple Blank A) e (St b*3: 1117 9 3 -2 11 33 8333773322 AX npnp`l e B l a n k A Centre Analytical Laboratories. Inc. Study 023^001 J Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 36 of69 Page 48 of 81 Page 114 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-57 Figure 2 (continued): Representative Chromatograms of a Control Whole Apple Sample (Set 111799 Apple, Centre ID 9S3732 Apple Blank A) Hane: 111799-211 . Taxi; 983732 Apple alai* 4: POAA ' 983732 Apple Blank A 111799^211 Sm (SG, 2x2) ' 1.C0n . 5.03' 2087 17-Ncv-1999 20:17:29* L C /M S/M S unit~6 MRM of 5 C hannels ES- 413 >359 3.01 e4 Area 1.00 ' 2.C0 ` 3 00 4.00 5,00 6.00 7.CO aI 9837 32 Apple ElankA _ ' ' 17-Nov-1999 20:17:29 i LC/MS/MS unit 6 1117 39-211 Sm (SG, 2x2) ' .g MRMof 5 C hannels E3- 100- 7.93 498 > 73 ' 2.72 e 4'09 4.24 5' 1'4--25 j 6-.246.53 7.47 1 728 o/a/. fe jf e . 1 b o ' 200 3-bo 4.00 5.00 6.C0 7.0 H b o ' Centre Analytical Laboratories, Inc. Study #f 023-001 Centre Analytical Laboratories, Inc. ; Centre Analytical Laboratories, Inc. . Page 37 of 69 - 1 j' Page 49 of |81 Page 115 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 I figure 3: Representative Chromatograms from the Injection of Control Wh i Apple Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH P F n s P n 7 , PFOSA (Set 111799Apple, Centre ID 983732Spk A) ' 5' P0AA,and N a a a : 111799-214 ' : T e x : 383722 S p i A, A pple 500 ppe S. Centre Analytical Laboratories. Inc. Study 023-001 Centre Analytical Laboi dories, Inc. Centre Analytical Laboratories, Inc. I Page 38of69 Page 50 of 81 Page 116 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 Figure 3 (continued): Representative Chroniatograms from the In iection nf r , ' , Whole Apple Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE OH Pirnc0"1TM POAA, and PFOSA (Set 111799 Apple, Centre ID 983732 Spk A )'' ' ^ M arne: 1 1 1 7 9 9 -2 H ' Taat: 583732 Spk A, Apple S00 ppt Centre Analytical Laboratories. Inc. Study # 025*001 ! Centre Analytical Laboratories, Inc. . Centre Analytical Laboratories, Inc. Page 39 o f 69 Page 51 of 81 Page 117 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 ` ........... -- - - Centre Protocol No..00P,023-05.7.... .. Figure 4: Representative Chromatograms of a Control Green Bean Sample (Set 111299 Beans, Centre ID 990S032 Green Beans Blank A) H a i : U 1 2 3 9 -6 0 1 1 - tx z ; 3908032 G raea B eans S ia s i * C-rOSZ-CH 990E 032 G reen B e a n s B lank A ' 1112S9-6011 Sm (SG, 2*2) 100 . `. . _ . . . %- . ' H 7 3.43 . . - _. I B -l 6f 12-N ov-1999 20:08:46 LC/M S/M S u n it 6 MRM of 5 C hannels ES 630 >59 w 7s - s-07 f f e 7- 1 3 7 B9 " 1-3963 ' 1.00 ' 2 00 . 3.00 2 : He-rOSE-OH 9908 032 G reen B ean s B lank A . 111239-6011 Sm [SG, 2x2) 100n o/ _ .' . .. 4.00 5.00 6.00 7.00 ' ann l `" `c . ' . _ 2131 . . .- d 12-N ov-1S99 20:08:46 LC /M S/M S u n it 6 MRM of 5 Channels ES '' . 616 >59 2.75e3 Area 1 0 ` 2 00 3.00 3: Pros " 9908 032 G reen B ean s Blank A 1112'19-6011 Sm (SG, 2x2) 100a -' . %- 4.00 1.-00 __ 6.00 :- 51J 24S4" . ' ______ _ --- ' 1 0 ' 2 00 ' 3.00 4.00 5.00 6.00 7.00 n no ' -. 12-N o v -1S99 20:08:46 LC/M S/M S u n it 6 MRM of 5 Channels E3- 499 > 99 2.25e4 Area 7.0 ' a.OO Centre Analytical Laboratories, Inc. Study If 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 40 o f 69 Page 52 of 81 Page 118 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 Figure 4 (continued): Representative Chromatograms of a Control Green Bean Sample (Set 111299 Beans, Centre ED9908032 Green Beans Blank A) B -aa : 111239-6011 9903032 G reen B ean s B la n k X ?OAA 9208032 Green Beans Blank A 111299-5011 Sm (SG, 2x2) 10th 5.05 4602 . 12-Nov-1999 2 0 :08:4 - LC/MS/MS unit L MRM of 5 Channels ES- 413 >369 ' 6.32S4 Area ________1.00 ' 2.00 ' 3.00 ' 4.0 0 ' ` 5.00 ' 6.0 ' 7 op 00 5 : PFOSA " S908032 Green Beans Blank A 12-Nov-1999 20:08:46 111299-6011 Sm (SG, 2x2) 1C " MRMLoCf 5/MCSh/aMnnSe unit ls ES 6 ' ' 498 > 78 647 j 0.23>0.-5~2 , l o - 2.08 --^7 3.50 4 694, S0 8.28 8.Ss 8,S5 ' 1.00 J 2.00 ' 3.00 ` 4.00 ' 5.i)0 ' 6.00 ' 700 ' m " 1 ' ,r ~Tin1e Coltre Analytical Laboratories. Inc. Study # 023-001 Ceituc .*--tytt-nl Laboratr ;s, Inc. Centre Analytical Laboratories, Inc. Pngi 41 of 69 Page 53 of 81 Page 119 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 ! Figure 5:- Representative Chromatograms-from the Injection of Control Gree Beans Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH PFOS POA a* a PFOSA (Set 111299 Beans, Centre ID 990S032 Spk B)- ' ' ' ,and Haaa: 111299-6015 .T e x t: 3908032 Spk B, G ra s s B ean s 500 p p t l: Et-rosr-m sso 8032 Spk B, Green Beans . 500 pr rpt 111 239-6015S m (S G .2x2) 10Ch : ~. .. ' a/Ot .. ' . ' 6.69 4200") ." I 1 r , ' ------------------ 12-Nov-1999 21:11:23 LC/MS/MS unit 6 i 5 Channels ES - 630 > 59 4.05e4 Area 0 --------------^ ----------------------------- -------- _ -------- arte-------- - w -------- ^00 B.nn ` .............. 2 : Me-?OS-OH stru t 032 Spk B, Green Beans 5 0 0 ppt : . -. : T r r ;-- -- -- -------- - I 2-M0V-1999 21:11:231 111239-5015 Sm (SG, 2x2) . -. ( LC/MS/MS unit 6 100- ` 6,53 * Channels E3- _- 3c55~| - - 616 > 5 9 ' -! 3.55e4 atOt ' i0- . . . . . --------.--i.uu : 2.UU - R nnj .uu -----*--.-u-u-----------c-.-'j-u-----------6-.-0-0----------7---n-n---- ' -- -- nm e P?OS - S908 J32 Spk B, Green Beans 500 ppt ^ ---------------i--, T2-NOV-1999 21:11:23! 11123 3-6015 Sm (SG, 2x2) 100- ' ' 528 32051 ' k^D,L<T/M S/M S U nit 6 MRM of 5 Channels S - 499 >99 '. 2.53e4 aJQt ~ J :ty- ----- .C0 2.00 3.00 4.00 5.00 6.00 ? nn ' 0 qq ' ' ' llms Centre Analytical Laboratories. Inc. Study # 023--001 Centre Analytical Laboratories. Inc. Centre Analytical Laboratories, Inc. Page 42 o f 69 I Page 54 of 81 Page 120 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 ------ .... CentrePromcol.Na.QQP-Q23_-057 tFigure 5 (continued): Representative Chromatograms from the Injection of Co 1 Green Beans Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH PFOS ^ f POAA, and PFOSA (Set 111299 Beans, Centre ID 9908032 Spk B) ' ' ' Nana: 111253-5015 ' * . Text: 3908032 Spk B, Green Beans 5ff0 ppt 4: POAA ' 5903032Spk B, Green Beans 500ppt . - 1112S9-6015Sm (SG,2x2) 10O . 5.07 27512 ' - 0//O ' 12-Nov-l299 21:11:23 LC/MS/MS unit 6 MP`M of5ChannelsE5413>369 ' . 3.49e5 Area 0' 1.00 2.00 . 3.0 4.00 5: ?rOSA . ** 5908032 Spk B, Green Beans 500ppt 1112!:S-5015Sm {SG,2x2) 10On * . ` 0//O_ 5.00 -6.00 ' 6.C5 102C4" bn7 ' finn"-- . nme ` - 12-Nov-1S99 21:11323 LC/MS/MS unit 6 MRM of 5Channels93 '498>78 9.S6e4 . - Area - 1.00 ` '2.00 ' 3.C0 4.C0 .00 6.C0 7.00''" Rnn ' Time Centre Analytical Laboratories, Inc. Study # 023-001 Centre Analytical Laboratories, Inc.. Centre Analytical Laboratories, Inc. ' Page 43 of 69 Page 55 of 81 Page 121 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: OOP-023-057 Centre Protocol No. 00P-23-057 ' Figure 6: Representative Chromatograms of a Control Chicken Egg Sample (Set 122999 Egg, Centre ID 9912031 Egg Blank A) N an a: 122999-1011 T bx c: 9912031 Zgg B la n k X Centre Analytical Laboratories Inc. Study # 023-001 *. I - Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 44 o f 69 ! 1 Page 56 of 81| Page 122 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 Figure 6 (continued): Representative Chromatograms of a Control Chick r Sample (Set 122999 Egg, Centre ID-9912031 Egg Blank A) N ana; 12JS 99-1011 S912031 SSg M a sk A i C-ntrc Analytical Laboratories. Inc Study 3 023-001 Centre Analytical Laboratories, Inc. . D ' ., . i| ra g e 45 o f 69 . J Page 57 of 81 Centre Analytical Laboratories, Inc. Page 123 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: OOP-OIS^OS? .Centre Protocol N,00M 23-057 ____ Figure 7: Representative Chromatograms from the Injection of Control Chi k Egg Fortified at 500ppt each ofEt-FOSE-OH, Me-FOSE-OH PFOS pda e"> PFOSA (Set 122999Egg, Centre ID 9912031 SpkB) > UAA,and Koena: U 2 S 9 S -1 0 1 S SS12031 Spk B Big 500 ppc 1: Et-rosg-QH 1 ____'_____ S912031 S p k B Egg 500 ppt 122999-1015 Sm (SG, 2x2) 10Oi 1.00 . 2.00 3.00 7: Me-?osv-n 9912031 Spk B Egg 500ppt 122933-1015 Sm (SG, 2x2) ICO-i ' ' ^ c^ssg 19:18:19 6.37 MRM of 5 Channels ES1107 630 > S 9.SSe3 . . " - Area 683430-- 29-D ec-1S99 19:18:19 MRM of 5 Channels ES ' 616 >59 7.19e3 A re a .1 .0 0 2.00 3.00 4.00 5.00 6.00 7.00" ` a n n T ^ Time 3: ' Pros 9912031 Spk B Egg 500ppt 122993-1015 Sm (SG, 2x2) 100i ` 539 4971' 29-D ec-1999 19:18:1s1 MRM of 5 Channels ES I . 499 > E: 6.58e-i Area. 1.00 ? 00 3.00 4.00 5.00 6.00 7.00 A n n ' ` 9|,00 Time Centre Analytical Laboratories, Inc. Study # 023-001 j Centre Analytical Laboratories, Inc. !' Centre Analytical Laboratories, Inc. . Page 46 of 69 - Page 58 of 81 . Page 124 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. OOP-023-057 Figure 7 (continued): Representative Chromatograms from the Injection of Control ' Chicken Egg Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH PFOS POAA, and PFOSA (Set 122999 Egg, Centre ED9912031 Spk B) ' ' N ana: 122939-1015 a x t : 9 9 1 2 0 3 1 Spie B Z g g 5 0 0 j p t 4: PfO SA 9912 031 S p k B Egg 5 00 p p t 122959-1015 Sm(SG, 2x2) 100 - ; - ' %- - . 5.85 6336" ` 29-Dec-1999 19:18:19 MRM of 5 C hannels ES 498 >78 ' 7.51e4 Area o ; 1-bo 2.00 3.00 4.00 5.00 6.00 .. 7.00 finn ' n f,n ' ' ' ' m l e - 5 .- POAA 9912031 Spk B Egg 500 ppt 122999-1015 Sm (SG, 2x2) ' 29-Dec-1SS9 13:18:' MRM of 5 C h n r '- 's E A re . Centre Analytical Laboratories. Inc. Study ft 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 47 o f 69 Page 59 of 81 Page 125 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Cntre Protocol No. .00P-023:Q57 i Figure 8: Representative Chromatograms of a Control Chicken Muscle Sample (Set 111999 Chicken, Centre ID 9910292 Chicken Blank B ). tana: 1 1 1 9 9 9 - S 1 2 ex e: 9910292 C h ick en . B la n k B 1 : E E-FO SE-C H - . 991C 2 9 2 C h ic k e n B la n k B '1119 39-612 Sm (SG, 2x2) - .. ' 10O- o/Or j 53 1.80 . ' ` `` ' . .I 1 . ' 1. 1 6.40 5.32 6.11 . 20-Nov-1999 03:41:4C . LC/MS/MS u n it 6 MRM of S Channels ES630 > 59 1.1Se3 o - <2 " 1.00 ' 2 00 ' 3.00 2 : M e-FOSE-O H . 292 C hicken Blank B - t o 99-512 Sm (SG , 2x2) - ' - 4.0 5.0 6.C . -. 6.Go 1371 : 7-00 fl.bo " "q nn`,,,= 20-N ov-I 999 03:41:40 LC/MS/MS u n it 8 - MRM of 5 Channels ES 616 > 59 . 1.7253 Area - V ____ 9 to - . ,, ' 1 0 0 ' ? 00 ' 3.CO 4.00 3: PFOS 9910292 C hicken Blank B . 5.00 1119.39-612 SrnfSG, 2x2) 100n ' 4._/3 - 4401 % 6.00 1 7 m " r ' 1 . i i ime 7-00 8.00 P nn ' 2 0 -N o v -I 9 9 9 0 3 :4 1 :40i LC/MS/MS u n it MRM of 5 C hannels S499 > 99 ' 2003 " Area ' 1 bo ' 2 bo ' 3.00 4.0 5.00 6.00 '00 8.00 9 00 j . Centre Analytical Laboratories. Inc. Study # 023-001 i* . Centre Analytical Laboratories, Inc. . Centre Analytical Laboratories, Inc. Page 48 of 69 I Page 60 of 81 Page 126 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol-No-.00P-023-Q57-__ - Figure 8 (continued):. Representative Chromatograms of a Control i-v -i Sample (Set 111599 Chicken, Centre ID 9910292Chicken BlarSc B) ^>, 111999-S 2 ' T e x t: 9910292 C k ic k ea B la n k B Centre Analytical Laboratories. Inc. Study 023-001 Centre Analytical Laboratories, Iric. Centre Analytical Laboratories, Inc. != 49 of 69 Page 61 of 81 Page 127 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 " ------ - .Centre Protocol No. OQPrQ23-057 I Figure 9: Representative Chromatograms from the Injection of Control Ch' v Muscle Fortified at 500 ppt each of Et-FOSE-OH,Me-FOSE-OH PFOS p m a" and PFOSA (Set 111999 Chicken, Centre ID 9910292 Spk A) ' ' AA' Naaa: l l l s s s - s n ' Ta3ct: SS10232 S p k A , C S i c s n S 00 p p t 1 : t-?O S r-n w 9910292 Spk A, C hicken 500 ppt 111299-S14 Sm (SG, 2x2) 10<h - 6.23 "4S79~j 20-Nov-1999 04:13:01 LC/MS/MS unit S MRM of 5 Channeis ES- 630 > 9 . 4.36e4 Area 9910292 Spk A, Chicken 500 ppt 111S99-614 Sm {SG, 2x2) 10Oi . 6.05 3213^ 2?Nv7l999 04:13:0lj ' LC/MS/MS unit G MRM of 5 C hannels ES ' 616 > 59 ' - 3.02e4 . Areal 1.00 2.00 3.00 3 :.eros 9910292 Spk-A, Chicken 500 ppt. 111993-614 Sm (SG , 2x2) 100a . 4.34 22971 20-N ov-1999 04:13:011 LC/MS/MS unit 6 MRM of S Channels ES . 499 > 99 . 2.15e4 ' Area A 1.00 2.C0 3.00 4.00 5.00 6.00 7.00 8.00 urne 9.00 Centre .Analytical Laberetches. Inc. Seedy if 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 50 o f 69 Page 62 of 81 Page 128 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 tFigure 9 (continued): .Representative Chromatograms from the Injection nf r 1 Chicken Muscle Fortified at 00 ppt each of Et-FOSE-OH, Me-FOSE OH ptrnc ^ ^'POAA, and PFOSA (Set 111999 Chicken, Centre ID 9910292 Spk A) ' ' Na= = 111999-SU . . Taxa: 9910292 Spk X, Ctiekan 500 ppt 4: PCAA . - *910292 Spk A,'Chickeri 500 ppt 111999-514 Sm 100- * . 4.69 19253 %* . . ` _ . u-nov-1999 04:13:01 t-C/MS/MS unit 5 MRM of S Channels HS413 >369 2.42e5 ' Area (X S : PFOSA 1.0 ` 2.00 ` 3.00 ` 4.00 ` 5.00 ` B.bo ` 7 nn ' " ;---------------------- " ------------------------------------------ "-- `------------ - " '1 Time o00 Q nn 10292 Spk A, Chicken 500 ppt ~ - 2 ^ m =S9 4H H 111999-614 Sm (SG . 2x2)- in o , ' - O40/. Ch L l_ 1.00 2.00 '' ' 3.00 4.00 . 5.89 * 10023" Unil 5 MHM of o Channels E3- - 498 > 78 9.6ce4 Area i JL ' 5.00 - ^ 2 9 -------- L 2 2 J Z ~ M r ^ T n 7 me Centre Analytical Laboratories, Inc. Study 023-001 Centre Analytical Laboratories, Inc: Centre Analytical Laboratories, Inc. Page 51 o f 69 Page 63 of SI Page 129 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No.-00P.-023r052._ . Figure 10: Representative Chromatograms of a Control Cow Milk Samnle (Set 110999 Milk, Centre ID 972627 Milk Blank B) ; .` t:s u ; 1 1 0 9 9 9 -2 0 1 2 -a :: H ill 3 1 aa l B 3: P;0S Milk Blank B . . 110SS9-2012 Sm (SG, 2x2)' 1CO- JOt - . 1074] .' 1.00 ' 2.M ' 3.00 . 4X0 J 5X0 U9-NOV-1S29 2 0 :4 3 :5 a ` LC/MS/MS unit 6j ' MRMofS Channels ES. 499 > 99 . 1.39e4 Area ______________ 6.00 7.0 ' 6(30 ' l""c 1CentreAnalyticalLaboratories, Inc.Study 023-001 Centre Analytical Laoorau Ir.c. . Centre Analytical Laboratories, Inc. I Page 52 of 69 | 'i Page 64 of 81 ; Page 130 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 - ----------- Cntre ProtocoLNo. 0nP-QTV0S7 Figure 10 (continued): Representative Chromatograms of a Control Cow \Iiik Sample (Set 110999 Milk, Centre ID 972627 M ilk Blank B) ` Nana; 110999-2012 . T t : Hi.l i S l a c k S Centre Analytical Laboratories, Inc. Study if 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. 1 Page 53 of 69 i .- j Page 65 of 81 Page 131 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 , ........ r * ------------ Centre.Pt6tocoLNo.-00P-(ra-nr7 ' ; _ '' `* '' I Figure 11: Representative Chromatograms from the Injection of Control Cow v n t Fortified at 500 ppt each ofEt-FOSE-OH, Me-FOSE-OH, PFOS, POAA and PFOSA' (Set 110959 Milk, Centre ID 972627 Spk A) '' N asia: 1 1 0 9 3 9 -2 0 1 4 ' ' T e x t: 972627 S p iia X, M ili 500 p p t 1 : Et-FO SE -O H S72f'27 Spike A, Milk 500 ppt 110S99-2014 Sm (SG, 2x2) 'roo . " ' . /O/ '' ' 6.50 . ' '409S*1 I . I OS-Nov-1999 21:15:21 LC/MS/MS unit 6 MRMo(5 Channels E3- ' 630 > 59 . 3.54e4 Area 0- 2_: Me-?0S2-0H ` 372527.Spike A, Milk 500 ppt 1109S'3-2014 Sm(SG, 2x2) lOfr / .' Of0_ ' * 7-bo ' finn ' . 6.43 35121 OS-Nov-1999 21:15:21 LC/MS/MS unit 5 MRMof5 Channels ES ' . . 616 >59 3.45e4 ' Area Ch 1.00 ` ZOO ` 3.(30 ' 4.bo ` S.bo ' 6.00 ' 7.00 ~ s o n " ' ,Time 3: gros 9 /2 6 27 S p ik e A, Milk 500 p p t 1109 99-2014 Sm (SG. 2x2) 100- ' 0/O/ . . 5.03 - . 3438' : - US-Nov-1999 21:15:21 LC/MS/MS unit 6 of 5 Channels ES " 499 > 99 3.03e4 Area 0^____ _ ' __ _ t . 1.0 2.bo 3.bo 4.0 a.bci 6.00 7 bn ' gnn ' ' Centre Analytical Laboratories, Inc. Study If 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. .Page 54 of 69 I Page 6 6 of 81 Page 132 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No.-OOP-023-057 Figure 11 (continued): Representative;Chromatograms from theInjection of Control Cow M ilk Fortified at 500 ppt each ofEt-FOSE-OH, Me-FOSF n n p rn e POAA, and PFOSA (Set 110999 Milk, Centre ID 972627Spk A) '' N a n a :.110999-2014 .. 972627 S p ik e X, M ilk S00 p p t 4 : POAA S72S27 Spike A, Milk500 ppt 110999-2014 Sm (S u, 2x2) 10th . . Prosa 972627 Spike A, Milk 500 ppt 110999-2014 Sm (SG, 2x2) - 1C<h - 09-NoV-19S9 21:15:21| LC/MS/MS unit 6 MRM of 5 Channels E S :' 413 >359 ' ' 3.04e . Area OS-Nov-1999 21:15:21 LC/MS/MS unit 6 MRM of 5 Channels ES . ' ' ' 498 > 73 8.6Se4 Area Centre Analytical Laboratories. Inc. Study S 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. ! Page 55 of 69 i i Page 67 of 81 Page 133 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 , Centre Protocol No.J)0P:023-057 Figure 1 2 : Representative Chromatograms ofa Control Bread Sample (Set i n q q o Bread, Centre ID 9908031 Bread Blank A) ....... H a aaj 111399-511 T a x t: 9908031 B raad a l a r i A se-rosa-r_______ S908031 Bread Blank A 111999-511 Sm (SG,2x2) ioth 19-NoV-l9sg 20:21:5: LC/MS/MS u n it,, MRM o( S C hannels ES- 630 > 59| . 1.31e3 Area :-V~A 1.00 2.00 3.00 4.00 5.00 6.00. ' 7 C 0 ^ T ^ 7 ~ T7^ rr'` TTime 2: Me-?on:-OH ------------- :----------- .00______9.00 9908031 Bread Blank A 111999-311 Sm {SG, 2x2) iOOi ' ' 19-Nov-1999 20:21:5; LC/MS/MS unit i MHMof5 Channels E5- ' 616 > 5 9 ' ` 1.61e3 . . Area 1.00 ZOO 3.00 Pros_____ 4,00 5.00 6.00 7.00 ~2 * ' 1J 1i Time J*-00 QQO 9308031 Bread Blank A 111S99-511 Sm (SG,*2x2J 100y 4.73 296 19-N om S 9 9 20:21:581 . LC/MS/MS unit 6 MRM Of 5 Channels ES . 499 > 99 S.03e3 ' Area Centre Analytical Laboratories, Inc. Study " 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 56 o f 69 : ' Page 68 of 81 Page 134 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 ----------------Gentrg-Pmtocal-JJn. finP.n-nj1S7 figure 12 (continued): Representative Chromatograms of a Control Bread 9 i .(Set 111999 Bread, Centre ID 9908031 Bread Blank A) . sample Sana: 111599-511 . M t 9908031 B read B la s t X i: PCM . S90031 Bread BlankA 111959-511 Sm(SG, 2x2) 100- . /o .' ' . 4.67. 2233 ' . 1--NOV-1999 0:21:58 LC/MS/MS unit 6 MRMof 5 Channels ES . 413 >369 . 2.98e4 Area 0^ 1.00 2.0 3.0 5 : TTOSk 9908031 Bread Blank A 111999-511 Sm (SG, 2x2) . Of100/O" ' . 4.00 5.00 6.00 7 . b r ^ . 0 o " ' .o h iime l9-Nov-1999 20:21:53 " 5 >7 S'f5 LC/MS/MS unit 6 MRMof 5 Channels ES- 717 ` a-ss>4S8>78 - 1-bo 2.00 3.0 4.00 5.00 6.00 . 7.00 ` am `' " ! Centre Analytical Laboratories. Inc. Study ? 023-001 'I . Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. P ige 57 o f 69 Page 69 of 81 Page 135 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 ................- -- - Centre Protocol H o . QQP.-.023d25Z Figure 13: Representative Chromatograms from the Injection ofControl Bread Fortified at 500. ppt each of Et-FOSE-OH, Me-FOSE-OH, PFOS, POAA and PFOSA (Set 111999 Bread, Centre ED990S031 Spk-A) '' Kama: li! 3 S 9 - 5 1 i Txt * 9908031 SpJc 1 , B read 500 p p t 1: Zt-FOSZ-OH 290 8031 S p k A, Bread 500 p p t 111S99-514 Sm (SG, 2x2) 100 04/0 . . ' i s - iNov- 1 S 9 9 2 1 :0 9 :0 4 " LC/M S/M S u n it 6 6.24 MRM of 5 Channels E s ' 411s i ' ; 630 > 59 j 3.44e4 I . . Area 01.00 2.00 3.00 4.00 .5.00 2: Me-rOSZ-OH ud S pk A, Bread 500 p p t ; 6.00 1119 S-514-Sm (SG, 2x2) 10O -. 6.09 04/0* c4 L 1.00 2.00 3.00 3* P?0S 2 9 0 8 331 S p k A, B read 500 p p t 11199 9-514 S n (SG, 2x2) 100r ; . 0/O/ _ 1 4.00 5.00 6.0 ^ 1938" . . 7 nn ` ,, 1." " ^ .e . 12-NOV-1999 21:09:041 . LC/M S/M 3 u n it 6 MRM of 5 C hannels 5 3 616 >59 3.37e4 . . Area 7.00 ' 1"" = - _ --------- ly-N ov-1999 21:09:04 LC/M S/M S u n it 6 MRM of 5 C hannels S 499 > 99 ' 2.05e4 Area 1.00 2 00 3.00 ' 4.00 5.00 6.00 7.00 n o ' Centre Analytical Laboratoires, Inc. Study " 023-001 entre Analytical Laboratories', Inc. Centre Analytical Laboratories, Inc. Page 58 o f 69 Page 70 of 81'. Page 136 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 : Centre Protocol No. 00P-023-057. figure 13 (continued): Representative Chromatograms from the T,, ' Control Bread Fortified at 500 ppt each of Et-FOSE-OH M e-fn cro il-0" f FOAA, and PFOSA (Set 111999 Bread, Centre ID 9908031 Spk A.) ' S an a: 111999-5U . T e x t : 9 9 0 8 0 3 1 S p i A, B ro d 5 0 0 p p t Centre Analytical Laboratories, Inc..Study # 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 59 o f 69 Page 71 of 81 Page 137 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 ' ' Centre Protocol Nor-OOP-023^ 5 7 - Figure 14: Representative Chromatograms of a Control Hot Do Sanmle fSet 110899 Hot Dog, Centre ID 9908035 Hot Dog Blank B) rta a a : 110899-1012 E o t D o g H z t r i n B la n k B. Centre Analytical Laboratories. Inc. Study 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 60 o f 69 Page 72 of 81- Page 138 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 ...... -- ------------- r Centre PmtocollicL.QDt021-057 aFigure 14 (continued): Representative Chromatograms of a Control Hot D Sample (Set 110899 Hot Dog, Centre ID 9908035 Hot Dog Blank B) Centre Analytical Laboratories, Inc. Study # 023-001 . Centre Analytical Laboratories, Inc.. Centre Analytical Laboratories, Inc. Page 61 of 69 Page 73 of 81 Page 139 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 0QP-023-057 Centre ProtoeoiNch-Q0P-Q23-57_____ I figure 15: Representative Chromatograms from the Injection of Control w t n Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH, PFOS POAA h S PFOSA (Set 110899 Hot Dog, Centre ID 9908035 Spk B) ' ,and H s u : 110839-1015 T a x i; H ot Dog 500 p p t S p ik a B r1: Et-rosE-oH___________ _ Hot Dog 500 ppt Spike B 9908035 Spk B ' 110899-1015Sm (SG,2x2) 10CH ' '- 08-Nov-1929 2 0 :00:20 LC/MS/MS unit 6 MRMof 5 Channels ES . 630 > 59 Hot Dog 500 ppt Spike B 9908035 Spk B - 08-NOV-1999 2 0 :00:20 110899-1015 SmfSG. 2x2) . LC/MS/MS unit 6 100-1 -. 6.63 MRM of 5 Channels E5- 36841 ' 616 >59 1' . 3.464 Are: 6.001.00 2.00 3.00 4.00 5.00 7.00 - Time Hot Dog SOO.ppt Spike B . 9908035 Spk B 110899-1015 Sm(SG, 2x2) 100-1 ' L. 1.00 2.00 3.00 4.00 s -18- I .. j. 5.00 6-00. U8-N 0V -909 2 0 :0 0 :2 0 LC/MS/MS unit 6 MRM qf s C hannels E3- 499 > 99 . ' . 3.11e4 Area 7 .o T ~ T~ 7 i ^ ' ^ I Court Analytical Laboratories, Inc. Study It 023-001 Centre Analytical Laboratories, Inc. Page 62 o f 69 Page 74 of 81. j i i I Centre Analytical Laboratories, Inc. Page 140 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No.-OOP-023-.O57_ Figure 15 (continued): Representative Chromatograms from the Injection of Control Hot Dog Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH PFOS POAA, and PFOSA (Set 110899 Hot Dog, Centre ID 9908035 Spk B) ' ' N aa*; 110899-1015 - e t : H ot q 500 p p t S p iia 3 Hot Dog 5 0 0 ppt Spike B 9908 0 3 5 Spk B 110899-1015 Sm(SG, 2x2) 100 %*" . ' 5.03 27302" . - . .. ' 0 8 -f jo V.-] o S g 2 0 :0 0 :2 0 , LC/MS/MS unit 5 MRm of 5 C hannels ES ' .. 413 >359 3.33e5 Area 0----- 1.0 2.00 3.0 4.0 5.0 6 00 7 nn ' ,, j, ~ - Time Hot Dog'500 ppt Spike B 2908035 SpkB 110899-1015 Sm{SG, 2x2) 10(H ' 6.Q5 991^ 08-Nov-1999 2 0 :00:20 - LC/MS/MS unit 6 MRM 015 C hannels ES- 498 > 78 8.41 e4 . Area 0-- 6.001.00 2.00 3.00 .4 .0 0 5.00 7.00 8.00 Time Centre Analytical Laboratories. Inc. Study # 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. ! i Page 63 o f 69 ' ..Page 75 of 81 Page 141 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No.-GGP-02-3-05X Figure 16: Representative Chromatograms of a Control Catfish SamDle K m rtiojnn Catfish, Centre ID 9912032 Catfish Blank A) P (iet 012400 H aaa: 012400-1011 7 a x t: s j 12032 C a e iis h B la n k A . '. ' Centre Analytical Laboratories. Inc. Study # 023-001 Centre Analytical Laboratories, Inc. . -. i j i 1 ! Centre Analytical Laboratories, Inc. Page 64 o f 69 . : Page 76 of 81 Page 142 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 - Centre Protoeol-N,flQP.022-057. Figure 17: Representative Chromatograms from the Injection of Control c tr h Fortified at 500 ppt each of PFOS, POAA, and PFOSA (Set 012400 Catfich r 'S. ID 9912032 Spk A) ".Centre N aaa: 012400-101 . S e r e : SS12032 S ;k X , C a t f i s h 5 0 0 -p p t 1: PFOS ' 9912032 Spk A, Catfish 500 ppt . 012400-1014 Sm {SG, 2x2) 100i 5.24 - 13095^| 24-Jan-2000 20:13:1ll LC/MS/MS unit 6 MRM of 5 Channels ES429 > co . 1.00el Area 1.00 ZOO -3.00 9912 032 Spk-A, Catfish 500 ppt 0124100-1014 Sm (SG, 2x2) 100- .' o/Q/~ o-! 1.0 ZOO 3.00 4.00 5.00 6.00 7.00 8.00 ; Time 24-Jan-2000 20:13:11 ' 5.05 . . ' ' ` . L.C/MS/MS unit 6 MRMof 5 Channels ES- 60714- ` ` 413 >359 I. 6.09e5 4.00 -5.00 6.0 7(W ' nn' ' '*` ` lll"li 99.12032SpkA,Catfish500 ppt 012400-1014 Sm (SG, 2x2) 100-1 5.70 14937^ 24-Jan-2000 20:13:11 ' LC/MS/MS unit 6 MRMof 5 Channels ES- ' 498 > 7 8 1.31e5 Area 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Time Centre Analytical Laboratories! Inc. Study ff 023-001 Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 65 o f 69 Page 77 of 81 Page 143 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 Figure 18: Representative Chromatograms of a Control Pork Muscle Samni re , 122199 Pork, Centre ED99117S3 Pork Blank B) ' P N aa! 122199-212 9911783 P o ri B la n k B ! Centre Analytical Laboratories, Inc. Study # 023-001 Centre Analytical Laboratories, Iric. . , Centre Analytical Laboratories, Inc. Page 66 o f 69 Page 78 of 81 Page 144 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 -------- Centre Protocel-NW0QP-Q23=05.7____ Figure 18 (continued): Representative Chromatograms of a Control Pork Muscle Sample (Set 122139 Pork, Centre ID 9911783 Pork Blank B) . T s x t: S311783 P o ri. B lank B 4 : P'OSA___________ ;__________ S911783 Pork Blank B 122199-212 Sm (SG , 2x2) 100-1 5.89 ' 21-Dec-1999 18 :20:41 LC/MS/MS unit 6 MRM of 5 Channels ES ' 498 > 78 2.85e3 Area - A__ ' 1.00 2.00 3.00 4.00 5.00 6.00 7.00 . ' 8.00 * Time 5 ; PCAA 9911783 Pork Blank B 122199-212 Sm (SG , 2x2) IOO7 ' 21-Dec-1999 18:20:41 ' LC/MS/MS unit 6 5.07. 3738 MRM of 5 Channels ES413 > 363 4.81e4 Area 1.00 2.00 3.00 4.00 ' Centre Analytical Laboratories, Inc. Study #023-001 !. - ! Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 67 o f 69 Page 79 of 81. Page 145 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No...QQP-023-057 -_ Figure 19: Representative Chromatograms from the Injection of Control Pork Muscle Fortified at 500 ppt each of Et-FOSE-OH, Me-FOSE-OH, PFOS POAA and PFOSA (Set 122199 Pork, Centre ID 9911783 SpkB) . '' H aaa: 2222SS-225 ' T e x t: 3922783 Spk S , Pork 500 p p t 1: EC-FOSa-OH 9911783 SpkB, Pork 500 ppt 1221S9-215 Sm (SG, 2x2) Q0-} ' 1.00 2.00_ _ 3.00 2: Me-FOSE-OH_______ 9911783 Spk 3, Pork 500 ppt. 122199-215 Sm (SG, 2x2) 100n - 1.00 2.00 3.00 3; pros 9911 783 Spk B, Pork 500 ppt 1221 39-215 Sm (SG, 2x2) 100i . o4/_ 6.54 21-Dee-1999 19:07:39 ' LC/MS/MS unit 6 MRM of 5 Channels ES- 630 > 59 . 4.17e4 Area 4.00 5.00 6.00 7.00 '--^:iime 8.00 9.00 6.37 1 21-Dec-1999 19:07:39 . LC/MS/MS unit 6 MRM 0 /5 Channels ES 616 >59 ` 3 ' 8e4 . . ' Area 4.00 5.00 6.00 7.00 8.00 -1 Time 9.00 ' I960' J 21-D eo-1999 19:07:39 . LC/MS/MS unit 6 MRMof 5 Channels SS ! 499 > 99 ! 2.16e4 Area ' 1.00 ' 2 0 0 3.C0 4.0' 5.00 6-PO 7.00 ' : 8.n " o " W Centre Analytical Laboratories. Inc. Study # 023-001 i Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Page 68 of 69 Page 80 of 81 Page 146 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Protocol No. 00P-023-057 Figure 19 (continued): Representative Chromatograms from the Injection F Control Pork Muscle Fortified at 500 ppt each of Et-FOSE-OH Me FOST nrr PFOS, POAA, and PFOSA (Set 122199 Pork, Centre ID 9911783 Sp'k B) ' H aaa; 122199-215 99X1733 Spk 3 , P ork 500 p p t C en a ; Analytical Laboratories. Inc. Study if 023-001 ' I Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. D ' ,, Page 69 of 69- Page 81 of 81 Page 147 of 151 BACK TO MAIN Centre Study No.: 023-057Centre Protocol No: 00P-023-057 Centre Analytical Laboratories. Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 Page 1 of PROTOCOL AMENDMENT Amendment Number 1 Effective Date: March 26,2001 Centre Study Number 023-057 Centre Protocol Number 00P-023-057 DESCRIPTION OF AMENDED SECTION 1. Page 5 of Protocol: Assigns Susan Beach as the Study Director at 3M. AMENDED TO 1. Change the study director to Karen Smith at Centre Analytical Laboratories and make Susan Beach the Sponsor Representative at 3M. RATIONALE 1. Reassignment o f responsibilities for study at the request of Sponsor. IMPACT ON THE STUDY 1. There is no negative impact on the study. StW y DDiirreeccttir^ignature - J r ^ > Sponsor Representative Signature Centre Analytical Laboratories, Inc. Date loi Date CAI- QAU ReviewS L r ^ i ( J ,J n / February 12.1998/1 Page 148 of 151 BACK TO MAIN Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Analytical Laboratories, Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 Centre Analytical Laboratories, Inc. Page 149 of 151 BACK TO MAIN * Centre Study No.: 023-057' Centre Protocol No: 00P-023-057 Centre Analytical Laboratories. Inc. 3048 Research Drive State College, PA 16801 . www.centrelab com Phone: (814) 231-8032______________ Fax: (814) 231-1253 or (814) 231-1580 Page 1 of 1 PROTOCOL AMENDMENT Amendment Number: 3 Effective Date: June 15,2001 Centre Study Number: 023-057 Centre Protocol Number: 00P-023-057 DESCRIPTION OF AMENDED SECTION 1 . Study Title AMENDED TO 1. Analysis of PFOS, FOSA and PFOA From Various Food Matrices Using HPLC Electrospray/Mass Spectrometry. RATIONALE 1. Changed for clarity at the request of the sponsor. IMPACT ON THE STUDY 1. There is no negative impact on the study. Study DireciQjxSignature Sponsor Representative Signature 6 fe k lo l Date w /g Date CAL QAU Review k c u t / i s f a i _____ _____________________________ February 12, 1998/1 Centre Analytical Laboratories, Inc. Page 150 of 151 BACK TO Centre Study No.: 023-057 Centre Protocol No: 00P-023-057 Centre Analytical Laboratories, Inc 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231 -1580 PROTOCOL DEVIATION Page 1 of 1 Deviation Number: 1 Date of Occurrence: 1) January 19,2001; 2) February 15,2001 Centre Study Number 023-057 Sponsor Study Number NA DESCRIPTION OF DEVIATION 1) A recovery of 56% for FOSA is being accepted for the 10.0 ng/g Spk B in data set . _ 011901A Fish. 2) A CCV recovery of 143% is being accepted for the 2.5 ng/mL standard for FOSA in data set 021401A Beef. ACTIONS TAKEN i.e.. amendment issued. SOP revision, etc. 1) Deviation issued. 2) Deviation issued. Recorded By/Date: |~ t y 02-11 bio I IMPACT ON THE STUDY 1) There was no negative impact on the study since the 2.5 ng/mL Spk A for FOSA in data set 011901A Fish was within the 60-130% acceptable range and the levels of residue found in the fish samples were all below 2.5 ng/pd^ (S ^<*1 n|o j 2) There was no negative impact on the study since the CCV's before and the CCV run after the standard in question were acceptable. Principal Iitaesti gator Signature MPi Study Director Signature Sponsor Management Signature Centre QAU Review Centre Analytical Laboratories, Inc. o Date Date Date (7 January 5,2001/3 Page 151 of 151