Document ExYy7jwV47kx3xREB8ZD22ybj

INTERIM REPORT #3 - Analysis of Fish and Clam Samples STUDY TITLE Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, and Clams Using LC/MS/MS for the 3M Decatur Monitoring Program DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: 610-701-3761 INTERIM REPORT COMPLETION DATE June 17, 2008 TESTING FACILITY MPI Research, Inc. 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: 651-733-6374 PROJECT MPI Research Study Number: 0137.0219 MPI Project Number: P0003268 Total Pages: 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT MPI Project Number P0003268, entitled "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, and Clams Using LC/MS/MS for the 3M Decatur Monitoring Program," conducted for 3M Company, is being performed in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by MPI Research, Inc. Principal Investigator MPI Research, Inc. Jaisimha Kesari P.E., DEE Study Director Weston Solutions, Inc. Micha Sponsor Representative 3M Company Date Date MPI Research Page 2 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 QUALITY ASSURANCE STATEMENT MPI Research's Quality Assurance Unit reviewed MPI Project Number P0003268, entitled, "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, and Clams Using LC/MS/MS for the 3M Decatur Monitoring Program." All reviewed phases1were inspected for conduct according to MPI Research's Standard Operating Procedures, the Study Protocol, the Study Method, and all applicable Good Laboratory Practice Standards. All findings were reported to the MPI Principal Investigator, Management and to the Study Director. Phase Date Inspected Date Reported to Date Reported to Principal MPI Date Reported to Investigator Management Studv Director Raw Data, Protocol Amendments 5-8, and Draft Report Review 05/30/08 05/30/08 05/30/08 05/30/08 Final Interim Report Review 06/04/08 06/04/08 06/04/08 06/04/08 (j? - n - o 8 Lynann Porter Date Quality Assurance Research Group Leader, Quality Assurance Unit 'Note: All in-lab inspections and the protocol review will be documented in the QA statement for the final analytical report at the conclusion of the study. This QA statement involves only the review of the interim report and associated raw data. MPI Research Page 3 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 CERTIFICATION OF AUTHENTICITY This interim report, for MPI Project Number P0003268, is a true and complete representation of the raw data. Submitted by: MPI Research, Inc. 3058 Research Drive State College, PA 16801 (814) 272-1039 MPI Research, Inc. Date Director, Analytical Laboratory Operations MPI Research, Inc. Study Director, Weston Solutions, Inc. Jaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: Michael A. Santoro Director of Regulatory Affairs 3M Company MPI Research Date Date Date ' Page 4 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 STUDY IDENTIFICATION Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, and Clams Using LC/MS/MS for the 3M Decatur Monitoring Program MPI PROJECT NUMBER: P0003268 MPI STUDY NUMBER: 0137.0219 TYPE OF STUDY: Residue SAMPLE MATRIX: Fish and Clam TEST SUBSTANCES: Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) SPONSOR: 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 STUDY DIRECTOR: Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 SPONSOR REPRESENTATIVE: Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 TESTING FACILITY: MPI Research, Inc. 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: 06/14/07 Interim Analytical Start Date: 12/27/07 Interim Analytical Termination Date: 05/08/08 Interim Report Completion Date: 06/17/08 MPI Research Page 5 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 PROJECT PERSONNEL The Study Director for this project is Jaisimha Kesari at Weston Solutions, Inc. The following personnel from MPI Research, Inc. were associated with various phases of this interim portion of the study: Name Karen Risha Amy Sheehan Christine Edwards Krista Gallant Mark Neeley Gary Oden Ellen Dashem Stacey Orso Nancy Saxton Mark Ammerman Eric Edwards Title Manager Analytical, Principal Investigator Group Leader, LC/MS/MS Analysis Project Leader, LC/MS/MS Analysis Research Chemist Associate 2 Research Chemist Associate 2 Research Chemist Associate 2 Research Chemist Associate 1 Research Chemist Associate 1 Research Chemist Associate 1 Project Leader, Sample Control Sample Custodian 2 MPI Research Page 6 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 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.................................................................................................... 13 1.0 SUMMARY................................................................................................................ 14 2.0 OBJECTIVE............................................................................................................... 15 3.0 INTRODUCTION...................................................................................................... 15 4.0 ANALYTICAL TEST SAMPLES............................................................................. 16 5.0 REFERENCE MATERIAL........................................................................................ 17 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................ 18 6.1 Extraction Procedure for Fish and Clams................................................................ 18 6.1.1 Sample Preparation........................................................................................ 18 6.1.2 Sample Extraction.......................................................................................... 19 6.2 Preparation of Standards and Fortification Solutions.............................................. 19 6.3 Chromatography.......................................................................................................21 6.4 Instrument Sensitivity...............................................................................................21 6.5 Description of LC/MS/MS Instruments and Operating Conditions.........................21 6.6 Quantitation and Example Calculation....................................................................22 7.0 EXPERIMENTAL DESIGN......................................................................................24 8.0 RESULTS...................................................................................................................25 9.0 CONCLUSION...........................................................................................................26 10.0 RETENTION OF DATA AND SAMPLES.............................................................26 MPI Research Page 7 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples LIST OF TABLES MPI Study No.: 0137.0219 MPI Project No.: P0003268 Page Table I. Summary of PFBS, PFHS, and PFOS in Fish Fillet Samples...................... 28 Table II. Summary of PFOS in Re-extracted Fish Fillet Samples.......................... 31 Table III.Summary of PFBS, PFHS, and PFOS in Whole Body Fish Samples................ 32 Table IV. Summary of PFOS in Re-extracted Whole Body Fish Samples.....................35 Table V. Summary of PFBS, PFHS, and PFOS in Clam Samples.......................... 36 Table VI. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Fish Fillet Samples.......................................................................................................... 37 Table VII. Matrix Spike Recovery Summary of PFOS in Re-spiked Fish Fillet Samples..........................................................................................................41 Table VIII. Matrix Spike Recovery Summary of PFOS in Re-extracted Fish Fillet Samples..........................................................................................................43 Table IX. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Whole Body Fish Samples...................................................................................................44 Table X. Matrix Spike Recovery Summary of PFOS in Re-extracted Whole Body Fish Samples..........................................................................................................49 Table XI. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Clam Samples.......................................................................................................... 50 MPI Research Page 8 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples LIST OF FIGURES MPI Study No.: 0137.0219 MPI Project N o.: P0003268 Page Figure 1. Typical Extracted Calibration Curve for PFBS in Catfish Fillet Matrix.......52 Figure 2. Extracted Standards of PFBS in Catfish Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively......................................... 53 Figure 3. PFBS in a Control Catfish Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Catfish Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Catfish Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Catfish Fillet Spike C, Respectively.............54 Figure 4. Chromatogram Representing a Catfish Fillet Sample Analyzed for PFBS (ExyLIMS ID: C0227555, Data Set: 011108D)............................................ 55 Figure 5. Typical Extracted Calibration Curve for PFHS in Catfish Fillet Matrix.......56 Figure 6. Extracted Standards of PFHS in Catfish Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively......................................... 57 Figure 7. PFHS in a Control Catfish Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Catfish Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Catfish Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Catfish Fillet Spike C, Respectively.............58 Figure 8. Chromatogram Representing a Catfish Fillet Sample Analyzed for PFHS (ExyLIMS ID: C0227556, Data Set: 011108D)............................................ 59 Figure 9. Typical Extracted Calibration Curve for PFOS in Catfish Fillet Matrix.......60 Figure 10. Extracted Standards of PFOS in Catfish Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively......................................... 61 Figure 11. PFOS in a Control Catfish Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Catfish Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Catfish Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Catfish Fillet Spike C, Respectively.............62 Figure 12. Chromatogram Representing a Catfish Fillet Sample Analyzed for PFOS (ExyLIMS ID: C0227557, Data Set: 011108D)............................................ 63 Figure 13. Typical Extracted Calibration Curve for PFBS in Bass Fillet Matrix........... 64 Figure 14. Extracted Standards of PFBS in Bass Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively.................................................. 65 Figure 15. PFBS in a Control Bass Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Bass Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Bass Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Bass Fillet Spike C, Respectively........................ 66 Figure 16. Chromatogram Representing a Bass Fillet Sample Analyzed for PFBS (ExyLIMS ID: C0227544, Data Set: 122707B)............................................ 67 Figure 17. Typical Extracted Calibration Curve for PFHS in Bass Fillet Matrix........... 68 MPI Research Page 9 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 LIST OF FIGURES (Continued) Page Figure 18. Extracted Standards of PFHS in Bass Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively.................................................. 69 Figure 19. PFHS in a Control Bass Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Bass Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Bass Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Bass Fillet Spike C, Respectively........................ 70 Figure 20. Chromatogram Representing a Bass Fillet Sample Analyzed for PFHS (ExyLIMS ID: C0227546, Data Set: 122707B)............................................ 71 Figure 21. Typical Extracted Calibration Curve for PFOS in Bass Fillet Matrix...........72 Figure 22. Extracted Standards of PFOS in Bass Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively...................................................73 Figure 23. PFOS in a Control Bass Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Bass Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Bass Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Bass Fillet Spike C, Respectively........................74 Figure 24. Chromatogram Representing a Bass Fillet Sample Analyzed for PFOS (ExyLIMS ID: C0227544, Data Set: 122707B).............................................75 Figure 25. Typical Extracted Calibration Curve for PFBS in Whole Body Catfish Matrix............................................................................................................. 76 Figure 26. Extracted Standards of PFBS in Whole Body Catfish Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively................................77 Figure 27. PFBS in a Control Whole Body Catfish, a 0.075 ng/mL (0.30 ng/g) Fortified Whole Body Catfish Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Whole Body Catfish Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Whole Body Catfish Spike C, Respectively.....................................................................................78 Figure 28. Chromatogram Representing a Whole Body Catfish Sample Analyzed for PFBS (ExyLIMS ID: C0227612, Data Set: 032108C)...................................79 Figure 29. Typical Extracted Calibration Curve for PFHS in Whole Body Catfish Matrix............................................................................................................. 80 Figure 30. Extracted Standards of PFHS in Whole Body Catfish Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively.................................81 Figure 31. PFHS in a Control Whole Body Catfish, a 0.075 ng/mL (0.30 ng/g) Fortified Whole Body Catfish Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Whole Body Catfish Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Whole Body Catfish Spike C, Respectively..................................................................................... 82 MPI Research Page 10 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 LIST OF FIGURES (Continued) Page Figure 32. Chromatogram Representing a Whole Body Catfish Sample Analyzed for PFHS (ExyLIMS ID: C0227612, Data Set: 032108C).................................. 83 Figure 33. Typical Extracted Calibration Curve for PFOS in Whole Body Catfish Matrix............................................................................................................. 84 Figure 34. Extracted Standards of PFOS in Whole Body Catfish Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively.................................85 Figure 35. PFOS in a Control Whole Body Catfish, a 0.075 ng/mL (0.30 ng/g) Fortified Whole Body Catfish Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Whole Body Catfish Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Whole Body Catfish Spike C, Respectively..................................................................................... 86 Figure 36. Chromatogram Representing a Whole Body Catfish Sample Analyzed for PFOS (ExyLIMS ID: C0227612, Data Set: 032108C)...................................87 Figure 37. Typical Extracted Calibration Curve for PFBS in Whole Body Bass Matrix............................................................................................................. 88 Figure 38. Extracted Standards of PFBS in Whole Body Bass Matrix, 0.01 ng/mL (0.10 ng/g) and 0.02 ng/mL (0.20 ng/g), Respectively............................................ 89 Figure 39. PFBS in a Control Whole Body Bass, a 0.03 ng/mL (0.30 ng/g) Fortified Whole Body Bass Spike A, a 0.2 ng/mL (2.0 ng/g) Fortified Whole Body Bass Spike B, and a 4.0 ng/mL (40 ng/g) Fortified Whole Body Bass Spike C, Respectively................................................................................................... 90 Figure 40. Chromatogram Representing a Whole Body Bass Sample Analyzed for PFBS (ExyLIMS ID: C0227603, Data Set: 042208A).............................................91 Figure 41. Typical Extracted Calibration Curve for PFHS in Whole Body Bass Matrix............................................................................................................. 92 Figure 42. Extracted Standards of PFHS in Whole Body Bass Matrix, 0.01 ng/mL (0.10 ng/g) and 0.02 ng/mL (0.20 ng/g), Respectively............................................93 Figure 43. PFHS in a Control Whole Body Bass, a 0.03 ng/mL (0.30 ng/g) Fortified Whole Body Bass Spike A, a 0.2 ng/mL (2.0 ng/g) Fortified Whole Body Bass Spike B, and a 4.0 ng/mL (40 ng/g) Fortified Whole Body Bass Spike C, Respectively................................................................................................... 94 Figure 44. Chromatogram Representing a Whole Body Bass Sample Analyzed for PFHS (ExyLIMS ID: C0227601, Data Set: 042208A).............................................95 Figure 45. Typical Extracted Calibration Curve for PFOS in Whole Body Bass Matrix............................................................................................................. 96 MPI Research Page 11 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 LIST OF FIGURES (Continued) Page Figure 46. Extracted Standards of PFOS in Whole Body Bass Matrix, 0.01 ng/mL (0.10 ng/g) and 0.02 ng/mL (0.20 ng/g), Respectively........................................... 97 Figure 47. PFOS in a Control Whole Body Bass, a 0.03 ng/mL (0.30 ng/g) Fortified Whole Body Bass Spike A, a 0.2 ng/mL (2.0 ng/g) Fortified Whole Body Bass Spike B, and a 4.0 ng/mL (40 ng/g) Fortified Whole Body Bass Spike C, Respectively................................................................................................... 98 Figure 48. Chromatogram Representing a Whole Body Bass Sample Analyzed for PFOS (ExyLIMS ID: C0227600, Data Set: 042208A)............................................ 99 Figure 49. Typical Extracted Calibration Curve for PFBS in Clam Matrix.................100 Figure 50. Extracted Standards of PFBS in Clam Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively....................................................... 101 Figure 51. PFBS in a Control Clam, a 0.075 ng/mL (0.30 ng/g) Fortified Clam Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Clam Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Clam Spike C, Respectively......................................................... 102 Figure 52. Chromatogram Representing a Clam Sample Analyzed for PFBS (ExyLIMS ID: C0227658, Data Set: 050508A)............................................................. 103 Figure 53. Typical Extracted Calibration Curve for PFHS in Clam Matrix................. 104 Figure 54. Extracted Standards of PFHS in Clam Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively....................................................... 105 Figure 55. PFHS in a Control Clam, a 0.075 ng/mL (0.30 ng/g) Fortified Clam Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Clam Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Clam Spike C, Respectively......................................................... 106 Figure 56. Chromatogram Representing a Clam Sample Analyzed for PFHS (ExyLIMS ID: C0227658, Data Set: 050508A)............................................................. 107 Figure 57. Typical Extracted Calibration Curve for PFOS in Clam Matrix.................108 Figure 58. Extracted Standards of PFOS in Clam Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively........................................................ 109 Figure 59. PFOS in a Control Clam, a 0.075 ng/mL (0.30 ng/g) Fortified Clam Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Clam Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Clam Spike C, Respectively.......................................................... 110 Figure 60. Chromatogram Representing a Clam Sample Analyzed for PFOS (ExyLIMS ID: C0227658, Data Set: 042208A)............................................................. 111 MPI Research Page 12 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples LIST OF APPENDICES MPI Study No.: 0137.0219 MPI Project No.: P0003268 Page Appendix A Study Protocol and Amendments........................................................... 112 MPI Research Page 13 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 1.0 SUMMARY MPI Research, Inc. successfully extracted and analyzed fish and clam samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to 3M Environmental Laboratory Method ETS-8-049.2 (V0003970) detailed in MPI Protocol P0003268 Amendments #3 through #8 (Appendix A, pgs. 169-197). The limits of quantitation (LOQ) for the analytes in the fish fillet samples are listed in Table I. The target LOQ for the method for fish fillet samples was 0.20 ng/g. The limits of quantitation (LOQ) for the analytes in the whole body fish samples are listed in Table III. The target LOQ for the method for whole body fish samples was 0.20 ng/g. The limits of quantitation (LOQ) for the analytes in the clam samples are listed in Tables V. The target LOQ for the method for clam samples was 0.20 ng/g. After evaluation of the reagent blanks (method blanks) used for the analysis, the LOQ was determined. In some cases, the LOQ was raised due to the evaluation. A discussion of the process used to evaluate the reagent blanks can be found in section 6.3 of the report. The LOQ for the analyte in the re-extracted fish fillet samples is listed in Table II. The target LOQ for the method for the re-extracted fish fillet samples was 0.20 ng/g. The LOQ for the analyte in the re-extracted whole body fish samples is listed in Table IV. The target LOQ for the method for the re-extracted whole body fish samples was 0.20 ng/g. Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS found in the fish fillet samples are summarized in Table I. Fortification recoveries for PFBS, PFHS, and PFOS in the fish fillet samples are detailed in Table VI. The average percent recoveries standard deviations for PFBS, PFHS, and PFOS in the fish fillet samples were 103 21%, 100 19%, and 99 20%, respectively. The PFBS result for one fish fillet sample was not reported (NR) due to low spike recovery outside of the acceptance criteria range. The PFOS results for several fish fillet samples were not reported in the initial analysis due to low spike recovery or because endogenous concentrations greatly exceeded the fortification levels. The samples were re-spiked to obtain quality control recoveries with an appropriate fortification level. Fortification recoveries for PFOS in the re-spiked fish fillet samples are detailed in Table VII. The average percent recovery standard deviation for PFOS in the re-spiked fish fillet samples was 101 16%. The PFOS results for two re-spiked fish fillet samples were not reported (NR) due to spike recoveries outside of the acceptance criteria range. Three samples were re-extracted and reanalyzed for PFOS, and the analytical results and assessed accuracies for the analysis of PFOS found in the re-extracted fish fillet samples are summarized in Table II. Fortification recoveries for PFOS in the re-extracted fish fillet samples are detailed in Table VIII. The percent recovery standard deviation for PFOS in the re-extracted fish fillet samples was 93 11%. Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS found in the whole body fish samples are summarized in Table III. Fortification recoveries for PFBS, PFHS, and PFOS in the whole body fish samples are detailed in Table IX. The average percent MPI Research Page 14 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 recoveries standard deviations for PFBS, PFHS, and PFOS in the whole body fish samples were 95 15%, 98 17%, and 87 19%, respectively. The PFOS results for five whole body fish samples were not reported due to spike recoveries outside of the acceptance criteria range. The samples were re-extracted and reanalyzed, and the analytical results and assessed accuracies for the analysis of PFOS found in the re extracted whole body fish samples are summarized in Table IV. Fortification recovery for PFOS in the re-extracted whole body fish samples are detailed in Table X. The percent recovery standard deviation for PFOS in the re-extracted whole body fish samples was 84 10%. Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS found in the clam samples are summarized in Table V. Fortification recoveries for PFBS, PFHS, and PFOS in the clam samples are detailed in Table XI. The average percent recoveries standard deviations for PFBS, PFHS, and PFOS in the clam samples were 105 9%, 128 7%, and 102 23%, respectively. The PFHS results for two clam samples were not reported due to low spike recoveries outside of the acceptance criteria range. The assessed accuracy for the majority of the samples reported is +/- 30%. The accuracies were assessed for each sample by reviewing the matrix spike whose spiking level most closely matches the endogenous concentration found in the sample. 2.0 OBJECTIVE The objective of the analytical part of this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) in fish and clam samples according to Protocol P0003268 and Amendments #3 through #8 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFBS, PFHS, and PFOS in fish and clams using the 3M Environmental Laboratory analytical method ETS8-049.2 (V0003970) entitled, "Determination of Fluorochemicals via Solid-Phase Extraction of Fish Tissues (Fillet or Whole Body) by High Performance Liquid Chromatography with Tandem Mass Spectrometry" detailed in MPI Protocol P0003268 Amendments #3 through #8. The study was initiated on June 14, 2007, when the study director signed protocol number P0003268. The analytical start date for this interim report was December 27, 2007, and the analytical termination date for this interim report was May 8, 2008. MPI Research Page 15 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 4.0 ANALYTICAL TEST SAMPLES A total of one hundred and eighteen samples (ExyLIMS ID C0227544, C0227546 C0227656, and C0227658 - C0227663 from login ID L00010436), one hundred and twelve fish and six clams, were received on dry ice on December 20, 2006 from Charles Young at Weston Solutions, Inc. The one hundred and twelve fish samples represented six sample sites: DL1, DL2, DL3, DOU, DMC, and DBC. Each site consisted of twenty samples, with the exception of site DL2 which consisted of twelve samples. The twenty samples represented ten catfish samples (five catfish fillets and five whole body catfish samples) and ten bass samples (five bass fillets and five whole body bass samples). For the DL2 site, the twelve samples represented ten catfish samples (five catfish fillets and five whole body catfish samples) and two bass samples (two whole body bass samples). All samples were logged in by MPI personnel and placed in frozen storage. The sample IDs assigned by the client follow this formula: Dxx-x02-xxxxxx-x(x)-0612xx. The first string begins with D for Decatur, Alabama and the xx defines the sampling area where: DL3 = Up river Letter of Intent (LOI) sampling location LOC-3 DL2 = Cross river LOI sampling location LOC-2 DL1 = Down river Fox Creek mouth LOI sampling location LOC-1 DBC = Bakers Creek mouth DOU = Cove at 3M WWTP outfall DMC Downstream Mallard Creek mouth The second string defines the general category of the biota sample and the sampling round where: F02 = Fish sample second round 102 = Invertebrate sample second round The third string defines the species (first two characters), the sample type (third character) and the sample number (last three characters) within that sampling area and type of sample where: IP = channel catfish {Ictalurus punctatus) MS = largemouth bass (Micropterus salmoides) CF = Asiatic clam (Corbiculafluminea) F = Fillet tissue sample W = Whole body sample 001 through 005 = sample number within the sampling area and sample type MPI Research Page 16 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples The fourth string additionally defines the sample type where: 0 = Primary field sample 1 = Field duplicate sample RB = Equipment rinseate blank sample MPI Study No.: 0137.0219 MPI Project No.: P0003268 The fifth string defines the sample preparation date in YYMMDD format. Sample log-in and chain of custody information is located in the raw data package associated with this interim report. Storage records will be kept at MPI Research, Inc. (State College). 5.0 REFERENCE MATERIAL The requisition information, lot, purity, and expiration date for the reference material used in this study is listed below. The reference material was stored refrigerated. Compound ExvLIMS Inventory No. PFBS SP0008956 PFHS SP0008961 PFOS SP0002402 Supplier 3M Environmental Laboratory 3M Environmental Laboratory 3M Environmental Laboratory Lot # Puritv Expiration Received (%) Date Date 2 97.3 01/18/17 02/19/07 NB 120067- 98.6 02/12/17 02/19/07 69 217 86.9 08/31/16 01/20/03 The molecular structure of the standard is given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4F9SO3KC) Transitions Monitored: 299 - 80, 99 Structure: FF FF F SO3 FF FF MPI Research Page 17 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (C6Fi3S03'K+) Transitions Monitored: 399 - 80, 99 Structure: FFF FFF F SO3 FFF FFF PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CsFnSCVK*) Transitions Monitored: 499 -->80, 99 Structure: FFFF FFFF FF FFFF 3 6.0 DESCRIPTION OF ANALYTICAL METHOD The 3M Environmental Laboratory analytical method ETS-8-049.2 (V0003970) entitled, "Determination of Fluorochemicals via Solid-Phase Extraction of Fish Tissues (Fillet or Whole Body) by High Performance Liquid Chromatography with Tandem Mass Spectrometry" and details in Amendments #3 through #8 (Appendix A, pgs. 169-197) were used for the sample analysis in this study. 6.1 Extraction Procedure for Fish and Clams 6.1.1 Sample Preparation Before the samples could be weighed for the extraction, they had to be processed. To process, the frozen samples were placed into a food processor and homogenized with dry ice. The samples were transferred to one-gallon Ziploc bags and placed in frozen storage with bag left open to allow the dry ice to sublime. After sublimation, the sample bags were sealed and remained in frozen storage until time of analysis. Sample processing records are located in the Sample Information section of the MPI Research Page 18 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 raw data package except for those that were processed and supplied by the study sponsor as outlined in the protocol deviation found in the raw data package. 6.1.2 Sample Extraction A 3-gram portion of each sample was weighed into a graduated cylinder, and the samples were brought up to 30 mL with acetonitrile. Each sample was then homogenized for -2 minutes. 5-mL aliquots of each sample were transferred to 15-mL centrifuge tubes, fortified as appropriate, and vortex mixed. The samples were placed into a freezer for -1 hour, and then centrifuged for ~20 minutes at -3000 rpm at -5C. The supernatant was decanted into 100 mL of 2% H3PO4 (aq) and vortexed. Next, the samples (catfish fillets, bass fillets, catfish whole bodies and clams) were loaded onto preconditioned HLB SPE cartridges (conditioned with 2 mL of methanol followed by 2 mL of water) and drawn through under vacuum, discarding the eluent. The whole body bass samples were loaded onto preconditioned tC18 SPE cartridges (conditioned with 5 mL of methanol followed by 5 mL of water) and drawn through under vacuum, discarding the eluent. After the samples were drawn through the cartridges, the cartridges were washed with 2 mL of 5% methanol (aq) (for the fillet samples) followed by 2 mL of 2% formic acid (aq) and suctioned dry. The HLB SPE cartridges were then eluted with 2 mL of 5% NH4OH in methanol, and the tCl 8 SPE cartridges were eluted with 5 mL of 5% NH4OH in methanol. The samples were then transferred to HPLC autosampler vials and diluted as necessary in 5% NH4OH in methanol. Each sample was analyzed by LC/MS/MS electrospray. 6.2 Preparation of Standards and Fortification Solutions Stock standard solutions of PFBS, PFHS, and PFOS were prepared as specified in the method. The stock standard solutions were prepared at a concentration of 1000 pg/mL by dissolving 0.1 g of the appropriate standard (corrected for purity and salt content, if necessary) in acetonitrile. From those solutions, a 100 pg/mL mixed fortification standard solution was prepared by taking 10 mL of each of the stocks and bringing the volume up to 100 mL with acetonitrile. By taking 10 mL of the 100 pg/mL mixed fortification standard and bringing the volume up to 100 mL with acetonitrile, a 10 pg/mL mixed fortification standard was prepared. By taking 10 mL of the 10 pg/mL mixed fortification standard and bringing the volume up to 100 mL with acetonitrile, a 1.0 pg/mL mixed fortification standard was prepared. By taking 10 mL of the 1.0 pg/mL mixed fortification standard and bringing the volume up to 100 mL with acetonitrile, a 0.1 pg/mL mixed fortification standard was prepared. By taking 10 mL of the 0.1 pg/mL mixed fortification standard and bringing the volume up to 100 mL with acetonitrile, a 0.01 pg/mL mixed fortification standard was prepared. By taking 10 mL of the 0.01 MPI Research Page 19 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 pg/mL mixed fortification standard and bringing the volume up to 100 mL with acetonitrile, a 0.001 pg/mL mixed fortification standard was prepared. A set of extracted calibration standards was prepared for each type of matrix (catfish fillet, bass fillet, whole body catfish, whole body bass, and clams). The extracted calibration standards were processed through the same extraction procedure as outlined above. The following concentrations were prepared for the catfish fillet, bass fillet, whole body catfish, and clam samples: Cone, of Fort. Solution (ng/mL) 1.0 1.0 10 10 10 10 100 100 100 Aliquot Volume (mL) 50 100 25 50 100 125 25 50 100 Final Volume of Standard (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Final Cone, of Calibration Std. (ng/mL) 0.025 0.050 0.125 0.25 0.50 0.625 1.25 2.5 5.0 Final Cone, of Calibration Std. (ng/g) 0.10 0.20 0.50 1.0 2.0 2.5 5.0 10 20 The following concentrations were prepared for the whole body bass samples: Cone, of Fort. Solution (ng/mL) 1.0 1.0 10 10 10 100 100 100 1000 Aliquot Volume (mL) 50 100 25 50 100 25 50 100 25 Final Volume of Standard (mL) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Final Cone, of Calibration Std. (ng/mL) 0.01 0.02 0.05 0.10 0.20 0.50 1.0 2.0 5.0 Final Cone, of Calibration Std. (ng/g) 0.10 0.20 0.50 1.0 2.0 5.0 10 20 50 The stock standard solution was stored in a freezer (-20 5C) when not in use. All other fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. MPI Research Page 20 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 6.3 Chromatography Quantification of the analytes was accomplished by LC/MS/MS electrospray. The retention time of PFBS ranged from 8.3 to 8.8 minutes, the retention time of PFHS ranged from 9.7 to 10.2 minutes, and the retention time of PFOS ranged from 10.7 to 11.1. A method blank prepared for each data set was used to determine the LOQ. In instances where there were no peaks in the method blank, the LOQ was determined by the concentration of the lowest standard injected in the analytical run that met the 70-130% recovery range of its known value. In instances where there was a peak detected in the method blank, the blank was evaluated. If the response of the method blank was less than 50 % of the response of the lowest standard meeting the recovery criteria, then the LOQ was determined by the lowest standard. If the response of the method blank was greater than 50 % of the response of the lowest standard meeting the recovery criteria, then the LOQ was raised to the standard that met the less than 50 % criteria. 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 0.025 ng/mL for the catfish fillet, bass fillet, whole body catfish and clam samples, and a concentration of 0.010 ng/mL for the whole body bass samples. 6.5 Description of LC/MS/MS Instruments and Operating Conditions Instruments: API 5000 Biomolecular Mass Analyzer Interface: SCIEX Turbo Ion Spray Liquid Introduction Interface Computer: DELL Precision 360 DELL OptiPlex GX400 Software: PE SCIEX Analyst 1.4.1 HPLC: Hewlett Packard (HP) Series 1200 Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Electron Betasil C l8, 100 mm x 2.1 mm, 5pm Column Temp.: -30 C Injection Voi.: 10 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Acetonitrile Gradient: Time (min) %A % B Flow Rate Ipl/mLl 0.0 90 10 400 1.0 90 10 400 8.5 25 75 300 13.5 25 75 300 MPI Research Page 21 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples 14.0 90 17.0 90 25.0 90 MPI Study No.: 0137.0219 MPI Project No.: P0003268 10 300 10 400 10 400 Total run time: ~25 min Ions monitored: Analyte Mode PFBS PFHS PFOS negative negative negative Transition Monitored 299 -> 80, 99 399 -> 80, 99 499 -> 80, 99 Retention Time (min) --8.55 min. -9.95 min. -10.9 min. 6.6 Quantitation and Example Calculation Ten 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 fit weighted quadratic regression) by Analyst software using eight or nine concentrations of standards. The concentration was determined from the following equations. Equation 1 calculated the amount of analyte found (in ng/mL, based on peak area) using the standard curve (quadratic regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/mL) = (TV(B2+ 4AY - 4AC))-B) x Dilution Factor 2A Where: A = X variable 1 B = X variable 2 C = intercept Y = peak area Equation 2 was used to convert the amount of analyte found in ng/mL to ng/g (ppb). Equation 2: Analyte found (ppb) = ranalyte found (ng/mL) x final volumeAx aliquot factor(6)*1 sample weight (3 g) AFinal volume of 2mL for catfish fillet, bass fillet, whole body catfish, and clams. Final volume of 5mL for whole body bass. *Aliquot factor calculated by the following equation: [initial volume of sample (30mL)]/[extraction aliquot volume (5mL)] = 6 MPI Research Page 22 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 For samples fortified with known amounts of analyte prior to extraction, Equation 3 was used to calculate the percent recovery. Equation 3: Recovery (%) = (total analyte found (ng/g) - average analyte in sample (ng/g)) x l00% analyte added (ng/g) An example of a calculation using an actual sample follows: Bass fillet sample ExyLIMs ID: C0227581 Spike F (Set: 010308C), fortified at 2.0 ng/g with PFHS where: Y (peak area) 233684 A (X variable 1) 14600 B (X variable 2) 349000 C (intercept) 6640 dilution factor ng/g PFHS added (fort level) 2.0 ng/g average amt in corresponding sample 0.435 From equation 1: Analyte found (ng/mL) = ((V (3490002 + 4(146001(233684) - 4(14600)(6640)))-349000) x 1 2(14600) From equation 2: Analyte found, wet weight (ng/g) 0.634 ng/mL = (0.634 ng/mL x 2 mL x 6 ) % Recovery (2.54ng/g - 0.435 ng/g) x 100% 2.0 ng/g 105% NOTE: Numbers may differ slightly from raw data due to rounding. MPI Research Page 23 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 7.0 EXPERIMENTAL DESIGN For all fish and clam samples designated as laboratory matrix spikes, PFBS, PFHS, and PFOS were added at known concentrations to the samples in the laboratory after the samples were weighed, homogenized and aliquoted for the extraction. The fish samples were extracted in twenty seven sets. Each set included one reagent blank (method blank), two matrix controls, three matrix controls fortified at a lower level, three matrix controls fortified at a mid level, and three matrix controls fortified at a higher level of known concentrations. The bass fillet fish samples were extracted in five sets. All five sets contained five bass fillet fish samples, each from the same sample site. The catfish fillet samples were extracted in six sets. All six sets contained five catfish fillet samples, each from the same sample site. The whole body bass samples were initially partially extracted in four sets. The initial analysis was not reported due to quality control failures. The samples were then extracted using a modified version of the method (outlined in protocol amendment #6). The whole body bass samples were extracted in six sets. Five of the sets contained five whole body bass samples, each from the same sample site and one of the sets contained two samples, both from the same sample site. The whole body catfish samples were extracted in six sets. All six sets contained five whole body catfish samples, each from the same sample site. The clam samples were extracted in one set. The set included one reagent blank (method blank), two matrix controls, three matrix controls fortified at a lower level, three matrix controls fortified at a mid level, and three matrix controls fortified at a higher level of known concentrations. The set contained six clam samples, one sample from each of the six sample sites. Thirty fish fillet samples were re-spiked at higher fortification levels for PFOS due to endogenous levels of PFOS in the samples greatly exceeding the initial fortification levels. The re-spike fish fillet samples were extracted in two sets. The sets included one reagent blank (method blank), two matrix controls, three matrix controls fortified at a lower level, three matrix controls fortified at a mid level, and three matrix controls fortified at a higher level of known concentrations. One set contained ten catfish fillet re spiked samples. The second set contained twenty bass fillet re-spiked samples. Several fish fillet samples were re-extracted to obtain PFOS results that met quality control objectives. The fish fillet samples were re-extracted in two sets. The sets included one reagent blank (method blank), two matrix controls, three matrix controls fortified at a lower level, three matrix controls fortified at a mid level, and three matrix controls fortified at a higher level of known concentrations. Two catfish fillet sample were re-extracted in one set and one bass fillet sample was re-extracted in one set. Several whole body fish samples were re-extracted to obtain PFOS results that met quality control objectives. Five whole body bass samples were re-extracted in one set. MPI Research Page 24 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 The set included one reagent blank (method blank), two matrix controls, three matrix controls fortified at a lower level, three matrix controls fortified at a mid level, and three matrix controls fortified at a higher level of known concentrations and the five whole body bass samples. Accuracies were assessed for each sample by reviewing the individual QC results obtained for each sample. 8.0 RESULTS The limits of quantitation (LOQ) for the analytes in the fish fillet samples are listed in Table I. The target LOQ for the method for fish fillet samples was 0.20 ng/g. The limits of quantitation (LOQ) for the analytes in the whole body fish samples are listed in Table III. The target LOQ for the method for whole body fish samples was 0.20 ng/g. The limits of quantitation (LOQ) for the analytes in the clam samples are listed in Tables V. The target LOQ for the method for clam samples was 0.20 ng/g. After evaluation of the reagent blanks (method blanks) used for the analysis, the LOQ was determined. In some cases, the LOQ was raised due to the evaluation. A discussion of the process used to evaluate the reagent blanks can be found in section 6.3 of the report. The LOQ for the analyte in the re-extracted fish fillet samples is listed in Table II. The target LOQ for the method for the re-extracted fish fillet samples was 0.20 ng/g. The LOQ for the analyte in the re-extracted whole body fish samples is listed in Table IV. The target LOQ for the method for the re-extracted whole body fish samples was 0.20 ng/g. Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS found in the fish fillet samples are summarized in Table I. Fortification recoveries for PFBS, PFHS, and PFOS in the fish fillet samples are detailed in Table VI. The average percent recoveries standard deviations for PFBS, PFHS, and PFOS in the fish fillet samples were 103 21%, 100 19%, and 99 20%, respectively. The PFBS result for one fish fillet sample was not reported (NR) due to low spike recovery outside of the acceptance criteria range. The PFOS results for several fish fillet samples were not reported in the initial analysis due to low spike recovery or because endogenous concentrations greatly exceeded the fortification levels. The samples were re-spiked to obtain quality control recoveries with an appropriate fortification level. Fortification recoveries for PFOS in the re-spiked fish fillet samples are detailed in Table VII. The average percent recovery standard deviation for PFOS in the re-spiked fish fillet samples was 101 16%. The PFOS results for two re-spiked fish fillet samples were not reported (NR) due to spike recoveries outside of the acceptance criteria range. Three samples were re-extracted and reanalyzed for PFOS, and the analytical results and assessed accuracies for the analysis of PFOS found in the re-extracted fish fillet samples are summarized in Table II. Fortification recoveries for PFOS in the re-extracted fish fillet samples are detailed in Table VIII. The percent recovery standard deviation for PFOS in the re-extracted fish fillet samples was 93 11%. Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS found in the whole body MPI Research Page 25 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 fish samples are summarized in Table III. Fortification recoveries for PFBS, PFHS, and PFOS in the whole body fish samples are detailed in Table IX. The average percent recoveries standard deviations for PFBS, PFHS, and PFOS in the whole body fish samples were 95 15%, 98 17%, and 87 19%, respectively. The PFOS results for five whole body fish samples were not reported due to spike recoveries outside of the acceptance criteria range. The samples were re-extracted and reanalyzed, and the analytical results and assessed accuracies for the analysis of PFOS found in the re extracted whole body fish samples are summarized in Table IV. Fortification recovery for PFOS in the re-extracted whole body fish samples are detailed in Table X. The percent recovery standard deviation for PFOS in the re-extracted whole body fish samples was 84 10%. Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS found in the clam samples are summarized in Table V. Fortification recoveries for PFBS, PFHS, and PFOS in the clam samples are detailed in Table XI. The average percent recoveries standard deviations for PFBS, PFHS, and PFOS in the clam samples were 105 9%, 128 7%, and 102 23%, respectively. The PFHS results for two clam samples were not reported due to low spike recoveries outside of the acceptance criteria range. The assessed accuracy for the majority of the samples reported is +/- 30%. The accuracies were assessed for each sample by reviewing the matrix spike whose spiking level most closely matches the endogenous concentration found in the sample. 9.0 CONCLUSION Except as noted above, the fish and clam samples were successfully extracted and analyzed for PFBS, PFHS, and PFOS according 3M Environmental Laboratory analytical method ETS-8-049.2 (V0003970). 10.0 RETENTION OF DATA AND SAMPLES All original paper data generated by MPI Research, Inc. (State College) that pertains to this interim report will be shipped to the study director. This does not include facilityspecific raw data such as instrument or temperature logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the MPI Research, Inc. (State College) archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. MPI Research Page 26 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 TABLES MPI Research Page 27 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table I. Summary of PFBS, PFHS, and PFOS in Fish Fillet Samples ExyLIMS ID C0227544 C0227544 Rep C0227546 C0227546 Rep C0227547 C0227547 Rep C0227548 C0227548 Rep C0227549 C0227549 Rep C0227550 C0227550 Rep C0227551 C0227551 Rep C0227552 C0227552 Rep C0227553 C0227553 Rep C0227554 C0227554 Rep C0227555 C0227555 Rep C0227556 C0227556 Rep C0227557 C0227557 Rep C0227558 C02275548 Rep C0227559 C0227559 Rep C0227560 C0227560 Rep C0227561 C0227561 Rep C0227562 C0227562 Rep C0227563 C0227563 Rep C0227564 C0227564 Rep C0227565 C0227565 Rep C0227566 C0227566 Rep C0227567 C0227567 Rep Client Sample ID DL3-F02-MSF001-0-061207 DL3-F02-MSF001-0-061207* DL3-F02-MSF002-0-061207 DL3-F02-MSF002-0-061207* DL3-F02-MSF003-0-061207 DL3-F02-MSF003-0-061207* DL3-F02-MSF004-0-061207 DL3-F02-MSF004-0-061207* DL3-F02-MSF005-0-061207 DL3-F02-MSF005-0-061207* DL3-F02-IPF001-0-061211 DL3-F02-IPF001-0-061211* DL3-F02-IPF002-0-061212 DL3-F02-IPF002-0-061212* DL3-F02-IPF003-0-061212 DL3-F02-1PF003-0-061212* DL3-F02-IPF004-0-061212 DL3-F02-IPF004-0-061212* DL3-F02-IPF005-0-061212 D L 3-F02 -IP F0 05 -Q -0 61 2 12 * DL2-F02-IPF001-0-061209 DL2-F02-IPF001-0-061209* DL2-F02-IPF002-0-061209 DL2-F02-IPF002-0-061209* DL2-F02-IPF003-0-061209 DL2-F02-IPF003-0-061209* D L 2 - F 0 2 - IP F 0 0 4 - 0 - 0 6 1209 DL2-F02-IPF004-0-061209* DL2-F02-IPF005-0-061209 D L 2 - F 0 2 -IP F 0 0 5 -0-0 61 20 9 * DBC-F02-MSF001-0-061207 DBC-F02-MSF001-0-061207* DBC-F02-MSF002-0-061207 D B C -F 0 2 -M S F 0 0 2 -0 -0 6 1 2 0 7 * D B C -F 0 2 -M S F 0 0 3 -0 -0 6 1207 DBC-F02-MSF003-0-061207* DBC-F02-MSF004-0-061207 D8C-F02-MSF004-0-061207* D B C -F 0 2 -M S F 0 0 5 -0 -0 6 1 207 DBC-F02-MSF005-0-061207* DBC- F02-IP F001-0-061211 DBC-F02-IPF001-0-061211 * DBC- F02-IP F002-0-061211 DBC-F02-IPF002-0-061211* DBC- F02-1PF003-0-061211 D B C -F 0 2 -IP F 0 0 3 - 0 - 0 6 1 2 1 1* C4 Sulfonate PFBS C6 Sulfonate PFHS C6 Sulfonate PFOS P e r fl u o ro b u ta n e tu lfo n a te ___________________Pertluorohcxanesulfo nate ___________________P erfluoroocta nesulfo nate Analyte Found (ppb, ng/g) Acceptable LOQ (ng/g) Assessed Accuracy <+/- %) Analyte Found (ppb. ng/g) Acceptable LOQ (ng/g) Assessed Accuracy (./-%> Analyte Found (ppb, ng/g) Acceptable LOQ (ng/g) Assessed Accuracy {+/- %) ND 0.10 30 ND 0.10 30 12.5* 0.10 30 ND 0.10 30 ND 0.10 30 19.8A 0.10 30 ND 0.10 30 0.133 0.10 30 53.4 0.10 30 ND 0.10 30 0.127 0.10 30 52.2 0.10 30 0.118 0.10 30 0.139 0.10 30 66.4 0.10 30 0.109 0.10 30 0.142 0.10 30 66.2 0.10 30 0.110' 0.10 30 0.109 0.10 30 17.3A 0.10 30 ND1 0.10 30 0.100 0.10 30 13.3A 0.10 30 ND 0.10 30 ND' 0.10 30 17.9 0.10 30 ND 0.10 30 0.106' 0.10 30 21.2 0.10 30 ND 0.10 30 ND 0.10 30 NR* . - ND 0.10 30 ND 0.10 30 NR* - - ND 0.10 30 ND 0.10 30 11.4 A 0.20 50 ND 0.10 30 ND 0.10 30 8.90* 0.20 50 ND 0.10 40 ND 0.10 30 7.92 0.20 30 ND 0.10 40 ND 0.10 30 8.50 0.20 30 ND 0.10 50 ND 0.10 30 4.39 0.20 30 ND 0.10 50 ND 0.10 30 3.69 0.20 30 ND 0.10 40 ND 0.10 30 4.26A 0.20 30 ND 0.10 40 ND 0.10 30 5.49* 0.20 30 ND 0.10 30 ND 0.10 30 ND 0.10 30 ND 0.10 30 ND 0.10 30 ND 0.10 30 ND 0.10 50 ND 0.10 40 19.1* 0.10 40 ND 0.10 50 ND 0.10 40 10.1* 0.10 40 ND 0.10 30 ND 0.10 30 6.66* 0.10 30 ND 0.10 30 ND 0.10 30 8.33* 0.10 30 ND 0.10 40 ND 0.10 40 ND 0.10 40 3.70 0.10 30 ND 0.10 40 3.51 0.10 30 ND 0.10 50 ND 0.10 40 4.24 0.10 30 ND 0.10 50 ND 0.10 40 4.33 0.10 30 0.803* 0.300A 0.10 0.10 40 0.9B2A 0.10 40 0.615A 0.10 40 40 7290 7650 0.10 0.10 30 30 1.02* 0.10 30 0.456 0.10 30 328 0.10 30 1.27A 0.10 30 0.450 0.10 30 271 0.10 30 ND 0.10 30 0.473 0.10 30 221* 0.10 30 ND 0.10 30 0.391 0.10 30 274* 0.10 30 1.22 0.10 30 0.834A 0.10 30 5490 0.10 30 1.18 0.10 30 0.605A 0.10 30 5130 0.10 30 0.252 0.10 30 0.545 0.10 30 5670 0.10 30 0.284 0.10 30 0.624 0.10 30 4940 0.10 30 0.199 0.203 0.10 0.10 30 30 1.34 1.36 0.10 0.10 30 30 1070 1070 0.10 0.10 30 30 ND 0.10 30 0.575 0.10 30 894 0.10 30 ND 0.10 30 0.587 0.10 30 983 0.10 30 0.108 0.10 30 0.387 0.10 30 1030 0.10 30 0.106 0.10 30 0.375 0.10 30 1060 0.10 30 'Laboratory Duplicate *Retative Percent Difference > 20% 'Relative Percent Difference was not calculated due to the presence of a nondetect and resulting uncertainty. ND = Not detected at or above the acceptable LOQ. NR* * Not reported due to quality control failures; see Table II for re-extracted sample results. MPI Research Page 28 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table I. Summary of PFBS, PFHS, and PFOS in Fish Fillet Samples (continued) ExyLIMS ID C0227568 C0227568 Rep C0227569 C0227569 Rep C0227570 C0227570 Rep C0227571 C0227571 Rep C0227572 C0227572 Rep C0227573 C0227573 Rep C0227574 C0227574 Rep C0227575 C0227575 Rep C0227576 C0227576 Rep C0227577 C0227577 Rep C0227578 C022778 Rep C0227579 C0227579 Rep C0227580 C0227580 Rep C0227581 C02275581 Rep C0227582 C02275582 Rep C0227583 C0227583 Rep C0227584 C0227584 Rep C0227585 C0227585 Rep C0227586 C0227586 Rep C0227587 C0227587 Rep C0227588 C0227588 Rep C0227589 C0227589 Rep C0227590 C0227590 Rep Client Sample ID DBC-F02-IPF004-0-061211 DBC-F02-IPF004-0-061211* DBC-F02-IPF005-0-061211 D B C -F 0 2 -IP F 0 0 5 -0-0 61 21 1 * DOU-F02-MSF001-0-061209 D O U -F 0 2 -M S F 0 0 1 -0 -0 6 1 2 0 9 * DOU-F02-MSF002-0-061212 DOU-F02-MSF002-0-061212* DOU-F02-MSF003-0-061212 DOU-F02-MSF003-0-061212* DOU-F02-MSF004-0-061212 DOU-F02-MSF004-0-061212* DO U-F02-MSF005-0-061212 DOU-F02-MSF005-0-061212* DOU-F02-IPF001-0-061212 DOU-F02-IPF001-0-061212* D O U - F 0 2 -IP F 0 0 2 -0 - 0 6 1212 DOU- F02-1PF002-0-061212* DO U-F02-IPF003-0-061212 DOU-F02-IPF003-0-061212* DOU-F02-IPF004-0-061212 DOU-F02-IPF004-0-061212* DO U-F02-IPF005-0-061211 D O U - F 0 2 - IP F 0 0 5 - 0 - 0 6 1 2 1 1* DLI-F02-MSF001-0-061209 D L I-F 0 2 -M S F 0 0 1 -0 -0 6 1 2 0 9 * DLI-F02-MSF002-0-061209 DLI-F02-MSF002-0-061209* DU-F02 -M S F0 0 3-0-0 6 12 0 9 DLI-F02-MSF003-0-061209* DLI-F02-MSF004-0-061209 DLI-F02-MSF004-0-061209* DLI-F02-MSF005-0-061209 DLI-F02-MSF005-0-061209* D L I-F 0 2 -IP F 0 0 1 -0 -0 6 1 2 1 2 D L I-F 0 2 -IP F 0 0 1 -0 -0 6 1 2 1 2 * D L I-F 0 2 -IP F 0 0 2 -0 -0 6 1 2 1 2 D L I-F 0 2 -IP F 0 0 2 -0 -0 6 1 2 1 2 * D L I-F 0 2 -IP F 0 0 3 -0 -0 6 1 2 1 2 D L I-F 0 2 -IP F 0 0 3 -0 -0 6 1 2 1 2 * D U - F 0 2 -IP F 0 0 4 -0 -0 6 1 212 D L I-F 0 2 -IP F 0 0 4 -0 -0 6 1 2 1 2 * D L I- F 0 2 - IP F 0 0 5 - 0-0 61 21 2 D L I-F 0 2 -IP F 0 0 5 -0 -0 6 1 2 1 2 * D M C -F02-M S F001-0 -061209 D M C -F 0 2 -M S F 0 0 1 -0 -0 61 2 09 * C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Pertluorotoutanesulfonate___________________PcrlluorohcxanesulTonate___________________Perfluorooctanesulfonate Analyte Found (ppb, ng/g) Acceptable LOQ (ng/g) Assessed Accuracy (/- %) Analyte Found (ppb, ng/g) Acceptable LOQ (ng/g) Assessed Accuracy <*/- %) Analyte Found (ppb, ng/g) Acceptable LOQ (ng/g) Assessed Accuracy {+/- %) ND 0.10 30 0.364 0.10 30 600 0.10 30 ND 0.10 30 0.374 0.10 30 610 0.10 30 ND ND 2.35 2.61 0.10 0.10 0.10 0.10 30 30 30 30 0.541 0.10 30 NR* - - 0.603 0.10 30 NR* - - 4.71 5.03 0.10 30 1060* 0.20 30 0.10 30 836A 0.20 30 0.353' 0.10 30 1.37* 0.10 30 840* 0.20 50 ND' 0.10 30 1.04* 0.10 30 1380* 0.20 50 2.99 2.66 0.10 0.10 30 30 1.32 1.13 0.10 0.10 30 30 1030 1260 0.20 0.20 30 30 0.203A 0.10 30 0.299A 0.10 30 5010 0.20 30 0.264A 0.10 30 0 .4 13A 0.10 30 4630 0.20 30 0.842 0.851 0.10 0.10 30 0.893A 0.10 30 1.22* 0.10 30 30 NR* NR* - 1.10 0.10 30 0.307 0.10 40 260 0.10 30 1.25 0.10 30 0.347 0.10 40 267 0.10 30 NR NR - 0.678 0.10 40 230 0.10 30 0.774 0.10 40 217 0.10 30 0.447 0.10 30 0.354 0.10 40 877 0.10 30 0.447 0.10 30 0.387 0.10 40 900 0.10 30 1.39 0.10 30 0.401 0.10 40 1.45 0.10 30 0.441 0.10 40 424 0.10 30 466 0.10 30 1.08 0.10 30 0.398 0.10 40 255 0.10 30 1.16 0.10 30 0.392 0.10 40 258 0.10 30 ND 0.10 30 0.530 0.10 30 460* 0.50 30 ND 0.10 30 0.634 0.10 30 347* 0.50 30 0.128' 0.10 30 0.452 0.10 30 316 0.50 30 ND' 0.10 30 0.418 0.10 30 325 0.50 30 0.160' 0.10 30 0.709 0.10 30 266 0.50 30 ND' 0.10 30 0.714 0.10 30 242 0.50 30 0.109' 0.10 30 0.483A 0.10 30 ND' 0.10 30 0.754A 0.10 30 206* 290* 0.50 0.50 30 30 ND' 0.10 30 0.687 0.10 30 0.170' 0.10 30 0.714 0.10 30 256* 335* 0.50 0.50 30 30 ND 0.10 30 0.148 0.10 30 56.3 0.10 30 ND 0.10 30 0.157 0.10 30 59.4 0.10 30 ND 0.10 30 0.109 0.10 30 12.9 0.10 30 ND 0.10 30 0.114 0.10 30 12.5 0.10 30 ND 0.10 30 ND 0.10 30 21.0 0.10 30 ND 0.10 30 ND 0.10 30 22.7 0.10 30 ND 0.10 30 0.162 0.10 30 49.7 0.10 30 ND 0.10 30 0.171 0.10 30 45.4 0.10 30 ND 0.10 30 0.129 0.10 30 51.1 0.10 30 ND 0.10 30 0.135 0.10 30 53.7 0.10 30 0.171 0.10 30 0.358A 0.10 30 0.190 0.10 30 0.567A 0.10 30 389* 286* 0.10 0.10 30 30 'Laboratory Duplicate ARelative Percent Difference > 20% 'Relative Percent Difference was not calculated due to the presence of a nondetect and resulting uncertainty. ND Not detected at or above the acceptable LOQ. NR = Not reported due to quality control failures. NR* Not reported due to quality control failures; see Table II for re-extracted sample results. MPI Research Page 29 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table I. Summary of PFBS, PFHS, and PFOS in Fish Fillet Samples (continued) ExyLlMS ID C0227591 C0227591 Rep C0227592 C0227592 Rep C0227593 C0227593 Rep C0227594 C0227594 Rep C0227595 C0227595 Rep C0227596 C0227596 Rep C0227597 C0227597 Rep C0227598 C0227598 Rep C0227599 C0227599 Rep Client Sample ID DMC-F02-MSF002-0-061209 DMC-F02-MSF002-0-061209* DMC-F02-MSF003-0-061209 DMC-F02-MSF003-0-061209* DMC-F02-MSF004-0-061212 D M C -F 0 2 - M S F 0 0 4 -0 - 0 6 1212* DMC-F02-MSF005-0-061212 DMC-F02-MSF005-0-061212* D M C -F 0 2 -IP F 0 0 1 -0-0 61212 DMC-F02-IPF001 -0-061212* D M C -F02-IP F002-0-061212 D M C -F 0 2 -IP F 0 0 2 -0 -0 6 1 2 1 2 * D M C -F 0 2 -IP F 0 0 3 -0 -0 6 1 2 1 2 D M C -F02-IP F003-0-061212* DMC-F02-IPF004-0-061212 D M C -F 0 2 - IP F 0 0 4 -0 -0 6 1212* D M C -F 0 2 -IP F 0 0 5 -0 -0 6 1 2 1 2 D M C -F 0 2 - IP F 0 0 5 -0 -0 6 1212* ' Laboratory Duplicate ARelative Percent Difference > 20% ND Not detected at or above the acceptable LOQ. C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS P crflu o ro b u ta ne su lfo na te_____________________ P e rflu o ro h e x a n e tu lfo n a te _____________________ P e rflu oro octa ne au lfon ate ND Found (bpb, nq/q) Acceptable LOQ (nq/q) Assessed Accuracy (/-%) Analyte Found (PPb. nfl/fl) Acceptable LOQ (nq/q) Assessed Accuracy (+/-%) Analyte Found (ppb. nq/q) Acceptable LOQ (nq/q) Assessed Accuracy W - %) 0.156 0.156 0.10 0.10 30 30 0.604 0.520 0.10 0.10 30 30 327* 247* 0.10 0.10 30 30 0.152 0,10 30 0.447 0.10 30 0.138 0,10 30 0.513 0.10 30 262* 327* 0.10 0.10 30 30 0.131 0.10 30 0.393 0.10 30 226 0.10 30 0.135 0.10 30 0.381 0.10 30 253 0.10 30 ND 0.10 30 0.227 0.10 30 370* 0.10 30 ND 0.10 30 0.267 0.10 30 293* 0.10 30 ND 0.10 30 0.114 0.10 30 14.9 0.20 30 ND 0.10 30 0.119 0,10 30 16.2 0.20 30 ND 0.10 30 0.109 0.10 30 20.8 0.20 30 ND 0.10 30 0.114 0.10 30 19.3 0.20 30 ND 0.10 30 0.109 0.10 30 13.2 0.20 30 ND 0.10 30 0.108 0.10 30 13.5 0.20 30 0.100 0.10 30 0.169 0.10 30 0.101 0.10 30 0.166 0.10 30 12.8 0.20 30 13.3 0.20 30 0.232 0.10 30 0.128 0.10 30 0.245 0.10 30 0.127 0.10 30 12.0 11.7 0.20 0.20 30 30 MPI Research Page 30 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table II. Summary of PFOS in Re-extracted Fish Fillet Samples ExyLIMS ID C0227550 C0227550 Rep C0227569 C0227569 Rep C0227574 C0227574 Rep "Laboratory Duplicate Client Sample ID DL3-F02-IPF001-0-061211 DL3-F02-IPF001-0-061211* DBC -F02-IPF005-0-061211 DBC-F02-IPF005-0-061211 * D O U -F 0 2 -M S F 0 0 5 -0 -0 6 1 212 D O U -F 0 2 -M S F 0 0 5 -0 -0 6 1212* Analyte Found (PPb, ng/g) 3.17 3.10 1280 1180 631 576 C8 Sulfonate PFOS Perfluorooctanesulfonate Acceptable LOQ (ng/g) 0.10 0.10 0.10 0.10 0.10 0.10 Assessed Accuracy (+/- %) 30 30 30 30 30 30 MPI Research Page 31 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table III. Summary of PFBS, PFHS, and PFOS in Whole Body Fish Samples E xyL IM S ID C0227600 C 0227600 Rep C0227601 C0227601 Rep C0227602 C0227602 Rep C0227603 C0227603 Rep C0227604 C 0227604 Rep C0227605 C 0227605 Rep C0227606 C0227606 Rep C0227607 C0227607 Rep C0227608 C 0227608 Rep C0227609 C 0227609 Rep C0227610 C 0227610 Rep C0227611 C 0227611 Rep C0227612 C 0227612 Rep C0227613 C 0227613 Rep C0227614 C 0227614 Rep C0227615 C 0227615 Rep C0227616 C 0227616 Rep C0227617 C 0227617 Rep C0227618 C 0227618 Rep C0227619 C0227619 Rep C0227620 C0227620 Rep C0227621 C0227621 Rep C0227622 C 0227622 Rep C lie n t S am p le ID D L 3-F 02-M S W 001-0-061207 D L 3-F 02-M S W 001-0-061207 D L 3-F 02-M S W 002-0-061207 D L 3-F 02-M S W 002-0-061207 D L 3-F 02-M S W 003-0-061207 D L 3-F 02-M S W 003-0-061207 D L 3-F 02-M S W 004-0-061207 D L 3-F 02-M S W 004-0-061207 D L 3-F 02-M S W 005-0-061207 D L 3-F 02-M S W 005-0-061207 D L 3 -F 0 2 -IP W 00 1 -0-06 1 21 2 D L 3 -F 0 2 -IP W 00 1 -0-06 1 21 2 D L 3 -F 0 2 -IP W 0 0 2 -0 -0 6 1 2 1 2 D L 3-F 0 2 -IPW 0 0 2 -0 -0 6 1212 D L 3 -F 0 2 -IP W 0 0 3-0 -06 1 21 2 D L 3-F 02-IP W 003-0-061212 D L 3 -F 0 2 -IP W 0 0 4 -0 -0 6 1 2 1 2 D L 3 -F 0 2 -IP W 0 0 4 -0 -0 6 1 2 1 2 D L 3 -F 0 2 -IP W 00 5 -0-06 1 21 2 D L 3-F 0 2 -IP W 00 5 -Q -0 61 212 D L 2-F02-M S W 001-0-061211 D L 2 -F 0 2 -M S W 0 0 1 -0-061211 D L 2 -F 0 2 -M S W 0 0 2 -0 -0 6 1211 D L 2 -F 0 2 -M S W 0 0 2 -0 -0 6 1211 D L 2 -F 0 2 -IP W 0 0 1 -0 -0 6 1 2 0 9 D L 2-F 02-IP W 001-0-061209 D L 2 -F 0 2 -IP W 00 2 -0-06 1 20 9 D L 2 -F 0 2 -IP W 00 2 -0-06 1 20 9 D L 2 -F 0 2 -IP W 00 3 -0-06 1 20 9 D L 2-F 02-IP W 003-0-061209 D L 2-F 02-IP W 004-0-061209 D L 2-F 02-IP W 004-0-061209 D L 2-F 02-IP W 005-0-061209 D L 2 -F 0 2 -IP W 00 5 -0-06 1 20 9 D B C -F 0 2 -M S W O 0 1-0 -0 6 1 2 0 7 D B C -F 0 2 -M S W 0 0 1 -0-0 61 2 07 D B C -F02-M S W 002-0-061207 D B C -F02-M S W 002-0-061207 D B C -F 02-M S W 003-0-061207 D B C -F 02-M S W 003-0-061207 D B C -F 02-M S W 004-0-061207 D B C -F 02-M S W 004-0-061207 D B C -F 02-M S W 005-0-061207 D B C -F 02-M S W 005-0-061207 D B C -F 0 2 -IP W 0 0 1 -0 -0 6 1 2 1 1 D B C -F 0 2-IP W 0 0 1 -0-061211 C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS P erflu o ro b u ta n e su lfo n a te ___________ P e rfluorohexanesu lfonate______________P e rfluorooctanesulfo nate Analyte Found (ppb, ng/g) Acceptable LOQ (ng/g) Assessed Accuracy (+/- %) Analyte Found (ppb, ng/g) A c ce p ta b le LOQ (ng/g) Assessed Accuracy (+/- %) Analyte Found (ppb, ng/g) Acceptable LOQ (ng/g) Assessed Accuracy (+/- %) ND 0 .10 30 0 .128 0 .1 0 30 202 0.1 0 40 ND 0 .10 30 0 .128 0 .10 30 204 0.1 0 40 N D 0.10 N D 0.10 30 0.161 0 .1 0 30 0 .147 0 .10 30 30 108 0 .10 109 0 .10 30 30 ND 0 .1 0 30 0 .170 0 .10 30 ND 0 .1 0 30 0 .169 0 .10 30 196 0 .10 189 0 .10 30 30 N D 0.10 30 N D 0.10 30 ND 0 .10 30 8 6 .4 0 .10 30 ND 0 .10 30 88.1 0 .10 30 ND 0 .1 0 30 0 .7 4 4 0 .10 30 354 0 .10 30 ND 0 .1 0 30 0 .7 2 2 0 .10 30 327 0 .10 30 N D 0.10 30 N D 0 .10 30 ND 0 .1 0 30 8 .4 5 0 .10 30 ND 0 .1 0 30 8 .0 3 0 .10 30 ND 0 .10 30 N D 0 .10 30 ND 0 .1 0 30 9 .6 1 0 .1 0 30 ND 0 .1 0 30 9 .7 4 0 .1 0 30 ND 0.10 30 ND 0.10 30 ND 0 .1 0 30 1 6 .9 0 .10 30 ND 0 .1 0 30 1 6 .6 0 .1 0 30 ND 0 .1 0 30 0.321 0 .1 0 30 13.3 0 .10 30 ND 0 .1 0 30 0 .3 3 0 0 .10 30 13.2 0 .10 30 N D 0 .10 30 N D 0 .10 30 ND 0 .10 30 7 .68 0 .10 30 ND 0 .1 0 30 7 .8 3 0 .10 30 0 .1 8 3 0 .1 8 2 0 .10 0 .10 30 30 ND 0 .2 0 30 7 0.6A 0 .10 50 ND 0 .2 0 30 5 5.7 A 0 .10 50 0 .1 4 1 0 .1 5 1 0 .10 0 .1 0 30 30 ND 0 .2 0 30 7 5 .8 0 .1 0 30 ND 0 .2 0 30 7 9 .5 0 .1 0 30 ND 0 .10 30 ND 0 .10 30 ND 0 .10 30 4 0 .2 0 .10 30 ND 0 .10 30 3 9 .3 0 .10 30 ND 0 .10 30 ND 0 .10 30 ND 0 .10 30 17.2 0 .10 30 ND 0 .1 0 30 18.3 0 .10 30 ND 0 .10 30 0 .1 0 1 1 0 .1 0 30 4 0 .3 0 .10 30 ND 0 .10 30 ND1 0 .10 30 3 8 .4 0 .10 30 N D 0.1 0 30 ND 0.1 0 30 ND 0 .10 30 4 3 .1 0 .1 0 30 ND 0 .10 30 4 8 .7 0 .10 30 ND 0 .10 30 ND 0 .10 30 ND 0 .10 30 3 7 .2 0 .10 30 ND 0 .10 30 3 6 .7 0 .10 30 2 .7 7 0 .10 30 7 .58 0 .1 0 40 1610 0 .10 50 2 .7 1 0 .10 30 7 .6 2 0.1 0 40 1640 0 .10 50 0 .7 1 7 0 .10 30 5 .55 0 .10 30 NR* 0 .806 0.1 0 30 5 .5 2 0 .10 30 NR* - . - 0 .361A 0.1 0 30 1 .1 8 0 .10 30 4700 0 .10 40 0 .249A 0 .10 30 1 .0 4 0 .10 30 4120 0 .1 0 40 4 .7 1 0.1 0 30 3 .4 1 0 .1 0 30 1520 0 .10 30 5 .04 0 .10 30 3 .44 0.1 0 30 1460 0 .10 30 6 .54 0 .10 30 2 .7 1 0.1 0 30 2540 0 .10 30 6 .34 0 .10 30 2 .73 0.1 0 30 2260 0 .10 30 2 .36 0 .10 30 2 .6 7 0 .10 30 558 0 .10 30 2 .14 0.1 0 30 2 .8 7 0 .10 30 576 0 .10 30 ` Laboratory Duplicate ARelative Percent Difference > 20% 'Relative Percent Difference was not calculated due to the presence o f a nondetect and resulting uncertainty. ND = Not detected at or above the acceptable LOQ. NR* = Not reported due to quality control failures; see Table IV for re-extracted sample results. MPI Research Page 32 of 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table III. Summary of PFBS, PFHS, and PFOS in Whole Body Fish Samples (continued) E xyLIM S ID C0227623 C0227623 Rep C0227624 C0227624 Rep C0227625 C0227625 Rep C0227626 C0227626 Rep C0227627 C0227627 Rep 00227628 C0227628 Rep C0227629 C0227629 Rep C0227630 C0227630 Rep C0227631 C0227631 Rep C0227632 C0227632 Rep C0227633 C0227633 Rep C0227634 C0227634 Rep C0227635 C0227635 Rep C0227636 C0227636 Rep C0227637 C0227637 Rep C0227638 C0227638 Rep C0227639 C0227639 Rep C0227640 C0227640 Rep C0227641 C02276441 Rep C0227642 C0227642 Rep C0227643 C0227643 Rep C0227644 C0227644 Rep C0227645 C0227645 Rep Client S am ple ID D B C -F02-IPW 002-0-061211 D B C -F02-IPW 002-0-061211 D B C -F02-IP W 003-0-061211 D B C -F02-IP W 003-0-061211 D B C -F02-IP W 004-0-061211 D B C -F 02 -IP W 0 04 -0 -0 6 1211 D B C -F 0 2 -IP W 0 0 5 -0 -0 6 1 2 11 D B C -F 02 -IP W 0 05 -0 -0 6 1211 DO U-F02-M SW 001-0-061209 DO U-F02-M SW 001-0-061209 D O U-F02-M SW 002-0-061212 DOU-F02-M SW 002-0-061212 DOU-F02-M SW 003-0-061212 DOU-F02-M SW 003-0-061212 D O U -F02-M SW 004-0-061212 DO U -F02-M SW 004-0-061212 D O U-F02-M SW 005-0-061212 D O U-F02-M SW 005-0-061212 DOU-F02-IPW 001-0-061212 DOU-F02-IPW 001-0-061212 D O U -F02-IP W 002-0-061212 DOU-F02-IPW 002-0-061212 DO U -F02-IPW 003-0-061212 DOU-F02-IPW 003-0-061212 DO U-F02-IPW 004-0-061212 DO U-F02-IPW 004-0-061212 D O U -F 0 2 -IP W 0 0 5 -0 -0 6 1 2 12 DO U-F02-IPW 005-0-061212 D L1-F02-M SW 001-0-061209 D L1-F02-M SW 001-0-061209 D L1-F02-M SW 002-0-061209 DL1-F02-M SW 002-0-061209 DL1-F02-M SW 003-0-061209 DL1-F02-M SW 003-0-061209 DL1-F02-M SW 004-0-061209 D L1-F02-M SW 004-0-061209 D L1-F02-M SW 005-0-061209 D L1-F02-M SW 005-0-061209 DL1-F02-IPW 001-0-061212 DL1-F02-IPW 001-0-061212 DL1-F02-IPW 002-0-061212 DL1-F02-IPW 002-0-061212 DL1-FO2-IPW 0O 3-0-061212 D L 1-F 0 2 -IPW 0 0 3 -0 -0 6 1212 D L1-F02-IPW 004-0- 61215 DL 1-F 0 2 -IPW 0 0 4 -0 -0 6 1215 C4 Sulfonate PFBS Perfluorobutanesulfonate A n a ly te Found (PPb, ng/g) Acceptable LOQ (ng/g) Assessed Accuracy (+/- %) C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorohexanesulfonate_____________PerfluorooctanesuHonate Analyte Found (ppb, ng/g) Acceptable LOQ (ng/g) Assessed Accuracy (+/-% ) A n a ly te Found (ppb, ng/g) A c c e p ta b le LOQ (n g /g ) Assessed Accuracy <+/- %) 1.53 0.10 30 0.821 0.10 30 265 0.10 30 1.50 0.10 30 0 .7 6 6 0 .1 0 30 267 0.10 30 1.11 0.10 30 1.62 0 .1 0 30 4700 0 .1 0 30 1.06 0.10 30 1.58 0 .1 0 30 4770 0 .1 0 30 ND1 0.10 30 0 .1 0 5 * 0 .1 0 30 395 0.10 30 0 .2 5 5 1 0 .1 0 30 0 .2 2 9 * 0 .1 0 30 363 0.10 30 2.58 2.49 0.10 0.10 30 0.941 0 .1 0 30 0 .9 2 4 0 .1 0 30 3160 0 .1 0 30 3260 0 .1 0 30 30 2.86 0.10 30 2 .1 9 0 .1 0 30 2.60 0 .1 0 30 2 .0 6 0 .1 0 30 115 0.10 50 115 0.10 50 2.03 0 .1 0 30 5 .4 8 0 .1 0 30 4230 0.10 30 1.91 0 .1 0 30 5 .0 0 0 .1 0 30 4150 0.10 30 3.14 0.10 50 2.37 0.10 30 375* 0 .1 0 40 3.36 0.10 50 2.68 0.10 30 614* 0 .1 0 40 1.78 0.10 30 1.59 0.10 30 NR* 1.57 0.10 30 1.60 0.10 30 NR* . - - 5.02 0.10 30 3.02 0.10 30 NR* 4 .9 3 0.10 30 3.03 0 .1 0 30 NR* . . - 0.382 0.10 30 0 .5 5 9 0 .1 0 30 1190 0.10 30 0.364 0 .1 0 30 0.532 0 .1 0 30 1310 0 .1 0 30 1.47 0 .1 0 40 0.731 0 .1 0 50 1100 0 .1 0 30 1.45 0 .1 0 40 0.717 0 .1 0 50 1110 0 .1 0 30 1.40 0 .1 0 30 2 .5 6 0.10 30 2800 0 .1 0 30 1.48 0 .1 0 30 2 .5 8 0.10 30 2550 0 .1 0 30 0 .4 5 4 0 .4 5 5 0 .1 0 0.10 30 0 .1 5 9 0.10 30 0.167 0.10 30 299* 0 .1 0 30 7 9 .3 * 0 .1 0 30 30 7.42 0.10 30 1.39 0.10 30 1070 0 .1 0 30 7.27 0.10 30 1.45 0 .1 0 30 1150 0.10 30 0.429 0 .1 0 30 1.41 0 .1 0 30 NR* 0.499 0 .1 0 30 1.54 0 .1 0 30 NR* . - - 0.178 0.171 0 .1 0 0 .1 0 30 0 .7 2 8 0 .1 0 30 0.761 0.10 30 30 166 0.20 150 0.20 50 50 0 .4 6 5 0 .1 0 30 1.27 0.10 30 349 0.20 50 0 .4 4 5 0 .1 0 30 1.35 0 .1 0 30 303 0.20 50 0.137 0.10 30 1.29 0.10 30 NR* . . 0 .1 3 9 0 .1 0 30 1.33 0 .1 0 30 NR* - - 0.277 0 .1 0 30 1.70 0 .1 0 30 212 0.20 50 0.274 0 .1 0 30 1.75 0 .1 0 30 213 0.20 50 0.203 0 .1 0 40 0 .2 0 8 0 .1 0 40 32.3 0 .1 0 30 0.207 0 .1 0 40 0 .1 9 8 0 .1 0 40 30.3 0 .1 0 30 0.234 0.235 0 .1 0 0 .1 0 30 0 .2 4 0 0 .1 0 30 0 .2 3 9 0 .1 0 30 30 134 0.10 139 0.10 30 30 0 .2 4 0 0 .1 0 30 0.184 0 .1 0 30 23.8 0 .1 0 30 0 .2 0 9 0 .1 0 30 0.182 0 .1 0 30 23.9 0 .1 0 30 0 .6 4 3 0 .1 0 30 0 .3 4 6 0 .1 0 30 34.0 0 .1 0 30 0.624 0 .1 0 30 0.334 0 .1 0 30 34.2 0 .1 0 30 ` Laboratory Duplicate ARelative Percent Difference > 20% 'R elative Percent Difference was not calculated due to the presence of a nondetect and resulting uncertainty. ND = Not detected at or above the acceptable LOQ. NR* = Not reported due to quality control failures; see Table IV fo r re-extracted sam ple results. MPI Research Page 33 of 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table III. Summary of PFBS, PFHS, and PFOS in Whole Body Fish Samples (continued) E xyU M S ID C0227646 C0227646 Rep C0227647 C0227647 Rep C0227648 C0227648 Rep C0227649 C0227649 Rep C0227650 C0227650 Rep C0227651 C0227651 Rep C0227652 C0227652 Rep C0227653 C0227653 Rep C0227654 C0227654 Rep C0227655 C0227655 Rep C0227656 C0227656 Rep Client S am ple ID D L1-F02-IPW 005-0-061215 DL1-F02-IPW 005-Q -061215 DM C -F02-M SW 001-0-061209 DM C -F02-M SW 001-0-061209 DM C -F02-M SW 002-0-061209 DM C-F02-M SW 002-0-061209 DM C-F02-M SW 003-0-061209 DM C-F02-M SW 003-0-061209 DM C-F02-M SW 004-0-061212 DM C-F02-M SW 004-0-061212 DM C-F02-M SW 005-0-061212 DM C-F02-M SW 005-0-061212 D M C -F02-IP W 001-0-061212 D M C -F02-IP W 001-0-061212 D M C -F02-IP W 002-0-061212 D M C -F02-IP W 002-0-061212 D M C -F02-IP W 003-0-061212 DM C-F 02-IP W 003-0-061212 D M C -F02-IP W 004-0-061212 D M C -F02-IP W 004-0-061212 D M C -F02-IP W 005-0-061212 D M C -F02-IP W 005-0-061212 C4 Sulfonate PFBS C6 Sulfonate PFHS Perfluorobutanesulfonate____________ Perfluorohexanesulfonate C8 Sulfonate PFOS ______ Perfluorooctanesutfonate Analyte Found (PPb, ng/g) Acceptable LOQ (n g /g ) Assessed Accuracy <+/- % i A n a ly te Found (PPb, ng/g) Acceptable LOQ (n g /g ) Assessed Accuracy (+/- %) Analyte Found (PPb, ng/g) Acceptable LOQ (n g /g ) Assessed Accuracy (+/- %> 0.323 0.10 30 0 .3 4 5 0 .1 0 30 25.9 0 .1 0 30 0.301 0 .1 0 30 0 .3 4 6 0 .1 0 30 25.9 0 .1 0 30 0.237 0 .1 0 30 0 .4 5 2 0.20 30 241 0.50 30 0.246 0 .1 0 30 0 .4 1 0 0.20 30 245 0.50 30 0 .3 7 3 * 0 .1 0 30 0 .5 9 8 0 .2 0 30 128* 0 .5 0 30 0 .2 9 3 * 0.10 30 0 .5 0 5 0.20 30 195* 0 .5 0 30 0.396 0 .4 7 9 0.10 0.10 30 0 .9 2 3 0.20 30 0 .9 5 4 0.20 30 30 167 0.50 166 0.50 30 30 1 .1 6 * 0.10 30 1.76* 0 .2 0 30 222* 0.50 30 0 .3 3 1 * 0.10 30 0 .7 0 6 * 0 .2 0 30 130* 0 .5 0 30 0.154 0 .1 0 30 0 .5 8 3 0.20 30 147 0.50 30 0.156 0 .1 0 30 0.481 0.20 30 131 0 .5 0 30 0.208 0.10 30 0 .1 8 0 0.10 30 25.0 0 .1 0 30 0.201 0.10 30 0.173 0.10 30 23.5 0 .1 0 30 0 .2 0 9 0.10 30 0 .2 7 9 0 .1 0 30 31.6 0.10 30 0.188 0.10 30 0 .2 7 5 0 .1 0 30 31.8 0 .1 0 30 0.101 0.10 50 0.181 0 .1 0 50 12.1 0 .1 0 30 0.108 0.10 50 0.201 0 .1 0 50 12.4 0 .1 0 30 1.16 0 .1 0 30 0 .5 4 0 0.10 30 185 0.10 40 1.14 0 .1 0 30 0.582 0.10 30 184 0.10 40 0.127 0 .1 0 30 0.151 0.10 30 32.7 0 .1 0 30 0.131 0.10 30 0 .1 6 8 0.10 30 34.1 0.10 30 ' Laboratory Duplicate ARelative Percent Difference > 20% MPI Research Page 34 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table IV. Summary of PFOS in Re-extracted Whole Body Fish Samples ExyLIMS ID C0227618 C0227618 Rep C0227630 C0227630 Rep C0227631 C0227631 Rep C0227637 C0227637 Rep C0227640 C0227640 Rep ' Laboratory Duplicate Client Sample ID D B C -F02-M S W 002-0-061207 D B C -F02-M S W 002-0-061207 D O U -F02-M S W 004-0-061212 D O U -F02-M S W 004-0-061212 D O U -F02-M S W 005-0-061212 D O U -F02-M S W 005-0-061212 DL1-F02-MSW001 -0-061209 DL1-F02-MSW001 -0-061209 D L 1-F 02-M S W 004-0-061209 D L 1-F 02-M S W 004-0-061209 Analyte Found (ppb, ng/g) C8 Sulfonate PFOS Perfluorooctanesulfonate Acceptable LOQ (ng/g) 13500 11400 0.20 0.20 203 0.20 176 0.20 381 0.20 376 0.20 295 0.20 300 0.20 248 0.20 245 0.20 Assessed Accuracy (+/- %) 30 30 30 30 30 30 30 30 30 30 MPI Research Page 35 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table V. Summary of PFBS, PFHS, and PFOS in Clam Samples ExyLIMS id C0227658 C0227658 Rep C0227659 C0227659 Rep C0227660 C0227660 Rep C0227661 C0227661 Rep C0227662 C0227662 Rep C0227663 C0227663 Rep Client S am ple ID DL3-I02-CFW001 -0-061219 D L3-I02-C FWO01-0-061219* DL2-I02-CFW 001-0-061219 DL2-I02-CFW001 -0-061219* DBC-102-C FWO01-0-061219 DBC-I02-CFW 001-0-061219* D O L M 0 2-C FW 0 01 -0 -Q 6 12 19 DOU-I02-CF W001 -0-061219* D-I02-CFW 001-0-061219 DLI-I02-CFW001 -0-061219* DMC-I02-CFW 001-0-061219 DMC-I02-CFW 001-0-061219* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Periluorobutanesutfonate______________ P e r f l u o r o h e x a n e s u l f o n a t e ______Perfluorooctanesulfonate Analyte Found (ppb, ng/g) Acceptable LOQ (ng/g) Assessed Accuracy <'-*> Analyte Found (ppb. ng/g) Acceptable LOQ (ng/g) Assessed Accuracy (/-%> Analyte Found (ppb, ng/g) Acceptable LOQ (ng/g) Assessed Accuracy <+/- %) ND 0.10 30 ND 0.10 30 NR NR 0.750 0.10 30 - 0.785 0.10 30 ND 0.10 30 ND 0.10 30 NR NR . 0.751 0.10 40 0.791 0.10 40 ND 0.10 30 ND 0.10 30 ND 0.10 30 2.98 0.10 30 ND 0.10 30 3.23 0.10 30 0.233 0.236 0.10 0.10 30 30 ND 0.10 40 2.27 0.10 30 ND 0.10 40 2.35 0.10 30 0.163 0.169 0.10 0.10 30 30 ND 0.10 30 1.31 0.10 30 ND 0.10 30 1,37 0.10 30 ND 0.10 30 ND 0.10 30 ND 0.10 30 0.791 0.10 30 ND 0.10 30 0.909 0.10 30 'Laboratory Duplicate ND = Not detected at or above the acceptable LOQ. NR = Not reported due to quality control failures. MPI Research Page 36 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table VI. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Fish Fillet Samples Sample Description D L 3 -F 0 2 -M S F 0 0 1 -0 -0 61 2 07 (C0227S44 Spk O, 2.0 ppb spike) DL3-F02-MSF001-0-061207 (C0227S44 Spk E. 100 ppb spike) DL3-F02-MSF002-0-061207 (C0227S46 Spk F, 2.0 ppb spike) DL3-F02-MSF002-0-061207 (C0227S46 Spk G, 100 ppb spike) DL3-F02-MSF003-0-061207 (C0227547 Spk H. 2.0 ppb spike) DL3-F02-MSF003-0-061207 IC0227S47 Spk 1,100 ppb spike) DL3-F02-MSF004-0-061207 (C0227548 Spk J, 2.0 ppb spike) DL3-F02-MSF004-0-061207 (C0227548 Spk K, 100 ppb Spike) DL3-F02-MSF005-0-061207 (C0227S49 Spk L, 2.0 ppb spike) DL3-F02-MSF005-0-061207 (C0227549 Spk M, 100 ppb spike) DL3-F02-IPF001-0-061211 (C0227SS0 Spk O. 2.0 ppb spike) DL3-F02-IPF001-0-Q61211 (C0227SS0 Spk E. 100 ppb spike) D L 3 - F 0 2 -IP F 0 0 2 -0 -0 6 1212 (C0227SS1 Spk F, 2.0 ppb spike) DL3-F02-IPF002-0-061212 (C0227551 Spk G, 100 ppb spike) DL3-F02-IPF003-0-061212 (C0227552 Spk H, 2.0 ppb spike) DL3-F02-IPF003-0-061212 (C0227SS2 Spk 1.100 ppb spike) D L 3 - F 0 2 -IP F 0 0 4 -0 -0 6 1 212 (C02275S3 Spk J, 2.0 ppb spike) DL3-F02-IPF004-0-061212 (C0227SS3 Spk K, 100 ppb spike) D L 3 - F 0 2 -IP F 0 0 5 -0 -0 6 1 21 2 (C0227S54 Spk L. 2.0 ppb spike) DL3-F02-IPF005-0-061212 (C0227554 Spk M. 100 ppb spike) DL2-F02-IPF001-0-061209 (C0227SSS Spk O. 2.0 ppb spike) DL2-F02-IPF001-0-061209 (C0227555 Spk E, 100 ppb spike) D L 2 - F 0 2 -IP F 0 0 2 -0 -0 6 1209 (C0227SSS Spk F, 2.0 ppb spike) DL2-F02-IPF002-0-061209 (C02275S6 Spk G, 100 ppb spike) DL2-F02-IPF003-0-061209 (C0227S57 Spk H, 2.0 ppb spike) DL2-F02-IPF003-0-061209 (C0227SS7 Spk 1,100 ppb spike) DL2-F02-IPF004-0-061209 (C0227558 Spk 3. 2.0 ppb spike) DL2- F02-1PF004-0-061209 (C0227558 Spk K, 100 ppb spike) Amount Spiked (nq/q) C4 Sulfonate PFBS C6 Sulfonate PFHS CS S ulfonate PFOS Perfluorobutanesulfonate_________________ Perfluorohexanesulfonate_________________ Perfluorooctanesulfonate Avg Am t Found Amount Avg A m t Found Amount Avg A m t Found Amount in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery fnq/q) (nq/q) (%) (nq/q) (ng/g) <%) (ng/g) (ng/g) <%) 2.0 ND 2.08 104 ND 2.04 102 16.2 NA NA 100 ND NA NA ND NA NA 16.2 93.1 77 2.0 ND 1.77 89 0.130 1.79 83 52.8 NA NA 100 ND NA NA 0.130 NA NA 52.8 124 71 2.0 0.113 2.00 94 0.140 2.08 97 67.3 NA NA 100 0.113 NA NA 0.140 NA NA 67,3 162 115 2.0 0.105 2.03 96 0.105 2.04 97 15.3 NA NA 100 0.105 NA NA 0.105 NA NA 15.3 113 98 2.0 ND 2.28 114 0.103 2.01 95 19.5 NA NA 100 ND NA NA 0.103 NA NA 19.5 99.1 60 2.0 ND 2.47 124 ND 2.00 100 NR* NR* NR* 100 ND NA NA ND NA NA NR* NR* NR* 2.0 ND 2.56 129 ND 1.94 97 10.2 NA NA 100 ND NA NA ND NA NA 10.2 152 142 2.0 ND 2.76 138 ND 1.92 96 8.21 NA NA 100 ND NA NA ND NA NA 8.21 132 124 2.0 ND 2.81 141 ND 2.24 112 4.04 5.74 85 100 ND NA NA ND NA NA 4.04 NA NA 2.0 ND 2.70 135 ND 2.47 124 4.87 6.64 99 100 ND NA NA ND NA NA 4.87 NA NA 2.0 ND 2.14 107 ND 2.53 127 ND 2.52 126 100 ND NA NA ND NA NA ND NA NA 2.0 ND 2.92 146 ND 2.68 134 14.6 NA NA 100 ND NA NA ND NA NA 14.6 152 137 2.0 ND 2.55 128 ND 2.57 129 7.50 9.21 86 100 ND NA NA ND NA NA 7.50 NA NA 2.0 ND 2.69 135 ND 2.77 139 3.61 5.80 110 100 ND NA NA ND NA NA 3.61 NA NA ND = Not detected at or above the acceptable LOQ reported in Table I. NA a Not applicable. This matrix spike concentratrion was not used to assess the accuracy for this analyte. NR* a Not reported due to quality control failures; see Table VIII for re-extracted sample results. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research Page 37 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table VI. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Fish Fillet Samples (continued) Sample D e s c rip tio n DL2-F02-IPF005-0-061209 (C022755B Spk L, 2.0 ppb spike) DL2-F02-IPF005-0-061209 (00227550 Spk M, 100 ppb spike) DBC-F02-MSF001-0-061207 (C0227560 Spk D. 2.0 ppb spike) DBC-F02-MSF001-0-061207 (C0227560 Spk E, 100 ppb spike) D B C -F 0 2 -M S F 0 0 2 -0 -0 6 12 07 (C0227561 Spk F, 2.0 ppb spike) DBC-F02-MSF002-0-061207 (C0227561 Spk G, 100 ppb spike) DBC-F02-MSF003-0-061207 (C0227582 Spk H. 2.0 ppb spike) D B C -F 0 2 -M S F 0 0 3 -0 -0 6 1207 (C0227562 Spk 1.100 ppb spike) DBC-F02-MSF004-0-061207 (C0227563 Spk J, 2.0 ppb spike) DBC-F02-MSF004-0-061207 (C0227503 Spk K. 100 ppb spike) D B C -F 0 2 -M S F 0 0 5 -0 -0 6 1207 (C0227564 Spk L. 2.0 ppb spike) DBC-F02-MSF005-0-061207 (C0227564 Spk M, 100 ppb spike) DBC-F02-IPF001-0-061211 (C0227585 Spk 0, 2.0 ppb spike) DBC-F02-IPF001-0-061211 (C0227565 Spk E, 100 ppb spike) DBC-F02-IPF002-0-061211 (C0227566 Spk F, 2.0 ppb spike) DBC-F02-IPF002-0-061211 (C022756S Spk G, 100 ppb spike) D B C -F 0 2 -IP F 0 0 3 -0 -061211 (C0227567 Spk H. 2.0 ppb spike) D B C -F 0 2 -IP F 0 0 3 - 0 - 0 6 1 2 11 (C0227557 Spk 1,100 ppb spike) DBC-F02-IPF004-0-061211 (C0227S68 Spk J, 2.0 ppb spike) D B C-F02-IPF004-0-0612 1 1 (C0227568 Spk K, 100 ppb spike) DBC-F02-IPF005-0-061211 (C0227669 Spk L. 2.0 ppb spike) D B C -F 0 2 -IP F 0 0 5 -0 -061211 (C0227569 Spk M, 100 ppb spike) D O U - F 0 2 -M S F 0 0 1- 0 - 0 6 1209 (C0227S70 Spk D, 2.0 ppb spike) DOU-F02-MSF001-0-061209 (C0227570 Spk E. 100 ppb spike) DOU-F02-MSF002-0-061212 (C0227571 Spk F, 2.0 ppb spike) D O U - F 0 2 -M S F 0 0 2 -0 -0 6 1212 (C0227671 Spk G, 100 ppb spike) DOU-F02-MSF003-0-061212 (C0227572 Spk H, 2.0 ppb spike) DOU-F02-MSF003-0-061212 (C0227572 Spk 1.100 ppb spike) Amount Spiked (ng/g) C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS PerfluorobutanesuKonate_________________ Perfluorohexanesulfonate_________________ Pcrfluorooctanesulfonate Avg A m t Found Amount Avg Amt Found Amount Avg Am t Found Amount in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/g) (ng/g) <%) (ng/g) (ng/g) <%> (ng/g) (ng/g) (%> 2.0 ND 2.91 146 ND 2.72 136 4.28 6.23 98 100 ND NA NA ND NA NA 4.28 NA NA 2.0 0.551 1.86 65 0.798 2.16 68 7470 NA NA 100 0.551 NA NA 0.798 NA NA 7470 NA* NA* 2.0 1.15 2.75 80 0.453 2.33 94 299 NA NA 100 1.15 NA NA 0.453 NA NA 299 NA* NA* 2.0 ND 1.96 98 0.432 2.21 89 247 NA NA 100 ND NA NA 0.432 NA NA 247 NA* NA* 2.0 1.20 2.85 83 0.719 2.47 88 5310 NA NA 100 1.20 NA NA 0.719 NA NA 5310 NA* NA* 2.0 0.268 1.97 85 0.585 2.26 84 5300 NA NA 100 0.268 NA NA 0.585 NA NA 5300 NA* NA* 2.0 0.201 2.37 108 1.35 3.66 116 1070 NA NA 100 0.201 NA NA 1.35 NA NA 1070 NA* NA* 2.0 ND 2.46 123 0.581 3.02 122 939 NA NA 100 ND NA NA 0,581 NA NA 939 NA* NA* 2.0 0.107 2.66 128 0.381 2.78 120 1040 NA NA 100 0.107 NA NA 0.381 NA NA 1040 NA* NA* 2.0 ND 2.31 116 0.369 2.67 115 605 NA NA 100 ND NA NA 0.369 NA NA 605 NA* NA* 2.0 ND 2.32 116 0.572 2.89 116 NR* NR* NR* 100 ND NA NA 0.572 NA NA NR* NR* NR* 2.0 2.48 4.99 126 4.87 7.42 128 947 NA NA 100 2.48 NA NA 4.87 NA NA 947 NA* NA* 2.0 0.227 1.78 78 1.20 3.57 119 1110 NA NA 100 0.227 NA NA 1.20 NA NA 1110 NA* NA* 2.0 2.83 4.70 94 1.23 2.62 70 1140 NA NA 100 2.83 NA NA 1.23 NA NA 1140 NA* NA* ND ** Not detected at or above the acceptable LOQ reported in Table I. NA a Not applicable. This matrix spike concentratrion was not used to assess the accuracy for this analyte. NA* Not applicable. This matrix spike concentratrion was not used to assess the accuracy for this analyte. See Table VII for re-spiked sample results. NR* Not reported due to quality control failures; see Table VIII for re-extracted sample results. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research Page 38 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table VI. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Fish Fillet Samples (continued) Sample Description DOU-F02-MSF004-0-061212 (C0227973 Spk J, 2.0 ppb spike) DOU-F02-MSF004-0-061212 {C0227973 Spk K, 100 ppb splke) D O U -F 02 -M S F 0 05 -0 -0 6 1212 (C0227S74 Spk L, 2.0 ppb splke) DOU-F02-MSF005-0-061212 (C0227S74 Spk M, 100 ppb splke) DOU-F02-IPF001-0-061212 (C0227S78 Spk D. 2.0 ppb apike) DO U-F02-IPF001-0-061212 (C0227S7S Spk E. 100 ppb splke) DOU-F02-IPF002-0-061212 (C0227976 Spk F, 2.0 ppb spike) DOU-F02-IPF002-0-061212 (C022787S Spk 6,100 ppb splke) DOU-F02-IPF0Q3-0-061212 (C0227S77 Spk H, 2.0 ppb splke) D O U -F 02 -I P F 00 3 -0 -06 1212 (C0227577 Spk 1,100 ppb splke) DOU-F02-IPF004-0-061212 (C022787S Spk J, 2.0 ppb splke) DO U-F02-IPF004-0-0 61212 (C022797S Spk K, 100 ppb splke) DO U-F02-IPF005-0-061211 (C0227870 8pk L, 2.0 ppb spike) DOU-F02-IPF005-0-061211 (C0227679 Spk M, 100 ppb splke) D U -F 02 -M SF 0 0 1-0-0 6 12 0 9 (C0227990 Spk O, 2.0 ppb splke) DLI-F02-MSF001-0-061209 (C02278B0 Spk E, 100 ppb splke) DLI-F02-MSF002-0-061209 (C0227991 Spk F, 2.0 ppb splke) D U -F 02 -M SF 0 0 2-0-0 6 12 0 9 (C0227891 Spk G, 100 ppb splke) D U -F 02-M SF 003-0-0 61209 (C0227882 Spk H, 2.0 ppb splke) DLI-F02-MSF003-0-061209 (C0227892 Spk 1, 100 ppb spike) DLI-F02-MSF004-0-061209 (C02279S3 Spk J, 2.0 ppb splke) D U -F 02 -M S F 0 0 4-0-061 20 9 (C02278S3 8pk K, 100 ppb splke) DLI-F02-MSF005-0-061209 (C02275B4 Spk L, 2.0 ppb spike) DLI-F02-MSF005-0-061209 (C0227SM Spk M, 100 ppb splke) DU-F02-IPF001-0-061212 (C0227886 Spk O, 2.0 ppb splke) DLI-F02-IPF001-0-061212 (C0227989 Spk E. 100 ppb splke) DU-F02-IPF002-0-061212 (C0227536 Spk F, 2.D ppb splke) DLI-F02-IPF002-0-061212 (C02278CS Spk G, 100 ppb spike) Amount Spiked (np/q) C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS PerfluorobuUnesulfonate_______________________________________________________PerSuorooctanesulTcnate Avg Amt Found Amount Avg Amt Found Amount Avg Amt Found Amount in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/g) (ng/g) (%) (ng/g) (ng/p) (%) (ng/g) (ng/g) (%) 2.0 0.234 2.11 94 0.356 2.79 122 4820 NA NA 100 0.234 NA NA 0.356 NA NA 4620 NA* NA* 2.0 0.847 3.08 112 1.06 3.30 112 NR* NR* NR* 100 0.847 NA NA 1.06 NA NA NR* NR* NR* 2.0 1.17 2.61 72 0.327 1 .6 8 66 263 NA NA 100 1.17 NA NA 0.327 NA NA 263 NA* NA* 2.0 NR NR NR 0.726 2.01 64 224 NA NA 100 NR NR NR 0.726 NA NA 224 NA' NA* 2.0 0.447 1.91 73 0.371 1.68 65 889 NA NA 100 0.447 NA NA 0.371 NA NA 689 NA* NA* 2.0 1.42 2.86 72 0.421 1.69 63 445 NA NA 100 1.42 NA NA 0.421 NA NA 445 NA* NA* 2.0 1.12 2.56 72 0.395 1.73 67 256 NA NA 100 1.12 NA NA 0.395 NA NA 256 NA* NA* 2.0 ND 2.15 106 0.582 2.86 114 403 NA NA 100 ND NA NA 0.582 NA NA 403 NA* NA* 2.0 0.114 2.17 103 0.435 2.54 105 320 NA NA 100 0.114 NA NA 0.435 NA NA 320 NA* NA* 2.0 0.130 1.60 74 0.712 2.47 86 254 NA NA 100 0.130 NA NA 0,712 NA NA 254 NA' NA* 2.0 0.105 2.16 103 0.619 2.97 118 248 NA NA 100 0.105 NA NA 0.619 NA NA 248 NA* NA* 2.0 0.135 2.26 106 0.700 2.50 90 296 NA NA 100 0.135 NA NA 0.700 NA NA 296 NA* NA* 2.0 ND 2.02 101 0.152 2.12 96 57.9 NA NA 100 ND NA NA 0.152 NA NA 57.9 139 81 2.0 ND 2.04 102 0.111 2.01 95 12.7 NA NA 100 ND NA NA 0.111 NA NA 12.7 101 68 ND = Not detected at or above the acceptable LOQ reported in Table I. NA - Not applicable. This matrix spike concentration was not used to assess the accuracy for this analyte. NA* Not applicable. This matrix spike concentration was not used to assess the accuracy for this analyte. See Table VII for re-spiked sample results. NR = Not reported due to quality control failures. NR* = Not reported due to quality control failures; see Table VIII for re-extracted sample results. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research Page 39 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table VI. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Fish Fillet Samples (continued) Sample D e s c rip tio n DU-F02-IPF003-0-061212 (C0227S87 Spk H, 2.0 ppb spike) D L I-F 0 2 -IP F 0 0 3 -0 -0 6 1 2 1 2 (C0227S87 Spk 1.100 ppb spike) D L I-F 0 2 -IP F 0 0 4 -0 -0 6 1 2 1 2 (C0227S88 Spk J, 2.0 ppb spike) D L I-F 0 2 - IP F 0 0 4 -0 -0 6 1212 (C0227S88 Spk K, 100 ppb spike) D L I-F 0 2 -IP F 0 0 5 -0 -0 6 1 2 1 2 (C0227589 Spk L. 2.0 ppb spike) D L I-F 0 2 -IP F 0 0 5 -0 -0 6 1 2 1 2 (C0227S89 Spk M, 100 ppb spike) D M C -F 0 2 -M S F 0 0 1-0 -0 61 2 09 {C0227590 Spk O, 2.0 ppb spike) DMC-F02-MSF001 -0-061209 (C0227S90 Spk E. 100 ppb spike) D M C -F 0 2 - M S F 0 0 2 -0 - 0 6 1209 (C0227591 Spk F, 2.0 ppb spike) DMC-F02-MSF002-0-061209 (C0227S91 Spk G, 100 ppb spike) DMC-F02-MSF003-0-061209 (C0227592 Spk H, 2.0 ppb spike) DMC-F02-MSF003-0-061209 (C0227S92 Spk 1,100 ppb spike) DMC-F02-MSF004-0-061212 (C0227S93 Spk J. 2.0 ppb spike) DMC-F 02-M SF004-0-061212 (C0227S93 Spk K, 100 ppb spike) D M C -F 0 2 -M S F 0 0 5 -0 -0 6 1212 (C0227S94 Spk L, 2.0 ppb spike) D M C -F 0 2 -M S F 0 0 5 -0 -0 6 1212 (C0227S94 Spk M. 100 ppb spike) D M C -F 0 2 -IP F 0 0 1 -0 -0 6 1 2 1 2 (C0227S9S Spk D. 2.0 ppb spike) D M C -F 0 2 -IP F 0 0 1 -0 -0 6 1 2 1 2 (C0227S9S Spk E, 100 ppb spike) DMC-F02-IPF002-0-061212 (C0227S96 Spk F, 2.0 ppb spike) D M C -F 0 2 -IP F 0 0 2 -0 -0 6 1 2 1 2 (C0227596 Spk G, 100 ppb spike) D M C -F 0 2 - IP F 0 0 3 -0 -0 6 1212 (C0227S97 Spk H, 2.0 ppb spike) DMC-F02-IPF003-0-061212 (C0227597 Spk 1,100 ppb spike) D M C -F 0 2 -IP F 0 0 4 -0 -0 6 1 2 1 2 (C0227598 Spk J, 2.0 ppb spike) D M C -F 0 2 -IP F 0 0 4 -0 -0 6 1 2 1 2 (C0227598 Spk K. 100 ppb spike) DM C-F02-1PF005-0-061212 (C0227599 Spk L, 2.0 ppb spike) DMC-F02-IPF005-0-061212 (C0227599 Spk M, 100 ppb spike) Amount Spiked (ng/8) C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulfonata_________________ Perfluorohsxancsulfonate_________________ Perfluorooctanesulfonate Avg A m t Found Amount Avg Am t Found Amount Avg Am t Found Amount in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/g) (ng/g) <%) (ng/g) (ng/g) (%) (ng/g) (ng/g) <%> 2.0 NO 1.79 90 ND 1.96 98 21.8 NA NA 100 ND NA NA ND NA NA 21.8 107 85 2.0 ND 1.76 88 0.166 2.09 96 47.6 NA NA 100 ND NA NA 0.166 NA NA 47.6 124 76 2.0 ND 1.92 96 0.132 1.98 92 52.4 NA NA 100 ND NA NA 0.132 NA NA 52.4 137 65 2.0 0.181 2.69 125 0.462 2.31 92 338 NA NA 100 0.181 NA NA 0.462 NA NA 336 NA* NA* 2.0 0.156 1.90 87 0.562 2.30 87 287 NA NA 100 0.156 NA NA 0.562 NA NA 287 NA* NA* 2.0 0.145 2.18 102 0.480 2.22 87 294 NA NA 100 0.145 NA NA 0.460 NA NA 294 NA* NA* 2.0 0.133 1.56 71 0.387 2.06 84 239 NA NA 100 0.133 NA NA 0.387 NA NA 239 NA* NA* 2.0 ND 1.53 77 0.247 1.87 81 332 NA NA 100 ND NA NA 0.247 NA NA 332 NA* NA* 2.0 ND 2.07 104 0.117 2.04 96 15.5 NA NA 100 ND NA NA 0.117 NA NA 15.5 130 115 2.0 ND 2.13 107 0.112 2.05 97 20.1 NA NA 100 ND NA NA 0.112 NA NA 20.1 117 97 2.0 ND 2.05 103 0.108 2.07 98 13.3 NA NA 100 ND NA NA 0.108 NA NA 13.3 114 101 2.0 0.100 2.36 113 0.168 2.23 103 13.0 NA NA 100 0.100 NA NA 0.168 NA NA 13.0 122 109 2.0 0.238 2.22 99 0.128 2.16 102 11.9 NA NA 100 0.238 NA NA 0.128 NA NA 11.9 118 106 Average: Standard Deviation: 103 21 Average: Standard Deviation: 100 19 NO = Not detected at or above the acceptable LOQ reported in Table I. NA = Not appficable. This matrix spike concentratrion was not used to assess the accuracy for this analyte. NA* = Not applicable. This matrix spike concentratrion was not used to assess the accuracy for this analyte. See Table VII for re-spiked sample results. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Average: Standard Deviation: 99 20 MPI Research Page 40 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table VII. Matrix Spike Recovery Summary of PFOS in Re-spiked Fish Fillet Samples Sam pie D e s c rip tio n DBC-F02-MSF001-0-061207 (C0227560 Spk D, 20000 ppb splke) D B C -F 0 2 -M S F 0 0 2 -0 -0 61 2 07 (C0227561 Spk E, 1000 ppb splke) DBC-F02-MSF003-0-061207 (C0227562 Spk F, 1000 ppb splke) DBC-F02-MSF004-0-061207 (C0227583 Spk O, 20000 ppb splke) D BC-F02- MSF005-0-061207 (C0227S64 Spk H, 20000 ppb spike) DBC-F02-IPF001-0-061211 (C022756S Spk D, 5000 ppb spike) D BC-F02-IPF002-0-061211 (C0227566 Spk E, 2000 ppb spike) DBC-F02-IPF003-0-061211 (C0227S67 Spk F. 5000 ppb spike) D B C -F 0 2 -IP F 0 0 4 -0-0 61211 (C0227568 Spk G. 2000 ppb spike) D B C -F 0 2 -IP F 0 0 5 -0 -0 6 1 211 (C0227569 Spk H. 5000 ppb spike) DOU-F02-MSF001-0-061209 (C0227570 Spk 1, 2000 ppb spike) D O U -F 02 -M S F 0 02 -0 -0 6 1 212 (C0227571 Spk J, 2000 ppb spike) D O U -F 02 -M S F 0 03 -0 -0 6 1 212 (C0227572 Spk K, 5000 ppb spike) D O U -F 02 -M S F 0 04 -0 -0 6 1 212 (C0227S73 Spk L, 20000 ppb spike) DOU-F02-MSF005-0-061212 (C0227574 Spk M. 1000 ppb spike) DOU-F02-IPF001 -0-061212 (C0227575 Spk 1,1000 ppb spike) DO U-F02-IPF002-0-061212 (C0227576 Spk J. 1000 ppb spike) DOU-F02-IPF003-0-061212 (C0227577 Spk K, 2000 ppb spike) DOU-F02-IPF004-0-061212 (C0227576 Spk L, 1000 ppb spike) DO U- F02-IP F005-0-061211 (C0227579 Spk M, 1000 ppb spike) DLI-F02-MSF001-0-061209 (C0227560 Spk N, 1000 ppb spike) DLI-F02-MSF002-0-061209 (C0227S81 Spk 0 , 1000 ppb spike) D -F 02-M SF 003-0-061209 (C02275B2 Spk P, 1000 ppb spike) D L I-F 0 2-M S F 00 4-0-06 1209 (C0227583 Spk Q, 1000 ppb spike) DLI-F02-MSF005-0-061209 (C0227584 Spk R, 1000 ppb spike) Amount Spiked (ng/g) 20000 1000 1000 20000 20000 5000 2000 5000 2000 5000 2000 2000 5000 20000 1000 1000 1000 2000 1000 1000 1000 1000 1000 1000 1000 Avg Amt Found in Sample (ng/g) C8 Sulfonate PFOS Perfluorooctanesulfonate Amount Recovered (ng/g) Recovery (%> 7470 21800 72 299 1280 98 247 1370 112 5310 23300 90 5300 23900 93 1070 6070 100 939 2670 87 1040 6490 109 605 2470 93 NR* NR* NR* 947 3200 113 1110 3980 144 1140 7270 123 4820 23900 95 NR* NR* NR* 263 1310 105 224 1020 80 889 2740 93 445 1410 97 256 1150 89 403 1490 109 320 1480 116 254 1350 110 248 1510 126 296 1310 101 NR* = Not reported due to quality control failures; see Table VIII for re-extracted sample results. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research Page 41 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table VII. Matrix Spike Recovery Summary of PFOS in Re-spiked Fish Fillet Samples (continued) Sample Description DMC-F02-MSF001-0-061209 (C0227590 Spk S, 1000 ppb spike) DMC-F02-MSF002-0-061209 (C0227591 Spk T, 1000 ppb spike) D M C -F 02-M S F 003-0-061209 (C0227592 Spk U, 1000 ppb spike) DMC-F02-MSF004-0-061212 (C0227593 Spk V, 1000 ppb spike) D M C -F 0 2 -M S F 0 0 5-0 -06 1212 (C0227594 Spk W, 1000 ppb spike) Amount Spiked (ng/g) 1000 1000 1000 1000 1000 Avg Amt Found in Sample (ng/g) C8 Sulfonate PFOS Perfluorooctanesulfonate Amount Recovered (ng/g) Recovery (%) 338 1090 75 287 1190 90 294 1280 99 239 1170 93 332 1590 126 Average: Standard Deviation: 101 16 Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. MPI Research Page 42 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table VIII. Matrix Spike Recovery Summary of PFOS in Re-extracted Fish Fillet Samples Sample Description DL3-F02-IPF001 -0-061211 (C0227550 Spk D, 5.0 ppb spike) DBC-F02-IPF005-0-061211 (C0227569 Spk E, 5000 ppb spike) DOU-F02-MSF005-0-061212 (C0227574 Spk D, 1000 ppb spike) Amount Spiked (ng/g) 5.0 C8 Sulfonate PFOS Perfluorooctanesulfonate Avg Amt Found in Sample Amount Recovered (ng/g) (ng/g) Recovery (%) 3.14 7.30 83 5000 1230 5810 92 1000 603 1650 105 Average: Standard Deviation: 93 11 Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research Page 43 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table IX. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Whole Body Fish Samples S am ple Description DL3-F02-M SW 001 -0-061207 (C0227600 Spk D. 2.0 ppb spike) D L3-F02-M SW 001-0-061207 (C0227600 Spk E. 400 ppb spike) C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS P e rfiu o ro b u ta n e s u lfo n a te ________________ Perfluorohexanesutfonate__________________ Pertiuorooctanesuifonate Amount Avg Amt Found Amount Avg Am t Found Amount Avg Amt Found Amount Spiked in S am ple R ecovered R ecovery in S am ple R ecovered R ecovery in S am ple R ecovered R ecovery (ng/g) (ng/g) (ng/g) (%> (ng/g) (ng/g) (%) (ng/g) (ng/g)______ (% ) 2.0 ND 400 ND 1.98 NA 99 NA 0.128 0.128 2.04 NA 96 NA 203 203 NA NA 464 65 DL3-F02-M SW 002-0-061207 (C0227601 Spk F. 2.0 ppb spike) 2.0 ND 2.06 103 0.154 2.12 98 108 NA NA D L3-F02-M SW 002-0-061207 (C0227601 Spk G, 400 ppb spike) 400 ND NA NA 0.154 NA NA 108 435 82 DL3-F02-MSW 003-0-061207 (C0227602 Spk H, 2.0 ppb spike) DL3-F02-MSW 003-0-061207 (C0227602 Spk 1. 400 ppb spike) 2.0 400 ND ND 2.14 NA 107 NA 0.169 0.169 2.29 NA 106 NA 193 193 NA NA 553 90 DL3-F02-MSW 004-0-061207 (C0227603 Spk J, 2.0 ppb spike) 2.0 ND 2.16 108 ND 2.44 122 87.2 NA NA DL3-F02-MSW 004-0-061207 (C0227603 Spk K. 400 ppb spike) 400 ND NA NA ND NA NA 87.2 470 96 DL3-F02-MSW 005-0-061207 (C0227604 Spk L, 2.0 ppb spike) DL3-F02-MSW 005-0-061207 (C0227604 Spk M. 400 ppb spike) 2.0 400 ND ND 2.30 NA 115 NA 0.733 0.733 3.04 NA 115 NA 341 341 NA NA 694 88 D L3-F02-IPW 001-0-061212 (C02276Q5 Spk D, 2.0 pp b spike) 2.0 ND 2.11 106 ND 2.03 102 8.24 NA NA D L3-F02-IPW 001-0-061212 (C0227605 Spk E. 50 ppb spike) 50 ND NA NA ND NA NA 8.24 48.2 80 DL3-F02-IPW 002-0-061212 (C0227606 Spk F, 2.0 ppb spike) 2.0 ND 1.93 97 ND 1.98 99 9.68 NA NA DL3-F02-IPW 002-0-061212 (C0227606 Spk G. 50 ppb spike) 50 ND NA NA ND NA NA 9.68 56.8 94 DL3-F02-IPW 003-0-061212 (C0227607 Spk H, 2.0 ppb spike) 2.0 ND 2.12 106 ND 2.09 105 16.7 NA NA D L3- F02-1PW 0 0 3 -0 -0 6 1212 (C0227607 Spk 1. 50 ppb spike) 50 ND NA NA ND NA NA 16.7 51.8 70 DL3-F02-IPW 004-0-061212 (C0227608 s p k J. 2.0 ppb spike) DL3-F02-IPW 004-0-061212 (C0227608 Spk K, 50 ppb spike) 2.0 50 ND ND 2.00 NA 100 NA 0.326 0.326 2.32 NA 100 NA 13.2 13.2 NA 53.5 NA 81 DL3-F02-IPW 005-0-061212 (C0227609 Spk L, 2.0 ppb spike) 2.0 ND 2.03 102 ND 2.21 111 7.75 NA NA DL3-F02-IPW 005-0-061212 (C0227609 Spk M, 50 ppb spike) 50 ND NA NA ND NA NA 7.75 51.1 87 DL2-F02-M SW 001 -0-061211 (C0227610 Spk D. 2.0 p pb spike) 2.0 0.183 1.99 90 ND 2.53 127 63.2 NA NA D L2-F02-M SW 001-0-061211 (C0227610 Spk E, 400 ppb spike) 400 0.183 NA NA ND NA NA 63.2 275 53 D L2-F02-M SW 002-0-061211 (C0227611 Spk F, 2.0 ppb spike) 2.0 0.146 1.93 89 ND 2.17 109 77.6 NA NA D L2-F02-M SW 002-0-061211 (C0227611 Spk G. 400 ppb spike) 400 0.146 NA NA ND NA NA 77.6 388 78 DL2-F02-IPW 001-0-061209 (C0227612 Spk D, 2.0 p pb spike) 2.0 ND 1.91 96 ND 1.93 97 39.8 NA NA DL2-F02-IPW 001-0-061209 (C0227612 Spk E, 200 ppb spike) 200 ND NA NA ND NA NA 39.8 188 74 ND = Not detected at or above the acceptable LOQ reported in Table III. NA = Not applicable. This matrix spike concentratrion was not used to assess the accuracy for this analyte. Note: Since th is sum m ary table sh ow s rounded results, recovery values m ay vary s lig h tly fro m the values in the raw data. MPI Research Page 44 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table IX. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Whole Body Fish Samples (continued) S am ple Description DL2-F02-IPW 002-0-061209 (C0227613 s p k F, 2.0 ppb spike) DL2-F02-IPW 002-0-061209 (C0227613 Spk G, 200 ppb spike) C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulfonate__________________ Perfluorohexanegulfonate__________________ Perfluorooctanesulfonate Amount Avg Am t Found Amount Avg Amt Found Amount Avg Amt Found Amount S piked in S am ple R ecovered Recovery in S am ple R ecovered R ecovery in S am ple R ecovered Recovery (ng/g) (ng/g) (ng/g) (%) (ng/g) (ng/g)______ (% ) (ng/g) (ng/g) (%) 2.0 ND 200 ND 2.09 NA 105 NA ND ND 2.17 NA 109 NA 17.8 17.8 NA NA 188 85 DL2-F02-IPW 003-0-061209 (C0227614 Spk H, 2.0 p pb spike) DL2-F02-IPW 003-0-061209 (C0227614 Spk 1, 200 ppb spike) 2.0 200 ND ND 1.89 NA 95 NA 0.100 0.100 1.98 NA 94 NA 39.3 39.3 NA NA 218 89 DL2-F02-IPW 004-0-061209 (C0227615 Spk J. 2.0 ppb spike) 2.0 ND 1.97 99 ND 1.86 93 45.9 NA NA DL2-F02-IPW 004-0-061209 (C022761S Spk K, 200 ppb spike) 200 ND NA NA ND NA NA 45.9 192 73 DL2-F02-IPW 005-0-061209 (C0227616 sp k L, 2.0 ppb spike) DL2-F02-IPW 005-0-061209 (C0227616 s p k M. 200 ppb spike) 2.0 200 ND ND 2.07 NA 104 NA ND ND 2.08 NA 104 NA 37.0 37.0 NA NA 224 94 DBC-F02-MSW 001-0-061207 (C0227617 Spk D, 2.0 p pb spike) D B C -F 02-M S W 001-0-061207 (C0227617 Spk E, 10000 p pb spike) 2.0 10000 2.74 2.74 4.63 NA 95 NA 7.60 7.60 NA 6270 NA 63 1620 1620 NA 7320 NA 57 DBC-F02-MSW 002-0-061207 (C0227618 Spk F. 2.0 ppb spike) DBC-F02-MSW 002-0-061207 (C0227616 Spk G. 10000 ppb spike) 2.0 10000 0.761 0.761 2.60 NA 92 NA 5.54 5.54 7.39 NA 93 NA NR* NR* NR* NR* NR* NR* DBC-F02-MSW 003-0-061207 (C0227619 Spk H. 2.0 ppb spike) DBC-F02-MSW 003-0-061207 (C0227619 Spk 1,10000 ppb spike) 2.0 10000 0.305 0.305 2.13 NA 91 NA 1.11 1.11 2.96 NA 93 NA 4410 4410 NA 18200 NA 138 DBC-F02-MSW 004-0-061207 (C0227620 Spk J. 2.0 ppb spike) D B C -F 02-M S W 004-Q -061207 (C0227620 Spk K, 10000 ppb spike) 2.0 10000 4.87 4.87 6.71 NA 92 NA 3.42 3.42 5.32 NA 95 NA 1490 1490 NA 10500 NA 90 DBC-F02-MSW 005-0-061207 (C0227621 Spk L. 2.0 ppb spike) DBC-F02-MSW 005-0-061207 (C0227621 Spk M, 10000 ppb spike) 2.0 10000 6.44 6.44 NA 9080 NA 91 2.72 2.72 4.64 NA 96 NA 2400 2400 NA 15200 NA 128 D BC -F02-IPW 001-0-061211 (C0227622 Spk D, 2.0 ppb spike) 2.0 2.25 4.39 107 2.77 5.08 116 567 NA NA D BC -F02-IPW 001-0-061211 (C0227622 Spk E. 5000 ppb spike) 5000 2.25 NA NA 2.77 NA NA 567 5220 93 DBC-F02-IPW 002-0-061211 (C0227623 Spk F, 2.0 ppb spike) 2.0 1.52 3.33 91 0.793 2.70 95 266 NA NA D B C -F 02 -IP W 0 02 -0 -0 61 211 (C0227623 Spk G. 5000 ppb spike) 5000 1.52 NA NA 0.793 NA NA 266 4510 85 D B C -F 02 -IP W 0 03 -0 -0 61 211 (C0227624 Spk H. 2.0 p pb spike) D B C -F 02 -IP W 0 03 -0 -0 61 211 (C0227624 Spk 5000 p pb spike) 2.0 5000 1.09 1.09 2.78 NA 85 NA 1.60 1.60 3.53 NA 97 NA 4730 4730 NA 9150 NA 88 D B C -F 02-IP W 0Q 4-0-Q 61211 (C0227625 Spk J. 2.0 ppb spike) 2.0 0.177 2.70 126 0.167 2.67 125 379 NA NA D BC -F02-IPW 004-0-061211 (C0227625 Spk K. 5000 ppb spike) 5000 0.177 NA NA 0.167 NA NA 379 5170 96 ND = Not detected at or above the acceptable LOO reported in Table III. NA = Not applicable. This matrix spike concentratrion was not used to assess the accuracy for this analyte. NR* = Not reported due to quality control failures; see Table X for re-extracted sample results. Note: Since th is sum m ary table shows rounded results, recovery values may vary slig h tly from the values in the raw data. MPI Research Page 45 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table IX. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Whole Body Fish Samples (continued) S am ple Description DBC-F02-IPW 005-0-061211 (C0227626 Spk L, 2.0 ppb spike) DBC-F02-IPW 005-0-061211 (C0227628 Spk M. 5000 ppb spike) C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulfonate__________________ Perfiuorohexanesutfonate__________________ P erfluorooclanesuifonate Amount Avg Amt Found Amount Avg Amt Found Amount Avg Am t Found Amount Spiked in S am ple R ecovered R ecovery in S am ple R ecovered Recovery in S am ple R ecovered R ecovery W 9) (ng/g) W 9 ) ______ [ % L _ (nq/q) ___w g > (% > (ng/g) (ng/g) (% ) 2.0 5000 2.54 2.54 4.12 NA 79 NA 0.933 0.933 2.76 NA 91 NA 3210 3210 NA 8010 NA 96 D O U -F02-M SW 001-0-061209 (C0227627 Spk D. 2.0 ppb spike) DOU-F02-MSW 001-0-061209 (C0227627 Spk E. 25000 ppb spike) 2.0 25000 2.73 2.73 4.72 NA 100 NA 2.12 2.12 3.73 NA 81 NA 115 NA NA 115 14400 57 DOU-F02-MSW 002-0-061212 (C0227628 Spk F. 2.0 ppb spike) D O U-F02-M SW 002-0-061212 (C0227628 Spk G, 25000 ppb spike) 2.0 25000 1.97 1.97 3.93 NA 98 NA 5.24 5.24 7.02 NA 89 NA 4190 4190 NA 21900 NA 71 DOU-F02-MSW 003-0-061212 (C0227629 Spk H. 2.0 ppb spike) DOU-F02-MSW 003-0-061212 (C0227629 Spk I, 25000 ppb spike) 2.0 25000 3.25 3.25 4.38 NA 57 NA 2.53 2.53 4.89 NA 118 NA 495 NA NA 495 15800 61 DOU-F02-MSW 004-0-061212 (C0227630 Spk J. 2.0 ppb spike) DOU-F02-MSW 004-0-061212 (C0227630 Spk K, 25000 ppb spike) 2.0 25000 1.68 1.68 3.14 NA 73 NA 1.59 1.59 3.58 NA 100 NA NR* NR* NR* NR' NR' NR* DOU-F02-MSW 005-0-061212 (C0227631 Spk L, 2.0 ppb spike) DOU-F02-MSW 005-0-061212 (C0227631 Spk M, 25000 ppb spike) 2.0 25000 4.97 4.97 7.17 NA 110 NA 3.03 3.03 5.37 NA 117 NA NR* NR* NR* NR* NR* NR* DOU-F02-IPW 001-0-061212 (C0227832 Spk D, 2.0 ppb spike) D O U -F02-IPW 001-0-061212 (C0227632 Spk E, 5000 ppb spike) 2.0 5000 0.373 0.373 2.33 NA 98 NA 0.546 0.546 2.58 NA 102 NA 1250 1250 NA 6980 NA 115 DOU-F02-IPW 002-0-061212 (C0227633 Spk F. 2.0 ppb spike) DOU-F02-IPW 002-0-061212 (C0227633 Spk G, 5000 ppb spike) 2.0 5000 1.46 1.46 2.71 NA 63 NA 0.724 0.724 1.90 NA 59 NA 1100 1100 NA 5910 NA 96 DOU-F02-IPW 003-0-061212 (C0227634 Spk H. 2.0 p pb spike) DOU-F02-IPW 003-0-061212 (C0227634 Spk 5000 ppb spike) 2.0 5000 1.44 1.44 3.57 NA 107 NA 2.57 2.57 4.55 NA 99 NA 2680 2680 NA 8080 NA 108 DOU-F02-IPW 004-0-061212 (C0227635 Spk J, 2.0 ppb spike) 2.0 0.455 2.44 99 0.163 2.17 100 189 NA NA DOU-F02-IPW 004-0-061212 (C022743S Spk K. 5000 ppb spike) 5000 0.455 NA NA 0.163 NA NA 189 5760 111 DOU-F02-IPW 005-0-061212 (C0227636 Spk L. 2.0 ppb spike) DOU-F02-IPW 005-0-061212 {C0227636 Spk M, 5000 ppb spike) 2.0 5000 7.35 7.35 NA 5870 NA 117 1.42 1.42 3.16 NA 87 NA 1110 1110 NA 7030 NA 118 DL1-F02-MSW 001-0-061209 (C0227637 Spk D, 2.0 ppb spike) D L 1-F02 -M SW 0 0 1-0 -061209 (C0227637 Spk E, 1000 ppb spike) 2.0 1000 0.464 0.464 2.48 NA 101 NA 1.48 1.48 3.80 NA 116 NA NR* NR* NR* NR' NR* NR* DL1-F02-MSW 002-0-061209 (C0227638 Spk F, 2.0 ppb spike) DL1-F02-MSW 002-0-061209 (C0227638 Spk G. 1000 p pb spike) 2.0 1000 0.175 0.175 2.17 NA 100 NA 0.745 0.745 3.30 NA 128 NA 158 158 NA NA 669 51 NA - Not applicable. This matrix spike concentratrion was not used to assess the accuracy for this analyte. NR* = Not reported due to quality control failures; see Table X for re-extracted sample results. Note: Since th is sum m ary table sh ow s rounded results, recovery values m ay vary s lig h tly fro m the values in the raw data. MPI Research Page 46 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table IX. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Whole Body Fish Samples (continued) Sam ple Description DL1-F02-MSW 003-0-061209 (C0227639 Spk H, 2.0 ppb spike) DL1-F02-M SW Q Q 3-0-061209 (C0227639 Spk I. 1000 ppb spike) C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobu taneulto nate_________________ Perfluorohexanesuifonate__________________ Perfluorooctanesulforiate Amount Avg Amt Found Amount Avg Am t Found Amount Avg Am t Found Amount Spiked in S am ple R ecovered R ecovery in S am ple R ecovered R ecovery in S am ple R ecovered Recovery (ng/g) (ng/g) (ng/g) (%> (ng/g) (ng/g) (%) (ng/g) (ng/g) (% ) 2.0 1000 0.455 0.455 2.60 NA 107 NA 1.31 1.31 3.71 NA 120 NA 326 326 NA NA 858 53 DL1-F02-MSW 004-0-061209 (C0227640 Spk J, 2.0 ppb spike) DL1-F02-M SW 004-0-061209 (C0227640 Spk K, 1000 ppb spike) 2.0 1000 0.138 0.138 2.19 NA 103 NA 1.31 1.31 3.65 NA 117 NA NR* NR* NR* NR* NR* NR* DL1-F02-MSW 005-0-061209 (C0227641 Spk L. 2.0 ppb spike) DL1-F02-MSW 005-0-061209 (C0227641 Spk M, 1000 ppb spike) 2.0 1000 0.275 0.275 2.46 NA 109 NA 1.73 1.73 4.15 NA 121 NA 212 212 NA NA 708 50 DL1-F02-IPW 001 -0-061212 (C0227642 Spk D, 2.0 ppb spike) DL1-F02-IPW 001-0-061212 (C0227642 Spk E, 200 ppb spike) 2.0 200 0.205 0.205 1.51 NA 65 NA 0.203 0.203 1.54 NA 67 NA 31.3 31.3 NA NA 194 81 D L 1-F02-1PW 0 0 2 -0 -0 6 1212 (C0227643 Spk F, 2.0 ppb spike) 2.0 0.234 2.35 106 0.239 2.26 101 136 NA NA D L 1- F02-1PW 0 0 2 -0 -0 6 1212 (C0227643 Spk G, 200 ppb spike) 200 0.234 NA NA 0.239 NA NA 136 331 98 DL1-FQ2-IPW 003-0-061212 (C0227644 Spk H. 2.0 p pb spike) DL1-F02-IPW 003-0-061212 (C0227644 Spk 1, 200 ppb spike) 2.0 200 0.224 0.224 1.69 NA 73 NA 0.183 0.183 1.61 NA 71 NA 23.7 23.7 NA NA 213 95 DL1-F02-IPW 004-0-061215 (C0227645 Spk J. 2.0 ppb spike) DL1-F02-IPW 004-0-061215 (C0227645 Spk K. 200 ppb spike) 2.0 200 0.633 0.633 2.40 NA 88 NA 0.340 0.340 2.05 NA 86 NA 34.1 34.1 NA NA 210 88 D L1-F02-1PW 0 05-0-061215 (C0227646 Spk L, 2.0 ppb spike) DL 1-F02-IPW 005-0-061215 (C0227646 Spk M, 200 p pb spike) 2.0 200 0.312 0.312 1.86 NA 77 NA 0.345 0.345 1.79 NA 72 NA 25.9 25.9 NA NA 207 91 DM C-F02-MSW 001-0-061209 (C0227647 Spk O, 2.0 ppb spike) DMC-F02-MSW 001-0-061209 (C0227647 Spk E, 1000 p pb spike) 2.0 1000 0.242 0.242 2.18 NA 97 NA 0.431 0.431 2.43 NA 100 NA 243 243 NA NA 978 74 DMC-F02-MSW 002-0-061209 (C0227648 Spk F. 2.0 ppb spike) 2.0 0.333 2.77 122 0.552 2.59 102 161 NA NA DMC-F02-MSW 002-0-061209 (C0227648 Spk G. 1000 ppb spike) 1000 0.333 NA NA 0.552 NA NA 161 1050 89 DMC-F02-MSW 003-0-061209 (C0227649 Spk H. 2.0 ppb spike) 2.0 0.438 2.68 112 0.938 2.87 97 167 NA NA DM C-F02-MSW 003-0-061209 (C0227649 Spk I, 1000 ppb spike) 1000 0.438 NA NA 0.938 NA NA 167 1110 94 DMC-F02-M SW 004-0-061212 (C02276S0 Spk J. 2.0 ppb spike) DMC-F02-MSW 004-0-061212 (C0227650 Spk K. 1000 p pb spike) 2.0 1000 0.746 0.746 2.34 NA 60 NA 1.23 1.23 2.71 NA 74 NA 176 NA NA 176 1100 92 DMC-F02-MSW 005-0-061212 (C0227651 Spk L. 2.0 ppb spike) 2.0 0,155 2.08 96 0.532 2.98 122 139 NA NA D M C-F02-M SW 005-0-061212 (C0227651 Spk M. 1000 ppb spike) 1000 0.155 NA NA 0.532 NA NA 139 1100 96 NA = Not applicable. This matrix spike concentratrion was not used to assess the accuracy for this analyte. NR* = Not reported due to quality control failures; see Table X for re-extracted sample results. Note: Since th is sum m ary table shows rounded results, recovery values may vary slig h tly from the values in the raw data. MPI Research Page 47 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table IX. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Whole Body Fish Samples (continued) Sam ple Description DM C-F02-1PW001-0-061212 (C0Z276S2 Spk 0 , 2.0 ppb spike) D M C-F02-1PW 0 0 1-0-061212 (C0227652 Spk E. 200 ppb spike) DM C-F02-IPW 002-0-061212 (C0227653 Spk F, 2.0 ppb spike) DM C-F02-IPW 002-0-061212 (C0227653 Spk G. 200 ppb spike) D M C -F02-IPW 003-0-061212 (C0227654 Spk H. 2.0 ppb spike) DM C-F02-IPW 003-0-D61212 (C0227654 Spk I, 200 ppb spike) DM C-F02-IPW 004-0-061212 (C022765S Spk J. 2.0 ppb spike) DM C-F02-IPW 004-0-061212 (00227655 Spk K, 200 ppb spike) DM C-F02-IPW 005-0-061212 (C0227656 Spk L. 2.0 ppb spike) D M C -F02-1PW 0 0 5 -0 -0 6 1212 (C0227656 Spk M. 200 ppb spike) C4 S ulfonate PFBS C6 S ulfonate PFHS C8 S ulfonate PFOS Perfluorobutanesulfonate__________ Perfluorohexanesutfonate__________________________ perfluorooctanesulfonate Amount Spiked (ng/g) Avg Am t Found in Sample (ng/g) Amount R e co ve re d (ng/g) R e cove ry (% ) Avg Am t Found in Sample <ng/g) Amount Recovered Recovery (ng/g)______ ( % L _ Avg Am t Found in Sample (nq/g) Amount Recovered Recovery (ng/g)_____ i s a _ 2.0 0.204 200 0.204 1.65 NA 72 NA 0.176 0.176 1.75 NA 79 NA 24.3 24.3 NA NA 192 84 2.0 0.199 200 0.199 1.97 NA 89 NA 0.277 0.277 2.07 NA 90 NA 31.7 31.7 NA NA 213 91 2.0 0.105 200 0.105 1.18 NA 54 NA 0.191 0.191 1.35 NA 58 NA 12.3 12.3 NA NA 187 87 2.0 1.15 200 1.15 3.18 NA 102 NA 0.561 0.561 2.56 NA 100 NA 184 184 NA NA 447 132 2.0 0.129 200 0.129 1.52 NA 70 NA 0.159 0.159 1.57 NA 71 NA 33.4 33.4 NA NA 225 96 A verag e: S tandard D eviation: 95 15 A verag e: S tandard D eviation: 98 17 NA = Not applicable. This matrix spike concentratrion was not used to assess the accuracy for this analyte. Note: Since th is sum m ary table show s rounded results, recovery values may vary s lig h tly fro m the values in the raw data. A verag e: Standard D eviation: 87 19 MPI Research Page 48 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table X. Matrix Spike Recovery Summary of PFOS in Re-extracted Whole Body Fish Samples Sample Description DBC-F02-MSW002-0-061207 (C0227618 Spk D, 20000 ppb spike) DO U-F02-MSW004-0-061212 (C0227630 Spk F, 1000 ppb spike) DOU-F02-MSW005-0-061212 (C0227631 Spk G, 2000 ppb spike) DL1-F02-MSW001-0-061209 (C0227637 Spk H, 1000 ppb spike) DL1-F02-MSW004-0-061209 (C0227640 Spk I, 1000 ppb spike) Amount Spiked (ng/g) 20000 1000 2000 1000 1000 Avg Amt Found in Sample (ng/g) C8 Sulfonate PFOS Perfluorooctanesulfonate Amount Recovered (ng/g) 12500 27600 Recovery (%) 76 189 1190 100 378 1920 77 297 1110 81 247 1110 86 Average: Standard Deviation: 84 10 Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research Page 49 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Table XI. Matrix Spike Recovery Summary of PFBS, PFHS, and PFOS in Clam Samples S am ple Description DL3-I02-CFW 001-0-061219 (C0227658 Spk D, 2.0 ppb Spike) DL3-I02-CFW001 -0-061219 (C0227658 Spk E. 20 ppb Spike) C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulfonate___________________ Perfluorohexaneaulfonate___________________ Perfluorooctanesulfonate Amount Avg A m t Found S piked in S am ple (ng/g) (ng/g) Amount Recovered (ng/g) Avg Amt Found Recovery in S am ple (%) (ng/g) Amount Recovered (ng/g) Avg Amt Found Recovery in Sample (%> (ng/g) Amount Recovered (ng/g) Recovery <%) 2.0 ND 2.22 111 NR NR NR 0.767 3.10 117 20 ND NA NA NR NR NR 0.767 NA NA DL2-I02-CFW 001-0-061219 (C0227659 Spk F. 2.0 ppb Spike) DL2-I02-CFW 001-0-061219 (C0227659 Spk G. 20 ppb Spike) 2.0 20 ND ND 2.28 NA 114 NA NR NR NR NR 0.771 NR NR 0.771 3.50 NA 136 NA DBC-I02-CFW001 -0-061219 (C0227660 SpK H. 2.0 ppb Spike) DBC-I02-CFW001 -0-061219 (C0227660 Spk I, 20 ppb Spike) 2.0 20 ND ND 2.21 NA 111 NA ND ND 2.47 NA 124 NA 3.10 3.10 4.82 NA 86 NA DOU-I02-CFW 001 -0-061219 (C0227661 Spk J, 2.0 ppb Spike) DOU-I02-CFW 001 -0-061219 (C0227661 Spk K, 20 ppb Spike) 2.0 20 0.234 0.234 2.34 NA 105 NA ND ND 2.77 NA 139 NA 2.31 2.31 4.44 NA 107 NA DLI-I02-CFW 001-0-061219 (C0227662 Spk L. 2.0 ppb Spike) DLI-I02-CFW 001-0-061219 (C0227662 Spk M. 20 ppb Spike) 2.0 20 0.166 0.166 2.24 NA 104 NA ND ND 2.52 NA 126 NA 1.34 1.34 3.28 NA 97 NA DM C- I02-C FW 0 0 1-0-061219 (C0227603 Spk N, 2.0 ppb Spike) DMC-I02-CFW001-0-061219 (C0227663 Spk O. 20 ppb Spike) 2.0 20 ND ND 1.76 NA 88 NA ND ND 2.45 NA 123 NA 0.850 0.850 2.29 NA 72 NA Average: Standard Deviation: 105 9 Average: Standard Deviation: 128 7 ND = Not detected at or above the acceptabte LOG reported in Table V. NA = Not applicable. This matrix spike concentratrion was not used to assess the accuracy for this analyte. NR - Not reported due to quality control failures. Note: Since this sum m ary table show s rounded results, recovery values may vary slig h tly from the values In the raw data. Average: Standard Deviation: 102 23 MPI Research Page 50 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 FIGURES MPI Research Page 51 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 1. Typical Extracted Calibration Curve for PFBS in Catfish Fillet Matrix 0 1 1 1 0 8 0 Catfish F ille t S e t2 .rd b (PFBS): "Q u a dra tic" Regression C` 1 / * ' w e ig htin g ): y * -0.69e+OO4 x *2 + 1.48e+006 x + 4 13 (r = 0 .0 9 94 ) Area, counts MPI Research Page 52 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 2. Extracted Standards of PFBS in Catfish Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively I XC910909-1 - PFBS (Standard) 299.0/80.0 am u,299.0/99.0 amn -sam pfe 1 o f 55 from 011109D.wiff A rea: 39226counts H eight: 8. 6Se+003c /tt RT: 9.61 m in 8.61 Tim e, min I X C 0 10908-2 - PFBS (S ta n d a rd )2 9 9 .0/8 0.0 am u.299 0 /9 9 .0 a m u - sam ple 2 of 66 from 0 1 1 108D.iwiff Area: 76439 counts Height: 1.69e+004cps R T :8 .6 0 min 8.60 MPI Research Page 53 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 3. PFBS in a Control Catfish Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Catfish Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Catfish Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Catfish Fillet Spike C, Respectively C0292603 Coirtrot 1 - PFBS (Unknown) 299.0/90.0 amu,299.0/99.0 amu - sample 12 o f 55 from 011109fXwiff Area: 1993counts Height: 1.91e+002cps RT: 9.56min Tim e, min I C0202603 Spk A1 PFBS (QC) 299 .0/8 0.0 a m u.299.0/99.0 a m u - sam ple 13 of 55 from 0 1 1 108D.wiff Area: 128796 counts Height: 2.89e+004 cps RT: 8.60 min 8.60 Tim e, min I C0292603 Sph B 3-P F B S (QC) 299.0/90.0 amu,299.0/99.0 amu -sample 20 o f 55 from 011109D.wiff Area: 792013couuts Height: 1.79e+005cps RT: 9.57min 8.57 1.6 e5 (a0. i.oe5- CO c oe " 5.0e4 - 0.0 J------ ------- .-------------1------ ------- 1------ ------ 1------ ------- 1------ ------- 1------------- 1------ ------- .------ ----1------ '------ 1-------------------- ------- i------------- 1------------- ,------------ ------- ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Tim e, min I C0292603 Spk C1 PFBS (Q C )299 .0/8 0.0 a m u.299.0/99.0 a m u - sam ple 21 of 55 from 0 11108D .w iff Area: 6 53349 counts Height: 1.49e+005 cps RT: 8.59 m in 1.4e5 -I 8.59 1.2 5 " 1.0e5 o 8.0 e4 to m .0e4 4.0 e4 2 .0 e 4 - 0.0 J------------- 1-------------.------------- .------------- ------------- -------------- ------------- 1------------- ------- -------------------- ---------------------------- .------------- .------------- 1 - .... .------------- r1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Tim e, min MPI Research Page 54 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 4. Chromatogram Representing a Catfish Fillet Sample Analyzed for PFBS (ExyLIMS ID: C0227555, Data Set: 011108D) C0227555-PFBS (Unknown) 299.0/80,0amu,299.0/99.0amu -sample 28o f 55 from 011108D.wiff A n a : 3120counts Height: 4.5te*002cps RT: 8.58 min Intensity, cps MPI Research Page 55 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 5. Typical Extracted Calibration Curve for PFHS in Catfish Fillet Matrix 011 10 8 D Catfish F ille t S e t2 .rd b (PFHS): "Q uadratic/' Regression ("1 / * ' w e ig htin g ): y = 6 .5 5 e + 0 0 4 x A2 + 1.63e+006 x + l.<W e+003 (r = 0 .9 9 94 ) Area, counts Concentration, ng/mL MPI Research Page 56 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 6. Extracted Standards of PFHS in Catfish Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively I XC010908- J -Pf=HS (Standard) 399. 0/90.0 ama,399. 0/ 99.0 amu -sam pfe 1 o f 55 fro m O M O M Xw iff Area: 39858 counts H eight: &7?e+003cp* RT: 9.95 m itt Tim e, min XC010908-2 PFHS (S tandard)399.0/80 0 amu,399.0/99.0 am u - sample 2 of 55 from 011108D.wiff Area: 76901 counts H eight: 1.60e+OO4 cps RT: 9 .9 4 m in MPI Research Page 57 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 7. PFHS in a Control Catfish Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Catfish Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Catfish Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Catfish Fillet Spike C, Respectively I CO292603 Control 1 - PFHS (Unknown) 399L0/80L0 affftf,399.0/99l0 amn amp/ 12 o f 55 from 011109D.wiff (freak not foand) 11.47 Tim e, min I C0292603 Spk A1 - PFHS (Q C )399.0/80.0 amu.399.0/99.0 amu sample 13 of 55 from 0 11 108D.wiff Area: 128106 counts Height: 2 .6 7 e+ 00 4 cps RT: 9.91 min 9.91 Tim e, min I C0292603 Spk B3 - PFHS (QC) 39910/0A 0 amn,39%0/99.0 amn - sample 20 o f 55 from 911109D.wiff Area: 853409 counts Height: f.80e+005cp* RT: 9.90 min 9.90 Tim e, min I C 0292603 Spk C1 - PFHS (QC) 3 99,0/80.0 am u.399.0/99.0 a m u -s a m p le 21 of 55 from 011108D .w iff A rea: 7 22 17 2 counts Height: 1.57e+005 cps RT: 9 .9 4 min MPI Research Page 58 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 8. Chromatogram Representing a Catfish Fillet Sample Analyzed for PFHS (ExyLIMS ID: C0227556, Data Set: 011108D) I C0227556 - PFHS (Unknown) 399.0/90.0 amu,399.0/93.0 amu -sampfe 3 4 o f 55 from 011109D.wiff A n a : 22170counts tteigfit: 4.43e+093cps RT: 9.09 min 11.41 Intensity, cps MPI Research Page 59 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 9. Typical Extracted Calibration Curve for PFOS in Catfish Fillet Matrix 0 11 10 8 D Catfish F ille t S et 2 .rdb (PFOS): "Q u a dra tic" Regression C'1 w e ig htin g ): y *7.2 9 e + 0 0 4 x *2 + 1.44e+006 x + -3.9e+003 ( r * 0 .9 9 97 ) Area, counts Concentration, ng/m L MPI Research Page 60 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 10. Extracted Standards of PFOS in Catfish Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively I XC 010908-1 P F O S (S ta n d a rd )499.0/80.0am u ,499.0/99.0a m a -s am p le 1 o f 55 fro m 011108D.w iff A re a : 3 2 4 3 6 c o n n ts H e ig h t: 5.35e +003c p s H T : 1 0 .9 m in 1 0 .9 4 T im e , m in I X C 010908-2 - PFOS (S tandard) 4 99 .0/8 0.0 a m u,4 99 .0/9 9.0 am u sam ple 2 of 5 5 from 011 10 8 D .w iff A rea: 5 37 18 counts H eight: 1 .0 3 e+ 00 4 cps R T :1 0 .9 m in 1.00 4 4 8000.00 CO Q. 6000.00 CO g 4000.00- 2000.00 - 0 .0 0 J------ ------- 1------ ------- 1------ ------- 1------ ------- 1------ ------- ------ ------- r 123 456 10.93 T im e, m in 1 2 .6 6 ^ 13.09 1 4 .5 8 0 4 .9 2 1 6 .7 3 ^ r ' 'I " I B | *1 ' I r r 12 13 14 15 16 MPI Research Page 61 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 11. PFOS in a Control Catfish Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Catfish Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Catfish Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Catfish Fillet Spike C, Respectively I C0292603 C o n tr o l 1 - P F O S (U n k n o w n ) 499 .0/00 .0 a m u ,499.0/99 .0 a m u -s a m p le 1 2 o f 55 fro n t 0 1 1 1 0 9 D .w iff A re a : 1190 counts H eig h t: 1.24e+002cps R T: 10.9 m in T im e, m in I C 0 292603 Spk A1 - PFO S (Q C) 4 99 .0/8 0.0 a m u,499.0 /9 9 .0 am u - sam ple 13 of 5 5 from 0 1 1 108D .w iff A rea: 107546 counts H eight: 1 .7 1 e + 0 0 4 c p s RT: 10.9 m in 1 0 .8 9 T im e , m in I C0292603 S p k B 3 - P F O S (Q C ) 4 9 9 .0/90 .0 a m u ,4 9 9 .0 /9 9 .0 a m u -s a m p le 20 o f 55 fro m 0 1 1 1 0 9 D .w iff A rea: 706102con nt9 H eight: 1.13e+005 cps R T: 10.9 m in 10.88 Tim e, m in I C 0 2 9 2 6 0 3 Spk C1 P F O S ( Q C ) 4 9 9 .0 /8 0 .0 a m u .4 9 9 .0 /9 9 .0 a m u - s a m p le 2 1 o f 5 5 fro m 0 1 1 1 0 8 D .w iff A rea: 5 5 6 5 5 4 counts H eight: 9 .0 6 e + 0 0 4 cps RT: 10.9 m in 10.91 MPI Research Page 62 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 12. Chromatogram Representing a Catfish Fillet Sample Analyzed for PFOS (ExyLIMS ID: C0227557, Data Set: 011108D) C0227557 PFOS (lktAaowa)499,W8(K0amu,499.(V99.Qamu -sampin 3 9 o f 55 from 011109D.wtfT A n a : 219306Bcoortts H&ig&t: 4.B5e+05cps RT: 10,Bmin Intensity, cps MPI Research Page 63 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 13. Typical Extracted Calibration Curve for PFBS in Bass Fillet Matrix 1227Q7B Bass F ille t S et l.rd b (PFBS): "Q u a dra tic" Regression C'1 / * ' w e ig htin g ): y = 3 .7 2 e + 0 0 4 x A2 + 5 .0 6 e+ 00 5 x + -8 .94e+003 (r = 0.9 9 69 ) Area, counts Concentration. ng/m L MPI Research Page 64 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 14. Extracted Standards of PFBS in Bass Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively I XC122707-1 - PFBS (Standard) 299.0/90.0 amu,299.0/99.0 ama -sample 1 o f 55 from 122707B.wiff Area: 5909 counts Heigftt: 1.22e+003cps RT: 9.75 min 8.75 Tim e, min I XC122707-2 - PFBS (S tan d ard)299.0/80.0 amu,299.0/09.0 amu - sample 2 of 55 from 122707B.wiff A rea: 18013 counts H eight: 4 .0 0 e+ 00 3 ops RT; 8 .7 4 min 8 .7 4 MPI Research Page 65 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 15. PFBS in a Control Bass Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Bass Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Bass Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Bass Fillet Spike C, Respectively 1 C0294954 Control 1 PFBS (Unknown) 299.0M0.0 antu,299.0/99.0 amu -sample 12 o f 55 from 122707B.wiff peak not found) C 0284954SpkA 1 - PFBS (QC) 209.0/80.0 amu.299.0/99.0 am u - sample 13 of 55 from 122707B.wiff Area: 26605 counts H e ig h t 5.48e+003 cps RT: 8.73 min 8.73 Tim e, min I C0204954Spk B 3-PFB S (QC) 299.0/S0.0 amu,299.0/99.0 amu -sample 20o f 55 from 122707B.wiff Area: 211111 counts Height: S05e+0(Wcps RT: 8.73 mitt 8.73 4 .0 e 4 CO 3.0e4 s CO 2.0e4 1.0 e4 0.0 1 1 1 1J--------------- -------------- ------- -------- .------- -------- --------------- ------- -------- .--------------- .---------------.--------- * - i ---------------.-------------- ---------------i---------------.-------------- .-------------- .---------------r 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time, min I C 0284954 Spk C1 PFBS (0 0 )2 9 9 ,0 /8 0 .0 amu,2 99.0/99.0 amu - sample 21 of 55 from 122707B.iAriff Area: 147003 counts Height: 3.4 0 e+ 00 4 cps RT: 8.72 min 8.72 3.0e4 2.5e4 CO Q. 2.0e4 co . _ , c 1.5e4 OJ ~ 1.0 e4 5000.0 0.0 -1-------------1-------------1-------------.------ - ------------- --------------------- .------ - 1 ------------------ 1------- 1------ - 1-------------------- .------- .------ - 1 1 2 3----------- 4 5 6 -----------7----------- 8----------- 9 10--------- 11---------- 12 13----------14--------- 15----------16 Time, min MPI Research Page 66 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 16. Chromatogram Representing a Bass Fillet Sample Analyzed for PFBS (ExyLIMS ID: C0227544, Data Set: 122707B) C0227544 PFBS (Unknown) 299.O/80L0 amu,299.0/99*0 amu sampte 29 o f 55 from 1227Q7B.wiff A n a : 3124count* Height: 4.46e+Q02cps RT: 9.69 min Intensif/, cps MPI Research Page 67 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 17. Typical Extracted Calibration Curve for PFHS in Bass Fillet Matrix 1 22 7 0 7 B Bass F ille t S et t .idb (P FH S ):-Q u ad ra tic/' Regiesslon C` 1 I X ' w e ig htin g ): y = -2.B 9e+004 *2 + 4 ,7 5 e+ 00 5 x + -8 26e +0 03 (r = 0.6 9 37 ) Area, counts MPI Research Page 68 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 18. Extracted Standards of PFHS in Bass Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively I XC122707-1 -PFH S (Standard)399.0/V0.0amu,399.0/99,0amu -sampte 1 o f 55 from 122707B.wiff Area: 5293 counts Height: 1.09e+003cps RT: 10*1 min 15.05 Tim e, min X C 122707-2 - PFHS (Standard) 399.0/80.0 am u.399.0/99.0 amu - sample 2 of 55 from 122707B.wiff Area: 15246 counts Height: 2.94e+003 cps RT: 10.1 min 10.07 MPI Research Page 69 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 19. PFHS in a Control Bass Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Bass Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Bass Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Bass Fillet Spike C, Respectively C0294954 Control 1 ' PFHS (Unknown)399.0/90.0 amu,399.0/99.0 amu -sample 12o f 55 from 122707B.wiff freak not found) I C 0284954 SpkA1 - PFHS (QC) 399.0/80.0 amu.399.0/89.0 amu sam ple 13 of 55 from 122707B.wiff Area: 2 95 94 counts Height: 6.06e+003 cps RT: 10.1 min 10.05 Tim e, min C0294954Spk B3 - PFHS (QC) 399,0/90.0 amu,399.0^9.0 amu -sample 20 o f 55 from 122707B.wiff Area: 228037 counts Height: 4.95e+Q04cps RT: 10.0 min 10.04 Tim e, min I C0284Q54 Spk C1 PFHS (QC) 3 9 9 .0/8 0.0 a m u.399.0/99.0 a m u -s a m p le 21 of 5 5 from 122707B.w iff Area: 173336 counts Height: 3 .8 9 e + 0 0 4 cps RT: 10.0 min 10.04 MPI Research Page 70 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 20. Chromatogram Representing a Bass Fillet Sample Analyzed for PFHS (ExyLIMS ID: C0227546, Data Set: 122707B) I C0227546 PFHS (Unknown) 399.0/80.0 am u,399.0/99.0 am u -sa m p le 3 4 o f 55 from 122707B.wiff Area: 7499counts Height: 1.64e+003cps RT: 10.0min Intensity, cps MPI Research Page 71 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 21. Typical Extracted Calibration Curve for PFOS in Bass Fillet Matrix 1 2 2 7 0 7 B Bass F ille t S et l. t d b (PFOS): "Q u a dra tic" Regression (" 1 / * ' w e ig htin g ): y = -2 .e e + 0 0 4 x "2 + ` l. `W e+005 x + 4 .2 9 e + 0 0 4 (r = 0 .9 9 84 ) Area, counts MPI Research Page 72 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 22. Extracted Standards of PFOS in Bass Fillet Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively I XC122707*1 - PFOS (Standard) 499.0/8 0l0 amu,499. 0/99l0 ama sample 1 o f 55 from 122707B.wiff Area: 511B5count9 teigftt: 7.47&+03 cps RT: 11.1 min Tim e, min XC122707-2 PFOS ( S t a n d a r d ) ^ .0/80.0 amu,4Q9.0/99.0 a m u -s a m p le 2 of 55 from 122707B.wiff Area: 69405 counts Height: 1.14e+004 cps RT: 11.1 min 11.05 MPI Research Page 73 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 23. PFOS in a Control Bass Fillet, a 0.075 ng/mL (0.30 ng/g) Fortified Bass Fillet Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Bass Fillet Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Bass Fillet Spike C, Respectively I CQ2949S4 Control 1 - PFOS (Uhknown) 4M .M 0.Q atnu,499*m M amu -sample 12 o f 55 from 122707B.wiff Area: 10200 counts Height: 2.4Se*003cps RT: 11.1 min 15.22 Tim e, min C0284954 SpkA1 - PFOS (QC) 460.0/80.0 am u.499.0/99.0 amu - sam ple 13 of 55 from 122707B.wiff Area: 90071 counts Height: 1.60e+004 cps RT: 11.0 min 11.05 I C0294954 SpA B3 PFOS (QC) 499.0/90.0 amu,499.0/991Offmu sample 20 o f 55 from 122707B.wiff Area: 265397 counts Height: 4.6e+004cps RT: 11.0 min 11.04 Tim e, min I C 0284954 Spk C1 PFOS (QC) 499 .0/8 0.0 a m u.499.0/99.0 a m u -s a m p le 21 of 55 from 122707B.w iff Area: 161031 counts Height: 2.90e+OO4 cps RT: 11.0 min 11.03 MPI Research Page 74 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 24. Chromatogram Representing a Bass Fillet Sample Analyzed for PFOS (ExyLIMS ID: C0227544, Data Set: 122707B) I C0227544 - PFOS (Unknown) 499.0/90.0 amu,499.0/99.0 amu -sampte 28 o f 55 from 122707B.wtff A n a : 1714267counts Height: 3.26e+005cps RT: 11.0min 11.02 Intensity, cps MPI Research Page 75 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 25. Typical Extracted Calibration Curve for PFBS in Whole Body Catfish Matrix 0 3 2 1 0 8 C W ho! Catfish S et 2.rdb (PFBS): "Q u a dra tic" Regression ("1 w e ig htin g ): y = -6 .44 e + 0 04 x*2 + 2 .17e+006 x + 6 .16e+003 (r = 0 .9 9 98 ) Area, counts Concentration. ng/m L MPI Research Page 76 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 26. Extracted Standards of PFBS in Whole Body Catfish Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively I XC3210-r-PfrBS (Standard) 299.0/90.0 amu,299.0/99,0 amu -sampfe 1 o f 55 from 032109C.w/ff Area: 61549 counts Height: 1.43e+004cps RT: 8.53 min 8.53 Tim e, min I XC032108-2 PFBS (Standard) 299.0/80.0 am u.299.0/99.0 a m u - sample 2 of 55 from 032108C.wiff A rea: 117106 counts H eight: 2.75e+ C 04 cps RT: 8 .5 2 mir 8.52 MPI Research Page 77 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 27. PFBS in a Control Whole Body Catfish, a 0.075 ng/mL (0.30 ng/g) Fortified Whole Body Catfish Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Whole Body Catfish Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Whole Body Catfish Spike C, Respectively I C0292558 Control 1 - PFBS (Unknown) 299L0/8AO am u,299.0/99.0 am u -sa m p le 12 o f 55 from 032109C,wiff (peak not found) 13.59 T im e, m in I C 0 2 9 2 5 5 8 S p k A 1 - P F B S (Q C ) 2 9 9 .0 /8 0 .0 a m u .2 9 9 .0 /9 9 .0 a m u - s a m p le 13 o f 5 5 fro m 0 3 2 1 0 8 C.uuiff A rea: 174683 counts H eight: 4 .0 6 e + 0 0 4 cps RT: 8 .4 9 m in T im e, m in I C0292558 S p k B2 - PFBS (QC) 299.0/90.0 a m u ,299.0/99.0 am u -sam ple 20 o f 55 from 032109C.wtff Area: 1025592counts Heigkt: Z49e+005cps RT: 8.48min T im e, m in I C 0292558 Spk 01 - PFBS (Q C) 2 99 .0/8 0.0 am u.299.0 /9 9 .0 amu - sam ple 21 of 55 from 032 10 8 C .w iff A rea: 7 274491 counts H eight: 1 .73e+006 cps RT: 8 .4 7 m in 1.5e6 - CO Q. 1.0e6 & CO c 0) c 5.0e5 8 .4 7 10 .0 I ' I------ '-------I------ ------I------ '------ -------1------ I------------ I--- I I ' I "! I , . I- 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 T im e, m in MPI Research Page 78 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 28. Chromatogram Representing a Whole Body Catfish Sample Analyzed for PFBS (ExyLIMS ID: C0227612, Data Set: 032108C) C0227612 PFBS (Uahnowa) 299LA/90LO am a,299.0/99,0 amu -sampfe 3 0o f55 fro m 932109C.wiff TM Area: 41740 counts Height: &4te+0O3c;wr RT: 9.52 mitt 13.40 Intensity, cps MPI Research Page 79 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 29. Typical Extracted Calibration Curve for PFHS in Whole Body Catfish Matrix 0 3 2 1 0 8 C W hole Catfish S e t2 .id b (PFHS): "Q u a dra tic" Regression C'1 / * ' w e ig htin g ): y = -4 .8e + 0 0 4 x *2 + 2 .1 3 e + 0 0 6 x + 2 .7 3 e + 0 0 4 (r = 0 .9 9 9 8 ) Area, counts MPI Research Page 80 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 30. Extracted Standards of PFHS in Whole Body Catfish Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively XC032109-1 - PFHS (Standard) 399.0/90.0 amuf399.0/99.0 amu -sampte 1 o f 55 from 032109C.wiff Area: 79144counts Height: 1.28e+004cps RT: 9.95 min 9.85 Tim e, min X C 032108-2 - PFHS (Standard) 3 9 9 .0 0 .0 amu.399.0/99.0 a m u -sa m p le 2 of 55 from 032108C.wiff Area: 143788 counts Height: 2 .59e+004 cps R T :9 .8 5 min 9.85 MPI Research Page 81 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 31. PFHS in a Control Whole Body Catfish, a 0.075 ng/mL (0.30 ng/g) Fortified Whole Body Catfish Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Whole Body Catfish Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Whole Body Catfish Spike C, Respectively I C0292S58 Control 1 -PFHS (Unknown)399.0/80.0atrtti,399.0/99.0amn -sample 12o f 55 from 032108C.wiff A n a : 23814counts Height: Z f9 e +003 cp RT: 9.86 mitt 11.38 I C0292558 SpkA1 PFHS (QC) 399 0/80.0 amu.399.0/99.0 amu - sample 13 of 55 from 032108C.rwiff Area: 198780 counts Height: 3.58e+004 cps RT: 9.82 min 9.82 I C0292558 Sp* B3 - PFHS (QC) 399.0/80.0amu,399.0/99.0 amu -sample 20 o f 55 from 032108C.wiff A n a : 1007866couats Height: 2.07e+005 cp RT: 9.82 mia 9.82 Tim e, min I C0292558 Spk C1 PFHS (QC) 399.0/80.0 amu,399.0/99.0 a m u - sam ple 21 of 65 from 032108C.wiff Area: 7135083 counts Height: 1.48e+008 cps R T:9.81 min MPI Research Page 82 of 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project N o.: P0003268 Figure 32. Chromatogram Representing a Whole Body Catfish Sample Analyzed for PFHS (ExyLIMS ID: C0227612, Data Set: 032108C) I C02276T2 - PFHS (Unknown) 399LOWO.0 amu,399.0/9910 amu - sample 30 o f 55 from O3208C.w f f Area: 45263 counts Height: 6.49e+003cp* RT: 9.91 min Intensity, cps MPI Research Page 83 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 33. Typical Extracted Calibration Curve for PFOS in Whole Body Catfish Matrix 0 3 2 1 0 8 C W hole Catfish S e t2 .rd b (PFOS): "Q u a dra tic" Regression p w eighting): y -3.54e+004>c*2 + 1,78e+006 x + 9.1 e+004 (r = 0.9995) Area, counts MPI Research Page 84 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 34. Extracted Standards of PFOS in Whole Body Catfish Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively I XC032108-1 PFOS (Standard) 499.0/90.Oamn,499.0/99.0 am n -sam ple 1 o f 55 from 032100C.wiff Area: 733799co u n ts Height: 1.91e+004cps RT: 10.9min T im e, m in I X C 0 3 2 1 0 8 -2 - PFOS (S tan d ard) 4Q 9.0/80.0 a m u,499 .0/9 9.0 am u - sam ple 2 o f 55 from 032108C .iw iff Area: 187578 counts H e ig h t 2 .7 8 e + 0 0 4 cps RT: 10.8 m in 3 .0 e 4 2 .5 e 4 - CO Q. 2 .0 e 4 O & CO 1 .5 e 4 c 0) c 1.0e4 5 0 0 0 .0 0 . 0 J------ ------ 1------ ------- 1------ ------- .------------- 1------------- 123 45 W 7 8 9 10 11 12 13 1 4 15 16 T im e, m in MPI Research Page 85 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 35. PFOS in a Control Whole Body Catfish, a 0.075 ng/mL (0.30 ng/g) Fortified Whole Body Catfish Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Whole Body Catfish Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Whole Body Catfish Spike C, Respectively I C0292558 Contro t 1 - PFOS (Unknown) 499.0/80.0 a/??i/,499.0/99.0 am u -sam ple 12 o f 55 front 032109C.wiff Area: 105409counts Height: 1.36e+004cps RT: 10.9 min T im e, m in I C 0292558 S pkA 1 - PFOS (Q C) 4 9 9 .0 /8 0 .0 a m u,4 99 .0 /9 9 .0 am u - sam ple 13 o f 55 from 0 3 2 1 0 8 C .wiff A rea: 2 37 77 0 counts H eight: 3.51 e +0 04 cps RT: 10.8 m in 3.0 e 4 0 .0 -Al L. 123456789 10 11 12 Tim e, m in C0292558S p k B 3 -P F O S (QC)499.0M0.0amu,499.0/99.Oamu -sa m p le 2 0 o f 55 from 032109C.wiff Area: 904606 co u n ts Height: 1.45e+005cps RT: 10.9 min T im e , m in I C 0 2 9 2 5 5 8 Spk C1 P F O S ( Q C ) 4 9 9 .0 /8 0 .0 a m u .4 9 9 .0 /9 9 .0 a m u - s a m p le 21 o f 5 5 fro m 0 3 2 1 0 8 C .w iff A rea: 611 26 9 6 counts H eight: 9 .9 5 e+ 00 5 cps RT: 10.8 m in MPI Research Page 86 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 36. Chromatogram Representing a Whole Body Catfish Sample Analyzed for PFOS (ExyLIMS ID: C0227612, Data Set: 032108C) I C022762 -P F O S (Unknow n) 499KV8G.0 a m u ,499.0/99.0 am u -s am p le 30 o f 55 from 032109C.WH A rea: 14126900 counts H eight: 2.90e+006 cps RT: 10.9 m in Intensity, cps MPI Research Page 87 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 37. Typical Extracted Calibration Curve for PFBS in Whole Body Bass Matrix 0 4 2 2 0 8 A W hole Bass S et # 1.fdb (PFBS): " Quadratic?' Regression ("1 w e ig htin g ): y = .5 .2 6 e + 0 0 4 x * 2 + 1.9e+006 x + 4 .0 4 e+ 00 3 (r = 0 .9 9 98 ) Area, counts Concentration. ng/mL MPI Research Page 88 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 38. Extracted Standards of PFBS in Whole Body Bass Matrix, 0.01 ng/mL (0.10 ng/g) and 0.02 ng/mL (0.20 ng/g), Respectively I XC042208-1 - PFBS (Standard) 299.0/80.0 amu,299.0/99.0 amt/ -sample 1 o f 67 from 042208A.wiff Area: 21537 counts Height: 5.19e*003 cps RT: 8.75 min 8.75 Tim , min I X C 042208-2- PFBS (Standard) 299.0/80.0 amu.299.0/99.0 amu - sample 2 of 67 from 042208A.w iff Area: 4 14 63 counts H eight: 1.0 2 e+ 00 4 ops R T :8 .7 1 min 8.71 MPI Research Page 89 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 39. PFBS in a Control Whole Body Bass, a 0.03 ng/mL (0.30 ng/g) Fortified Whole Body Bass Spike A, a 0.2 ng/mL (2.0 ng/g) Fortified Whole Body Bass Spike B, and a 4.0 ng/mL (40 ng/g) Fortified Whole Body Bass Spike C, Respectively I C0305046 C o n tro / 1 - PFBS (Unknown) 299.0/30.0 a m u ,299.0/99.0 amu -sample 12 o f 6 7 from 042208A.wff (peak not found) Tim e, min I C0305046 Spk A1 - PFBS (QC) 200.0/80.0 mu,200.0/00.0 mu sample 13 of 67 fiom 042208A.w iff Area: 61830 counts Height: 1.54e+004cps R T:8.65 min 8.65 Tim e, min I CO305O46Spk B 3-PFBS (QC) 299.0/80.0 a m u ,299.0/99.0antu -sample 2Oof 67 from 04220dA.wiff Area: 396270 c ounts Height: 9.94e+004cps RT: 8.72 min 8.72 8.0e4 B .0 e 4 r C 4,0e4 2.0e4- 0 .0 -I-------------1------ -------1------------- 1-------------1------ ------- 1------------- 1----- ------- .------------- 1------ - ^ - i -------------------------- .----------------------------------------- .------------ r 1 2 3 4 5 6 7 8 0 10 11 12 13 14 15 Time, min I C0305046 Spk C1 - PFBS (0 0 )2 0 0 .0 /8 0 .0 am u.200.0/e0.0 a m u -s a m p le 21 of 67 from 042208A.iw iff Area: 6805321 counts Height: 1.72e+00Q cps R T :8 .7 0 min i 16 8.70 1.5e6 U) Q. 1 .0 .B - V) c 22 ~ 5.0e5 0.0 J------ ------- 1-------------1------ ------- .------ ------- 1------------- 1------------- 1------------ 1------------- 1------- * - i ------ ------- 1------------- 1------------- 1------ ------- 1-------------1----- ------- 1------------- r 1 2 3 4 5 6 7 8 0 10 11 12 13 14 15 16 Time, min MPI Research Page 90 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 40. Chromatogram Representing a Whole Body Bass Sample Analyzed for PFBS (ExyLIMS ID: C0227603, Data Set: 042208A) I C022763 - PFBS (Unknown) 299.0/30.0 am a,29a0/99.0 amu -sampte 56 o f 67 from 04220BA.w/ff freak not found) Intensity, cps MPI Research Page 91 of 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 41. Typical Extracted Calibration Curve for PFHS in Whole Body Bass Matrix 0 4 2 2 0 8 A W h o le Bass S et #1.rdb (PFHS): "Q u a dra tic" Regression C'1 / * ' w e ig htin g ): y = -4 .3e + 0 0 4 x *2 + 1 .8 3 e + 0 0 6 x + 1.92e+003 (r = 0 .9 9 99 ) Area, counts Concentration, ng/m L MPI Research Page 92 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 42. Extracted Standards of PFHS in Whole Body Bass Matrix, 0.01 ng/mL (0.10 ng/g) and 0.02 ng/mL (0.20 ng/g), Respectively XC04220S-1 PFHS (Standard)399.0/80.0am a,399.M 9.0amu -sampto 1 o f 7 front 042200A.wiff Area: 20959 counts Height: 4.46c+003c#w RT: 10.0 min 10.00 Tim e, min XC042208-2 - PFHS (S tandard)399.0/80.0 amu,399.0/99.0 amu sample 2 of 67 from 042208A.w iff Area; 39648 counts Height: 8 ,6 7 e+003 cps R T :9 .9 0 min 9.96 MPI Research Page 93 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 43. PFHS in a Control Whole Body Bass, a 0.03 ng/mL (0.30 ng/g) Fortified Whole Body Bass Spike A, a 0.2 ng/mL (2.0 ng/g) Fortified Whole Body Bass Spike B, and a 4.0 ng/mL (40 ng/g) Fortified Whole Body Bass Spike C, Respectively C0305046 Control 1 - PFHS (Unknown) 399.0/80,0 amu,399.0/99.0 amu -sample 12 o f 67 from 042209A.wiff freak not found) 12.07 Tim e, min I C0305046 Spk A1 PFHS (QC) 399.0/80.0 am u.399.0/99.0 amu sample 13 of 67 from 0 42208A.wiff Area: 56938 counts Height: 1.22e+004 cps RT: 9.91 min 9.91 Time, min 1 C0305046 Spk 03 - PFHS (QC) 399.0800 amu,399.<V99.0 amu -sample 20 o f 67 from Q4220M.wiff Area: 366398 counts Heigkt: 7.94e*004cps RT: 9.99 min 9.98 Tim e, min I C0305046 SpkC1 - PFHS (QC) 399.0/80.0 amu.399.0/99.0 am u-sam ple 21 of 67 from 042208A.wiff Area: 6764270 counts Height: 1.43e+006 cps R T:9.9S min MPI Research Page 94 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 44. Chromatogram Representing a Whole Body Bass Sample Analyzed for PFHS (ExyLIMS ID: C0227601, Data Set: 042208A) I C0227601 -PFH S (Unknown)399.0/00.0 amut299.0/99.0amu -sam ple 4 0 o f 67 from 042200A.wiff A n a : 31415 count Height: 7.31e+Q03cps RT: 9.95mitt 9.95 Intensity, cps MPI Research Page 95 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 45. Typical Extracted Calibration Curve for PFOS in Whole Body Bass Matrix 0 4 2 2 0 8 A W hole Bass Set # 1.rdb (PFOS): "Q u a dra tic" Regression ( " t / x " w eig htin g ): y = -1 .3 8 e + 0 0 4 x A2 + 1.15e+006 x + 1.1e+005 (r = 0.9 9 98 ) Area, counts MPI Research Page 96 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project N o.: P0003268 Figure 46. Extracted Standards of PFOS in Whole Body Bass Matrix, 0.01 ng/mL (0.10 ng/g) and 0.02 ng/mL (0.20 ng/g), Respectively XC042208-1 - PFOS (Standard) 499.0/80.0 am u,499.0/99.0 am u -sa m p le 1 o f 67 from 042208A.mff Area: 122754counts Height: 2.46e+004cps RT: 10.9 m in 9.08 Tim e, min XC422Q8-2 - PFOS (S tandard) 4 9 9 .0/8 0.0 amu,499.Q>99.0 am u sam ple 2 of 67 from 0 4 2 2 0 8 A .w iff Area: 1358S8 counts Height: 2.7 3 e+ 00 4 cps RT: 10.9 min 9.01 MPI Research Page 97 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 47. PFOS in a Control Whole Body Bass, a 0.03 ng/mL (0.30 ng/g) Fortified Whole Body Bass Spike A, a 0.2 ng/mL (2.0 ng/g) Fortified Whole Body Bass Spike B, and a 4.0 ng/mL (40 ng/g) Fortified Whole Body Bass Spike C, Respectively 1 C0305046 Control 1 - PFOS (Unknown) 499.0/99.0 amu,499.0/99.0 amu -sample 12 o f 67 from 042209A.wiff Area: 99962 counts Height: Z14e+004cps RT: 70.9 min 8.96 I C0305046 Spk A1 PFOS (QC) 409.0/80.0 amu.499.0/99.0 a m u - sample 13 of 87 from 042208A.w iff Area: 138974 counts Height: 2.86e+004 cps RT: 10.9 min 8.96 1 C0305046 Spk B3 PFOS (QC) 499.0/90.0 amu,499.0/99.0 ama -sample 20 o f 67 from 042209A.wiff A n a : 343163 counts Height: 6.39e+004cps RT: 10.9 min 9.03 Tim e, min C0305046 Spk C1 - PFOS (0 0 )4 0 9 .0 /8 0 .0 am u.499.0/99.0 amu - sample 21 of 67 from 042208A.w iff Area: 4431077 counts Height: 7.52e+005 cps RT: 10.9 min 10.91 MPI Research Page 98 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 48. Chromatogram Representing a Whole Body Bass Sample Analyzed for PFOS (ExyLIMS ID: C0227600, Data Set: 042208A) I 1C022760 - PFOS (Unknown) 499.0/8010 a nut,499.0/99 0 antn sample 30 o f 67 front 042209A.wiff A n a : 2374133 coants Height: 5.23e+0O5 cps RT: 70.8 min 10.85 Intensity, cps MPI Research Page 99 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 01,37.0219 MPI Project No.: P0003268 Figure 49. Typical Extracted Calibration Curve for PFBS in Clam Matrix 0 5 0 5 0 8 A Clam s S et riM.rdb (PFBS): "Q u a dra tic" Regression p / x" w e ig htin g ): y = -2 .3 4 e + 0 0 4 x *2 + 1 .1 6 e + 0 0 8 x + 138 (r = 0 .9 9 9 1 ) Area, counts MPI Research Page 100 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 50. Extracted Standards of PFBS in Clam Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively XC0S0509-1 -PFBS (Standard) 299,0/90.0amu,299.0/99.0 amu -sample 1 o f SI from OSQSOdA.wiff Area: 27922 counts Height: 7.ite+003cp$ RT: 9,49 min 8.48 Tim e, min I XC050508-2 - PFBS (Standard) 299.0/80.0 amu.299.0/09.0 a m u - sample 2 of 61 from 060508A.iwiff Area: 57408 counts H e ig h t 1.42e+004 cps RT: 8.52 min MPI Research Page 101 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 51. PFBS in a Control Clam, a 0.075 ng/mL (0.30 ng/g) Fortified Clam Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Clam Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Clam Spike C, Respectively CO259278 Control 1 - PFBS (UttAnowtt) 299L(VftlL0 am ir,299.0/99.0 amir -sample 12 o f 61 from 05050BA.wtff Area: 1493 counts Height: 2.95e+002cps RT: 8.50 mr Tim e, min C0259278 Spk A1 PFBS (QC) 299.0/80.0 amu,299.0/99.0 amu sample 13 of 61 from 050508A.w iff Area: 101601 counts Height: 2.55e +0 04 cps R T :6 .4 7 min 2.5e4 A 8.47 2 .0 e 4 CO Q. 1.5e4 CO c a> 1.0e4 c 5000.0 0.0 J------ ------- 1-------------.------------------- ------ 1------------- 1------------- 1------------- 1------------- 1-- ---- 1------ ------- ----- ------- ------------- e 12 3 4 5 6 7 8 0 10 11 12 Tim e, min C0259278 SpA B 3-PFBS (QC) 299.0t90.0amu,299.M9.0ama sample 20 o f 61 from 050509A.wiff Area: 595199 counts Height: 1.45e+005cps RT: 9.46 min ' `I 13 I------------- 1------ ------- r 14 15 16 1 4e5 -I 8 46 1.2e5 - to 1,0e5 Q. 8.0e4 <o 6 ,0 .4 C " 4.0e4 2.0e4 0.0 J------ ------- .------ -------1------------- 1-------------i-------------1------ ------- .------------ 1-------------1---- ----- .-------------1------------- 1------ ------- 1------ ------- 1------ ------- 1------------- 1------------- r 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Tim e, min I C0259278 Spk C1 - PFBS (QC) 299.0/80.0 am u.200.0/89.0 amu sample 21 o f6 1 from 050508A.w iff Area: 4 45 0952 counts Height: 1.10e+008 cps RT: 8.47 min 1.00e6 8.47 to 8.00 e5 0-OOeS <0 c 4.00 e5 2.00e5 0.00 J-------1-------.------ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Tim e, min MPI Research Page 102 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 52. Chromatogram Representing a Clam Sample Analyzed for PFBS (ExyLIMS ID: C0227658, Data Set: 050508A) I C022765B - PFBS (Unknown) 299.9/90.9 amu,299.0M9.0 amu -sample 30 o f 61 from 959509A.wiff Area: 3903 counts Height: 9.35e+002 cps RT: 9.45 min 10.33 Intensity, cps MPI Research Page 103 o f 197 Interim Report #3 --Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 53. Typical Extracted Calibration Curve for PFHS in Clam Matrix 0 5 0 5 0 8 A Clam s S e tiM .rd b (PFHS): "Quadratic?' Regression C'1 w e ig htin g ): y = -1 .77e+003 x *2 + 2 .0 2 e+ 00 5 x + 651 (r = 0 .9 9 86 ) Area, counts Concentration. ng/m L MPI Research Page 104 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 54. Extracted Standards of PFHS in Clam Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively I jTC0S0S08-7 - PFHS (Standard) 399L0/80.0 amu,399.0/99.0 amu -sample 1 o f 61 from 050509A.wiff Area: 5027 counts Height: 1.04e+003cps R7: 9.77 min 1000 9.77 800 to Qo . 000 400 - / S i i f t -- Yr*i 12 13 14 Tim e, min I XC050508-2 - PFHS (S tandard)399.0/80.0 amu,399.0/99.0 amu - sample 2 of 61 from 050508A.i/uiff Area: 11764 counts Height: 2.64e+003 cps RT: 9.82 min 9.82 MPI Research Page 105 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 55. PFHS in a Control Clam, a 0.075 ng/mL (0.30 ng/g) Fortified Clam Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Clam Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Clam Spike C, Respectively I C0259278 Contro1 1 - PFHS (Unknown) 399.0/80.0 ama,399.0/99.0 amu -sample 12 o f 61 front 05050OA.wiff freafs not found) 16.21 V) Q<_>. & Time, min I C0259278 Spk A1 - PFHS (QC) 399.0/80.0 am u.399.0/99.0 amu sam ple 13 of 61 from 050508A .w iff Area: 16419 counts Height: 3.61 e+003 cps R T :9 .7 6 m in 9.76 I C0259278 Spit B3-PFHS (QC) 39910/80.0 firmer,399l0/99,0 amu -sample 20 o f 61 front 050509A.wtff Area: 105600counts Hefgftt: 2.25e+004cps RT: 9.75m/ff 9.75 Time, min 00259278 Spk C1 PFHS (QC) 399.0/80.0 arnu.399.0/99.0 amu sample 21 of 61 from 050508A.iAiiff Area: 826601 counts Height: 1.76e+005 cps R T :9 .7 6 min 9.76 MPI Research Page 106 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 56. Chromatogram Representing a Clam Sample Analyzed for PFHS (ExyLIMS ID: C0227658, Data Set: 050508A) I C0227658 - PFHS (Unknown)399.0/80,0 amu,399.0/99.0 amu -sampie 30 o f S1 from QSOSOZA.wiff Area: 1201 counts Height: Z28e+002cp* RT: 9.77min Intensity, cps MPI Research Page 107 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 57. Typical Extracted Calibration Curve for PFOS in Clam Matrix I 05Q 508A Clams S e t# 1 .rd b (PFOS): " Q uadratic" Regression C'1 w e ig htin g ): y = *2.79e+Q03 x*2 + 5 .3 8 e+ 00 5 x + 1.92e+0Q3 (r = 0 .9 9 96 ) Area, counts MPI Research Page 108 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 58. Extracted Standards of PFOS in Clam Matrix, 0.025 ng/mL (0.10 ng/g) and 0.050 ng/mL (0.20 ng/g), Respectively I XC050508-7 - PFOS (Standard) 499.V80.O amu,499.0/99.0 amu -sample 1 o f 61 from 050509A.wiff Area: 14999counts Heigftt: 2LS6e+003cps RT: 10.7mitt 10.72 Tim e, min I XC050508-2 - PFOS (S tandard)409.0/80.0 amu,409.0/99.0 am u* sample 2 of 61 from 050508A.iwitf A rea:287S 1 counts Height: 4.90e+003 cps RT: 10.8 min 10.78 MPI Research Page 109 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 59. PFOS in a Control Clam, a 0.075 ng/mL (0.30 ng/g) Fortified Clam Spike A, a 0.50 ng/mL (2.0 ng/g) Fortified Clam Spike B, and a 4.0 ng/mL (16 ng/g) Fortified Clam Spike C, Respectively C02S9279 Contro/ 1 PFOS (Unknown) 499.M 0 .0 amot499.0/99.0 ami/ -sample 12 o f 61 from 050509A.wiff Area: 3267 count* Height: &78e+002cps RT: 19.7 min I C0259278 SPkA 1 PFOS (QC) 409.0/80.0 am u.499.0/99.0 a m u - sample 13 of 81 from 050508A.w iff Area: 50796 counts Height: 8.59e+003 cps RT: 10.7 min J55s 12.03 . nfs r 1 23456789 10 11 Tim e, min 0259278 Spit 0 3 - PFOS (QC) 49910/80.0 amo,499.0/99.0 amu -sampfe 20 o f 61 from 05050BA.wiff Area: 305491 counts Height: S.14e+004cps RT: 70L7nv/n 10.70 Time, min I C0259276 SpkC1 - PFOS (QC) 499.0/80.0 am g.499.0/99.0 a m u - sample 21 of 61 from 050S08A.wiff Area: 2224443 counts Height: 3.82e+005 cps RT: 10.7 min 10.71 MPI Research Page 110 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Figure 60. Chromatogram Representing a Clam Sample Analyzed for PFOS (ExyLIMS ID: C0227658, Data Set: 042208A) I CO227658 - PFOS (UnAnown) 499.0/80.0 amu,499.0/99.0 amu -sampte 30 o f 61 from 050508A.wiff A n a : 102697 counts Height: 2.33e+0O4cp$ RT: 10.7 min 10.71 Intensity, cps MPI Research Page 111 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 APPENDIX A Study Protocol and Amendments MPI Research Page 112 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 STUDY PROTOCOL Study Title: Analysis of Perfluorooctanoic Acid (PFOA) in Water, Soil, Sediment, Fish, and Clams Using LC/MS/MS for the 3M Decatur Monitoring Program MPI Research Study Number.: 0137.0219 0 ExyLIMS Protocol Number: P0003267 Performing Laboratory: M PI Research, Inc. State College 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 Sponsor Representative: Michael A. Santoro Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651)733-6374 Page o f 56 MPI Research Page 113 of 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions, Inc. 2) Karen Risha, Principal Investigator, MPI Research, Inc. 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) MPI Research Quality Assurance Unit MPI Research Page 2 o f 56 Page 114 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 PROTOCOL APPROVAL Study Title: Analysis o f Perfluorooctanoic Acid (PFOA) in Water, Soil, Sediment, Fish, and Clams Using LC/MS/MS for the 3M Decatur '/trnitorir.g P'-og' '!^' MPI Research Study Number: 0137.0219 ExyLIMS Protocol Number: P0003267 APPROVALS EQ u Jaisimha Kesari, Study Director Weston Solutions, Inc. Michael A. Santoc, Sponsor Representative 3M Company / aren Risha, Principal Investigator MP1 Research, Inc. Lydia Chaffer, Qualify Coi Assurance Manager MPI Research, Inc. Date a i/tf/oi Date Page 3 of 56 MPI Research Page 115 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 TABLE OF CONTENTS TITLE PAGE......................................................................................................................................................1 DISTRIBUTION................................................................................................................................................ 2 PROTOCOL APPROVAL................................................................................................................................. 3 TABLE OF CONTENTS................................................................................................................................... 4 INTRODUCTION.............................................................................................................................................. 5 TEST MATERIAL............................................................................................................................................. 5 SURROGATE FIELD SPIKE COMPOUND.................................................................................................... 5 OBJECTIVE...................................................................................................................................................... 6 TESTING FACILITY........................................................................................................................................ 6 STUDY DIRECTOR.......................................................................................................................................... 7 SPONSOR REPRESENTATIVE....................................................................................................................... 7 PRINCIPAL INVESTIGATOR......................................................................................................................... 7 PROPOSED EXPERIMENTAL START AND TERMINATION DATES..................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM..........................................................7 SAMPLE PROCUREMENT. RECEIPT AND RETENTION..........................................................................8 SAMPLE IDENTIFICATION........................................................................................................................... 8 ANALYTICAL PROCEDURE SUMMARY.................................................................................................... 9 VERIFICATION OF ANALYTICAL PROCEDURE....................................................................................... 10 METHOD FOR CONTROL OF BIAS.............................................................................................................. 11 STATISTICAL METHODS.............................................................................................................................. 11 GLP STATEMENT............................................................................................................................................II R EPO R T............................................................................................................................................................. 12 SAFETY AND HEALTH.................................................................................................................................. 13 AMENDMENTS TO PROTOCOL................................................................................................................... 13 DATA RECORD KEEPING............................................................................................................................. 13 QUALITY ASSURANCE................................................................................................................................. 14 RETENTION OF DATA AND ARCHIVING.................................................................................................. 14 APPENDIX I, ANALYTICAL METHODS...................................................................................................... 15 Page 4 o f 56 MPI Research Page 116 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 INTRODUCTION The purpose o f this study is to perform analysis for perfluorooctanoic acid (PFOA) in water, soil, sediment, fish, and clams using LC/MS/MS for the 3M Decatur Monitoring Program. The study will be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit o f MPI Research. TEST MATERIAL The test material is perfluorooctanoic acid (PFOA) purchased from Oakwood Product, Inc. PFOA Chemical Name: Perfluorooctanoic acid Molecular Weight: 414 Transitions Monitored: 413 - 369 (for quantification) and 413 -->219 (for confirmation) Structure: FFF FFFF OH SURROGATE FIELD SPIKE COMPOUND The surrogate field spiking compound is l3C labeled perfluorooctanoic acid (13C PFOA) provided by the sponsor. The surrogate field spiking is optional. I3C PFOA Chemical Name: 1,2-I3C perfluorooctanoic acid Molecular Weight: 416 Transition Monitored: 415 --> 370 Structure: FFF FFF C ^ ^OHF I I3 p _ 13 F F FI F Page 5 o f 56 MPI Research Page 117 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 OBJECTIVE The purpose o f this study is to perform analysis for perfluorooctanoic acid (PFOA) in water, soil, sediment, fish, and clams for the 3M Decatur Monitoring Program using the current versions o f the following 3M Company and MPI Research analytical methods: ETS-8-012: "Method of Analysis for the Determination o f Perfluorobutanoic Acid (PFBA), Perfluoropentanoic Acid (PFPeA), Perfluorohexanoic Acid (PFHA), Perfluoroheptanoic Acid (PFHpA), Perfluorooctanoic Acid (PFOA), Perfluorononanoic Acid (PFNA), Perfluorodecanoic Acid (PFDA), Perfluoroundecanoic Acid (PFUnA), Perfluorododecanoic Acid (PFDoA), Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil and Sediment by LC/MS/MS" V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781 "Method o f Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782 "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783 "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" The primary analytical method for the analysis of the water, soil, and sediment samples is the ETS-8-012 method. In instances where quantitative analytical results are not obtained by this method, the Study Director may direct the performing laboratory to analyze the samples by alternate methods (V0001780 for water samples, V0001781 for soil samples, and V0001782 for sediment samples). TESTING FACILITY MPI Research, Inc. (formerly Exygen Research, Inc.) State College 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 Fax: (814)231-1580 Page 6 o f 56 MPI Research Page 118 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax: (610)701-7401 j.kesari@westonsolutions.com SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director of Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone:(651)733-6374 PRINCIPAL INVESTIGATOR Karen Risha MPI Research, Inc. State College 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 karen.risha@mpiresearch.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion of this study be conducted from June 14, 2007 to December 31, 2007. The actual experimental start and termination dates will be included in the interim reports and the final report. IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: Water (groundwater and surface water) Soil Page 7 o f56 MPI Research Page 119 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Sediment Fish Clams The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the appropriate interim report and/or final report associated with this study. The test systems were chosen to comply with the 3M Letter o f Intent (LOI) with the U.S. EPA to collect PFOA data. Additional samples not part of the LOI may be analyzed at the discretion o f the Study Director. SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, and clam samples are being received at MPI Research, State College directly from Weston Solutions. The details o f sample procurement procedures for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media for PFOA at the 3M Decatur AL Plant." The number and types o f samples received and analyzed will vary depending on data needs for additional characterization of PFOA concentrations in various environmental media and availability in the field. The total number o f samples received and analyzed for each matrix will be documented in the appropriate interim report associated with this study. Water, soil, and sediment samples will be used as received at MPI Research. These samples will be homogenized before the extraction process by vigorously shaking the sample bottles. These samples will be stored refrigerated at 2C-8C. Fish and clam samples will be processed according to the appropriate analytical method (see Appendix I). These samples will be stored frozen at < -10C. The receipt and processing of the samples will be documented in the appropriate interim report and raw data associated with the study. SAMPLE IDENTIFICATION Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Page 8 o f 56 MPI Research Page 120 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Sample storage conditions and locations will be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: ETS-8-012: "Method of Analysis for the Determination o f Perfluorobutanoic Acid (PFBA), Perfluoropentanoic Acid (PFPeA), Perfluorohexanoic Acid (PFHA), Perfluoroheptanoic Acid (PFHpA), Perfluorooctanoic Acid (PFOA), Perfluorononanoic Acid (PFNA), Perfluorodecanoic Acid (PFDA), Perfluoroundecanoic Acid (PFUnA), Perfluorododecanoic Acid (PFDoA), Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil and Sediment by LC/MS/MS" V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" The primary analytical method for the analysis o f the water, soil, and sediment samples is the ETS-8-012 method. In instances where quantitative analytical results are not obtained by this method, the Study Director may direct the performing laboratory to analyze the samples by alternate methods (V0001780 for water samples, V0001781 for soil samples, and V0001782 for sediment samples). The above referenced methods are to be followed for the analysis o f the samples with the following exceptions: If sludge samples are received by MPI Research to be included in the study, the sludge samples will be extracted and analyzed following the method for soil samples. If necessary for the preparation o f appropriate standards levels, alternate weights of test material and alternate volumes o f suitable solvent (acetonitrile, methanol, etc.) may be used. If necessary, the duplicate bottle o f a water sample may be used in an extraction if the primary bottle does not have enough volume available. Page 9 o f 56 MPI Research Page 121 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sample will be used for the preparation of fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, MPI Research will supply one bottle per sample collected. The bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for PFOA sample collection at the testing facility and have been shown to be free o f PFOA. All containers used for water sample collection will be shipped to the sample location and may or may not contain l3C PFOA, as l3C PFOA is not a requirement for this study, but may be incorporated at the discretion o f the Study Director. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike, a mid field spike (optional, at the discretion of the Study Director), and a high field spike of each sample will be collected. The sample and the field duplicate may or may not contain 100 ng of l3C PFOA (equivalent to 0.50 ng/mL in the final sample). The low, mid (optional), and high field spikes will contain PFOA and l3C PFOA (optional) as well as perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS). These compounds are included in the solutions used to spike the samples. The results for PFBS, PFHS and PFOS will not be reported in this study. MPI Research will supply one field blank (control water fortified with 13C PFOA, optional) and two or three field blank spikes (control water fortified with PFOA and 13C PFOA (optional) at a low, mid (optional), and high level) for every twenty aqueous samples collected at a minimum. At the testing facility, each water sample (excluding field duplicates and field spikes) may be extracted in duplicate and may also be fortified at a low, mid (optional), and high concentration and processed through the described procedure. The use of the in lab duplicate and the in lab fortified spikes is not a requirement but may be requested to determine method accuracy and to check for bias at the discretion o f the Study Director. For soil, sediment, fish, and clams, MPI Research will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containers/bags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration with PFOA at a low, mid (optional, at the discretion o f the Study Director), and high level and processed through the described procedure to determine method accuracy and to check for bias. ,3C-PFOA may or may not be added to each sample in the laboratory prior to extraction as 13C PFOA is not a requirement for this study, but may be incorporated at the discretion of the Study Director. Page JO o f 56 MPI Research Page 122 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLlMS Protocol Number: P0003267 Low, mid, and high spiking levels for each matrix are defined below: M atrix Low PFOA Mid PFOA High PFOA Spiking Levels Spiking Levels Spiking Level Water 0.25 ng/mL 5.0 ng/mL 100 ng/mL Soil 2ng/g 40 ng/g 800 ng/g Sediment 2 ng/g 40 ng/g 800 ng/g Fish 2 ng/g 40 ng/g - Clams ____ 2 n S/g____ 40 ng/g - Low, mid and high spiking levels of the analytes for each matrix may be altered depending on sample size, sample availability, and/or to cover analyte concentrations expected in the samples. In instances where the expected analyte concentrations exceed the relevant range o f the lower level spikes, the lower level spikes may be deferred as for the analysis will not provide any usable data. Recoveries are anticipated to be between 70% and 130% o f the fortified levels; however, the exact precision and accuracy will be determined by the analysis o f the quality control samples described above. A statement o f accuracy will be included in the appropriate interim report. METHOD FOR CONTROL OF BIAS Control of bias will be addressed by taking representative sub-samples from a homogeneous mixture o f each matrix from untreated control samples, and by analyzing at least two levels of fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation o f average recoveries, as applicable. GLP STATEMENT All aspects o f this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative. Page II o f 56 MPI Research Page 123 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 REPORT Interim reports will be prepared by the principal investigator or their designee for specific sampling sites and sample matrices due to the size o f the data sets and the phased nature o f sample collection activities. A final report will be prepared by the principal investigator or their designee at the conclusion o f the study. All reports will include, but will not be limited to, the following: The name and address o f the Study Director, Sponsor Representative, and of the testing facility. A statement o f GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). The signed and dated statement by the MPI Research Quality Assurance Unit regarding dates o f study inspections and dates findings were reported to the Study Director and Management. A description o f the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy of the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page. A description of the instrumentation used and operating conditions. All results from all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level. Representative chromatograms for each analyte in each matrix, including chromatograms o f a standard and a control sample, and a chromatogram at a fortification level. The location o f the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity o f the data will be documented in the report. Locations where raw data, interim reports, and the final report are to be archived. Additions or corrections to the final report shall be in the form o f an amendment signed by the Study Director. The amendment shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Representative. Page 2 o f 56 MPI Research Page 124 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 SAFETY AND HEALTH Laboratory personnel will practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure of personnel and the environment to the test or reference substance(s). AMENDMENTS TO PROTOCOL All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation of study plan change. However, when a change is required without sufficient time for the issue o f a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date of the written amendment will be the date o f the documented change. Copies o f the signed amendments will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description o f any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing o f study personnel, signatures and initials Chronological presentation of all study correspondence Any other documentation necessary for the reconstruction o f the study Chromatograms- All chromatograms will contain the following: Sample identification, injection date, arrow or other indication o f the area o f interest, and injection number corresponding to the run. Page 13 o f 56 MPI Research Page 125 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Additionally, fortifications will include the amount o f analyte added and the sample number o f the sample that was fortified. Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL). . As part o f the documentation the following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. All applicable requirements for reporting o f study results as per 40 CFR 792.185. QUALITY ASSURANCE The QA Unit of MPI Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings of audits to the Study Director, MPI Management at State College, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period o f time specified in 40 CFR 792.195. An exact copy o f the materials submitted to the study director will also be kept at MPI Research State College. MPI Research will obtain permission from the study director before discarding or returning samples. Page 4 o f 56 MPI Research Page 126 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 APPENDIX I ANALYTICAL METHODS ETS-8-012: "Method of Analysis for the Determination o f Perfluorobutanoic Acid (PFBA), Perfluoropentanoic Acid (PFPeA), Perfluorohexanoic Acid (PFHA), Perfluoroheptanoic Acid (PFHpA), Perfluorooctanoic Acid (PFOA), Perfluorononanoic Acid (PFNA), Perfluorodecanoic Acid (PFDA), Perfluoroundecanoic Acid (PFUnA), Perfluorododecanoic Acid (PFDoA), Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil and Sediment by LC/MS/MS" V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" MPI Research Page 15 o f 56 Page 127 o f 197 Interim Report #3 --Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 3NI Environmental Laboratory . Method . Method o f A n a ly s is f o r th e D e te rm in a tio n o f P e r flu o ro b u ta n o ic A d d (P F B A ), P erfluo ro p e n ta n o ic A d d (PFPeA), P erfluo ro he xa no ie A d d (PFHA), Ferfluoruftepfano/c A d d (P FH pA ), P e r flu o ro o c ta n o lc A d d (P F O A ), P erfluo ro no n an oic A d d fP F N A ), P erfluorodecanolc A d d (P F D A ), Perfluoroundecano/c A d d (P F U n A ), P e r fiu o ro d o d e c a n o ic A d d (P F D o A ), P e rflv o ro b v ta n e s u ifo n a te (PFBS), P erflv o ro h e x a n e s u llo n a te fPFHS), a n d P erfiu o ro o cta n e su tfo n a ie (PFOS) in W ater, S o il a n d S e d im e n t b y LC /M S /M S . Method Number; 7S*8*012.t A d o p tio n D ate: U pon S igning Effective Date: Approved Gy: tesseri, Technical Manager Oste 6TS--012.1 Page I o f 12 uodtod efA naida 1 tho OaionrtnaUcn o(P artodotvunde Add (PFSA), PtcGuorepcrrtanofc Add (PFPAJ. PrffluwWMxandC Add (PFHA). PtrdvwohepunpK Add (P F ^A ). Pvftuorwdanota Add (PFOA). Partuerananande Add (PFHA). Pcsfuorodcanoe Add (PFOA). P*rtuMoaCnole Add (PFUnA|.PtduorododocandcAdd (PFOoA), PoreMroOvlanowdenato (PFBS). Pirthw rem anM dtow W (PFHS). and Pdfueroodancodtonat (PFOS)In W aif, Sod and Sodbm * by LC/MS/MS Page 16 o f 56 MPI Research Page 128 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 1 Scope and Application This method is to be used to quantify Perftuorobutanoic Add (PFBA), Perftuoropenlanoic Add (PFPeA), Perfluorohexanoic Add (PFHA), Perfluoroheptanoic Add (PFHpA), Perfluorooctanoic Acid (PFOA). Perfluorononanob Add (PFNA), Perfluorodecanoic Acid (PFDA). Perfluoroundecanoic Add (PFU rt), Perfluorododecanoic Add (PFDoA), Perfluorobutanesutfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesultonate (PFOS) by High Performance Uquid Chromatography coupled to a tandem Mass Speoromethc Detector (LCIMS/MS) in water, so) and sediment The method is designed to target a towerBmiJ of quantitation (LOQ) of0.025 ng/mL (water) and 0.20 ng/g (soil and sediment). 2 M e th o d S u m m a ry____________________________________ ________________ _____ Aqueous samples are mixed wfth equal volumes of acetonitrfle, thoroughly mixed, centrifuged if necessary, and 3flquoted for analysis by LC/MS/MS. Similarly, one-gram aliquots of sofl and sediments are mixed with 80:20 acdonM awater mixture, thoroughly mixed, centrifuged, and quoted for analysis by LC/MS/MS, This is a performance-based method. Method accuracy is determined for each sample set using multiple laboratory control spikes at multiple concentrations. This method also requires that the precision and accuracy for each sample be determined using field matrix spikes (aqueous samples) or laboratory matrix spires (sol and sediment) to verify that the method b appScable to each sample matrix. Sample results for spikes outside of 70% to 130%, win not be reported due to non-compSant quality control samples. Fortification levels for field matrix spikes and for laboratory matrix spikes should be at least 50% of the endogenous level and less than 10 limes the endogenous level to be used to determine the statement of accuracy foranalytical results. ' 3 Definitions 3.1 Calibration Standard . . A solution prepared by spiking a known volume of the Working Standard (WS) into a predetermined amountof ASTM type I or HPtC grade water, diluted with acetonitrile, and analyzed according to Ihb method. Calibration standards are used to calibrate the instrument response with respect to snalyta concentration. 3.2 Laboratory Duplicate Sample (LDS, or Lab Oup) A laboratory duplicate sample is a separate aliquot of e sample taken in the analytical laboratory that b extracted and analyzed separately with identical procedures. Analysis of LDSs compared to that of the first. aliquot give a measure of the precision associated with laboratory procedures, but not wtth sample collection, preservation, or.storage procedures. . 3.3 Field Blank (FBjmip Blank . ASTM Type I or HPLC grade waterplaced in a sample containerin the laboratory and treated as a sample in all respects. Including exposure to sampling site conditions, storage, preservation and 8 l analytical procedures. The purpose of the F8 b to determine if test substances or other interferences are present in the field environment Thb sample b also referred to as a Trip Blank. Trip blanks are not a requirement for soil or sediment samples. ' 3.4 Field Duplicate Sample (FDS, Field Dup) A sample collected In duplcate at the same time from the same location as the sampb. The FDS b placed under identical circumstances and treated exactly the same throughout field and laboratory procedures. ETS-8-012.1 Page 2 o f 12 Method o fAnalysis to r ft DetcrmftsUon o f PertuorobUenelc Add (PFBA). Pwfluoropenranolc Add (PFPeA). PvSuoroheunofc Add (PFHA), Porfluorohepianoic Add (PFHpA), PorflueroocUneJc Add (PFOA). Portucrononanole Add (PFNA).Parfluorodacanoic Add (PFDA). ParSuorouidacanoteM d (PFUnA). PartuoradodacanoleAdd (PFDoA). PtrtuoroO uunesdfonala (PFBS). P erfciorotw unesU lonjie (PFHS). and Perfluorooetanaueonata (PFOS) In W atar. S o l and Satfmam by LC/MS/MS Page ] 7 o f 56 MPI Research Page 129 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Analysis of the FD5 compared to that of the first sample gives a measure of the precision associated with sample collection, preservation and storage, asweDas with laboratory procedures. 3.5 Reid Matrix Spike (FMS) A sample to which known quantities of the target analytes are added to the sample bottle in the laboratory before the bottles are sen! to the field for collection of aqueous samples. A known, specific volume of sample must be added to the sarryile container without rinsing. This may be accomplished by making a TO to this leveT fine on the outside of the sample container, The FMS should be spiced between approximately 50% and 10 times the expected analyte concentration in the sample. If the expected range of anaiyte concentrations is unknown, multiple spikes at varying levels may be prepared to increase the likelihood that a spike at an appropriate level is made. The FMS Is analyzed lo ascertain If any matrix effects, interferences, or stability issuesmay oompfcate the interpretation of the sample analysis. 3.6 Trip Blank Spike (Field Spike Control Sample, FSCS) An aliquot ofASTM Type I.or HPLC grade waterto which known quantities of the target analytes are added in the laboratory prior to the shipment of the colection bottles. The FSCS is extracted and analyzed exactly Bee a sample to help determine if the method Is in control and whethera loss of analyte could be attributed to holding time, sample storage and/or shipment issues. A low and high FSCS are appropriate when expected sample concentrations are not known or may vary. At least one separate, urvspiked sample must be taken at the same time and place ss each FMS. 3.7 Laboratory Control Sample (LCS) An aJiquol of control matrix to which known quantities of the target analytes are added in the laboratory at the time of sample extraction. At least two levels are included, one generally at the low end o f the calibration curve and one near the mid to upper range of the curve. The ICSs are extracted and analyzed exactly tike a laboratory sample to determine whetherthe method is in control LCSs should be prepared each day samples are attracted. - . 3.8 Laboratory Matrix Spike (LMS) A laboratory matrix spike i$ an 8Qquotof a sampleto which known quantities of target analytes are added In the laboratory. The LMS is extracted and analyzed exactly Bee a laboratory sample to determine whether the sample matrix contributes bias to the analytical results. The endogenous concentrations of the analytes In the sample matrix must be determined in a separate aliquot and the measured values in the LMS corrected for these concentrations. LMSs are required for soils end sediments and are optional for analysis of aqueous samples. 3.9 Laboratory Sample . A portion of aiquot of8 sample received from the field for testing. 3.10 Limit of Quantitation (LOQ) The tower firrtl of quantitation (LLOQ) for a dataset is (he lowest concentration that can be reliably quantitated within the specified limits of precision and accuracy during,routine operating conditions. To simplify data reporting, the LLOQ is generally selected as the lowest non-zero standard In the calbralton curve that meets method criteria Sample LLOQs are matrix-dependent The upper(unit of quantitation (ULOQ) for a dataset is the highestconcentration that can be reliably quantitated within the specified limits of precision and accuracy during routine operating conditions. The highest standard in the cafibratton curve that meets method criteria is defined as the ULOQ. ETS-8-012.1 Page 3 o f I2 Method ofAnalysis for the Determination ofPeriluorotutanblc Add (PFBA), Perftuoropentenoic Add (PFPeA), Perduorohoxanofe Add (PFHA). PeriluoretepUngic Add (PFHpA). Pertuoreodanoic Add (PFOA). Pertuoronorande Add (PFNA). ParSuprodecanele - Add (PFOA). Perttuoroundeceholc Add (PFUnA), Periuorododecanoic Add (PFDoA), PerftjoroOUanesdtonate (PfB S). PertuorotacanesuHonate (PFKS), and Perfluoroodaneiulfbnata (PFOS) In VW er, Sod n d Sediment by LC/MSMS Page IS o f 56 MPI Research Page 130 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 3.11 Method Blank An alquot of contra! m att* that is (rested exactly UKe a laboratory sample including exposua to a l glassware, equipment, solvents, and reagents (hat are used wfth other laboratory samples. The method blank is used to determine If test substances or other interferences ere present In the laboratory environment, the reagents, or the apparatus. 3.12 Sample A sample is an aliquot removed from a larger quantity of material Intended to represent the original source material 3.13 Stock Standard Solution (SSS) A concentrated solution ofa single-analyte prepared In the laboratory with an assayed reference compound. 3.14 Surrogate A compound similar to the target anafyte(s) in chemical composition and behaviorthat is not normally found in the santotofs). A surrogate compound b typicaly a target analyte with at least one atom containing an boloptcady labeled substitution. If used, surrogate^) are added to a l samples and qua&ty control samples (except solvent blanks and half of the prepared method blanks}. Surrogate^) are added to quantitatively evaluate the entire analytical procedure including sample colection, extraction, and analysis, inclusion of a surrogate analyte b an optionalquality contra!measure and b NOT required. 3.15 Working Standard (WS) A solution of several analytes prepared in the laboratory from SSSs and diluted as needed to prepare calibration standards and otherrequired analyte solutions. . 4 Warnings and Cautions___________________________________________ 4.1 Health and Safety The acute and chronic toxicity of the standards for this method have not been precisely determined; however, each should be treated as a potential health hazard. The analyst should wear gloves, a lab coat, and safety glasses to prevent exposure to chemicals that mightbe present. The laboratory is responsible for maintaining a safe work enviranmer* and a current awareness o1 local regulations regarding the handEng of the chemicals used in this method. A reference file of material safety data sheets (MSDS) should be available to all-personnel involved in these analyses. 4.2 Cautions The analyst must be iamiKar with the laboratory equipment end potentialhazards including, but not limited to, the use of solvents, pressurized gas and solvent fines, high voltage, and vacuum systems. Refer to the appropriate equipmentprocedure or operatormanualfor additionalInformation and cautions. S Interferences During extraction and analysis, major potential contaminantsources are reagents and glassware. ASmaterials used to the analyses th a t be demonstrated to be free from interferences under condftorts of enalysb by running method blanks. ETS-6-012.1 Page 4 Of12 Method o fA n afyitt lo r ihe DetenninaSon o fPeifluorodutenote Add (pFBA). PtfSioropenU noic Add (pFPeA}, Pertugrahenndc . Add (PFHA). P e rih ro fttp ttrtd c Add (PFHpA). Pertuorooetfnotc Add (PfOAJ, Pertluorenonencic Acid (PFNAJ, Perfluprodecanoic Add (PfD A). Pertuoraundecendc Add (PFtlnA). P ertooratodecvuic Add (PFDoA). PertuoroPutenewXfeneta (PFBS), PW fluorotaxenetudende (PFKS). end P efltooodineeU lQ nM (PF05) In W iter. S o iand SedimentOyIC/MS/MS Page 19 o f 56 MPI Research Page 131 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Parts and supplies that contain Teflon should be avoided or minimized due to the possfoiitty of interference and/or contamination. These may include, but are not Unruled to: wash bottles. Teflon* lined caps, autonal caps. KPLC parts, etc. The use of disposable micropipettes or pipettes to aliquot standard solutions is recommended to make calibration standards and matrix spikes. 6 Instrumentation, Supplies, and Equipment____________________ _________ 6.1 Instrumentation and Equipment A high performance liquid chromatograph capable Of pumping up to 2 solvents and equipped with a variable volume injectorcapable of injecting 5-200 p i connected to a tandem Mass Spectrometer (LC/MS/MS). Applied Blosystems SdexAPI 5000 instrumentation is required to meetthe LOQsof 0.025 ng/mL (water) and 0.20 ng/g (soil and sediment). If analyte concentrations requre dilutions lor one or more analytes that preclude the targeted LOOs from being reached, Appied Biosystems Sciex API 4000 instrumentation may be utilized since the LOQswill already be raised. Analytical balance capable of reading to 0.0001 gram. - A device lo collect raw data for peak integration and quantitation. 15-mL and 50-mL disposable polypropylene centrifuge tubes. Disposable micropipe&es (10-20 p i, 25-50 pl_ 50-100 pL. 100-200 p i). 125-mL LDPE narrow-mouth bottles. 2-mL dear HPLC vial k it Disposable pipettes, polypropylene or glass as appropriate. Ultrasonic bath. Centrifuge capable of spinning 15-mL and 50nlpolypropylene tubes at 3000 rpm. 6,2 Chromatographic System ' Analytical Column: Luna 3 pm C6 (2) Mercury (Phenomenex), 2 mm x 4 mm. 3 pm (P/N: OOM-4248-DO-CE) Temperature: 35*C Mobile Phase (A): 2 mM Ammonium Acetate in Water Mobile Phase (B): Methanol Gradient Program: Time fm inl %A 0.0 90 10 0.5 90 10 2.0 10 90 5.0 10 90 5.1 0 100 6.0 0 100 6.1 90 10 10.0 90 10 injection Volume: 5 pL (can be increased to as much es 50 pL). Flow Rate ' fmL/min) ' 0.7S 0.75 0.75 0.75 0.75 0.75 0.75 0.75 Quantitation: Pea*Area-external standard eafibration curve, 1/Xweighted. ETS-a-012.1 Pages o f 12 Method o fAnalysis far the Determination ofPetSuorcbuanolc Add (PFBA). Peffluorepantanoie Add (PFPeA). Partuowhastandc Add (PFHA), PtfftuarohepCanoic Add (PFKpA). Perfluoroodaralc Add (PfO A ). Parfluorononanoie Add (PFNA). Peftiuoredeesnoic Add (PFQA). Perfuorotm decande Add (PFUnA), Parfuorododaeandc Add (PFDoA). PertuoroeutanaaUfenati (PFBS). Perduorohexanasilanata (PPKS). and Parttuoroodaneauflonata (PFOS) In W ktar. S o l and Sediment by LCATSMS Page 20 o f 56 MPI Research Page 132 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Run Time: - 10 minutes. The previous Information Is Intended as a guide: alternate conditions and equipment may be used provided (hat data quality objectives are met 6.3 MS/MS System 6.3.1 Mode Bedrospn# Negato MRMmode,mentoring to fbtowtytransftions: Analvte Transition Monitored PFBA 213 - 169 PFPoA 263-4 219 PFHA 313 - 269 and 313-4 119 PFHpA . 363 -a 319. 363 -a 169 and 383 - 119 PFOA 413 -4 369.413 -> 219 and 413 -* 169 PFNA 463 -a 419.463 - 169 and 463 - * 219 PFOA 513 ^ 469.513 - 219 and 513 - 269 PFUnA 563 - 519.563 -4 269 and 563 -4 219 PFOoA 613-4 569.613 >4 169 and 613-4 319 PFBS 299 -4 60 and 299 - 99 PFHS 399-4 80 and 399-99 PFOS 499 - 80.499 - 99 and 499 -4 130 Multiple transitions ter monitoring the analytes is an option because summing m ultiple transitions may provide quantitation of isomers that more closely matches NMR data and may have the added benefit of increased anaiyte signal The use of one daughter ion is acceptable if method sensitivity is achieved, provided that retention time criteria are met to assure adequate specificity. The previous information is intended as a guide, alternate instruments and equipment may be used. ' 7 Reagents and Standards W ater-HPLC grade Methanol- HPUC grade Ammonium Acetate-A.C.S. Reagent Grade Acetonitrile - HPLC grade Perfluorobutanoic Acid (PFBA) - Oakwood Products, Inc Perfluoropentanoic Acid (PFPeA) - Sigma-Aldrich Perfluorohexanoie Add (PFHA) - OaJcwoodProducts. Inc PerftuorotoptanpJcAdd (PFHpA) - OaJcwoodProducts, Inc Perftuoroodanote Acid (PFQA) - Oakwood Products, Inc PerfluorononanoicAdd (PFNA)- OaJcwoodProducts, Inc Perfluorodecanoic Add (PFQA)- Oakwood Products, Inc Perfluoroundecanoic Add (PFUnA) - Oakwood Products, Inc Perfluorododacanoic Add (PFDoA)- OaJcwoodProducts, Inc ' ETS-WM2.1 Paged o f 12 Method o fAneiyal tar the Determination of RwHuoroOuUnoJc Add (PFBA). Peifluoropentanoie Add (PFPeA), ParSuorohnsnoic Add (PFHA), Parituorotieptanolc Add (PFHpA). Partuoroodanoic Add (PFOA). PerfluorondnanolcAdd (PFNA), PtrfluorodaeanoJe Add (PFQA), Perluorcundecandc Add (PFUnA). Pertworododeeande Add (PFOoA). PertuorobuUneauironale (PFBS), Perfluorphaxeneautfonete (PFHS), and PerttwroecUneaulfonate (PFOS) In W ater, S o l and Sediment by kC/M&MS Page 21 o f 56 MPI Research Page 133 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Nuniber: P0003267 Perfluorobutanesuffonate (PFBS) - 3M Perttuorohexanesulfonate (PFHS)- 3M Perfluoroodamsuifonate (PFOS) - Fluka . . The previous information Is intended as a guide. Reagents and standards from alternate sources may be used. 8 Sample Handling _____________________ __ ,____________________ 8.1 Water Sample Extraction 8.1.1 Measuie10mLofsampiekntoa50fflLpotypfopytenecentnfugetube. Fortificationsare to bedone at this point,!necessary. Othervolumesandcontainers maybe used asapproprie. 6.1.2 AddlOmLofacetonitrSetothesampieinthecentrifugetuba Captightly and shake. 8.1.3 Sonicate sample for-2 hours atroomtemprature. This step is optional, butis recommended if particulates appearto be present 8.1.4 Centrifugefor --10minutesel-3000 rpm Thisstep is optional, butIs recommended if samplesare sonicated. 8.1.5 Transferaportfonofthesupemalarttoanautosamplerviaiforanalysls. ' 8.1.6 oautesample,if necessary.wth 50:50 aoetonbitewater. 8.2 Soli and Sediment Extraction 821 Weigh 1g ofsampleirtoa15-mLpoiypfCpytenecertrifuge tute. Fortificationsare to be done atthis point,! 82 2 Add8 mLof8020 acetongriie.-watertothe sampled the centrifoge tube. Capbghflyandshaka 82.3 -Sonicate sampletbr*2 hoursat roomtemperature. 824 Centrifuge for'1 0 minutesat-3000 rpm 825 Transfera portion ofthe siaiemstBrltoan autosarrplervialforanalysis. 826 08ute sample,if necessary,with 5050 acetonWewater. 6 2 7 Analyze samples using eiectrosprayLC/MSMS Other weights end volumes can be u$8d as long as the QC elements' specified in this method are satisfied and e!i sample preparation procedures may be reconstructed. 9 Sample Analysis - LC/MS/MS ETS-8-Q12.1 Page 7 o ft 2 M elted o fA ntTyttstor (he DetemtfnaOon of Pefftorobutanoic Add(PFBA). PeSuoropentanolcAdd(PFPeA). ParSuorohetande Add (P fK A ). PerSuorotepttndc Add (PFHpA), Perfboraocunoic Add {PFOA), Peduerononendc Add (PFNA). Pettuorodacanoie Add (PFOA). PcrtuorouteacanaicAdd (PFUnA), Peduoredodecanofc Add (PfOaA). PerSuorateUnewItens (PF6S). PteSuorotexaneMtfonata {PFHS). m d P affluoroocunatefendt (P fOS)in W uw , Sa a te S a d ro n t Oy IfMSfMS Page 22 o f 56 MPI Research Page 134 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 9.1.1 A minimumofsix calibrationlevelsmustbepresent in the finalcafbration curve. 9.1.2 Anentire setofcalibration standardsis injected atthe beginningofa sampleset priorto sample analysis. As analternative,an entire setofcalfcration standards may be injectedatthe beginningofa samplesetfcfcwed bycalbrationstandardsinterspersed evety5-10 samptesCoeceountfcrasecondsetcfstandards). In eithercase,cassation Stanfordsora corihuing cassation verification s&idard mustbracketthe first sampleandthe last sample. Analyticalstudes mayonly require that anHfialca&xation curve be analyzed priorto samplesandthatthe cortiwed accuracyofthe initialcaEbratbncurve be confirmed by the analysisof c o n in g captation verifcaSon standards. The sameapproach should beusedthroughoutthe entire study unlessreasons are documentedandtechnicaljustification to changeis approved priorto sampleanalysis. 9.1.3 The standard cwveis plotted byquadratics (y * a ^+ tuc* c),weighted 1/k orunweighted,or using a near fit, weighted lo using suitable software. The calibrationcunresmayinclude butshould notbeforced througfizero. ThemathemalicalmethodusedtocalculatethecaEbratjoncuiveshoLJdbeappGed consistentlythroughouta study. Anychange should be documentedin the rawdata' 9.1.4 Samplescontaininganalytesthatamquantitatedabovethe concentrationofthe higheststandard in the curve should be forther(fluted and reanalyzedor reinjected using a smaDervolume. 10 Quality Control 10.1 Data Quality Objectives. This method and required quaity control samples is designed to generate data accurate to +/-30% with a targeted LOQ of 0.025 ng/mL (water) or 0.20 ng/g, Wet weight (sol and sediment). Any deviations from the quafity control measures spelled out belowwiObe documented in the raw data and footnoted In the finalreport 10.2 Blanks Method Blank Method blanks mustbe prepared with each extraction batch. A range ofthree to seven method blanks must be prepared. These method blanks must be Interspersed throughout the extraction batch and analyzed interspersed throughoutthe analytical sequence. ' The mean area count for each analyte in the method blanks must be less than 50% of the area count of the LOQ standard. The standard deviation of the area counts of these method blanks should be calculated and reported. If the mean erea counts of the method blanks eweed 50% of the LOQ standard, then .the LOQ must be raised to the first standajd level in the curve that meets criteria, or alternatively, the method blanks must be evaluated statistically to determine outtfere or technical justification to eliminate one or more results should be made. 10.22 Solvent Blank ' Solvent blanks must be analyzed throughout the analytical sequence. Solvent blanks that show area counts greater than 50% of the area counts of the LOQ standard must be evaluated lo determine if analytical results are significantly impacted by sample carry-over or an unacceptable buldup of analyte in the instrumental background. 10.3 Sample Replicates Samples should be prepared In duplicate In the tab (soil and sediment) or colected In duplicate in the field (water). The relative percent difference, RPD, should be reported. RPD results greater than 20% (water) or ETS-8-012.1 Page 8 o f 12 M tinod ofAnalysis tor too DotormbuSon ofPwfluorcbvUnolc Add (PF0A),ParfluaroptnUnoic Add (PFPaA). Pw&joreheunofc Add (PFHA).PtrtuorohepU nefc Add (PFHpA). PvftuoroocUneic Add (PFOA). Pw tugrvtonanote Add (PFNA). Pufluoredeanoie Add (PFOA). Pcrttoorom ddcinolc Add (PFUnA). PsrSuwododtcanok Add (PFDoA). Pcrihrareouttnesuttonito (PFBS). P ortow rohearm titonal* (FFMS), tn d PerfluoroodandSUtomto (PFOS) in W sitf, Soiland S * * r* rt by LOUS/MS Page 23 o f 56 MPI Research Page 135 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 30% (soO and sediment) w il be flagged In the report but w fl not be excluded from repotting. The requirement for replicates excludes field blanks and rinse blanks. 10.4 Surrogate Spikes Surrogate spaces are not required butmay be usedon project specific requirements. 10.5 Lab Control Sample Triplicate tab control spices at e minimum of two different concentrations are to be prepared w*h each extraction batch. Low lab control spikes should be prepared et concentrations in the range of five to ten times higher than the targeted LOO and high lab control spikes should be prepared at concentrations near the mid point of the curve. The relative standard deviation of the control spices for these six pooled control samples must be less than or equal to 20% and the average recovery must be 80-120%. If the above criteria are not met, the entire setof samples should be re-injected or reextra& d as appropriate. 10.6 Field Matrix Spikes / Lab Matrix Spikes Recoveries of field matrix spikes (aqueous samples) and laboratory matrix spSces (sofls and sediments) are anticipated to be between 70% and 130% of the fortified levels. If the recoveries of the spikes fan outride the acceptable range, the sample resultwill be reported as 'NR* (not reported due lo non-compUant QC results). The targeted fortification levels should bq at least 50% of the endogenous level and less than 10 times the endogenouslevelto be used withoutjustification to determine the statementof accuracyfor analytical resuis. The average ofthe sample and the lab duplicate (soBs and sediment) or field duplicate (water) should be used to calculate the recovery. . 10.7 Standard Preparation 10.7.1 Standard Stock Solutions Prepare individual stock solutions of each analyte at *1000 pg/m l by weighing 100 mg of analytical standard (corrected for purity and salt content, If necessary) and dilute to too m l with methanol or acetonitrile in separale 100-mL volumetric flasks. For purity correction, take the amount of analyte that Is needed for weighing (i.e.100 mg) and divide by the purity in decimal form (ie. 99.6% B 0.996). The result is the weight that is needed to make the solution corrected for purity (l.e. weigh 100.4 mg). For salt correction, calculate the u t content by taking the molecularweight of the target compound (Le. PFOS 499) and dividing by the molecular weight of the entire compound (Le. PFOS potassium salt (CaFirSOrK* 538). The result is the salt content in decimal form (i-e. 0.9275), Take the amount of analyte that is needed for weighing (i.e. 100 mg) and divide by th salt content in decanal form. The result Is the weight that is needed to make the solution corrected for salt content (l.e. weigh 107.8 mg). Store all stock solutions in appropriate container* and at appropriale storage . conditions for up to 6 monthsfrom the date of preparation. ' - Alternate stock solution concentrations can be made, if necessary, using alternative masses and volumes. 10.72 Standard Fortifatfion Solutions A 10 pg/tnL meted fortification solution containing a l of the analytes is prepared by bringing 1 m l of each o fthe 1000 pg/m l stock solutions to a final volume of 100 mL wfth ecetonitrie in a 100-mL volumetric flask. A 1.0 pg/m l mixed fortification solution containing aHof the analytes is prepared by bringing 10 mL of the 10 pg/mL mixed solution to a final volume of 100 wkh acetonitrie in a 10O-mL volumetric flask. A 0.1 pgTnL mixed fortification solution containing all of the analytes is prepared by bringing 10 m l of the 1.0 pgftnl mixed solution to a final volume of 100 w ih acetonitrile in a 100-mL volumetric flask. A 0 01 pg/mL mixed fortification solution containing eOof the analytes is prepared by bringing 10 m l of the 0.1 pg/m l solution to a final volume of 100 with acetonitrile in a 10O-mL volumetric flask. Store a l fortification standards up to 6 months from foe data of preparation . E T S -8-01 2.1 Page 9 o f 12 Method ofAnelyili for the Oateminetlonef Partuorebutande Add (PFBA), PerikjoropenlaroleAdd(PFPoA). Pofluorahasnole Add (PFHA). ParHuoreheptandcAdd(FFHpA), Pwfluomocunotc Add (PFOA). Perfluomnonanofo Add (PFNA), Pertuorodecande Add (PFOA). Perduoroundeeanote Add (PFUnA), Pertwododecvwtc Add (PFDdA). PerSuorotatanewflonsta (PF8S). Par*uoraheaananJtoraia (PFHS), and Pertuorooctannulfonata (PFOS) In w ater. SoO end Sediment by LCTUS1M5 Page 24 o f 56 MPI Research Page 136 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Alternata fortification solution concentrations can be made, if necessary, likewise, alternative volumes may be used. 10.8 Instrument Calibration 10.8.1 IQMS/MScalibration standards arepreparedeademallyin 50:50acetonitrtewaterand are usedto construct the cassation curvesforthe water,soi, and sedimenlprocedures. 10.6.2 The WtowtngIs a typical exampleofa calibration sei Atonale or additer concentrationsmaybe prepared as.needed. . Concentration Initial Solution Final Concentration of of Initial Solution Aliquoted Volume Final Solution Calibration Standard (ng/mL) 100 (m l) Volume (m l) 5 100 (ng/mL) 5.0 100 2.5 100 2.5 10 10 100 1.0 5 10 100 0.5 2.5 10 100 0.25 1 10 100 0.1 0.5 10 100 0.05 t 2.5 100 0.025 Store all calibration standards for up to three months. Alternate volumes and concentrations of standards may be prepared as needed. . 11 Data Analysis and Calculations_____________________________________ Use the following equation to calculate the amount of analyte found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by an appropriate software program: Analyte foundW mL) - <Peal1 , DF OF - factorby which the final volume was diluted, if necessary. For samples fortified with known amounts of analyte prior to extraction, use the following equation to calculate the percent recovery. _ Total analyte found (ng/mL) -Average analyte found in sample (ng/mL) KOCOveiy 1 ---- .. -- - , 1- ..... xlUU Analyte added(ng/m l) Use the following equation to convert the amountofanalyte found in ngfinLto ng/g (ppb). Analyte found (ppb)> Analyte found(ria/m l) * Volume extracted (m l) Sample weight (g) . Use the following equation to calculate the amountof analyte found in ppb based on dry weight Analyte found (ppb) dry weight a Analyte found (ppb) x [100% / totalsolids (%)] 12 M ethod Perform ance ETS-8-012.1 Paga 10 of 12 Method et A natrila fo r 9 Determination ot PerfluoroM anete Add (PFBA). Pettiuoropeniande Add IPFPeAJ, Perfluorohexande Add (PFHA). Perfluorohcptanoic Add (PFHpA). Pe/fluorooaanote Add (PFOA), Perfluororienanote Add (PFNA). Perfluorodocanoie Add (PFQA). Perfluoroundecande Add (PFUnA). Perfluorododdcanole Add (PFOcA), PefBuofobUanegulfonale (PF8S). Perfluerohexanesuionite (PFHS). and PefluvoocUnesutfonato (PFOS) in W ater. SODand Sedimenl by LC/MS/MS Page 25 o f 56 MPI Research Page 137 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Any method performance parameter that are not achieved must be considered in the evaluation of the data Nonconformance to any specified parameters mustbe described and discussed Hthe Technical Manager(nonGLP study) or Study Director(GLP study) chooses to report the data. If criteria listed in this method performance section are not met maintenance may be performed on the system end samples reanalyzed, or otheractions taken as appropriate. Documentall actions in the raw data. . If data are to be reported when performance criteria have not been met the data must be footnoted on tables and discussed in the text of the report. 12.1 System Suitability . .System Suitability standards are not a required component of this method. If required by protocol or by the technical manager, a minimum of three system suitability samples are injeded st the beginning of each analytical run priorto the calibration cuve. Typically these samples are at a concentration nearthe mid level of the calibration curve and are repeated Ejections from one autosampler vial. The system suitability irfcetions musthave area counts with an RSD ofSS%and a retention time RSD of S2%to be compSant 12.2 Quantitation . Calibration Curve: The coefficient of determination (r2) value for the caSbration curve must be greater than or equal to 0.985. The concentration of each point in the curve must back calculate to be within t25% of the theoretical concentration with the exception of the LLOQ. which may be within 30%. Calibration curve points that are not at the high or low end ofthe curve maynot be deectivated. CCV Performance: The continued accuracy of the calibration curve must be demonstrated by analyzing continuing calibration verification (CCV) standards. Each CCV may be a calibration curve point that is reinjected or a separately prepared standard, and is typically near the middle of the calibration curve. Alternative concentrations or multiple concentrations may be chosen depending on project requirements. Not more than ten samples or spires may be analyzed between the initial calibration and a CCV or bracketing CCV#. The accuracy of each analyte must be within 25% of the theoretical value. Samples that are bracketed by CCVs notmeeting these criteria mustbe reanalyzed. Demonstration o f Specificity: Specificity is demonstrated by chromatographic retention time (within 4% of standard) and the mass spectral response ofunique ions. 12.3 Sensitivity The targeted lim it of quantitation for aRanalytes Is 0.025 ng/mL (water) and 0.20 ngfg (soils and sediments, wet weight). The LOO for any specific analyte may vary depending on the evaluation ofappropriate blanks and the accuracyof the low-levelcaBvation curve points. Referto Section 10for additional details. 12.4 A cc u ra c y This method and required qualify control samples are designed to generate data that are accurate to /-30%. Section 10 contains additional ^formation regarding the required accuracy of laboratory control spires,'field matrix spikes (watersamples) and laboratorymatrix spires (soils and sediments). 12.5 Precision Samples should be prepared in duplcate in the tab (sol and sediment) or coSecled in duplicate In the field (aqueous). The relative percent difference, RPD, should be reported. RPD results greaterthan 20% (water) or 30% (soli and sediment) wobe flagged in the report, but wQ not be excluded from repotting. The requirement for replicates excludes field blanks or rinse blanks. Section 10 contain additional information regarding the required precision of laboratory control spires. 13 Pollution Prevention and Waste Management ETS-8-012.1 Page It o f 12 Method of A natyitetortheO etcrm ineligflof Pe'fluoretMtsnolCAdd{PFBA). PeteuoropentanolC Add (PFPeA). Pefluorohexanolc Acid (PFHA), Perfluofoheptinolc Add (PFHpA), PeriluoraoctanoicAddtPFOA), PerituoranonanolcAdd (PFNAJ, Ptrtuorodocanoic Add (PFOA). Pfluoroundecande Add (PFUnA), Petfluorododecanoic Add (PFOoA), Pcrtuorobutanuuifonale (PFBS), PeifluorohexsnetuRorato (PFHS), and Perftuw oodeneivtfeneiilPFO S) in W ater, Son and Sedimentby IC/MS/VtS Page 26 o f 56 MPI Research Page 138 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Wbste generated when performing this methodwill be disposed of appropriately. The original samples will be archived atthe3M EnvironmentalLaboratory In accordancewith internal procedures. 14 Records_____________________________________________________ ___ Each data package generated for a study mustinclude all supporting information for reconstruction of the data Information for the data package must include, but is not limited to the following items: study or project number, sample and standard prep sheets/records, instrument run log (instrument batch records, instrument acquisition method, summary pages), instrument results files, chromatograms, calibration curves, and data calculations. 15 References Exygen Research Analytical Method V0003305-1 detais method development and verification of recovery for multiple analytes in groundwater, surface water, soil, and sediment This wiltbe archived as part of E06-0549. 16 Affected Documents_______ None. ' 17 Revisions Revision Number Revision Description Revision Date The sonication and centrifugation stepsofthe watersample preparation were 1V1Q/Q6 made optional The accuracy requirements of the analytical balance were updated. The methodwas updated to allow the use of a linear 1/x weighted calibration curve. The table of required daughterions for the analyteswas updated. The method was updated to allow different sample volumesto be extracted andtoaDow different standard bottles and storage conditions to be used. Calibration requirements were updated to show that analyzing continuing calibration verification standards at one concentration was an acceptable alternative to reinjecting allcurve points used to construct the calibration curve. Minorwording changes were made throughout. ETS-8-012.1 Page 12o f 12 Method e l Anelyl ter the Determination oi Perflucrobutanolc Add (PFBA), Perfluorcpenundc Add (PFPeA), PerSuaroheunoic Add (PFHA). Perfluoraheptenoie Add (PFHpA). Perftuorooctenele Add (PFOA). Perfliwrononanoie Add (PFNA). Perfuorodecandc Add (PFOA), P erivoroundc a r'd : Add (PFUnA). Perfluorododecande Add (PFDoA). PerfluorobutanesuMbnate (PFBS). PtrftnrohexaneM M onate (PFH5), and PertkioroeoaneauHonate (PFOS) in W ater. So* and SedimenttoyLOMSIMS Page 27 o f 56 MPI Research Page 139 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 ANALYTICAL METHOD Method Number: V0001780 M ethod o f Analysis for the D eterm ination of P erfluorooctanoic Acid (PFO A ) in W ater by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 A pproved By: C,-- ______________ Paul Connolly * Technical Leader, LC-M S, Exygen Research %ilfl? r fh L c f /7 o'Johhn F laherty / / VV iirce President, O perations, E xygen Research ioIz-Woif Date M i D ate Total Pages: 7 Page 28 o f 56 MPI Research Page 140 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygen Research Method Number V0001780 ANALYTICAL METHOD M ethod o f Analysis for the Determination o f Perfiuorooctanoic Acid (PFO A ) in W ater by LC /M S/M S 1.0 Scope This m ethod is to be employed for the isolation and quantitation o f perfiuorooctanoic acid by High Performance Liquid Chrom atography coupled to a tandem M ass Spectrom trie Detector (LC /M S/M S) in water. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chem ical for proper safety precautions. Sample Requirement 3.1 A t least 40 m L o f test sam ple for extraction. 3.2 No sam ple processing is needed for water samples. 3.3 Sam ples stored refrigerated should b e allow ed to equilibrate to room tem perature. 3.4 AJ1 sam ples m u st be thoroughly m ixed before bein g sam pled for extraction. 3.5 Any sam ples containing particles should be centrifuged at -3 0 0 0 rpm for -5 m inutes and the supernatant used for the extraction. 3.6 Sample collection procedures will be specified in the sam pling plan for this project. R eagents and Standards 4.1 W ater -H P L C grade 4.2 M ethanol - HPLC grade 4.3 Am m onium Acetate - A.C.S. Reagent Grade 4.4 Perfiuorooctanoic Acid - Sigma-Aldrich 5.0 Instrum ent and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.S 5.9 5.10 5.11 A high perform ance liquid chromatograph capable o f pum ping up to 2 solvents equipped with a variable volum e injector capable o f injecting 5-200 p L connected to a tandem M ass Spectrom eter (LC/M S/M S). A device to collect raw data for peak integration and quantitation. A nalytical balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene centrifuge tubes. 1S m L disposable polypropylene centrifuge tubes. D isposable m icropipets (50-1 OOuL, 100-200uL). 12S-mL LDPE narrow-mouth bottles. 2 m L clear HPLC vial kit. Disposable pipettes. Autopipettes (100-1000 pL and 10-100 pL), w ith disposable tips. W aters Sep Pak Vac 6 cc (lg ) tC l8 SPE cartridges. Page 2 o f 7 Page 29 o f 56 MPI Research Page 141 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygcn Research Method Number VO00I780 ANALYTICAL m e t h o d M ethod o f A nalysis for the Determination o f Periluorooctanoic Acid (PFO A ) in W ater by L C /M S/M S 5.12 SPE vacuum manifold. 5.13 Centrifuge capable o f spinning 50 mL polypropylene tubes at 3000 rpm. 6.0 Chrom atographic System 6.1 Analytical C olum n: Fluophase RP (K eystone Scientific), 2.1 m m x 50 m m , 5p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 M obile Phase (A) : 2 mM Am m onium Acetate in W ater M obile Phase (B) ; Methanol Gradient Program: Tim efm in) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B fm L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volum e: 15 p L (can b e increased to as m uch as 50 pL), 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 R unT im e: - 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: Electrospray N egative M R M m ode, m onitoring 413 -> 369 m/z. T h e ab o v e conditions are intended as a guide and m ay b e changed in order to optim ize the M SMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM am m onium acetate in w ater is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. A lternate volum es m ay be prepared. Page J o f 7 Page 30 o f 56 MPI Research Page 142 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygen Research Method Number V0001780 ANALYTICAL METHOD M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in W ater by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 P repare a stock solution o f - 1 0 0 p g /m L o f PFO A b y w eighing 10 m g o f analytical standard (corrected for purity) and dilute to 100 m L with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/m L fortification solution o f P FO A is prepared by bringing 10 m L o f the 100 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 1.0 pg/m L fortification solution o f PFO A is prepared by bringing 10 m L o f the 10 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.4 A 0.1 pg/m L fortification solution o f P FO A is prepared b y bringing 10 m L o f t h e 1.0 pg/m L solution to a final vo lu m e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.5 A 0.01 pg/m L fortification solution o f PFO A is prepared by bringing 10 raL o f the 0.1 pg/m L solution to a final volum e o f 100 with methanol in a 125 m L LDPE bottle. 9.1.6 The stock and fortification solutions are to b e stored in a refrigerator at approximately 4C and are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions LC/M S/M S calibration standards are prepared in H PLC water. The calibration standards are processed through the extraction p rocedure,. identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Concentration o f F ortification Solution (ro b ) 0 10 10 10 100 too 100 Fortification Volume o f Volume Fortified Control (PL) Sample (m L) 0 40 100 40 200 40 400 40 100 40 200 40 400 40 Final Concentration o f C alibration Standard (p p tl* 0 25 50 100 250 500 1000 C a lib ra tion Standard D (exam ple) XCipm ddyy-0 XCm m ddyy-l XCm m ddyy-2 X C m m ddyy-3 XCm m ddyy-4 XCm m ddyy-5 X C m m ddyy-6 * The extracted concentration o f the calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC extracted calibration standard. Page 4 of? Page 3 o f 56 MPI Research Page 143 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygen Reiesrch Method Number VOOO1780 ANALYTICAL METHOD M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in W ater by L C /M S /M S 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) m ust be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene tubes at 2C to 6C, up to two weeks. Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f sam ples extracted (typically 20 o r less) m ust include at least one reagent control (method blank using H PLC water) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 R equirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 M easure 40 m L o f sam ple o r a portion o f sam ple d iluted to 4 0 m L w ith w ater into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 C ondition the C n SPE cartridges (1 g, 6 m L ) by p assin g 10 m L m ethanol followed by 5 m L o f HPLC water (~ 2 drop/sec). Do not let colum n run dry 11.3 Load sam ple on conditioned C is S PE cartridge. D iscard eluate. 11.4 Elute w ith " 5 m L 100% m ethanol. C ollect 5 m L o f eluate into graduated 15 m L polypropylene centrifuge tubes (final volum e = 5 mL). 11.5 A nalyze sam ples using electrospray LC/M S/M S. 12.0 Chrom atography 12.1 Inject th e sam e am ount o f each standard, sam ple and fortified sam ple into the LC /M S /M S system . A calibration standard m ust p recede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels m ust be included in an analytical set. 12.3 A n entire set o f extracted calibration standards m ust b e included at the beginning and at the end o f a sam ple set. Extracted standards m ust be interspersed between every 5-10 samples. A s an alternative, an entire set of extracted calibration standards m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5*10 sam ples (to account for a second set o f extracted standards). In either case, extracted calibration standards m ust be the first and last injection in a sam ple set. 12.4 U se linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x w eighting o f peak area Page 5 o f 7 Page 32 o f 56 MPI Research Page 144 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: PQQQ3267 Exygeo Resctrch Method Number V000I7IQ ANALYTICAL METHOD M ethod o f A nalysis for the Determination o f Periluorooctanoic A cid (PFO A ) in W ater by L C /M S /M S versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 C hrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. The 413 amu parent corresponds to the PFO A anion, w hile the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 M ethod blanks m ust not contain P FO A at levels g re ater than th e LOQ . If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample m ust be obtained and the entire set must be re-extracted. 13.3 R ecoveries o f control spikes and m atrix spikes m ust b e b etw een 70-130% o f their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated b y th e analyst to determ ine i f re-extraction is w a rranted. 13.4 A ny calibration standard found to be a statistical outlier b y using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve. However, the total num ber o f extracted calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f extracted standards injected. 13.5 T he correlation coefficient (R ) for calibration curves generated m ust be 20.992 (R2 20.985). I f calibration results fall outside these limits, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention tim es betw een standards and sam ples m ust not drift m ore than 4 % within an analytical run. If retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the am ount o f P F O A found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program: PFOA found (ng/L) = (Peak area - intercept) x DF slope DF = factor by w hich the final volum e was diluted, i f necessary. Pgc 6 o f 7 Page 33 o f 56 MPI Research Page 145 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLlMS Protocol Number. P0003267 Exygcn Research Method Number V0001780 | ANA LYTICAL M ETH O D M ethod o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in W ater by L C /M S /M S 14.2 F or sam ples fortified w ith known am ounts o f PFO A prio r to extraction, use the following equation to calculate the percent recovery. Recovery (% )- ( total analyte found (ng/L) analyte found in control (ng/L)] analyte added (ng/L) MPI Research Page 7 o f 7 Page 34 o f 56 Page 146 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 ANALYTICAL METHOD Method Number: V0001781 M ethod o f A nalysis fo r th e D eterm ination o f P erfluorooctanoic A cid (P F O A ) in Soil by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 A pproved B y: C-HL P aul C on n o lly ................. Technical Leader, LC-M S, Exygen Research iqlz-fc/o-t D ate D a te Total Pages: 7 Page 35 o f 56 MPI Research Page 147 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygeo Research Method Number VOOOI78I | ANA LYTICAL M ETH O D M ethod o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Soil by L C /M S /M S 1.0 Scope This m ethod is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrom etric D etector (LC/M S/M S) in soil. 2.0 Safety 2.1 Alw ays observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 15 g o f test sam ple for extraction. 3.2 N o sam ple processing is needed for soil samples. 3.3 Sam ples stored refrigerated should be allow ed to equilibrate to room tem perature. 3.4 All samples m ust be thoroughly m ixed before being sam pled for extraction. 3.5 Sam ple collection procedures will be specified in the sam pling plan for this project. 4.0 Reagents and Standards 4.1 W a te r-H P L C grade 4.2 M ethanol - HPLC grade 4.3 Am m onium Acetate - A.C-S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrum ent and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 A high perform ance liquid chromatograph capable o f pum ping up to 2 solvents equipped with a variable volum e injector capable o f injecting 5*200 pL connected to a tandem M ass Spectrom eter (LC/M S/M S). A device to collect raw data for peak integration and quantitation. A nalytical balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene centrifuge tubes. IS m L disposable polypropylene centrifuge tubes. D isposable m icropipets (SO-lOOuL, 100-200uL). 125-mL LDPE narrow-mouth bottles. 2 m L clear HPLC vial kit. D isposable pipettes. Autopipettes (100-1000 pL and 10*100 pL), w ith disposable tips. W aters Sep Pak Vac 6 cc (lg ) tC I8 SPE cartridges. SPE vacuum manifold. Ultrasonic bath. P ig e 2 o f7 Page 36 o f 56 MPI Research Page 148 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygcn Research Method Number VOOO1781 ANALYTICAL METHOD M ethod o f Analysts for the Determination o f Periluorooclanoic A cid (PFO A ) in Soil by LC /M S/M S 5.14 W rist-action shaker. 5.15 Centrifuge capable o f spinning 50 m L polypropylene tubes at 5000 rpm . 6.0 Chrom atographic System 6.1 Analytical C olum n: F lu o p h aseR P (K eystone Scientific), 2.1 m m x 5 0 m m ,5 p (P/N: 82505-052130) 6.2 Tem perature: 30eC 6.3 M obile Phase (A ) : 2 m M Am monium Acetate in W ater 6.4 M obile Phase (B) : Methanol 6.5 Gradient Program: lim e (ffinl 0.0 1.0 8.0 20.0 22.5 . %A 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0,3 35 0.3 6.6 Injection Volum e: 15 p L (can be increased to as m u ch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Tim e: - 23 minutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: Electrospray N egative M R M m ode, m onitoring 413 - 369 m /z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optim ize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w ater is prepared by adding 0.1S4 g o f am m onium acetate to 1000 tnL o f water. Alternate volum es m ay be prepared. P ige3of7 Page 37 o f 56 MPI Research Page 149 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygcn Research Method Number V000178I ANALYTICAL METHOD ] M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by L C /M S /M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/m L o f PFOA by w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 m L with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/m L fortification solution o f P FO A is p repared by bringing 10 m L o f th e 100 p g /m L solution to a final volum e o f 100 w ith methanol in a 125 m L LD P E bottle. 9.1.3 A 1.0 pg/m L fortification solution o f PFO A is prepared by bringing 10 m L o f the 10 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.4 A 0.1 pg/m L fortification solution o f P F O A is prepared b y bringing 10 m L o f Jhe 1.0 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LDPE bottle, 9.1.5 A 0.01 pg/m L fortification solution o f PFO A is prepared by bringing 10 m L o f the 0.1 p g /m L solution to a final volum e o f 100 with methanol in a 125 m L LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a m axim um period o f 6 months from the date o f preparation. Standard Calibration Solutions 9.2.1 9.2.2 LC/M S/M S calibration standards are prepared in H PLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations m ay be prepared as needed. Final Concentration Fortification Volume o f Concentration o f Calibration o f Fortification Volume Fortified Control C alibration Standard ID Solution (nob) 0 (u L ) 0 Santole (m L) 40 Standard {port* 0 (example) X C m m ddyy-0 10 100 40 25 XCmm ddyy-I 10 200 40 50 XCmmddyy-2 10 400 100 100 too 200 100 400 40 40 40 40 100 250 500 1000 XCmmddyy-3 X Cm m ddyy-4 XCm m ddyy-5 XCmmddYY-6 * T he extracted concentration o f the calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC = extracted calibration standard. Page 4 o f 7 Page 38 o f 56 MPI Research Page 150 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygcn Research Method Number V000178! ____________________________ A N A L Y T IC A L M E T H O D ____________________________ M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Soil by L C /M S /M S 9.2.3 9.2.4 9.2.3 A zero standard solution (reagent blank) m ust be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene tubes at 2C to 6C, up to two weeks. A lternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f sam ples extracted (typically 20 o r less) m ust include at least one reagent control (method blank using 3 m L o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 11.2 11.3 11.4 11.5 11.6 11.7 n.8 11.9 W eigh 5 g o f sample into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Add 5 m L o f methanol and shake on a wrist action shaker for ~ I5 minutes. Transfer the tubes to an ultrasonic bath and sonicate for ~15 minutes. Bring the volume up to 40 m L with water in the 50 m L polypropylene centrifuge tube. C entrifuge for -1 0 m inutes at -3 0 0 0 rpm. C ondition the C u SPE cartridges (1 g , 6 m L ) b y p assin g 10 m L m ethanol followed by 5 m L o f HPLC water ( - 2 drop/sec). Do not let colum n run dry Load (decant) the sample on the conditioned C u SPE cartridge. Discard eluate. Elute w ith -S m L 100% m ethanol. Collect 5 m L o f eluate into graduated 15 m L polypropylene centrifuge tubes (final volum e = 5 m L). Analyze samples using electrospray LC/MS/MS. 12.0 Chrom atography 12.1 Inject the sam e am ount o f each standard, sam ple and fortified sam ple into the LC /M S/M S system. A calibration standard m ust precede and follow all analyzed samples. 12.2 S tandards o f P FO A corresponding to at least five or m o re concentration levels m ust be included in an analytical set. 12.3 A n entire set o f extracted calibration standards m ust b e included at the beginning and at the end o f a sam ple set. Extracted standards m ust be interspersed between every 5-10 samples. A s an alternative, an entire set o f Page 5 o f 7 Page 39 o f 56 MPI Research Page 151 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygeo Research Method N uirtw r V00017B1 ANALYTICAL METHOD M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A ) in Soil by L C /M S /M S ] extracted calibration standards m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards m ust be the first and last injection in a sam ple set. 12.4 Use linear standard curves for quantitation. L inear standard curves are generated for the analyte by linear regression using 1/x w eighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 A cceptance Criteria 13.1 C hrom atogram m ust show a peak o f a d aughter ion at 36 9 am u from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, white the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 M ethod blanks m ust not contain P FO A a t levels g re ater than th e LO Q . I f a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set m ust be re-extracted. 13.3 R ecoveries o f control spikes and m atrix spikes m u st be betw een 70-130% o f their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any m atrix spike outside 70 130% should b e evaluated b y th e analyst to d ete rm in e i f re-extraction is warranted. A ny calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total num ber o f extracted calibration standards that could be excluded must not exceed 20% o f the total num ber o f extracted standards injected. 13.5 T he correlation coefficient (R ) for ca libration curves generated m ust be 0.992 (R2 0.985). If calibration results fall outside these lim its, then appropriate steps must be taken to adjust instrum ent operation, and the standards or the relevant set o f sam ples should be reanalyzed. 13.6 R etention tim es betw een standards and sam ples m ust not drift m ore than 4 % within an analytical run. If retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. Page 6 o f 7 Page 40 o f 56 MPI Research Page 152 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygeo Research Method Number V000I78) ANALYTICAL METHOD M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A ) in Soil by L C /M S/M S 14.0 Calculations 14.1 U se the follow ing equation to calculate the am ount o f P FO A found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program: PFOA found (ng/L) - (Peak area - intercept) x DF slope DF - factor by which the final volume was diluted, if necessary. 14.2 F or sam ples fortified w ith know n am ounts o f PFO A prio r to extraction, use the following equation to calculate the percent recovery. Recovery (% ) = [ total analyte found (ng/L) - analyte found in control (ng/L)] ^ analyte added (ng/L) 14.3 U se th e follow ing equation to convert the am ount o f P F O A found in ng/L to ng/g (ppb). P FO A found (ppb) fPFO A found (ng/L ) x volum e extracted (0.04LV| sample w eight (3 g) 14.4 Use the following equation to calculate the am ount o f PFO A found in ppb based on dry weight. P FO A found (ppb) d ry w e ig h t=>P FO A found (ppb) x [ 100% / total solids(% )) Page 7 o f 7 Page 41 o f 56 MPI Research Page 153 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 ANALYTICAL METHOD Method Number: V0001782 M ethod o f A nalysis fo r th e D eterm ination o f P erfluorooctanoie A cid (P F O A ) in Sedim ent by LC/M S/M S . ' Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 1680) Approved By: C jL Paul Connolly I Technical Leader, LC-M S, Exygen Research ____ lO k b fa f D a te ohn Flaherty . Vice President, Operations, Exygen Research D ate MPI Research Total Pages: 7 Page 42 o f 56 Page 154 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygco Research Method Number VOGOI782 ) ANA LYTICAL M ETH O D | M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Sediment by L C /M S /M S 1.0 Scope This m ethod is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid C hrom atography coupled to a tandem M ass Spcctrom ctric Detector (LC/M S/M S) in sediment. Safety 2.1 A lw ays observe safe laboratory practices. 2.2 C onsult the appropriate M SDS before handling any chem ical for proper safety precautions. Sample Requirement 3.1 A t least 30 g o f test sam ple for extraction. 3.2 N o sam ple processing is needed for sedim ent sam ples. 3.3 Sam ples stored refrigerated should be allowed to equilibrate to room tem perature. 3.4 All sam ples m ust be thoroughly m ixed before being sam pled for extraction. 3.5 Sam ple collection procedures w ill be specified in the sam pling plan for this project. Reagents and Standards 4 .t W a ter-H P L C grade 4.2 M ethanol - HPLC grade 4.3 Acetic Acid - Reagent grade 4.4 Am m onium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrum ent and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 A high performance liquid chromatograph capable o f pum ping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 p L connected to a tandem M ass Spectrom eter (LC/M S/M S). A device to collect raw data for peak integration and quantitation. A nalytical balance capable o f reading to 0.00001 g. 50 mL disposable polypropylene centrifuge tubes. 15 m L disposable polypropylene centrifuge tubes. D isposable m icropipets (50-1 OOuL, 100-200uL). 125-mL LDPE narrow-mouth bottles. 2 m L clear HPLC vial kit. . D isposable pipettes. Autopipettes (100-1000 pL and 10-100 pL), w ith disposable tips. W aters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. SPE vacuum manifold. Page 2 o f 7 Page 43 o f 56 MPI Research Page 155 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygen Research Method Number VOOQJ782 [______________________________ A N A L Y T IC A L M E T H O D _________________ | M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A ) in Sedim ent by L C /M S /M S . 5.13 Vortexer. 5.14 W rist-action shaker. 5.15 C entrifuge capable o f spinning 50 m L polypropylene tubes at 3000 rpm . 6.0 C hrom atographic System 6.1 Analytical Colum n: Fluophase RP (K eystone Scientific), 2 .1 m m x 50 m m , 5p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 M obile Phase ( A ) : 2 mM A m m onium Acetate in W ater 6.4 M obile Phase ( B ) : M ethanol 6.5 G radient Program: Tim e (mini 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate (mL/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volum e: 15 p L (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: E lectrospray Negative M R M m ode, m onitoring 413 - 369 m /z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optim ize the M SMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w ater is prepared b y adding 0.154 g o f am m onium acetate to 1000 mL o f water. Page 3 o f 7 Page 44 o f 56 MPI Research Page 156 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygen Research Method Number VOOOI782 | ANA LYTICAL M ETH O D | M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Sediment by L C /M S/M S 8.2 Extraction Solutions 8.2.1 1% acetic acid in water is prepared by adding 10 m L o f acetic acid to 1000 mL o f water. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/m L o f PFO A by w eighing 10 m g o f analytical standard (corrected for purity) and dilute to 100 m L with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/m L fortification solution o f P F O A is prepared b y bringing 10 m L o f the 100 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 1.0 pg/m L fortification solution o f P FO A is prepared b y bringing 10 m L o fth e 10 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.4 A 0.1 pg/m L fortification solutioo o f P F O A is prepared b y bringing 10 m L o f th e 1.0 pg /m L solution to a final volum e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.5 A 0.01 pg/raL fortification solution o f PFO A is prepared by bringing 10 m L o f the 0.1 pg/m L solution to a final volum e o f 100 with methanol in a 125 m L LD PE bottle. 9.1.6 T h e stock and fortification solutions are to b e stored in a refrigerator at approximately 4C and are stable for a m axim um period o f 6 months from the date o f preparation. Standard Calibration Solutions 9.2.1 LC/M S/M S calibration standards are prepared in m ethanol via dilution o f the 0.1 pg/m L fortification solution. 9.2.2 Tbe following is a typical exam ple: additional concentrations m ay be Concentration ofFortification Solution inx/mL) 100 100 100 10 5 2 Volume (mL) 10 5 2 10 10 10 Diluted to (m L ) 100 100 100 100 100 100 Final Concentration (ng/mL) 10.0 5.0 2.0 1.0 0.5 0.2 Page 4 o f 7 Page 45 o f 56 MPI Research Page 157 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygen Research Method Number V0001782 | ANA LYTICAL M ETH O D | M ethod o f A nalysis for the Determination o f Perfluorooctanoic Acid (PFO A ) in Sediment by LOM S/M S 9.2.3 9.2.4 Store all calibration standards in 125-mL LDPE narrow -m outh bottles at 2C to 6C, up to six months. Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 E ach batch o f sam ples extracted (typically 20 o r less) m ust include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for held and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 11.2 11.3 11.4 U .S 11.6 11.7 1 1.8 11.9 11.10 11.11 11.12 11.13 W eigh 5 g o f sam ple into SO m L polypropylene ce ntrifuge tubes (fortify as needed, replace lid and mix well). Add 3S mL o f 1% acetic acid, cap, vortex and shake on a wrist action shaker for *-60 m inutes. Centrifuge the tubes at -3 0 0 0 rpm for -2 0 m inutes. Condition the C n SPE cartridges ( l g, 6 m L) by passing 10 m L methanol followed by 20 mL o f HPLC water ( - 2 drop/sec). Do not let colum n run dry Load (decant) the sam ple on the conditioned C u SPE cartridge. Discard eluate. Add 20 mL o f methanol to the sediment left in the bottom o f the 50 mL centrifuge tube. Cap, vortex and shake on a wrist action shaker for -3 0 m inutes. Centrifuge the tubes at -3 0 0 0 rpm for -2 0 minutes. Decant the methanol onto the sam e SPE cartridge. Collect the eluate. W ash the column w ith 4 mL o f methanol. Collect the eluate and add it to the eluate collected in step 11.8. C ondition a second C n S PE cartridge (1 g, 6 m L ) b y passing 10 m L m ethanol followed by 20 m L o f HPLC water ( - 2 drop/sec). Do not let colum n run dry Add the methanol to -2 0 0 mL o f water and load on the second conditioned SPE cartridge. Elute with - 5 m L 100% methanol. Collect S m L o f eluate into graduated IS m L polypropylene centrifuge tubes (final volum e S mL). Analyze samples using electrospray LC/MS/MS. Page 5 o f 7 Page 46 o f 56 MPI Research Page 158 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygen Research Method Number VOOOI782 | ANA LYTICAL M ETH O D 1 M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A ) in Sedim ent by L C /M S/M S 12.0 C hrom atography 12.1 Inject the sam e am ount o f each standard, sam ple and fortified sam pte into the LC/M S/M S system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to al least five o r m ore concentration levels m ust be included in an analytical set. 12.3 A n entire set o f extracted calibration standards m u st b e included at the beginning and at the end o f a sam ple set. Standards m ust be interspersed between every S '10 sam ples. As an alternative, an entire set o f calibration standards m ay be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards m ust be the first and last injection in a sample set. 12.4 U se linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x w eighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 A cceptance Criteria 13.1 C hrom atogram m ust show a peak o f a d aughter ion at 369 am u from a parent o f 413 amu. The 413 am u parent corresponds to the PFO A anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 M ethod blanks m ust not contain P FO A at levels g re ater than th e LOQ . I f a blank contains PFOA at levels greater than 0.2 ng/m L, then a new blank sam ple m ust be obtained and the entire set m ust be re-extracted. 13.3 R ecoveries o f control spikes and m atrix spikes m ust b e betw een 70-130% o f their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated b y th e analyst to d ete rm in e i f re-extraction is w a rranted. A ny calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve. However, the total num ber o f extracted calibration standards that could be excluded must not exceed 20% o f the total num ber o f extracted standards injected. The correlation coefficient (R ) for calibration curves generated m ust be 0 .9 9 2 (R* 0.985). I f calibration results fall ou tsid e these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f sam ples should be reanalyzed. Page 6 o f 7 Page 47 o f 56 MPI Research Page 159 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygca Research Method Number V00017S2 | ____________ANALYTICAL METHOD | M ethod o f A nalysis for the Determination o f Perfluorooctanoic Acid (PFO A ) in Sediment by L C /M S/M S 13.6 Retention times between standards and sam ples m ust not drift m ore than 4 % w ithin an analytical nin. If retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 C alculations 14.1 U se the follow ing equation to calculate the am ount o f P FO A found (in ng/m L, based on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program; PFOA found (ng/m L) fPcak area - intercept) x DF slope D F factor by which the final volume was diluted, if necessary. 14.2 F or sam ples fortified w ith know n am ounts o f PFO A prio r to extraction, use the following equation to calculate the percent recovery. Recovery (% ) [ total analyte found (ng/m L) - analyte found in control (ng/m L )] 100 analyte added (ng/mL) 14.3 U se the follow ing equation to convert the am ount o f P FO A found in ng/m L to ng/g (ppb). P FO A found (p p b ) fP F O A found (ng/m L ) x final volum e (S mLV) sample weight (3 g) 14.4 U se the following equation ( if necessary) to calculate the am ount o f PFOA found in ppb based on dry weight. PFO A found (ppb) dry weight = PFOA found (ppb) x [100% / total soiids(% )] Pige 7 o f 7 Page 48 o f 56 MPI Research Page 160 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 ANALYTICAL METHOD Method Number: V0001783 Method of Analysis for the Determination of Perfluorooctanolc Acid (PFOA) in Fish and Clams by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: v U C-JL Paul C onnolly 1 Technical Leader, LC-M S, Exygen Research 2 lL/ * ? / y Vohn Flaherty ' Vice President, O perations, Exygen Research D a te D a le Total Pages: 8 Page 49 o f 56 MPI Research Page 161 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygea Reiearch Method Number V0Q0I783 ANALYTICAL m e t h o d ] M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A ) in Fish and Clams by LC/MS/MS 1.0 Scope This m ethod is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Perform ance Liquid C hrom atography coupled to a tandem Mass Spectrom etric Detector (LC/M S/M S) in fish and clams. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 C onsult the appropriate MSDS before handling any chem ical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 2 0 g o f test sam ple for extraction. 3.2 Sam ples should be processed before extraction. Place the frozen sam ple in a food processor and hom ogenize w ith dry ice. Place the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time o f analysis. 3.3 Sam ple collection procedures will be specified n the sam pling plan for this project. Reagents and Standards 4.1 4.2 4.3 4.4 4.3 4.6 4.7 4.8 4.9 4 .10 4 .11 4.12 4.13 W ater - HPLC grade Acetonitrile - HPLC grade Carbon ( 12(M 0 0 m esh) - Reagent grade M ethanol - HPLC grade Silica gel (60-200 mesh) - Reagent grade Florisil (60-100 m esh) - Reagent grade Superclean LC-NHj - Reagent grade 1-O clanol - H P L C grade L-A scotbic acid - Reagent grade Dimethyldichlorosilane - Reagent grade Toluene - Reagent grade Ammonium A c etate- A.C.S. Reagent Grade Perfluorooctanoic Acid - Sigma-Aldrich 3.0 Instrument and Equipment ` 3.1 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 p L connected to a tandem M ass Spectrom eter (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 A nalytical balance capable o f reading to 0.00001 g. Page 2 of 8 Page 50 o f 56 MPI Research Page 162 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exyges Research Method Number V0001783 ANALYTICAL METHOD M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Fish and Clams by LC/MS/MS 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14 5.15 5.16 Rotary evaporator. T issum izer. 125 m L pear-shaped flasks. 50 mL disposable polypropylene centrifuge tubes. 15 m L disposable polypropylene centrifuge tubea. D isposable m icropipets (50-1OOuL, 100-200uL). 125-mL LDPE narrow-mouth bottles. 2 mL clear HPLC via] kit. D isposable pipettes. A utopipettes(100-1000pL and 10-100 pL), w ith disposable tips. SPE tubes (20mL) (Supelco c a t no. N057177). W rist action shaker. C entrifuge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 C hrom atographic System 6.1 A nalytical Colum n: Fluophase R P (K eystone S cientific), 2.1 m m x 50 m m , 5n (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 M obile Phase (A ) : 2 mM Am monium Acetate in W ater 6.4 M obile Phase (B) : M ethanol 6.5 Gradient Program: Time (m ini 0.0 1.0 8.0 20.0 22.5 Vo A 65 65 25 25 65 Flow Rate % B fm L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection V olum e: 15 p L (can be increased to as m u ch as 50 pL). 6.7 Quantitation: Peak A rea - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and m ay b e changed in order to optim ize the HPLC system. Pge 3 of8 Page 51 o f 56 MPI Research Page 163 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygcn Research Method Number VOOOI783 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A ) in Fish and Clams by LC/MS/MS 7.0 M S/M S System 7.1 M ode: Electrospray N egative M R M m ode, m onitoring 413 -* 369 m /z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optim ize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM am m onium acetate in water is prepared by adding 0.154 g o f am m onium acetate to 1000 m L o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in m ethanol is prepared b y dissolving 2 g o f ascorbic acid in 100 m L o f methanol. 30% D im ethyldichlorosilane in toluene is prepared b y brin g in g 3 m L o f dimethyldichlorosilane to a final volum e o flO m L w ith toluene. A lternate volum es m ay be prepared. Standard Preparation 9.1 Standard Stock/Fortification S olution 9.1.1 9.1.2 9.1.3 9.1.4 9 .1.5 Prepare a stock solution o f -1 0 0 pg/m L o f PFOA by w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-raL LDPE bottle. A 1.0 pg/m L fortification solution o f PFOA is prepared by bringing 1 m L o f the 100 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LDPE bottle. A 0.1 pg/m L fortification solution o f P FO A is prepared by bringing 10 m L o f th e 1.0 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LDPE bottle. A 0.01 pg/m L fortification solution o f PFO A is prepared by bringing 10 m L o f the 0.) p ^ m L solution to a final volum e o f 100 with methanol in a 125 m L LDPE bottle. T he stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a m axim um period o f 6 months from the date o f preparation. Page 4 o f 8 Page 52 o f 56 MPI Research Page 164 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygen Research Method Number V0001783 _____________________________A N A L Y T IC A L M E T H O D _____________________________ M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A ) in Fish and Clams by LC/MS/MS 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/M S calibration standards are prepared in methanol via dilution o f the 1.0 ng/m L fortification solution. The following is a typical example: additional concentrations m ay be prepared as needed. Concentration o f Fortification Solution fue/mL) Volume (m L) Diluted to (mL) Final Concentration (lig /m L ) 1.0 1.0 1.0 0.05 0.02S 0.1 o.oos S.O 2.5 1.0 10 10 10 10 100 100 100 100 100 100 100 0.05 0.025 0.01 0.005 0.0025 0.001 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-m outh bottles at 2C to 6C, up to six months. 9.2.4 Alternate volum es and concentrations o f standards m ay b e prepared as needed. 10.0 Batch Set Up Each batch o f samples extracted (typically 20 or less) m ust include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 W eigh 5 g o f frozen sam ple into SO m L p o lypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 30 m L o f acetonitrile and shake on a w rist action shaker for ~1 S m inutes. 11.3 Place the tubes in a freezer for - 1 hour. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 P ack th e 20 m L SPE tubes in sequence w ith 2 g florisil, 2 g silica gel, 2 g carbon, and l g LC-NHj. Condition the colum ns w ith 20 mL o f methanol, then 20 m L o f acetonitrile. D iscard all w ashes. D o not allow the colum n to dry. 11.6 Silanize the 125 m L pear-shaped flasks b y rinsing w ith the 30% dim ethyldichlorosilane in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks com pletely before use, either by air-drying or with a stream o f nitrogen. Page 5 o f 8 Page 53 o f 56 MPI Research Page 165 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygcn Research Method Number VOOO1783 | ANA LYTICAL M ETH O D 1 M ethod o f Analysis for the Deteimination o f Perfluorooctanoic Acid (PFO A ) in Fish and Clams by LC/MS/M S 11.7 11.8 11.9 11.10 11.11 11.12 11.13 11.14 11.15 11.16 11.17 11.18 C entrifuge the SO m L polypropylene tubes containing sam ple at -2 0 0 0 rpm for -1 0 minutes. Decant the extract on to a conditioned SPE colum n fitted inside the m outh of the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape flask. Add 10 mL o f acetonitrile to the sam ple in the 50 m L centrifuge tube. Homogenize the frozen fat phase using a tissum izer for -3 0 seconds and rinse the tissum izer with -1 0 mL o f acetonitrile into the tube. Shake the sam ple again fo r-1 0 minutes on a wrist-action shaker. Place the tubes in a freezer for - 1 hour more. C entrifuge the 50 mL polypropylene tubes containing sam ple at -2 0 0 0 rpm fo r -1 0 minutes. Decant the extract onto the sam e SPE column. C ollect the eluate into the sam e pear-shaped flask and com bine w ith the eluent from the initial extraction. P ass 20 m L o f acetonitrile through the S P E colum n and c o m b in e th e eluate in the sam e pear-shaped flask. A dd 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40*C). M ake th e final volum e, by adding 2 m L o f 2% ascorbic acid in m ethanol to the pear-shaped flask and swirl to mix/dissolve. Transfer the extracts to HPLC vials using disposable pipels. Analyze sam ples using electrospray LC/MS/MS. 12.0 C hrom atography 12.1 Inject th e sam e am ount o f each standard, sam ple and fortified sam ple into the LC /M S /M S system . A calibration standard m ust p recede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels m ust be included in an analytical set. 12.3 A n entire set o f calibration standards m ust b e included at the beginning and at the end o f a sample set. Standards m ust be interspersed betw een every 5-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 sam ples (to account for a second set o f standards). In either case, calibration standards m ust be the first and last injection in a sam ple set. 12.4 U se linear standard curves for quantitation. Linear standard curves are generated for the analyte b y linear regression using 1/x w eighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. Page 6 o f 8 Page 54 o f 56 MPI Research Page 166 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygco ReiMJxh Method Number V000I783 ANA LYTICAL M ETH O D M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Fish and Clams by LC/MS/MS 12.5 Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance C riteria 13.1 C hrom atogram m ust show a peak o f a d aughter ion at 369 am u from a parent o f 413 am u. T h e 413 am u p aren t corresponds to the P F O A anion, w hile (he daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 M ethod blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.5 ppb, then a new blank sam ple m ust be obtained and the entire set must be re-extracted. 13.3 R ecoveries o f control spikes and m atrix spikes m ust b e betw een 70-130% o f their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. 13.4 A ny ca libration standard found to b e a statistical ou tlie r by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could b e excluded m ust not exceed 20% o f the total number o f standards injected. 13.5 T he correlation coefficient (R ) for ca libration cu rv es generated m ust be 20.992 (RJ 20.98$). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrum ent operation, and the standards or the relevant set o f sam ples should be reanalyzed. 13.6 Retention tim es between standards and sam ples m ust not drift m ore than 4 % w ithin an analytical run. If retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 C alculations 14.) U se the following equation to calculate the am ount o f PFO A found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program: PFOA found (ng/mL) (Peak area - intercept) slope 14.2 U se the following equation to convert the am ount o f P FO A found in ng/m L to ng/g (ppb). P FO A found (ppb) ** fPFO A found fn e /m U x final volum e f m D x DF1 sample weight (g) D F * factor b y w hich the final volum e was diluted, i f necessary. Page 7 o f 6 Page 55 o f 56 MPI Research Page 167 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 ExyLIMS Protocol Number: P0003267 Exygen Research Method Number V0001783 ANA LYTICAL M ETH O D M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Fish and Clams by LC/MS/MS 14.3 F or sam ples fortified w ith know n am ounts o f P FO A p rio r to extraction, use the following equation to calculate the percent recovery. Recovery (% ) TM [ total analyte found (ng/g) - analyte found in control (n g /g )] ^ ^^ analyte added (ng/g) MPI Research Page 8 o f 8 Page 56 o f 56 Page 168 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 CHEM EHSR 2 3 6 1B 651-733 1958 ' 1 2 /2 0 '0 7 1 2 :1 4 N O .2 7 9 0 7 /1 1 Amendment Number: Effective Date: MPI State College Project Number PROTOCOL AMENDMENT 3______ i 12/18/07 ' Other Study P0003268 Number(s): Page 1 of 0137.0219 2 : DESCRIPTION OF AMENDED SECTION 1) Identification and Justification of the Test System Section - 'The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included In the appropriate interim report and/or final report associated wilh this study." page 8 of 57 of the protocol ' 2) Objective and Analytical Procedure Summary Sections - list of methods referenced, pages 6 and 9 of 57 of the protocol 3) Analytical Procedure Summary Section - page 9 of 57 of the protocol ' I AMENDEDTO 1) The control samples will be purchased and prepared by the'testing facility, with the exception of bass and catfish fillet control samples, which will be processed and supplied by the sponsor (3M). Purchase and processing details for the control samples will be included in the appropriate interim report and/or final report associated with this study, with the exception of fish fillet control sample purchase and processing details, which will remain with the sponsor (3M). 2) ETS-8-049.2 (V0003970-2): `Determination of Fluorochemicals Via Solid-Phase Extraction of Fish Tissues (Fillet or Whole Body) by High Performance Liquid Chromatography with Tandem Mass Spectrometry" 3) The primary analytical method for the analysis of fish and clam samples is theV0001783 method; however, in instances where the fish samples to be analyzed are fish fillets, method ETS-3-049.2 (V0003970-2) Is to be used. ! RATIONALE' --' 1) Client requests that, we use their species-specific fillet controls when analyzing fish fillet samples. 2) Fish fillet samples will be.analyzed using method ETS-3-049.2 (V0003370-2), see method attached. , 3) Fish fillet samples analyzed using method ETS-8-049.2 (V0003970-2) yield more accurate results due to matrix effects noted when using method VQQ01733 for fish fillet analysis. IMPACT ON STUDY 1) No negative impact. 2) No negative impact.___________________________ LIB R AR Y IDV0001226-12 AD M INISTRATIVE FORM MPI Research Page 169 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 CHEI1 EHSR 2 3 6 IB ' 651 733 1958 1 2 /2 0 '0 7 1 2 :1 4 N O .2 7 9 0 8 /1 1 i Amendment Number: Effective Date: MPI State College Project Number PROTOCOL AMENDMENT 3______ . 12/18/07 . ' Other Study P0003268 Numbers): Page 2 of 0137.0219 2 I LIB R A R Y ID V000122S-12 AD M IN ISTRATIVE FORM MPI Research Page 170 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 . Amendment Number: Effective Date: MPI State College Project Number PROTOCOL AMENDMENT ______ 3______ 12/18/07 . Other Study P0003268 Number(s): Page 1 of _ 2 0137.0219 DESCRIPTION OF AMENDED SECTION 1) Identification and Justification of the Test System Section - "The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the appropriate Interim report and/or final report associated with this study." page 8 of 57 of the protocol 2) Objective and Analytical Procedure Summary Sections - list of methods referenced, pages 6 and 9 of 57 of the protocol 3) Analytical Procedure Summary Section - page 9 of 57 of the protocol AMENDED TO 1) The control samples will be purchased and prepared by the testing facility,, with the exception of bass and catfish fillet control samples, which will be processed and supplied by the sponsor (3M). Purchase and processing details for the control samples will be included in the appropriate interim report and/or final report associated with this study, with the exception of fish fillet control sample purchase and processing details, which will remain with the sponsor (3M). 2) ETS-8-049.2 (V0003970-2): "Determination of Fluorochemicals via Solid-Phase Extraction of Fish Tissues (Fillet or Whole Body) by High Performance Liquid Chromatography with Tandem Mass Spectrometry" 3) The primary analytical method for the analysis of fish and clam samples is the V0001783 method; however, in instances where the fish samples to be analyzed are fish fillets, method ETS-8-049.2 (V0003970-2) Is to be used. RATIONALE 1) Client requests that we use their species-specific fillet controls when analyzing fish fillet samples. 2) Fish fillet samples will be. analyzed using method ETS-8-049.2 (V0003970-2), see method attached. ' . .. 3) Fish fillet samples analyzed using method ETS-8-049.2 (V0003970-2) yield more accurate results due to matrix effects noted when using method V0001783 for fish fillet analysis. IMPACT ON STUDY 1) No negative impact. . 2) No negative impact. ___________________ LIB R A R Y ID V0001226-12 ADM INISTRATIVE FORM MPI Research Page 171 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Amendment Number: Effective Date: MPI State College Project Number PROTOCOL AMENDMENT ______ 3______ 12/18/07 . Other Study P0003268 . Number(s): Page 2 of _2_ 0137.0219 LIB R AR Y ID V0001226-12 ., ADM INISTRATIVE FORM MPI Research Page 172 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 3M Environmental Laboratory M eth o d D e te rm in a tio n o f F lu o ro c h e m ic a ls via S o lid -P h a s e E x tra c tio n o f F ish T issu es (F ille t o r W h o le B o d y) b y H ig h P e rfo rm a n c e L iq u id C h ro m a to g ra p h y w ith T a n d em M ass S pectro m etry M e th o d N u m b er: E T S -8-049.2 A d o p tio n D a te : 9/26/07 . . R e v is io n D ate : U p o n S ig n in g E ffective D ate: I l / l ' i f O ') A pproved By: William K. Ragen, Ph.D. Technical Manager <0? Date E T S-8-049.2 P a g e ) o f 13 D ete rm in a tio n o f Fluorochem icals in Fish T issu e (F ille t or W h o le B o d y ) by L C /M S /M S MPI Research Page 173 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 1 Scope and Application This method describes the extraction of fluorochemicals.from fish tissues using solid-phase extraction . 1 followed by separation, identification, and quantification using high performance liquid chromatography tandem mass spectrometry. These procedures are appropriate for perfluorinated acids, alcohols, amides, sulfonates and other liuorinated compounds in fish fillets, whole bodies, or other biota such as clams, provided that quality control sample results meet the precision and accuracy requirements of the study. ' ' 2 Method Summary _____ This method describes the procedure for the extraction and quantification of fiuorochemicals. Initially, whole fish or fish fillets are processed before extraction by placing the frozen sam ple in a food processor and hom ogenizing with dry ice. Th e dry ice is allowed to sublimate overnight. A n aliquot of frozen, ' hom ogenized tissue is accurately weighed and analytes of interest are extracted from the fish tissues by homogenizing in acetonitrile, cooling for at least one hour at -2 0 ` C ., centrifuging cold (approximately - 5 ` C .) at approximately 3000 rpm for at least 20 minutes, dilutinginto water, and solid-phase extraction using a W aters Oasis H LB solid-phase extraction cartridge. Quantification is accomplished by high performance liquid chromatography tandem mass spectrometry. Extracted calibration.standards are prepared in suitable commercial fish tissue. If suitable fish tissue (or an appropriate surrogate tissue) is not available, study sample(s) may be semi-quantitatively screened against a solvent curve to identify a sample to use for. matrix-matched calibration. T h is extracted curve may be used to quantify study samples directly if the method blanks support an acceptable limit of quantitation (L O Q ). Alternately, the method of standard addition may be used to adjust calibration curve concentrations and samples quantified accordingly. Lastly, a solvent curve m ay be used to quantify . samples if an appropriate matrix is unavailable. Expanded method uncertainty may be required if a solvent-curve is the only.option for study samples. . 3 Definitions 3.1 Solid-Phase Extraction (SPE) S P E is a separation method that uses a solid phase and a liquid phase to isolate analytes from a solution. S P E is based on the preferential affinity of desired or undesired solutes for the stationary (solid) phase of the S P E cartridge. 4 Warnings and Cautions . 4.1 Health and Safety W arnings Always w ear appropriate personal protective equipment such as protective gloves, e ye protection, and appropriate clothing w hen working with biological matrices, solvents, chemicals and instrumentation. For potential hazard information refer to material safety data sheets, packing material, the 3M Environmental . Laboratory's Chem ical H azard Review, the 3M G uide to Laboratory Practices or other resources as ' ' appropriate: 4.2 Cautions Th e analyst must be familiar with the laboratory equipment and potential hazards including, but not limited to, the use of biological materials (refer to E T S -2 -5 ), solvents, high temperatures, pressurized gas and solvent lines, high voltage, and vacuum systems. Refer to the appropriate equipment procedure or operator manual for additional Information and cautions. E T S -8 -0 4 9 .2 P a g e 2 o fl3 D eterm in atio n o f Fiuorochem icals in Fish Tissue (F ille t o r W h o le B o d y ) by L C /M S /M S MPI Research Page 174 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 5 Interferences _______ __________________________________________ T o minimize interferences, polytetrafluoroethylene (P T F E , e.g. Teflon) should not be used for sam ple .. storage or any part of instrumentation that com es in contact with the sample or extract. '' Co-extracted matrix components m ay enhance or suppress the measured analyte signal in the mass spectrometer. The se effects are minimized using this extraction technique, but the precision and accuracy of spike results must be evaluated for possible effects of co-extracted matrix interferences that m ay be present. 6 Instrumentation, Supplies, and Materials Th e following instrumentation, supplies, and materials are used while performing this method. Equivalent or other instrumentation, supplies, and materials may be used in place of those listed. 6.1 Instrum entation . Balance, analytical (display at least 0.0001 g) Homogenizer, P O L Y T R O N , Om niPrep or similar Agilent H P LC 1200 system: . Pump: Quaternary Pump, Agilent, Model G1311A; or Binary Pump, Agilent, Model 1312 Solvent Degasser, Agilent, Model G1322A ' Autosampler, Agilent, Model G1313A, or Thermostated Autosampler, Agilent, Model G1329A - Autosampler thermostat (optional), Agilent, Model G1330A . ' Column Compartment (Temperature Controlled), Agilent, Model G1316A ' Diode Array Detector, (D A D ) Agilent, Model # G 1 315A ' Controller, Hand Held, Agilent, Model # G1323A Applied Biosyslems M D S S C IE X A P I 5000 Biomolecular M ass A n a lyze r . S C IE X Turbo Ion Spray Liquid Introduction Interface Other instrumentation as needed, document as appropriate . 6.2 Supplies and Materials Hamilton gas tight syringes (capable of dispensing at least 50 pL) Cla ss A volumetric flasks Eppendorf or plastic disposable pipettes Therm o Fischer Scientific Betasil C , 8l 2.1 x 100 mm, 5 pm particle size Therm o Fischer Scientific Prism TM R P 2x50 mm, 5 pm particle size . H P L C vials capable of containing approximately 2 mL extract W aters Oasis H LB S P E cartridge (3cc, 60mg, W aters, WAT094226). Disposable Extraction Column (J.T . Baker, 7120-03 or equiv.) Adaptor (Agilent, 5185-5794 or equiv.) Centrifuge tubes, polypropylene, various sizes . ." ETS-8-049.2 Page 3 of 13 . Determination of Fluorochemicals in Fish Tissue (Fillet or Whole Body) by LC/MS/MS MPI Research Page 175 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Other,supplies and materials as needed, document as appropriate 7 Reagents and Standards! . W hen preparing different amounts or concentrations than those listed, adjust accordingly. 7.1 Reagents . Acetonitrile, H P L C grade or equivalent . Methanol, H P L C grade or equivalent A S T M T y p e I W ater or equivalent. All water, used in this method should be A S T M T yp e I or equivalent, and may be provided by a Milli-Q Gradient system or by another system or vendor, provided there is adequate documentation of the source. ' Ammonium acetate, Mallinckrodt 98% pure or equivalent Formic acid, 8 8 % or equivalent (other acids may be used) ' Ammonium H ydroxide, NH4OH, 30% or equivalent '- Sodium Hydroxide, A C S Grade, 98% pure or better . Phosphoric acid, H3PO4, 85% or equivalent . Various fish tissues from supplier or study sponsor for use as control matrix Note: T h e concentrations of all acid and base reagent solutions are not corrected for the reagent strength. F or example, a 2% H 3 P O 4 (aq) solution might be prepared by adding 2 mL of the 85% a dd to water and brought up to volume of 100 mL. Similarly, a solution of 5% N H 4 O H in methanol might be prepared b y adding 5 mL of the 30% reagent up to a volume of 100 mL of m ethanol.. 7.2 Standards N ote: Due to the likely possibility of ester formation between carboxylic acids and alcohols,.when preparing stock solutions in methanol as in this method, it is important that the methanol be treated with an e xcess of base. In this method, when preparing the primary stock solution of 1ppm periluorocarboxylic a d d in methanol, the methanol needs to be treated with base to an approximate concentration of 10 uM. If stock solutions are created at a higher concentration, then the concentration of N a O H should be scaled up appropriately. Upon making dilutions of the primary stock solutions, it is not necessary to use caustic methanol, as the base will still be present in e xce ss to the acids. The following is an example for preparing spiking solutions for a matrix-matched calibration curve with a lower limit of quantitation of 0.025 ng/mL in extract following the extraction procedure described in ' ' sedion 11. N O T E : If suitable fish tissue is not available to construct the matrix-matched calibration ' curve, it is acceptable to quantify the sample extracts versus a solvent-based calibration curve. W hen performing extractions with different volumes, or analyses with a different required lower limit of quantitation, adjust accordingly. Prepare individual stock solutions of 100 pg/mL (stock solution A) by , weighing out 10 mg of analytical standard (corrected for purity and salt form) and dilute to 100 mL with 10 uM N a O H in methanol or other appropriate solvent in a 100 mL volumetric flask. Each stock solution may be stored in a refrigerator at 2 C to 6 "C and has an expiration date of a maximum period of six months from the date of preparation (unless determined otherwise). Next, prepare the following solutions in water. W ater is the recommended solvent for the final spiking solutions, how ever other solvents or solutions with water may be used. At least six calibration standards E T S -8 -0 4 9 .2 P a g e 4 o f 13 D e te rm in a tio n o f F lu o ro ch em ica ls in F ish T issu e (F ille r o r W h o le B o d y ) by L C /M S /M S MPI Research Page 176 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 should be prepared (nine are recommended). T h e final calibration curve must consist of at least six calibration points after analysis. If the analyst suspects that some curve points may be deactivated, more calibration standards should be prepared. B 1000 r SW 10 B 1000 | 250 10 B 1000 100 10 B 1000 50 10 B 1000 10 . 10 . B 1000 5.0 10 SB 50 C 25 D 10 E 5.0 ! F 1.0 ! G 0.5 - H - -- l... . .. ng F in a l c o n c e n t r a t i o n ----------= ------- = S p ik in g ng S o lu tio n -- x V o lu m eo f sp ------------------------ -i-k--i-n--g=-s--o--l--u--t-i-o---n--1( m L ). . m L extract m L F inal E x tra ct V o lu m e (m L ) Following the procedure as described In section 11 using solution H: F in a l C o n c e n t r a t i o n ------- -- ------- = 0. 5^ - x -- = 0 . 0 2 5 ----------- ------- . m L extract m L 2.0 m L m L extract ' 8 Sample Handling After the initial processing outlined in Section 11.1, additional sample processing is needed for fish fillet or whole body samples. All aliquoting and accurate weighing of tissue samples should be done while ensuring that the bulk tissues samples remain frozen. After accurately weighing the tissue samples, the samples must be allowed to completely thaw, un-aided, at room temperature. Sam ples stored ' refrigerated should also be allowed to equilibrate to room temperature. If at any time, the bulk tissue samples thaw, or appear to be non-homogenous, they should be reprocessed by regnnding with dry ice in a sample processor. .. 9 Quality Control Refer to section 13 for acceptance critena. Each extraction batch (i.e. each day samples are extracted) may include the following: 9.1 Blanks 9.1.1 Matrix Blank At least two blank matrtx controls should be extracted with each batch. If a different matnx is used for the calibration curve than the samples, duplicate matnx blanks should be prepared in the .calibration curve matrix and the sample matrtx (if blank matnx is available). If an internal standard/ surrogate are used, one matnx blank should be with the internal standard/surrogate and one without. . 9.1.2 Method Blank . At least two method controls (e.g. water blank) should be extracted with each batch. This is accomplished by extracting an equal volume of A S T M T y p e I water instead of the sample matrix. ETS-8-049.2 . Page 5 of 13 Determination of Fluorochemicals in Fish Tissue (Fillet or Whole Body) by LC/MS/MS MPI Research Page 177 o f 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 9.2 Quality Control (QC) Samples Prepare triplicate spikes using the control fish tissue that w as used for the calibration standards. At least . 3 levels (levels at approximately 3 x lower limit of quantitation (L L O Q ), mid range, and 80% of the upper limit of quantitation (U L O Q )) should be prepared. T h e total number of Q C samples should be at least 5% of the total number of samples extracted or at least 9 total; w hichever is greater. It is recommended to add additional Q C samples near the L L O Q if multiple analytes are extracted and significantly' different L L O Q s are anticipated. Specifically when a low level spike may not be appropriate for a particular analyte.because it is less than three times the L L O Q . . 9.3 Matrix Spike . At least one aliquot of homogenate may be separately fortified at a known concentration of each.analyte and carried through the procedure to verify recovery. T h e fortification level will be determined on an individual basis depending on the amount of analyte that is suspected in the sample. Th e endogenous level of each analyte in that specific sample will be subtracted from the determined concentration of the fortified sample prior to calculations of recovery. 9.4 Sample Duplicate - At least one sample may be prepared in duplicate. ' 9.5. Surrogates . Isotopically labeled surrogates are required to be spiked into each sample (prior to extraction) if conventional matrix spiking is not performed. 9.6 Internal Standard Internal standards may be used as determined by each project/study. 9.7 Sample Dilution A ny sample with an analyte area greater than that of the highest acceptable standard will need to be diluted and reanalyzed. If samples are diluted into the range of the curve during analyses and enough . sample remains, a post-run dilution validation may be performed to verify sample values. T o perform the dilution validation, one sample will be separated into two representative samples (e.g. two 50 y L aliquots, or other amount as determined b y the analyst and documented in the raw data) then diluted using'two procedures. T h e first procedure consists of diluting the sample with additional blank matrix prior to extraction (fluid adding fluid), while, the second procedure consists of diluting the extract ' with solvent post-extraction. 10 Calibration and Standardization 10.1 Instrument Calibration . Analyze the standard curves prior to each set of samples. Th e standard curve may be plotted by linear regression (y = mx + b) or quadratic fit (y = ax2 + b x + c); weighted 1 /x or unweighted, using suitable software. The calibration curves m ay include but should not be forced through zero. 10.1,1 Method o f Standard Addition If endogenous levels of analyte are present in the matrix used to prepare the matrix-matched calibration curve, the Method of Standard Addition should be used (provided that the level of analyte in the matrix blank is at least twice the level of analyte in the method blanks). T o calculate the endogenous amount of analyte in the matrix, create a calibration curve using the theoretical values. Keep all calibration points that meet the accuracy requirements as per the ETS-8-049.2 . . Page 6 o f 13 Determination o f Fluorochemicals in Fish Tissue (Fillet or Whole Body) ' by LC/MS/MS MPI Research Page 178 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 method and are at least twice the method (not matrix) blanks. Calculate the value of the calibration curve for the x intercept (when y = 0), this should be a negative number. T h e absolute value of this number is the amount of endogenous analyte in the matrix used and this result should be added to the theoretical value for each standard level. Reconstruct the calibration curve using the new values plus adding the matrix blanks (for now-the endogenous value is known) as standards also. Apply the acceptance criteria from section 13.2 as would be used fo ra standard curve not using the Method of Standard Addition. If the endogenous level of analyte in the matrix is higher than the desired L L O Q , a solvent curve may be prepared to estimate the value of analyte In samples (provided that adequate Q C demonstrates that this is feasible). 10.2 Continuing Calibration Verification (CCV) '. Continuing calibration verifications (C C V s ) are analyzed to verify the continued accuracy of the calibration curve. Analyze a mid-range calibration standard (at a minimum) after every tenth sample, not including solvent blanks, with a minimum of one per sample set. Sam ples must be bracketed by passing C C V s . Multiple C C V levels may be used.' .. . ' . ' 10.3 System Suitability Check Five Injections of a mid-range standard are run prior to the calibration curve to verify the system performance. T h is standard may be one of the extracted standards or a standard prepared in. solvent. The peak area and retention time are monitored. ' 10.4 Acetonitrile Blanks Acetonitrile blanks should be run after calibration standards and C C V s . It is also recommended to run a blank after sam ples if the analyst suspects a high concentration of the analyte. If consecutive blanks are run, use the last blank In the series to determine if it passes method performance criteria. 11 Procedures , 11.1 Sam ple Preparation - Initial Fish Processing Once obtained, the initial fish tissue (fillets or w hole bodies) must be processed before extraction. Place the frozen samples in a food processor and hom ogenize with dry ice. Place the sam ples in polyethylene containers and leave open In frozen storage overnight to allow for. carbon dioxide sublimation. O nce the carbon dioxide has sublimated, seal the containers and place the samples in frozen storage until the time of analysis. " Note: While an optimum size distribution .for fish particle size has not been determined, practical. experience suggests that an approximate fish particle size of >5 mm allow for sufficient homogenization in later steps of the method. 11.2 Sam ple Preparation - Homogenization Accurately weigh approximately 1 gram offish tissue into an appropriate container for homogenization. Add acetonitrile up to a homogenate volume of 10 mL| homogenize for 2 minutes o r until a uniform consistency is a chieved .' Larger aliquots of tissue may be used. If, for example. 3 gram s of fish are . weighed, the final homogenate volume before homogenization will be 30 mL. .. . Transfer 2 mL aliquots to 15 mL polypropylene centrifuge tubes. If larger aliquots of homogenate are used, the amount of fish matrix in the homogenate should not exceed two grams to ensure that the HLB S P E cartridge capacity is not exceeded. For example; a 5 mL aliquot of the homogenate would contain 0.5 grams of fish tissue. ' ETS-8-049.2 P a g e 7 o f 13 Determination of Fluorochemicals in Fish Tissue (Fillet or Whole Body) ' by LC/MS/MS MPI Research Page 179 of 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Spike homogenate aliquots with up to 100 pL of an appropriate surrogate or internal standard, vortex mix. . Place homogenate In freezer (approximately -20 C.) for a minimum of 1 hour. Centrifuge homogenized sample-solution at approximately -5 "C for 20 minutes at approximately-3000 ' rpm. Th e sample must be stored at approximately -20 C and re-centrifuged if there are delays prior to ' decanting into 2% H 3PO< (aq) in the next step. Decant each 2-m L (nominal) supernatant Into 40 mL of 2% H 3PO< (aq). Note that the 40-mL volume of 2% H 3 PO< (aq) needs to be scaled for different volumes of homogenate used. For example, a 5 mL aliquot of homogenate would be decanted into 100 mL of 2% H 3PO< (aq). .. Mix by vortexing and transfer onto pre-conditioned H L B S P E cartridges. - 11.3 Solid-Phase Extraction Cartridge Conditioning Pre-condition the S P E cartridge prior to sample extraction by first passing a minimum of a 2 m L aliquot of methanol through the cartridge followed by a minimum of a 2 mL aliquot of A S T M T y p e 1 water. 11.4 Solid-Phase Extraction Pour the 2% H 3 PO< (aq)/acetonitrlle/fish tissue mixture described a bove onto a prepared H L B S P E cartridge. P a ss the liquid through the cartridge, under vacuum as needed. T h e cartridge then sh ou ld 'b e w ashed with a 2 mL aliquot of 5% methanol (aq) followed by 2 mL of 2% formic acid (aq) and suctioned dry. A 2 mL aliquot of 5% N H O H in methanol then should be used to extract the cartridge. '. ' .11.5 S am ple Analysis Reference the equipment procedure or manual that pertains to the specific Instrument as appropriate. Conditions may be adjusted'provided adequate Q C is used. Conditions should be consistent throughout the study/projed as much as possible. 11.5.1 Liquid Chromatograph Parameters O n e of two settings may be used: O ne : Recommended for sulfonates, longer chain acids and other analytes Colum n Tem perature (C): Injection Volum e (pL): Mobile Phase A: Mobile Phase B: G uard Colum n (placed after purge valve) Analytical Column Gradient: 30 5 to 10 2 mmol/L ammonium acetate In w ater Acetonitrile Therm o Fischer Scientific Prism TM R P 2x50 mm, 5 pm particle size Th e rm o Fisch e r Scientific Betasil C is , 2.1 x 100 mm, 5 pm particle size Tim e 0 .0 0 . 1 .0 0 1 1 .0 13.5 14.0 am 90 90 10 10 90 B (%) . 10 10 90 90 1,0 Flow (mL/min) 0.3 0.3 0,3 . 0.3 ' 0.3 ETS-8-049.2 . Page 8 o ft 3 Determination of Fluorochemicals in Fish Tissue (Fillet or Whole Body) by LC/MS/MS MPI Research Page 180 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 17.0 90 10 .0.3 . ' Tw o: Recommended for shorter chain acids (C< - C j): Column Temperature (C): Injection Volum e (p L ): 30 . . 5 to10 ' ' Mobile Phase A: 5 mmol/L ammonium acetate plus 0.01% Acetic a d d in water Mobile Phase B: Methanol ' G uard Colum n (placed after, purge valve) ' Analytical Column Gradient: Th e rm o Fisch e r Scientific P rism TM R P 2x50 mm, 5 pm particle size Therm o Fischer Scientific Prism TM RP 2x50 mm, 5 pm particle size . Tim e 0 .0 0 1 .0 0 3.00 13.5 14.0 19.0 Am 55 55 5.0 5.0 55 55 B (%) 45 45 95 95 45 45 F lo w fmL/min) 0.3 . ' 0.3 0.3 0.3 0.3 0.3 11.5.2 Mass Spectrometer Parameters ' Mode: E le dro sp ra y Negative M RM mode, monitoring the following transitions: Analyte Tra n sition s M onitored PFBA 213>169 PFPeA .' 263 >2 19 PFHA 313 > 269 and 313 > 119 PFHpA ' 363 > 319, 363 > 1 6 9 and 363 > 119 PFOA 413 > 369, 413 > 2 1 9 and 413 > 169 PFNA 463 > 419, 463 > 2 1 9 and 463 > 169 PFDA 513 > 469, 513 > 269 and 513 > 219 PFUnA 563 > 519, 563 > 269 and 563 > 219 PFDoA 613 > 569, 613 > 3 1 9 and 613 > 169 PFBS 299 > 60 and 299 > 99 PFHS 399 > 80 and 399 > 99 PFOS 499 > 80, 499 > 99 and 499 > 130 PFDS 599 > 80, 599 > 99 and 599 > 1 30 FOSA 498 > 78 Monitoring multiple transitions is a desirable option because summing multiple transitions may provide quantitation of isom ers that more closely matches N M R data and may have the added benefit of increased analytical signal. T h e use of one daughter ion is acceptable if method sensitivity requirements are achieved, provided that retention time criteria are met to assure adequate specificity, .. ETS-8-049.2 Page 9 of 13 Determination of Fluorochemicals in Fish Tissue (Fillet or Whole Body) by LC/MS/MS MPI Research Page 181 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 12 Data Analysis and Calculations Calculate the amount of analyte in extract from the calibration curve generated. Calculate the amount of analyte in fish tissue as follows: Sample Concentration ng Final Extract V olum e (mL) = A nalytical R esult------ -------- x m L matrix mL extract Sample-Volume (mL) - Include other dilution factors as appropriate. .- Calculate the percent relative standard deviation (% R S D ) for the system suitability as follows: % R S D = S la n d a rd D e via liO n xlQ 0 A verage '" ly - g * ) 2 Where the standard deviation (s) is: s = and average is: average = -- S ' x n ( n - 1) n" . The relative percent difference should be calculated as follows: A b so lu te V alue (X 2 - X , ) R elative P ercen t D iffere n ce = A verage o f X 2 and X , Calculate the percent recovery for the Q C samples as follows: ,,. Percent recovery A m o u n t o f an a ly te d etected (n g /m L ) , ,, ,, ----------------------------------------- :-------- -- -- - x 10 0 A m o u n t o f an a ly te sp ik ed (n g /m L ) For the matrix spike, the percent recovery is calculated as: A m o u n to f an a ly te d etected (n g /m L )-A m o u n t o f a n a ly te in n o n sp ik e d sam p le (n g /m L ) P ercent reco v ery = xlO O A m o u n t o f an aly te sp ik ed (n g /m L ) 13 M ethod Perform ance 13.1 System S u itability The system suitability Injections must have peak area counts with an R S D 5% and a retention time R S D 2%. If the system suitability falls, the sample set must be reanalyzed. It Is recommended to verily the beginning system suitability passes prior to the start of the analysis. 13.2 Q uantitation . The coefficient of determination (r2) value for the calibration curve must be greater than or equal to 0.990 (or a correlation coefficient (r) of 0.995). Seventy five percent, or a minimum of six standards, when back-calculated (Including U L O Q ) shall fall within 25%, except for th L L O Q , when it shall be 30% of the nominal value. V a lu es falling outside these limits must be discarded. ETS-8-049.2 ' Page IOof 13 Determination of Fluorochemicals In Fish Tissue (Fillet or Whole Body) by LC/MS/MS MPI Research Page 182 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 13.3 Continuing Calibration Verification (CCV) - C C V s should be within 75% -125%. Samples not bracketed b y passing C C V s shall be rerun. 13.4 Sensitivity Th e analyte response of the low level of quantitation (L L O Q ) for each analyte shall be at least two times the average response of that analyte In the method blanks. T h e average response Is used only if the method blank responses have a % R S D of s 30%. If the % R S D Is greater than 30%, the largest method blank response will be used for comparison, l,e,, the response of the L L O Q must be greater than two times the highest method blank response. 13.5 Accuracy 13.5.1 QC Samples . At least six of every nine (i.e. two-thirds) Q C samples shall be within 30% of their respective nominal value. Three of the nine Q C samples may be outside the 30% of their respective nominal value, but not all at the same concentration. If more than three levels of Q C sam ples are prepared, a level that is less than three times the L L O Q may be disregarded provided that there are three levels at higher concentrations that meet the required spiking.levels In section 9.2. 13.5.2 Matrix Spikes Matrix spikes shall be within 30% of their respective nominal value. . 13.5.3 Analytical Method Uncertainty Th e analytical uncertainty should be determined based on historical Q C data to evaluate method accuracy and precision. The method uncertainty should be calculated following ETS-12-012. T h e uncertainty is determined by statistical evaluation of the individual analyte recovery in laboratory matrix spike samples. The standard deviation is calculated for the set of recovery results (in % ). The expanded uncertainty is calculated b y multiplying the standard deviation by a . factor of 2, corresponding to a confidence limit of 95%. A minimum of twenty data points should be used to determine method uncertainty by this method. . If there is insufficient data to determine the uncertainty using the method above, matrix spikes or quality.control samples will be used to determine analytical uncertainties for each analyte. Quality control samples meeting acceptance criteria of 100%30% will be assigned an analytical uncertainty of 100%30% . 13.6 Precision Q C sam ples shall be within 20% relative standard deviation. 13.7 Sam ple Duplicates Th e two sam ples should have a relative percent difference within 20%. If the relative percent difference is not within 20% for these two samples, additional testing m ay be performed to determine which value is a correct representation of the sample concentration as determined by the analyst and documented in the raw data. 14 Pollution Prevention and Waste Managem ent Biological sample waste Is disposed of In infectious biohazard w aste containers. Flammable solvent waste is disposed of in high B T U containers. G la ss pipette waste is disposed of In broken glass containers located in the laboratory. E T S -8 -0 4 9 .2 P a g e 11 o f 13 D e te rm in a tio n o f F lu o ro ch em ica ls in F ish T issu e (F ille t or W h o le B o d y ) by L C /M S /M S MPI Research Page 183 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 15 Records Use the appropriate prep sheet or equivalent to record the pertinent data. Other information should be. documented as appropriate. . 16 References Guidance for Industry: Bioanalytical Method Validation: U .S . Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research (C D E R ), Center for Veterinary Medicine (C V M ); M ay 2001 ETS-8-049.2 Page 12 of 13 Determination of Fluorochemioals in Fish Tissue (Fillet or Whole Body) . by LC/MS/MS . MPI Research Page 184 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 17 Revisions Revision . Number 1. Summary of Changes 1. S ection 6.1 w a s modified to add a hom ogenizer to the list of supplies required. . 2. Section 7.1 w as modified to add sodium hydroxide to the list of reagent required. 3. A note w as added to Section 7.2 indicating the need to use caustic methanol as the solvent for the preparation.of the prim ary stock solutions.. 4. Section 10.3 w as am ended to indicate that only the system suitability at the beginning of the run will be analyzed. 5. A note w a s added to Section 11.1 to indicate a desired particle size after processing of the w hole fish or fish fillet tissues. .. 6. S ection 13.4 w as modified to better describe the com parison of responses for the L L O Q sample relevant to the responses obtained from the method blank samples. ' 1 7. A s p e c ts of control charting of quality control re coveries for determination of method . control and analyte stability were rem oved from Section 13.5.1. 8. S ection 13.5.3 w as. added to discu ss the determ ination of analytical method uncertainties and control charting of matrix spike and quality control recoveries. 9. Individual typos in version 049.0 w ere corrected. 10. Section 8 w as amended to stress the im portance of keeping the bulk sam ples frozen at all times, and to indicate the need to reprocess the bulk sam ples if they appear to be non-hom ogenous. ' 2 1. Section 7.1 w as amended to clarify that the concentrations of acid a n d 'b a se reagent solutions are not corrected for the reagent strength. 2. Section 9.1.2 now states that the volum e of A S T M w ater used for method blanks should be equal to the volume of hom ogenate used for extraction. Previously, a 1 mL aliquot was designated. ' 3. S ection 10.3 w as amended to eliminate re dundancy o f statements. 4. S ection 11.2 w as modified to allow va ryin g volum es o f hom ogenate to be used provided that other ratios of reagents are scaled appropriately. Added requirem ent to store the hom ogenate cold after centrifugation if delays are encountered prior to adding to the 2% H 3P O 4 (aq). 5. Section 11.4 w as modified to eliminate the requirem ent to suction dry the HLB cartridge prior to the rinse steps. 6. Section 11.5.2 w as corrected for one transition for P FD o A . 7. T h e Q C accuracy acceptance criterion in S e c tio n -13.5 w as changed to 30% to better reflect our general assay acceptance criteria. . . 8. T h e Q C precision acceptance criterion in S ection 13.6 w as changed to 20% to better reflect our general a ssa y acceptance criteria. 9. S ection 13.6 w as amended to rem ove sentence redundant with Section 13.7. 10. S ection 13.7 section header w as corrected. .. 11. T h e sam ple duplicate precision acceptance criterion in S ection 13.7 w as changed to 20% to better reflect our general assay acceptance criteria. 12. M inor w ording changes throughout. ETS-8-049.2 Page 13 of 13 Determination of Fluorochemicals in Fish Tissue (Fillet or Whole Body) byLC/MS/MS . MPI Research Page 185 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 CHEI1 EHSR 2 3 6 ' 1B 651 733 1958 ' 1 2 / 2 0 10 7 1 2 : 1 4 N O .2 7 9 0 9 / 1 1 Amendment Number Effective Date: MPI State College Project Number PROTOCOL AMENDMENT ______ 4 , 12/18/07 ' Other Study P0003268 N u m b ers): Page 1 of _ 3 ' 0137.0219 DESCRIPTION OF AMENDED SECTION 1) Protocol Amendment 3, Analytical Method ETS-8-049.2 (V0003970-2), Section 9.1.2 Method Blank - "At least two method controls (e.g. wa'ter blank) should be extracted with each batch." . 2) Protocol Amendment 3, Analytical Method ETS-8-049.2 (V0003970-2), Section 10.3 System Suitability Check 3) Protocol Amendment 3, Analytical Method ETS-8-049.2 (V0003970-2), Section 11.5.1 Liquid Chromatograph Parameters ' 4) Protocol Amendment 3, Analytical Method ETS-8-049.2 (V0003970-2), Section 13.2 Quantitation - "The coefficient of determination (r2) value for the calibration curve must be greater than or equal to 0.990 (or a correlation coefficient of 0.995)." 5) .Protocol Amendment 3, Analytical Method ETS-8-049.2 (V0003970-2), Section 13.4 Sensitivity . 6) Protocol Amendment 3, Analytical Method ETS-8-049.2 (V0003970-2), Section 13.5.2 Matrix Spikes . '. 7) Protocol Amendment 3, Analytical Method ETS-8-049.2 (V0003970-2), Section 13.5.3 Analytical Method Uncertainty '. 8) Protocol Amendment 3. Analytical Method ETS-8-049.2 (V0003970-2), Section 13.6 Precision . AMENDED TO ' 1) At least one method control (e.g. water blank) should be extracted with each batch. 2) System Suitability standards are not a required component of this method. 3) Guard Column (placed after purge valve): Two Thermo Fischer Scientific 10x4 mm, ' 5 pm Hypercarb Drop-In Guard Cartridges in tandem Gradient: Time A (% ) B <%) Flow CmL/minl 0.00 90 10 0.4 1.00 90 10 0.4 . 8.50 25 75 0.3 13.5 25 75 0.3 14.0 90 ' 10 0.3 17.0 90 TO 0.4 25.0 90 10 0.4 LIB R A R Y ID V00O1226-12 a d m in is t r a t iv e f o r m MPI Research Page 186 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 CHEM HSR 2 3 6 1B 651 733 '1958 1 2 /2 0 '0 7 1 2 :1 4 N O .2 7 9 1 0 /1 1 Amendment Number: Effective Date: MPI State College Project Number PROTOCOL AMENDMENT 4 12/18/07 Other Study P0003268 Numbers): Page 2 of 0137.0219 3 4) The coefficient of determination (r*) value for the calibration curve must be greater than or. equal to 0.985 (or a correlation coefficient of 0.9925). ' , 5) The analyte response of the low level of quantitation (LLOQ) for each analyte shall be at least two times the average response of that analyte in the method blank(s). If the response of the lowest standard is more than two times the response of the method blank (or the average of the responses of all method blanks), then the lowest standard is the LLOQ. If the response of lowest standard is less than two times the response of the method blank, then the LLOQ is raised to the next lowest standard that meets the criteria. 6) Matrix spikes shall be within 30% of their respective nominal value. 7) . Matrix spikes will be used to determine analytical uncertainties for each analyte. Quality control samples meeting acceptance criteria of 100% 30% will be assigned an analytical uncertainty of 30%. 8) QC samples shall be within 15% relative standard deviation, RSD. Sample duplicates shall be within 20% relative percent difference, RPD. RSD and RPD results outside of the acceptable range will be flagged in the report, but will not be excluded from reporting. . RATIONALE / 1) As per the sponsor, it is not necessary to extract more than one method control per batch for this project. 2) As with other analytical methods used in this investigation, system suitability cheeks are not required for this project. ' 3) Section 11.5 Sample Analysis states that instrument conditions may be adjusted. These updated conditions have produced the most consistent, accurate results in the past and will likely be the instrument conditions used most often for this method. 4) The correlation coefficient requirements for analyses using method ETS-8-049.2 (V0003970-2) need not be stricter than requirements of other methods in this project. Correlation coefficients of greater than of equal to 0.9925 are sufficient for determining accurate sample results. 5) LLQQ determination requirements for analyses using method ETS-S-049.2 (V0003970-2) shall be consistent with the requirements of other methods in this project. S) Matrix spike requirements for analyses using method ETS-8-049.2 (V000397Q-2) need not be stricter than requirements of other methods in this project. Matrix spike recoveries within 30% of their nominal value are sufficient for determining accurate sample results.____________ _ _ _ L IB R A R Y ID V0001226-12 AD M IN ISTRATIVE FORM MPI Research Page 187 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 CHEM EHSR 2 3 6 1B '651 7 3 3 195 8 1 2 /2 0 '0 7 1 2 :1 5 N O .2 7 9 1 1 /1 1 Amendment Number: Effective Date:' MPI State College Project Number PROTOCOL AMENDMENT .4 1 2 /1 8 /0 7 P0003268 Other Study Numbers): Page 3 o f, 3 .0137.0219 7) Analytical uncertainties for "the samples analyzed using this method shall be determined In a manner consistent with uncertainty determinations of other methods used in this project. 8) As with other analytical methods used for this project, all RSD and RPD data will be reported, and those values exceeding the precision requirements will be flagged. IMPACT ON STUDY 1) No negative impact. 2) No negative impact. 3) No negative impact. 4) No negative impact. 5) No negative impact. 6) No negative impact. 7) No negative impact. B) No negative impact. S tu d i D irector S ig n a tu re ^ r/c ip a l Investigator S ig nature ' D ate Men D a te S lu ^ v DLpctf M a n g ^ m e n t S ig n a tu re MPI State College Managerism Sgiytture I/Um^G i fav S p o n s o r S i g n a t u r aa j(jfpf ef gg u i r e a ) D a te Ixfrtl?- Datea 1 D ate LASM P I State College QAU fn/t/Oate / l2//VtT7 LIB R A R Y IO V000122S-12 AD M IN ISTRATIVE FORM MPI Research Page 188 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Amendment. Number: Effective Date: MPI State College. Project Number PROTOCOL AMENDMENT 4' 12/18/07 Other Study P0003268 Numbers): Page 3 of 0137.0219 3 7) A nalytical uncertainties for the sam ples ana lyzed using this m ethod shall be determ ined in a m anner consistent with uncertainty determ inations of other m ethods u se d in this project. 8) A s w ith other analytical m ethods used for this project, all R S D an d R P D data will be re p o rte d , a n d th o s e v a lu e s e x c e e d in g the p re c isio n re q u ire m e n ts w ill b e fla g g e d ._________ IM P A C T O N S TU D Y 1) N o n e g a tive Impact. 2) N o negative im pact. 3) N o negative Impact. 4 ) N o negative im pact, 5) N o negative impact. 6) N o negative im pact. 7) N o negative Impact. 8) N o negative im pact. S p o n so r S ig nature (if required) D ate LASM P I State C o lle g e Q A U Init./D ate / 12-/r?/ff? LIB R A R Y ID V0001226-12 AD M INISTRATIVE FORM MPI Research Page 189 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Amendment Number: Effective Date: MPI State College Project Number PROTOCOL AMENDMENT 5 02/04/08 Other study ID: MPI Study P0003268 Number: ' Page _ 1 _ of 1 0137.0219 DESCRIPTION OF AMENDED SECTION 1) Analytical Procedure Summary Section - page 9 of 57 of the protocol 2) Protocol Amendment 3, Analytical Method ETS-8-049.2 (V0003970-2), Section 10.1.1 Method of Standard Addition AMENDED TO 1) The primary analytical method for the analysis of fish and dam samples is the V0001783 method; however, in Instances where the fish samples to be analyzed are fish fillets and whole body fish, method ETS-8-049.2 (V0003970-2) is to be used. 2) If the endogenous level of analyte in the matrix used to prepare the calibration curve is higher than the endogenous level of analyte in a study sample, a calibration curve may be prepared using the study sample as the matrix. RATIONALE 1) Fish fillet and whole body fish samples analyzed using method ETS-8-049.2 (V0003970-2) yield more accurate results due to matrix effects noted when using method V0001783 for fish fillet and whole body fish analysis. 2) In order to obtain the requested LOO, the matrix used to create the calibration curve needs to have the lowest level of analyte present when creating the curve for quantification. 1 IMPACT ON STUDY ' : 1) No negative impact. 2) No negative Impact. Date O2josic0 Date Date eilollob Date" I / OdiosiMPI State College QC Init./Date L J J fi? LIBRARY ID V0001225-13 ADMINISTRATIVE FORM MPI Research Page 190 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples 0 2 -U -2 0 0 8 06:48pm F ront-llESTON SOLUTIONS + MPI Study No.: 0137.0219 MPI Project No.: P0003268 T-855 P ,002/002 F-889 Amendment Number Effective Date: MPI State College Project Number PROTOCOL AMENDMENT 5 02/04/08 Other study ID: . MPI Study P0003268 Number Page 1 of 0137.0219 1 DESCRIPTION OF AMENDED SECTION 1) Analytical Procedure Summery Section -- page 9 of 57 of the protccjl 2) Protocol Amendment 3, Analytical Method ETS-8-0492 (V0003970-2), Section 10.1.1 Method of Standard Addition AMENDED TO 1) The primary analytical method for Ihe analysis of fish and dam namples 3 the V0001783 method; however, in Instances where the fish samples to be analyzed are fish fillets and whole body fish, method ETS-8-049.2 (V0003970-2) Is to be used. .. 2) If the endogenous level of analyte in the matrix used to prepare the calibration curve Is higher than the endogenous level of analyte In a study sample, a eallbreticn curve may be prepared using (he study sample as the matrix. . RATIONALE 1) Fish fillet and whole body fish samples analyzed using method ETS-8-049.2 (V0003970-2) yield more accurate results due to matrix effects noted when using method V0001783 for fish fillet and whole body fish analysis. 2) In order to obtain the requested LO Q . Ihe matrix used to create the calibration curve needs to have the lowest level of analyte present when creating the curve for quantification. IMPACT ON STUDY 1) No negative Impact 2 ) No negative Impact I recior Signature Da ` I Ittjog0 Dote Dote S p o n s o r S ignature (if required) MPI State Collage QC tnit/Data L 7 J / Q llo s ls r ' LIBRARY ID V0001225-13 .. ADMINISTRATIVE FORM MPI Research Page 191 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Amendment Number: Effective Date: MPI State College Project Number PROTOCOL AMENDMENT 6 03/11/08 Other study'ID: MPI Study P0003268 Number: . Page 1 of _1 0137.0219 DESCRIPTION OP AMENDED SECTION Protocol Amendment 3, Analytical Method ETS-8-049.2 (V0003970-2), Section 11.4 Solid-Phase Extraction. . AMENDED TO The cartridge then should be washed with a 2 mL aliquot of 5% methanol (aq) (not to be used with whole body tissue samples) followed by 2 mL of 2% formic acid (aq) and suctioned dry. RATIONALE The 5% methanol wash is suspected to wash analytes off the cartridge during the extraction of the whole body tissues. . IMPACT ON STUDY No negative impact, more usable results may be obtained. y Director Signature Date lPIa illi o's.. D a te S tu d y D irecto r M a n a g em e n t Sig nature D ata M P I S tate C o lleg e ^ n a g p m e n t Signature VamL S p o n s o r S ig n a tu ree ^(if r e q u ire d ) D a te D ate' ' ( P P PMPI State College QC Init./Datl 13/13/08 LIBRARY ID V0001226-13 ADMINISTRATIVE FORM MPI Research Page 192 of 197 Interim Report #3 - Analysis of Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 03-ZO-ZOOB IZ z n Ft w -WSION solutions T-895 P 003/003 F-OZS Amendment Number. Effective Date MPI State College Project Number PROTOCOL AMENDMENT 6 0301/08 Other study ID MPI Study P0003268 Number Page _J_ of 0137 0219 1 D ESCR IP TIO N O F AM ENDED S E C T IO N P rotocol Am endm ent 3, Analytical M ethod E T S -8 -0 4 9 2 (V0D0397Q-2), S ection 114 Solid-Phase Extraction AMENDED TO T h e cartridge then should be w ashed with a 2 m L aliquot or 5/o m ethanol (a q ) (not to be used with whole body nssue sam ples) followed by 2 mL of 2% formic acid (aq) and suctioned dry R ATIO N ALE Th e 5% methanol w ash is suspected to w ash analytes off the cartridge during the extraction of the whole body tissues IM PACT O N S TU D Y No negative impact, more usable results m ay be obtained Study Director Sig nature (nopal Inv[eestimgaattor Siignature VIA Study O tre d o r M a n a g em e n t Sig natura PI S ta te C o lleg e M anagem ent Si|g nature mm Dale -Minies. D a ta Dite Sponsor S ignature (if required) D ite M PI State C o lle g e Q C Imi a t i ' ^ g P / z j i i j o L IB R A R Y ID VO Q 01226-13 ADM INISTRATIVE FORM MPI Research Page 193 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 CHEM EHSR 2 3 6 1B 651 733 1958 ' 0 4 /2 1 '0 8 1 0 :2 4 NO.311 0 3 /0 3 Amendment Number Effective Date: ' MPI State College Project Number PROTOCOL AMENDMENT 7 Page _ 1 _ of 04/18/08 Other study ID: MPI Study . P0003268 Number. 0137,0219 1 D ESCR IPTIO N O F AM EN D ED S E C TIO N Protocol Amendment 3, Analytical Melhod ETS-fl-049,2 (V0003970-2), Section 2 Melhod Summery, Section 112 Sample Preparation - Homogenization, Section 11.3 Solid-Phase Extraction Cartridge Conditioning, end Section 11.4 Solid-Phase Extraction. AMENDED TO Section 2 Add clarification to the last sentence In first paragraph: An aliquot.......... diluting into water, and solid-phase extraction using a W aters Oasis HLB solid phase extraction cartridge (with the exception of whole body bass samples which will be extracted using a Waters Sep-Pak Vac 6oc (1g) tC18 solid phase extraction cartridge). Section 11.2 Add clarification: Mix by vortexlng and transfer onto pre-conditioned HLB S P E cartridge, except for whole bass samples which must be transferred onto a pre-conditioned IC18 cartridge. Section 11.3 Addition of paragraph Pre-condition the tC10 S P E cartridge prior to sample extraction by first passing a minimum of 10 mL of methanol through the cartridge followed by a minimum of 10 mL of A S TM Typ e 1 water. Section 11.4 Change wording i Pour t h e ........onto a prepared HLB S P E cartridge (tC l8 cartridge for whole body bass samples). A 2 mL aliquot (for the HLB) or a 5 mL aliquot (for the IC18) of 5% N H jO H m methanol then should be used to extract the S P E cartridge._____________________________________________ RATIO N ALE Whole body bass samples can be extracted with greater accuracy using a |C18 S P E cartridge. IM PACT ON S TU D Y No negative impact, more usable resulls may be obtained. Sturdy D irector Signature incipal liw estigator Signature Osta oM[a-iIo6> D s te Study Director M a n a g em e n t Sig nature Date P I S tate C o l l p M a n a g em e n t SSignature Sponsor Signature ffoQUirod) O ale *jfjo t D a le f M PI State C o lle ge Q C Inlt./Date A l i L IB R A R Y ID V 00 0 12 Z 6-13 ADM INISTRATIVE FORM MPI Research Page 194 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 0 4 -2 1 -2 0 0 ! 01:33pm Fron-WESTON SOLUTIONS P 003/003 F-0T6 Amendment Number: Effective Date: MPI State College Project Number PROTOCOL AMENDMENT 7 04/18/08 Other study ID: MPI Study P0003268 Number: Page _ J _ of 0137.0219 1 D ES CR IP TIO N O F AM EN DED S E C T ION Protocol Amendment 3, Analytical Method ETS-B-049.2 (V0003970-2), Section 2 Melhod Summary, Section 11.2 Semple Preparation - Homogenization, Section 11.3 Solid-Phase Extraction Cartridge Conditioning, and Section 11.4 Solid-Phase Emraclion. AMENDED TO Section 2 Add clarification to the last sentence in first paragraph: An aliquot........diluting Into water, and solid-phase extractor using a W aters O a sis H IB solid phase extraction cartridge (with the exception of whole body bass samples which will be extracted using a W aters Sep-PaK V a c 6cc (1g) tC (8 solid phace extraction cartridge) Section 11.2 Add clarification: Mix by vortexing and transfer onto pre-conditioned HLB S P E cartridge, except for whole bass samples which must be transferred onto'a pre-conditioned IC16 cartridge. Section 11.3 Addition of paragraph Pre-condition the 1C18 S P E cartridge prior lo sample extraction by first passing a minimum of 10 mL of methanol through the cartridge followed by a minimum of 10 mL of A S T M Typ e 1 water. Section 11.4 Change warding Pour the ........onto a prepared HLB S P E cartridge (tC18 cartridge for whole body bass samples). A 2 mL aliquot (for the H LB) or a 5 mL aliquot (for the tC18) of 5% N H O H m methanol then should be used lo extract the S P E cartridoe.______________________________________________ R A TIO N A LE Whole body bass samples can be extracted with orealer accuracy using a IC 1 8 S P E cartridge. IM P A C T O N S T U D Y . No negative impact, more usable resells may be obtained. U k M i ur Director Signature ipal Investigator S ignature Amm D ale1 1 flJb l oft Date Study Director M anagem ent Signature M PI S late Collagp M anagem ent Signature Date 9/zi /pg Date Sponsor Signature (if required) MPI Stale C o lle g e Q C Imt./Date A l S LIBRARY ID V0001226-13 ADM INISTRATIVE FORM MPI Research Page 195 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project No.: P0003268 Amendment Number Effective Date: MPI State College Project Number PROTOCOL AMENDMENT 6 05/05/08 Other study ID: MPI Study P0003268 Number: Page _ J _ of 0137.0219 1 DESCRIPTION OF AMENDED SECTION 1) Analytical Procedure Sum m ary Section - page 9 of 56 of the protocol 2) Protocol Am endm ent 3. Analytical M ethod ETS -8 -0 4 9 .2 (V0003970-2), Section 11.4 Solid Phase Extraction. . AMENDED TO 1) Th e prim ary analytical method for the analysis of fish and clam sam ples is method E T S -8 049.2 (V0003970-2). 2) The cartridge then should be w ashed with a 2 mL aliquot of 5% methanol (a q ) (not to be used with w hole b o d y tissue o r clam sam ples) followed b y 2 mL of 2% form ic acid (aq) and suctioned dry. RATIONALE 1) Fish fillet, w hole b o d y fish, and clam sam ples analyzed using m ethod ETS -8 -0 4 9 .2 (V0003970-2) yield more accurate results due to matrix effects noted when using method V0001783 for fish fillet, w hole b od y fish, and clam analysis. [ 2) The 5% methanol wash is suspected to wash analytes off the cartridge during the extraction of the whole body tissues and dam samples. IMPACT ON STUDY 1) N o negative Impact. 2) N o negative impact. Sponsor Signature (^ req u ired ) D a te tftlpSSb. Date ' -s'/r/og. D ate //8 5-, D ate MPI State College QC Init./Date _ f t / t f k j i r LIBRARY ID V0001226-13 ADMINISTRATIVE FORM MPI Research Page 196 o f 197 Interim Report #3 - Analysis o f Fish and Clam Samples MPI Study No.: 0137.0219 MPI Project N o.: P0003268 Amendment Number: Effective Date: MPI State College Project Number . PROTOCOL AMENDMENT ______8_____ 05/05/08 Other study ID: MPI Study P0003268 Number: Page 1 of 0137.0219 1 DESCRIPTION OF AMENDED SECTION 1) Analytical P rocedure Sum m ary Section - page 9 of 56 of the protocol 2) P rotocol Am endm ent 3, Analytical Method ETS -8-04 9.2 (V0003970-2), Section 11.4 Solid Phase Extraction. AMENDED TO 1) T h e prim ary analytical method for the analysis of fish and clam sam ples is method E T S -8 049.2 (V0003970-2). 2) T h e cartridge then should be w ashed with a 2 mL aliquot of 5% methanol (aq) (not to be used with whole body tissue or clam samples) followed by 2 mL of 2% formic acid (aq) and suctioned dry. ; RATIONALE .1) Fish fillet, w hole body fish, and clam sam ples analyzed using method ETS -8 -0 4 9 .2 (V0003970-2) yield more accurate results due to matrix effects noted w hen using method V0001783 for fish fillet, whole body fish, and clam analysis. 2) Th e 5% methanol w ash is suspected to w ash analytes off the cartridge during the extraction of the w hole body tissues and clam samples. - IMPACT ON STUDY : 1 ) N o negative Impact. . S p o n s o r S ig n a tu re {if re q u ire d ) D ate MPI State College QC Inlt./Date _ LIBRARY ID V0001226-13 ADMINISTRATIVE FORM MPI Research Page 197 of 197