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Project / Study Archive Request Shaded areas for Archivist use only R equestor Name Kent Lindstrom Phone 651 778-5352 Date 09/22/06 Request fo r; 3 Archival Retrieval Report IZIFC LIMS number: E05-0209 Title/Summary: Interim Report #24 - Analysis of Water, Sludge, and Sediment Samples Study Title: Analysis of Perfluorooctanoic Acid (PFOA) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program________________________________________ Executive Summary__________________________________________________________________ Group: 3M Decatur_________________________________________________________ Group Project Number: Exygen Protocol Number P0000760____________________________________ Project Lead/ Study Director: Gary Hohenstein (data management), Mike Santoro (Sponsor), John Flaherty (Principal Investigator), Jaisimha Kesari P.E., DEE (Study Director, Weston Solutions, Inc)_______________________________________________________________ Contract: Exygen Research Contract Project #: P0000760_________________________________________________ Analyte(s): PFOA Test System (Sample Matrix): Water, Sludge, Sediment Project Type 13 Analysis 1 1Degradation 1 1Duct Residue Analysis 1 1Ecotox I~1 Method Validation NA 1 1Phys/Chem Properties 1 1Research/Method Development Regulatory Status: _| Non-GLP | 3 GLP Cther: Initiation Date: 11/05/04 (Protocol Signed by Study Director), Analytical Start Date for Interim Report: 05/09/06 Completion Date: Analytical Termination Date for Interim Report #24: 07/07/06 Signed by Study Director: See Report, Signed by Sponsor: See Report Electronic Media: Data CD: EW P ) FTIR Data Instrument Data None (raw data files archived at Page 1of2 Project / Study Archive Request Archive Date Scan Date Off-Site Storage Date Key Words: Decatur Memorandum of Understanding, MOU, GLP, Protocol P0000760, PFOA, E05-0209, E05-0210, Interim Report #24______________ Comments: Note to Archivist. This is Interim Report #24 for this LIMS project (GLP). There will be multiple Interim reports. Please include the Interim Report number in the title of the PDF. Please send this entire report including the raw data via web to Jai Kesari. Please send a copy of the report without raw data to Michael Santoro and to Gary Hohenstein. Distribution Point Gary Hohenstein (report without raw data) Jai Kesari (report with raw data) Michael Santoro (report without raw data) Date ~ 09/22/06 - 09/22/06 ~ 09/22/06 Page 2 of2 INTERIM REPORT #24 - Analysis of Waten Sludge, and Sediment Samples STUDY TITLE Analysis of Perfluorooctanoic Acid (PFOA) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum 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 September 13, 2006 PERFORMING LABORATORY Exygen Research 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 Protocol Number: P0000760 Exygen Study Number: P0000760 Total Pages: 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number P0000760, entitled "Analysis of Perfluorooctanoic Acid (PFOA) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum 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 Exygen Research. John Flaherty Principal Investigator Exygen Research Weston Solutions, Inc. ^ [tUoC> Date k k - atQ' Exygen Research Page 2 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen Study Number P0000760, entitled, "Analysis of Perfluorooctanoic Acid (PFOA) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program". All reviewed phases1 were inspected for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Exygen Principal Investigator and Management and to the Study Director. Phase 34) Raw Data and Interim Report Review 36) Raw Data and Interim Report Review Date Inspected 6/26-27/06 Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Study Director 07/06/06 07/07/06 07/07/06 09/12/06 09/13/06 09/13/06 09/13/06 'Note: All in-lab inspections 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. Exygen Research Page 3 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 CERTIFICATION OF AUTHENTICITY This interim report, for Exygen Study Number P0000760, is a true and complete representation of the raw data. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Principal Investigator, Exygen: John Flahesoy Vice President Exygen Research Exygen Research Facility Management: Date )3-JzP Date Study Direoior, Weston Solutions, Inc. hJaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: AfaJd a Michael A. Sahtoro Director of Regulatory Affairs Exygen Research Page 4 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 STUDY IDENTIFICATION Analysis of Perfluorooctanoic Acid (PFOA) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program PROTOCOL NUMBER: P0000760 EXYGEN STUDY NUMBER: P0000760 TYPE OF STUDY: Residue SAMPLE MATRIX: Water, Sludge, and Sediment TEST SUBSTANCE: Perfluorooctanoic acid (PFOA) 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 STUDY MONITOR: Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 PERFORMING LABORATORY: Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: 11/05/04 Interim Analytical Start Date: 05/09/06 Interim Analytical Termination Date: 07/06/06 Interim Report Completion Date: 09/13/06 Exygen Research Page 5 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 PROJECT PERSONNEL The Study Director for this project is Jaisimha Kesari at Weston Solutions, Inc. The following personnel from Exygen Research were associated with various phases of this interim portion of the study: Name John Flaherty Karen Risha Chrissy Edwards Mark Ammerman Amy Sheehan Eric Edwards Mindy Cressley Brian McAllister Frances Crespi Sharareh Zolghadr Scott Crain Krista Gallant Brittany Kravets Kim Hall Title Vice President Laboratory Supervisor Technician Sample Custodian Associate Scientist Sample Custodian Technician Sample Custodian Technician Technician Technician Technician Technician Technician Exygen Research Page 6 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 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....................................................................................................10 1.0 SUMMARY................................................................................................................ 11 2.0 OBJECTIVE............................................................................................................... 12 3.0 INTRODUCTION....................................................................................................... 12 4.0 ANALYTICAL TEST SAMPLES..............................................................................12 5.0 REFERENCE MATERIAL........................................................................................ 12 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................13 6.1 Extraction Procedure for Water................................................................................ 14 6.2 Extraction Procedure for Sludge............................................................................... 14 6.3 Extraction Procedure for Sediment........................................................................... 14 6.4 Percent Solids Determination For Sludge and Sediment.......................................... 15 6.5 Preparation of Standards and Fortification Solutions...............................................15 6.6 Chromatography....................................................................................................... 16 6.7 Instrument Sensitivity............................................................................................... 17 6.8 Description of LC/MS/MS Instrument and Operating Conditions...........................17 6.9 Quantitation and Example Calculation..................................................................... 18 7.0 EXPERIMENTAL DESIGN......................................................................................21 8.0 RESULTS...................................................................................................................22 9.0 CONCLUSIONS........................................................................................................ 23 10.0 RETENTION OF DATA AND SAMPLES............................................................ 24 Exygen Research Page 7 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table I. LIST OF TABLES Page Summary of PFOA in Water Samples............................................................ 26 Table II. Summary of PFOA in Re-extracted Water Samples....................................... 29 Table El. Summary of PFOA in Sludge Samples........................................................... 30 Table IV. Summary of PFOA in Sediment Samples....................................................... 31 Table V. Matrix Spike Recovery of PFOA in Water Samples................................. 32 Table VI. Matrix Spike Recovery PFOA in Re-extracted Water Samples................39 Table VH. Matrix Spike Recovery of PFOA in Sludge Samples................................40 Table VIE. Matrix Spike Recovery PFOA in Sediment Samples..................................... 41 Table IX. Surrogate Spike Recovery of 13C PFOA in Water Samples........................... 42 Table X. Surrogate Spike Recovery of 13C PFOA in Re-extracted Water Samples......49 Table XI. Surrogate Spike Recovery of 13C PFOA in Sludge Samples.......................... 50 Table XE. Surrogate Spike Recovery of 13C PFOA in Sediment Samples...................... 51 Table XEI. Total Percent Solids for Sludge and Sediment Samples................................ 52 Exygen Research Page 8 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFOA in Reagent Water................................ 54 Figure 2. Extracted Standards of PFOA in Reagent Water, 25 ng/L and 50 ng/L, Respectively....................................................................................................55 Figure 3. PFOA in Reagent Control, 50 ng/L Fortified Reagent Spike A, and 500 ng/L Fortified Reagent Spike B, Respectively....................................................... 56 Figure 4. Chromatogram Representing a Water Sample Analyzed for PFOA (Exygen ID: C0169336, Data Set: 042806B)................................................ 57 Figure 5. Chromatogram Representing a Sludge Sample Analyzed for PFOA (Exygen ID: C0171106, Data Set: 050906BR)...............................................58 Figure 6. Chromatogram Representing a Sediment Sample Analyzed for PFOA (ExygenID: C0171108, DataSet: 051106A).................................................59 Figure 7. Typical Calibration Curve for 13C PFOA in Reagent W ater......................... 60 Figure 8. Extracted Standards of 13C PFOA in Reagent Water, 25 ng/L and 50 ng/L, Respectively.....................................................................................61 Figure 9. 13C PFOA in Reagent Control, 50 ng/L Fortified Reagent Spike A, and 500 ng/L Fortified Reagent Spike B, Respectively................................. 62 11 Figure 10. Chromatogram Representing a Water Sample Analyzed for C PFOA (Exygen ID: C0169336, Data Set: 042806B).................................................63 Figure 11. Chromatogram Representing a Sludge Sample Analyzed for 13C PFOA (Exygen ID: C0171106, Data Set: 050906B).................................................64 Figure 12. Chromatogram Representing a Sediment Sample Analyzed for C PFOA (Exygen ID: C0171108, Data Set: 051106A)................................................ 65 Exygen Research Page 9 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 LIST OF APPENDICES Page Appendix A Study Protocol P0000760 (Exygen Study No. P0000760) with Analytical Methods and Protocol Amendments....................................... 66 Exygen Research Page 10 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 1.0 SUMMARY Exygen Research extracted and analyzed water, sludge, and sediment samples for the determination of perfluorooctanoic acid (PFOA) according to Exygen Methods V0001780, V0001781, and V0001782, respectively (Appendix A). The limit of quantitation for PFOA in water was 25 ng/L. The limit of quantitation for PFOA in sludge and sediment was 0.2 ng/g (wet weight). Analytical results for the analysis of PFOA in water samples are summarized in Table I. Analytical results for the analysis of PFOA in re-extracted water samples are summarized in Table II. Analytical results for the analysis of PFOA in sludge samples are summarized in Table III. Analytical results for the analysis of PFOA in sediment samples are summarized in Table IV. Quantitative results were obtained for all samples and analytes except for samples from sites DAL-GW-138R-0-060412, DAL-GW-138S-0060412, DAL-SD-OSPO1-0-0000, and DAL-SD-OSP02-0-0000 that are not reported (NR) due to quality control failures. Fortification recoveries for PFOA in the water samples are detailed in Table V. The average percent recovery standard deviation for PFOA in the water samples was 75 22%. Fortification recoveries for PFOA in the re-extracted water samples are detailed in Table VI. The average percent recovery standard deviation for PFOA in the re extracted water samples was 99 16%. Fortification recoveries for PFOA in the sludge samples are detailed in Table VII. The average percent recovery for PFOA in the sludge sample was 69%. A standard deviation was not calculated for PFOA in sludge because only one sample was analyzed. Fortification recoveries for PFOA in the sediment samples are listed as NR in Table VIII. The average percent recovery standard deviation for PFOA in the sediment samples was not calculated due to quality control failures. Fortification recoveries for 13C PFOA in the water samples are detailed in Table IX. The average percent recovery standard deviation for 13C PFOA in the water samples was 75 + 26%. Fortification recoveries for 13C PFOA in the re-extracted water samples are detailed in Table X. The average percent recovery standard deviation for 13C PFOA in the re-extracted water samples was 97 34%. Fortification recoveries for 13C PFOA in the sludge samples are detailed in Table XI. The average percent recovery standard deviation for 13C PFOA in the sludge samples was 49 16%. Fortification recoveries for 13C PFOA in the sediment samples are listed as NR in Table XII. The average percent recovery standard deviation for 13C PFOA in the sediment samples was not calculated due to quality control failures. Percent recoveries have not been reported for samples that contained analyte levels three times or greater than the fortification level. Percent solids for sludge and sediment samples are detailed in Table XIII. Exygen Research Page 11 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 2.0 OBJECTIVE The objective of the analytical part of this study was to determine level of perfluorooctanoic acid (PFOA) in water, sludge, and sediment samples according to Protocol P0000760 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFOA, in water samples using the analytical method entitled, "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS," in sludge using the analytical method entitled, "V0001781: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS," and in sediment using the analytical method entitled, "V0001782: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS." The study was initiated on November 05, 2004, when the study director signed protocol number P0000760. The analytical start date for this interim report was May 9, 2006 and the analytical termination date for this interim report was July 07, 2006. 4.0 ANALYTICAL TEST SAMPLES In total, one hundred and sixty water samples, one sludge sample, and two sediment samples were received from the client. Thirty-eight water samples (Exygen ID: C0169333 - C0169370) were received on wet ice on March 13, 2006 from Tim Frinak at Weston Solutions, Inc. One hundred and twenty-two water samples, one sludge sample, and two sediment samples (Exygen ID: C0170984 - C0171108) were received on wet ice on March 18, 2006 from Tim Frinak at Weston Solutions, Inc. All samples were logged in by Exygen personnel and placed in refrigerated storage. 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 Exygen Research. 5.0 REFERENCE MATERIAL The analytical standard, PFOA, was purchased from Oakwood Products, Inc. and was received at Exygen on March 17, 2005. The surrogate spiking standard, 13C labeled perfluorooctanoic acid (13C PFOA SP0004184), was received at Exygen on April 15, 2004 from the 3M Company. Exygen Research Page 12 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 The available information for the reference materials is listed below. PFOA was stored ambient and 13C PFOA was stored frozen. Compound PFOA 13CPFOA Exygen Inventory No. Lot # SP0005444 203 SP0004184 3507-195 Purity (%) Expiration Date 99 03/31/07 97 03/29/09 The molecular structures of PFOA and 13C PFOA are given on the following pages: PFOA Chemical Name: Perfluorooctanoic acid Molecular Weight: 414 Transitions Monitored: 413 -- 369 (for quantification) and 413 -->219 (for confirmation) Structure: 13C PFOA Chemical Name: 1,2-13C perfluorooctanoic acid Molecular Weight: 416 Transition Monitored: 415 - 370 Structure: FFF FF F FFF FF F OH 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical methods "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS," "V0001781: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS," and "V0001782: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" were used for this study. Exygen Research Page 13 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 6.1 Extraction Procedure for Water A 40 mL aliquot of the water sample was used for the extraction procedure. After fortification of appropriate samples, the samples were loaded onto a Qg SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.2 Extraction Procedure for Sludge Before the samples were weighed for the extraction, they were mixed thoroughly by vigorous shaking the sample container. A 5-gram portion of sludge was weighed into a fifty-milliliter centrifuge tube for the extraction. After fortification of appropriate samples, 5 mL of methanol was added to the samples. The samples were allowed to shake on a wrist action shaker for -15 minutes and were then sonicated in an ultrasonic bath for -15 minutes. The volume was taken to 40 mL with water and the samples were then centrifuged for -10 minutes at -3000 rpm. The supernatant was then loaded onto a Ci8 SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.3 Extraction Procedure for Sediment Before the samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A 5-gram portion of sediment was weighed into a fiftymilliliter centrifuge tube for the extraction. After fortification of appropriate samples, 35 mL of 1% acetic acid in water was added to the samples. The samples were vortexed and allowed to shake on a wrist action shaker for ~60 minutes. The samples were centrifuged for -20 minutes at -3000 rpm. The supernatant was then loaded onto a Qg SPE cartridge conditioned with 10 mL of methanol and 20 mL of water. The eluate was discarded. Twenty milliliters of methanol was added to the sediment samples left in the centrifuge tube. The samples were vortexed and allowed to shake on a wrist action shaker for another 30 minutes. The samples were centrifuged again for ~20 minutes at -3000 rpm. The supernatant was then loaded onto the same Cis SPE cartridge. The eluate was collected into a 500 mL Nalgene Bottle. The column was washed with 4 mL of methanol. The wash was collected in the same bottle as the eluate. Approximately two hundred milliliters of water was added to the bottles. The samples were mixed by shaking and loaded onto another Cis SPE cartridge conditioned with 10 mL of methanol and 20 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a Exygen Research Page 14 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.4 Percent Solids Determination For Sludge and Sediment Percent solids were determined using the procedure indicated in Exygen method V0000427. Approximately 20 grams of sample was weighed into a pan. The weight of the sample plus the pan was recorded. The sample was then dried in an oven overnight at 104 2 C. Then the sample was transferred to a dessicator and allowed to cool for ~15 minutes. Each sample was then weighed again, including the weight of the pan. The percent solid for each sample was then calculated. 6.5 Preparation of Standards and Fortification Solutions A mixed stock standard solution of PFOA and 13C PFOA was prepared at a concentration of 1000 pg/mL by dissolving 100 mg of each of the standards (corrected for purity and salt content) in 100 mL of methanol. From this solution, the following fortification standards were prepared: Cone, of Fort Stock or Fort. Volume Solution (mL) (pg/mL)1 1000 10 100 10 10 10 1.0 10 0.1 10 1of PFOA and 13C PFOA Final Volume (mL) 100 100 100 100 100 Final Cone, of Fortification Std. (pg/mL) 100 10 1.0 0.1 0.01 A stock solution of only 13C PFOA was also prepared at a concentration of 100 pg/mL by dissolving 10 mg the standard (corrected for purity and salt content) in 100 mL of methanol. From this solution, the following fortification standards were prepared: Cone, of Stock or Fort. Solution (pg/mL)1 100 10 1.0 1of 13C PFOA Fort Volume (mL) 10 10 10 Final Volume (mL) 100 100 100 Final Cone, of Fortification Std. (pg/mL) 10 1.0 0.1 Exygen Research Page 15 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 i -j A set of non-extracted calibration standards containing PFOA and C PFOA (for the sediment samples) was prepared in methanol, as specified in Exygen method V0001782. The following concentrations were prepared: Cone, of Fort Fort Solution Volume (ng/mL)1 (mL) 100 1 100 0.5 100 0.2 10 1 51 21 1of PFOA and 13C PFOA Volume of Fortified Sample (mL) 10 10 10 10 10 10 Final Cone, of Calibration Std. (ng/mL) 10.0 5.0 2.0 1.0 0.5 0.2 A set of extracted calibration standards containing PFOA and 13C PFOA (for the sludge and water samples) was prepared in water before extraction, as specified in Exygen methods V0001780 and V0001781. Cone, of Fort Fort Solution Volume (ng/mL)1 (mL) - 10 0.1 10 0.2 10 0.4 100 0.1 100 0.2 100 0.4 1 of PFOA and 13C PFOA Volume of Fortified Sample (mL) 40 40 40 40 40 40 40 Final Cone, of Calibration Std. (ng/L) 0 25 50 100 250 500 1000 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.6 Chromatography Quantification of PFOA and 13C PFOA was accomplished by LC/MS/MS electrospray. The retention time of PFOA and 13C PFOA was ~11.5 min. Peaks above the LOQ were not detected in any of the reagent blank samples corresponding to the analyte retention time. Exygen Research Page 16 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 6.7 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 25 ng/L of PFOA and 13C PFOA for water samples and a concentration of 0.2 ng/mL of PFOA and 13C PFOA for sludge and sediment samples. 6.8 Description of LC/MS/MS Instrument and Operating Conditions Instrument: Interface: Computer: Software: HPLC: API 4000 Biomolecular Mass Analyzer Turbo Ion Spray Liquid Introduction Interface DELL OptiPlex GX400 Windows NT, Analyst 1.4.1 Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: 30 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time (mini 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: ~18min Flow Rate: 0.3 mL/min Ions monitored: %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Analvte PFOA PFOA Confirm Ion ,3C PFOA Mode negative negative negative Transition Monitored 413->369 413 -->219 415 --> 370 Approximate Retention Time (min') -11.5 min. -11.5 min. -11.5 min. Exygen Research Page 17 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 6.9 Quantitation and Example Calculation Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations of standards. The concentration was determined from the equations below. For water and sludge samples: Equation 1 calculated the amount of analyte found in sludge and water (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/L) = (Peak area - intercept) x DF slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. For samples fortified with known amounts of PFOA and 13C PFOA prior to extraction, Equation 2 was used to calculate the percent recovery. Equation 2: Recovery (%) = (analyte found (ng/L) - analyte in control (ng/L)) xl00% amount added (ng/L) Note: For the PFOA recovery calculation, the "control" is the unspiked aliquot of the primary field sample. Equation 3 was used to convert the amount o f sludge PFOA found in ng/L to ng/g (ppb). Equation 3: PFOA found (ppb) = rPFOA found (ng/L) x volume extracted (0.04L)1 sample weight (5 g) Equation 4 was then used to calculate the amount of PFOA found in ppb based on dry weight. Equation 4: PFOA found (ppb) dry weight = PFOA found (ppb) x [100% / total solids(%)] NOTE: Total solids (%) = [wet weight (g) / dry weight (g)] x 100% Exygen Research Page 18 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 An example of a calculation using an actual sample follows: Sludge sample Exygen ID C0171106 Spk D (Set: 050906B), fortified at 50,000 ng/L with PFOA where: peak area = 406453 intercept = 6660 slope = 456 dilution factor = 100 ng/L PFOA added (fort level) = 50,000 ng/L in corresponding sample (ng/L) = 53000 volume extracted (L) = 0.04 sample weight (g) 5 total solids (%) = 22.83 From equation 1: Analyte found (ng/L) = r406453- 66601 x 100 456 = 87700 ng/L From equation 2: % Recovery = (87700 ng/L - 53000 ng/L) x 100% 50000 ng/L = 69% From equation 3: PFOA found (ppb) = 187700 ng/L x 0.04L) 5g = 702 ppb From equation 4: PFOA found (ppb) dry weight = 702 ppb x (100% / 22.83%) = 3070 ppb Note: Numbers may vary slightly due to rounding. For sediment samples: Equation 5 calculated the amount of analyte found in sediment (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Exygen Research Page 19 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Equation 5: Analyte found (ng/mL) = (Peak area - intercept) x DF slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. For samples fortified with known amounts of PFOA and 13C PFOA prior to extraction, Equation 6 was used to calculate the percent recovery. Equation 6: Recovery (%) = (analyte found (ng/mL) - analyte in control (ng/mL)) xl00% amount added (ng/mL) Note: For the PFOA recovery calculation, the "control" is the unspiked aliquot of the primary field sample. Equation 7 was used to convert the amount of sediment PFOA found in ng/mL to ng/g (ppb). Equation 7: PFOA found (ppb) = [PFOA found (ng/mL) x volume extracted (5 mL)1 sample weight (5 g) Equation 8 was then used to calculate the amount of PFOA found in ppb based on dry weight. Equation 8: PFOA found (ppb) dry weight = PFOA found (ppb) x [100% / total solids(%)] NOTE: Total solids (%) = [wet weight (g) / dry weight (g)] x 100% An example of a calculation using an actual sample follows: Sediment sample Exygen ID C0171107 Spk C (Set: 051106A), fortified at 0.5 ng/mL with PFOA where: peak area = 327495 intercept = 1130 slope = 190000 dilution factor =1 ng/mL PFOA added (fort level) = 4.0 ng/mL in corresponding sample (ng/mL) = 0.440 volume extracted (L) =5 sample weight (g) =5 total solids (%) = 58.39 Exygen Research Page 20 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 From equation 5: Analyte found (ng/mL) T327495 - 11301 190000 1.72 ng/mL From equation 6: % Recovery (1.72 ng/mL - 0.440 ng/mL) x 100% 4 ng/mL 32% From equation 7: PFOA found (ppb) = (1.72 ng/mL x 5mL) 5g = 1.72 ppb From equation 8: PFOA found (ppb) dry weight = 1.72 ppb x (100% / 58.39%) = 2.95 ppb Note: Numbers may vary slightly due to rounding. 7.0 EXPERIMENTAL DESIGN 1-5 1 1 C PFOA was used as a surrogate for all the samples except the rinse blanks. C PFOA was added to the sludge and sediment samples and sample replicates in the laboratory after collection. 13C PFOA was added to the water sample collection bottles in the laboratory before being shipped to the field for sampling. For water samples designated as field matrix spikes, PFOA was also added at a known concentration to the bottles in the laboratory before being shipped to the field. The water sample bottles were filled to a 200 mL volumetric fill line in the field except for C0169347 and C0169354, which were filled to 250 mL, and C0171088, which was filled to 230 mL. The water samples and trip blank samples were analyzed in thirteen sets. Each set included one reagent blank and two reagent blanks fortified at known concentrations. The first water set contained two sample sites and one trip blank and its associated spikes. Water sets two through twelve contained three sample sites each. The thirteenth water set contained one sample site and two trip blanks and their associated spikes. For each site, a sample, a field duplicate and two or three matrix field spikes were collected. For each site, a laboratory duplicate of the primary sample was extracted and two laboratory matrix Exygen Research Page 21 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 spikes were also extracted. For the two laboratory matrix spikes, two 40 mL portions of the primary sample collected for the site was poured from the bottle and fortified. Not only was PFOA added in the laboratory prior to extraction, but C PFOA was also added. The additional 13C PFOA was added because the levels of PFOA spiked into the samples were known to exceed the calibration ranges and were not analyzed without dilution; therefore, 13C PFOA levels were adjusted to require the same dilution as the other analytes. The re-extracted water samples were analyzed in three sets. Each set included one reagent blank and two reagent blanks fortified at known concentrations. The first set contained eighteen samples with appropriate spiking levels. The second set contained fourteen samples with appropriate spiking levels. The last set contained six samples and the appropriate spiking levels. For the two laboratory matrix spikes, two 40 mL portions of the primary sample collected for the site was poured from the bottle and fortified. Not only was PFOA added in the laboratory prior to extraction, but 13C PFOA was also added. The additional 13C PFOA was added because the levels of PFOA spiked into the samples were known to exceed the calibration ranges and were not analyzed without dilution; therefore, 13C PFOA levels were adjusted to require the same dilution as the other analytes. The single sludge sample was extracted in one set. The set included one reagent blank and two reagent blanks fortified at known concentrations. For each sample, a laboratory duplicate of the sample and two laboratory matrix spikes were also extracted. The laboratory spikes were fortified with known concentrations of PFOA and 13C PFOA. The two sediment samples were extracted in one set. The set included one reagent blank and two reagent blanks fortified at known concentrations. For each sample, a laboratory duplicate of the sample and two laboratory matrix spikes were also extracted. The laboratory spikes were fortified with known concentrations PFOA and 13C PFOA. 8.0 RESULTS Analytical results for the analysis of PFOA in water samples are summarized in Table I. Analytical results for the analysis of PFOA in re-extracted water samples are summarized in Table II. Analytical results for the analysis of PFOA in sludge samples are summarized in Table III. Analytical results for the analysis of PFOA in sediment samples are summarized in Table IV. Quantitative results were obtained for all samples and analytes except for samples from sites DAL-GW-138R-0-060412, DAL-GW-138S-0060412, DAL-SD-OSPO1-0-0000, and DAL-SD-OSP02-0-0000 that are not reported (NR) due to quality control failures. Accuracies were assessed for each sample by reviewing the individual QC results obtained for each sample site. For the water samples, there were two laboratory and two or three field spike recovery results available for each sample site that were used to assess Exygen Research Page 22 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 the accuracy. In instances of failed laboratory or field spikes, recoveries associated with other spikes were used to assess sample accuracy. For the sludge and sediment samples, there were two laboratory spike recovery results available for each sample site that were used to assess the accuracy. In instances of failed laboratory spike recoveries, the samples were not reported due to the quality control failure. Fortification recoveries for PFOA in the water samples are detailed in Table V. The average percent recovery standard deviation for PFOA in the water samples was 75 22%. Fortification recoveries for PFOA in the re-extracted water samples are detailed in Table VI. The average percent recovery standard deviation for PFOA in the re extracted water samples was 99 + 16%. Fortification recoveries for PFOA in the sludge samples are detailed in Table VII. The average percent recovery for PFOA in the sludge sample was 69%. A standard deviation was not calculated for PFOA in sludge because only one sample was analyzed. Fortification recoveries for PFOA in the sediment samples are listed as NR in Table VIII. The average percent recovery standard deviation for PFOA in the sediment samples was not calculated due to quality control failures. Fortification recoveries for 13C PFOA in the water samples are detailed in Table IX. The average percent recovery standard deviation for 13C PFOA in the water samples was 75 26%. Fortification recoveries for C PFOA in the re-extracted water samples are detailed in Table X. The average percent recovery standard deviation for 13C PFOA in the re-extracted water samples was 97 34%. Fortification recoveries for 13C PFOA in the sludge samples are detailed in Table XI. The average percent recovery standard deviation for C PFOA in the sludge samples was 49 16%. Fortification recoveries for 13C PFOA in the sediment samples are listed as NR in Table XII. The average percent recovery standard deviation for 13C PFOA in the sediment samples was not calculated due to quality control failures. Percent recoveries have not been reported for samples that contained analyte levels three times or greater than the fortification level. Percent solids for sludge and sediment samples are detailed in Table XIII. 9.0 CONCLUSIONS Except as noted above, the ground water, sludge, and sediment samples were successfully extracted and analyzed for PFOA according to analytical methods V0001780, V0001781, and V0001782 respectively. There were no circumstances that may have affected the data quality or integrity. Exygen Research Page 23 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 10.0 RETENTION OF DATA AND SAMPLES All original paper data generated by Exygen Research that pertains to this interim report will be shipped to the study director. This does not include facility-specific raw data such as instrument or temperature logs. Exact copies of all raw data, as well as a signed copy of the interim analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. Exygen Research Page 24 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 TABLES Exygen Research Page 25 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table I. Summary of PFOA in Water Samples Exygen ID C0169333 C0169336 C0169336 Rep C0169337 C0169341 C0169341 Rep C0169342 C0169346 C0169346 Rep C0169347 C0169351 C0169351 Rep C0169352 C0169355 C0169355 Rep C0169356 C0169359 C0169359 Rep C0169360 C0169363 C0169363 Rep C0169364 C0169367 C0169367 Rep C0169368 C0170984 C0170984 Rep C0170985 C0170988 C0170988 Rep C0170989 C0170992 C0170992 Rep C0170993 C0170996 C0170996 Rep C0170997 C0171000 C0171000 Rep C0171001 Client Sample ID DAL-GW-TRIP1-0-060411 DAL-GW-138L-0-060411 DAL-GW-138L-0-060411* DAL-GW-138L-DB-060411 DAL-GW-138R-0-060412 DAL-GW-138R-0-060412* DAL-GW-138R-DB-060412 DAL-GW-138S-0-060412 DAL-GW -138S-0-060412* DAL-GW-138S-DB-060412A DAL-GW-605R-0-060412 DAL-GW-605R-0-060412* DAL-GW-605R-DB-060412 DAL-GW-605L-0-060412 DAL-GW-605L-0-060412* DAL-GW-605L-DB-060412 DAL-GW-603S-0-060412 DAL-GW-603S-0-060412* DAL-GW-603S-DB-060412 DAL-GW-604L-0-060412 DAL-GW-604L-0-060412* DAL-GW-604L-DB-060412 DAL-GW-604S-0-060412 DAL-GW-604S-0-060412* DAL-GW-604S-DB-060412 DAL-GW-601R-0-060413 DAL-GW-601R-0-060413* DAL-GW-601R-DB-060413 DAL-GW-601S-0-060413 DAL-GW-601 S-0-060413* DAL-GW-601 S-DB-060413 DAL-GW-602R-0-060413 DAL-GW-602R-0-060413* DAL-GW-602R-DB-060413 DAL-GW-601 L-0-060413 DAL-GW-601L-0-060413* DAL-GW-601 L-DB-060413 DAL-GW-602S-0-060413 DAL-GW-602S-0-060413* DAL-GW-602S-0-060413 Analyte Found (ppt, ng/L) C8 Acid PFOA Perfluorooctanoic Acid ND 219 239 238 NR NR NR NR NR NR 92.1 106 66.1 ND ND ND 317 321 392 1580 1490 1710 913 937 1070 NR NR NR NR NR NR 584 563 627 NR NR NR 557 523 563 Assessed Accuracy <+/-%) 30 50 50 50 NR NR NR NR NR NR 50 50 50 50 50 50 30 30 30 40 40 40 30 30 30 NR NR NR NR NR NR 30 30 30 NR NR NR 50 50 50 'Laboratory Duplicate ASample was bottle was filled to 250 mL. ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures, see Table II for re-extraction data Exygen Research Page 26 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table I. Summary of PFOA in Water Samples Continued Exygen ID C0171004 C0171004 Rep C0171005 C0171008 C0171008 Rep C0171009 C0171012 C0171012 Rep C0171013 C0171016 C0171016 Rep C0171017 C0171020 C0171020 Rep C0171021 C0171025 C0171025 Rep C0171026 C0171030 C0171030 Rep C0171031 C0171035 C0171035 Rep C0171036 C0171039 C0171039 Rep C0171040 C0171043 C0171043 Rep C0171044 C0171047 C0171047 Rep C0171048 C0171051 C0171051 Rep C0171052 C0171055 C0171055 Rep C0171056 Client Sample ID DAL-GW-602L-0-060413 DAL-GW-602L-0-060413* DAL-GW-602L-DB-060413 DAL-GW-604R-0-060413 DAL-GW-604R-0-060413* DAL-GW-604R-DB-060413 DAL-GW-603R-0-060413 DAL-GW-603R-0-060413* DAL-GW-603R-DB-060413 DAL-GW-603L-0-060413 DAL-GW-603L-0-060413* DAL-GW-603L-DB-060413 DAL-SW-BPP01-0-060413 DAL-SW-BPP01-0-060413* DAL-SW-BPP01-0-060413 DAL-SW-BPP02-0-060413 DAL-SW-BPP02-0-060413* DAL-SW-BPP02-DB-060413 DAL-SW-BPP03-0-060413 DAL-SW-BPP03-0-060413* DAL-SW-BPP03-DB-060413 DAL-SW-BCT01 -0-060412 DAL-SW-BCT01-0-060412* DAL-SW-BCT01 -DB-060412 DAL-SW-BCT02-0-060412 DAL-SW-BCT02-0-060412* DAL-SW-BCT02-DB-060412 DAL-SW-BCT03-0-060412 DAL-SW-BCT03-0-060412* DAL-SW-BCT03-DB-060412 DAL-SW-BC01-0-060412 DAL-SW-BC01 -0-060412* DAL-SW-BC01-DB-060412 DAL-SW-BC02-0-060412 DAL-SW-BC02-0-060412* DAL-SW-BC02-DB-060412 DAL-SW-BC03-0-060414 DAL-SW-BC03-0-060414* DAL-SW-BC03-DB-060414 Analyte Found (ppt, ng/L) C8 Acid PFOA Perfluorooctanoic Acid 2820 2940 3070 Assessed Accuracy (%) 30 30 30 699 40 694 40 737 40 545 50 551 50 574 50 145 30 148 30 145 30 NR NR NR NR NR NR 248000 250000 249000 30 30 30 231000 248000 352000 30 30 30 NR NR NR NR NR NR 132000 126000 125000 24700 24700 28000 40 40 40 50 50 50 103 50 104 50 111 50 126 50 133 50 140 50 2370 2530 2660 30 30 30 ' Laboratory Duplicate ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures, see Table II for re-extraction data Exygen Research Page 27 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table I. Summary of PFOA in Water Samples Continued Exygen ID C0171059 C0171059 Rep C0171060 C0171063 C0171063 Rep C0171064 C0171067 C0171067 Rep C0171068 C0171071 C0171071 Rep C0171072 C0171075 C0171075 Rep C0171076 C0171079 C0171079 Rep C0171080 C0171084 C0171084 Rep C0171085 C0171088 C0171088 Rep C0171089 C0171092 C0171092 Rep C0171093 C0171096 C0171096 Rep C0171097 C0171100 C0171103 Client Sample ID DAL-SW-BC04-0-060414 DAL-SW-BC04-0-060414* DAL-SW-BC04-DB-060414 Analyte Found (ppt, ng/L) C8 Acid PFOA Perfluorooctanoic Acid NR NR NR Assessed Accuracy (%) NR NR NR DAL-SW-BC05-0-060414 DAL-SW-BC05-0-060414* DAL-SW-BC05-DB-060414 NR NR NR NR NR NR DAL-SW-OSP01 -0-060413 DAL-SW-OSP01 -0-060413* DAL-SW-OSP01 -DB-060413 DAL-SW-OSP02-0-060413 DAL-SW-OSP02-0-060413* DAL-SW-OSP02-DB-060413 335 307 313 NR NR NR 40 40 40 NR NR NR DAL-SW-OSP03-0-060413 DAL-SW-OSP03-0-060413* DAL-SW-OSP03-D B-060413 NR NR NR NR NR NR DAL-GW-BPWELL-0-060413 DAL-GW-BPWELL-0-060413* DAL-GW-BPWELL-DB-060413 NR NR NR NR NR NR DPWS-WWI-DCTP01-0-060413 DPWS-WWI-DCTP01-0-060413* DPWS-WWI-DCTP01-DB-060413 4200 4230 4390 50 50 50 DPWS-WWE-DCTP01 -0-060413A DPWS-WWE-DCTP01 -0-060413*A DPWS-WWE-DCTP01 -DB-060413 7030 7180 7020 50 50 50 DAL-WW-EFF01 -0-060413 DAL-WW-EFF01 -0-060413* DAL-WW-EFF01-DB-060413 4650 4460 4790 40 40 40 DAL-LCH-MCLF01-0-060414 DAL-LCH-MCLF01-0-060414* DAL-LCH-MCLF01-DB-060414 NR NR NR NR NR NR DAL-GW-TRIP1 -0-060412 ND 30 DAL-WW-TRIP1 -0-060413 ND 30 'Laboratory Duplicate ASample was bottle was filled to 230 mL. ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures, see Table II for re-extraction data Exygen Research Page 28 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table II. Summary of PFOA in Re-extracted Water Samples Exygen ID C0169341 C0169347 C0170984 C0170989 C0170997 C0171021 C0171035 C0171060 C0171063 C0171071 C0171076 C0171079 C0171097 Client Sample ID DAL-GW-138R-0-060412 DAL-GW-138S-DB-060412 DAL-GW-601R-0-060413 DAL-GW-601S-DB-060413 DAL-GW-601 L-DB-060413 DAL-SW-BPP01-DB-060413 DAL-SW-BCT01-0-060412 DAL-SW-BC04-DB-060414 DAL-SW-BC05-0-060414 DAL-SW-OSP02-0-060413 DAL-SW-OSP03-DB-060413 DAL-GW-BPWELL-0-060413 DAL-LCH-MCLF01-DB-060414 NR = Not reported due to quality control failures. Analyte Found (ppt, ng/L) C8 Acid PFOA Perfluorooctanoic Acid NR NR 107 121 19800 526000 29000 27700 782 2660 2650 641000 48700 Assessed Accuracy (+/-%) NR NR 30 30 30 30 30 30 30 30 30 30 30 Exygen Research Page 29 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table III. Summary of PFOA in Sludge Samples Analyte Found (ppb, ng/g) Dry Weight Assessed Client C8 Acid PFOA Accuracy Exygen ID____________ Sample ID_________________ Perfluorooctanoic Acid_____________ (+/- %) C0171106 C0171106 Rep DPWS-SL-DCTP01-0-0000 DPWS-SL-DCTP01-0-0000* 1860 1890 40 40 'Laboratory Duplicate Exygen Research Page 30 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table IV. Summary of PFOA in Sediment Samples Exygen ID C0171107 C0171107 Rep C0171108 C0171108 Rep Client Sample ID DAL-SD-OSP01-0-0000 DAL-SD-OSP01-0-0000* DAL-SD-OSP02-0-0000 DAL-SD-OSP02-0-0000* Analyte Found (ppb, ng/g) C8 Acid PFOA Perfluorooctanoic Acid NR NR NR NR Assessed Accuracy (+/-%) NR NR NR NR Laboratory Duplicate NR = Not reported due to quality control failures. Exygen Research Page 31 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table V. Matrix Spike Recovery of PFOA in Water Samples S a m p le D e s c rip tio n DAL-GW -TRIP1-LS-060411 (C0169334,1.0 ppb Field Spike) DAL-GW -TRIP1-HS-060411 (C0169335,10.0 ppb Field Spike) DAL-GW -138L-0-060411 (C0169336 Spk C, 10.0 ppb Lab Spike) D A L -G W -138L -0-060411 (C0169336 Spk D, 1000 ppb Lab Spike) DAL-GW -138L-LS-060411 (C0169336,10.0 ppb Field Spike) D A L -G W -13 8L -M S -06 0 4 11 (C0169339,100 ppb Field Spike) D AL-G W -138 L-H S -0 6 0 4 11 (C0169340,1000 ppb Field Spike) Amount Spiked (ng/L) 1000 10000 10000 1000000 10000 100000 1000000 Amt Found in Sam ple (ng/L) ND ND 219 219 219 219 219 C8 Acid PFOA Amount Recovered (ng/L) 1040 10800 7990 896000 5760 38900 190000 Recovery (% ) 104 108 78 90 55 39 19 D AL-G W -138R -0-060412 (C0169341 Spk E, 10 ppb Lab Spike) DAL-GW -138R-0-060412 (C0169341 Spk F, 1000 ppb Lab Spike) D A L -G W -138R -LS -060412 (C0169343,10 ppb Field Spike) D AL-G W -138R -M S-060412 (C0169344,100 ppb Field Spike) DAL-GW -138R-HS-060412 (C0169345,1000 ppb Field Spike) 10000 1000000 10000 100000 1000000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR DAL-GW -138S-0-060412 (C0169346 Spk C, 10 ppb Lab Spike) D A L -G W -138S -0-06Q 412 (C0169346 Spk 0,1000 ppb Lab Spike) D A L -G W -138S -L S -060412 (C0169348,10 ppb Field Spike) D A L -G W -138S -M S -060412 (C0169349,100 ppb Field Spike) D A L -G W -13 8 S -H S -0 6 04 12 (C0169350,1000 ppb Field Spike) 10000 1000000 10000 100000 1000000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR DAL-GW -605R-0-06Q412 (C0169351 Spk E, 0.1 ppb Lab Spike) DAL-G W -605R-0-060412 (C0169351 Spk F, 1.0 ppb Lab Spike) DAL-GW -605R -LS-060412 (C01693S3,0.1 ppb Field Spike) DAL-G W -6 0 5 R -H S -0 6 0 4 12 (C0169354,1.0 ppb Field Spike)* 100 1000 100 800 92.1 92.1 92.1 92.1 175 83 833 74 138 46 566 59 DAL-G W -6 0 5L -0-0 6 0 4 12 (C0169355 Spk G, 0.1 ppb Lab Spike) DAL-G W -6 0 5L -0-0 6 0 4 12 (C0169355 Spk H, 1.0 ppb Lab Spike) DAL-GW -605L-LS-060412 (C0169357,0.1 ppb Field Spike) DAL-G W -605L-HS-060412 (C0169358,1.0 ppb Field Spike) 100 1000 100 1000 ND ND ND ND 79.5 839 56.8 487 80 84 57 49 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated A Sample was bottle was filled to 250 mL instead of 200 mL, causing the spiking level to be modified. NO = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures, see Table VI for re-extraction data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 32 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table V. Matrix Spike Recovery of PFOA in Water Samples Continued Sample Description DAL-G W -603S -0-060412 (C0169359 Spk C, 0.1 ppb Lab Spike) D AL-G W -603S-0-060412 (C0169359 Spk D, 1.0 ppb Lab Spike) DAL-GW -603S-LS-060412 (C0169361, 0.1 ppb Field Spike) DAL-G W -6 0 3 S -H S -0 6 0 4 12 (C0169362,1.0 ppb Field Spike) D AL-G W -604L-0-060412 (C0169363 Spk E, 0.1 ppb Lab Spike) D AL-G W -604L-0-060412 (C0169363 Spk F, 1.0 ppb Lab Spike) DAL-G W -604L-LS-060412 (C0169365, 0.1 ppb Field Spike) DAL-G W -604L-HS-060412 (C0169366,1.0 ppb Field Spike) D A L-G W -604S-0-060412 (C0169367 Spk G, 0.1 ppb Lab Spike) D A L-G W -604S-0-060412 (C0169367 Spk H, 1.0 ppb Lab Spike) D AL-GW -604S-LS-060412 (C0169369,0.1 ppb Field Spike) DAL-G W -604S -H S -060412 (C0169370,1.0 ppb Field Spike) D A L -G W -6 0 1R -0-0 6 0 4 13 (C0170984 Spk C, 0.1 ppb Lab Spike) D A L -G W -6 0 1R -0-0 6 0 4 13 (C0170964 Spk D, 1.0 ppb Lab Spike) D A L -G W -6 0 1R -L S -0 60 4 13 (C01709S6,0.1 ppb Field Spike) DAL-GW -601R-HS-060413 (C0170987.1.0 ppb Field Spike) D A L -G W -601S -0 -06 0 4 13 (C0170988 Spk E, 0.1 ppb Lab Spike) D A L-G W -601S -0-060413 (C0170986 Spk F, 1.0 ppb Lab Spike) D A L -G W -6 0 1S-LS -060413 (C0170990,0.1 ppb Field Spike) DAL-GW-601 S-HS-060413 (C0170991,1.0 ppb Field Spike) DAL-GW -602R-0-060413 (C0170992 Spk G, 0.1 ppb Lab Spike) D A L -G W -602R -0-060413 (C0170992 Spk H, 1.0 ppb Lab Spike) D A L -G W -602R -L S -060413 (C0170994,0.1 ppb Field Spike) D A L -G W -602R -H S -060413 (C0170995,1.0 ppb Field Spike) Amount Spiked (ng/L) 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 Amt Found in Sample (ng/L) C8 Acid PFOA Amount Recovered (ng/L) Recovery (%) 317 317 317 317 1580 1580 1580 1580 913 913 913 913 NR NR NR NR NR NR NR NR 584 584 584 584 418 1130 458 1040 1680 2350 1670 2210 987 1800 1300 2040 NR NR NR NR NR NR NR NR 656 2010 762 1880 81 72 77 * 63 e 89 113 NR NR NR NR NR NR NR NR e 143 . 130 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR - Not reported due to quality control failures, see Table VI for re-extraction data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 33 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table V. Matrix Spike Recovery of PFOA in Water Samples Continued S a m p le D e s c rip tio n D AL-G W -601L-0-060413 (C0170996 Spk C, 0.1 ppb Lab Spike) D A L-G W -601L-0-060413 (C0170996 Spk D, 1.0 ppb Lab Spike) DAL-GW -60 L-LS-060413 (C0170998,0.1 ppb Field Spike) DAL-GW -60 L-H S-060413 (C0170999,1.0 ppb Field Spike) D AL -G W -602S -0-060413 (C0171000 Spk E, 0.1 ppb Lab Spike) D AL -G W -602S -0-060413 (C0171000 Spk F, 1.0 ppb Lab Spike) DAL-GW -602S-LS-060413 (C0171002,0.1 ppb Field Spike) D A L-G W -602S-H S-060413 (CQ171003,1.0 ppb Field Spike) D A L -G W -602L -0-060413 (C0171004 Spk G, 0.1 ppb Lab Spike) D AL-G W -602L -0-060413 (C0171004 Spk H, 1.0 ppb Lab Spike) D A L -G W -602L -LS -060413 (00171006, 0.1 ppb Field Spike) D AL-GW -602L-HS-060413 (C0171007,1.0 ppb Field Spike) D A L -G W -6 0 4R -0 -06 0 4 13 (C0171008 Spk C, 0.1 ppb Lab Spike) DAL-GW -6 0 4 R -0-0 6 0 4 13 (C0171008 Spk D, 1.0 ppb Lab Spike) D A L -G W -604R -L S -060413 (C0171010, 0.1 ppb Field Spike) D A L -G W -6 0 4R -H S -06 0 4 13 (C0171011,1.0 ppb Field Spike) DAL-GW -603R-0-060413 (C0171012 Spk E, 0.1 ppb Lab Spike) DAL-G W -6 0 3 R -0 -0 6 0 4 13 (C0171012 Spk F, 1.0 ppb Lab Spike) DAL-G W -6 0 3R -L S -0 6 0 4 13 (C0171014.0.1 ppb Field Spike) DAL-G W -6 0 3R -H S -06 0 4 13 (C0171015,1.0 ppb Field Spike) D AL-G W -603L-0-060413 (C0171016 Spk G, 0.1 ppb Lab Spike) D AL-G W -603L-0-060413 (00171016 Spk H, 1.0 ppb Lab Spike) DAL-GW -603L-LS-060413 (C0171018,0.1 ppb Field Spike) DAL-G W -6 0 3L -H S -0 6 0 4 13 (C0171019,1.0 ppb Field Spike) Amount Spiked (ng/L) 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 Amt Found in Sam ple (ng/L) C8 Acid PFOA Amount Recovered (ng/L) NR NR NR NR NR NR NR NR 557 697 557 1280 557 573 557 1120 2820 2820 2820 2820 3250 3720 2870 4030 699 780 699 1440 699 789 699 1340 545 623 545 1230 545 682 545 1100 145 249 145 1050 145 214 145 882 Recovery <%) NR NR NR NR * 72 * 56 * 90 . 121 . 74 64 * 69 * 56 104 91 69 74 "Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures, see Table VI for re-extraction data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 34 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table V. Matrix Spike Recovery of PFOA in Water Samples Continued S a m p le D e s c rip tio n D A L-S W -B P P 01-0-060413 (C0171020 Spk C, 1.0 ppb Lab Spike) DAL-SW-BPP01 -0-060413 (C0171020 Spk 0,10 ppb Lab Spike) D A L -S W -B P P 01-LS-060413 (C0171022,1.0 ppb Field Spike) D A L -S W -B P P 01-M S -0 6 0 4 13 (C0171023,10 ppb Field Spike) DAL-S W -B P P 01-H S-060413 (C0171024,100 ppb Field Spike) D AL-SW -BPP02-0-060413 (C0171025 Spk E. 1.0 ppb Lab Spike) DAL-S W -B P P 0 2-0 -0 6 04 13 <00171025 Spk F, 100 ppb Lab Spike) D A L -SW -B PP02-LS-060413 (C0171027,1.0 ppb Field Spike) DAL-SW -BPP02-MS-060413 (C0171028,10 ppb Field Spike) D A L -S W -B P P 02-H S -060413 (CQ171029,100 ppb Field Spike) DAL-SW -BPP03-0-060413 (C0171030 Spk G, 1.0 ppb Lab Spike) DAL-SW -B P P 03-0-060413 (C0171030 Spk H, 100 ppb U b Spike) D A L -S W -B P P 03-LS -060413 (C0171032,1.0 ppb Field Spike) DAL-SW -BPP03-MS-060413 (C0171033,10 ppb Field Spike) D AL -SW -B PP03-H S-060413 (C0171034,100 ppb Field Spike) D AL-SW -BCT01-0-060412 (C017103S Spk C, 1.0 ppb U b Spike) DAL-SW -B C T01-0-060412 (C 0 17 1 03 5 S p k D , 10 p p b L ab S p ike) DAL-SW-BCT01 -LS-060412 (C0171037,1.0 ppb Field Spike) D A L -S W -B C T01-H S -060412 (C0171038,10 ppb Field Spike) DAL-SW -BCT02-0-060412 (C0171039 Spk E, 1.0 ppb U b Spike) DAL-SW -BCT02-0-060412 (C0171039 Spk F, 10 ppb Lab Spike) D AL-S W -B C T02-LS -060412 <00171041,1.0 ppb Field Spike) DAL-SW -BCT02-HS-060412 <00171042,10 ppb Reid Spike) Amount Spiked (ng/L) 1000 100000 1000 10000 100000 1000 100000 1000 10000 100000 1000 100000 1000 10000 100000 1000 10000 1000 10000 1000 10000 1000 10000 Amt Found in Sam ple (ng/L) C8 Acid PFOA Amount Recovered (ng/L) NR NR NR NR NR NR NR NR NR NR 248000 248000 248000 248000 248000 224000 319000 226000 219000 324000 231000 231000 231000 231000 231000 229000 352000 231000 206000 346000 NR NR NR NR NR NR NR NR 132000 132000 132000 132000 144000 138000 119000 138000 Recovery (V.) NR NR NR NR NR * 71 * 76 * 121 . 115 NR NR NR NR * 60 . 60 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures, see Table VI for re-extraction data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 35 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table V. Matrix Spike Recovery of PFOA in Water Samples Continued S a m p le D e s c rip tio n D AL-S W -B C T03-0-060412 (C0171043 Spk G, 1.0 ppb Lab Spike) D AL-S W -B C T03-0-060412 (C0171043 Spk H, 10 ppb Lab Spike) DAL-SW -BCT03-LS-060412 (C0171045,1.0 ppb Field Spike) D A L -S W -B C T 03-H S -060412 (C0171046,10 ppb Field Spike) D A L-SW -B C01-0-060412 (C0171047 Spk C. 1.0 ppb Lab Spike) DAL-S W -B C 01-0-060412 (C0171047 Spk D, 10 ppb Lab Spike) DAL-S W -B C 0 1-L S -0 6 04 12 (C0171049,1.0 ppb Field Spike) D A L-SW -B C 01-H S -06 0 4 12 (C0171050,10 ppb Field Spike) D AL-SW -BC02-0-060412 (C0171051 Spk E, 1.0 ppb Lab Spike) DA L-SW -B C 0 2 -0-0 6 0 4 12 (C0171051 spk F, 10 ppb Lab Spike) DAL-SW -BC02-LS-060412 (C0171053,1.0 ppb Field Spike) D A L-S W -B C 02-H S -O 60412 (C0171054,10 ppb Field Spike) D A L -S W -B C 03-0-060414 (C0171055 Spk G, 1.0 ppb Lab Spike) DAL-S W -B C 03 -0 -06 0 4 14 (C0171055 Spk H, 10 ppb Lab Spike) DAL-S W -B C 03 -L S -0 6 04 14 (C0171057,1.0 ppb Field Spike) DAL-SW -BC03-HS-060414 (C017105S,10 ppb Field Spike) DAL-SW -BC04-0-060414 (C0171059 Spk C. 1.0 ppb U b Spike) D A L -S W -B C 04-0-060414 (C0171059 Spk D, 10 ppb U b Spike) D A L -S W -B C 04-LS -060414 (C0171061,1.0 ppb Field Spike) DAL-S W -B C 04 -H S -06 0 4 14 (C0171062.10 ppb Field Spike) D A L -S W -B C 05-0-060414 (C0171063 Spk E, 1.0 ppb U b Spike) DAL-SW -B C 05-0-060414 (C0171063 Spk F, 10 ppb U b Spike) D A L -S W -B C 05-LS -060414 (C0171065,1.0 ppb Field Spike) D A L -S W -B C 05-H S -060414 (C0171066,10 ppb Field Spike) Amount Spiked (ng/L) 1000 10000 1000 10000 1000 10000 1000 10000 1000 10000 1000 10000 1000 10000 1000 10000 1000 10000 1000 10000 1000 10000 1000 10000 Amt Found in Sam ple (ng/L) C8 Acid PFOA Amount Recovered (ng/L) 24700 24700 24700 24700 103 103 103 103 26000 32100 26500 30400 863 7760 647 4780 126 850 126 8090 126 654 126 5500 2370 2370 2370 2370 NR NR NR NR 3360 9900 3300 9200 NR NR NR NR NR NR NR NR NR NR NR NR Recovery (%) * 74 . 57 76 77 54 47 72 80 53 54 99 75 93 68 NR NR NR NR NR NR NR NR Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures, see Table VI for re-extraction data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 36 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table V. Matrix Spike Recovery of PFOA in Water Samples Continued Sample Description DAL-SW-OSP01 -0-060413 (C0171067 Spk G, 0.1 ppb Lab Spike) DAL-SW-OSP01 -0-060413 (C0171067 Spk H, 1.0 ppb Lab Spike) DAL-SW-OSP01 -LS-060413 (C0171069, 0.1 ppb Field Spike) DAL-SW-OSP01-HS-060413 (C0171070,1.0 ppb Field Spike) D AL-S W -O S P 02-0-060413 (C0171071 Spk C, 1.0 ppb Lab Spike) D AL-S W -O S P 02-0-060413 (C0171071 Spk D, 10 ppb Lab Spike) DAL-SW-OSP02-LS-060413 (C0171073,1.0 ppb Field Spike) D AL-S W -O S P 02-H S -060413 (C0171074,10 ppb Field Spike) DAL-SW-OSP03-0-060413 (C0171075 Spk E, 1.0 ppb Lab Spike) D A L -S W -O S P 0 3 -0-0 6 0 413 (C0171075 Spk F, 10 ppb Lab Spike) D AL-SW -O S P 03-LS -060413 (C0171077,1.0 ppb Field Spike) D A L -S W -O S P 03-H S -060413 (C0171078,10 ppb Field Spike) DAL-GW-BPWELL-0-060413 (C0171079 Spk G, 10 ppb Lab Spike) DAL-GW-BPWELL-0-060413 (C0171079 Spk H, 1000 ppb Lab Spike) DAL-GW-BPWELL-LS-060413 (C0171081,10 ppb Field Spike) DAL-GW-BPWELL-MS-060413 (C 0171M2,100 ppb Field Spike) DAL-GW-BPWELL-HS-060413 (C0171083,1000 ppb Field Spike) DPW S-W W I-DCTP01-0-060413 (C0171M4 Spk C. 10 ppb Lab Spike) DPW S-W W I-DCTP01-0-060413 (C0171084 Spk D, 100 ppb Lab Spike) DPW S-W W I-DCT P01-LS-060413 (C01710M, 10 ppb Field Spike) DPW S-W W I-D C TP 01-HS-060413 (C0171087,100 ppb Field Spike) DPW S-W W E-DCTP01-0-060413 (C01710M Spk E, 10 ppb Lab Spiked DPW S-W W E-DCTP01-0-060413 (C01710M Spk F. 100 ppb Lab Spike)A DPWS-WWE-DCTP01 -LS-060413 (C0171000,10 ppb Field Spike) D P W S -W W E -D C TP 01-H S -060413 (C0171091,100 ppb Reid Spike) Amount Spiked (ng/L) 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 10000 1000000 10000 100000 1000000 10000 100000 10000 100000 10000 100000 10000 100000 Amt Found In Sample (ng/L) 335 335 335 335 C8 Acid PFOA Amount Recovered (nfl/L) 375 1070 402 971 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 4200 4200 4200 4200 NR NR NR NR NR 13100 96500 9220 46700 7030 7030 7030 7030 11700 95100 12600 64300 Recovery (%> 74 64 NR NR NR NR NR NR NR NR NR NR NR NR NR 89 92 50 43 47 88 56 57 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ASample was bottle was filled to 230 mL instead of 200 mL. ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures, see Table VI for re-extraction data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. Exygen Research Page 37 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table V. Matrix Spike Recovery of PFOA in Water Samples Continued Sample Description DAL-W W -E F F 0 1-0 -060413 (C0171092 Spk G, 1.0 ppb Lab Spike) DAL-W W -EFF01-0-060413 (C0171092 S pk H, 10 ppb Lab Spike) D A L -W W -E F F 0 1-LS -0 6 0413 (C0171094,1.0 ppb Field Spike) D A L -W W -E FF 01-H S -060413 (C0171095,10 ppb Field Spike) D A L-LC H -M C LF01-0 -0 6 0 4 14 (C0171096 S pk C, 10 ppb Lab Spike) DAL-LC H-M C LF01-0 -060414 (C0171096 S pk D. 100 ppb Lab Spike) DAL-LCH-MCLF01 -LS-060414 (C0171098,10 ppb Field Spike) DAL-LCH-MCLF01 -HS-060414 (C0171099,100 ppb Field Spike) DAL-GW -TRIP1-LS-060412 (C0171101,1.0 p pb Field Spike) DAL-GW-TRIP1 -HS-060412 (C0171102,10 ppb Field Spike) DAL-W W -TRIP1 -LS-060413 (C0171104,1.0 p pb Field Spike) D A L -W W -T R IP 1-H S -0 6 0 4 13 (C0171105,10 ppb Field Spike) Amount Spiked (ng/L) 1000 10000 1000 10000 10000 100000 10000 100000 1000 10000 1000 10000 Amt Found in Sample (ng/L) C8 Acid PFOA Amount Recovered (ng/L) Recovery (% ) 4650 4650 4650 4650 NR NR NR NR ND ND ND ND 5250 11700 5890 11200 NR NR NR NR 776 7950 857 9500 71 * 66 NR NR NR NR 78 80 86 95 Average: Standard Deviation: 75 22 'Sam ple residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NO - Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures, see Table VI for re-extraction data. N ote: S ince th is sum m ary tab le s h o w s rounded re su lts, re co very values m ay va ry s lig h tly fro m th e va lue s in th e ra w data. Exygen Research Page 38 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table VI. Matrix Spike Recovery PFOA in Re-extracted Water Samples Sample Description DAL-GW-138R-HS-060412 (C0169345,1000 ppb Field Spike) DAL-GW-138S-DB-060412 (C0169347 Spk E, 100 ppb Lab Spike) DAL-GW-138S-MS-060412 (C0169349,100 ppb Field Spike) DAL-GW-601R-LS-060413 (C0170986,0.1 ppb Field Spike) DAL-GW-601 S-DB-060413 (C0170989 Spk G, 0.1 ppb Lab Spike) D A L -G W - 6 0 1S -LS -0 6 0 4 1 3 (C0170990,0.1 ppb Field Spike) DAL-GW-601 L-DB-060413 (C0170997 Spk E, 10 ppb Lab Spike) DAL-SW-BPP01 -DB-060413 (C0171021 Spk E, 1000 ppb Lab Spike) DAL-SW-BCT01 -HS-060412 (C0171038,10 ppb Field Spike) D A L -S W -B C 0 4 -H S -0 6 0 4 14 (C0171062,10 ppb Field Spike) DAL-SW-BC05-LS-060414 (C0171065,1.0 ppb Field Spike) DAL-SW-OSP02-HS-060413 (C0171074,10 ppb Field Spike) DAL-SW-OSP03-DB-060413 (C0171076 Spk G, 1.0 ppb Lab Spike) DAL-GW-BPWELL-HS-060413 (C0171083,1000 ppb Field Spike) DAL-LCH-MCLF01 -HS-060414 (C0171099,100 ppb Field Spike) Amount Spiked (ng/L) 1000000 100000 100000 100 100 100 10000 1000000 10000 10000 1000 1000 1000 1000000 100000 Amt Found in Sample (ng/L) C8 Acid PFOA Amount Recovered (ng/L) NR NR NR 107 121 121 19800 526000 29000 27700 NR NR NR 222 223 220 30100 1790000 37300 39700 782 2660 2650 641000 48700 1520 3610 3620 1540000 132000 Recovery (%) NR NR NR 115 102 99 103 126 83 120 74 95 97 90 83 Average: Standard Deviation: 99 16 'Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NR = Not reported due to quality control failures. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 39 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table VII. Matrix Spike Recovery of PFOA in Sludge Samples Sample Description DPWS-SL-DCTP01-0-0000 (CQ171106 Spk C, 4 ppb Spike) DPWS-SL-DCTP01 -0-0000 (C0171106 Spk D, 400 ppb Spike) Amount Spiked (nfl/fl) 4 400 Amt Found in Sample (nq/q) wet weight C8 Acid PFOA Amount Recovered (ng/g) wet weight 424 439 424 701 Recovery (%) * 69 Average: Standard Deviation: 69 NA 'S a m p le residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cann ot be calculated. Note: Since this sum mary table show s rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 40 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table V ili. Matrix Spike Recovery PFOA in Sediment Samples Sample Description DAL-SD-OSP01-0-0000 (C0171107 Spk C, 4 ppb Lab Spike) DAL-SD-OSP01-0-0000 (C0171107 Spk D, 400 ppb Lab Spike) DAL-SD-OSP02-0-0000 (C0171108 Spk E, 4 ppb Lab Spike) DAL-SD-OSP02-0-0000 (C0171108 s p k F, 400 ppb Lab Spike) Amount Spiked (ng/g) Amt Found in Sample (ng/g) wet weight C8 Add PFOA Amount Recovered (ng/g) wet weight 4 NR 400 NR NR NR 4 NR 400 NR NR NR NR = Not reported due to quality control failures. Recovery (%) NR NR NR NR Exygen Research Page 41 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table IX. Surrogate Spike Recovery of 13C PFOA in Water Samples Exygen ID C0169333 C0169334 C0169335 C0169336 SpkC C0169336 Spk D C0169336 C0169336 Rep C0169337 C0169338 C0169339 C0169340 C0169341 Spk E C0169341 Spk F C0169341 C0169341 Rep C0169342 C0169343 C0169344 C0169345 C0169346 SpkC C0169346 Spk D C0169346 C0169346 Rep C0169347 C0169348 C0169349 C0169350 C0169351 Spk E C0169351 Spk F C0169351 C0169351 Rep C0169352 C0169353 C 0169354 C0169355 Spk G C0169355 Spk H C0169355 C0169355 Rep C0169356 C0169357 C0169358 Sample Description DAL-GW-TRIP1-0-060411 DAL-GW-TRIP1-LS-060411 DAL-GW-TRIP1 -HS-060411 DAL-GW-138L-0-060411 DAL-GW-138L-0-060411 DAL-GW-138L-0-060411 DAL-GW-138L-0-060411 DAL-GW-138L-DB-060411 DAL-GW-138L-LS-060411 DAL-GW-138L-MS-060411 DAL-GW-138L-HS-060411 DAL-GW-138R-0-060412 DAL-GW-138R-0-060412 DAL-GW-138R-0-060412 DAL-GW-138R-0-060412 DAL-GW-138R-DB-060412 DAL-GW-138R-LS-060412 DAL-GW-138R-MS-060412 DAL-GW-138R-HS-060412 DAL-GW-138S-0-060412 DAL-GW-138S-0-060412 DAL-GW-138S-0-060412 DAL-GW-138S-0-060412 DAL-GW-138S-DB-060412A DAL-GW-138S-LS-060412 DAL-GW-138S-MS-060412 DAL-GW-138S-HS-060412 DAL-GW-605R-0-060412 DAL-GW-605R-0-060412 DAL-GW-605R-0-060412 DAL-GW-605R-0-060412 DAL-GW-605R-DB-060412 DAL-GW-605R-LS-060412 D A L-G W -605R -H S -060412A DAL-GW-605L-0-060412 DAL-GW-605L-0-060412 DAL-GW-605L-0-060412 DAL-GW-605L-0-060412 DAL-GW-605L-DB-060412 DAL-GW-605L-LS-060412 DAL-GW-605L-HS-060412 Amount Spiked (ng/L) 13c-pfo a Amount Recovered (ng/L) Recovery (%) 500 1000 10000 546 1070 10700 109 107 107 10500 1000500 500 500 500 10000 100000 1000000 8240 861000 413 450 466 5020 37000 166000 78 86 83 90 93 50 37 17 10500 1000500 500 500 500 10000 100000 1000000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 10500 1000500 500 500 400 10000 100000 1000000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 600 1500 500 500 500 100 800 478 1090 371 401 393 53.8 440 80 73 74 80 79 54 55 600 1500 500 500 500 100 1000 546 1230 403 438 512 54.2 449 91 82 81 88 102 54 45 ASample was bottle was filled to 250 mL, causing the 13C-PFOA spiking level to be modified. NR = Not reported due to quality control failures, see Table X for re-extractlon data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 42 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table IX. Surrogate Spike Recovery of 13C PFOA in Water Samples Continued Exygen ID C0169359 SpkC C0169359 SpkD C0169359 C0169359 Rep C0169360 C0169361 C0169362 C0169363 Spk E C0169363 Spk F C0169363 C0169363 Rep C0169364 C0169365 C0169366 C0169367 Spk G C0169367 Spk H C0169367 C0169367 Rep C0169368 C0169369 C0169370 C0170984 SpkC 00170984 SpkD C0170984 C0170984 Rep C0170985 C0170986 C0170987 C0170988 Spk E C0170988 Spk F C0170988 C0170988 Rep C0170989 C0170990 C0170991 C0170992 SpkG C0170992 Spk H C0170992 C0170992 Rep C0170993 C0170994 C0170995 Sample Description DAL-GW-603S-0-060412 DAL-GW-603S-0-060412 DAL-GW-603S-0-060412 DAL-GW-603S-0-060412 DAL-GW-603S-DB-060412 DAL-GW-603S-LS-060412 DAL-GW-603S-HS-060412 DAL-GW-604L-0-060412 DAL-GW-604L-0-060412 DAL-GW-604L-0-060412 DAL-GW-604L-0-060412 DAL-GW-604L-DB-060412 DAL-GW-604L-LS-060412 DAL-GW-604L-HS-060412 DAL-GW-604S-0-060412 DAL-GW-604S-0-060412 DAL-GW-604S-0-060412 DAL-GW-604S-0-060412 DAL-GW-604S-DB-060412 DAL-GW-604S-LS-060412 DAL-GW-604S-HS-060412 DAL-GW-601R-0-060413 DAL-GW-601R-0-060413 DAL-GW-601 R-0-060413 DAL-GW-601 R-0-060413 DAL-GW-601 R-DB-060413 DAL-GW-601 R-LS-060413 DAL-GW-601 R-HS-060413 DAL-GW-601 S-0-060413 DAL-GW-601 S-0-060413 DAL-GW-601 S-0-060413 DAL-GW-601 S-0-060413 DAL-GW-601 S-DB-060413 DAL-GW-601 S-LS-060413 DAL-GW-601 S-HS-060413 DAL-GW-602R-0-060413 DAL-GW-602R-0-060413 DAL-GW-602R-0-060413 DAL-GW-602R-0-060413 DAL-GW-602R-DB-060413 DAL-GW-602R-LS-060413 DAL-GW-602R-HS-060413 Amount Spiked (ng/L) " C-PFOA Amount Recovered (ng/L) Recovery (%) 600 1500 500 500 500 100 1000 520 1220 395 411 511 72.9 656 87 81 79 82 102 73 66 600 1500 500 500 500 100 1000 547 1350 431 440 446 62.7 536 91 90 86 88 89 63 54 600 1500 500 500 500 100 1000 491 1270 430 420 472 66.2 538 82 85 86 84 94 66 54 600 1500 500 500 500 100 1000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 600 1500 500 500 500 100 1000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 600 1500 500 500 500 100 1000 530 2280 974 948 941 120 1050 88 152 195 190 188 120 105 NR = Not reported due to quality control failures, see Table X for re-extraction data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 43 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table IX. Surrogate Spike Recovery of 13C PFOA in Water Samples Continued Exygen ID C0170996 Spk C C0170996 Spk D C0170996 C0170996 Rep C0170997 C0170998 C0170999 C0171000 Spk E C0171000 Spk F C0171000 C0171000 Rep C0171001 C0171002 C0171003 C0171004 Spk G C0171004 Spk H C0171004 C0171004 Rep C0171005 C0171006 C0171007 C0171008 Spk C C0171008 Spk D C0171008 C0171008 Rep C0171009 C0171010 C0171011 C0171012 Spk E C0171012 Spk F C0171012 C0171012 Rep C0171013 C0171014 C0171015 C0171016 Spk G C0171016 Spk H C0171016 C0171016 Rep C0171017 C0171018 C0171019 Sample Description DAL-GW-601L-0-060413 DAL-GW-601 L-0-060413 DAL-GW-601 L-0-060413 DAL-GW-601 L-0-060413 DAL-GW-601 L-DB-060413 DAL-GW-601 L-LS-060413 DAL-GW-601 L-HS-060413 DAL-GW-602S-0-060413 DAL-GW-602S-0-060413 DAL-GW-602S-0-060413 DAL-GW-602S-0-060413 DAL-GW-602S-DB-060413 DAL-GW-602S-LS-060413 DAL-GW-602S-HS-060413 DAL-GW-602L-0-060413 DAL-GW-602L-0-060413 DAL-GW-602L-0-060413 DAL-GW-602L-0-060413 DAL-GW-602L-DB-060413 DAL-GW-602L-LS-060413 DAL-GW-602L-HS-060413 DAL-GW-604R-0-060413 DAL-GW-604R-0-060413 DAL-GW-604R-0-060413 DAL-GW-604R-0-060413 DAL-GW-604R-DB-060413 DAL-GW-604R-LS-060413 DAL-GW-604R-HS-060413 DAL-GW-603R-0-060413 DAL-GW-603R-0-060413 DAL-GW-603R-0-060413 DAL-GW-603R-0-060413 DAL-GW-603R-DB-060413 DAL-GW-603R-LS-060413 DAL-GW-603R-HS-060413 DAL-GW-603L-0-060413 DAL-GW-603L-0-060413 DAL-GW-603L-0-060413 DAL-GW-603L-0-060413 DAL-GW-603L-DB-060413 DAL-GW-603L-LS-060413 DAL-GW-603L-HS-060413 Amount Spiked (no/L) 1sC-PFOA Amount Recovered (ng/L) Recovery (%) 600 1500 500 500 500 100 1000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 600 1500 500 500 500 100 1000 598 1210 473 449 469 53.9 581 100 81 95 90 94 54 58 600 1500 500 500 500 100 1000 557 1330 423 437 448 67.6 710 93 89 85 87 90 68 71 600 1500 500 500 500 100 1000 508 1160 433 428 448 68.3 575 85 77 87 86 90 68 58 600 1500 500 500 500 100 1000 541 1140 436 433 445 68.6 486 90 76 87 87 89 69 49 600 1500 500 500 500 100 1000 555 1350 434 438 439 76.6 676 93 90 87 88 88 77 68 NR = Not reported due to quality control failures, see Table X for re-extraction data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 44 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table IX. Surrogate Spike Recovery of 13C PFOA in Water Samples Continued Exygen ID C0171020 SpkC C0171020 Spk D C0171020 C0171020 Rep C0171021 C0171022 C0171023 C0171024 C0171025 Spk E C0171025 Spk F C0171025 C0171025 Rep C0171026 C0171027 C0171028 C0171029 C0171030 Spk G C0171030 Spk H C0171030 C0171030 Rep C0171031 C0171032 C0171033 C0171034 C0171035 SpkC C0171035 Spk D C0171035 C0171035 Rep C0171036 C0171037 C0171038 C0171039 Spk E C0171039 Spk F C0171039 C0171039 Rep C0171040 C0171041 C0171042 C0171043 Spk G C0171043 Spk H C0171043 C0171043 Rep C0171044 C0171045 C0171046 Sample Description DAL-SW-BPP01-0-060413 DAL-SW-BPP01 -0-060413 DAL-SW-BPP01 -0-060413 DAL-SW-BPP01 -0-060413 DAL-SW-BPP01 -DB-060413 DAL-SW-BPP01-LS-060413 DAL-SW-BPP01 -MS-060413 DAL-SW-BPP01 -HS-060413 DAL-SW-BPP02-0-060413 DAL-SW-BPP02-0-060413 DAL-SW-BPP02-0-060413 DAL-SW-BPP02-0-060413 DAL-SW-BPP02-DB-060413 DAL-SW-BPP02-LS-060413 DAL-SW-BPP02-MS-060413 DAL-SW-BPP02-HS-060413 DAL-SW-BPP03-0-060413 DAL-SW-BPP03-0-060413 DAL-SW-BPP03-0-060413 DAL-SW-BPP03-0-060413 DAL-SW-BPP03-DB-060413 DAL-SW-BPP03-LS-060413 DAL-SW-BPP03-MS-060413 DAL-SW-BPP03-HS-060413 DAL-SW-BCT01 -0-060412 DAL-SW-BCT01 -0-060412 DAL-SW-BCT01 -0-060412 DAL-SW-BCT01 -0-060412 DAL-SW-BCT01-DB-060412 DAL-SW-BCT01 -LS-060412 DAL-SW-BCT01 -HS-060412 DAL-SW-BCT02-0-060412 DAL-SW-BCT02-0-060412 DAL-SW-BCT02-0-060412 DAL-SW-BCT02-0-060412 DAL-SW-BCT02-DB-060412 DAL-SW-BCT02-LS-060412 DAL-SW-BCT02-HS-060412 DAL-SW-BCT03-0-060412 DAL-SW-BCT03-0-060412 DAL-SW-BCT03-0-060412 DAL-SW-BCT03-0-060412 DAL-SW-BCT03-DB-060412 DAL-SW-BCT03-LS-060412 DAL-SW-BCT03-HS-060412 Amount Spiked (ng/L) "C-PFOA Amount Recovered (ng/L) Recovery <%) 1500 100500 500 500 500 1500 10500 100500 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 1500 100500 500 500 500 1500 10500 100500 770 84400 146 134 134 366 3820 45000 51 84 29 27 27 24 36 45 1500 100500 500 500 500 1500 10500 100500 811 86100 152 144 138 404 4230 59800 54 86 30 29 28 27 40 60 1500 10500 500 500 500 1000 10000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 1500 10500 500 500 500 1000 10000 935 7620 152 147 143 379 4980 62 73 30 29 29 38 50 1500 10500 500 500 500 1000 10000 1110 7610 276 266 267 585 5130 74 72 55 53 53 59 51 NR = Not reported due to quality control failures, see Table X for re-extraction data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 45 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table IX. Surrogate Spike Recovery of 13C PFOA in Water Samples Continued Exygen ID C0171047 SpkC C0171047 SpkD C0171047 C0171047 Rep C0171048 C0171049 C0171050 C0171051 Spk E C0171051 Spk F C0171051 C0171051 Rep C0171052 C0171053 C0171054 C0171055 Spk G C0171055 Spk H C0171055 C0171055 Rep C0171056 C0171057 C0171058 C0171059 SpkC C0171059 SpkD C0171059 C0171059 Rep C0171060 C0171061 C0171062 C0171063 Spk E C0171063 Spk F C0171063 C0171063 Rep C0171064 C0171065 C0171066 C0171067 Spk G C0171067 Spk H C0171067 C0171067 Rep C0171068 C0171069 C0171070 Sample Description DAL-SW-BC01 -0-060412 DAL-SW-BC01-0-060412 DAL-SW-BC01-0-060412 DAL-SW-BC01-0-060412 DAL-SW-BC01 -DB-060412 DAL-SW-BC01 -LS-060412 DAL-SW-BC01 -HS-060412 DAL-SW-BC02-0-060412 DAL-SW-BC02-0-060412 DAL-SW-BC02-0-060412 DAL-SW-BC02-0-060412 DAL-SW-BC02-DB-060412 DAL-SW-BC02-LS-060412 DAL-SW-BC02-HS-060412 DAL-SW-BC03-0-060414 DAL-SW-BC03-0-060414 DAL-SW-BC03-0-060414 DAL-SW-BC03-0-060414 DAL-SW-BC03-DB-060414 DAL-SW-BC03-LS-060414 DAL-SW-BC03-HS-060414 DAL-SW-BC04-0-060414 DAL-SW-BC04-0-060414 DAL-SW-BC04-0-060414 DAL-SW-BC04-0-060414 DAL-SW-BC04-DB-060414 DAL-SW-BC04-LS-060414 DAL-SW-BC04-HS-060414 DAL-SW-BC05-0-060414 DAL-SW-BC05-0-060414 DAL-SW-BC05-0-060414 DAL-SW-BC05-0-060414 DAL-SW-BC05-DB-060414 DAL-SW-BC05-LS-060414 DAL-SW-BC05-HS-060414 DAL-SW-OSP01 -0-060413 DAL-SW-OSP01 -0-060413 DAL-SW-OSP01 -0-060413 DAL-SW-OSP01 -0-060413 DAL-SW-OSP01-DB-060413 DAL-SW-OSP01 -LS-060413 DAL-SW-OSP01-HS-060413 Amount Spiked (ng/L) 1sC-PFOA Amount Recovered (ng/L) Recovery <%) 1500 10500 500 500 500 1000 10000 1200 8240 389 389 429 509 4680 80 78 78 78 86 51 47 1500 10500 500 500 500 1000 10000 1240 8430 421 450 433 499 5300 83 80 84 90 87 50 53 1500 10500 500 500 500 1000 10000 1230 8100 419 420 451 589 6300 82 77 84 84 90 59 63 1500 10500 500 500 500 1000 10000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 1500 10500 500 500 500 1000 10000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 600 1500 500 500 500 100 1000 561 1250 479 477 440 78.3 650 94 83 96 95 88 78 65 NR = Not reported due to quality control failures, see Table X for re-extraction data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 46 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table IX. Surrogate Spike Recovery of 13C PFOA in Water Samples Continued Exygen ID C0171071 SpkC C0171071 Spk D C0171071 C0171071 Rep C0171072 C0171073 C0171074 C0171075 Spk E C0171075 Spk F C0171075 C0171075 Rep C0171076 C0171077 C0171078 C0171079Spk G C0171079 Spk H C0171079 CO171079 Rep C0171080 C0171081 C0171082 C0171083 C0171084 SpkC C0171084 Spk D C0171084 C0171084 Rep C0171085 C0171086 C0171087 C0171088Spk E C0171088 Spk F C0171088 C0171088 Rep C0171089 C0171090 C0171091 C0171092 Spk G C0171092 Spk H C0171092 C0171092 Rep C0171093 C0171094 C0171095 Sample Description DAL-SW-OSP02-0-060413 DAL-SW-OSP02-0-060413 DAL-SW-OSP02-0-060413 DAL-SW-OSP02-0-060413 DAL-SW-OSP02-DB-060413 DAL-SW-OSP02-LS-060413 DAL-SW-OSP02-HS-060413 DAL-SW-OSP03-0-060413 DAL-SW-OSP03-0-060413 DAL-SW-OSP03-0-060413 DAL-SW-OSP03-0-060413 DAL-SW-OSP03-DB-060413 DAL-SW-OSP03-LS-060413 DAL-SW-OSP03-HS-060413 DAL-GW-BPWELL-0-060413 DAL-GW-BPWELL-0-060413 DAL-GW-BPWELL-0-060413 DAL-GW-BPWELL-0-060413 DAL-GW-BPWELL-DB-060413 DAL-GW-BPWELL-LS-060413 DAL-GW-BPWELL-MS-060413 DAL-GW-BPWELL-HS-060413 DPWS-WWI-DCTP01-0-060413 DPWS-WWl-DCTP01-0-060413 DPWS-WWI-DCTP01-0-060413 DPWS-WWI-DCTP01-0-060413 DPWS-WWI-DCTP01-DB-060413 DPWS-WWI-DCTP01-LS-060413 DPWS-WWI-DCTP01-HS-060413 DPWS-WWE-DCTP01 -0-060413A DPWS-WWE-DCTP01 -0-060413A DPWS-WWE-DCTP01 -0-060413A DPWS-WWE-DCTP01 -0-060413A DPWS-WWE-DCTP01-DB-060413 DPWS-WWE-DCTP01-LS-060413 DPWS-WWE-DCTP01-HS-060413 DAL-WW-EFF01-0-060413 DAL-WW-EFF01-0-060413 DAL-WW-EFF01-0-060413 DAL-WW-EFF01-0-060413 DAL-WW-EFF01-DB-060413 DAL-WW-EFF01 -LS-060413 DAL-WW-EFF01-HS-060413 Amount Spiked (ng/L) 13c -pfo a Amount Recovered (ng/L) Recovery (%) 600 1500 500 500 500 100 1000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 600 1500 500 500 500 100 1000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 10500 1000500 500 500 500 10000 100000 1000000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 10500 100500 500 500 500 10000 100000 8680 96200 391 393 426 4600 42500 83 96 78 79 85 46 43 10435 100435 435 435 500 10000 100000 8260 92400 343 350 391 4870 48800 79 92 79 80 78 49 49 1500 10500 500 500 500 1000 10000 1180 7880 360 335 425 744 6690 79 75 72 67 85 74 67 NR = Not reported due to quality control failures, see Table X for re-extraction data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 47 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table IX. Surrogate Spike Recovery of 13C PFOA in Water Samples Continued Exygen ID C0171096 SpkC C0171096 SpkD C0171096 C0171096 Rep C0171097 C0171098 C0171099 C0171100 C0171101 C0171102 C0171103 C0171104 C0171105 Sample Description DAL-LCH-MCLF01 -0-060414 DAL-LCH-MCLF01 -0-060414 DAL-LCH-MCLF01 -0-060414 DAL-LCH-MCLF01-0-060414 DAL-LCH-MCLF01-DB-060414 DAL-LCH-MCLF01 -LS-060414 DAL-LCH-MCLF01-HS-060414 DAL-GW-TRIP1 -0-060412 DAL-GW-TRIP1 -LS-060412 DAL-GW-TRIP1 -HS-060412 DAL-WW-TRIP1 -0-060413 DAL-WW-TRIP1 -LS-060413 DAL-WW-TRIP1 -HS-060413 Amount Spiked (ng/L) " C-PFOA Amount Recovered (ng/L) Recovery (%) 10500 100500 500 500 500 10000 100000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 500 1000 10000 412 748 8150 82 75 82 500 1000 10000 501 808 8130 100 81 81 Average: Standard Deviation: ASample was bottle was filled to 230 mL, causing the 13C-PFOA spiking level to be modified. NR = Not reported due to quality control failures, see Table X for re-extractlon data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. 75 26 Exygen Research Page 48 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table X. Surrogate Spike Recovery of 13C PFOA in Re-extracted Water Samples Exygen ID C0169341 C0169345 C0169347 Spk E C0169347 C0169349 CO170984 C0170986 C0170989 Spk G C0170989 C0170990 C0170997 Spk E C0170997 C0171021 Spk E C0171021 C0171035 C0171038 C0171060 C0171062 C0171063 C0171065 C0171071 C0171074 C0171076 Spk G C0171076 C0171079 C0171083 C0171097 C0171099 Sample Description DAL-GW-138R-0-060412 DAL-GW-138R-HS-060412 DAL-GW-138S-DB-060412 DAL-GW-138S-DB-060412 DAL-GW-138S-MS-060412 DAL-GW-601R-0-060413 DAL-GW-601R-LS-060413 DAL-GW-601 S-DB-060413 DAL-GW-601 S-DB-060413 DAL-GW-601 S-LS-060413 DAL-GW-601 L-DB-060413 DAL-GW-601 L-DB-060413 DAL-SW-BPP01-DB-060413 DAL-SW-BPP01-DB-060413 DAL-SW-BCT01-0-060412 DAL-SW-BCT01 -HS-060412 DAL-SW-BC04-DB-060414 DAL-SW-BC04-HS-060414 DAL-SW-BC05-0-060414 DAL-SW-BC05-LS-060414 DAL-SW-OSP02-0-060413 DAL-SW-OSP02-HS-060413 DAL-SW-OSP03-DB-060413 DAL-SW-OSP03-DB-060413 DAL-GW-BPWELL-0-060413 DAL-GW-BPWELL-HS-060413 DAL-LCH-MCLF01 -DB-060414 DAL-LCH-MCLF01-HS-060414 Amount Spiked (ng/L) 13c -p f o a Amount Recovered (ng/L) Recovery (%) 500 1000000 NR NR NR NR 100500 500 100000 NR NR NR NR NR NR 500 639 128 100 114 114 600 777 130 500 772 154 100 106 106 10500 500 14600 540 139 108 1000500 500 1180000 243 118 49 500 10000 475 6850 95 69 500 10000 432 7270 86 73 500 1000 607 761 121 76 500 1000 1500 500 669 641 1590 630 134 64 106 126 500 1000000 341 230000 68 23 500 100000 398 53600 80 54 Average: Standard Deviation: NR = Not reported due to quality control failures. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. 97 34 Exygen Research Page 49 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table XI. Surrogate Spike Recovery of 13C PFOA in Sludge Samples Exygen ID Sample Description C0171106 C0171106 Rep C0171106 Spk C C0171106 Spk D DPWS-SL-DCTP01 -0-0000 DPWS-SL-DCTP01 -0-0000 DPWS-SL-DCTP01 -0-0000 DPW S-SL-DCTP01 -0-0000 Amount Spiked (ng/g) 4 4 4 400 13c - p f o a Amount Recovered W et Weight (ng/g) 1.82 1.67 1.48 291 Recovery <%) 46 42 37 73 Average: Standard Deviation: 49 16 Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 50 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table XII. Surrogate Spike Recovery of 13C PFOA in Sediment Samples Exygen ID C0171107 C0171107 Rep C0171107 SpkC C0171107 SpkD C0171108 C0171108 Rep C0171108 SpkE C0171108 Spk F Sample Description DAL-SD-OSP01 -0-0000 DAL-SD-OSP01-0-0000 DAL-SD-OSP01-0-0000 DAL-SD-OSP01-0-0000 DAL-SD-OSP02-0-0000 DAL-SD-OSP02-0-0000 DAL-SD-OSP02-0-0000 DAL-SD-OSP02-0-0000 NR = Not reported due to quality control failures. Amount Spiked (ng/g) 1sC-PFOA Amount Recovered (ng/g) Recovery <%) 4 NR 4 NR 4 NR 400 NR NR NR NR NR 4 NR 4 NR 4 NR 400 NR NR NR NR NR Exygen Research Page 51 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Table XIII. Total Percent Solids for Sludge and Sediment Samples Exygen ID C0171106 C0171107 C0171108 S a m p le Description D P W S -S L-D C TP 01-0-0000 DAL-SD-OSP01-0-0000 DAL-SD-OSP02-0-0000 Total Solids (%) 22.83 58.39 53.33 Exygen Research Page 52 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 FIGURES Exygen Research Page 53 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 1. Typical Calibration Curve for PFOA in Reagent Water 0 4 2 8 0 6 B P760-1131 W ater.rdb (PFOA): "Lineai" Regression C'1 / x " w e ig h tin g ): y = 3 .7 7 e+003 x + 0 .0 4 2 ( r = 0 .9 9 8 2 ) Area, counts Exygen Research Page 54 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 2. Extracted Standards of PFOA in Reagent Water, 25 ng/L and 50 ng/L, Respectively I XC05106-1 -PFOA (Standard)41X 0^369.0 amn -sample 2 o f 3* from 042906B .w iff Area: 120797 counts Height: 7.61e+003 cps RT: 11.5 min Tim e, m in XC 051006-2 - PFOA (Standard) 413.0/368.0 amu - sam ple 3 of 38 from 04 2 8 0 6 B.wiff Area: 2 37 159 counts Height: 1.48e+Q04 cps RT: 11.5 min 11.51 Intensity, cps Intensity, cps Exygen Research Page 55 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 3. PFOA in Reagent Control, 50 ng/L Fortified Reagent Spike A, and 500 ng/L Fortified Reagent Spike B, Respectively I Reagent Control - PFOA (Unknown) 473L0/369L0 amu -sample 9 o f 39 from 042906B.wiff freak not found) Tim e, m in I R e a g e n t Spk A - P FO A (0 0 )4 1 3 .0 /3 6 9 .0 a m u - sam ple 10 of 38 from Q42806B.uuiff Area: 209 542 counts Height: 1.49e+ 004 cps R T :1 1 .5 min 11.52 T im e, min R e agent Spk B - P FO A (Q C )4 1 3 .0 /3 6 9 .0 am u - sam ple 11 of 38 from 0 4 2 8 0 6 B.w iff A rea: 1872882 counts Height: 1.35e+005 cps RT: 11.5 min 11.51 Exygen Research Page 56 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 4. Chromatogram Representing a Water Sample Analyzed for PFOA (Exygen ID: C0169336, Data Set: 042806B) Intensity, ops I C0769336 - PFOA {Unknown) 412.0/^69.0 M ir -santpte 20 o f 39 from 042906B.wiff A n a : 924652 count Height: 5L85e+O04cp* RT: 11.5 min 11.48 5.5 e4 - 5.0 e4 - 4.5 e4 4 .0 e 4 - 3.5 e4 3.0 e42.5e4 2.0 4 1 .5 *4 1 .0 *4 5000.0 0 ,0 -1----- ------ 1....... >.....,> "................ 123 4 .| 5 i 0 i 7 i -- i i i-------------r i ' i ............. 1------'------ i----- ------r 8 0 10 11 12 13 1 4 15 16 17 T im e, min Exygen Research Page 57 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 5. Chromatogram Representing a Sludge Sample Analyzed for PFOA (Exygen ID: C0171106, Data Set: 050906BR) I C0171106 PFOA (Unknown) 413.0/369.0 amu -sample 74o f 21 from 05090SBR.wifT Area: 275395 counts Height: Z49e+O04cps RT: 70.7 min 10.73 Intensity, cps Exygen Research Page 58 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 6. Chromatogram Representing a Sediment Sample Analyzed for PFOA (Exygen ID: C0171108, Data Set: 051106A) Intensity, cps I C0171109 - PFOA (Unknown)473.01369.0 amn -sample 21 o f 24 from 051106A .w iff Area: 270507 counts Height: 1.47e+004cps RT: 10.9 min 10.77 1.4*4 1 .3 *4 - 1 .2 e 4 - 1 .1 *4 - 1 .0 *4 - 9000.0 - 8000.0 - 7000.0 - 6000.0 - sooo.o- 4000.0 - 3000.0 - 2000.0 1000.0 i i 9 10 Tim e, min i 11 ,-- - -- 12 13 --r i -- 14 15 16 17 Exygen Research Page 59 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 7. Typical Calibration Curve for 13C PFOA in Reagent Water 0 4 2 8 0 6 B P 760-1131 W ater.rdb (13C PFOA): "L in e a i" Regression C'1 / * ' w e ig h tin g ): y = 3 .8 6 e+003 x + 0 .0 1 6 8 (r = 0.S 994 ) Area, counts Exygen Research Page 60 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 8. Extracted Standards of 13C PFOA in Reagent Water, 25 ng/L and 50 ng/L, Respectively I XC051006-1 - 13CPFOA {Standard)415.0/370.0amn-sampte 2 o f 39 from 042B06B.wiff Area: 102310 counts Height: 8.55e+003cp* RJ: 11.5 min 11.52 T im e, min X C 0 5 1 0 0 6 -2 - 13C P F O A (S ta n d a rd ) 41 5 .0 /3 7 0 .0 a m u - s a m p le 3 of 3 8 from 0 4 2 8 0 6 B.uviff Area: 212002 counts Height: 1.77+004 cps RT: 11.5 m in 11.52 Intensity, cps Intensif/, cps Exygen Research Page 61 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 9. 13C PFOA in Reagent Control, 50 ng/L Fortified Reagent Spike A, and 500 ng/L Fortified Reagent Spike B, Respectively I Reagent Control - 13CPFOA (Unknown) 41S.0/370.0 amn sample 9 o f 3d from 04299B.wiff p e a k not found) Intensity, cps Intensity, cps Area: 2 0 7 0 7 4 counts Height: 1 .7 3 e+004 cps RT: 11.5 min 11.53 A rea: 193 2996 counts H e ight: 1 .56e+ 005 cps RT: 11.5 m in Intensity, cps Exygen Research Page 62 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 10. Chromatogram Representing a Water Sample Analyzed for 13C PFOA (Exygen ID: C0169336, Data Set: 042806B) COf9336 J3C PFOA (Unknown) 415.0^70.0 am a sam ple 20 o f 3# from 042006B.wiff Area: *595574count* Height: J.34e+005cp* RT: 11.5 min 1 1 .4 8 Intensity, cps Exygen Research Page 63 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 11. Chromatogram Representing a Sludge Sample Analyzed for 13C PFOA (Exygen ID: C0171106, Data Set: 050906B) Intensity, cps I C0171106 - 13C PFOA (IbkitowH) 415.0/370*Oama -santpte 16 o f 21 from 050906B.wiff A na: 131520 counts Haight: 1.13*+064cps RT: 10.7 mia 1.10 e4 10.73 1.00 e4 9000.00 8000.00 - 7000.00 - 6000.00 5000.00 - 4000.00 3000.00 - 2000.00 - 1000.00 0.00 -I---------- ----------- ----------- .---------- ----------- ----- ------ .---- ------ 12 3 4 5 6 7 Tim e, min Exygen Research Page 6 4 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Figure 12. Chromatogram Representing a Sediment Sample Analyzed for 13C PFOA (Exygen ID: C0171108, Data Set: 051106A) Intensif/, cps I C0171100 - 13C-PFQA (Unknown) 415.0/370.0 ama -tam p ft 21 o f 24 from 05110A.wiff Area: 306027 counts Height: 1.09e+004cps AT: 10.0 min 1.8 4* 1 .7 e 4 - 10.77 1 .6 e 4 - 1 .5 e 4 - 1 .4 e 4 - 1 .3 e 4 - 1 .2 e 4 - 1 .1 e 4 1.0e4 5 0 0 0 .0 8000.0 - 7000.0 6000.0 - 5 000.0 4 000.0 3 000.0 2 000.0 1 0 0 0 .0 0 .0 * - ......r i i ------ 1 i i i i 12 34 567 g 10 11 12 13 1 4 15 16 17 Tim e, min Exygen Research Page 65 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 APPENDIX A Study Protocol P0000760 (Exygen Study No. P0000760) with Analytical Methods and Protocol Amendments Exygen Research Page 66 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 STUDY PROTOCOL Study Title: Analysis of Perfluorooctanoic Acid (PFOA) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0000760 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 Sponsor Representative: Michael A. Santoro Director o f Regulatory Affairs 3M Building 02 3 6 -0 1-B -10 St. Paul, MN 55144 Phone: (651)733-6374 Page l of 65 Exygen Research Page 67 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 of 65 Page 68 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 PROTOCOL APPROVAL Study Title: Analysis o f Perfluorooctanoic Acid (PFOA) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0000760 APPROVALS Jaisimha Kesari, Study Director Weston Solutions Michael A. Santoro, Sponsor Representative 3M Company M. Flaherty, Principal Investigator Exygen Research ^ / R i c h a r d A. Gfazzini,president, Facility Management Exygen Resea LydS Shaffer, T echnip^lead, Quality Assurance Unit gen Research mi 0L ' Date >Af/SY Date 'TAX)1-/' Date Date Page 3 of 65 Exygen Research Page 69 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 TABLE OF CONTENTS TITLE PAGE.............................................................................................................................................. 1 DISTRIBUTION.........................................................................................................................................2 PROTOCOL APPROVAL.......................................................................................................................... 3 TABLE OF CONTENTS............................................................................................................................ 4 INTRODUCTION....................................................................................................................................... 5 TESTMATERIAL...................................................................................................................................... 5 SURROGATE FIELD SPIKE COMPOUND............................................................................................... 5 OBJECTIVE...............................................................................................................................................6 TESTING FACILITY................................................................................................................................. 6 STUDY DIRECTOR................................................................................................................................... 6 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...................................................................................9 METHOD FOR CONTROL OF BIAS.........................................................................................................11 STATISTICAL METHODS........................................................................................................................ 11 GLP STATEMENT.....................................................................................................................................11 REPORT..................................................................................................................................................... 11 SAFETY AND HEALTH............................................................................................................................ 12 AMENDMENTS TO PROTOCOL..............................................................................................................13 DATA RECORD KEEPING....................................................................................................................... 13 QUALITY ASSURANCE........................................................................................................................... 14 RETENTION OF DATA AND ARCHIVING..............................................................................................14 APPENDIX I, ANALYTICAL METHODS................................................................................................. 15 Page 4 of 65 Exygen Research Page 70 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P000760 INTRODUCTION The purpose o f this study is to perform analysis for perfluorooctanoic acid (PFOA) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum 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 Exygen Research. TEST MATERIAL The test material is perfluorooctanoic acid (PFOA) and was purchased from Sigma-Aldrich. PFOA Chemical Name: Perfluorooctanoic acid Molecular Weight: 414 Lot Number: 23116HB Purity: 97.64% Transitions Monitored: 413 -> 369 (for quantification) and 413 -> 219 (for confirmation) Structure: F FFFF SURROGATE FIELD SPIKE COMPOUND The surrogate field spiking compound is 13C labeled perfluorooctanoic acid (13C PFOA) and was purchased by 3M from Perkin-Elmer Life and Analytical Sciences. ,3C PFOA Chemical Name: 1.2-13C perfluorooctanoic acid Molecular Weight: 416 Lot Number: 3507-195 Purity: 97% Transition Monitored: 415 - 370 Page 5 of 65 Exygen Research Page 71 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 OBJECTIVE The purpose o f this study is to perform analysis for perfluorooctanoic acid (PFOA) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions o f the following Exygen analytical methods: V0001780: "Method of 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" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 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 Page 6 of 65 Exygen Research Page 72 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone:(651)733-6374 PRINCIPAL INVESTIGATOR John M. Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 john.flaherty@exygen.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion o f this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and termination dates will be included in 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 Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing Page 7 of 65 Exygen Research Page 73 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 details for the control samples will be included in the final report associated with this study. The test systems were chosen to access the environmental impact o f PFOA in the Decatur, Alabama area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples will be received at Exygen directly from Weston Solutions. The details o f sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types o f samples collected will vary depending availability in the field. The total number o f samples received and analyzed for each matrix will be documented in the final report associated with this study. Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at 2C-8C. Fish, clam, vegetation and small mammal liver samples will be processed according to the appropriate analytical method (see Appendix I). These samples will be stored frozen at < -10C. Small mammal whole blood samples will be centrifuged in the field at the time o f collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at < -10C. The receipt and processing of the samples will be documented in the final 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. Sample storage conditions and locations will be documented throughout the study. Page 8 of 65 Exygen Research Page 74 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No. : P0000760 Exygen Protocol Number: P0000760 ANALYTICAL PROCEDURE SUMMARY References: V0001780: "Method of 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 of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" The above methods use analytical conditions capable o f separating the isomers o f PFOA. The final report will include the isomers summed into total PFOA found. 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, Exygen 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 containing 100 ng o f l3C PFOA (equivalent to 500 ng/L in the final sample). Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike o f each sample will be collected. The low and high field spikes will contain PFOA 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 z*" * will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFOA at a low Page 9 oj 65 Exygen Research Page 75 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFOA and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one 500 raL 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 both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. 13C-PFOA will also be added to each sample in the laboratory prior to extraction at the level specified below. For small mammal liver, Exygen will supply a 50 mL polypropylene centrifuge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with PFOA at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. l3C-PFOA will also be added to each sample in the laboratory prior to extraction at the level specified below. Low and high spiking levels for each matrix are defined below; M atrix Low PFOA High PFOA ,3c -p f o a Spiking Level Spiking Level Spiking Level Water 500ng/L 5000 ng/L 500 ng/L Soil 4 ng/g 40 ng/g 40 ng/g Sediment 4ng/g 40ng/g 40 ng/g Fish 10ng/g 100 ng/g 100 ng/g Clams 10ng/g 100 ng/g 100 ng/g Vegetation 10ng/g 100 ng/g 100 ng/g Small Mammal Liver 10ng/g 100 ng/g 100 ng/g Small Mammal Serum 10ng/g 100 ng/mL 100 ng/mL 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 of accuracy will be included in the final report. Prior to sample analysis, a laboratory control spike study will be conducted in water to justify the use o f 13C PFOA results to establish precision and accuracy o f PFOA samples. The following samples are to be prepared: Page 10 of 65 Exygen Research Page 76 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Two control water blanks Two control water samples fortified at 250 ng/L with 13C PFOA Two control water samples fortified at 250 ng/L with 13C PFOA and at 250 ng/L with PFOA. Two control water samples fortified at 5000 ng/L with I3C PFOA and at 5000 ng/L with PFOA. The above samples are to be extracted and analyzed according to method V0001780 entitled "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS." METHOD FOR CONTROL OF BIAS Control o f 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 o f fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation of 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. REPORT A final report will be prepared by the principal investigator or their designee at the conclusion o f the study. The report will include, but will not be limited to, the following: Page II of 65 Exygen Research Page 77 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 The name and address o f the Study Director, Sponsor Representative, and o f 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 Exygen Research Quality Assurance Unit regarding dates o f study inspections and dates findings were reported to the Study Director and Management. A description of the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy o f 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. Description o f 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 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. SAFETY AND HEALTH Laboratory personnel will practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environment to the test or reference substance(s). Page 12 of 65 Exygen Research Page 78 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 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 o f the written amendment will be the date o f the documented change. Copies of 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 o f 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. 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). Page 13 of 65 Exygen Research Page 79 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P000076 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 Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings o f audits to the Study Director, Exygen Management, 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 Exygen Research. Exygen will obtain permission from the study director before discarding or returning samples. Exygen Research Page 14 of 65 Page 80 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 APPENDIX I ANALYTICAL METHODS 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 of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" Exygen Research Page 15 of 65 Page 81 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD Method Number: V 0001780 M ethod o f A nalysis for the Determ ination o f Perfluorooctanolc A d d (PFO A ) in W ater by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Pau) Connolly 1 Technical Leader, LC-MS, Exygen Research lO llV gif Date 'P ? /ih c f 'John Flaherty / Vice Prsidait, Operations, Exygen Research M i Date Exygen Research Total Pages: 7 Page 16 of 65 Page 82 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number VOOO1780 I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Water by L C /M S/M S 1.0 Scope This method is to be em ployed for the isolation and quantitation o f perfluorooctanoic acid by H igh Performance Liquid Chromatography coupled to a tandem Mass Spectromtrie Detector (LC/MS/MS) in water. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 40 mL o f test sample for extraction. 3.2 N o sample processing is needed for water samples. 3.3 Sam ples stored refrigerated should b e allow ed to equilibrate to room temperature. 3.4 All samples must be thoroughly m ixed before being sampled for extraction. 3.5 A n y samples containing particles should be centrifuged at -3 0 0 0 rpm for - 5 minutes and the supernatant used for the extraction. 3.6 Sample collection procedures w ill be specified in the sam pling plan for this project. 4 .0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volum e injector capable o f injecting $-200 pL connected to a tandem Mass Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 IS mL disposable polypropylene centrifuge tubes. 5.6 D isposable micropipets (50-10QuL, 100*200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 p L and 10-100 p L ), w ith disposable tips. 5.11 Waters Sep Pak V a c 6 c c ( lg )t C 1 8 SPE cartridges. Pige 2 u f1 Page 17 of 65 Exygen Research Page 83 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Resesrch Method Number V0O0I780 [_____________________________ a n a l y t i c a l m e t h o d _____________________________ Method o f Analyst for the Determination o f Perfluorooctanoic A cid (PFO A ) in Water 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 Chromatographic System 6.1 Analytical Column: Fluophase RP (K eystone Scien tific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 m M Am m onium Acetate in Water 6.4 Mobile Phase (B ) : Methanol 6.5 Gradient Program: Tim e (min) 0.0 1.0 8.0 20.0 22.5 `A A 65 65 25 25 65 AS 35 35 75 75 35 Flow Rate (m U raia) 0.3 0.3 0.3 0.3 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. T he above conditions are intended as a guide and m ay b e changed in order to optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative M RM m ode, m onitoring 413 -+ 369 m/z The above conditions are intended as a guide and may be changed in order io optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o f water. Alternate volum es may be prepared. Exygen Research Page 18 of 65 Page 84 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number VOOO1780 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Water 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 p g/m L o f PFO A b y w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125*raL LDPE bottle. 9.1.2 A 10 pg/m L fortification solution o f PFO A is prepared by bringing 10 mL o f the 100 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFO A is prepared by bringing l(> mL o f tiie 10 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0 .1 pg/m L fortification solution o f PFO A is prepared by bringing 10 mL o f the 1.0 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL 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 maxim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/M S calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The follow ing is a typical exam ple: additional concentrations may be prepared as needed. Final Concentration Fortificati! Volume o f Concentration o f Calibration o f Fortification Solution (nob) 0 Volume ivU 0 Fortified Control Sample (mL) 40 Calibration Standard (ppt)* 0 Standard ID (example) XCtranddyy-0 10 100 40 25 XCmmddyy-1 10 200 40 50 XCmmddyy*2 10 400 40 100 XCmmddyyO 100 100 40 250 XCmmddyv-4 100 200 40 500 XCmxnddyy-5 100 400 40 1000 XCmmddw-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). X C " extracted calibration standard. Pa*e 4 01"' Page 19 of 65 Exygen Research Page 85 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0O0I780 | ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Water by L C /M S/M S 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) must be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-m L polypropylene tubes at 2C to 6*C, up to two weeks. Alternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least on e reagent control (method blank using HPLC water) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 11.2 11.3 11.4 1 1.5 Measure 40 mL o f sample or a portion o f sample diluted to 40 mL with water into 5 0 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Condition the C n SPE cartridges (1 g , 6 m L) by p assing 10 mL methanol follow ed b y 5 mL o f HPLC water (~ 2 drop/sec). D o not let colum n run dry Load sample on conditioned C ji SPE cartridge. Discard eluate. Elute with - 5 mL 10054 methanol. C ollect 5 mL o f eluate into graduated 15 m L polypropylene centrifiige tubes (final volum e - 5 mL). Analyze samples using electrospray LC/MS/M S. 12.0 Chromatography 12.1 Inject the sam e amount o f each standard, sam ple and fortified sample into the LC/M S/M S system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical se t 12.3 An entire set o f extracted calibration standards m ust be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed betw een every 5*10 samples. A s an alternative, an entire set o f extracted calibration standards m ay be injected at the beginning o f a set follow ed b y extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the first and last injection in a sam ple set. 12.4 U se linear standard curves for quantitstion. Linear standard curves are generated for the analyte by linear regression using 1/x w eighting o f peak area 5 of 7 Page 20 of 65 Exygen Research Page 86 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number VQ001780 [_____________________________ A N A L Y T IC A L M E T H O D _____________________________ Method o f Analysis for the Determination o f Periluorooctanoic A cid (PFOA) in Water 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 n y sam ples that exceed standard responses should b e further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram must show a peak o f a daughter ion at 36 9 amu from a parent o f 413 amu. T he 413 amu parent corresponds to the PFO A anion, w hile the daughter ion (369 amu) represents the loss o f carbon dioxide. Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L , then a new blank sample must b e obtained and the entire set m ust b e re-extracted. Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike falls outside the acceptable lim its, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated b y the analyst to determine 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. H owever, the total number o f extracted calibration standards that could be excluded m ust not exceed 20% o f the total number o f extracted standards injected. The correlation coefficient (R ) for calibration curves generated must be 0.992 (R* 0.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f sam ples should b e reanalyzed. Retention tim es between standards and sam ples must not drift more than 4 % within an analytical run. I f retention tim e drift exceed s this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the amount o f PFO A found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated b y the M ass Lynx software program: PFOA found (ng/L) (Peak area - intercept) x DF slope D F factor b y which the final volum e w as diluted, i f necessary. Pige 6 of 7 Page 21 of 65 Exygen Research Page 87 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Rcacaich Method Number V0001780 | ANALYTICAL M ETHOD ~] Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A) in Water by L C /M S/M S 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. R ecovery (%) * [ total analyte found (ng/L) - analyte found in control (ng/L)] analyte added (ng/L) Exygen Research Page 7 o f 7 Page 22 of 65 Page 88 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD Method Number: V0001781 M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Soil by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C_nL Paul Connolly * Technical Leader, LC-MS, Exygen Research Date Date Exygen Research Total Pages: 7 Page 23 of 65 Page 89 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygeo Reaesrch Method Number VOOO1781 ____________________________ A N A L Y T IC A L M E T H O D ____________________________ Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Soil by LC /M S/M S 1.0 Scope This method is to be em ployed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in soil. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult the appropriate M SD S before handling any chem ical 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 sample processing is needed for soil samples. 3.3 Sam ples stored refrigerated should be allow ed to equilibrate to room temperature. 3.4 All samples must be thoroughly m ixed before being sampled for extraction. 3.5 Sample collection procedures w ill be specified in the sam pling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 M ethanol-H PL C grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-AIdrich 5.0 Instrument 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 performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volum e injector capable o f injecting 5*200 pL connected to a tandem M ass Spectrometer (L C /M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 mL disposable polypropylene centrifuge tubes. 15 mL disposable polypropylene centrifuge tubes. Disposable micropipets (50-100uL, 100-200uL). 125-mL LDPE narrow-mouth bottles. 2 mL clear HPLC vial kit D isposable pipettes. Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. Waters Sep Pak Vac 6 c c ( l g ) tC 18 SPE cartridges. SPE vacuum manifold. Ultrasonic bath. Page 2 o f 7 Page 24 o f 65 Exygen Research Page 90 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 E xygen Protocol Num ber: P 0000760 Exygen Research Method Number V0001781 A N A L Y T IC A L M E T H O D ____________________________ Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Soil by L C /M S/M S 5.14 Wrist-action shaker. 5.15 CentriAige capable o f spinning SO mL polypropylene tubes at 5 0 0 0 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (K eystone S cientific), 2.1 mm x SO mm, Sn (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 M obile Phase (A ) : 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 2L 65 65 25 25 65 AS. 35 35 75 75 35 Flow Rate fm L /m inl 0.3 0.3 0.3 0.3 0.3 6.6 Injection Volume: 1S p L (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTime: - 2 3 minutes. The above conditions are intended as a guide and m ay b e changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray N egative MRM m ode, monitoring 41 3 -* 369 m/z for PFOA. T he above conditions are intended u a guide and m ay b e changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volum es m ay b e prepared. Page 3 o f 7 Page 25 of 65 Exygen Research Page 91 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0Q0I78I A N A L Y T IC A L M E T H O D ____________________________ Method o f Analysis for the Determination ofPerfluorooctanoic 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 p g/m L o f PFO A b y w eigh ing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/m L fortification solution o f PFO A is prepared b y bringing 10 mL o f the 100 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL 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 ft h e 10 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/m L fortification solution o f PFO A is prepared b y bringing 10 mL o f (he 1.0 pg/raL solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/raL solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.22 LC/M S/M S calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Concentration ofFoctification Solution (ppb) 0 10 10 10 100 100 100 Fortification Volume o f Volume Fortified Control (PL) Sample (mL) 0 40 100 40 200 40 400 40 100 40 200 40 400 40 Final Concentration o f Calibration Standard (p p O* 0 25 50 100 250 500 1000 Calibration Standard ID (example) XCmmddyy-0 XCmmddyy-1 XCmmddyy-2 XCmmddyy-3 XCmmddyy-4 XCmmddyy-5 XCmmddw-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. Pge 4 o f 7 Page 26 of 65 Exygen Research Page 92 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ExygraRMtarch Method Number VOOOI78I | ANALYTICAL METHOD | Method 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 9.2.5 A zero standard solution (reagent blank) m ust be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-m L polypropylene tubes at 2C to 6C, up to two weeks. Alternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least on e reagent control (m ethod blank using 5 raL o f methanol) and tw o reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality aaaurance plan for this project. 11.0 S im ple Extraction 11.1 11.2 1 1.3 11.4 11.5 11.6 11.7 11.8 11.9 W eigh 5 g o f sample into SO m L polypropylene centrifuge tubes (fortify as needed, replace lid and m ix well). Add 5 m L o f methanol and shake on a wrist action shaker fo r - 1 S minutes. Transfer the tubes to an ultrasonic bath and sonicate for - 1 5 minutes. Bring the volum e up to 4 0 m L with water in the 50 mL polypropylene centrifuge tube. Centrifuge fo r ~ 1 0 minutes at -3 0 0 0 ipm. Condition the C u SPE cartridges (1 g, 6 m L) b y passing 10 mL methanol follow ed b y 5 m L o f HPLC water (~ 2 drop/sec). D o not let colum n run dry Load (decant) the sample on the conditioned C u SPE cartridge. Discard eluate. Elute with - 5 mL 100% methanol. C ollect 5 mL o f eluate into graduated 1S mL polypropylene centrifuge tubes (final volum e - S mL). Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the sam e amount o f each standard, sam ple and fortified sample into the LC/M S/M S system . A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFO A corresponding to at least fiv e or more concentration levels must b e included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sam ple set. Extracted standards must be interspersed between every 5*10 samples. A s an alternative, an entire set o f Pge 5 o f 7 Page 27 of 65 Exygen Research Page 93 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygea Research Method Number V 000P81 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A) in S oil by L C /M S/M S 12.4 12.5 extracted calibration standards m ay be injected at the beginning o f a set followed b y extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the first and last injection in a sam ple set. U se linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression usin g 1/x w eighting o f peak area versus calibration standard concentration using M assL ynx 3.3 (or equivalent) software system. Sam ple response should not exceed standard responses. A ny samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram must show a peak o f a daughter ion at 3 6 9 amu 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. Method blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFO A at levels greater than SO ng/L, then a n ew blank sample must be obtained and the entire set must be re-extracted. Recoveries o f control spikes and matrix spikes must be between 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. A ny matrix spike outside 70 130% should b e evaluated b y the analyst to determine i f re-extraction is warranted. A n y calibration standard found to be a statistical outlier b y using the Huge Error T est, m ay be occluded from the calculation o f the calibration curve H owever, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. T he correlation coefficient (R ) for calibration curves generated must be 0.992 (R3 0.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f sam ples should be reanalyzed. Retention tim es betw een standards and sam ples m ust not drift more than 4 % within an analytical run. I f retention tim e drift exceed s this lim it within an analytical run then the set must b e reanalyzed. Page 6 of? Page 28 o f 65 Exygen Research Page 94 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0Q01781 ANALYTICAL M ETHOD Method o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Soil by LC /M S/M S 14.0 Calculations 14.1 U se the follow ing equation to calculate the amount o f PFO A found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated b y the M ass Lynx software program: PFOA found (ng/L) - /Peak area - intercept) x DF slope D F * factor by which the final volum e w as diluted, i f necessary. 14.2 For samples fortified with known amounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. R ecovery (%) [total analyte found (ng/L) analyte found in control (ng/L)] tQQ analyte added (ng/L) 14.3 U se the follow ing equation to convert the amount o f PFOA found in ng/L to ng/g (ppb). PFO A found (ppb) =fPFOA found fn u /D x volum e extracted (0.04LH sample weight (S g) 14.4 U se the follow ing equation to calculate the amount o f PFO A found in ppb based on dry weight. PFOA found (ppb) dry weight * PFO A found (ppb) x [ 100% / total solids(%)] Psgc 7 of 7 Page 29 of 65 Exygen Research Page 95 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD Method N um ber V001782 M ethod o f A nalysis for the Determ ination o f Perflnorooetanoie A cid (PFO A ) io Sedim ent by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C_jL Paul Connolly I Technical Leader, LC-MS, Exygen Research a yfoohhin Flaherty / Viccie President, Operations, Exygen Research ____ lOlz-bM Date />A^r Date Exygen Research Total Pages: 7 Page 30 of 65 Page 96 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygea Research Method Number VOOO1782 | A N A L V T IC A L M E T H O D ______________________ | Method o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Sedim ent by LC /M S/M S 1.0 Scope This method is to be em ployed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectromctric Detector (LC/M S/M S) in sediment. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult the appropriate M S D S before handling any chem ical for proper safety precautions. 3 .0 Sample Requirement 3.1 A t least 30 g o f test sample for extraction. 3.2 N o sample processing is needed for sediment samples. 3.3 Samples stored refrigerated should be allow ed to equilibrate to room temperature. 3.4 A ll samples must be thoroughly mixed before being sampled for extraction 3.5 Sample collection procedures w ill b e specified in the sam pling plan for this project. 4 .0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Methanol - HPLC grade 4.3 Acetic Acid - Reagent grade 4.4 Ammonium Acetate - A .C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance 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 Maas Spectrometer (L C /M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 IS mL disposable polypropylene centrifuge tubes. 5.6 D isposable micropipets (SO-lOOuL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 p L and 10-100 pL ), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc ( l g ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. Page 2 o f 7 Page 3J o f 65 Exygen Research Page 97 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Resevch Method Number V0001782 1 ANALYTICAL M ETHOD | Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Sedim ent by L C /M S/M S 5.13 Vortexer. 5.14 Wrist-action shaker. 5 .15 Centrifuge capable o f spinning SO mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (K eystone Scien tific), 2.1 mm x SO mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A ) : 2 raM Am monium Acetate in Water M obile Phase (B ) : Methanol Gradient Program: Tim e (mini 0.0 1.0 8.0 20.0 22.5 &A 65 65 25 25 65 Flow Rate K B (mlVminl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (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 optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray N egative M R M m ode, m onitoring 413 --36 9 m /z for PFOA. The above conditions are intended as a guide and m ay b e changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f . ammonium acetate to 1000 mL o f water. P*ge 3 o f 7 Page 32 o f 65 Exygen Research Page 98 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Rcaearcb Method Number VOOO1782 | ANALYTICAL M ETHOD | Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFO A) in Sediment by LC/MS/M S 8.2 Extraction Solutions 8.2.1 1% acetic acid in water is prepared b y adding 10 mL o f acetic acid to 1000 mL o f water. Alternate volum es m ay b e prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f - 1 0 0 p g/m L o f PFO A by w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/raL fortification solution o f PFO A is prepared b y bringing 10 mL o f the 100 pg/m L solution to a final volum e o f 100 with methanol in a 12S mL LDPE bottle. 9.1.3 A 1.0 pg/m L fortification solution o f PFO A is prepared by bringing 10 mL o f the 10 pg/m L solution to a final volum e o f 100 with methanol in a 123 mL LDPE bottle. 9.1.4 A 0 .1 pg/m L fortification solution o fP F O A is prepared b y bringing 10 m L o fth e 1.0 pg/m L solution to a final volum e o f 100 with methanol in a 123 mL LDPE bottle. 9.1.5 A 0.01 pg/m L fortification solution o f PFO A is prepared by bringing 10 mL o f the 0.1 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C end are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/M S/M S calibration standards are prepared in methanol via dilution o fth e O .l pg/mL fortification solution. 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Concentration Final ofFonification Solution (na/mL) 100 100 100 10 3 2 Volume (mL) 10 5 2 10 10 10 Diluted to (mL) too 100 100 100 100 100 Concentration (na/mL) 10.0 5.0 2.0 1.0 0.5 0.2 Page 4 o f 7 Page 33 o f 65 Exygen Research Page 99 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Rmarch Method Number VOOO1782 | ANALYTICAL M ETHOD | Method o f Analysis for the Determination ofPerfluorooctanoic A cid (PFO A ) in Sedim ent by LC/MS/M S 9.2.3 Store all calibration standards in 125-m L LDPE narrow-mouth bottles at 2CC to 6C , up to six months. 9.2.4 Alternate volum es and concentrations o f standards m ay be prepared as 10.0 Batch Set Up 10.1 Each batch o f sam ples extracted (typically 2 0 or less) must 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 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 W eigh 5 g o f sample into 30 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 33 mL o f 1% acetic acid, cap, vortex and shake on a wrist action shaker for --6 0 minutes. 11.3 Centrifuge the tubes at -3 0 0 0 rpm for - 2 0 minutes. 11.4 Condition the C u SPE cartridges (1 g , 6 m L ) b y p assing 10 mL methanol follow ed by 20 mL o f HPLC water ( - 2 drop/sec). D o not let colum n run dry 11.3 Load (decant) the sample on the conditioned C u SPE cartridge. Discard eluate, 11.6 Add 20 mL o f methanol to the sedim ent left in the bottom o f the SO mL centrifuge tube. Cap, vortex and shake on a wrist action shaker for - 3 0 minutes. 11.7 Centrifuge the tubes at -3 0 0 0 rpm for - 2 0 minutes. 11.8 Decant the methanol onto the sam e SPE cartridge. Collect the eluate. 11.9 W ash the column with 4 m L o f methanol. C ollect die eluate and add it to the eluate collected in step 11.8. 11.10 Condition a second C n SPE cartridge (1 g, 6 m L ) b y passing lO m L methanol follow ed b y 2 0 m L o f HPLC water ( - 2 drop/sec). D o not let colum n run dry 11.11 Add the methanol to -2 0 0 m L o f water and load on the second conditioned SPE cartridge. 11.12 Elute with - 3 mL 100% methanol. C ollect 3 mL o f eluate into graduated 13 mL polypropylene centrifiige tubes (final volum e = 3 mL). 11.13 Analyze samples using electrospray LC/MS/M S. Page 5 o f 7 Page 34 of 65 Exygen Research Page 100 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Reeearch Method Number VQ001782 I ANALYTICAL M ETHOD | Method o f Analysis for the Detcnnination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/M S 12.0 Chromatography 12.1 12.2 12.3 12.4 12.5 Inject the sam e amount 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. Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. An entire set o f extracted calibration standards m ust be included at the beginning and at the end o f a sam ple set. Standards must be interspersed between every 3-10 samples. A s an alternative, an entire set o f calibration standards m ay b e inject! at the beginning o f a set follow ed by calibration standards interspersed every 5-10 sam ples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in sam ple set. 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. Sam ple response should not exceed standard responses. A n y samples that exceed standard responses should be farther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.3 Chromatogram must sh ow a peak o f a daughter ion at 36 9 amu 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. Method blanks must not contain PFO A at levels greater than the LOQ. i f a blank contains PFOA at levels greater than 0.2 ng/m L, then a new blank sample must be obtained and the entire set must be re-extracted. R ecoveries o f control spikes and matrix spikes m ust be betw een 70-130% o f their known values. I f a control spike falls outside the acceptable lim its, the entire set o f samples should b e re-extracted. A ny matrix spike outside 70. 130% should be evaluated b y the analyst to determ ine i f re-extraction is warranted. A n y calibration standard found to b e a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. H owever, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. The correlation coefficient (R ) for calibration curves generated must be 2 0 .9 9 2 (R2 20.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and (he standards or the relevant set o f sam ples should be reanalyzed. Pig 6 o f 7 Page 35 o f 65 Exygen Research Page 101 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygea Research Method Number V0001782 I ANALYTICAL METHOD | Method o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Sedim ent by L C /M S/M S 13.6 Retention times betw een standards and sam ples m ust not drift more than 4% within an analytical run. Ifretention tim e drift exceeds this limit within an analytical ran then the set must b e reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the amount o f PFO A found (in ng/mL, baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (n g to L ) - (Peak area - intercept) x D F slope DF - factor b y w hich the final volum e w as diluted, i f necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) f total analyte found (ng/mL) - analyte found in control (ng/m L )] ^ analyte added (ng/mL) 14.3 U se the follow ing equation to convert the amount o f PFO A found in ng/mL to n g /g (p p b ). PFOA found (ppb) - fPFPA (ny/mIA x final volum e (S m U i sample weight (5 g) 14.4 U se the follow ing equation ( i f necessary) to calculate the amount o f PFOA found in ppb based on dry weight. PFO A found (ppb) dry weight " PFOA found (ppb) x [100% / total solids(%)] . PageT o f 7 Page 36 o f 65 Exygen Research Page 102 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No. : P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD Method Num ber V 0001783 M ethod o f A n alysis fo r th e D eterm ination o f P erfln orooctan oic A cid (P F O A ) in Fish and Clam s by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly Technical Leader, LC-MS, Exygen Research '/w / U / ihn Flaherty Vice President, Operations, Exygen Research Date Date Exygen Research Total Pages: 8 Page 37 of 65 Page 103 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0001783 a n a l y t ic a l m e t h o d Method o f A nalysis for the DeterminationofPerfluorooctanoic A cid (PFO A ) in Fish and Clams by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid b y H igh Perfonnance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/M S/M S) in fish and clams. 2.0 Safety 2.1 A lw ays observe safe laboratory practices, 2.2 Consult the appropriate M SD S before handling any chem ical for proper safety precautions. 3.0 Ssm ple Requirement 3.1 At least 20 g o f test sample for extraction. 3.2 Samples should b e processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place die sam ples in frozen storage until time o f analysis. 3.3 S sm ple collection procedures w ill be specified in the sam pling plan for this project. Reagents and Standards 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 Water - HPLC grade Acetonitrile - HPLC grade Carbon ( 120-400 mesh) - Reagent grade Methanol - HPLC grade Silica gel (60*200 mesh) -R ea g en t grade Florisil (60-100 mesh) -R ea g en t grade Superclean LC-NH2- Reagent grade 1-O ctanol- HPLC grade L-Ascorbic acid - Reagent grade Dimethyldichloroailane - Reagent grade Toluene - Reagent grade Ammonium Acetate - A.C.S. Reagent Grade Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high perfonnance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volum e injector capable o f injecting 5-200 pL connected to a tandem M ass Spectrometer (L C /M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Page 2 o f 8 Page 38 o f 65 Exygen Research Page 104 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V 0001783 1 ANALYTICAL METHOD \ Method o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Fish and Clams by LC/MS/M S 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. Tissuznizer. 125 mL pear-shaped flasks. 50 mL disposable polypropylene centrifuge tubes. IS m L disposable polypropylene centrifuge tubes. Disposable micropipets (50-100uL, 100-200uL). 125-m LLDPE narrow-mouth bottles. 2 mL clear HPLC vial kit. Disposable pipettea. Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. SPE tubes (20m L) (Supelco cat. no. N 057177). Wrist action shaker. Centriftige capable o f spinning 50 mL polypropylene tubes at 2 0 0 0 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophasc RP (K eystone Scien tific), 2.1 mm x 50 mm, 5p (P/N; 82505-052130) 6.2 Temperature: 30C 6.3 M obile Phase (A ): 2 mM Am m onium Acetate in Water 6.4 M obile Phase (B ) : Methanol 6.5 Gradient Program: Time fminl 0.0 1.0 8.0 20.0 22.5 AA 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 Volume: 15 pL (can b e increased to as much 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 optimize the HPLC system. Page 3 o f 8 Page 39 of 65 Exygen Research Page 105 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0001783 ANALYTICAL M ETHOD Method o f A nalysis for the Determination o f Periluorooctanoic A cid (PFO A ) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM m ode, m onitoring 413 PFOA. 369 m/z for The above conditions are intended as s guide and m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol is prepared by dissolving 2 g o f ascorbic acid in 100 mL o f methanol. 30% Dimethyldichlorosilane in toluene is prepared by bringing 3 mL o f dimcthyldichloroailane to a final volum e o f 10 m L w ith toluene. Alternate volum es m ay be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f - 1 0 0 p g/m L o f PFO A b y w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing I mL o f the 100 pg/m L solution to s final volum e o f 100 with methanol in a 125 mL LDPE bottle. A 0.1 pg/m L fortification solution o f PFO A is prepared by bringing 10 m L o f the 1.0 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. A 0.01 pg/m L fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/roL solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. T he 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. Page 4 o f 8 Page 40 of 65 Exygen Research Page 106 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Rcacarcb Method Number V0001783 _____________________________A N A L Y T IC A L M E T H O D _____________________________ Method o f A nalysis for the Detennination o f Perfluorooctanoic A cid (PFO A ) in Fish and Clams by LC/MS/MS 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/M S/M S calibration standards are prepared in methanol via dilution o f the 1.0 pg/mL fortification solution. The follow ing is typical exam ple: additional concentrations may be prepared as needed. Concentration Final o f Fortification Volume Diluted to Concentration Solution fua/mL) 1.0 1.0 1.0 imL) 5.0 2.5 1.0 imL) 100 100 100 (jig/mL) 0.05 0.025 0.01 0.05 0.025 0.1 0.005 10 10 10 10 100 100 100 100 0.005 0.0025 0.001 0.0005 9.2.3 Store all calibration standards in 125mL LDPE narrow-mouth bottles at 2C to 6C , up to fix months. 9.2.4 Alternate volum es and concentrations o f standards m ay be prepared as 10.0 Batch Set Up 10.1 Each batch o f sam ples extracted (typically 20 or less) m ust include at least on e untreated control and tw o untreated controls fortified at known concentrations (lab control spike) to verily procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in die quality assurance plan for this project. 11.0 Sample Extraction 11.1 11.2 11.3 11.4 11.5 11.6 W eigh 5 g o f frozen sam ple into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Add 30 mL o f acetonitrile and shake on a wrist action shaker for ~ 1 5 minutes. Place the tubes in a freezer for- 1 hour. Pack and condition the SPE tubes and lilan izc the pear-shaped flasks. Pack foe 20 mL SPE tubes in sequence with 2 g florisil, 2 g silica gel, 2 g carbon, and 1 g LC-NHj. Condition the colum ns with 20 mL o f methanol, then 2 0 m L o f acetonitrile. Discard all washes. D o not allow the column to dry. Silanize the 125 mL pear-shaped flasks b y rinsing with the 30% dimethyldichlorosilane in toluene solution. Rinse the flask with toluene once, follow ed b y methanol (three tim es). D ry the flasks com pletely before use, either by air-drying or with a stream o f nitrogen. Page 5 of X Page 4! of 65 Exygen Research Page 107 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0001783 A N A L Y T IC A L M E T H O D _____________________________ Method o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Fish and Clams by LC/MS/MS 11.7 Centrifuge the 5 0 mL polypropylene tubes containing sam ple at - 2 0 0 0 rpm for MO minutes. 11.8 Decant the extract on to a conditioned SPE colum n fitted inside the mouth o f the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape flask. 11.9 Add 10 mL o f acetonitrile to the sam ple in the 50 mL centrifuge tube. H om ogenize the frozen fat phase using a tissum izer for - 3 0 seconds and rinse the tissumizer with - 1 0 mL o f acetonitrile into the tube. 11.10 Shake the sample again f o r - 1 0 minutes on a wrist-action shaker. 11.11 Place the tubes in a freezer for - 1 hour more. 11.12 Centrifuge the 50 m L polypropylene tubes containing sam ple at -2 0 0 0 rpm fo r - 1 0 minutes. 11.13 Decant the extract onto the sam e SPE colum n. C ollect the eluate into the sam e pear-ihaped flask and com bine with the eluent from the initial extraction. 11.14 Pass 2 0 m L o f acetonitrile through the SPE colum n and com bine the eluate in the sam e pear-shaped flask. 11.15 Add 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 < 40C). 11.16 Make the final volum e, b y adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to m ix/dissolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze samples using electrospray LC/MS/M S. 12.0 Chromatography 12.1 Iqject the sam e amount o f each standard, sam ple and fortified sample into the LC/M S/M S system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 A n entire set o f calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed betw een every 5 - It) samples. A s an alternative, an entire te l o f calibration standards may be injected at the beginning o f a set follow ed b y calibration standards interspersed every 5 -10 sim p le s (to account for a second set o f standards). In either case, calibration standards must 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 b y linear regression using 1/x w eighting o f peak area versus calibration standard concentration using M assL ynx 3.3 (or equivalent) software system. Page 6 o f 8 Page 42 of 65 Exygen Research Page 108 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygra Research Method Number V0001783 ANALYTICAL METHOD Method o f Analytit for the Determination o f Perfluorooctanoic A cid (PFO A ) in Fish and Clams by LOM S/M S 12.5 Sample response should not exceed standard responses. A ny samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram must sh ow a peak o f a daughter ion at 369 amu 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. Method blanks must 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 sample must be obtained and the entire set must be re-extracted. Recoveries o f control spikes and matrix spikes must be between 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. 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. H owever, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. T he correlation coefficient (R ) for calibration curves generated must be 2 0.992 (R2 20.985). I f calibration results fall outside these limits, then appropriate steps must b e taken to adjust instrument operation, and the standards or A c relevant set o f sam ples should be reanalyzed. Retention times between standards and sam ples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the amount 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 follow ing equation to convert the amount o f PFO A found in ng/mL to ng/g (ppb). PFO A found (ppb) - (PFO A found (ng/m L ) x final volum e (m L) x DF1 sample weight (g) D F factor b y which the final volum e w as diluted, i f necessary. Page 7 o f 8 Page 43 of 65 Exygen Research Page 109 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen EUaearch Method Number VOOO1783 I ANALYTICAL M ETHOD ] Method o f Analysis for the Determination o f Periluorooctanoic A cid (PFO A ) in Fish and Clams by LC/MS/MS 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use the follow ing equation to calculate the percent recovery. Recovery (%) [ total analyte found (ng/g) - analyte found in control (ng/g)] analyte added (ng/g) Exygen Research page 8 of 8 Page 44 o f 65 Page 110 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD Method Number: V 0001784 M ethod o f A n alysis fo r th e D eterm ination o f Perflu orooctan oic A cid (P F O A ) in Vegetation by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: H -X c a L ___ Paul Connolly * Technical Leader, LC-MS, Exygen Research , / / f i U / ________ jfycoi Flaherty ' Vice Prsidait, Operations, Exygen Research ___ t a j i t M Date Date Exygen Research T out Pages: 7 Page 45 of 65 Page 111 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No. : P0000760 Exygen Protocol Number: P0000760 Exygen Rctwrch Method Number V0001784 I ANALYTICAL M ETHOD Method o f Analysts for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope This method is to be em ployed for the isolation and quantitation o f perfluorooctanoic acid b y H igh Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/M S/M S) in vegetation. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult the appropriate M SD S before handling any chem ical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 20 g o f test sample for extraction. 32 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the sam ples in frozen storage until time o f analysis. 3.3 Sam ple collection procedures w ill b e specified in the sampling plan for this project. 4 .0 Reagents and Standards 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 W ater-H P LC gride Acetonitrile - HPLC grade Carbon (120-400 mesh) - Reagent grade Methanol - HPLC grade Silica gel (60-200 mesh) - Reagent grade Florisi! (60-100 m esh) - Reagent grade Superclean LC-NHj - Reagent grade 1-Octanol - HPLC grade L-Ascoibic acid - Reagent grade DimethyldichlorosUa&e - Reagent grade Toluene - Reagent grade Ammonium Acetate - A.C.S. Reagent Grade Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with variable volum e injector capable o f injecting 5-200 pL connected to a tandem M ass Spectrometer (L C /M S/M S). 5.2 A d evice to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. P ige2 of 7 Page 46 of 65 Exygen Research Page 112 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0001784 ANALYTICAL M ETHOD | Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Vegetation byLC/M S/M S 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14 5.15 Rotary evaporator. 125 mL pear-shaped flaaks. 50 mL disposable polypropylene centrifuge tubes. 15 mL disposable polypropylene centrifuge tubes. D isposable micropipets (50-lOOuL, 100-200uL). 125-mL LDPE nairow-mouth bottles. 2 mL clesr HPLC vial kit. D isposable pipettes. Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. SPE tubes (20m L) (Supelco cat. no. N 0 5 7 177). Wrist action shaker. Centrifuge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (K eystone Scien tific). 2.1 mm x 50 mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 mM Am monium Acetate in Water 6.4 Mobile Phase (B ) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 AA 65 65 25 25 65 Flow Rate ImL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak A r ea -e x ter n a l 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 optimize the HPLC system. 7.0 M&/MS System 7.1 Mode: Electrospray N egative M RM m ode, monitoring 413 -* 3 6 9 m /z for PFOA. Page 3 of 7 Page 47 of 65 Exygen Research Page 113 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No. : P0000760 Exygen Protocol Number: P0000760 Exygen Rewareb Method Number V0001784 a n a l y t i c a l m e t h o d ______________________________ Method o f A nalysis for the Determination o f Periluorooctanoic A cid (PFO A) in Vegetation by LC/MS/M S The above conditions are intended as a guide and m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M ammonium acetate in water is prepared b y adding 0.154 g o f ammonium acetate to 1000 mL o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic a d d in methanol is prepared by dissolving 2 g o f ascorbic acid in 100 mL o f methanol. 30% Dimethyldichlorosilane in toluene is prepared by bringing 3 mL o f dimethyldichlorosilane to a final volum e o f 10 mL with toluene. Alternate volum es may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 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 PFO A b y w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 1 mL o f the 100 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. A 0 .1 pg/m L fortification solution o fP F O A is prepared b y bringing 10 mL o f the 1.0 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. A 0.01 pg/m L fortification solution o fP F O A is prepared by bringing 10 mL o f the 0.1 pg/m L solution to a final volum e o f 100 with methanol in a 125 m L L D PE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/M S/M S calibration standards are prepared in methanol via dilution o f the 1.0 pg/mL fortification solution. Page 4 o i 7 Page 48 of 65 Exygen Research Page 114 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0001784 ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A) in Vegetation by LC/MS/M S 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. C o n c e n tr a tio n Final o f Fortification Volum e Diluted to Concentration Solution (ua/mL) fm U (mL) (UR/mL) 1.0 5.0 100 0.05 1.0 2.5 100 0.025 1.0 1.0 100 0.01 0.05 10 100 0.005 0.025 10 100 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 Store all calibration standards in 125-m L LDPE nairow-m outh bottles at 2*C to 6 #C, up to six months. 9.2.4 Alternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and tw o untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in die quality assurance plan for this project. 11.0 Sample Extraction 11.1 W eigh 5 g o f d ozen sam ple into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 30 raL o f acetonitrile and shake o n a wrist action shaker for ~ l 5 minutes. 11.3 Centrifuge the 50 mL polypropylene tubes containing sam ple at -2 0 0 0 rpm for - 1 0 minutes. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in sequence with 2 g florisil, 2 g silica gel. 2 g carbon, and 1 g LC-NHj. Condition the colum ns with 20 mL o f methanol, then 2 0 mL o f acetonitrile. Discard all w ashes. D o not allow the column to dry. 11.6 Silanize the 125 mL pear-shaped flasks b y rinsing with the 30% dimethyldichlorosilane in toluene solution. Rinse the flask with toluene once, follow ed b y methanol (three tim es). D ry the flasks com pletely before use, either by air-drying or with a stream o f nitrogen. 11.7 Decant the extract on to a conditioned SPE colum n fitted inside the mouth o f the pear-shaped flask. Collect the eluate in the 125 mL silanized pear-shape flask. Pgc 5 o f 7 Page 49 of 65 Exygen Research Page 115 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0001784 ANALYTICAL M ETHOD Method o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Vegetation by LC/MS/MS 11.8 Add 2 0 mL o f acetonitrile to die sample in the 50 m L centrifuge tube. 11.9 Shake the sample again for - 1 0 minutes on a wrist-action shaker. 11.10 Centrifuge the 50 m L polypropylene tubes containing sam ple at -2 0 0 0 rpm for - 5 minutes. 11.11 Decant the extract onto the sam e SPE column. C ollect the eluate into the sam e pear-shaped flask and com bine with the eluent from the initial extraction. 11.12 R e p e a tste p sll.8 th ro u g b U .lla g a in . 11.13 Add 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 < 40C). 11.14 M ake the final volum e, b y adding 2 mL o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to m ix/dissolve. 11.15 Transfer the extracts to HPLC vials using disposable pipets. 11.16 A nalyze samples using electrospray L C/M S/M S. Chromatography 12.1 Inject the sam e amount o f each standard, sam ple and fortified sam ple into the LC/M S/M S system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 A n entire set o f extracted calibration standards must be included at the beginning and at the end o f a sam ple set. Extracted standards must be interspersed betw een every 5*10 samples. A s an alternative, an entire set o f extracted calibration standards m ay b e injected at the beginning o f a set followed b y 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 utt 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 versus calibration standard concentration using M assL ynx 3.3 (or equivalent) software system. 12.5 Sam ple response should not exceed standard responses. A ny samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must sh ow a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFO A anion, while the daughter ion (369 arou) represents the loss o f carbon dioxide. Page 6 of 7 Page 50 of 65 Exygen Research Page 116 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exy|en R n o ich Method Number V00017M ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Vegetation by LC/MS/MS 13.2 13.3 13.4 13.5 13.6 M ethod blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFOA at levels greater than O.S ppb, then a new blank sample must be obtained and the entire set must b e re-extracted. Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. A ny calibration standard found to b e a statistical outlier b y using the Huge Error Test, m ay b e excluded from the calculation o f the calibration curve. H owever, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. The correlation coefficient (R ) for calibration curves generated must be 0.992 (R3 0.985). I f calibration results fall outside these limits, then appropriate steps must b e taken to adjust instrument operation, and the standards or the relevant set o f sam ples should be reanalyzed. Retention tim es betw een standards and sam ples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must b e reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the amount o f PFO A found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) - (Peak area - intercept) slope 14.2 U se the follow ing equation to convert the amount o f PFO A found in ng/mL to ng/g (ppb). PFOA found (ppb) - rPFOA found fn c/m L l x final volum e (mU x DF1 sample weight (g) DF factor b y w hich the final volum e w as diluted, i f necessary. 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use the follow ing equation to calculate the percent recovery. Recovery (% )" [ total analyte found (ng/g) - analyte found in control (ng/g)] ^ analyte added (ng/g) Pge 7 o f 7 Page 51 o f 65 Exygen Research Page 117 of 140 Interim Report #24 - A nalysis o f Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD Method Number V0001785 M ethod o f A o ily ih for th e D eterm lnatloB o f Perfluorooctanoic A d d (PFO A ) in Sm all M am m al Liver by LC/M S/M S Analytical Teeting Facility: Exygen Reeeaich 3058 Research Drive State College, PA 16801 Approved By: _C-- LLh____ Paul Connolly t Technical Leader, LC-MS, Exygen Research /)? 6hn Flaherty ' Vice President, Operations, Exygen Research Date Date Exygen Research Total Pages: 7 Page 52 of 65 Page 118 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number VOOOI785 | ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Periluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/M S 1.0 Scope This method is to b e em ployed for the isolation and quantitation o f periluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectromctric Detector (LC/MS/MS) in small mammal liver. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sample Requirement 3.1 At least S g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and hom ogenize with dry ice. P lace the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the sam ples in frozen storage until time o f analysis. Alternately, i f there is an insufficient amount o f sam ple ( - le s s than 3 g), then no processing is necessary and the sam ple can be used as supplied. 3.3 Sample collection procedures w ill be specified in the sam pling plan for this project 4 .0 Reagents and Standards 4.1 W ater-H PL C grade 4.2 M ethanol-H PL C grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.3 Periluorooctanoic Acid - Sigma-Aldrich 3.0 Instrument and Equipment 3.1 A high performance 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 Maas Spectrometer (LC/M S/M S). 3.2 A device to collect raw data for peak integration and quantitation. 3.3 Analytical balance capable ofread in g to 0.00001 g. 3.4 30 mL disposable polypropylene centrifuge tubes. 3.3 13 mL disposable polypropylene centrifuge tubes. 3.6 Disposable micropipets (50-tOOuL, 100-200uL). 3.7 123-mLLDPEnazTOW-mouth bottles. 3.8 2 mL clear HPLC vial kit. Page 2 o f ' Page 53 of 65 Exygen Research Page 119 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0001785 | ANALYTICAL M ETH OD Method o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Small Mammal Liver by LC/M S/M S I 5.9 5.10 5.11 5.12 5.13 5.14 5.15 D isposable pipettes. Autopipettes (100*1000 pL and 10*100 pL), with disposable tips. Waters Sep Pak Vac 6 cc ( l g ) t C l8 SPE cartridges. SPE vacuum manifold. Tissuemizer. Wrist-action shaker. Centriftige capable o f spinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluop h aseR P (K eyston e S cientific), 2.1 mm x 5 0 mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 M obile Phase (A ) : 2 m M Am m onium Acetate in Water 6.4 Mobile Phase (B ) : Methanol 6.5 Gradient Program: T im e fmiril 0.0 1.0 8.0 20.0 22.5 ZA 65 65 25 25 65 ZA 35 35 75 75 35 Flow Rate fm L /m inl 0.3 0.3 0.3 0.3 0.3 6 .6 Injection Volume: 15 pL (can b e increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. T he above conditions are intended as a guide and m ay b e changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM m ode, monitoring 413 --* 369 m/z for PFOA. The above condition! are intended as a guide and m ay be changed in order to optimize the MSMS system. Page 3 o f 7 Page 54 of 65 Exygen Research Page 120 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number VOOO1785 ___________________________ A N A L Y T IC A L m e t h o d ____________________________ Method o f A nalytic for the Determination o f Perfluorooctanoic A cid (PFO A ) in Small Mammal Liver b y LC/M S/M S 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M ammonium acetate in water is prepared by adding 0 .154 g o f ammonium acetate to 1000 mL o f water. Alternate volum es m ay be prepared. 9.0 Standard Preparation 9.1 Standard Stoclc/Fortification Solution 9.1.1 Prepare a stock solution o f - 1 0 0 p g/m L o f PFO A by w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing I mL o f the 100 p ^ m L solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pg/m L fortification solution o f PFO A is prepared by bringing 10 m L o fth e 1.0 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 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 9.2.1 9.2.2 LC/M S/M S calibration standards are prepared in methanol via dilution o f the 0.1 pg/mL fortification solution. The follow ing is a typical exam ple: additional concentrations may be prepared as needed. Concentration o f Fortification Solution (na/mL) 100 100 100 5.0 2.0 1.0 Volume (mL) 5.0 2.0 1.0 10 10 10 Diluted to (mL) 100 too 100 100 100 100 Final Concentration (na/mL) 5.0 2.0 1.0 0.5 0.2 0.1 9.2.3 Store all calibration standards in 125-m L LDPE narrow-mouth bottles at 2C to 6*C, up to six months. 9.2.4 Alternate volum es and concentrations o f standards m ay be prepared as needed. Page 4 o f 7 Page 55 of 65 Exygen Research Page 121 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V000178J | ANALYTICAL METHOD Method o f Analyst for the Determination o f Perfluorooetanoic A cid (PFOA) in Small Mammal Liver by LC/MS/M S | 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and tw o untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 W eigh 1 g o f sample into a SO mL polypropylene centrifuge tubes (fortify as needed, replace lid and m ix w ell). N ote that alternate w eigh ts o f liver may be measured depending on the sample size available for use. 112 A dd water to the sam ple for a final volum e o f 10 mL. 11.3 H omogenize sample using a tissuemizer for - 1 minute. 11.4 Transfer 1 mL o f the sample using a disposable pipette into a IS mL disposable centriflige tube. 11.5 Add 5 mL o f acetonitrile and shake for - 2 0 m inutes on a wrist-action shaker 11.6 Centrifuge the tubes at -3 0 0 0 ipm for - 5 minutes. 11.7 Decant the supernatant into a 50 m L disposable centrifuge tube and add 35 mL o f water. 11.8 Condition the C is SPE cartridges (1 g, 6 m L) by passing 10 mL methanol follow ed by 5 m L o f HPLC water ( - 2 drop/sec). D o not let colum n run dry 11.9 Load the sample on conditioned C n SPE cartridge. Discard eluate. 11.10 Elute with - 2 m L o f methanol. Collect 2 m L o f eluate into a graduated 15 mL polypropylene centrifuge tube (final volum e - 2 mL). 11.11 A nalyze sam ples using electrospray L C/M S/M S. 12.0 Chromatography 12.1 Inject the sam e amount o f each standard, sam ple and fortified sam ple into the LC/M S/M S system . A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFO A corresponding to at least fiv e or m ore concentration levels must be included in an analytical act. 12.3 A n entire set o f calibration standards m ust b e included at the beginning and at the end o f t sample set. Standards must be interspersed between every 5-10 samples. A s an alternative, an entire set o f calibration standards may be injected at the beginning o f a set follow ed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in a cample 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 f7 Page 56 of 65 Exygen Research Page 122 of 140 Interim Report #24 - A nalysis o f Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygea Research Method Number VOOO1785 | ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Small Mammal Liver b y LC/M S/M S versus calibration standard concentration u sin g M assLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. A n y samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram must show a peak o f a daughter ion at 36 9 amu from a parent o f 413 amu. T he 413 amu parent corresponds to the PFO A anion, w hile the daughter ion (369 amu) represents the loss o f carbon dioxide. Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFO A at levels greater than 10 ng/g, then a new blank sample must be obtained and the entire set must be re-extracted. Recoveries o f control spikes and matrix spikes must be between 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. A ny matrix spike outside 70 130% should be evaluated b y the analyst to determ ine i f re-extraction is warranted. A n y 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. H owever, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards iryected. The correlation coefficient (R ) for calibration curves generated must be 0.992 (RJ 0.985). I f calibration results fall outside these limits, then appropriate steps must b e taken to adjust instrument operation, and the standards or the relevant set o f sam ples should be reanalyzed. Retention tim es betw een standards and sam ples m ust not drift more than 4 % within an analytical run. I f retention tim e drift exceed s this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the amount o f PFO A found (in ng/mL. based on peak area) using the standard curve (linear regression parameters) generated b y the M ass Lynx software program: PFO A found (ng/raL) - (Peak area - intercept) x D F x aliquot factor slope DF " factor b y which the final volum e was diluted, if necessary. Aliquot factor* 10 Page 6 o f ' Page 57 o f 65 Exygen Research Page 123 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0001785 I ANALYTICAL m e t h o d Method o f A nalysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Small Mammal Liver by LC/MS/MS 14.2 For samples fortified with known amounts o f PFO A prior to extraction, use the follow ing equation to calculate the percent recovery. R ecovery (/) [ total analyte found (ng/mL) - analyte found in control (ng/m L )] ^ ^ analyte added (ng/mL) 14.3 U se the follow ing equation to convert the amount o f PFOA found in ng/mL to n g /g (p p b ). pfoa found(ppb)- fPFOAfound(nrtni) final volume (mLll sample weight (g) Exygen Research Page 7 o f 7 Page 58 of 65 Page 124 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD Method Number: VQ001786 M eth od o f A nalysts for th e Determ inatloB o f P erflu orooctan olc A cid (P F O A ) In Sm all M am mal Serum by LC/M S/M S Analytical Testing Facility: Exygen Research 30S8 Research Drive State College, PA 16801 Approved By: c--iL) Paul Connolly 1 Technical Leader, LC-MS, Exygen Research Date a / - d J / _________ Joh n Flaherty s Vice President, Operations, Exygen Research Date Exygen Research Total Pages: 7 Page 59 o f 65 Page 125 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0001786 | ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Periluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/M S 1.0 Scope This method is to b e em ployed for the isolation and quantitation o f perfluorooctanoic acid b y H igh Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in small mammal serum. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult the appropriate M SD S before handling any chem ical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 1 mL o f test sample for extraction. 3.2 N o sample processing is needed for serum samples. However, frozen serum samples must to allowed to com pletely thaw to room temperature before use. 3.3 Sample collection procedures w ill b e specified in the sampling plan for this project. Reagents and Standards 4.1 W a ter-H P L C grade 4.2 M ethanol - HPLC grade 4.3 A ceton itrile-H P L C grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument 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 performance 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 Mass Spectrometer (LC/M S/M S). A device to collect raw date for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 mL disposable polypropylene centrifuge tubes. 15 mL disposable polypropylene centrifuge tubes. Disposable micropipets (50-lQOuL, 100-200uL). 125-m LLDPE narrow-mouth bottles. 2 m L clear HPLC vial kit. D isposable pipettes. Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. Waters Sep Pak Vac 6 cc (Ig ) tC U SPE cartridges. SPE vacuum manifold. Vortexer. Page 2 o f 7 Page 60 of 65 Exygen Research Page 126 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P000760 Exygeo Research Method Number V0001786 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Small Mammal Serum by LC/MS/M S $.14 Wrist-action shaker. $.1$ Centrifrge capable o f spinning 1$ mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (K eystone S cientific), 2 .1 mm x SO mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 mM Am m onium A cetate in Water Mobile Phase (Bj : Methanol Gradient Program: Tim e imin) 0.0 1.0 8.0 20.0 22.5 `A A 65 65 25 25 65 Flow Rate & fmL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volum e: 15 pL (can be increased to as much as 5 0 mL) 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTime: ~ 2 3 minute*. 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 Mode: E lectrosprayN egativeM 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 optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.1S4 g o f ammonium acetate to 1000 m L o f water. Alternate volum es m ay b e prepared. Pige 3 o f1 Page 61 of 65 Exygen Research Page 127 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number VOOOI786 | ANALYTICAL METHOD Method o f Analysis for the Determination o f Periluorooctanoic A cid (PFO A) in Small Mammal Serum by LC/MS/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 PFO A by w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125*mL LDPE bottle. 9.1.2 A 1.0 pg/m L fortification solution o f PFOA is prepared by bringing I m L o f the 100 pg/m L solution to a final volum e o f 100 with methanol in a 12S mL LDPE bottle. 9.1.3 A 0.1 pg/m L fortification solution o f PFO A is prepared b y bringing 10 m L o f the 1.0 p g ta L solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. 9 .1.4 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. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/M S/M S calibration standards are prepared in methanol via dilution o f the 0.1 pg/mL fortification solution. The follow ing is a typical exam ple: additional concentrations may be prepared as needed. Concentration Final o f Fortification Volume Solution (ne/mL) (mL) Diluted to (mL) Concentration (ne/mL) 100 5.0 100 100 2.0 100 100 1.0 100 5.0 2.0 1.0 5.0 10 100 0.5 2.0 10 100 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-m L LDPE narrow-mouth bottles at 2*C to 6C, up to six months. 9.2.4 Alternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 2 0 or less) m ust include at least one untreated control and tw o untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. Page 4 of? Page 62 o f 65 Exygen Research Page 128 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Rematch Method Number V0001786 I a n a l y t ic a l m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Small Mamina) Serum by LC/M S/M S | 11.0 Sam ple Extraction 11.1 Measure 1 mL o f sample into a 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and m ix w ell). N ote that alternate volum es o f serum may be measured depending on the sample size available for use. 11.2 Add water to the sample for a final volum e o f 2 0 mL. Cap tightly 11.3 Vortex for ~1 minute. 11.4 Transfer 1 m L o f the sam ple using a disposable pipette into a 15 mL disposable centrifuge tube. 11.5 Add 5 mL o f acetonitrile and shake for - 2 0 m inutes on a wrist-action shaker. 11.6 Centrifuge the tubes at - 3 0 0 0 rpm for - 5 minutes. 11.7 Decant the supernatant into a 50 mL disposable centrifuge tube and add 35 mL o f water. 11.8 Condition the C u SPE cartridge (1 g , 6 m L) by passing 10 mL methanol follow ed b y 5 mL o f HPLC water (~ 2 drop/sec). D o not let colum n run dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eluate. 11.10 Elute with - 2 mL o f methanol. C ollect 2 mL o f eluate into a graduated 15 mL polypropylene centriftige tube (final volum e - 2 mL). 11.11 Analyze samples using electrospray LC/M S/M S. 12.0 Chromatography 12.1 Inject the tam e amount o f each standard, sam ple and fortified sample 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 at least five or more concentration levels must be included in an analytical set. 12.3 A n entire set o f calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5-10 samples. A s an alternative, an entire set o f calibration standards may be injected at the beginning o f set follow ed b y calibration standards interspersed every 5-10 sam ples (to account for a second set o f standards). In either case, calibration standards must b e 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 b y linear regression using 1/x w eighting o f peak area versus calibration standard concentration using M assL ynx 3.3 (or equivalent) software system. 12.5 Sam ple response should not exceed standard responses. A n y sam ples that exceed standard response should b e further diluted and reanalyzed. Page 5 o f ^ Page 63 of 65 Exygen Research Page 129 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Reeevcb Method Number V0001786 ___________________________ A N A L Y T IC A L M E T H O D ____________________________ Method o f Analysis for the Determination o f Periluorooctanoic A cid (PFO A ) in Small Mammal Serum by LC/MS/M S 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram must show ap eak o f a daughter ion at 369 amu 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. Method blanks must not contain PFO A at levels greater than the LOQ. I f a blank contains PFOA at levels greater than 10 ng/rnL, then a new blank sample must be obtained and the entire set must be re-extracted. Recoveries o f control spikes and matrix spikes must be between 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. A ny matrix spike outside 70 130% should be evaluated by the analyst to determine i f re-extraction is warranted. A ny calibration standard found to b e a statistical outlier b y using the Huge Error Test, may b e excluded from the calculation o f the calibration curve. H owever, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. The correlation coefficient (R ) for calibration curves generated must be 2 0.992 (R3 20.985). I f calibration results fall outside 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. Retention tim es between standards and sam ples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the amount o f PFO A found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated b y the M ass Lynx software program: PFOA found (ng/mL) " (Peak area - intercept) x D F x aliquot factor slope D F " factor b y w hich the final volum e w as diluted, i f necessary. A liquot factor 20 14.2 For samples fortified with known amounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. R ecovery (%) [ total analyte found (ng/mL) - analyte found in control (ng/m L)] ^ ____________________ analyte added (ng/mL)_________________________________ Page 6 o f 7 Page 64 of 65 Exygen Research Page 130 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number VOQOI786 I a n a l y t ic a l m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Small Mammal Serum by LC/MS/M S 14.3 U se the follow ing equation to convert the amount o f PFO A found in ng/mL to ppb. PFOA found (ppb) - fPFOA found (ng/m L ) x final volum e O nD I sample volum e (mL) Exygen Research Pige 7 o f 7 Page 65 of 65 Page 131 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 n 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment Number. 1 Effective Date: 01/19/05 Exygen Study Number P0000760_______ Client Study Number Page 1 of 1 DESCRIPTION OF AMENDED SECTION 1) Analytical Procedure Summary V0001780:Section 9.1 2) Verification of Analytical Procedure None . AMENPEPIQ 1) Add to Section 9.1: Section 9.1.6, Alternate weights of standards may be used to prepare alternate concentrations of stock solutions as necessary. Alternate levels of fortification solutions may also be prepared. 2) Low and high spiking levels of the analytes for each matrix may be altered depending on sample size available for extraction and/or to cover analyte concentrations expected in the samples. RATIONALE 1) Higher concentrations of standards need to be prepared in order to spike the sample bottles at higher levels. 2) The sample size available for small mammal liver and serum was smaller than `'J expected. Spiking at the pre-determlned levels in the protocol puts the spiked concentration lower than the detection limit Also, the analyte levels in the ground water samples are expected to greatly exceed the pre-determlned spiking levels listed in the protocol. When the levels in the samples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the QC sample. Higher spiking levels in the bottles will cover the analyte concentrations expected in the water samples. IMPACT ON STUDY The LOQ is 100 ng/g for a 0.1 g sample of small mammal liver and Is 1000 ng/mL for a 0.01 mL sample of small mammal serum. Higher levels of spiking for the water samples will ensure that more QC recovery data can be used. 3hfoS .Principal Investigator Signature > vii. /* Study Director Mrfafegaraant Signature iIm U M m F Sponsor S ta tu re (if required) D a ta . vi\Ac Date / / 3hW _ Da / . ExvaenQAUReview L I S /ttloiInr LIBRARY IO: W 0001226-6 ADMINISTRATIVE FORM Exygen Research Page 132 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOLAMENDMENT 2 03/07/05 P0000760 Client Study Number: Page 1 of 1 None DESCRIPTION OF AMENDED SECTION Report, page 11 of 65 . AMENDEDTQ Instead of one final report, interim reports will be issued. RATIONALE Due to the excessive sizes of the data sets, interim reports will be issued to allow the client to receive data In a timelier mener. /htuUUt~ i$ j) IMPACT ON STUDY The client will be able to receive and review the data more quickly. Study Director Management Signature Exygen Manage lUlt'l t______ Sponsor Slgn^dre (ifrequired) LIBRARY ID: V0001226-8 Date f/lo/S ala ExygenrQAU Review JA (n ftr l S ADMINISTRATIVE FORM Exygen Research Page 133 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 08-02-200! 09:11 Frw-KSTON SOLUTIONS T-KT P.002/002 F-9SB IT RESEARCH 3 0 Research Drive Phene: 814-272-10 State College, PA 16801 F *c 814-231-1580 Amendment Number. Effective Date: Exygen Study Number PROTOCOL AMENDMENT _ 3_ 04/13/05 , F7SO Cfierrt Study N um ber ^ p"9* 1oM . d e sc r ip t io n o F AW-n d e d Se c t io n Antlyttarf Procedure Summary: V0001780^Mthod of A nalysis* the D e te n r in ^ Psrfluoroooctenolc Add (PFOA) In Water by LC/MS/MS.' Sections 8.2 end 11.0 of the method. SttonOJi. NoaeJdractBdcUbnlianeCandaida maybe prepared holder toquanUtate sam ples directly Weeted onto the LCMSMSeydem. ____ . Section n .o . Samples may be ansfyaed without going through * * * ? procedure, Instead, they may be dfiuted, iTappropriate, and directly Injected onto the LCMSMS system for anatysfs. * msssss------------- !----------- _______ ___ k Som e extracted results between leborewy end 1Wd s p fta s mm not M r a w w and/or were unusable. Directly irflecang the extracts may add sem e mslgm Wo me matrix effects on the date results. IMPACT ON gTUOY M ore usable d ata results m sy b e obtained. m 037 Bated i S' A Dm ExygerQAUReMewj^iL?2^iZiI UBHARVID! VD00122W RECEIVED TIME JUN. 2. 9:29f)M ADMMte(KATtVEFORM PRINT TIME JUN. 2. 9:30fiM Exygen Research Page 134 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 JUL.25-2085 8:55fiM EXYGEN RESEARCH NO.774 P.4 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814 231-1580 PROTOCOL AMENDMENT Amendment Number _____ 4 Effective Date: 07/18/05...' Exygen Study Number P760 Client Study Number Pag* 1 of 1 NA d e s c r ip t io n o f a m e n d e d s e c t io n V erification of Analytical Procedure, page 10 of protocol. AMENDED TO The field duplicate can be used for the laboratory spikes and replicate when the primary sample volume Is limited. RATIONALE The sample size for a water sample Is 200 mL If a sample site requires re-extraction for any reason, there would not be enough of the primary sample to repeat two laboratory spikes and a replicate. The field duplicate is technically the same sample as die primary sample and therefore, can be used for laboratory spikes and replicates as needed. IMPACT ON STUDY No negative impact on the study. Using the duplicate sample allows for the fui! QC of the sample sit to be completed. l L *uy i^ u ^ v r s i g n a t u r e , f t / A? / /^ C Principal Investigator Signature ..f b x f o t Date/ / Date Dato Ji/L CS /m /T S3 ? Exygen QAU Review fri./fv 7A x/ t'^~ LIBRARY ID; V0Q1226-S ADMINISTRATIVE FORM Exygen Research Page 135 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 t 3058 Research Drive Phone: 814-272-1039 S tate C ollege, PA 16801 Fax: 814-231-1580 RESEARCH Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 5 09/13/05 P760 Client Study Number: Page 1 of 1 NA . DESCRIPTION OF AM ENDED SECTION Analytical Procedure Sum mary: V0001780:"M ethod of Analysis fo r the D eterm ination of P erfluoroooctanoic A d d (P F O A ) in W ater by LC /M S /M S ." AMENDED TO The reanalysis o f tw o w ater samples, DF09-G W -131R-0-040126 and DFB-G W -135L-0040121 will be perform ed using the following procedure: An aliquot o f the w ater sam ple is spiked with the surrogate standard and/or other m atrix spike com pounds as appropriate. Add m ethanol to the fortified aqueous sam ple. Mix and aliquot for LC/MS/MS analysis. The LC conditions include the use of a Betasil C18, 2.1 x 50 m m colum n, a gradient o f 30% B to 100% B in 10 m inutes, w here A=2m M am m onium acetate in w ater and B=M ethanol (6 m in equilibration). U se a 5 pL injection volum e and a 0.3 m L/m in flow rate. LC conditions m ay be further m odified as appropriate for the system being used. An interim report will be issued that will include the exact details o f the reanalysis. R A T IO N A L E DF09-G W -131R-0-040126 and DF8b-GW -135L-0-040121 could not be reported quantitatively in interim report #3 due to quality control sam ple failures. T he study director and sponsor have requested reanalysis using the above conditions. IMPACT ON STUDY No negative im pact on the study. More usable data m ay be obtained. Study Director Signature Study Director Management Signature Exygen Management Signature Sponsor Signature (if required) Date Date Date Date Date Exygen Q A U R eview 0*.*^ 9 / / 3/t>S~ LIBRARY ID: V0001226-8 ADMINISTRATIVE FORM Exygen Research Page 136 of 140 Interim Report #24 Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 CHEM EHSR 236 1B WC* A I > EP. 13.2305 l :51Pn 65JJ733 1958 0CYOEN RESEARCH 09/14 '05 09:27 NO.915 02/02 &gSnRE5SEARCH 3058 Research Drive Phone. 814-273*1039 State College, PA 16801 Pa 814-231-1580 Amendment Number Effective Date; Exygen Study Number PROTOCOL AMENDMENT 5 09/13/05 P76Q Client Study Number Pagai on NA ' DESCRIPTION OF AMENDED ECTON ~" Analytical Procedure Summary: V0001760rMethod o f Analysis ter the Determination cf PerfluoroooctahofeAdd (PFOA) in Water by LQMS/MS,* " AMENDED TO The rearratysis or two water samples. DP09-GW-131R-0-040126 and OFB-GW-135L-0- 040121 will be performed using the following procedure: __ An aliquot of the water sample is spiked with the surrogate standard andter other matrix spike compounds as appropriate. Add methanol to the fortified aqueous sample. Mix and aliquot for LC/MS/MS analysis. The LC conditions indude the use of a Betasit Cl 8, Z.1 X 50 mm column, a gradient bf 30% S to 100% B in 10 minutes, where A2mM ammonium acetate in water and B=Metfianol (6 min equilibration}- Use a 5 (/L Injection volume and a 0.3 ml/rrfn flow rate. LC conditions may be further motflfled as appropriate ter tne system being used. An interim report will be issued that wHI indude the exact details of triereanalysis. . R A T IO N A L E DF09-GW-131R-0-040126 and DF8b-GW-13&-0-040121 could not be reported quantitatively in interim report #3 due to quality control sample failures. The study director and sponsor have requested reanalysis using the above conditions. . IM P A C T O N S T U D Y *" No negative impact on tee study. More usable data may be obtained. StudyDirectorSgrtetore Principal InvestigatorSignatum StudyDirectorNanagernursignature feryganManagern 1/l/bUtr SponsorS g i required) Date Dato Data Exvoen QAU Review (7h.tv LIBRARYID: VC00122M RECEIVED TIME SEP.14. 10:43AM Exygen Research ADMINISTRATIVEFORM PRINT TIME SEP.14. 10:44AM Page 137 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 - ' 5-2003 02:19 FR0M:3M ENU. LfiB 651 778 6176 SEP.13.2005 l :51Pn EXYGEN RESEARCH TO:*88142311580 NO.294 P.2 P :2 '2 3058 Research Drive Phone:814-272-1039 State College, PA 16801 Fax: 814-231 -1580 . Amendaient Number Effective Date: Exygen Study Number PROTOCOLAMENDMENT 5 09/13/05 P760 Client Study Number Page 1of 1 NA DESCRIPTION OF AMENDED SECTION Analytical Procedure Summaiy: V0001780;*Method of Analysis for the Determination of Perfluoraooctanoic Acid (PFOA) in Water by LC/MS/MS.' AMENDED TO The reanalysis of two water samples, DFO9-GW-131R-Q-040126 and DFB-GW-13SL-0W0121 will be performed using the following procedure: An aKquot of the water sample is spiked with the surrogate standard and/or other matrix spike compounds as appropriate. Add methanol to the fortified aqueous sample. Mix and aliquot for LC/MS/MS analysis.. The LC conditions include the use of a Beiasil C18, 2.1 x 50 mm column, a gradient of 30% 8 to 100% B in 10 minutes, where A=2mM ammonium acetate in water and B=Methanol (6 min equilibration). Use a 5 pL injection volume and a 0.3 mL/tnin flow rata LC conditions may be further modified as appropriate for the system being used. An interim report will be issued that will include the exact details of tne reanalysis. RATIONALE DF09-GW-131R-0-04Q12S and DF8b-GW-l35L-0-040121 could not be reported quantitatively in interim report #3 due to quality control sample failures. The study director and sponsor have requested reanalysis using the above conditions. IMPACT ON STUDY No negative impact on the study. More usable data may be obtained. ... W i k K L study a re c ^ V n a tu n f ^ Principal Investigator Signature Study Director Management Signature * Exygen Management Signature Sponsor Signature (if required) J it D a te / >' Date Date Date Date Exygen QAU Review /ft -*. LIBRARY ID: VD001228-8 RECEIVED TIME SEP. 14. 10:37811 Exygen Research ADMINISTRATIVE FORM PRINT TIME SEP. 14. 10:3801 Page 138 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 3058 Research Drive Phone: 814-272-1039 S ta te C ollege, PA 16801 Fax: 814-231-1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 6 09/20/05 P760 Client Study Number: Page 1 of 1 NA DESCRIPTION OF AM ENDED SECTION T he identification o f the tw o w ater sam ples listed in protocol am e n d m e nt No 5 are incorrect. The two w ater sam ples fo r DF8b-GW -135R-0-040121. AMENDED TO reanalysis should be DF09-GW -131R-0-040120 and R A T IO N A L E The correct sam ples are identified fo r reanalysis. IMPACT ON STUDY No negative im pact on the study. Study Director Signature . y ( / /, / '/ / Principal Investigator Signature Study Director M anagem ent Signature Exygen M artg^m eijL8tg nature Sponsor Signature (if required) D a te D a te D a te 2i~Jcp-0S D a te D a te Exvaen Q AU R eview ^ LIBRARY ID: V0001226-8 Exygen Research ADMINISTRATIVE FORM Page 139 of 140 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0000760 CHEM EHSR 236 1B SEP.3 0 .2 0 0 5 2 ! 29PM 651 733 1958 EXTGEN RESEARCH 09/20 '05 13:46 NO.920 02/02 m ,,, 3058 Research Drive Phone: 814*272-1039 5tate College, PA 16801 Fax: B14-23M580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 6 _ 09/20/05 P760 Oient Study Number Pasel of1 NA _________ _ ggSCgIEDONOFAMENDEDSECTION' The raentmcahon of the two water samples feted in protocol amendment No 5 are incorrect. 7 AMgN PE D T Q The two water samples for reaialysls should be DFO9-GW-131R-WWOT20 and DF8b-GW-13SR-0-040l2l. _. RATlPNaU The comet samplesaratefantiftetfforreanstysls. IMPACTONSTUDY nonegative impact onthestudy. atujyoir#Cwasnsur PUndpHWvssfigstorSlnatm -tIA.-- ' StudypraetorManagementSignatare Eq&tftMmsementSraMe SponsorSignatorygrrequired) Date CHIB -- Me bate . V*hs> 5S " r 7 -- ExyqanQAUReview /9u_**' 9/i.o fe LIBRARYID:V000122M RECEIVED TIME SEP.20. 3:01PM Exygen Research ADMINISTRATIVE PORM PRINT TIME .SEP.20. 3:02PM Page 140 of 140
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