Document DwmBBLpJvGEy6XLkGagLkZmd

INTERIM REPORT #24 - Analysis of Water. Stodge, and Sediment Samples STUDY TITLE Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) 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 27,2006 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: P0001131 Exygen Study Number: P0001131 Total Pages: 136 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number P0001131, entitled "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) 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. Exygen Research Jaisimha Kesari P.E., DEE Study Director Weston Solutions, Inc. I/M 6m Michael A. S Sponsor Representative 3M Company Exygen Research Date Date / / Page 2 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen Study Number P0001131, entitled, "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) 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 29. Interim Report and Raw Data Review Date Insoected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Studv Director 6/28/06 08/15/06 09/06/06 09/27/06 33. Raw Data Review 09/22/06 09/27/06 09/27/06 09/27/06 34. Final Interim Report 09/27/06 Review 09/27/06 09/27/06 09/27/06 '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 o f the interim report and associated raw data. Exygen Research Page 3 o f 136 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0001131 CERTIFICATION OF AUTHENTICITY This interim report, for Exygen Study Number P0001131, is a true and complete representation o f the raw data. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814)272-1039 Principal Investigator, Exygen: i l l ft) Xdhn Flaherty vice President Exygen Research / Date Exygen Research Facility Management: Exygen Research l> Date Study Director, Weston Solutions, Inc Jaisimha KesyHM., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: Michael A. Santoro Director of Regulatory Affairs Date Exygen Research Page 4 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 STUDY IDENTIFICATION Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) 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: P0001131 EXYGEN STUDY NUMBER: P0001131 TYPE OF STUDY: Residue SAMPLE MATRIX: Water, Sludge, and Sediment TEST SUBSTANCE: Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) SPONSOR: 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 STUDY DIRECTOR: Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 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: 07/13/06 Interim Report Completion Date: 09/27/06 Exygen Research Page 5 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 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 o f 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 Krista Gallant Scott Crain Brittany Kravets Cameala Graybill Kim Hall Title Vice President Laboratory Supervisor Technician Sample Custodian Associate Scientist Sample Custodian Technician Sample Custodian Technician Technician Technician Technician Technician Technician Technician Exygen Research Page 6 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 TABLE OF CONTENTS Page TITLE PAGE....................................................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................. 2 QUALITY ASSURANCE STATEMENT..........................................................................3 CERTFICATION 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........................................................ 14 6.1 Extraction Procedure for Water................................................................................ 14 6.2 Extraction Procedure for Sludge............................................................................... 14 6.3 Extraction Procedure for Sediment........................................................................... 15 6.4 Solvent Extraction Procedure For Sediment............................................................ 15 6.5 Percent Solids Determination For Sludge and Sediment.......................................... 15 6.6 Preparation of Standards and Fortification Solutions............................................... 15 6.7 Chromatography........ ........... 17 6.8 Instrument Sensitivity............................................................................... 17 6.9 Description of LC/MS/MS Instrument and Operating Conditions...........................17 6.10 Quantitation and Example Calculation................................................................... 18 7.0 EXPERIMENTAL DESIGN......................................................................................20 8.0 RESULTS.................................................................................................................. 21 9.0 CONCLUSIONS................................. 22 10.0 RETENTION OF DATA AND SAMPLES............................................................ 22 Exygen Research Page 7 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Table I. LIST OF TABLES Page Summary o f PFBS, PFHS, and PFOS in Water Samples............................... 24 Table EL Summary o f PFBS, PFHS, and PFOS in Re-extracted Water Samples..........28 Table HI. Summary of PFBS, PFHS, and PFOS in Sludge Samples.............................. 29 Table IV. Summary of PFBS, PFHS, and PFOS in Sediment Samples.......................... 30 Table V. Summary o f PFOS in Re-extracted Sediment Samples.................................31 Table VI. Matrix Spike Recovery of PFBS, PFHS and PFOS in Water Samples........ 32 Table VR Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Water Samples............................................................................................... 40 Table VIE. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sludge Samples....... 41 Table IX. Matrix Spike Recovery o f PFBS, PFHS and PFOS in Sediment Samples.......................................................................................................... 42 Table X. Matrix Spike Recovery of PFOS in Re-extracted Sediment Samples.......... 43 Table XI. Total Percent Solids for Sludge and Sediment Samples............................... 44 Exygen Research Page 8 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFBS in Reagent Water................................. 46 Figure 2. Extracted Standards of PFBS in Reagent Water, 25 ng/L and 50 ng/L, Respectively.................................................................................................. 47 Figure 3. PFBS in Reagent Control, 50 ng/L Fortified Reagent Spike A, and 500 ng/L Fortified Reagent Spike B, Respectively........ ................................48 Figure 4. Chromatogram Representing a Water Sample Analyzed for PFBS (Exygen ID: C0169336, Data Set: 042806B)................................................ 49 Figure 5. Chromatogram Representing a Sludge Sample Analyzed for PFBS (Exygen ID: C0171106, Data Set: 050906B)................................................ 50 Figure 6. Chromatogram Representing a Sediment Sample Analyzed for PFBS (Exygen ID: C0171108, Data Set: 051106A)............................................... 51 Figure 7. Typical Calibration Curve for PFHS in Reagent W ater................................ 52 Figure 8. Extracted Standards o f PFHS in Reagent Water, 25 ng/L and 50 ng/L, Respectively....................................................................................................53 Figure 9. PFHS in Reagent Control, 50 ng/L Fortified Reagent Spike A, and 500 ng/L Fortified Reagent Spike B, Respectively........................................ 54 Figure 10. Chromatogram Representing a Water Sample Analyzed for PFHS (Exygen ID: C0169336, Data Set: 042806B)................................................ 55 Figure 11. Chromatogram Representing a Sludge Sample Analyzed for PFHS (Exygen ID: C0171106, Data Set: 050906B)................................................ 56 Figure 12. Chromatogram Representing a Sediment Sample Analyzed for PFHS (Exygen ID: C0171108, Data Set: 051106A).................................................57 Figure 13. Typical Calibration Curve for PFOS in Reagent W ater................................ 58 Figure 14. Extracted Standards of PFOS in Reagent Water, 25 ng/L and 50 ng/L, Respectively..................... 59 Figure 15. PFOS in Reagent Control, 50 ng/L Fortified Reagent Spike A, and 500 ng/L Fortified Reagent Spike B, Respectively........................................ 60 Figure 16. Chromatogram Representing a Water Sample Analyzed for PFOS (Exygen ID: C0169336, Data Set: 042806B)................................................ 61 Figure 17. Chromatogram Representing a Sludge Sample Analyzed for PFOS (Exygen ID: C0171106, Data Set: 050906BR).............................................. 62 Figure 18. Chromatogram Representing a Sediment Sample Analyzed for PFOS (Exygen ID: C0171107, Data Set: 071106B).................................................63 Exygen Research . Page 9 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 LIST OF APPENDICES Page Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods, Protocol Amendments 1, 2, 8, and 10 and Deviations 5 and 8 .................................................................................... 64 Exygen Research Page 10 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o. : P0001131 1.0 SUMMARY Exygen Research extracted and analyzed water, sludge, and sediment samples for the determination o f perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Methods V0001780, V0001781, and V0001782, respectively (Appendix A). The limit of quantitation for PFBS, PFHS and PFOS in water was 25 ng/L. The limit of quantitation for PFBS, PFHS and PFOS in sludge and sediment was 0.2 ng/g (wet weight). The standard sediment method (V0001782) was originally used to extract the two sediment samples. Both o f the samples were not originally reported due to quality control failure for the analyte PFOS. Due to this reason, the samples were not re-analyzed using V0001782, but were solvent extracted and then analyzed, without further purification, through direct injection (Section 6.4). Analytical results for the analysis of PFBS, PFHS, and PFOS in water samples are summarized in Table I. Analytical results for the analysis o f PFBS, PFHS, and PFOS in re-extracted water samples are summarized in Table II. Analytical results for the analysis o f PFBS, PFHS, and PFOS in sludge samples are summarized in Table HI. Analytical results for the analysis of PFBS, PFHS, and PFOS in sediment samples are summarized in Table IV. Analytical results for the analysis o f PFOS in re-extracted sediment samples are summarized in Table V. Quantitative results were obtained for all samples and analytes except for PFBS in water samples from three sites and PFOS in sludge samples from one site, which are not reported (NR) due to quality control failures. Fortification recoveries for PFBS, PFHS and PFOS in the water samples are detailed in Table VI. The average percent recoveries standard deviations for PFBS, PFHS and PFOS in the water samples were 97 17%, 100 14% and 102 19%, respectively. Fortification recoveries for PFBS, PFHS and PFOS in the re-extracted water samples are detailed in Table VII. The average percent recoveries standard deviations for PFBS, PFHS and PFOS in the re-extracted water samples were 137 6%, 95 18% and 101 21%, respectively. Fortification recoveries for PFBS, PFHS and PFOS in the sludge samples are detailed in Table VIII. The average percent recoveries standard deviations for PFBS and PFHS in the sludge samples were 155 28% and 72 1%, respectively. PFOS results for the sludge samples were not reported due to quality control failures. Fortification recoveries for PFBS, PFHS and PFOS in the sediment samples are detailed in Table IX. The average percent recoveries standard deviations for PFBS and PFHS in the sediment samples were 60 18% and 85 10% respectively. Fortification recoveries for PFOS in the re-extracted sediment samples are detailed in Table X. The average percent recovery standard deviation for PFOS in the sediment samples was 70 Exygen Research Page 11 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 28%. The total percent solids for sludge and sediment samples are detailed in Table XL 2.0 OBJECTIVE The objective of the analytical part o f this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) in water, sludge, and sediment samples according to Protocol P0001131 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination o f PFBS, PFHS and PFOS 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" and a solvent extraction method, detailed in Section 6.4. The study was initiated on November 05, 2004, when the study director signed protocol number P0001131. The analytical start date for this interim report was May 9, 2006 and the analytical termination date for this interim report was July 14,2006. 4.0 ANALYTICAL TEST SAMPLES hi 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 Exygen Research Page 12 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o. : P0001131 The analytical standards, PFBS and PFHS, were supplied by 3M. PFBS was received from 3M at Exygen on May, 13, 2005. PFHS was received from 3M at Exygen on January, 20, 2003. PFOS was purchased from Fluka Corporation and was received at Exygen on April, 23, 2003. The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated. Compound Exygen Inventory No. Lot# Purity (%1 Expiration Date PFBS SP0005726 101 96.7 12/04/06 PFHS SP0002401 SE036 98.6 10/18/06 PFOS SP0002694 430180-1 101.2 10/31/07 The molecular structures of PFBS, PFHS and PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (GdFgSOa'K*) Transitions Monitored: 299 - 99 Structure: FF FF FF FF 3 PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFnSOaTC*) Transitions Monitored: 399 --> 80 Structure: FF FF FFF FF 3 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFnSOs'K*) Exygen Research Page 13 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Transitions Monitored: 499 - 80 Structure: 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 o f 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" and a direct injection method (Section 6.4) were used for this study. 6.1 Extraction Procedure for Water A 40 mL aliquot o f the water sample was used for the extraction procedure. After fortification o f appropriate samples, the samples were loaded onto a Cis SPE cartridge conditioned with 10 mL of methanol and 5 mL o f water. The eluate was discarded. Approximately five milliliters of methanol was added to file 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 in the sample container. The samples were then transferred back to the sampling container. A 5-gram portion o f sludge was weighed into a fifty-milliliter centrifuge tube for the extraction. After fortification o f 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 Cis 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. Exygen Research Page 14 o f 136 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0001131 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 o f sediment was weighed into a fiftymilliliter centrifuge tube for the extraction. After fortification o f 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 Cis SPE cartridge conditioned with 10 mL o f methanol and 20 mL o f water. The eluate was discarded. Twenty milliliters o f 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 o f methanol and 20 mL o f water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters o f eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.4 Solvent Extraction Procedure For Sediment Before the samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A one-gram portion o f sediment was weighed into a 15-milliliter centrifuge tube for the extraction. Ten milliliters o f 1% acetic acid in methanol was added to each sample. The samples were then shaken by hand, vortexed, and sonicated for thirty minutes. The samples were then centrifuged for ~10 minutes at -3000 rpm. Each sample was analyzed by LC/MS/MS electrospray. 6.5 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 o f the pan. The percent solid for each sample was then calculated. 6.6 Preparation of Standards and Fortification Solutions Exygen Research Page 15 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 A mixed stock standard solution o f PFBS, PFHS, PFOS was prepared at a concentration o f 1000 pg/mL by dissolving 100 mg of each of the standards (corrected for purity and salt content) in 100 mL o f 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 1o f PFBS, PFHS and PFOS Final Volume (mL) 100 100 100 100 100 Final Cone, of Fortification Std. (pg/mL) 100 10 1.0 0.1 0.01 A set of non-extracted calibration standards containing PFBS, PFHS, PFOS (for the sediment samples) was prepared in methanol, as specified in Exygen method V0001782. The following concentrations were prepared: Cone, o f Fort Fort Solution Volume (ng/mL)1 (mL) 100 1 100 0.5 100 0.2 10 1 51 21 1o f PFBS, PFHS and PFOS 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 o f extracted calibration standards containing PFBS, PFHS, and PFOS (for i sludge and water samples) was prepared in water before extraction, as specified Exygen method V0001780 and V0001781. Cone, of Fort Fort Volume of Solution Volume Fortified Sample (ng/mL)1 (mL) (mL) -- 40 10 0.1 40 10 0.2 40 10 0.4 40 100 0.1 40 100 0.2 40 100 0.4 40 1o f PFBS, PFHS, and PFOS Exygen Research Final Cone, of Calibration Std. (ng/L) 0 25 50 100 250 500 1000 Page 16 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation o f standard preparation is located in the raw data package associated with this interim report 6.7 Chromatography Quantification o f PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention time of PFBS, PFHS and PFOS was ~0.6 mins, ~9.1 mins, and ~11.6 mins, respectively. Peaks above the LOQ were not detected in any o f the reagent blank samples corresponding to the analyte retention time. 6.8 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 25 ng/L of PFBS, PFHS and PFOS for water samples and a concentration o f 0.2 ng/mL o f PFBS, PFHS and PFOS for sludge and sediment samples. 6.9 Description of LC/MS/MS Instrument and Operating Conditions Instrument: API 4000 Biomolecular Mass Analyzer Interface: Turbo Ion Spray Liquid Introduction Interface Computer: DELL OptiPlex GX400 Software: Windows NT, Analyst 1.4.1 HPLC : 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 %A 0.0 65 1.0 65 8.0 25 10.0 25 11.0 65 18.0 65 %B 35 35 75 75 35 35 Total run time: ~18 min Flow Rate: 0.3 mL/min Ions monitored: Exygen Research Page 17 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Analvte PFBS PFHS PFOS Mode negative negative negative Transition Monitored 299 -99 399 -> 80 499 - 80 Approximate Retention Time (miri) ~0.6 min. ~9.1 min. -11.6 min. 6.10 Quantitation and Example Calculation Fifteen microliters o f 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 o f standards. The concentration was determined from the equations below. Equation 1 calculated the amount of analyte found, in ng/L, based on peak area for water and sludge samples, using the standard curve (linear regression parameters) generated by the Analyst software program. For the sediment samples, the equation is the same but the units differ due to the use of non-extracted standards. The amount o f analyte found for sediment samples is in the units of ng/mL, based on peak area. 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 PFBS, PFHS, and PFOS prior to extraction, Equation 2 was used to calculate the percent recovery. Equation 2: Recovery (%) = (analyte found (ng/L) - analyte in control (ng/LV) xl00% amount added (ng/L) Note: For the analyte recovery calculation, the "control" is the unspiked aliquot of the primary field sample. Equation 3 was used to convert the amount of analyte found in ng/L to ng/g (ppb). Equation 3: analyte found (ppb) = fanalvte found (ng/L) x volume extracted (0.04D1 Exygen Research Page 18 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 sample weight (5 g) Note: Equation three is altered for calculation o f sediment in ppb. The extracted volume is equal to 5 mL in place of the 0.04 L amount shown in the equation. The equation below is the alternate equation to calculate ppb for sediment by the direct solvent injection method. Analyte found Direct Inject (ppb) = iAnalyte found (ng/mLl x final volume HO mLll sample weight (1 g) Equation 4 was then used to calculate the amount o f analyte found in ppb based on dry weight. Equation 4: analyte found (ppb) dry weight = analyte found (ppb) x [100% / total solids(%)] NOTE: Total solids (%) = [wet weight (g) / dry weight (g)] x 100% An example o f a calculation using an actual sample follows: Sludge sample Exygen ID C0171106 Spk C (Set: 050906B), fortified at 500 ng/L with PFHS where: peak area = 120539 intercept = 0.000229 slope = 229 dilution factor =1 ng/L PFHS added (fort level) ng/L in corresponding sample (ng/L) volume extracted (L) sample weight (g) total solids (%) = = = = = 500 161 0.04 5 22.83 From equation 1: Analyte found (ng/L) = f120539 - 0.0002291 x 1 229 = 526 ng/L From equation 2: % Recovery = (526 ng/L - 161 ng/L) x 100% 500 ng/L = 73% From equation 3: PFHS found (ppb) Exygen Research = (526ne/Lx 0.04L1 Page 19 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o. : P0001131 5g = 4.21 ppb From equation 4: PFHS found (ppb) dry weight = 4.21 ppb x (100% / 22.83%) = 18.4 ppb Note: Numbers may vary slightly due to rounding. 7.0 EXPERIMENTAL DESIGN For water samples designated as field matrix spikes, PFBS, PFHS, and PFOS was 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. One water sample bottle was filled to 230 mL, and three water sample bottles were filled to 250 mL in the field. Deviation 8 addresses the volumetric difference. 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 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 with PFBS, PFHS, and PFOS. 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 o f the sample and two laboratory matrix spikes were also extracted. The laboratory spikes were fortified with known concentrations o f PFBS, PFHS, and PFOS. 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 PFBS, PFHS, and PFOS. 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. Exygen Research Page 20 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 The re-extracted sediment samples were analyzed in one set. The set included one reagent blank, and two reagent blanks fortified at known concentrations. For each sample, a sample and two laboratory matrix spikes were also extracted by the direct solvent injection method. The laboratory spikes were fortified with known concentrations PFBS, PFHS, and PFOS. 8.0 RESULTS Analytical results for the analysis of PFBS, PFHS, and PFOS in water samples are summarized in Table I. Analytical results for the analysis o f PFBS, PFHS, and PFOS in re-extracted water samples are summarized in Table II. Analytical results for the analysis of PFBS, PFHS, and PFOS in sludge samples are summarized in Table III. Analytical results for the analysis of PFBS, PFHS, and PFOS in sediment samples are summarized in Table IV. Analytical results for the analysis of PFOS in re-extracted sediment samples are summarized in Table V. Quantitative results were obtained for all samples and analytes except for PFBS in water samples from three sites and PFOS in sludge samples from one site, which 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 the accuracy, hi instances o f 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 o f failed laboratory spike recoveries, the samples were not reported due to the quality control failure. The standard sediment method (V0001782) was originally used to extract the two sediment samples. Both o f the samples were not originally reported due to quality control failure for the analyte PFOS. Due to this reason, the samples were not re-analyzed using V0001782, but were solvent extracted and then analyzed, without further purification, through direct injection (Section 6.4). Fortification recoveries for PFBS, PFHS and PFOS in the water samples are detailed in Table VI. The average percent recoveries standard deviations for PFBS, PFHS and PFOS in the water samples were 97 17%, 100 14% and 102 19%, respectively. Fortification recoveries for PFBS, PFHS and PFOS in the re-extracted water samples are detailed in Table VII. The average percent recoveries standard deviations for PFBS, PFHS and PFOS in the re-extracted water samples were 137 6%, 95 18% and 101 21%, respectively. Fortification recoveries for PFBS, PFHS and PFOS in the sludge samples are detailed in Table VIII. The average percent recoveries standard deviations for PFBS and PFHS in the sludge samples were 155 28% and 72 1%, respectively. Exygen Research Page 21 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 PFOS results for the sludge samples were not reported due to quality control failures. Fortification recoveries for PFBS, PFHS and PFOS in the sediment samples are detailed in Table IX. The average percent recoveries standard deviations for PFBS and PFHS in the sediment samples were 60 18% and 85 10% respectively. Fortification recoveries for PFOS in the re-extracted sediment samples are detailed in Table X. The average percent recovery standard deviation for PFOS in the sediment samples was 70 28%. The total percent solids for sludge and sediment samples are detailed in Table XL 9.0 CONCLUSIONS Except as noted above, the ground water, sludge, and sediment samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical methods V0001780, V0001781, V0001782, and a solvent direct injection method, respectively. There were no circumstances that may have affected the data quality or integrity. 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 o f the interim analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period o f time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. Exygen Research Page 22 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 TABLES Exygen Research Page 23 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Table I. Summary of PFBS, PFHS, and PFOS in W ater Samples E xva e nID CO169333 C0169336 CO169336 R ep C0169337 C0169341 C0169341 Rep C0169342 C0169346 C 0169346 Rep CO169347 C0169351 C0169351 Rep C0169352 C0169355 CO169355 Rep C0169356 C0169359 CO169359 R ep C0169360 C0169363 C 0169363 Rep C0169364 C0169367 C0169367 Rep C0169368 C lie n t Sam ple ID D AL-G W -TR IP1-0-060411 D A L-G W -138L-0-060411 D A L-G W -138L-0-060411* D AL-G W -138L-D B-060411 D A L-G W -138R -0-060412 D A L-G W -138R -0-060412* D AL-G W -138R -D B-060412 D AL-G W -138S -0-060412 D AL-G W -138S -0-060412* D A L-G W -138S -D B -060412'1 D A L-G W -605R -0-060412 D A L-G W -605R -0-060412* D A L-G W -605R -D B -060412 D A L-G W -605L-0-060412 D AL-G W -605L-0-060412* D A L-G W -605L-D B -060412 D A L-G W -603S -0-060412 D AL-G W -603S -0-060412* D AL-G W -603S-D B-060412 D AL-G W -604L-0-060412 D AL-G W -604L-0-060412* D A L-G W -604L-D B -060412 D AL-G W -604S -0-060412 D A L-G W -604S -0-060412* D AL-G W -604S-D B-060412 C4 Sulfonate PFBS PsrfluorobutinesuHonsts Analyte Found (no/U Assessed Accuracy (+/-% > ND 30 8 1 .7 79.3 8 2 .2 30 30 30 7400 7750 7780 30 30 30 4650 4710 4750 30 30 30 6 5 .7 73.1 6 4 .3 30 30 30 ND 30 ND 30 ND 30 9 6 .2 104 101 30 30 30 186 40 177 40 170 40 112 108 95.6 30 30 30 C6 Sulfonate PFHS C8 Sulfonate PFOS Pffluorohsxanssulfonats_____ Psrfluorooetsnssulfonats Analyte Assessed Analyte Assessed Found (no/L) Accuracy <+/-%> Found (ng/L) Accuracy <+/-% ) ND 30 ND 30 8 5 .0 9 1 .0 9 0 .6 30 30 30 152 30 164 30 162 30 32800 30 330000 30 36400 30 330000 30 34300 30 357000 30 22000 30 156000 30 21200 30 161000 30 23900 30 172000 30 4 5 .4 5 2 .4 2 7 .7 30 30 30 ND 2 5 .7 ND 30 30 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 103 30 240 30 9 8 .2 30 236 30 118 30 288 30 260 30 850 50 256 30 824 50 310 30 1160 50 217 30 242 30 206 30 242 30 228 30 314 30 Laboratory Duplicato ASample was botila was (Med to 250 mL. ND = Not detected at o r above the Lim it o f Quantitation (LOQ) o f 25 ng/L. Exygen Research Page 24 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Table I. Summary of PFBS, PFHS, and PFOS in W ater Samples C on tin u ed Exygen ID C 0170984 C0170984 Rep CO170985 C 0 70988 CO170988 Rep C 0170989 CO170992 C0170992 Rep C0170993 C0170996 C0170996 Rep C0170997 C0171000 C0171000 Rep C0171001 C 0171004 C0171004 Rep C 0171005 C 0171008 C0171008 Rep C 0171009 C 0171012 C 0171012R ep C0171013 C 0171016 C0171016 Rep C0171017 C0171020 C0171020 Rep C0171021 C0171025 C0171025 Rep C 0171026 C lie n t S am ple ID D AL-G W -601R -0-060413 D AL-G W -801R -0-060413* D A L-G W -601R -D B -060413 D AL-G W -601S -0-060413 D AL-G W -601S -0-060413` D AL-G W -601S -D B -060413 D AL-G W -602R -0-060413 D A L-G W -602R -0-060413* D A L-G W -602R -D B -060413 D AL-G W -601 L-0-060413 D AL-G W -601L-0-060413* D A L-G W -601L-D B -060413 D A L-G W -602S -0-060413 D A L-G W -602S -0-060413* D AL-G W -602S -0-06413 D A L-G W -602L-0-060413 D A L-G W -602L-0-060413* D AL-G W -602L-D B -060413 D AL-G W -604R -0-060413 D AL-G W -604R -0-060413* D AL-G W -604R -D B-060413 D AL-G W -603R -0-060413 D AL-G W -603R -0-060413* D AL-G W -603R -D B-060413 D A L-G W -603L-0-060413 D AL-G W -603L-0-060413* D A L-G W -603L-D B -060413 D A L-S W -B P P 01-0-060413 DAL-SW -BPPO1-O-O0O413* D A L-SW -BPP01-0-060413 D AL-SW -BPP02-0-060413 D AL-SW -BPP02-0-060413* D AL-SW -BPP02-D B-060413 C4 Sulfonate PFBS P rfluprobutanesutfonat Analyte Found (n o fl.) A--eased Accuracy <+/-% ) NR NR NR NR NR NR 218 50 218 50 228 50 NR NR NR NR NR NR 117 30 111 30 117 30 117 30 121 30 118 30 201 30 215 30 195 30 44.1 4 3 .2 4 4 .9 30 30 30 7630 7320 7510 5030 5250 5110 30 30 30 30 30 30 CS Sulfonate PFHS C* Sulfonate PFOS Perftucrphaxaneiutfcnata_____ P eriluprppctan--utfpnata Analyte Found (no/L) Assessed Accuracy (/-% ) Analyte Found (no/L) A--eased Accuracy </-% ) ND 30 ND 40 ND 30 ND 40 2 5 .4 30 ND 40 ND 2 9 .2 ND 30 30 30 2 5 .8 3 6 .7 4 0 .8 30 30 30 112 30 188 50 113 30 171 50 121 30 220 50 NR - 2350 30 NR - 2380 30 NR - 2890 30 141 30 558 30 130 30 528 30 139 30 645 30 454 30 469 30 460 30 NR NR NR - 137 30 893 30 149 30 845 30 152 30 1020 30 323 30 120 50 333 30 108 50 332 30 133 50 4 8 .4 30 170 50 4 2 .0 30 166 50 4 8 .0 30 210 50 60400 61800 60100 40100 40000 39000 30 30 30 30 30 30 NR NR NR NR NR NR - . - ` Laboratory Duplicate ND > Not detected a t or above the Lim it o f Quantitation (LOQ) o f 25 nfl/L. NR Not Reported due to Q uality Control Failures, See Table II fo r re-extract data. Exygen Research Page 25 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Table I. Summary o f PFBS, PFHS, and PFOS in W ater Samples C on tin u ed Exvnen ID C0171030 C0171030 Rep C0171031 C0171035 C0171035 Rep C0171036 C 0171039 C0171039 Rep C0171040 C 0171043 C0171043 Rep C0171044 C 0171047 C 017 1 0 4 7 R ep C0171048 C0171051 C0171051 Rep C0171052 C0171055 C0171055 Rep C 0171056 C 0171059 C0171059 Rep C 0171060 C0171063 C0171063 Rep C0171064 C0171067 C0171067 Rep C 0171068 C lie n t S am ple ID D A L-S W -B P P 03-0-060413 D AL-S W -B P P 03-0-060413* D AL-SW -BPP03-D B-060413 D AL-SW -BC T01-0-060412 D A L-S W -B C T01-0-060412* D AL-SW -BCT01 -D B -060412 D AL-S W -B C T02-0-060412 D A L-S W -B C T02-0-060412* D A L-S W -B C T02-D B -060412 D A L-S W -B C T03-0-060412 D A L-S W -B C T03-0-060412* D A L-S W -B C T03-D B -060412 D A L-S W -B C 01-0-060412 D A L-S W -B C 01-0-060412* D A L-S W -B C 01-D B -060412 D A L-S W -B C 02-0-060412 D A L-S W -B C 02-0-060412* D AL-S W -B C 02-D B -060412 D AL-S W -B C 03-0-060414 D A L-S W -B C 03-0-060414* D AL-S W -B C 03-D B -060414 D AL-S W -B C 04-0-060414 D AL-S W -B C 04-0-060414 * D A L-S W -B C 04-D B -060414 D AL-S W -B C 05-0-060414 D A L-S W -B C 05-0-060414* D A L-S W -B C 05-D B -060414 D AL-SW -O SP01-0-060413 D AL-SW -O S P 01-0-060413* DAL-SW -O SP01 -D B -060413 C4 Sulfonate PFBS CS Sulfonate PFHS C l Sulfonate PFOS Partluorobutanaaulfonala_____ParfluofolKxanaaulfonaf_____ Psrtluorooctanasulfonate Analyte Found Assessed Accuracy Analyte Found Assessed Accuracy Analyte Found Assessed Accuracy (no/U (/-% ) (non.) (/-% ) (n o fl.) (/-% ) 5300 5280 5040 30 39100 50 30 38700 50 30 36900 50 NR NR NR - 492 30 2250 30 16100 30 455 30 1990 30 12600 30 489 30 2290 30 16500 30 2290 50 14000 30 2210 50 13400 30 2290 50 13300 30 NR NR NR - - 1010 957 948 30 30 30 4390 4290 4720 30 30 30 NR NR NR - - 65.3 30 31.5 30 65.1 30 31.7 30 64.1 30 31.6 30 6 4 .0 59.1 65.1 30 30 30 67.1 6 8 .3 6 5 .4 30 30 30 3 6 .3 3 7 .9 3 8 .9 30 30 30 8 5 .8 9 5 .7 93.1 30 30 30 150 30 430 30 3630 30 155 30 446 30 3820 30 155 30 451 30 4310 30 508 30 2260 30 481 30 2150 30 491 30 2120 30 NR NR NR . * 69.0 30 64.0 30 716 30 6 1 .6 30 6 6 .8 30 693 30 65.2 30 56.5 30 696 30 196 30 46.7 30 602 30 182 30 44.8 30 522 30 189 30 47.0 30 555 30 Laboratory Duplicate ND = Not detected at o r above the Lim it o f Quantitation (LOQ) of 25 ng/L. NR a Not Reported due to Q uality Control Failures, See Table II fo r re-exlract data. Exygen Research Page 26 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Table I. Summary o f PFBS, PFHS, and PFOS in W ater Samples C o n tin u ed E xva e nID C lie n t S am ple ID C4 Sulfonate PFBS Psrlluorobutanasultenste Analyte Assessed Found Accuracy (ng/L) <*-% > C 0171071 C0171071 Rep C 0171072 D A L-S W -O S P 02-0-060413 D AL-SW -O S P 02-0-060413* D A L-S W -O S P 02-D B -060413 343 350 334 30 30 30 C0171075 C0171075 Rep C0171076 D A L-S W -O S P 03-0-060413 D AL-SW -O S P 03-0-060413* D A L-S W -O S P 03-D B -060413 319 311 319 30 30 30 C0171079 C0171079 Rep C0171080 D AL-G W -BPW ELU -0-080413 D A L-G W -B P W E LL-0-060413 * D A L-G W -BPW ELL-D B-060413 18800 19700 20400 50 50 50 C0171084 C0171084 Rep C 0171085 D P W S -W W I-D C TP 01-0-060413 D P W S -W W I-D C TP 01-0-060413* D PW S-W W I-D C TP01-D B-060413 66.8 7 0 .3 6 6 .0 30 30 30 C 0171088 C0171088 Rep C 0171089 D PW S-W W E -D C TP 01-0-060413* D PW S-W W E -D C TP01-0-060413*A D PW S-W W E -D C TP01-D B-060413 8 5 .3 8 2 .8 7 8 .7 30 30 30 C 0171092 C0171092 Rep C0171093 D AL-W W -E FF01-0-060413 D AL-W W -EFF01-0-060413* D A L-W W -E FF01-D B -060413 312 312 303 30 30 30 C0171096 C0171096 Rep C0171097 D AU-LCH-M CLF01-0-060414 DAL-LC H-M C LF01 -0-060414* D AL-LC H -M C LF01-D B-060414 5180 4650 5010 30 30 30 C 0171100 D A L-G W -TR IP 1-0-060412 ND 30 C0171103 D A L-W W -TR IP 1-0-060413 ND 30 C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorohsxaneaulfonata_____Periluorooctanesutfonata Analyte Assessed A n a lyte Assessed Found ("0 -) Accuracy (+/-% > Found <no/L) Accuracy (+/-% ) 555 30 2670 30 526 30 2600 30 605 30 3180 30 501 30 2290 30 482 30 2490 30 502 30 2640 30 136000 40 834000 30 160000 40 957000 30 195000 40 1190000 30 102 30 698 30 100 30 733 30 104 30 765 30 106 30 574 30 106 30 575 30 101 30 565 30 815 30 4390 30 779 30 4090 30 842 30 4960 30 17600 16400 17800 30 30 30 27200 26600 29500 30 30 30 ND 30 ND 30 ND 30 ND 30 'Laboratory Duplicate Sampte was bottle was lilted to 230 mL. ND * Not detected a t or above the Lim it o f Quantitation (LOQ) of 25 ng/L. Exygen Research Page 27 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Table II. Summary o f PFBS, PFHS, and PFOS in Re-extracted W ater Samples E xygenID C0169367 C 0170984 C0170988 Rep C0170989 C0170997 C0171000 C0171004 C0171012 C0171021 C0171026 C0171030 C0171040 C0171044 C0171060 C0171063 C0171076 C0171097 C lient Sam ple ID D AL-G W -604S-0-060412 D AL-G W -601R -0-060413 D AL-G W -601S-0-060413* D AL-G W -601S-D B-060413 D AL-G W -601L-D B-060413 DAL-G W -602S -0-060413 D AL-G W -602L-0-060413 D AL-G W -603R -0-060413 D AL-SW -BPP01-D B-060413 D AL-SW -BPP02-D B-060413 D AL-SW -BPP03-0-060413 D AL-SW -BC T02-D B-060412 D AL-SW -BC T03-D B-060412 DAL-SW -BC 04-D B-060414 DAL-SW -B C 05-0-060414 DAL-SW -O SP03-D B-060413 D AL-LC H-M C LF01-DB-060414 C4 Sulfonate PFBS Pyfluorobutanesulfonet Analyte Found (ng/L) Assessed Accuracy (+/-% ) CS Sulfonate PFHS PfSuoroht.n--ulfo n .fr Analyte Found (ng/L) Assessed Accuracy (+/-% > C8 Sulfonate PFOS Ifrftluoroocfrrfrw jifoffrfr Analyte Found (ng/L) Assessed Accuracy <+/-% ) 156 30 199 30 287 30 NR - - - NR - NR . . .. NR - - " - - NR - 20800 30 - - 192 50 - - - - - - - - 1770 30 - - - - NR - - - - - 437000 30 7680 40 - - 354000 30 - - - - 326000 50 - - - - 279000 30 - - - - 44200 30 - - - - 27700 30 91.7 30 59.8 30 640 30 7130 30 - - 2100 30 - 26400 30 * Laboratory Duplicate ND > Not detected at o r above the Lim it of Quantitation (LOQ) o f 25 ng/L. NR * Not Reported due to Q uality Control Failures. Exygen Research Page 28 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Table III. Summary o f PFBS, PFHS, and PFOS in Sludge Samples E xvnen ID C 0171108 C 0171106 Rap C lie n t S am ple ID C4 Sulfonate PFBS Partluorobutencsultoaate Analyte Found Assessed (ppb, ng/g) Drv W eight A ccu racy (+ /-% ) C6 Sulfonate PFHS Parlluorohsxanasutfonate Analyte Found Assessed (ppb. ng/g) Dry W eight A ccu ra cy </-% ) C8 Sulfonate PFOS PatBuorooctencsuHonali Analyte Found Assessed (ppb, ng/g) D ry W eight A ccu racy (+ /-% ) D PW S-SL-D C TP01 <1-0000 3 .5 4 40 5 .6 4 30 D PW S-SL-D C TP01 -0-0000* 3 .0 6 40 4 .8 4 30 NR NR * Laboratory Duplicate NR = Not Reported dua to Q uality C ontrol Failures. Exygen Research Page 29 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Table IV. Summary o f PFBS, PFHS, and PFOS in Sediment Samples Exygen ID C0171107 C0171107Rep C0171108 C0171108 Rep C lie n t Sam ple ID D AL-SD -O SP01-0-0000 D AL-SD -O SP01-0-0000* D AL-SD-O SP02-Q -0000 D A L-S D -O S F '0 2 -0 -0 0 0 0 * C4 Sulfonate PFBS CSSulfonate PFHS C t Sulfonate PFOS Pwfluofobutanwulfonrt.______ PwfluofohaMMHBiite_______Psrtluorooclanssulfonat Analyte Found (ppb. ng/g) Dry W eight Asaessed Accuracy (/-% ) Analyte Found (ppb, ng/g) Dry W eight Assessed Accuracy (*/ % ) Analyte Found (ppb. ng/g) Dry W eight Assessed Accuracy (*/-% ) 0.981 ND 50 0.368 30 50 ND 30 NR NR NR NR ND 30 0.806 30 NR NR ND 30 1.11 30 NR NR ` Laboratory Duplicate ND = Not detected at or above the Lim it o f Quantitation (LOQ) o( 0.2 ng/g (wet weight). NR Not Reported due to Qulatty Control failures, see Tabla V. Exygen Research Page 30 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Table V. Summary o f PFOS in Re-extracted Sediment Samples Exygen ID C0171107 C0171108 Client Sample ID DAL-SD-OSP01-0-0000 DAL-SD-OSP02-0-0000 C8 Sulfonate PFOS P a rflu o ro o c ta n M u lfb n a to Analyte Found (PPb. ng/g) Dry W eight Assessed A c cu ra cy (+/-% ) 10.3 50 75.9 30 Exygen Research Page 31 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Table VI. M atrix Spike Recovery of PFBS, PFHS and PFOS in W ater S am p les Sam plt a scrip tion DAL-GW-TRIP1-LS-060411 (C01SSU4,1.0ppb Field Spfce) DAL-GW-TRIP1-HS-060411 (C01SS335,10.0ppb Field Spike) DAL-GW-138L-0-060411 (CS1SSSMSpkC, 10.0ppbLabSpite) DAL-GW-138L-0-060411 (COieSSMSpkD, 1000ppb LabSpite) DAL-GW-138L-LS-060411 (C0100SM, 10.0ppb Field Spite) DAL-GW-130L-MS-O6O411 (C01000SO, 100ppb Field Spike) DAL-GW-138L-HS-060411 (C0100S40.1000ppb Field Spite) DAL-GW-138R-0-060412 (C0100S41 SpkE, 10ppbLabSpite) DAL-GW-138R-0-060412 (C0100041 SpkF. 1000ppb LabSpite) DAL-GW-138R-LS-060412 (00100943,10ppb Field Sptite) DAL-GW-138R-MS-060412 (001*9944,100ppb FWdSpike) DAL-GW-138R-HS-060412 (C0100940,1000ppb Field Spite) DAL-GW-138S-0-060412 (00100940SpkC, 10ppb LabSpike) DAL-GW-138S-0-060412 (00100940SpkD, 1000ppb LabSpite) DAL-GW-138S-LS-060412 (coioo9a. io ppb FWdspace) DAL-GW-138S-MS-060412 (ceieesee. iee ppb Field Spine) DAL-G W -138S-H S-060412 (C01SS9S0,1000ppb FWdSpike) Amount Spiked (nort.) C4 Sulfonate PFBS___________ C6 Sulfonate PFHS___________ CB Sulfonate PFOS Amount Found In Amount Amount Found In Amount Amount Found In Amount Semple Recovered (0-) (no/L> Rec Samplt R tcovtrtd <%) (ng/L) Rec Samplt Racowrad <%> (ng/L) (ngA.) Rec <*> 1000 10000 ND ND 997 9870 100 ND 99 ND 1050 10100 105 ND 101 ND 1040 10100 104 101 10000 81.7 1000000 81.7 10000 81.7 100000 81.7 1000000 81.7 10000 7400 1000000 7400 10000 7400 100000 7400 1000000 7400 10000 4650 1000000 NR 10000 4650 100000 4650 1000000 4650 10300 1000000 10000 99100 976000 17100 1020000 17700 112000 963000 12200 NR 12300 104000 895000 102 85.0 100 85.0 99 85.0 99 85.0 98 85.0 9760 988000 10400 107000 999000 97 32800 101 32800 103 32800 105 32800 96 32800 41500 1030000 44700 138000 984000 76 22000 NR NR 77 22000 99 22000 89 22000 28800 NR 28400 140000 1120000 97 152 99 152 103 152 107 152 100 152 9910 975000 10600 114000 1250000 330000 295000 100 330000 * 330000 1350000 289000 105 330000 345000 95 330000 1160000 68 156000 169000 NR NR NR 64 156000 154000 118 156000 279000 110 156000 1360000 98 97 104 114 125 102 83 NR 123 120 Sanple residue exceeds the spiking level significantly: 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 FaRures, See Table VII for re-extract data. Note: Since this summary table shows rounded results, recoveryvalues mayvary slightly from the values In the raw data. Exygen Research Page 32 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Table VI. M atrix Spike Recovery o f PFBS, PFHS and PFOS in W ater Samples Continued Sample D escription DAL-GW-605R-0-060412 (C01MU1 SpkE,0.1 ppb LabSptfca) DAL-GW-605R-0-060412 (C01CM81 SpkF, 1.0ppb LabSpHca) DAL-GW-605R-LS-060412 (C010MSS,0.1ppb FlaWSpEta) DAL-GW-605R-HS-060412 (C0100M4,14 ppb FiaMSpika)A DAL-GW-605L-0-060412 (C0100SSSSpk0 ,0.1 ppb LabSpllw) DAL-GW-605L-0-060412 (C0100M0SpkH, 14 ppbLabSpNta) DAL-GW-605L-LS-060412 (C01I03I7,0.1ppb FWWSpHca) DAL-GW-605L-HS-060412 (C0100U0.1.0ppb FWWSpEta) DAL-GW-603$"0"060412 (COIPOMOSpkC.0,1 ppbLabSpNta) DAL-GW-603S-0-060412 (COI00900SpkD, 1.0ppbLabSpNta) DAL-GW-603S-LS-060412 (C0100301,0.1 ppb FWWSpan) DAL-GW-603S-HS-060412 (C0100902,1.0ppb FWWSpNta) DAL-GW-604L-0-060412 (COI00909SpkE,0.1 ppb LabSplha) DAL-GW-604L-0-060412 (00100909 SpkF, 1.0ppbLabSplha) DAL-GW-604L-LS-060412 (C0100905,0.1 ppb FWWSpNta) DAL-GW-604L-HS-060412 (coiaasaa, 1.0 ppb mamspun) DAL-GW-604S-0-060412 (C0100907SpkG,0.1 ppb LabSplha) DAL-GW-604S-0-060412 (C0100907spk H, 1.0ppb LabSpNta) DAL-GW-604S-LS-060412 (C0100900,0.1 ppbFWWSpNta) DAL-GW-604S-HS-060412 (COI00970,1.0ppb FWWSpNta) Amount Splkod ingfl.) C4 Sulfonate PFBS___________ C6 Sulfonate PFHS___________ C6 Sulfonate PFOS Amount Amount Amount Found In Amount Found In Amount Found In Amount Sampla Racovarad ("9-) Rac SampW Racovarad l* f (ngn.) (n tjfij Rac Sampla Racovarad (%) (no/U Ino/L) Rac (%1 100 1000 100 800 65.7 65.7 65.7 65.7 164 98 45.4 917 85 45.4 166 100 45.4 861 99 45.4 138 93 ND 990 94 ND 140 95 ND 938 112 ND 108 1000 90.5 891 108 100 91 111 100 1000 100 1000 ND ND ND ND 104 104 ND 925 93 ND 97.3 97 ND 800 60 ND 103 917 110 1020 103 ND 92 ND 110 ND 102 ND 91.2 91 944 94 112 112 935 94 100 1000 100 1000 96.2 96.2 96.2 96.2 100 1000 100 1000 186 166 186 186 210 1190 183 1060 278 1120 248 1150 114 103 109 103 87 103 96 103 92 260 93 260 62 260 96 260 216 1110 223 1170 376 1260 353 1260 113 240 101 240 120 240 107 240 116 850 100 850 93 850 100 850 374 1110 392 1150 1180 1910 1180 2300 134 87 152 91 106 145 100 1000 100 1000 112 112 NR 112 191 1070 NR 1070 79 217 96 217 NR NR 96 217 298 1270 NR 1250 81 242 105 242 NR NR 103 242 341 1260 NR 1330 99 102 NR 109 'Sample residue exceeds die spiking level significantly; therefore, an accurate recovery value cannot be calculated * Sample was bottle was filled to 250 mL, causing the spiking level to be modified. ND Not detected at or above the Limit of Quantitation (LOO) of 25 ng/L. NR Not Reported due to d ua lly Control Failures, See Table VII for reextract data. Note: Since this summary table shows rounded results, recovery values mayvary slightly from the values In the raw data. Exygen Research Page 33 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Table VI. M atrix Spike Recovery o f PFBS, PFHS and PFOS in W ater Samples Continued Sample D escription DAL-GW-601R-0-060413 (CJH70M4 Spk C, 0.1 ppb U b S p ite) DAL-GW-601R-0-060413 (CO170M4 Spk D. 1.0 ppb U b S p i ) DAL-GW-601R-LS-060413 (C0170U0, o.i ppb FMd Span) DAL-GW-601R-HS-060413 (C0170007,1.0 ppb FM d Spa) DAL-GW-601S-0-060413 (00170000 Spk E, 0.1 ppb U b S pite) DAL-GW-601S-0-060413 (C0170000 Spk P . I * ppb U b S pite) DAL-GW-601S-LS-060413 (C0170000,0.1 ppb FM d SpOw) DAL-GW-501S-HS-060413 (C0170001,1.0 ppb FMd S pite) DAL-GW-602R-0-060413 (COITOMI Spk 0 ,0.1 ppb U b S pite) DAL-GW-602R-0-060413 (C0170M2 Spk H. 1.0 ppb U b SpiM) DAL-GW-602R-LS-060413 (C0170004,0.1 ppb FteM S pite) DAL-GW-602R-HS-060413 (COI70000,1.0 ppb FM d SpNtO) DAL-GW-601L-0-060413 (C0170000 Spk C, 0.1 ppb Ub SpNca) DAL-GW-601L-0-060413 (C0170000Spk D, 1.0ppb U b Spfco) DAL-GW-601L-LS-080413 (COI70000,0.1 ppb Flow S pa) DAL-GW-601 L-HS-060413 (COITO000,1.0ppb FMdSpite) DAL-GW-602S-0-060413 (C0171000 SpkE, 0.1 ppb Ub Spite) DAL-GW-602S-0-060413 (C0171000Spk F, 1.0 ppb Ub sp ite ) DAL-GW-802S-LS-060413 (C0171002,0.1 ppb Flotd Spite) DAL-GW-602S-HS-060413 (C0171000,1.0ppb FMd Spite) Amount Spited (ngfl.) C4 Sultanat PFB8___________ C8 Sulfonate PFHS___________ C3 Sulfonate PFOS Amount Amount Amount Found in Amount Found in Amount Found In Amount Sampls Rscovsrsd (ng/L) (ng/L) Rc Samplp Rscovbisd n ("O-) (ng/L) Rpc Sample Recovered w (ng/L) (naff.) Rk (%) 100 NR NR NR ND 115 115 ND 107 107 1000 100 1000 NR NR NR NR NR ND NR NR ND NR NR ND 940 94 ND 948 95 127 127 ND 140 140 870 87 ND 1020 102 100 1000 100 1000 NR NR NR NR NR NR NO NR NR ND NR NR ND NR NR ND 107 107 25.8 905 91 25.8 120 120 25.8 546 55 25.8 123 97 939 91 154 128 659 63 100 1000 100 1000 218 218 218 218 100 1000 100 1000 100 1000 100 1000 NR NR NR NR NR NR NR NR 344 1920 365 1720 NR NR NR NR NR NR NR NR 126 112 170 112 147 112 150 112 NR NR NR NR NR NR NR NR NR 141 NR 141 NR 141 NR 141 187 1010 207 983 NR NR NR NR 252 1120 222 1190 75 188 90 188 95 188 87 188 NR 2350 NR 2350 NR 2350 NR 2350 111 558 98 558 81 558 105 558 269 1090 332 1250 2430 3280 2990 3510 772 1480 867 1830 81 90 144 106 93 * 116 . 62 127 Sample residue exceeds the spiking level slgnMcanfly; therefore, an accurate recovery value cannot Ire calculated ND Not detected at or above the Umit of Quantitation (LOQ) of25 ng/L. NR Not Reported due to Quality Control Failures, See Table VII for reextract data. Nose: Since this summary table shows rounded results, recovery values mayvary slightly from the values In the raw data. Exygen Research Page 34 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Table VL M atrix Spike Recovery o f PFBS, PFHS and PFOS in W ater Samples Continued Sample D escrip tio n DAL-GW-602L-0-060413 (00171004Spk Q,0.1 ppb Ub Spite) DAL-GW-602L-0-060413 (C0171004 SpkH, 1.0 ppb Lib Spite) DAL-GW-602L-LS-060413 (C0171000,0.1 ppb FMdSpOw) DAL-GW-602L-HS-060413 (C0171007, i.o ppb FMd Span) DAL-GW-604R-0-060413 (C0171000Spk0,0.1 ppb Ub Spite) DAL-GW-604R-0-060413 (C0171000Spk D. 1.0ppbUb SpBw) DAL-GW-604R-LS-060413 (C0171010,0.1 ppb FMdSpBco) DAL-GW-6O4R-HS-O0O413 (C0171011,1.0ppb FMd Spite) DAL-GW-603R-0-060413 (C0171012SpkC,0.1 ppbUb Spite) DAL-GW-603R-0-060413 (C0171012SpkF, 1.0ppb Ub Spite) DAL-GW-603R-LS-060413 (C0171014, 0.1 ppb FMdSpa) DAL-GW-603R-HS-060413 (C0171018,1.0ppb FMdSpati) DAL-GW-603L-0-060413 (00171010Spk0 ,0.1 ppb Ub Spite) DAL-GW-603L-0-060413 (00171010SpkH, 1.0ppbUb Spiti) DAL-GW-603L-LS-060413 (00171010,0.1 ppb FMd Spato) DAL-GW-603L-HS-060413 (C017101, 1.0ppb FMd Spite) DAL-SW-BPP01-0-060413 (C0171020SpkC. 1.0ppbUb Spiti) DAL-SW-BPP01-04)60413 (COI71020SpkD, 10ppb Ub Spite) DAL-SW-BPP01-LS-060413 (C0171022,1.0ppbFMd Spite) DAL-SW-BPP01-MS-060413 (C0171020,10ppb FMdSpa) DAL-SW-BPP01-HS-060413 (00171024.100 ppb FMdSpite) Amount Spited (ngrt.) C4 SuHonate PFBS___________ C6 Sulfonate PFHS___________ eg Sulfonate PFOS Amount Amount Amount Found in Amount Found in Amount Found in Amount Sampti Rtcovarsd (ngrt-l (ngrt.) Rsc Sampit Recovered (* t (ng/L) (ng/L) Ree Sampit Recovered ( * | (ngrtj (ng/L) Rtc (%) too 117 1000 117 100 117 1000 117 212 1090 207 1190 95 454 97 454 90 454 107 454 601 1480 534 1690 NR 103 NR NR 124 NR NR NR NR NR NR NR NR NR 100 1000 100 1000 117 117 117 117 192 1280 230 1150 75 137 116 137 113 137 103 137 209 1190 256 1220 72 893 105 893 119 893 106 893 1250 2030 1430 2130 6 114 * 124 100 1000 100 1000 201 201 201 201 295 1240 298 961 94 323 104 323 97 323 76 323 461 1380 461 1170 * 120 106 120 120 85 120 206 1100 275 1060 86 98 155 94 100 1000 100 1000 44.1 44.1 44.1 44.1 1000 7630 100000 7630 1000 7630 10000 7630 100000 7630 141 1040 130 944 8720 124000 7540 15000 90400 97 48.4 100 46.4 66 48.4 90 48.4 * . 60400 116 60400 60400 74 60400 83 60400 144 1160 157 1120 63300 193000 58500 65100 130000 96 170 m 170 109 170 107 170 . NR 133 NR NR NR 70 NR 324 1230 312 1400 NR NR NR NR NR 154 106 142 123 NR NR NR NR NR Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated NR Not Reported due to Quality Control Failures, See Table VII for reextract 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 o f 136 Interim Report #24 - Analysis ofW ater, Sludge, and Sediment Exygen Study No.: P0001131 Table VL M atrix Spike Recovery o f PFBS, PFHS and PFOS in W ater Samples Continued Sample D e scrip tio n DAL-SW-BPP02-0-060413 (C017102SSpkE, 1.0ppbUb Spite) DAL-SW-BPP02-0-060413 (C017102S SpkF, 100ppb Ub Spite) DAL-SW-BPP02-LS-060413 (C0171027,1.0ppbFMd Spin) DAL-SW-BPP02-MS-060413 (C0171020,10ppb FMd Spin) DAL-SW-BPP02-HS-060413 (C0171020,100ppb FMdSpite) DAL-SW-BPP03-0-060413 (C0171030Spk0 ,1.0ppb LabSpite) DAL-SW-BPP03-0-060413 (C01710MSpkM, 100ppbUb Spite) DAL-SW-BPP03-LS-060413 (C01710S2,1.0ppb Flakf Spin) DAL-SW-BPP03-MS-060413 (C01710SS, 10ppbFMd Spin) DAL-SW-BPP03-HS-060413 (C01710J4.100ppb FMd Spite) DAL-SW-BCT01-0-060412 (C017109SSpkC, 1.0ppbLabSpite) DAL-SW-BCT01-0-060412 (C0171000Spk0.10 ppb Ub Spite) DAL-SW-BCT01-LS-060412 (00171007,1.0ppb FMd Spin) DAL.-SW-BCT01-HS-060412 (C01710M, 10ppb FMd Spin) DAL-SW-8CT02-0060412 (co in SpkE, t.o ppo Labspifca) DAL-SW-BCT02-0-060412 (C01710MSpkF, 10ppbLabSpite) DAL-SW-BCT02-LS-060412 (C0171041,1.0ppbFMd Spin) DAL-SW-BCT02-HS-060412 (C0171042,10ppbFMd Spin) DAL-SW-BCT03-0-060412 (C0171043Spk0,1.0 ppbUb Spite) DAL-SW-BCT03-0-060412 (00171040SpkH, 10ppb Ub Spite) DAL-SW-BCT03-LS-060412 (C0171045,1.0ppb FMd Spin) DAL-SW-BCT03-HS-060412 (C0171040,10ppbFMd Spin) Amount Spited (ng/L) C4 Sulfonate PFBS___________ C6 Sulfonate PFHS__________ C8 Sulfonate PFOS Amount Amount Amount Found in Amount Found in Amount Found In Amount Sampia Recovered (ng/L) (ng/L) Rae Sampia Recovered (%) (ng/L) (ng/L) Rae Sample Recovered (%) (ng/L) (ng/L) Rae (%) 1000 5030 100000 5030 1000 5030 10000 5030 100000 5030 5640 111000 5190 14100 116000 * 40100 106 40100 40100 91 40100 113 40100 38600 150000 39100 45700 161000 NR 110 NR NR * NR 121 NR NR NR NR NR NR NR NR NR NR NR 1000 5300 100000 5300 1000 5300 10000 5300 100000 5300 5600 107000 5220 13100 132000 39100 102 39100 * 39100 78 39100 127 39100 40200 161000 40500 43400 188000 . NR 122 NR NR NR 149 NR NR NR NR NR NR NR NR NR NR NR 1000 10000 1000 10000 492 492 492 492 1610 10300 1500 9620 112 2250 98 2250 101 2250 91 2250 3270 11800 3050 11700 102 16100 96 18100 80 16100 95 16100 16600 22500 16500 25300 64 92 1000 10000 1000 10000 2290 2290 2290 2290 2980 11300 2860 11300 69 14000 90 14000 57 14000 90 14000 16300 23500 14500 23000 NR 95 NR NR 90 NR NR NR NR NR NR NR NR NR 1000 10000 1000 10000 1010 1010 1010 1010 1970 10100 I860 10400 96 4390 91 4390 85 4390 94 4390 5650 13300 5510 14500 . NR 89 NR NR 101 NR NR NR NR NR NR NR NR NR Sample residue exceeds die spddng level slgnldcandy; therefore, an accurate recoveryvalue cannot be calculated NR * Not Reported due to Quality Control Failures, See Table VII for reextract data. Note: Since this summary table shows rounded results, recoveryvalues mayvary slightly from tha values In the raw data. Exygen Research Page 36 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Table VI. M atrix Spike Recovery o f PFBS, PFHS and PFOS in W ater Samples Continued Sample D M criptlon DAL-SW-8C01-0-060412 (C0171047SpkC. 1.0ppbLabSpite) DAL-SW-BC01-0-060412 (CS171S47Spk D, 10ppb LabSpike) DAL-SW-BC01-LS-060412 (C0171S4S, 1.0ppb FMtfSpa) DAL-SW-BC01-HS-060412 (C01710M. 10ppbField Spfce) DAL-SW-BC02-0-060412 (00171001 Spk E, 1.0ppb LabSpike) DAL-SW-BC02-0-060412 (C01710S1Spk F, 10ppb LabSpite) DAL-SW-BCO2-LS-06O412 (C0171089.1.0ppb FMtfSpite) DAL-SW-BC02-HS-060412 (C0171004,10ppb FMtf Spite) DAL-SW-8C03-0-060414 (C01710S0Spk0,1J ppbLabSpike) DAL-SW-BC03-0-060414 (C01710SSSpk H, 10ppb LabSpite) DAL-SW-BC03-LS-060414 (C017107,1.0ppb FMtfSpite) DAL-SW-BC03-HS-060414 (C01710SS, 10ppb FMtfspite) DAL-SW-BC04-0-060414 (C0171000SpkC, 1.0ppbLabSpike) DAL-SW-BC04-0-060414 (C01710MSpk D, 10ppbUb Spike) DAL-SW-BC04-LS-060414 (C0171001,1.0ppb FMtfSpite) DAL-SW-BC04-HS-060414 (C01710S2, 10ppb FMtfSpin) DAL-SW-BC05-0-060414 (C017100SSpkE. 1.0 ppb LabSpi) DAL-SW-BC05-0-060414 (C01710SSSpkF. 10ppbLabSpite) DAL-SW-BC05-LS-060414 (C01710M. 1.0ppb FMtfSpite) DAL-SW-BC05-HS-060414 (C01710M. 10ppb FMtfSpite) Amount Spiked (non.) C4 S uH bniti PFBS___________ C6 Sulfonsts PFHS___________ C I SuHbnats PFOS Amount Amount Amount Found in Amount Found In Amount Found In Amount Sample Recovered (nan.) (nan.) Rac Sampla Racovarad (%> (non.) (na/L) Ree Sampla Racovarad <% (na/L) (na/U Ree %) 1000 10000 1000 10000 65.3 65.3 65.3 65.3 1030 9360 1000 9590 96 31.5 93 31.5 93 31.5 95 31.5 997 9370 1040 9470 97 64.0 93 64.0 101 64.0 94 64.0 949 9110 1020 8420 89 90 96 84 1000 10000 1000 10000 67.1 67.1 67.1 67.1 1000 10000 1000 10000 150 150 150 150 1070 9900 973 10200 1120 10100 1150 9390 100 36.3 980 98 36.3 9770 'V 91 36.3 971 101 36.3 9890 97 430 100 430 100 430 92 430 1490 10300 1490 9920 94 65.8 97 95.6 93 85.8 99 85.8 106 3630 99 3630 106 3830 95 3630 978 9540 1170 10300 5430 14600 5500 14600 89 95 108 102 110 110 1000 10000 1000 10000 508 508 508 508 1540 10100 1410 9660 103 2260 96 2260 90 2260 92 2260 3330 11800 3190 10700 107 NR 95 NR 93 NR 64 NR NR NR NR NR NR NR NR NR 1000 10000 1000 10000 69.0 69.0 NR 69.0 1020 10300 NR 6560 95 64.0 102 64.0 NR NR 65 64.0 1060 9330 NR 8010 100 716 93 716 NR NR 79 716 1570 9860 NR 9850 85 92 NR 91 "Sample residue exceeds the spiking level significantly. therefore, an accurate recoveryvaluecannot be calculated NR Not Reported due to QualityControl Fattures, SeeTable VII for re-extract data. Note: Since this summary table shows rounded results, recovery values mayvary slightly from the values In the raw data. Exygen Research Page 37 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Table VI. M atrix Spike Recovery o f PFBS, PFHS and PFOS in W ater Samples Continued Sample D escription DAL-SW-OSP01-0-060413 (CS171SS7Spk0,0.1 ppb LabSpite) DAL-SW-OSP01-0-060413 (C01710S7Spk H, 1.0ppb LabSpike) DAL-SW-OSP01-LS-060413 (CS1710SS,0.1 ppb Field SpNce) DAL-SW-OSP01-HS-060413 (Cei71070,1.0ppb Flewspite) DAL-SW-OSP02-0-060413 (C0171071 SpkC, 1.SppbLabSpfite) DAL-SW-OSP02-0-060413 (CO171071 Spk0,10 ppb LabSpike) DAL-SW-OSP02-LS-060413 (CS171073,1.0ppb Flew Space) DAL-SW-OSP02-HS-060413 (C0171074,10ppb FMdSpite) DAL-SW-OSP03-0-060413 (C017107SSpkS, i j ppb LabSpite) DAL-SW-OSP03-0-060413 (C0171079SpkF, 10ppbLabSpite) DAL-SW-OSP03-LS-060413 (C0171077,1.0ppb FISWSpfiiS) DAL-SW-OSP03-HS-060413 (C017107S, 10ppb FieldSpate) DAL-GW-BPWELL-0-060413 (CS171S79Spk0 ,10ppb LabSpite) DAL-6W-BPWELL-0-060413 (C017107SSpkH, 1000ppb LabSpite) DAL-GW-8PWELL-LS-060413 (CS1710S1,10ppb Floid Spots) DAL-GW-BPWELL-MS-060413 (C0171002,100ppb FMdSpNw) DAL-GW-BPWELL-HS-060413 (C01710S3,1000ppb FMd Spite) DPWS-WWI-DCTP01*0-060413 (C0171004SpkC. 10ppb LobSpite) DPWS-WWI-OCTP01-0-060413 (C0171004SpkD, 100ppb LabSpite) DPWS-WWI-DCTP01-LS-060413 (C0171000,10ppb FMd Spite) DPWS-WWI-DCTP01-HS060413 (C0171007,100ppb FieldSpite) Amount Spited (ngd.) C4 Sulfonate PFBS___________ C6 Sulfonate PFHS___________ C8 Sulfonate PFOS Amount Amount Amount Found m Amount Found In Amount Found In Amount Sample Recovered ("on.) (nan.) Rec Sample Recovered ( * f (non.) (non.) Rec Sample Recovered Is ! (ng/L) (ng)L) Rec <%) 100 1000 100 1000 196 196 196 196 100 1000 100 1000 343 343 343 343 100 1000 100 1000 319 319 319 319 10000 18800 1000000 18800 10000 18800 100000 18800 1000000 18800 270 1230 284 1180 418 1340 426 1290 384 1510 400 1340 25100 1240000 23900 123000 1030000 74 46.7 103 46.7 88 46.7 98 48.7 555 100 555 . 555 95 555 * 501 119 501 501 102 501 138 1060 139 1060 665 1590 718 1480 576 1430 611 1500 63 136000 144000 122 136000 1350000 51 136000 148000 104 136000 270000 101 136000 1180000 91 602 101 602 92 802 101 602 665 1590 745 1540 2870 104 2670 * 2670 93 2870 2950 3550 3010 3490 * 2290 93 NR 2290 100 2290 2340 NR 2790 3520 * 834000 753000 121 834000 834000 2030000 829000 134 834000 1010000 104 834000 1890000 99 . 94 * 88 82 NR 123 . 120 106 10000 66.8 100000 66.8 10000 66.8 100000 66.8 9990 110000 9570 108000 99 102 110 102 95 102 108 102 9940 107000 9600 100000 98 698 107 698 95 696 100 698 10100 98200 9310 89200 94 96 86 89 "Sample residue coeds the spiking level significantly, therefore, an accurate recovery value cannot be calculated NR * Not Reported due to Quality Control Failures, See Table VII for reextract data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. Exygen Research Page 38 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Table VI. M atrix Spike Recovery of PFBS, PFHS and PFOS in W ater Samples Continued Sanpto Description DPWS-WWE-DCTP01-0-000413 (CM71MSpkE, 10ppb LabSpite)* DPWS-WWE-DCTP01-0-060413 (C01710MSpk F. 100 ppb LabSpite) DPWS-WWE-DCTP01-LS-060413 (CM710SO,10ppbFieldSplice) DPWS-WWE-DCTP01-HS-060413. (GO1710S1.100ppbFieMSpite) Amount Spiked (non.) C4 Sulfonate PFBS Amount Found In Amount Sample Recovered Ree (non.) (na/L) M 10000 85.3 100000 85.3 10000 85.3 100000 85.3 9460 112000 10600 141000 94 112 105 141 C8 Sulfonate PFHS Amount Found In Amount Sample (non.) Recovered (ngft.) 106 9170 106 106000 106 10200 106 144000 DAL-WW-EFF01-0-060413 (00171002Spk0,1.0 ppbLab8pike) DAL-WW-EFF01*0-060413 (C01710S2SpkH, 10ppb LabSpNie) DAL-WW-EFF01-LS-060413 (C01710S4.1.0ppbFieldSpike) DAL-WW-EFFO1-HS-O0O413 (CM710S6,10ppbFieMSplka) 1000 10000 1000 10000 312 312 312 312 1330 11000 1450 10300 102 815 107 815 114 815 100 815 1600 10900 1980 11600 DAL-LCH-MCLF01-0-000414 (CM71000Spk C, 10ppb LabSpite) DAL-LCH-MCLF01-0-060414 (CM710S0SpkD,100ppbLabSpite) DAL-LCH-MCLF01-LS-000414 (CM7100S, 10ppb FWdSpite) DAL-LCM-MCLFO1-HS-O0O414 (CM71OSS, 100ppb Raid Spite) 10000 NR 100000 5180 10000 5180 100000 5180 NR 110000 13800 87400 NR 17600 105 17600 86 17600 82 17600 27500 118000 25600 95100 DAL-GW-TRIP1-LS-060412 (CM711M, 1JO ppb FieldSpite) 1000 ND 696 70 ND DAL-GW-TRIP1-HS-060412 (CM71102,10ppbFWdSpA) 10000 NO 6340 63 ND 992 9650 DAL-WW-TRIP1-LS-060413 (CM71104,141ppbFieldSpA) 1000 NO 789 79 ND DAL-WW-TRIP1-HS-060413 (CM71109,10ppb FWdSpite) 10000 ND 7020 70 ND 1000 9510 CSSulfonate PFQ8 Amount Found In Amount Ree Sample i* i (no-) Recovered (nfl/L) Ree i* t 91 574 106 574 101 574 144 574 9910 101000 10200 140000 93 100 96 139 99 4390 101 4390 117 4390 108 4390 5280 12500 6170 14100 81 . 97 99 NR 100 27200 80 27200 78 27200 NR 119000 34800 105000 NR 92 76 78 99 ND 97 ND 887 9680 89 97 100 ND 95 ND 917 9950 92 100 A verage: 07 Standard Deviation: 17 Average: Standard Deviation: *Sampleresidua exceeds the epRdnglevel elonMcantly;therefore, an accurate recoveryvaluecannotbe calculated NO Notdetected atorabovetheLimitofQuantitation (LOQ)of25 ng/l. NR * NotReporteddue to QuaNtyControlFailures, SeeTableVWforre-extractdata. Note:Sincethis summarytable showe rounded results, recoveryvalues mayvarysUghtty fromthe values in the raw data. ASample filled to 250 mL 100 14 Average: 102 Standard Deviation: 19 Exygen Research Page 39 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Table VII. M atrix Spike Recovery o f PFBS, PFHS and PFOS in Re-extracted W ater Samples Sampl* DMcrintlon DAL-GW-604S-LS-060412 (C010*M*.0.1ppbFMdSpi) C4Sulfonate PFBS C6ottonate PFH8 C8Sulfonate PFOS Amount Amount Amount Amount Found in Amount Found In Amount Found in Amount Spiked Semple (gnj (ntfL) Recovered Ree Sample (ihKL) n Recovered (no/L) Ree Semple W "`s s ir Ree W 100 156 296 140 199 314 115 287 406 119 DAL-GW-601R-LS-060413 (COITO***,0.1 ppb FlatoSpila) 100 NR NR NR - - - NR NR NR DAL-GW-601S-LS-080413 (COI70*00,0.1 ppbFlatoSpOia) 100 NR NR NR - - -- -- DAL-GW-0O1L-DB-060413 (CdTOOOTSpkE,10ppbLabSpira) DAL-GW-601L-DB-060413 (00170007SpkF,N ppbLabSpila) 10000 50000 NR - NR NR . . .. - - 20800 63700 86 - .. -- DAL-GW-602S-LS-060413 (COI71002,0.1 ppbFiatoSpila) 100 162 335 143 - - -- DAL-GW-602L-HS-060413 (Cd71047,1J ppbFMdSpi) 1000 -- -' 1770 2970 120 DAL-GW-603R-LS-060413 (CdTIOU, 0.1 ppbFMdSpila) 100 -- - NR NR NR DAL-SW-BPP01-DB-060413 (C0171021pkE,1000ppbLabSpi) 1000000 -- - 437000 1410000 97 DAL-SW-BPP02-DB-060413 (C*17102SSpk0,100ppbL* Spi) DAL-SW-BPP02-D8-060413 (C017102* SpkH,1000ppbLabSpi) 100000 1000000 7680 148000 - 140 - . .. - 354000 1560000 121 DAL-GW-BPP03-HS-060413 (C0171004,100ppbFlatoSpka) 100000 -- 326000 380000 54 DAL-SW-BCT02-DB-060412 (CdTlAoSpk0.000ppbLteSpi) 500000 - '- 279000 805000 105 DAL-SW-BCT03-DB-060412 (C0171044SpkLISOppbLabSpi) 100000 -- - 44200 136000 92 DAL-SW-BC04-DB-060414 (COI714*0SpkE,100ppbLabSpi) 100000 -- 27700 136000 108 DAL-SW-BC05-LS-060414 (CdTIOSO, 1.0ppbFlatoSpi) 1000 91.7 1380 129 50.8 893 83 640 1670 103 DAL-SW-QSP03-DB-060413 (Cd7107lSpkQ,1.0ppbLabSpi) 1000 - - -- - - 2100 2800 78 DAL-LCH-MCLF01-DB-060414 (Cd710*7SpkE,10ppbUb SpB) DAL-LCH-MCLF01-LS-060414 (Cd71000,10ppb FiatoSpi) 10000 10000 7130 - 20500 - 134 . -- .. .. - - 26400 38100 117 Average: 137 Standard Deviation: NR NotReporteddueto QualityContra!Falwee. Note:Sincethia summarytable shown rounded results, recoveryvalue* mayvarysightly fromthe values Inthe rawdata. Average: 96 Standard Oevladen: 18 Average: 101 Standerd Deviation: 21 Exygen Research Page 40 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Table VIII. M atrix Spike Recovery o f PFBS, PFHS and PFOS in Sludge S am p les Sample Description C4 Sulfonate PFB3 WetWeight C6 8utf6MrtePFH8 WetWeight Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery (nfl/Q) (nolo) wet w t (na/a) wet wt. <%> fna/a) wet wL (na/a) wet w t (%> C Sulfonate PFOSWetWeight Amt Found in Sample Amount Recovered Recovery (%> DPWS-SL-DCTP01-0-0000 (C017110SSpk C, 4 ppbSpike) DPWS-SL-DCTP01-0-0000 (C017110SSpk0,400 ppbSpike) 4 400 0.808 0.808 6.20 135 1.29 690 175 1.29 4.22 73 288 72 NR NR NR NR NR NR Average: 188 Standard Deviation: 28 Average: 72 Standard Deviation: 1 NR NotReported due to Quality Control Failures. Nota: Sincethis summarytable shows rounded results, recoveryvalues mayvarysHghtlyfromthe values Inthe raw data. Exygen Research Page 41 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Table IX. M atrix Spike Recovery o f PFBS, PFHS and PFOS in Sediment Samples Sample Description DAL-SD-OSP01-0-0000 (00171197 Spk C, 4 ppb LabSpite) DAL-SD-OSP01-0-0000 (C0171107 Spk D, 400ppb LabSpite) DAL-SD-OSP02-0-0000 (C017110SSpkE,4 ppbLabSpite) DAL-SD-OSP02-0-0000 (C017110SSpk P, 400 ppb LabSpite) Amount Spiked (natal C4 Sulfonate PFB8 WetWeight Amt Found Amount In Sample Recovered Rec. (natal wet wt. (natal wet wt. (%) C6 Sulfonate PFH3WotWeight Amt Found Amount in Sample Recovered (na/alw etw t (no(al wet wt. Rec. (%) C8 Sulfonate PFOS WetWeight Amt Found Amount In Sample Recovered (natal wet wt. (natal wet w t Rec. (*) 4 0.573 400 0.573 2.25 42 0.215 203 51 0.215 4.11 97 NR 353 88 NR NR NR NR NR 4 ND 400 ND 3.34 84 0.430 260 65 0.430 3.50 77 NR 308 77 NR NR NR NR NR Aversgs: 0 Standard Deviation: 18 ND * Notdetected at or above the Limitof Quantitation (LOQ)of 0.2 ng/g (wetweight). NR * Not Reported due to Quality Control failures, tee Table X Note: Since this summarytable shows rounded results, recovery values mayvary slightly from the values In the raw data. Average: S Standard Deviation: 10 Exygen Research Page 42 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Table X. M atrix Spike Recovery o f PFOS in Re-extracted Sediment S am p les S am ple D e s c rip tio n D A L -S D -O S P 01-0-0000 (C0171107 Spk E, 4 ppb Lab Spike) D A L-S D -O S P 01-0-0000 (C0171107 Spk F, 400 ppb Lab Spike) D A L -S D -O S P 02-0-0000 (C0171108 Spk G, 4 ppb Lab Spike) D A L -S D -O S P 02-0-0000 (C0171108 Spk H, 400 ppb Lab Spike) Am ount S p ik e d (n g/g ) C S u lfo n rte _ P F O S W e tW e lg h t Am t Found in S am ple (n g /g ) w et w t Am ount R e c o v e re d (n g /g ) w et w t. R ecovery (% ) 4 5 .9 9 4 0 0 5.9 9 4 4 0 .5 40 0 4 0 .5 7 .5 6 387 4 2 .9 344 39 95 * 76 A v erag e: Standard D eviation: 70 28 ` Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated Note: Since th is sum m ary tab le show s rounded results, recovery values m a fv a ry sligh tly from th e values in th e raw data. Exygen Research Page 43 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Table XI. Total Percent Solids for Sludge and Sediment Samples Exygen ID C0171106 C0171107 C0171108 Sample Description DPWS-SL-DCTP0 1-0-0000 DAL-SD-OSP0 1-0-0000 DAL-SD-QSP02-0-0000 Total Solids (%) 22.83 58.39 53.33 Exygen Research Page 44 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 FIGURES Exygen Research Page 45 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Figure 1. Typical Calibration Curve for PFBS in Reagent W ater 042808B P700-1131 W jttr.rd b (PFBS): 'U n ta r R tg ro c lo n C1 /* m tig h tln g ): y 324 x + 0.001 (r 0.0004) Area, counts Exygen Research Page 46 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Figure 2. Extracted Standards of PFBS in Reagent W ater, 25 ng/L and 50 ng/L, Respectively I XC *51M f-1 - P F B S (Standard ) 2M .W M l.Baaw -aam pfa 2 o f 3$ from A n a : 94S2 eoaata H tig h t: 5.TS#+002 cps R T: 9.555 m ia Tim, min XC051000-2 - PFBS (S tndjtd)2ee.0g.0 im ii - u rn p l 3 of 3S from 042800B.wiff A t. : 10S25 oounls H eight 1.08.+003 cps RT: 0.553 min 0.55 Intensity, cps Intensity, cps Exygen Research Page 47 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Figure 3. PFBS in Reagent Control, 50 ng/L Fortified Reagent Spike A, and 500 ng/L Fortified Reagent Spike B, Respectively I flM fM rt C o a tro f -P F B S Uakaovm) 299.A M 9 arar ampi* 9 o f 3 from 0429tiB .w fff t> *at n o t fon ati) 'S I A na: 10888 oounto Hoight: 1.11p*003 ops RT:0.SS3 min 0.93 Tim *, min Exygen Research Page 48 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Figure 4. Chromatogram Representing a W ater Sample Analyzed for PFBS (Exygen ID: C0169336, Data Set: 042806B) C 1 W 3 3 t-P F B S (U a k a o * a )2 t.W 9 l a m a -tam pt* 2o f 3from A n a : 26477 coaat* H H g tt: 7.37*+003 cp* RT: *S53 fa O.SS 1300 1200 1100 1000 000 800 700 000- 1.01 Intensity, cps I, " .y--. ....... f*m 11 P 1 1 2 3 4 0 0 7 8 0 10 11 12 13 14 15 18 17 Tima, min Exygen Research Page 49 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Figure 5. Chromatogram Representing a Sludge Sample Analyzed for PFBS (Exygen ID: C0171106, Data Set: 050906B) C tT m tf - PTBS (IM taona) 2 9 i.v n .ta m a .ampta I t o f 21from MMNB.WW At h ; 9435coaatt H H gtt: 1.92t*0*2cpa HT: 0.529mia Intensity, cp s Exygen Research Page 50 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Figure 6. Chromatogram Representing a Sediment Sample Analyzed for PFBS (Exygen ID: C0171108, Data Set: 051106A) 0*1111*0 PFBS (Unknown) i m i f H -ta m p it 21 o f 24 from 05110SA.wtff A rt : 5351 count* H aigkt: 1.92**002 ep* AT: t.S25m i* Intensity, cps Exygen Research Page 51 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Figure 7. Typical Calibration Curve for PFHS in Reagent W ater 042800B P700-1131 W ji.u d b (PFHS): " L in .ii" R tg rts io n ( " I / ^ WMighting): y - S41 x + 43.000164( r - 019007) Area, counts Exygen Research Page 52 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Figure 8. Extracted Standards of PFHS in Reagent W ater, 25 ng/L and 50 ng/L, Respectively 2 2xcoaiooe-i pfhs (stin su d ) see.cueo.o im u u m p it * 38 from <M eoee.wM A n i: 13715 M untf HtigM: 592**002 opt RT: 8.13 min 9.13 X C tS ttM -Z- PFH t (StMMdsnl)3n.V U .ttm * -ttm p b 3 o f3 from M tN O M T A n s : aM com rir HSIgtto 1JU ***3ct* R7i X U m is Intensity, cps Intensity, cps Exygen Research Page 53 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Figure 9. PFHS in Reagent Control, 50 ng/L Fortified Reagent Spike A, and 500 ng/L Fortified Reagent Spike B, Respectively I flM fM t (M M -PfHS (UPtaowa)39SI0/IM ama -tam ph of3$ from HtHUt-wm P*a* aotfoaad) Intensity, cps Intensity, c p s A rta: 26074 oounts H tig h t 1 ^7 +003 ops RT: S.14 min 0.14 T im t, min RaagantSpkB - PFHS(QC)388.0A0.0 tm u s jm p lt 11 of 38from042806B.wHf A lta :271382 counts H tig h t 133t+004ops RT:8.11 min 1.0t4< 0.11 14.31 , .A < ^ ^ 13 14 15 10 17 5000.0 70X)J---------- r -I---"'" "T ----- r 1 2 3 46 0 7 8 0 10 12 13 14 18 16 17 T im t, min Intensity, c p s Exygen Research Page 54 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Figure 10. Chromatogram Representing a W ater Sample Analyzed for PFHS (Exygen ID: C0169336, Data Set: 042806B) I C W fX X -P fH S (U U M * m )3 9 U M .$ m -JMpfc 21of38to M28MftwfVf *rM;439StcoMte 2ffe+M3cpt RT: Ittm iM 0.05 2800 2000 2400 2200 2000 1800 1000 1400 1200 1000 800 000 400 200 0 A .,. 45 10 11 12 13 14 15 10 17 Tim, min Intensity, cp s Exygen Research Page 55 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Figure 11. Chromatogram Representing a Sludge Sample Analyzed for PFHS (Exygen ID: C0171106, Data Set: 050906B) C91711$f-Pf=MS < y*taow a)3fS.V te.t ama -ta m p f t t o f 21 from tSt99SB.witf A n a : 3$24co*t* H tfg tt: lta*003epa RT: ILC3ri Exygen Research Page 56 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Sediment Sample Analyzed for PFHS (Exygen ID: C0171108, Data Set: 051106A) C t n t m PFH S fJakaaw a) 3HLMM.0 ama -ttm p f* 21 o f 24 from 65116tA.wM A n a : 25298 coamt* H a ig tt: 1.24**883cp* R7: 8.66m in 8.00 Exygen Research Page 57 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Figure 13. Typical Calibration Curve for PFOS in Reagent W ater 042800B P760-1131 W jtti.rd b (PFOS): " L in .ii" R ig iw lo n f i /xT H lghting): y 4BS x + 0.00111 (>>0.0007) Area, counts Exygen Research Page 58 o f 136 Intensity, cps Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Figure 14. Extracted Standards o f PFOS in Reagent W ater, 25 ng/L and 50 ng/L, Respectively X C 9 S tm -1 PFOS fS U od ard) 4MLOMLO - tim p h 2 o f 3$ from M2MCB.wHf A n : f272CcMHrtr fh ig k t: 7.i7+(d2cps RT: ll.S m m 11.00 700 800 soo- T im ., min XC051000-2 - PFOS (Standard) 400.0/80.0 amu - sampla 3 of 38 from 042S00B.wiff A n a : 24772 counts H aight 1.57*+003 ops RT: 11.0 min 1400 1200 1000- 1180 10.44 i 15 10 17 T im t, min Intensity, cps Exygen Research Page 59 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Figure 15. PFOS in Reagent Control, 50 ng/L Fortified Reagent Spike A, and 500 ng/L Fortified Reagent Spike B, R espectively A t# * * Cow trol ` P F O S (Uf*m >w>;4MftW LNr -tam p to t o f 38 from 842.80SB.witf t> tak a o t found) Intensity, cps Intensity, cp s Tima, min RaagantSpk A - PFOS (QC) 400.0/80.0 mu - s jm p lt 10 of 38 from 042800B.wlff A tta: 2S090 count Haight: 1.40a+003 cps RT: 11.0 min 11.01 Tima, min Raagant Spk B - PFOS (00)400X1/80.0 amu -sampla 11 of 38 from 042800B.MHf Araa: 243078 oounts Halght 1.44.+0O4 cps RT: 11.0 min 11.00 Intensity, cps Exygen Research Page 60 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Figure 16. Chromatogram Representing a W ater Sample Analyzed for PFOS (Exygen ID: C0169336, Data Set: 042806B) 1 0 1 0 3 3 $ P FO S {Unknown) 4M .O TM am * -ram p i* 20 o f 39 from M2M60.wff7 A rta : 74194t o r r i* H *igkt: 427**003 o p t KT: U .t m ir 11.57 4000 3900- 3000 2500- If c 2000 Sc 1500- 1000- 11J33 500- 0 --A .t 1 1 ................... . 234 5 1-- T 57 Tima, min 10 17 Exygen Research Page 61 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Figure 17. Chromatogram Representing a Sludge Sample Analyzed for PFOS (Exygen ED: C0171106, Data Set: 050906BR) I C $1711tt- P fO S ( t t o t o o v m ) - u m p f * H o f 21 from tS tW iB H w M A n a : 144ttScornot* H tig tt: SL95t+M3cp* R li 11.3m i* 1120 Exygen Research Page 62 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Figure 18. Chromatogram Representing a Sediment Sample Analyzed for PFOS (Exygen ID: C0171107, Data Set: 071106B) C*TT11tT PFOS ( U k t i n m * ) -n m p lt o f 22fro *711*tB.wtff A n a : 2*37coaMt* H H gt 1.tS*M 3cpa RT: 11.2m l* 11.28 Exygen Research Page 63 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods and Protocol Amendments Exygen Research Page 64 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number P0001131 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) 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: P0001131 P erform ing Laboratory: E xygen R esearch 30S8 Research Drive State C ollege, PA 16801 Phone: (814)272-1039 Sponsor R epresentative: M ichael A . Santoro Director o f Regulatory Affairs 3M B u ild in g 0 2 3 6 -0 1 -B -10 S t. P aul, M N 5 5 1 4 4 Phone: (651) 733-6374 Page I oj CS Exygen Research Page 65 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 DISTRIBUTION: 1) Jaisim ha K esari, Study Director, W eston Solutions 2) John M . Flaherty, Principal Investigator, Exygen Research 3) M ichael A . Santoro, Sponsor Representative, 3M Company 4 ) Exygen Research Q uality Assurance Unit Exygen Research Page 2 o f65 Page 66 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PROTOCOL APPROVAL Stu dy T itle: A nalysis o f PcrQuorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFH S), and Perfluorooctanesulfonate (PFOS) in Water, S oil, Sedim ent, Fish, Clam s, V egetation, Sm all Mammal Livers and Sm all M ammal Serum U sing LC/M S/M S for the 3M Decatur M onitoring Program Exygen Protocol Number: P0001131 APPROVALS Jaisim halK esan, S< W eston Solutions M ichael A. S 3M Comparfy John M. Flaherty, Prinicciippial Investigator X Exygen Research D ate S Richard A . < Exygen Resi ident, Facility M anagement Q uality Assurance Unit D ate u h kL D at/ Page 3 o f 65 Exygen Research Page 67 o f 136 Interim Report #24 - Analysis ofW ater, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 TABLE OF CONTENTS TITLE PAGE...............................................................................................................................................1 DISTRIBUTION..........................................................................................................................................2 PROTOCOL APPROVAL........................................................................................................................... 3 TABLE OF CONTENTS.............................................................................................................................4 INTRODUCTION........................................................................................................................................5 TEST MATERIALS....................................................................................................................................5 OBJECTIVE............................................................................................................................................... 6 TESTING FACILITY..................................................................................................................................6 STUDY DIRECTOR....................................................................................................................................7 SPONSOR REPRESENTATIVE..................................................................................................................7 PRINCIPAL INVESTIGATOR...................... ............................................................................................ ^ PROPOSED EXPERIMENTAL START AND TERMINATION DATES................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM....................................................... 8 SAMPLE PROCUREMENT, RECEIPT AND RETENTION...................................................................... 8 SAMPLE IDENTIFICATION..................................................................................................................... 9 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 o f 65 Exygen Research Page 68 o f 136 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number P0001131 INTRODUCTION The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFO S) in water, soil, sedim ent, fish, clam s, vegetation, sm all mammal livers and sm all mammal serum using LC/M S/M S for the 3M Decatur M onitoring Program. The study w ill be audited for com pliance w ith EPA TSCA G ood Laboratory Practice Standards 40 CFR 792 b y the Q uality Assurance U nit o f Exygen Research. TEST MATERIALS The test m aterials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFO S) and are all supplied by 3M . PFBS Chem ical Name: Perfluorobutanesulfonate M olecular W eight: 338 supplied as the potassium salt (C4FSO]*K+) Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 -+ 99 Structure: PFHS Chem ical Name: Perfluorohexanesulfonate M olecular W eight: 438 supplied as the potassium salt (C iF n S O jlO Lot Number: SE036 Purity: 98.6% Transitions M onitored: 3 9 9 - 8 0 Structure: FFF FFF F S03 Page S o f65 Exygen Research Page 69 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PFOS Chem ical Name: Perfluorooctanesulfonate M olecular W eight: 538 supplied as the potassium salt (CFi7SOaTC+) Lot Number: 217 Purity: 86.9% Transitions M onitored: 4 9 9 -* 99 Structure: OBJECTIVE The purpose o f this study is to perform analysis for periluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, so il, sedim ent, fish, clam s, vegetation, sm all mammal livers and sm all mammal serum for the 3M Decatur M onitoring Program using the current versions o f the follow ing Exygen analytical m ethods: V 0001780: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Water by LC/M S/M S" V 0001781: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in S oil by LC/M S/M S" V 0001782: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Sedim ent by LC/M S/M S" V 0001783: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Fish and Clam s by LC/M S/M S" V 0001784: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in V egetation by LC/M S/M S" V 0001785: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Sm all Mammal Liver by LC/M S/M S" V 0001786: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Sm all Mammal Serum by LC/M S/M S" TESTING FACILITY Exygen Research 3058 Research D rive State C ollege, PA 16801 P h o n e:(8 1 4 )2 7 2 -1 0 3 9 Page 6 o f 65 Exygen Research Page 70 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 STUDYDIRECTOR Jaisimha Kesari P.E., DEE W eston Solutions, Inc. 1400 W eston W ay W est Chester, PA 19380 Phone: (610) 701-3761 F ax:(610)701-7401 j .kesari@ westonsolutions.com SPONSOR REPRESENTATIVE M ichael A Santoro 3M Company Director o f Regulatory A flairs 3M Building 0236-01-B -10 St. Paul, M N 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M . Flaherty Exygen Research 3058 Research Drive State C ollege, PA 16801 Phone: (814) 272-1039 john.flaherty@ exygen.com Exygen Protocol Number P0001131 DATES It is proposed that the analytical portion o f this study be conducted horn October 01, 2004 to Decem ber 31, 2005. The actual experim ental start and termination dates will be included in the final report. Page 7 o f 65 Exygen Research Page 71 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 IDENTIFICATIONANDJUSTIFICATION OF THE TEST SYSTEM The follow ing are the test system s for this study: Water (groundwater and surface water) S oil Sedim ent Fish Clams V egetation Sm all Mammal Liver Sm all Mammal Serum The sam ples w ill be collected b y W eston Solutions. The control sam ples w ill be purchased and prepared by the testing facility. Purchase and processing details for the control sam ples w ill be included in the final report associated w ith this study. The test system s were chosen to access the environm ental im pact o f PFBS, PFHS and PFOS in the Decatur, Alabama area. ^ SAMPLEPROCUREMENT, RECEIPTANDRETENTION W ater, so il, sedim ent, fish, clam , vegetation, sm all mammal liver and sm all mammal serum sam ples w ill be received at Exygen directly from W eston Solutions. The details o f sam ple procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sam pling Environmental M edia." The number and types o f sam ples collected w ill vary depending availability in the field. The total number o f sam ples received and analyzed for each matrix w ill be docum ented in foe final report associated w ith this study. W ater, so il, and sedim ent sam ples w ill be used as received w ithout further processing at Exygen. These sam ples w ill be stored refrigerated at 2C -8C . F ish, clam , vegetation and sm all mammal liver sam ples w ill be processed according to foe appropriate analytical m ethod (see Appendix I)- These sam ples w ill b e stored fro ze n at S >10*C. S m a ll m a m m al w h o le b lo o d sam ples w ill be centrifuged in foe field at foe tim e o f collection and foe serum fraction will be used for the study. Small mammal serum will be stored frozen at -IC C . The receipt and processing o f foe sam ples w ill be docum ented in foe final report and raw data associated w ith the study. Page 8 o f 65 Exygen Research Page 72 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 SAMPLEIDENTIFICATION Prior to analysis, each sam ple w ill be assigned a laboratory sam ple reference number. The reference number w ill be unique and w ill distinguish each laboratory sam ple that is processed throughout the analytical procedure. Chromatographic data w ill be identified by the laboratory sam ple reference number. Sam ple storage conditions and locations w ill b e docum ented throughout the study. ANALYTICAL PROCEDURE SUMMARY R eferen ces: V 0001780: "Method o f A nalysis for die Determ ination o f Perfluorooctanoic A cid (PFOA) in W ater by LC/M S/M S" V 0001781: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Soil by LC/M S/M S" V 0001782: "Method o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Sedim ent by LC/M S/M S" V 0001783: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Fish and Clam s by LC/M S/M S" V 0001784: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in V egetation by LC/M S/M S" V 0001785: `M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Sm all Mammal Liver by LC/M S/M S" V 0001786: `M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Sm all Mammal Serum by LC/M S/M S" The above m ethods use analytical conditions capable o f separating the isom ers o f PFBS, PFHS and PFOS. The final report w ill include the isom ers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sam ple w ill be used for the preparation o f fortified control sam ples. The test substance w ill be made into solutions as per the m ethod, and added to the m atrices via a m icropipette. For water sam pling, Exygen w ill supply one bottle per sam ple collected. The bottles w ill be 500 mL precleaned Sci/Spec Prem ier w ide m outh HDPE bottles. These bottles have been routinely used for fluoiochem ical sam ple Page 9 o f65 Exygen Research Page 73 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P000I131 collection at the testing facility and have been show n to be free o f PFBS, PFHS and PFOS. Sam ples w ill be added to each container to a volum etric fill line at 200 mL. A field duplicate, a low field spike and a high field spike o f each sam ple w ill be collected. The low and high field spike bottles w ill contain PFBS, PFHS and PFOS as w ell as perfluorooctanoic acid (PFO A) and 1.2-13C perfluorooctanoic acid (13C PFOA). PFOA and l3C PFOA are included in the solutions used to spike the sam ples. The results for PFOA and l3C PFOA w ill not be reported in this study. Exygen w ill supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high lev el) for every tw enty sam ples collected. At file testing facility, each water sam ple (excluding field duplicates and field spikes) w ill be extracted in duplicate and w ill also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determ ine method accuracy and to check for bias. For so il, sedim ent, clam s, and vegetation, Exygen w ill supply one 500 mL precleaned Sci/Spec Premier w ide mouth HOPE bottle per sam ple collected or a zip-seal bag. A ll containers/bags used for sam ple collection w ill be shipped to the sam ple location. Sam ples w ill be added to each container or bag in the field. A t die testing facility, each sam ple w ill be extracted in duplicate and w ill also be fortified at a known concentration w ith PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determ ine m ethod accuracy and to check for bias. For sm all mammal liver, Exygen w ill supply a 50 mL polypropylene centrifuge tube. For sm all mammal serum, Exygen w ill supply a collection kit for each sam ple containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. A t the testing facility, each liver and serum sam ple w ill be extracted in duplicate and w ill also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determ ine m ethod accuracy and to check for bias. Low and high spiking levels for each matrix are defined below: M atrix Low Spiking L evel H igh Sp iking L evel W ater 500 ng/L 5000 ng/L S o il Sedim ent 4 ng/g 4 na/a 40 ng/g 40 ng/g Fish 10 ng/g 100 ng/g C lam s 10 ng/g 100 ng/g V egetation 10 ng/g 100 ng/g Sm all Mammal Liver 10 ng/g 100 ng/g Sm all M ammal Serum lO ng/m L lO O ng/m L Page 10of65 Exygen Research Page 74 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 R ecoveries are anticipated to be between 70% and 130% o f the fortified levels; how ever, the exact precision and accuracy w ill be determ ined by the analysis o f the quality control sam ples described above. A statem ent o f accuracy w ill be included in the final report. METHOD FOR CONTROL OF BIAS Control o f bias w ill be addressed by taking representative sub-sam ples from a hom ogeneous mixture o f each matrix from untreated control sam ples, and by analyzing at least two levels o f fortifications. STATISTICAL METHODS Statistics w ill be lim ited to those specified in the subject m ethods and to the calculation o f average recoveries, as applicable. GLP STATEMENT A ll aspects o f this study shall be performed and reported in com pliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statem ent that the study w as conducted in com pliance w ith current and applicable GLP standards and w ill outline any deviations in the study from those standards. This statem ent w ill be signed by the Study Director and Sponsor R ep resen tative. REPORT A final report w ill be prepared by the principal investigator or their designee at the conclusion o f the study. The report w ill include, but w ill not be lim ited to, foe follow ing: The name and address o f foe Study Director, Sponsor Representative, and of the testing facility. A statem ent o f GLP com pliance (any related docum entation, such as chain-of-custody records, must be in the study records). P a g t I I o f 65 Exygen Research Page 75 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number P0001131 The signed and dated statement by the Exygen Research Quality Assurance U nit regarding dates o f study inspections and dates findings were reported to the Study Director and M anagement. A description o f the exact analytical conditions em ployed in the study. If the subject method was follow ed exactly, it is necessary to include only a copy o f the analytical method. A ny m odifications to is m ethod w ill be incorporated into the report. I f the method is photo-reduced, the project number and page number m ust be included on each page. Description o f the instrumentation used and operating conditions. A ll results from all sets analyzed. Control and fortified sam ples w ill be identified and the data table w ill include sam ple number and fortification le v e l. Representative chromatograms for each analyte in each m atrix, including chromatograms o f a standard and a control sam ple, and a chromatogram at a fortification level. The location o f the analyte peaks w ill be clearly identified in all chromatograms. A ll circum stances that m ay have affected the quality or integrity o f the data w ill 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 foe report that is being altered and foe reasons for foe alterations. The amendment w ill be signed and dated by foe Study Director and the Sponsor R epresentative. A ll applicable requirements for reporting o f study results as per 40 CFR 7 9 2 .1 8 5 . 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 foe environm ent to foe test or reference substance(s). Page 12 o f 6$ Exygen Research Page 76 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number; POOOI131 AMENDMENTS TO PROTOCOL AU significant changes to the analytical protocol outlined here w ill be expressed in writing, signed and dated by the Study Director and Sponsor Representative. Amendments usually w ill be issued prior to initiation o f study plan change. H owever, when a change is required without sufficient tim e for the issue o f a written amendment, that change m ay be effected verbally with supporting documentation signed and dated by the Study Director and follow ed w ith a written amendment as soon as possible. In this case, the effective date o f the written amendment w ill be file date o f the docum ented change. C opies o f the signed amendments w ill be appended to all distributed study plan copies. The original amendment w ill be m aintained w ith the original study plan. Any deviations from the study plan or from the analytical m ethod as provided w ill be docum ented and reported prom ptly to the Sponsor R ep resen tative. DATA RECORD KEEPING Records to be maintained include the follow ing (as appropriate): Sam ple tracking sheet(s) Sam ple receipt records, storage history, and chains o f custody H istory and preparation o f standards (stock, fortification, calibration) D escription o f any m odifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables A ll chromatographic and instrumental conditions Sam ple extraction and analysis dates A com plete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence A ny other documentation necessary for the reconstruction o f the study Chromatograms-A ll chromatograms w ill contain the follow ing: Sam ple identification, injection date, arrow or other indication o f the area o f interest, and injection number corresponding to the run. A dditionally, fortifications w ill include the amount o f analyte added and the sam ple number o f the sam ple that w as fortified. Analytical standard chromatograms w ill additionally include the concentration (e.g ., pg/m L). Page H o f 65 Exygen Research Page 77 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 A s part o f the docum entation the follow ing sheets w ill be included in each analytical set: a run sheet listing the sam ples to be run in die set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITY ASSURANCE The QA U nit o f Exygen Research w ill inspect the study at intervals adequate to assure com pliance w ith GLP's, and w ill report the findings o f audits to the Study D irector, Exygen M anagement, and die Sponsor Representative. RETENTION OF DATA AND ARCHIVING A ll hard copy raw data, including, but not lim ited to, the original chromatograms, worksheets, correspondence, and results shall be included w ith die data package submitted to the Study Director. T hese w ill be archived w ith the original study plan, amendments, final report, and all pertinent inform ation from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period o f tim e specified in 40 CFR 792.195. An exact copy o f the m aterials submitted to the study director w ill also be kept at Exygen Research. Exygen w ill obtain perm ission from the study director before discarding or relum ing sam ples. Exygen Research Page 14 o f 65 Page 78 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number P0001131 APPENDIX I ANALYTICAL METHODS V 0001780: `M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Water by LC/M S/M S" V 0001781: "M ethod o f A nalysis for die Determ ination o f Perfluorooctanoic A cid (PFOA) in Soil by LC/M S/MS" V 0001782: "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Sedim ent by LC/M S/M S" V 0001783: "M ethod o f A nalysis for the D etennination o f Perfluorooctanoic A cid (PFOA) in Fish and Clam s by LC/M S/M S" V0001784: "M ethod o f A nalysis for foe Determ ination o f Perfluorooctanoic A cid (PFOA) in V egetation by LC/M S/M S" V 0001785: "Method o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Sm all Mammal Liver b y LC/M S/M S" V0001786: "M ethod o f A nalysis for the D etennination o f Perfluorooctanoic A cid (PFOA) in Sm all M ammal Serum b y LC/M S/M S" Exygen Research Page IS o f 65 Page 79 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: POOOt131 AN ALYTIC AL METHOD MethodNumber V0001780 Method efAnalyst! for the Determtnatien of Perflaorooctanoic Acid (PFOA) lo Water byLG/MS/MS Analytical TestingFacility: Exygen Research 3058 Research Drive StateCollege, PA 16801 Approved By: P ul Connolly ' Technical Leader, LC-MS, Exypn Retouch iQltuAW Due JMohn*Frla(he(rtmy t l/d __ V ic Preckknt, Oparatioej, ExygnnRetiieith Exygen Research Total Pagati 7 Page 16 o f 65 Page 80 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 EsyptlUaMMh MaltedNumterVOOOPIQ ANALYTICAL METHOD Methodo fAnalytic ftt the Dctenmnatiano fPerfluocooctanoic Add (PFOA) in Water by LOMS/MS 1.0 Scope U ni methodis to be employed for 0m isolation and quantitation of parfluoiooctanoic add by Kid) Pcribrmaaoc Liquid Chromatography eoopJad to a tandem Mass Spectromctric Detector(LC/MS/MS) in water. 2.0 Saftty 2.1 Always observesaft laboratorypractices. - 22 Consulttiie appropriateMSDS beftia handHnganychemical for proper safety 3.0 SamplefteqdrsnMot 3.1 At least40nL oftert sampleferextraction. 3.2 No sampleprocessingis needed ftr watersamples. 3J Samples stored refrigerated eboold be allowed to equilibrate to room temperature. 3.4 A ll samplesmustbethoroughlymixed beforebeing sampled for extraction. 3J Any samplescontaining pvticlee should be centrifriged at 3000 rpm for -5 minutes andfoesupernatantused for theextraction. 3.6 Sample collection procedures w ill be specified in the sampling plan for this project 40 ReagentsendStandards 4.1 Water-HPLC grade 4.2 Methanol-HPLC grade 4.3 AmmoniumAcetate-A.C.S. ReagentGrade 4.4 Perftaorooctmoic Acid - Sigma-Aldrich 5.0 InstrumentandEquipment 3.1 A high performance liquid efaromatograph enable o f pumping up to 2 solvants equipped with e variable volume injector capable o f injecting 5-200 pL connected to atandemMaesSpectrometer (LC/M&MS). 5.2 A deviceto oolloettaw dataforpeak integration andquantitation. $.3 Analyticalbalaooecapableo freadingto 0.00001 g. 5.4 50mL diy osablo polypropyleneosntriftige tubea. 5.5 13mL dftposablopolypropylene centrifuge tubas. 5.6 DisposablemkiopipMs(50-100uU 100-200uL). 5.7 125-mLLDPEnarrow-mouthbottles. 5.8 2 mLclearHPLCvial kit. 5.9 Disposablepipettes. 5.10 Autopipettes(100*1000 pL and 10*100pLL w itii disposable tips. 5.11 WatersSepPakVac6 cc (Ig) tCU SPEcartridges. Pa2of? Page 17 o f65 Exygen Research Page 81 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number; P0001131 BxyjwK----th MhOdNumberV0001780 [ ANALYTICAL m e th o d Methodo fAnatyria for the Determinationo fFerfluorooctanoie Acid (PFOA) in Waterby LC/MS/MS 9.12 SPBvtoum manifold. 9.13 Caotrifogecapableo ftpuuring 50raL polypropylene tubeeat 3000rpm. 6.0 Chromatographic System 6.1 Analytical Column: FtoophtaaRP(KeyefooeScientific), 2.1 mm x SOmm, $m (P/N: 2909-092130) 6.2 Tcmperatigr 30*C 6.3 Mobile Phaac(A ): 2 mM Ammonium Acetate in Water Mobile Phase(B ): M U M Gradient Program: Trnia fmiiA 0.0 1.0 8.0 20.0 22.9 &A 65 65 25 25 65 Flow Rate 21fi fmL/mint 35 0J 3$ 0J 79 0J 79 0.3 3$ 0.3 16 Injection Votame: 15|*L (cm be increasedto asmuch at 50 pL). 17 Quantitation: PeakAna - external standardcalibration curve. 6.8 RunTime: -2 3 manual. Theaboveconditions areintendedas aguideand maybechanged in order to optimize theHPLCsystem. 7.0 MS/MS System 7.1 Mode: ElactroeprayNegativeMRM mode,monitoring 413 ->369m/z. The above conditiona am Intended aa a guide and may be changed in order to optimize the MSMS syitem. 8.0 Prepetition o fSolutions 8.1 Mobile Pham 8.1.1 2 mM ammonium acetate in water it prepared by adding 0.154 g of ammoniumaeetateto 1000raL o fwater. Alternatevolume! maybeprepared Exygen Research Page 8 o f 65 Page 82 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number P0001131 BaygmKraeawli MetoadNubir VOOOITtt ANALYTICAL METHOD i MefoodofAmdyeiifcrfoeDeUrminetHmofPerfluorooctappic Arid (PFOA) in Waterby LC/MS/MS 9.0 StandardPreparation 9.1 StandardStock/FoctificatiooSolution 9.1.1 Piepm* a stock solution of~100|igfaiL o f PFOA by weighing 10 mg o fanalytical standard(corrected for purity) and dilute to 100 mL with methanol in a 125-L LOPEbottle. 9.12 A 10p tfnL fortification solution o fPFOA is prepared by bringing 10 mL o fthe 100pgtaL solution to a final volume o f 100 with methanol in a 129mL LDPBbottle. 9.1.3 A 1.0pfftnL fortification solution o fPFOA is prepared by bringing 1<> mLoffoe 10 pgfaL solution to a final volume o f 100 with methanol in a 125mL LDPBbottle. 9.1.4 A0.1 pgtaL fortification solution o fPFOA is prepared by bringing 10 mLoftho 1.0pgftnL eolation to afinal volumeo f 100with methanol in a 125mL LDPBbottle. 9.1.9 A 0.01 pgfniL fortification aolution o f PFOA ia prepared by bringing 10 mL o f die 0.1 p ffa L solution to a find volume of 100 with methanolin a 125mL LDPB bottle. 9.1.6 Thestock andfortification solutions erato bestored in a refrigerator at approximately 4*C sad are stable fix' a maximum period of 6 months from foedateo fpreparation. 92 StandardCalibrationSolutions 9.2.1 LC/MS/MS calibntion standards are prepared in HPLC water. The calibration cteadarda are prooeseed through the extraction procedure, identical to eamplea. . 922 The following is a typical ample: additional concentrations may be Final CMMBUlthM Fortifiration Volumeof Concentrationof Calibration ofPertificatkMt Volume FortifiedCoatiel Calibration StandardID Solution(cob) (liL) Sacrale(mL) Standardfax>* (example) 0 0 40 0 XCmmddyy-0 10 100 40 25 XCmraddyy-1 10 200 40 50 XCmmddyy>2 10 400 40 100 XCmmddyyO 100 to o 40 100 200 40 250 XCmmddyy4 500 XCmmddyy-5 100 400 ____ & _____ 1000 XCmmddvY-6 * Theextractedconcentrationo fthecalibration tendard is equal to 8 its initiai concentration,dueto theconcentration o fthe steadardduring the extraction (SPE). XC extractedcalibrationstandard. pBs*4ori> Page 19 o f 65 Exygen Research Page 83 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 Wwwwiti M ated Numb* V000I7I0 [ ANALYTICAL M IT H O D Method ofAnalyte for foe DeterminationofPcrfluorooctanoic Acid (PFOA) in Waterby LC/MS/MS 9.2.3 A mk>stendard solution (reagent blmk) mum ha Hrm n d with etch m ofstandard! SHlraulcd. 9.1A Storeall extracted calibration standardsin 15-mL polypropylene tube at 2*Cto <*C,up to two weeks. 9.2.5 Alternate volumes andconcentrations o fatandardi may beprepared ai 10.0 Batch SetUp 10.1 Each batch of samples extracted (typically 20 or tea) must include at least ooe reagent control (method blank using HPLC water) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural reooveryfor thebatch. 10.2 Requirements for field and laboratory duplicates and spikea w ill be specified in foequality assuranceplan for this project 11.0 Sople Extraction 11.1 Measare40 mLo fsampleor aportion o fsimple diluted to 40 mL with water into SOmL polypropylene ccntrifoge tubes (fortify as nosded, replace lid and mix wall). 11.2 Condition the Cu SPB cartridges (1 g, 6 mL) by passing 10 mL methanol followed by3 mLo fHPLC water(*2 dro^sec). Do not let column nut dry 11.3 LoadsampleonoonditionadCh SPEcartridge. Discard eluate. 11.4 Elute with -5 mL 100H methanol. Collect 5 mL of shiate into graduated 15mLpolypropyhpc centrifogetubes(final volume 5 mL). 11.5 Analyze samplesusing etectraspnyLC/MS/MS. 12.0 C liomaiogrephy 1Z1 Uyect foe samemount o feach stendard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzedsamples. 12.2 StandardsofPFOA eofreapcftdmgto at laaatfive or moreconcentration levels mastbe iachidedfatanaatfytioal set 1Z3 An entire sat of extracted calibration standards must be included at the beginning and at the end of a sample sat Extracted standards mini be intanpen sd bitween every 5-10 eatiptos. Aa an alternative, an entire set of extracted calibration standards may be iiyceted at the beginning of a set followed by extracted calibration standards interspersed every 5-10 simples (to account for s second set o f extracted stadards). In either case, extracted calibrationstandardsmustbefoe first andtost injection in asampleset. 12.4 Use linear stendard curves for quantitation. Linear standard curves we generatedfor theanalyteby linrer regression using 1/x weighting ofpeakarea Pa* 5 o n Page 20 o f6S Exygen Research Page 84 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number: POOOt 131 BxyftaKMMCch MKbodHwabwVOOOITtO ANALYTICAL METHOD ] Method o fAnalysis farthe Determinationo fPerihiorooctwiolc Add (PFOA) in Waterby LC/MS/MS versuscatibnikn attndard concentration using MasaLynx 3.3 (or equivalent) oftw tniyM B . 12.5 Sample response should not exceed sfondai* responses. Any samples that exceedstandardresponsesshouldbe farther diluted andreanalyzed. 13.0 AooaptaneeCriteria 13.1 Chranatopn must stow a peako ft daughter ion at 369 am from a parent o f413 amu. The413 aam parent corresponds to the PFOA anion, while the Aantf***ion (369asm) representsthe lossofcarbondioxide. 13J Method blanks mast 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 mustbeobtainedaadtheentire setmost bere-extracted. 13J Recoveries of control spikes and matrix spikes moat be between 70-130% of their known values. I f a control spike fa lli outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by die analyst to determine if re-extraction is warranted. 13.4 Any calibration atandard fouod to be a statistical outlier by using the Huge Error Test, may b t excluded from the calculation of the calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f die total number o f extracted standards (3.5 The corralation coefficient 90 9 * calibration curves generated must be 0.992 (R] 0.915). I f calibration reeults toll outside tbcae limits, then appropriate amps must be taken to adjust intirument operation, and die standard!ortherelevant aatofeanplas should bereanalyzed. 13.6 Retention rimes between standards and samples must not drift more than 4% within ananalytical run. I f retention time drift exceedsthis limit within ananalytical run thenthe setmintbereanalyzed. 14.0 Calculations 14.1 Use the Mowing aquation to calculate the amount of PFOA found (in ng/L, based on peak ares) using the standard curve (linear regression parameters) generatedby dieMassLynx softwareprogram: PFOAfound (ng/L) - (Peakarea-intercept) x DF slope DF - Actorby which foe final volume wasdiluted, if necessary. Ragffof? Page 21 o f63 Exygen Research Page 85 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 EiygMnwMfeh M*ewdNunbwVOOOI7IO I AjVALVnCAL M1THOD | MothodofAjuUyi lir tin Dmnntiuttan ofPwfluoroocunok Acid <PPOA)in Witer by LC/MS/MS 142 For ample Jbrtifiod with known amount o f PFOA prior to extraction, ute the fbnowing equationto m lculle Um percentraeovery. Recovery(H )- [ total-- tytefound(agfl.) - eaelytefoundin control(ng/L)] pp analyteadded(ng/L) Exygen Research Pat7 of? Page 22 o f 65 Page 86 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD MethodNumber V00017I1 MethodofAeelyih far the Dllm lelrtoe o fPerfinorooclooetc Add (PFOA) I Soil by LC/MS/MS Analyticel TootingFeeiHty: Exygen Reeeerch 3051 ReeeerchDrive SteloCollege, PA 16101 Approved By: T t l C J i,____ PoulCoenoDy ' Technical Leader, LC-MS, ExygenReeeerch w l* * * > * Dote J&mFtahoty 7 /v ic a Freddato, Operations,ExygenResearch Dato Exygen Research Total Pafto: 7 Page 23 o f 65 Page 87 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number P0001131 IqgnU m R k MhedNiaab*V00017tl I ANALYTICAL METHOD I Methodo fAnelyeil for theDetermination o fPnrfluorooctaooic Acid (PFOA) in Soil by LC/MS/MS 1.0 Scop* Thk math) to beemployed tar the isolation andquantitation o fperfluorooctanoic add by High PeribnnaDCo Liquid Chromatography coupled to a tandem Mass SpectromtrieDetector(LC/MS/MS)in soil. 2.0 Saftty 2.1 Alwaysobservesaft laboratorypraoticee. 2.2 Coasult the appropriateMSDSbefore handlingany chemical for proper safety precautions. 3.0 SampleRequirement 3.1 At least 15g oftestsamplefar amotion. 30 No sampleptooeetiiig is neededfor to il aamples. 3.3 Samples meed reftigerated should be allowed to equilibrate to mom temperature. 3.4 A ll aamplesmuttbethoroughly mixed beforebeingsampled for extraction. 3.5 Sample collection procedures w ill be specified in the sampling plan for this project 4.0 ReagentsandStandards A t Water-HPLCgmde 4.2 Methanol- HPLCgrade A3 Amtntathim Acetate- A.C.S. ReagentGrade A4 Periluorooctanoic Add - Sima-Aldrich 5.0 InUnaMot Etju^BNnt 5.1 A high performance liquid chromatograph capable o f pumping up to 2 adventa qnippfd with t variable volume injector capable o f injecting 5-200 pL comectadto atandemMam Spectrometer(LC/MS/MS). 5.2 A deviceto collectrawdata for peakintegration andquantitation. 5.3 Analyticalbalancecapableo freedingto 0.00001 g. 5.4 50mLdupoeablepolypropyleneeentrifligetubea. SJ 15nd-di^weaMepolypropyleneeentriAigetubea5.6 DiapottMe mtcropipeU (SO-lOOuL, 100-200uL). 5.7 125-mLLDPEnarrow-mouthbottle. 5.8 2 mLclearHPLCvial kit. 5.9 DiipftiiM t pipettea. 5.10 Autopipettes(100-1000tiLsnd 10-100pL), with disposable tips. 5.11 WstenSepPaltVac6 cc(lg) tC l8 SPEcartridges. 5.12 SPEvaotnanmanifold. 5.13 Ultrasonic bath. P stslo t' P a g e of6} Exygen Research Page 88 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number P0001131 ANALYTICAL MITHOD ] Methodo fAnalyaie feetheDetermination ofPwfluoroocitPoic Acid (PFOA) in Soil by LC/MS/MS 5.14 Wrict-aetioa shaker. 5.15 Centrifuge capableofepinring 50 mL polypropylene tubesal 5000rpm. 6.0 Chromatographic System 6.1 AnalyticalColumn: FluophaseHP(Keystone Scientific), 2.1 mmx 50mm. 5p (P/N: 625054)52130) 62 Temperature; 30*C 6.3 Mobile Phaae(A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Pbaec(B): Methanol 6.5 GradientPropun: XB8B2 0.0 1.0 8.0 20.0 22.5 *4 65 65 25 25 65 Flow Rate fmlVmiiri 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15pL (canboincweod to aamuch at 50 pL). 6.7 Quantitation: PeakAna -external standardcaHbcatioacurve. 6.t RunTime: ~23 tninutee. Theaboveoonditioaaareintendedaaaguideandmaybechanfed in order to optimise (heHPLCayaten. 7.0 MS/MS System 7.1 Mode: Blectro^vay NegativeMRM node, momtoring 413- 369 mfr for PPOA. Theaboveconditioiii areintendedaaaguide and maybechanged in order to optimisethe MSMSayatcm, 1.0 PrepmstxmofSolutions 8.1 Mobile Phaae y 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0.154 g of ammoniumacetateto 1000mL o fwater. Alternatevolumes maybeprepared. pejn Page 25 o f 65 Exygen Research Page 89 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygMtUamcfe MohadManbarVeOOlttl ANALYTICAL METHOD Methodo fAnalysis forfoeDotennintlooofPerfhiorooctanoic Acid (PFOA) In Soil by LOMS/MS 9.0 StandardPreparation 9.1 StandardStoeWFoitiflcation Solution 9.1.1 Preparea stock solution o fMOO pgtaL o f PFOA by weighing 10mg ofanalytical standard (connoted fin purity) and dilute to 100 mL with msfonol in a 123-mLLDPEbottle. 9.1.2 A 10 p jfa L fiwtificatioo tollmen o fPFOA ia prepared by bringing 10 mL o f the 100 ug/mL solution to a final volume o f 100 with methanol in a 125mL LDPE bottle. 91J A 1.0pgtaL fortification eohitiQnofPFOA is prepared by bringing 10 mL of the 10pgftnL solution to a final volume of 100 with methanol in a 125mL LDPEbottle. 9.1.4 A 0.1 pgftnL fortification solution o fPFOA is prepared by bringing 10 mLofjhe 1.0pgfaiL solution to a final volume of 100with methsnoiin a 125mLLDPB bottle. 9.1.5 A 0.01 pgtaiL fttctification aolution o f PFOA ia prepared by bringing 10 mL o f the 0.1 pgfaL solution to a final volume of 100 with methanolin a 125mL LDPEbottle. 9.1.6 Thestock and fortification solutions areto be stored in arefrigerator at approximately 4*C and am stable Ibr a maximum period o f 6 months from thedataofpreparation. 9.2 StandardCalibration Solntiona 9.2.1 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through tbs extraction procedure, identical to sample. 922 The following is a typical example; additional concentrations may be prepend ssneeded. Final Omoaattatien Fortification Vetunwof Concentrationof Calibration efFwtifieatian Volume FortifiedControl Calibration Standoti ID SolutionfooM (uL) laamlefruL Standardfeet)4 (examok) 0 0 40 0 XCmmddyy-0 19 100 40 25 XCmmddyy-l 10 200 40 SO XCmmddyy-2 10 400 40 100 XCnundyy-3 100 100 40 250 XCnunddyy-4 100 200 40 500 XCramddyy-S to o 400 40 1000 XCmmddw<6 * Theextractedoooosntraticn o ffits caUbntion standard is equal to 8x its initial concentration,duoto foeconcentrationo ffoestandardduring foe extraction (SPE). XC extractedcalibrationstandard. Pa| 4 of7 Page 26 o f 65 Exygen Research Page 90 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bayganfoams! Mattel Nu*trV000l78] ______________ ANALYTICAL METHOD Methodo fAnalysis for theDetsnninationofPerfluarooctsnoic Add (PFOA) in Soil by LC/MS/MS 9.2J A sera itenderd solution (resgent blank) mua he prepared with each eel o f atandarde extracted. 9wL4 Storeall extracted calibration standard in 13-mL polypropylene tube at2*C to 6"C, upto two week*. 9.2.5 Ahetnatovolumes endconcentrations o f standards may be prepared as 10.0 Batch SetUp 10.1 Each batch o f samples extracted (typically 20 or less) must include at least onereagent control (method blade using SmL o f methanol) and two reagent controls fortified as known concentrations (lab control spike) to verify proceduralnoovsty for thebatch. 10.2 Requirements for Add and laboratory duplicates and spikes w ill be specified is thequality aaauranoeplan for th ii project. 11.0 11.1 Weigh S g o f ample into 50 mL polypropylene centrifoge tubes (fortify ss needed,replacelid nodmix we. 11.2 Add5 mLofmetfaaaoi andshakeon awrist action shakerfor-15 minutes. 11J TYwn&rthetubmtoroutaascckbsihtnd sonicatefor-15 minutes. 11.4 Bring tits volume up to 40 mL with under in the 50 mL polypropylene centrifogetube. 11.5 Centriftige for-10 mutatesat-3000 rpm. 11.6 Condition the C SPB cartidgee (1 g, 6 mL) by passing 10 mL methanol followedby 5mLofHPLC water(- 2 drop/sec). Do not let column run dry 11.7 Load (decant) the sample on die conditioned Cu SPE cartridge. Discard ekiate. 11.8 Elute with -5 mL 100% methanol. Collect 5 mL o f eiuate into graduated IS mLpolypropylene centrifoge tubes(final volume - SmL). 11.9 Analyze samplesusingeleotitMpny LC/MS/MS. 12.0 Chronutopm'hy 12.1 Iijflo t the sameamountofeach standard, wnple and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow ell malyxed samples. 1X2 Standards ofPFOAcorrespondingto at least five or more concentration levels mustbeincluded in ananalytical set. 12.3 An entire set o f extracted calibration atandarde mint be included at the beginning and at the end of a ample set Extracted standards must be interspersedbetweenevery 5*10samples. As an alternative, anentire setof PaftSof7 Page 27 o f 65 Exygen Research Page 91 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number P0001131 MMkodNuato VW17<I I ............... ANALYTICAL METHOD | Methodo fAnilyflafthPcrtcm55ooo7K?iooctaolc Acid (PFOA) in Soilby LC/MS/MS extracted calibration standards may bo injected at the beginning of a set followed by extracted calibration standards interspersed every 3-10 sample* (to accountfor a second seto fextracted standards). Ia either case, extracted calibration standardsmustbethe first id last iryeetion in a sampleset. 12.4 Use line standard curves for quantitation. Line standard curves arc tenanted for foe analyteby line regression using 1/x weightingo fpeak area versus calibration standard conesntration using MaasLynx 3 3 (or equivalent> software system. 12.3 Sample responee should not exceed standard responses. Any samples that exceedstandardresponsesshould be furtherdiluted sndreanalyzed. 13.0 AcoeptanoeCriteria 13.1 Cfaromaiognmta u t ritow a peako f a daughter ion at 369 amu from a parent o f 413 amu. The 413 emuparent corresponds to the PFOA anion, while the daughterion (369 amu) iwprssHs foe loss ofcarton dioxide. 13.2 Method Maaks moat not cootew PFOA at levels greater than the LOQ. If a blank contahn PFOA at levels peelerthan SOng/L, then new blank sample mustbeobtainedandfoeentire s mu bere*cxtiacted. 13J Reeowiee o fcontrol qpficeaand matrix spikes must be between 70*130% of their known vahtes. If a control spike foUsoutside foe acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70* 130% should be evaluated by foe analyst to detannine if re-extraction is 13.4 Any calibration standard found to be a statistical outtiar by using the Huge Error Test, may be excluded from foe calculation o f foe calibration curve. However, the total numb o f extracted calibration standards that could be excluded must not exceed 20% o f the total numb o f extracted standards unacted. 13.3 The correlation coefficient (R) for calibration curves generated must be 20.992 (R2 20.963). If calibration results foil outside these limits, then appropriate step must be taken to adjust instrument operation, and the standardsor therelevantsetofsamplesshouldbereanalysed. 13.6 Retention times bstwesn standards snd ssmples must not drift more then 4 %within snanalytical ran. I f retention time drift exceedsthis lim it within ananalyticalranthan foe setmustbereanalyzed. Pag* $ o f? Page 28 o f 65 Exygen Research Page 92 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number; P0001131 Bargeel euewh M e d a * N e a t e r VOOOI7II ANALYTICAL MCTHOP | Methodo fAnalysis fortheDetermination o fPerfhtatooctanoie Acid (PFOA) in Soil by LC/MS/MS 14.0 Caknlatioas 14.1 Uic the following equation to calculate 0 amount o f PFOA found (in ng/L, baud on peak n ) m ini foe aundaid wive (linear regreceion panmeten) fenented byfoeMeet Lynx softwareprogram: PFOA found (ng/L) xDF DF- (eotorbywhichthe final volumeweediluted, if nooceeary. 14.2 For lamplee fortified with loom amotion of PFOA prior to extraction, ute foe following etpiatioo to calculate the percentrecovery. Recovery(14) f totalanalytefixmd(ng/L) analytefoundin cootiol(ng/L)l analyteadded(ng/L) * 14.3 Use foe following equation to coovst foe amount o f PFOA found in ng/L to ug(g(ppb). PFOAfound foobl- fPFQA found(n/L1 x volumee---" -t m r^r ampleweight (3 g) 14.4 U u the following equation to calculate foe amount o f PFOA found in ppb basedondty weight. PFOA found(ppb) dry weight " PFOA found (ppb) x [100%/ total aolide(14)] P U e7of7 Page 29 o f 65 Exygen Research Page 93 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD MethodNumber: V00017I2 Metked e fAaalyela fer Ike Decermtaedea o f Perflaeroectaaeic Add (N O A) la edlamal by LC/MS/MS Analytical Teuinj Facility: ExygntReaeaich * 3031 ReeearehDrive StateCollate, PA 16(01 ApprovedBy: Paul Connolly I Technical Leader, LC-MS, ExygenRaaaaich 10k b lv l Date CL' / m MIbhin Flaherty r Viecet Preaidenl, Operation,ExygenReaearoh Date Exygen Research Total Pafae: 7 Page 30 o f 65 Page 94 o f 136 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Mtffeod Num ber VOOOS7I2 I AIULYTICAL METHOD | Methodo fAnalysis forth Datemiination ofPerfluorooctsnoic Add (PFOA) in Sediment by LC/MS/MS 1.0 Soope Tba methodis to be employedftr the isolation endquantitation o fparfluorooctanoic eeid by Higb Performance Liquid Chromatography oouplod to a tandem Mail SpedrometrieDetector(LC/MS/MS) in sediment 2.0 Safety 2.1 Alwaysobservesafelaboratorypractices. 2.2 Consultthe appropriateMSDSbeferohandling anycbemioalfor propersafety precaution. 3.0 Senile Requirement 3.1 At leaat301 oftaetample fbr extraction. 32 tin ampin p rn riiih n 1nntriirt fhr leitim m t amplni 3.3 Sample noted reftigerated hould be allowed to equilibrate to room 3.4 A ll amplemustbethoroughly mixed beforebeing sampled for extraction. 3.5 Smple collection procedural w ill be pacified in the sampling plan for this 4.0 4.1 Watar-HPLC grade 4.2 Methanol-HPLCgade 4.3 Acetic Acid-Reagent grade 4.4 AmmoniumAcetate-A.GS.Reifant Grade 4.5 Perfiuorooctanok A d d -Sigma-Aldrich S.0 5.1 A high perfotmmoe liquid chromatograph capable o f pumping up to 2 ohranteequippedwith a variable volume injector capable o f injecting 5-200 pL contactedto atandemMam Spectrometer(LC/MS/MS). 5.2 A deviceto collectraw data forpeakintegration andquantitation. 53 Analytical balancecapableo fraiding to 0.00001 g 5.4 50 mLdiapoaabkpolypropylene cantrifiipB tubes. 5.5 15u L disposablepolypropylene eentrifege tubes. 5.6 Disposablemicropipets (50-lOOuL, 100-200uL). 5.7 125-mLLDPBnarrow-mouthbottles. 5.8 2 mLclearHFLC vial Idt. 5.9 DienonbleW pfflft 3.10 i^ ^ m n O o T io O O |iL a d 10-100nL). w di divouble lij. 3.11 W a n S*> PkVc6 oe(lg ) tCU SPBcutridga. 3.12 SPEvkcuoramtniEold. Page 31 o f 65 Exygen Research Page 95 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 foygmltaoencb MeMNim*trVOOOI7l2 I ANALYTICALMETHOD . Methodo fA u ly n i for theDeurannition ofPerfliiorooctanoic Acid (PFOA) in Sediment bv LC/MS/MS ' 5.13 Vortezer. 5.14 WriaMstko ahdmr. 5.15 CetnrUhgtcapableofapimring50 mL polypropylene tube* el 3000ipm. 6.0 ChromatographicSyetera 6.1 Analytical Column: nuophaaoRPQCeyatofie Scientific), 2.1 nun x 30mm.V (WN: 125054)52150) 6.2 Temperature: 30*C 6.3 Mobile Fhaaa(A ): 2 mM Ammonium Acetate in Water Mobil Phase(B ): Methanol Gradient Program: Tima /m ini 0.0 1.0 S.0 20.0 22.5 &A 65 65 25 25 65 Flow Rate 2LB (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 traction Volume: 15pL (can behtrraaaadto aamachaa50 pL). 6.7 Qaastitatiaa: PeakA m - external itandard calibration carve. 6.1 RunTime: - 23 minutaa. Theaboveconditiont areIntendedaaa guide andmaybechangedin order to opdmltetheHPLC cyatem. 7.0 MS/MSSyatam 7.1 Mode: EtaeMapny NegativeMUM mode,monitoring 413 -a 369m /i for PFOA. Theaboveeondlllenaareintended aaaguide andmaybechanged in order to optimizethe MSMSayatam. 1.0 Preparationo fSolution! 5.1 Mobile Phaae 1.1.1 2 mM ammonium acetate in water la prepared by adding 0.154 g of ammoniumacetateto 1000mL o fwater. PIC*) of7 Page 32 o f 65 Exygen Research Page 96 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen P rotocol N um ber P0001131 SqidlaM R k M e te d N u m b * VOOOI7I2 j ANALYTICAL METHOD ~~ Methodo fAnalyiu fa ihc Dtmmimtion ofParfluoroocUnoic Acid (PFOA) in Sediment by LC/MS/MS g.2 ExtractionSolutions 8.2.1 1H noetic add m wateris prepared by adding 10 mL ofacetic acid to 1000mLo fwater. Alternate volumes maybeprepared. 9.0 StandardPreparation 9.1 Stvdard Stock/Fortifictfkn Solution 9.1.1 Preparea stock solution o f-100 pg/mL o f PFOA by w eiring 10 mg ofanalytical standard (comctod fin purity) and dilute to 100 mL with methanolin a 125-mLLOPEbottle. 9.1.2 A 10 p^mL fortification solution o fPFOA ia prepared by bringing 10 mL o f the 100 pgtaL eolutioo to a final volume of 100 with methanol in a 123mL LDPBbottle. 9.1.3 A 1.0 pgfaiL fortification solution o f PFOA ia prepared by bringing 10 mL ofthe 10jigtaL solution to e final voltnne o f 100 with methanol in a 125mL LOPEbottle. 9.1.4 A0.1 pg/mL fortification solution ofPFOA ia prepared by bringing 10 mLoftbe l.Ojig/mL solution to a final volumeof 100with methanol in a 125mL LDPBbottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pgtaL solution to a final volume of 100 with methanolin a 125mL LDPB bottle. 9.1.6 TheHock andfixtification solutionsare to be stored in a reftigerator at spproximatdy 4*C and are stable for a maximum period o f 6 months fromdie dareo fpreparation. 92 ft Snlntbm* 9.2.1 LC/MS/MS calibration standards areprepared in methanol via dilution oftbeO.l pg/mL fortification aotutioo. 922 The following ia a typical example: additional concentrations may be Chneantration ofFortification SotutolaateD too 100 100 10 5 2 Vohme (mL)..... 10 5 2 10 10 10 Dilutedto (mL) 100 100 100 100 100 100 pirai Concentration fna/mL) 10.0 S.O 2.0 1.0 0.S 0.2 papson Pag* 33 o/65 Exygen Research Page 97 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number P0001131 M a te d N o te V00017I2 I ANALYTICAL MTTHOD | Methodo fAnalytic fortheDeterminationo fPerfiuomoetanalc Acid (PFOA) in Sediment by LC/MS/MS 9.2.3 Stan all calibration atandarda in 123-mL LDPC narrow-month bottles K 2*C to 6*C. upto elx tnantlu. 9.2.4 Alternate volumes andconcentratione o fstandard. maybo prepared ai 10.0 Batch Satlip 10.1 Bach M elt o f tamplaa ntnetad (typically 20 or loaa) muat inchide at lout one untreated control a d two untreated console fortified at known conoansttfooe (lah eonnot rpfke) to verify procedural recovery for the batch. 10.2 Requhetnenta for field tad laboratory duplicator and epiket w ill he epecifled in thequality aantmiceplan forthie project 11.0 SampleExtraction 11.1 Weigh J g o f temple into 50 mL polypropylene centrifoge tubea (fortify as needed,replaceUdtnd mix well). 11.2 Add 35 mL o f IS acetic acid, cap, vortex and ahake on a w rit! action shaker fbr-60 raitunee. 11.3 Centrifogethetubeaat -3000 rpm for -20 minutes. lM Condition the Cn SPE CKtridaa (1 g, 6 mL) by patting 10 mL methanol followedby20mLofHHiC water(~ 2 drop/eec). Do not let column nmdty 11.J Load (decant) foe eample cn foe conditioned Cn SPB cartridge. Diacard lid Add 20 mL o f methanol to the aedinicnt left in the bottom of the }0 mL centrifoge tube. Cep, vortex end Shake on a wriat action tinker for -30 mteuert-- 11.7 Centrifogethetubesat-3000ipm for-20 minutes. 11.1 Decantthemedunol ontotheaamcSPEcartridge. Collect the eluata. 11.9 Waahthe columnwith 4 mLo fmethanol. Collectthe eluate and add it to the ehtalecollectedin atop 11.1. 11.10 Condition asecondCu SPEcartridge (1 g. 6 mL) by paaeing 10mL methanol followedby 20mLofHPLC water( - 2 drop/eec). Do not let column run dry 11.11 Add dn methanol to -200 mL o f water and load on the aecond conditioned SPEcartridge. 11.12 Elute with ~J mL 100% methanol Collect 5 mL of eluate into graduated 15mLpolypropylene eantrifitgatub (final volume - 5 mL). 11.13 Analyse samplesusing eleetroepreyLC/MS/MS. PattSM? Page 34 o f 65 Exygen Research Page 98 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 M a tte d NtxuberVOOO 17S2 I ANALYTICAL M ITH O P | Methodo fAmlyaia fortheD etnilim lon ofPcrfluotooclanolc Acid (PFOA) In Sedimentby LC/MS/MS 12.0 Chromatography 12.1 Iqjeotthe sameamountofcach standard, sample and fortified sample mo ihc LC/MS/MS system. A celibretion standard muat precede end follow all analyxed samples. 12.2 Standardso fPFOAceaievoodiagto el loot five or moreconcemntion levels muetbeincluded in ananalytical act. 12.2 An audio eat o f extracted calibration atandarda muat be included at the beginning and at foe and o f a temple act. Standarda mutt be intenperaed between every 3-10 lempire. Ac an alternative, an entire act o f calibration atandarda may be ityocted at the beginning o f a cat followed by calibration atandarda intataperaad every 3-10 aamplca (to account Sir a aecond act of atandMda). In cither case, calibration atandarda muat be the firtt and let! injection in atempleact. 12.4 Uae --* standard enrvet for quantitation. Linear atandanl curvet arc generatedforfoe analyteby linearragreaeionwing 1/x weightingo fpealt area venue calibration atandard concentration uaing MaaaLynx 3.3 (or equivalent) softwareayatem. 12.5 Sample napenae abotdd not exceed atandard responses. Any temples that exceedstandardrcaponaaathouldbe Antherdiluted endreanalyzed. 13.0 AcceptanceCriteria 13.1 Chromatogrammuat show a peak o fa daughter ion at 369 amu from a patent o f 413 amu. The 413 amu patent corresponds to the PFOA anion, while the daughterkm(3fl> amu)loprcaantsthe Iocso fcarbondioxide. 132 Method blanks muat not oentain PFOA at levels greater than the LOQ. If a blank cootaina PFOA at levela greeter than 0.2 ng/tnL. than a new blank aampiamustbeobtainedsodtheentire actmuatbeiwxtracted. 133 Rscovariaa o fcentral spikes and matrix spikes muat be between 70-130% or their known values. I f a central apike folia outside the acceptable limita, the entiie set o f aamplra should be re-extracted. Any matrix apike outside 70 130% ahonld be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibntion atandard found to be a statistical outlier by using the Huge Error Teat, may be excluded tom the calculation o f the calibration curve. However, foe total number of extracted calibration atandarda that could be .hiawa must not exceed 20% o f the total number o f extracted standards injected. 13.3 The conelation coefficient (R) for calibration curves generated must be 10.992 (R1 20.9S5). I f oahbratsco results foil outside Ibase limits, then appropriate trope muat be taken to adjust Instrument operation, and the atandardaorthe relevant actofsamplesthouId be reanalyzed. Piaadef7 Page JS o f 65 Exygen Research Page 99 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number P0001131 M eted Numb V00017I2 I a n a l y t ic a l m e t h o d I Methodo fAnalysis fortheDetsnninstiono fPerfhrorooctsnolc Acid (PFOA) in Sediment by LC/MS/MS 13.6 Retention times between stsndards end samples must not drift more than 4 % within ananalytical ran. If retention time drift exceedsthis limit within ananalyticaltun thanthe tat mustbereanalyzed. 14.0 Calculations 14.1 UaeIlia following aquationto calculatetits amount of PFOA found (in nglmL. baaed on peak area) Using the standard curve (linear regression parameters) generatedby dasMassLynx softwareprogram: PFOA foundfaatinL) (Peekarea- intercept) xDF slope DF - fitte r bywhich the final tmlianc wasdiluted, i f necessary. 143 For samples fcttillcd with loom amounts o f PFOA prior to extraction, use thefollowing equationto calculatethepercent recovery. Recovery(% )- [totalanalytefound(mfmL) - analytefoundin conlrel(ng/mL)] i[[[m analytaadded(ng/mL) I4J Uaethe following equation to convert tha amotmto fPFOA ibund in ng/mL to n*fg(ppb). PFOA found(ppb)- [] sampleweight (3 g) 14.4 Use the fallowing equation (If necessary) to calculate the amount or PFOA found in ppbbasadon diy weight. PFOA found(ppb) dry weight - PFOA found(ppb)x [100% / total aolids(%l] Paae7af7 Page 36 o f 65 Exygen Research Page 100 o f 136 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number P0001131 ANALYTICAL METHOD MethodNumber V00017I3 Method o fA aliyah to rth r Petanmlaetica e fPortinero tta ce le Add (PFOA) la Fhh ad ClameVy LC/MS/MS Analytical Teating Facility; Exygen Reeeerch 305S Reeeereh Drive StateCollege, PA 16*01 Approved B y Paul Cotmoy Technical Leader,LCMS, Exygen Kccoatch Dale 3 tFlaherty ' Vice Preeident, Operation, ExygenReaeaich Date Exygen Research Total Pagaa: 8 Page 37 o f 65 Page 101 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen P rotocol N um ber: P0001131 BijfW tiW Ch MMMNuBtarVQOQItt) | ANaLYTICALM ITHO P I Method ofAnalysis for theDeterminationo fPerfluorooctanoicAcid (PFOA) in Pish and Claim by LC/MS/MS 1.0 Scope Thii method is to beemployed for the isolation andquantitation o fperfluorooctanoic add by High Peribnasaca Liquid Chromatography coupled to t tandem Mass Spectrometric Detector(LC/MS/MS)in fish anddams. 2.0 Safety 2.1 Alwaysobservesafelaboratorypractices. 22 Consulttheappropriate MSDS beforehandling anychemical for proper safely 3.0 SampleRequirement 3.1 At least20g o fteatsamplefor extraction. 3.2 Samples tixadd he processedbefore extraction. Place the frozen sample in foodprocessorandhomogenise 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 samples in frozen storage until time of analysis. 3J Sample collection procedures win be ipocifred la foe sampling plan for this ! standards 4.1 Water-HPLC grade 4.2 Acetonitrile-HPLC grade 4.3 Carbon(120-400mesh)- Reagentpads 4.4 Methanol- HPLC grade 4.3 Silica gel (60-200mesh)- Reagentpad* 4.0 Florisil (60*100mash)-Reagent grade 4.7 SuperdsanLC*NHj - Reagentpide 44 l*Octanol-HPLC grade 4.9 L-Aicocbie add-Reagent grade 4.10 Diroefoytdichlorostiene--Reagent grade 4.11 Toluene- Reagantgrade 4.12 Anunonium Acetate-A.C.S. ReagentGrade 4.13 Perfluorooctanoic Add - Sigme-Aldrich 5.0 InstrumentandEquipment 5.1 A Ugh peribrmaaoe liquid chromatograph enable o f pumping up to 2 solvents equipped with a variable volume iqjeetor capable o f injecting 5*200 pL contactedto atandemMaasSpectrometer(LC/MS/MS). 32 A devioelo oolleet raw dataforpeak integration andquantitation. 54 Analytical balancecapableo freadingto 0.00001 g. 2or a Page 38 o f 65 Exygen Research Page 102 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number P0001131 Biyjm R u MBCh r ANALYTICAL MfcTHOD M&od Number V001713 MethodOfAmlyU forthetlim nnlm iliw ofPwfluoroocttnoic Acid (PPOA) in Fish wid CtvnibyLC/MS/MS 5.4 Rotaryevaporator. 5.5 Tlamndmr. 5.6 125mLpear-staaped flasks. 5.7 50mL disposablepolypropytma ceotriftigc tubes. 5.8 15raL disposablepolypropykoecentrifage tuber. 5.9 D iyoitbto micropipots (SO-lOOuL, 100-200uL). 5.10 12S>mI.LDPBnarrow-mouthbottlea. 5.11 2 mLdrar HPLCvUlldt 5.12 Dispossbiepipettea. 5.13 Aotopipettes (100>1000|iL and 10-100fxL), with dupoeable tips. 5.14 SPEtubes(20mL)(Sopolcocat. no.N057177). 5.15 Wristactiondiakar. 5.16 Centxiftigecapableofspiantai50nLpolypropylene tubes at 2000rpm. 6.0 ChromatographieSystem 6.1 Analytical Column: FfcwpbaseRP(Keystone Scientific), 2.1 mm x 50mm, 5m (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase(A) : 2 mM AmmoniumAcetatein Water MobilePhase(B ): Methanol GradientProgram: X D iilltiB l 0.0 1.0 8.0 20.0 22.5 &A 65 65 25 25 65 Flow Rara 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 litfectioa Volume: IS (canbe increasedto as much as50 pL). 6.7 Quantitation: PeakArea- extents! standardcalibration curve. 6.8 RunTime: - 23minute*. Theaboveoonditions an intended asaguide andmaybe changed in order to optimize theHPLC system. PtatiofS Page 39 of6$ Exygen Research Page 103 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number P0001131 M tfbodN unfecr VOOOI7S3 ... .... ANALYTICAL m e t h o d fA n tlyU th e Dtannfautioo ofPirftoorooctonok Acid (PFOA) in Pih tnd CltmsbyLC/MS/MS 7.0 MS/MSSystam 7.1 Mode: ElocnoaprayNegative MKM mode,monitoring 413-369 m(z for PFOA. Theaboveooaditioos areintendedast guide andmaybe changed in order [0 optimize the MSMS system. 8.0 Pnpanuioao fSolutions 8.1 MobilePhase 8.1.1 2 b M ammonium acetate in water U prepend by adding 0.134 |o f ammoniumacetateto 1000mLo fwater. 82 Extraction Sohuiaoe 82.1 2%asoorbtcacid in methmol Upnpm d by dissolving 2 g o fascorbic acidm 100mLo fmethanol. 82.2 30% Dimedtyidichlororilane in toluene if prepared by bringing 3 mL o fdimethyldichlototilane to final volume o f 10mL with toluene. Alternate votumeemaybe pnparad. 9.0 StandardPrtperatioa 9.1 StandardStodt/Portification Solution 9.1.1 Prepare a stock aolulloo o f-100 pgfrnL o f PFOA by weighing 10 mg ofanalytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LOPEbottle. 9.12 A 1.0 pg/mL fbrtificatioa eolutioa o f PFOA i l prepared by bringing [ mL o fdie 100pgtmL aolutioo to a final volume o f 100 with mednool in a 123mL LOFBbottla. 9.1.3 A 0.1 pgfatL fbrtificatioo aolutiooo fPFOA ia prepared by bringing Id mLo fthe 1.0pgtaL solution to afinal volume o f 100with methanol in a123mL LOPEbottle. 9.1.4 A 0.01 pgfmL notification eolutioa o f PFOA ia prepared by bringing 10 mL of the 0.1 pghaL notation to a final volume o f 100 with methanol in a 123mL LDPE bottle. 9.13 Theitock and foctlflcarioc aolutiona erato be stored in a refrigeratorat approximately 4*C and am stable for a maximum period of 6 months from thedateo fpreparation. PaeeeefS Page 46 o f 65 Exygen Research Page 104 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E x y y ItwMcch M h o d Num ber VOOOtTU ANALYTICAL METHOD Methodo fAnalysis fbrtseDetermination o^Perfluorooctanoic Acid (PFOA) in Fiih and Clara by LC/MS/MS 9.2 StandardCalibration Solutions 9.2.1 LC/MS/MS calibration standards an pnparad in methanol via dilution ofthe 1.0pgtaL fortification solution. 922 The fbUowing is a typical example: additional concentrations may be pnparad aaneeded. CononOration ofFortification Solution(uafaL) 1.0 \A \X> 9 .0 5 0025 0.1 0.005 Volume (mL) 5.0 24 1JO 10 10 to 10 Diluted to (mL) 100 to o to o 100 100 100 to o Final Concentration (M taL) 0 .0 5 0 .0 2 5 0 .0 1 0.00S 0.002$ 0.001 0 .0 0 0 5 at 2"C to d*C, tv to six months. 9.2.4 Alternate volumeandconcentration ofetandardamay be prepared aa 10.0 Batch SetUp 10.1 Each batch o f samples extracted (typically 20 or leas) emit include at least one trotted central and two untreated controls fortified at known concentration(lab control apihe)to verityprocedural recovery forthe batch. 10.2 Requinmnots for field and laboratory duplicate* and tpikec w ill be specified in foequality aasuranoeplan for thi* project. 11.0 SampleExtraction 11.1 Weigh 5 g o f fiosen sample into 50 mL polypropylene cemrifoge tubes (fortify asneeded,replaceUdandmix well). 11.2 Add 30mL o facetonitrile endshakeoqa wriat action tinker for **13 minutes. 11.5 Placethetubein afleecerfor-1 hour. 114 Peckandcondition theSPBtubeeandsilanize the pear-shapedflaeks. 11.5 Pack the 20 mL SPE tubes in sequence with 2 g Oorisil, 2 g cilice pel, 2 g cartwn, and 1 g LC-NHj. Condition foe columns with 20 mL of methanol, than 20 mL o f acetonitrile. Discard ell washes. Do not allow the column to dry. 11.0 Sitaise the 125 mL pear ahaped flasks by rinsing with the 30% dnnetbyldiehtofoaibne in toluene solution. Ulnaefoe flask with toluene once, followed by methanol (throe times). Dry the flasks completely before use, fitt by air-dryingor with a streamo fnitrogen. Page 5 o f * Page 4! o f65 Exygen Research Page 105 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygea Protocol Number. P0001131 B tm rah esete M e t e d Number VOOOI1IJ I ANALYTICAL METHOD I Methodo fAnalysis for the Detennination ofPerfluorooctanolc Acid (PFOA) in Fish and Clamaby LCA4S/MS 11.7 Csntrifliga tha 30 n L polyprepylane tubas eonuiniai sample tt -2000 tpm foe-10 minutaa. I I . t Deeanl the axtractonto acoaditionsdSPEcolumnfitted incide the mouthor tbe pear-ahaped flaak. Collact tha aluate in tha 125 mL ailanized pear-shape flide. 11.9 Add 10 ral. o f acatonitiils to tha lasla in tha JO mL contrifuge taba. Homopanhs tha posan thtphaastsainga liasmnlrnr for -30 aacondi andnnae tha desandarwiih -10 mL o faestoainlla jato thatoba. 11.10 Shalcethasampleagatfoc-10 minutas en a wrist-action ahaker. 11.11 Placethatubasin aBoalar6a - ihour mora. 11.12 Centrifuga tha 50 mL polypropylene tubas cootaining sample al -2000 rpin for-10 minutos. 11.13 Docaat tha tn ct oato tha ana SPB column. Collact tha eluata into the asina paar-ahapad flash and combine with tha ahiant from tha initial sxtractioo. 11.14 Pasa20 mL ofacetnnitrik throtigh the SPE colontn and combine the alucie in tha asmapeerahqtmi flash. 11.15 Add 34 drops of l-eetanol to tha aatraet in tha pcsr-shsped flash and evaporaraatraducedpressuia ueingarotary vaprate*(at < 40"C). 11.16 Maka tha final volutas, by adding 2 mL o f 2K aaccibic acid in mathanol to thepeaMhapsdflashandswtrl lo mla/diaaolvc. 11.17 Tranafarthasxtncta lo HPLCvala uaiagdiapaaablapipete. 11.10 Anelyzt sanlplaausina dectroapny LC/MS/MS. 12.0 Chnnaiootaphy 12.1 htjaot fi araaaaraoualo f eachstandard, sample and fortified sample into tha LC/MS/MS ayataoL A calibration standard must praesds and fellow all m lyM d m p b i. 12.2 StandardsofPFOAccmapandfef to at least five or momconcentration levels ratiatbeincludedin ananalytical sat 12.3 An entire satofcalibration standardsmust ha included at tha beginning and at tha and of s sample sat. Standards must be interspersed between every 510 samples. As an alternative, an entire act of calibration standard! may be injected at tha beginning o f a act followed by calibration standards interspersed every J-10amples (to account for a second set ofstandards). In eite r case, calibration standards muat be tha first and last injection in a sampleact 12.4 Usa linear standard curves for quantitation. Linear standard curves am generatadfor theanalyteby linearregreasionusing 1/x weighting o fpeak area versus calibration standard concentration using MassLynx 3.3 (or quivalent) software system. Page 6 o f I Page 42 o f 65 Exygen Research Page 106 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number P000U31 Bajfw Kw wt MttfcodNumber VOOQ1713 I ANALYTICAL M ITHOD Method o fAnalyaia for the Determinationo fPerfluorooctanoic Acid (PFOA) in Filh and Clami by LC/MS/MS 12.3 Sample raaponae ihould not exceed rtattdard rcaponaea. Any aamplea thai exceedetxnderdrceponeeeihould bo Author diluted ind teanalyzed. 12.0 AccepuncoCritorio 13.1 Chromatogramn u it thow i peako f daughter ion it 3d9 n t from parent of 413 tow. The 413 amu parent oorraapoods to dw PFOA anion, while the daughterion <349emu) ripreaeuli the loopo fcaitoondioxide. 13.2 Method blaidte mtiat not ooatain PFOA at levole greater than the LOQ. If a blank containPFOA at levaia fuater than 0J ppb, then a new blank aample muatbeobtainedandtheentire aetmuatbeiwextraclcd. 13.3 Raooveriaao f ooatrol apUteeand matrix apikea muat be between 70-130% of their known vahtaa I f a ootwol apika (b ill ouulde the aoceptable limit, the entire aetofaamplaeihould bere-extracted. 13.4 Any calibration atandard Ibund to be a atatiatieal outlier by tiling the Huge Error Teat, may be excluded tram the calculation o f the calibration curve. However, the total number o f calibration atendarda that could be excluded mint notexceed20H ofthe total numbero fatandarda injected. 13.3 The eonakdlou ooefBciaat (R) lbr cahbratioo curve generated muat be 20.992 (R1 k0.9S3). I f calibration reeulta A ll outaide theae lim iu, then apprapriam atapa muat be taken to adjuat inatnnnem operation, and the anndardaor therelevant aato faamplaeihould be reaaalyxed. 13.6 Retention timei between atanderda and aamplae muat not drift more than 4% wititin M analytical ran. I f retention time drift exceed! thir lim it within ananalytical run thenthe actmuatboreanalyzed. 14.0 Calculation! 14.1 Uaethe Allowing equationto calculate the amount ofPFOA found (in ngtml, baaad on pack ana) uaing the atandard cane (linear regreaaion parameter!) geneniadby theM ill Lynx aoftwereprogram: PFOAfound(ngfaO.) - (Peakarea-intercom) elope 14.2 Ueethe ibtlawmg equationto oonvart the amount ofPFOA found in ngimL to ng(g(ppb). pfoa fctmd (ppb) fppQA found(n tte D g fim l volume fal.1 it DP) sampleweight (g) DP (actorbywhich Iba final volumewasdilutad, if nacassary. Pm*? o f s Page 43 o f65 Exygen Research Page 107 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exypa to rn e i feU dm l Number VOOOI7S.3 I ANALYTICAL METHOD I Method ofnaljr fw theDetermination ofPerfluorooctanoic Acid (PFOA) in Fish and Clamsby LC/MS/MS 14.3 For samples fortified with known amounts of FFOA prior to extraction, use thefollowing equationto catari thepercentrecovery. Recovery(% )- [ totalanafaXefound(m fr) analytefoundin control(n j/j)] ][1Q0 analyteadded(iif'g) X Exygen Research P ap S fi Page 44 o f65 Page 108 o f 136 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL MBTHOD MethodNumber V0001714 Method ofAeelyiie for the Determieetloo e f Perflaorooctaaoic Acid (PFOA) In Vecciatioe hy LC/M8/MS Analytical TeetlngFacility: ExyjanReaoarcfc 3058 ReaearchDrive SlateCollege, PA 16801 Approved By: Paul Connolly Technical Leader, LC-MS, ExygenReaearch . ..io Ea M Date Flaherty ' Vice PtecMant,O pentte, Bxypa Retouch Exygen Research TotalPagee: 7 Page 45 o f 65 Page 109 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen P rotocol N um ber: P0001131 B lJfN kM M ft MethodNuabtr V00UPN4 I ANALYTICAL METHOD ] MethodorAnelytil lor the Dcterminttian o fPerfluorooctenoic Add (FFOA) in Vegetation by LC/MS/MS 1.0 Scope Thu method it to be employed to the notation u d quantitation o fperfluorooctenoic acid by High Paefbenuioe Liquid Chromatography coupled to a tandem Meet SpectromctricDetector(LC/MS/MS) in vegetation. 2.0 Softly 2.1 Alwayaobearveaaft labctatory practical. 22 Coneult theappropriateMSDS before handling anychemical for properufety precaution!. 3.0 SampleRequirement 3.1 At laaat20g often ample for extraction. 3.2 Sampteeafaouidbeprcceeeed before extmottan. Place the Omen tample in a food preceeeorandhmnopeniie with dry ice. Place the eamplee in container! and leave open in ftoaan atcrepe ovwmight to allow for cerbon dioxide iuhlhnation. Seal and place tho lampIce in frozen atoregc until time of analyse. 3.3 Sunple collection piooeduree w ill be tpecifled in the templing plan for this project. 4.0 ReaganaandStandard! 4.1 Water- HPLC grade 42 Acetonitrile- HPLC pede 4.3 Cttbon (120200 truth) - Reagentgrade 4.4 Methanol-HPLC grade 4.3 Silica gel (60-200meeh)- Reagentgrade 4.6 Floriall (60-100 meeh)- Reagent grade 4.7 SupercieanLC-KHi- Reagentgrade 4.8 lO ctinol-H PLC gride 4.9 L-Aecorbic add- Reagentgrade 4.10 Dimelhyldichloroailane- Reagentgrade 4.11 Toluene- Reagonlgrade 4.12 Ammonium Acetate- A.CS. ReagentOrada 4.13 Perfhlorooctanok Acid- Sigma-Aldrich 3.0 InatnxrrantandEquipmatK 3.1 A h l^ i performance liquid chromatograph capable o f pumping up to 2 eohwda equippedwith a variable volume iqjeetor capable of injecting 3-200 pLeomtactndton madamMaaeSpectrometer(LC/MS/MS). 32 A deviceto collectrawdatafor peakintegratron andquantitation. 32 Analyticalbalancecapableo frewling to 0.00001 g P atri of 7 Page 46 o f65 Exygen Research Page 110 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen P rotocol N um ber: P0001131 fttJfMI lUmwcb MMkedNw V00017M ANALVnCAL METHOD MethodofAnalysis for tbaDctcrnimatioo ofPerfluorooctanoic Acid (PFOA) m Vegetation by LC/MS/MS 5.4 Rotarye v ira to . 5.5 125mLpear-eluped flasks. 5.6 SOmL dispossMapolypropylenecwtrifoge tab. 5.7 13mLdiipoi i bla polypropyleneoeolrifog tubes, 5.1 Dispaaablemicropipet (50-lQQoL. 10020(foL). 5.9 12J-L LDPEnarrow-mouthbottles. 5.10 2 mLdeerHPLCvial k it 5.11 p i m w hle pipettet . 5.12 Autopipctu (100-1000 |iL md 10-100|iL), with dupoub). tip. 5.13 SPBtub (20mL)(Supelcoc*. no. N057177). 5.14 W riit actionthaktr. 5.15 C tn ttilb it cipbblt o fpirating50mLpolypropylue lubM <t 2000 ipm 63) CbnmalognpUeSyaMai 6.1 Analytical Column: FluophaaaRP(Kayatone Scientific), 2.1 mm x 50mm. 5p (P/N: 125053)52130) 6.2 TS|Mrature: 30*C 6.3 Mobil Pham(A) : 2mM Ammonium Acoltte in Water Mobile Ptaas(B): Methanol Gradient Propani: n w /a h l 0.0 1.0 1.0 20.0 22J SLA 65 65 25 25 65 Flow Rate SLfi (mL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Iigoctiou Vohtrnr 15pL (oqboincnoaod to u much ai 50 pL). 6.7 Qiuntitation: PMk Area-niai m odini calibration cum. 6.6 RunTbnt: ~ 23 atinuton Th. aboveconditionsareintandodasguideid maybechanged in order to optimi IhHPLC ontani. 7.0 MS/MSSyataaa . 7.1 Mode ElactroaprayNegativaMRM mate, monitoring 4]3 - 369nv'z for PFOA. h t . ) at? Page 47 o f 65 Exygen Research Page 111 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Nutriber: P0001131 B * ) f R m ticli fclated NmAar V000I7S4 ANALYTICAL METHOD Methodo fAnalytic far the Detennioedoao fPerflunrooctinotc Acid (PFOA) in Vegetation byLC/MS/MS Tbeaboveccndihoiu areintendedasaguideendmaybo changed in order to optimizetheMSMS eyateni. (.0 Preparati o fSolution 5.1 MobilePhoto 1.1.1 2 eeM mmomum eoet*e in water le prepered by adding 0.154 g of anunottlumcaletelo 1000mLofwater. 12 Extracti Solution 1.2.1 2%eeoaitic ecid in wtheool tipreperod bydeolving 2 g o feecoibic eddinlOOrnLofmetheneL 122 M S Dimethytdichloraeilaiie in toluene io prepaed by bringing 3 mL o fdnelhyldichlerotilanelo i final votame o f 10mL with toluene. Alternate vohuneemeybe prepared. 2.0 StandardPieganti 9.1 Standard Stock/Fortiflcition Soluti 9.1.1 Prepare aetocfc eoluti ofMOO pgtmL o f PFOA by weighing 10 mg o f analytical atandard(coneoted tor putity) anddilute to 100 mL with methanol in e 125-mLLDPE botile. 9.12 A 1.0 pg/mL fortificati eohjti o f PFOA i l prepered by bringing I mL o fth 100pg'mL soluti to a flu volume o f 100 with methanoi in a 125mL LDPEbotile. 9.12 A 0.1 pgbnL fcftidcation coluti o fPFOA it preparai by bringing 10 raLofthe 1.0 pgtaL eoluti to e final voltaneo f 100with mettami in a 125mL LDPEbotile. 9.1.4 A 0.01 p^mL fortificati anturi of PFOA is prepared by bringing 10 mL o f die 0.1 pgitoiL aolotka to a linai volume o f 100 with Butheaol in a 125mL LDP8 bottll. 9.12 Theatoclcand Ibitiflca ti eolntiou areto be etored in aroftigtrator at pproxiraaraty 4'C and are tibie fbr a maximum period of 6 monti fiomtbe dateo fpreparati . 92 StandardCalibeati SolulioM 9.2.1 LOM8IM8 calibrali Mandarti areprepered in methanol via d ilu ii ofthe 1.0pgtaL ibttfleatim coluti. Page4o l' Page d i o f 65 Exygen Research Page 112 o f 136 Interim Report #24 - Analysis ofW ater, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 Bit)gw l i i w t h M M b o d N sa* V00017S4 I ANALYTICAL M ETHOD Methodo fAnstyris for theDetermination ofPerfhiorooctsnoic Add (PFOA) in Vegetation byLOMS/MS 92.2 The following ia a typical example: additional concentration may be CooeaMian ofFortifiaatien Solution(ufttaL) 1.0 1.0 1.0 0.05 0.025 0.1 0.005 Vohnaa (mL) S.0 22 1.0 10 10 10 10 Dilutedto (mL) 100 100 100 100 100 too 100 Find Concentration (usfaL) 0.05 0.025 0.01 0.005 0.0025 0.001 0.0005 at2*C to 6*C, up to aixmonths. 92.4 Alternatevohimeaandconcentrations o f itandarda may be prepared as 10.0 BatchSetUp 10.1 Bach batch o f aamplea extrarted (typically 20 or leaa) mint include it least one unhealed control and two untreated controls fortified at known concentrations(labcontrol spike) to verity procedural recovery for the batch. 102 Requirement! for field and laboratory duplicates and spikes w ill be specified in the quality assuranceplea for this project 11.0 Snp)e Extraction 11.1 Weigh 5 f o f fiocen ample into 50 mL polypiopyleoe centrifuge tubes (fortifyasseeded,replacelid andmix well). 112 Add 30mL ofaoetonfaileandshakeon awristaction shakerfor M 5 minutes. 11.3 Centrifoge the 50 mL polypropylene tuba containing sample at -2000 rpm for~10 minutes. 11.4 Fadeandcondition die SPBtuba endtilenize dmpeer-shaped flasks. 11.5 Pack the 20 mL SPB tubes in sequence with 2 g floristi, 2 g silica gel, 2 g carbon, and 1 g LC-NHj. Condition the column with 20 mL o f methanol, then 20 mL o f acetonitrile. Diacerd all washes. Do not allow the column to 11.6 Silaniae the 125 mL pear-shaped flasks by nosing with the 30% dimetiiytdicltioroeUenein toiMoe solution. Rinsethe flask with toluene once, followed by methanol (three tim a). Dry the flaks completely before use, eitherby air-dryingor with astreamofnitrogen. 11.7 Decantthe extract on to a conditioned SPBoolumn fitted inside the mouth of the pear-duped flask. Collect the eluate ia die 125 mL stlanized pear-shape Page 49 o f 65 Exygen Research Page 113 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen P rotocol N um ber: P0001131 ExypalMtarcfc M eted Nueter V00017*4 ANALYTICAL METHOD MethodofAnalysis fortheDeterminationo fPcrfluorooctanoic Add (PFOA) in Vegeuuion byLC/MS/MS 11.1 Add20 mL ofacstomfriloto thesamplein foe SOmL ceotrifligo tube. 11.9 Shakefoe nuapleagain for-10 minutesoo awrist-action shaker. 11.10 Ccntriftge foe SOn L polypropylene tub containing ample at -2000 rpm for-S minute. 11.11 Decant the extract onto the tame SPE column. Collect the eluate into the me peara h y ri flmk and combine with die eluent from the initial 11.12 Repeatrtapa U .t through 11.11 fain. 11.13 Add 3-4 drop* o f 1-octanol to the tract in the pear-shaped flask and evaporateat reducedpreeiurc usingarotaryevaporator(at < 40*C). 11.14 Make die final volume by adding 2 mL o f 7% aacorbic acid in methanol to thepear-shapedfistic andewirlto mix/diiaotve. 11.13 TMna&rfoeextracts to HPLC vials using disposablepipets. 11.16 Analyte aampleiusingelectrc*pry LC/MS/MS. 12.0 Chromatography 12.1 Iqfect die sameamount o feachstandard, sample and fortified sample into the LC/MS/MS system. A calibration atandard must precede end follow ell analysedsamples. 12.2 StandardscfPFOA correspondingto at least five or moreconcentration levels metbe included in ananalytical set. 12.3 An entire set of extracted calibration standards must be included at the beginning and at foe cod o f a sample set Extracted standards must be interspersed between every 9*10 samples. As an alternative, an entire set of extracted calibration standards may be btfeeted at the beginning of a set followed by extracted calibration standards intersperaed every 3*10 samples (to acoount for a aacoad set o fenframed standards), hi either case, extracted calibration standmdsmustbethe firm and last injection in a sampleset. 12.4 Use Ktmr standard curves for quantitation. Linear standard curves are generated for theanalyte by linearregression using 1/x weighting ofpeak tree venui calibration standard concentration using MassLynx 3.3 (or equivalent) softwaresystem. 12.3 Sample rmponae should not exceed atandard responses. Any aamplei that exoeedstendardregnsia tim id be Anther diluted and reanalyzed. 13.0 AcceptanceCriteria 13.1 Chromatoyam musttiww apeak o fa daughter ion at 369 amu from a parent o f 413 amu. Ihe 413 amupannt correspond to the PFOA anion, while (he dnaghlcrkm(369 amu)representthe lorn o fcarbondioxide. N e**r7 Page SO o f 6} Exygen Research Page 114 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen P rotocol N um ber P0001131 MatfaDdNuwbtr V000I7M | ANALYTICAL METHOD 1 Methodo fAnalysis fortheDetermination o^Perfluorooctanoic Acid (PFOA) in Vegetation byLC/MS/MS 13.2 Method Mania moat not oootain PFOA at levels greater than the LOQ. If a blank oootaincPFOA at levala graatar than 0.9 ppb, than a new blank sample mastbsobtainedand theentire setmustbere-extracted. 133 Recoveries o fcontrol spikesand matrix spikes must be between 70-130% of their known vahaa. I f a control spike fhUs outside the acceptable limits, the entire sato fsamplesshouldber*ctraeted. 13.4 Any calibration standard (bond to be a statistical outlier by using the Huge Error Test, nay be excluded from the calculation of the calibration curve. However, the total number o f calibration standards that could be excluded rauit not exceed2096ofthetotal numbero fetandeids injected. 13.5 The correlation coefficient (R) fbr calibration curves generated must be 0.992 (RJ 0.985). If calibration result* fid) outride these limits, then appropriate step* must be taken to adjust instrument operation, and the standardsor therrievant seto fsamplesshould be reanalyzed. 13.6 Retention times between standard* and aample* must not drift more than 4 % within ananalytical nm. I f retentiontime drift exceedsthis lim it within ananalytical runthenthe setmustbe reanalyzed. 14.0 Calculations 14.1 Uaaft fbUowingequation tocalculatelha mount o fPFOA found(in ngfaiL. baaed on peak m i) Bring the standard curve (linear regression parameters) gsnaatedbytheMaae Lynx softwareprogram: PFOA found fnaftmU (Peakam - jmmpgpi) slope 14.2 Ueethefollowing equationto convert the amountofPFOA found in ng/mL to ng/g(ppb). PFOA found(ppb)* [FTOAfound(BltaU Xflail YElWttfmU XPF1 samplewright (g) DF Actorby whichthe final volumewas diluted, if necessary. 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use the following equationto calculate thepercent reoovery. Recovery(96) [ totalnalyts found(n^g) analytefoundin control(ng/g)] |[|QQ analyteadded(ng/'g) Pap 7of 7 Page 5 / o f 65 Exygen Research Page 115 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number: P000I13I ANALYTICAL METHOD MethodNumber V00017IJ Method of Aaalyale fcr Du Dotoraalaatiao of ParilMreoclaaak AcM (PFOA) ta Snail M u n i U n r by LOM1/MS Analytical TeetingFacility: BxygaaHaaaaich 3058 ItMiawh Drive StateCollege, PA 15801 Approved By: Paul Connolly I Technical Leader,LC-MS, Exyjen Itri ewch // / i / Z J6bnFlaherty / Vice Prendd, Operation, Exygn Reaemh ___lA l & M Date a &/* Dale Exygen Research Total Pegee: 7 Page 52 o f 65 Page 116 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 laygwi Itiraw h MWfce*N u o to V000I7SJ I ... a n a l y t ic a l m e t h o d ............. MediodofAnalyshfortheDctcnmnitionofPcS uoroocS ^ Acid(PFOA) in Small Mammal Uvwby LC/MS/MS 1.0 Soup* This method is to be employed fix die isolation andquantitation ofperfluorooctanok acid by High Performance Liquid Chromatography coupled to a tandem Mau SpectrometncDetector(LC/MS/MS) in small mammal liver. 2.0 Ssftty 2.1 Alwaysobservesafelaboratorypractio**. 2.2 Consult die appropriate MSDSbefore haodling anychemical for proper safety precautions. 3.0 SampleRequirement 3.1 At leastSf o fteetaampleforextraction. 3.2 Sample* should be processed before extraction. Plaoe die frozen sample in a food prooeaaorandhomogenizewith dry ice. Place the sample* in container* and leave open hi frozen storage overnight to allow for cazboo dioxide sublimation- Seal and place die temples in frozen storage until time of analysis. Altwnstely, if them is an insufficient amount of aample Hess than 5 g),thenno processingis necessary sod the samplecanbe usedaasupplied. 3.3 Sample colleetioa procedures w ilt be specified in the campling plan for this pntfMt. 4.0 ReagentsandStudents 4.1 Water-HPLC grad* 4.2 Methanol-HPLC grad* 43 Acetonitrile-HPLC grade 4.4 AmmoniumAoetate-A.CS. ReagentGrade 4 i Perflucrooctradc Acid-Sigma-AlAich 9.0 InatrumantandEquiparai S.l A Ugh performance liquid chromatograph capable o f pumping up to 2 advents equipped with a variable vduma injector capable of injecting 5-200 pL connected to atsndep MassSpectrometer(LC/MS/MS). SJ A deviceto collectrawdataforpeakintegration andquantitation. 3J Anslytieal balancecapableofreading to 0.00001 g. 5.4 50mL disposablepolypropylenecentriftige tubes. 9.9 15mLdisposablepolypropylenecentriftige tubes. 5.6 Disposablemicropipets (SO-lOOuL, l00-200uL). 5.7 125-mLLIVE namm-moothbottles. 5. 2 mL dam HPLC vial Ul faction Page 53 o f 65 Exygen Research Page 117 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number; POOOI131 bygm luM unii M eflwd N um ber VOOOI7I5 | ANALYTICAL METHOD MutfaodofAnulymlbrthcDctunninitkoofPwfhiorooctiaoteAdd (PFOA) In Small Mammal Liver by LC/MS/MS 5.9 Dippoaablepipanaa. 5.10 AulopipeUee(100-1000 pL and 10-100pL), wllh diipoubte tipi. 5.11 WatereSapPak Vac6oc(lg)C16SPEcmridgei. 5.12 8PEvacuummanifold. 5.13 Tiiw m iiw r. 5.14 Wriat-actioo ihakar. 5.15 Ontriftiga capableofapinulog 15mL polypropylen tub at 3000ipm. 6.0 Ouanaieinphic Syatam 6.1 Analytical Column: Flnophue RP(Keyitone Scientific), 2.1 mm x SOmm. 5p (PM; 62505452130) 62 Temperature: 3CPC 6.3 MobilePhaaa(A ): 2 tnM Aononiiint Acetate in Water Mobil Phaue(B) : Mtffaaaol Gradtait Program: T h w (m m \ 0.0 1.0 t.0 20.0 22.5 ZA 65 65 25 25 65 Flow Rute SU fmL/mhrt 35 OJ 35 03 73 0.3 75 0.3 35 03 6.6 iojectian Volume: 15 pL(canbeincreaiid lo c i nmchai 30pL). 6.7 Quantition: PeakArea- ntenal andarticalibraticn curve. 6.1 RunTime; - 23 minulee. Theaboveoooditiooa aie inHndadai a guida andmaybeehangedin order to optimi th HPtC eymem. 7.0 MS/MSSyitan 7.1 Mode; Ebctioeplty NegativoMRM mode,monitoring 413 - 369 m/z for PFOA. The above canditiona aie inteodad aa a guida and may be changad in ordor io optimi tha MSMSlyatam. Paga J o r i Page 54 o f65 Exygen Research Page 118 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 bypalUaaafak MetodNmUt VOOO17ti I' AM/vLYUCAL m e t h o d MU>odofAnalysis fortbDeterminationo fPerfluorooclanoic Add (PFOA) in Small Msnmal Liverby LC/MS/MS 1.0 Preparationo fSolutions 1.1 Mobile Phme 1.1.1 2 mM i m w n h i M acetate in wets is prepared by adding 0154 g of ammonhxnacetateto 1000mL ofwater. Alternate volumesmaybepupated. 9.0 StandardPreparation 9.1 Standard Stock/Fortifleatiori Solution 9.1.1 Prepare a stock aohitien o f-100 pgtaL o f PFOA by weighing 10mg o fanalytical standard (corrected for purity) and dilute to 100mL with methanolin a 125-mLLDPSbottle. 9.1.2 A 1.0 pgfaiL fortification solution o f PFOA ii prepared by bringing 1 mLofthe 100jigfaaL solution torn fowl volume of 100 with methanol in a 125mL LDPBbottle. 9.1.3 A 0.1 pgfaL fortification solution ofPFOA is preparedby bringing 10 mLofthe 1.0pgftnL solution to afinal volumeo f 100with methanol in a 125mL LDPB bottle. 9.14 Theitoek andfortification solution an to beitorod in a refrigerator at approximately 4*C and as stable for a maximum period of 6 months from tiie dateo fpreparation. 92 StandardCalibration Solution 9.2.1 LC/MSAIS calibration tfndards an preparedin methanol via dilution o fthe0.1 pgfaL fortification solution. 92 2 The following is a typical ample: additional concentrations may be preparedasneeded. Final ofFortifloition Velum DOuMlta Concentration Solutiontonfati.) (mL) (mL) (na/mL) 100 5.0 100 5.0 100 24 100 24 100 14 100 14 54 10 100 04 24 10 too 02 ____ m_______ IS_______ 122___ 0.1 9X3 S toni a 2*C to 6*C, up to six months. 9X4 Alternate volumes andconcentrai i o fstandardsmay be prepared as P|*4 o f 7 Page 55 o f 65 Exygen Research Page 119 o f 136 M erini Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen P rotocol N um ber: P0001131 4 -* la n ta n i MtAod MemberV00017I5 I ANALYTICAL METHOD ~~ Methodo fAnalyoii for theDetemhutioo ofPeeiluoroaetenoic Add (PFOA) in Smell Memmi Liverby LC/MS/MS 10.0 Belch SetUp 10.1 Eeoh botch of temples extracted (typically 20 or leee) muet include at lent cm untreated coohol and two untreated cootrola foetified at known concenttaliom(lab control epike)to verify procedural recovery for the batch. 10.2 Requimnente for Sold and laboratory duplicator and epikee w ill be ipecified in the quality aaauranceplan forthie project. 112) SempleExtraction 11.1 Weigh 1 1 of temple into a SOraL polypropylene centrifuge tubes (fortify as needed,replace Bdandmix wall). Note that alternate weights o f liver may be moesureddependingonthecamplesize available for use. 112 Addwaterto the eamplefor aBnal volume of 10mi ll. } Horoogenfootempleuemgatieeoemiwrfor -1 minute. 11.4 Tranefor 1 mL o f die eample uainf a diepoeable pipette into a 13 mL di^O llbllC M lri(kl|t tub. 11.5 Add 5mLo facetonitrile andshakefor~20nriirotea on a wrist-action shaker. 11.6 Centrifuge die tubesat*3000rpm for-3 minutes. 11.7 Decant the supernatant into a 50 mL disposable centrifoge tube and add 35 mL ofwater. U.S Condition the C SPE cartridges ( l g, 6 mL) by passing 10 mL methanol followed by 5mLo fHPLCwater(~ 2 drop/see). Do not let column rundry 11.9 Loadtile campleon conditioned Cis SPEcartridge. Diecard eluate. 11.10 Bhtie with -2 mL of methanol. Collect 2 mL o f eluate into a graduated lSmL polypropylenecentrifogelobe(final volume 2 mL). 11.11 Analyzemapleusingdoctrapray LC/MS/MS. 12.0 Chwmatoyaphy 12.1 Inject the sameamounto feachstandard, sample and fortified sample into the LOMSMS system. A calftsaticn standard naut precede and follow all analysed eamplee. 12.2 StandaideofPFOAcosrevondintto at leaat five or more concentration levels mustbe included in ananalytical set 12J An entire setofeatibretien atandaitii mustbe included at the beginning and at die end o f a sample set. Standards must be inlerspeteed between every S-lo samples. As an alternative, so entire set o f calibration standards may be injected at the begmwhig o f a set followed by calibration standards interspersedvm y 5-10 samples(to account for a second set of standards). In either case, calibration standards must be the first and last injection in a sampleact 12.4 Use linear standard owes for quantitation. Linear standard curves are generatedforthe analyteby linearregression using lAt weightingo fpeakarea PagiSef? Page 56 o f 65 Exygen Research Page 120 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o. : P0001131 Exygen Protocol Number: P0001131 Eiyf M m r dl M e a d N lrie c V000I7H I ANALYTICAL MWHOD MethodofAnalyria fcrtin Detatminetlono fParftuccooctenoic Acid (PFOA) in Smal~ M a niail i n by LOMS/MS vereuecalibration atandardconcentration uring MaaeLynx 3.3 (or equivalent) aoftwara ayetaan. 12.3 Sample reqxxue ahould not end atandard reapoaaaa. Any umplea that exceedatandardreaponaeaahouldbe furtherdiluted andreanalyzed. 13.0 AcceptanceCriteria 13.1 Chrotnalognu mint thaw epeeko f edaughter ion at 369 emu faun aparent of413 emu. The 413 emu parentconaepowla to the PFOA anion, while the daughterion (309 ana) leptcecuu the loeaofcarbondioxide. 13.2 Method blanka n u t not contain PFOA a levela greater than the LOQ. if a Maniccontain! FFOA a levela greeter than 10 ngfg, then a new blank temple mua beobtained andthaentire aa moa bere-extracted. 13.3 Ractrveriee o f control apikaaand matrix apikaa nun be between 70-13034 of their known valuaa. If a control epilce bile outalda tha acceptable lim iu, the entile aa o f aantpiae ahonld be re-extracted. Any matrix tpike ontiide 70130K ahould be evaluated by die analyat to determine if rwoxtnctioa ia warranted. 13.4 Any calibration atandard bond a bo a riatiatical outlier by uxing tha Huge Error Teat, may be excluded ftmn the calculation o f the calibration curve However, the total number of calibration atandarda tha could be excluded mua notexceed20K oftheIota numbero fataadarda injected. 13.3 The correlation coefficient (R) for calibration corvee generated mua be 20.992 (R1 20.985). I f eabbratioa reaulti fhll outride thete limiu, then appropriate atepe mua be taken to adjuat iaatrument operation, and the atandardaortherelevantactofaampleeahould bereanalyzed. 13.6 Retention timea between rtandada and aamplee mum not drift more than 4% within ananalytical ran. Ifietention lime drift exceedethie lim it within ananalyticalrunthenthe aetmuetbereanalyzed. 14.0 Calculation 14.1 Uaathe followingaquationto calmlatetheamounto fPFOA (bund (in ngimL, baaed on peek area) tiling tha atandard curve (linear regrcaaion parameter!] generatedby theMaaaLynx aoftwareprogram: nena c-ut t--ii" i i n . imareantl x DF x allmioi thnrnr DF* fintarby which tha ifaul volumeweadiluted, if neoeeeary. Aliquot factor-10 h |iie f7 Page ST o f 65 Exygen Research Page 121 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen P rotocol N um ber: POOOI131 ExyimlMNRh MethodNiunbcrVOOO17t$ I ...............................A N Al.V llC ALM fTB O P Method ofAoelyeU farthe Detenninftliofio fPerflucrooctinok Acid (PFOA) in Small Meminal Liver by LC/MS/MS 14.2 For umploe Imtilled whb known amounts o f PFOA prior to extraction, use thefollowing eqoetion to calculatethepcrceol recovery. Recovery(%) [ totalenelytefound(n tta l.) - enelytefoundin control(ng/mL)] <100 analyteadded(nf/mL) 14J Uaethe following equationto convert the amounto f PFOA found in ng/mL 10 ntf*(ppb). PFOA found fe ta l I jl ^ ) 4 BMlYOlMiafaLH empioweight (g) Exygen Research Page 7 o n Page S8 o f 65 Page 122 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen P rotocol N um ber P0001131 A N A L Y T IC A L M ETH O D Mtfhod Number V000I7W Method ofAnalysts for the Dstanrieadoa ofPerOoorooctaneic Arid (FFOA) io Smalt Momiool Serumby LC/MS/MS Analytieri TestingFacility; Exygen Roteanti 3058 ResearchDrive StateCollege, PA 16801 Approved By: C J lL Paul Connolly I Technical Leader,LC-MS, ExygenResearch John Flaherty / Vice Pieskkot, Operations,BxyganReaeerch ___a if c M Data /'A /* Daw Exygen Research TotalPaga: 7 Page 59 o f 65 Page 123 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen Protocol Number P0001131 BaratoXaaeanll Mated Nuotar V000in I ANALiTICaL METHOD MethodofAoalyaiifirllieDetenninatinn o fParfluorooctanoic Acid (PFOA) in Small Mamma!Sannoby LC/MS/MS 1.0 Seopo TU mathod i l to be amployed fot the lactation adquantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mart Spactromethc Defector(LC/MS/MS) in email mammal renim. 2.0 Safety 2.1 Alwaya obaarvaaafe laboratorypraclicea. 2.2 Couault theappropriateMSDS before handling any chemical for properaalety 3.0 SampleRequirement 3.1 Atleaet I mL oftootcample foreatraotioa. 32 No aamplepropeering ia Deeded for actum maples. However, Dozen lerum aaaqileamuatto aliowad to complatalythaw to room temperaturebefore uae. 3.3 Sample ooDectioo pcoeeduna w ill be apecidad in die campling plan for this project. 4.0 Reagent!aod Stendarda 4.1 Waler-HPLC grade 4.2 Methanol-HPLC grade 4J Acetonitrile - HPLCgrade ^ 4.4 AmmoniumAcetate - A.C.S. ReagentGrade 4.1 PerfkiorooctenoicAcid - Sigma-AIdrich 5.0 Instrument andEquipment 5.1 A Ugh performance liquid chromatograph capable o f pumping up to 2 aolventa equipped with a variable volume iqjeetor capable o f injecting 5-200 pL connectedto atandemMela Spectrometer(LC/MS/MS) 5.2 A device to collectraw dataforpeakintegration endquantitation. 5.3 Analytical balancecapableo f(codingto 0.00001 g. 5A 50mL dupocabiepolypropylenecentrifoge tubca. 5.5 15mLdiipoeablepolypropylenecaotrilhge tubee. 5.6 Diapoeablemicropipet! (5iM00uL, 100*200uL). 5.7 125-mLLDPBnerTow-mouthbottles. 5.5 2 mLdearHPLC vial IdL 5.9 Disposablepipettes. 5.10 Autoplpettee(100-1000 pL end 10-100pL), with disposabletips. 5.11 Watcra SepPakVac6 cc (Ig) tCIS SPEcartridge!. 5.12 SPEvacuummanifold. 5.13 Vortexer. Plg2t>P Page 60 o f 65 Exygen Research Page 124 o f 136 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen P rotocol N um ber: P000I131 Mrthod Nuahtr V0WI71I6 I A1WVT1CAL METHOD | Method ofAm lyiU far Ihc Dacrnitnitlon o fPwfhioroocaooic Acid (PFOA) in Snail MammalSerumby LC/MS/MS 5.14 Wrist-actionahafcer. 5.15 Centrifugecapableo fginning 15 mLpoiypropyleoe tubes at 3000tpm 6.0 Chromatop ephicSystem 6.1 Analytical Column: FHwptaaaeRP(Keystone Scientific), 2.1 mm x 50 mm. Sp (?/N: S2S05432130) 62 Temperatura: 30*C 6J Mobile Phase(A) : 2mM Amaoroum Acetatein Water Mobile Phase(B): Methanol Grattisi* Propani: Tima fru lli 0.0 1.0 S.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate & (ml/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 byoctionVolume: 15pL (canbemenacedto acmuchas50 pL). 6.7 Quantitation: PeakAnn-external andaricalibration curve. 6J RonTime: - 23 minutt. Theaboveconditions va minded h aguide rod may bechanged in order to optimi theHPLC syctem. 7.0 MS/MS System 7.1 Mode: HlectroiprayNegative MRM mode,monitoring 413 - 369 m/z for PFOA. The above conditane are intended as a guide and may be changed in order to optimi theMSMS eyatem. 1.0 Preparationo fSolutions I.I Mobile Phase S.1.1 2 mM ammonium acetate in wiser is prepared by adding 0.1S4 g of ammonimnacetateto 1000mL ofwater. Ahamate votame maybe prepared. Page 61 o f 65 Exygen Research Page 125 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen P rotocol Num ber: P000U 31 ftw l um rt a w NmabwVH00I7I6 I ANALYTICAL METHOD Mtthod ofAmlyiU for theDetMrmnUion o fParfluorooctmoic Add (PFOA) nSmall MammalStrum by LC/MS/MS 9.0 StandardPreparation 9.1 StandardStock/Fortificatioo Solution 9.1.1 Preparea stock solutkm o f-100 pg/mL o f PFOA by weighing 10 mg of analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mLLDPE bottle. 9.1.2 A 1.0 p^mL fortification eolation of PFOA it prepared by bringing i mL o fdie 100 p fta L eolation to a final volume of 100 with methanol in a 125mL LDPEbottle. 9.1J A 0.1 pgfaiL fortification solution o fPFOA it prepared by bringing 10 mLo ffoe 1.0pgfaiL aolution to afinal volume of 100with methanol in 125mL LDPBbottle. 9.1.4 Theatoekend fortification eohitiona areto be itored in arefrigerator at approximately 4*C end an table for a maximum period o f 6 month* from thedateo fpreparation. 9.2 StandardCalibration Solution 9.2.1 LCMS/MS calibration aundarda an prepared in methanol via dilution o ffoe 0.1 p^mL fortification rotation. 9JL2 The following ia a typical example: additional concentrations may be Conseetrstion ofFortificatien Votane Diluted to Final Concentration SolutionfaetaL) (mL) (mL) (ne/mU 100 5.0 100 S.0 100 2.0 100 2.0 100 1.0 100 5.0 10 100 1.0 0.3 2.0 10 100 0.2 1.0 10 100 0.1 9.2.3 Store all calibration atandards io 125-mL LDPE narrow-mouth bottles at2*Cto fr*C, up to tix months. 9.2.4 Atonale volumes andconcentrationso f standards maybe prepared as needed. 10.0 BatchSetVp 10.) Bach batch o f aamplai extracted (typically 20 or leai) mnat include at least one untreated control aod two untreated controls fortified at known concentration(lab oootrolspike)to verify pcooedural recovery for the batch. 10.2 ReqidnnMnti for field and laboratory dedicates and spikes w ill be specified in die quality saauranceplan for this project. Pape 4 o f 7 Pagi 62 o f 65 Exygen Research Page 126 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Exygen P rotocol Nutrsber: P0001131 ExyewSsssatsS MsthedNaeWsrVOOOPtO I ANALYTICAL m e t h o d Method ofAnalyais fortheDetesrainaticeio fPerfluorooctanoic Acid (PFOA) in Small Mamma]Serumby LC/MS/MS 11.0 SampleExtraction 11.1 Moaura 1mL ofumple into a SOmL polypropylene cenlrifoge tuba (fortlTy aa needed, itplaoe lid and mix wall). Note that alternate volumes of serum maybemeasureddependingon thesamplesite available for use. 11.2 Addwaterto thesampletorafinal volumeo f20 mL. Cap tightly 11.3 Vottea for-1 minute. 11.4 TMnaftr 1 mL of the sample using a disposable pipette into a I I mL disposable oantrifhge tube. 11.3 Add 3 mLo faoetonitrileand shake for-20 minutes on swrist-action shaker 11.6 Centrifugeths tubesat-3000ipm for ~3 minutes. 11.7 Decant the supernatant into a SOmL disposable centriftige tube and add 33 mLo fwater. 11.6 Condition the Cti SPE eemidges (1 g, 6 mL) by passing 10 mL methanol followed by 3 mLo fHPLCwater( - 2 drop/see). Do not let column tun dry 11.9 Load thesampleonconditioned Cis SPEcartridge. Discard dualc. 11.10 Elute with -2 mL of methanol. Collect 2 mL o f eluate into a graduated IS mLpolypropylenecemriAigetube(final volume 2 mL). 11.11 Analyse samplesusingelactroepray LC/MS/MS. 12.0 Chromatography 12.1 Inject thesamemount o feach atandard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzedsamples. 12.2 StandardsofPFOA correspondingto at least five or moreconcentration levels mustbe included in ananalytical set. 12.3 An entire seto fcalibration standardsmust beincluded at the beginning and at the end of a sample set. Standardsmust be interspersed between every 5-10 samples. As an alternative, an entire eat of calibration standards may be injected at the beginning o f a set followed by calibration standards intan*!sod every 3-10 samples(to account for a second seto fstandards). In sithsr ease, calibration standards must bo the drat and last injection in a sample set. 12.4 Use linear atandard earns for quantitation. Linear standard curves are generated fortheanalyte by linear regressionusing 1/x weighting o fpeak area versus calibration standard concentration using MaasLynx 3.3 (or equivalent) softwaresystem. 12.3 Sample response should not exceed standard responses. Any samplea that exceedstandardresponsesshouldbe furtherdiluted andreanalyzed. Plea 3 of? Page 63 o f 65 Exygen Research Page 127 o f 136 Interim Report #24 - Analysis ofW ater, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 EicywU m net MflfcodNumberV0001716 I ANALYT1CALMICTHOD Methodo fAnilym ihr the Drt>rmintion o fPeriluorooctanoic Acid (PPOA) in Small Mammal Seramby LC/MS/MS 13.0 AcceptanceCriteria 13.1 Chromatogram muit show apeak ofadsugbtar ion at 369 emu from a parent of413 mm. The 413 anui parent corresponds to the PFOA anion, while tlie daughterion (369 amt) luprasaati thekteeo fcarbon dioxide. 13.3 Method blaaka mint not cootaia PFOA at level* greater (hen the LOQ. If a blank contain! PFOA at leveia greater than 10 ng/mL, then a new blank simple meetbeobtainedid theeotiie setmoatbe re-extracted. 13.3 Recoveriee o f oontrol spikes and matrix pike* must be between 70-130% of their known value*. If e control apike foUs outaide the acceptable limits, the entire eel o f atmplei should be rx xtracted. Any matrix spike outside 70 130% shouM be evehiated by foe analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Teat may be excluded font foe calculation o f the calibration curve. However foe total number o f calibration standards that could be excluded mustnotexoecd20%ofthe total numbero fstandards injected. 13.5 The correlation coefficient (IQ for calibration curves generated must be *0.992 (RJ *0.985). I f calibration results Gill outside these limits, then ^jprapriate steps mist be taken to adjust instrument operation, and the stmdanUorfoerelevant setofsamplesshould be reanalyzed, 13.6 Retention time* between standard* and samples must not drift more than 4 %within ananalytical run. I f retention time drift exceedathis limit within ananalytical run thenthe setmustbereanalyzed. 14.0 Calculations 14.1 Usethe foflowjng aquationto calculate foe mount o fPPOA found (in ng/mL. baaed on peak area) using foe standard curve (linear regression parameters) gensntedby theMan Lynx softwareprogram: PFOA found(ng/mL) (Peeks-intercept) x DF x aliquot factor slope DF* Actorby whichfoe final volumev u diluted, if neoessary. Aliquot foctor20 14.2 For samples fortified with known amounts o f PFOA prior to extraction, um the following equationto calculatethepercentraoovery. Recovery(% )* [ totalenalytefound(na/mL) - analytefound in oontrol(ng/mL)] ^ analyteadded(M faL) PagcS of? Page 64 o f 65 Exygen Research Page 128 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 BQ V M X aM m h MMbod NiuabtrVOOO1716 I ANa LVUCAI. M1THOP Methodo fAnalyiii for the Detorminrkm o fPerfluoroocunoic Acid (PFOA) in Small Mammal Scran by LC/MS/MS 14.3 Ucethe following equationto eeevm the amounto f PFOA found in ng/mL to ppb. PPOAmadfnnM - IPPQA foundtna teLl. Itn.1 velum ln .n l aample volume (mL) Exygen Research Pa*t T o f t Page 65 o f65 Page 129 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o. : P0001131 VT RESEARCH 3058 Research Drive Phone: 814-272*1039 State College, PA 16801 Fax: 814-231 -1580 PROTOCOL AMENDMENT Amendment Number 1 Effective Pate: 01/10/05 ,,MangExygen Study Number P0001131 Client Study Number Pagai of 1 " BESCBIPTIQNOFAMENDEDSECTION 1) Analytical Procedure Summary V0001780:Section 9.1 2) Verification of Analytical Procedure AMENP6PTP 1) Add to Section 9.1: Section 9.1.8, 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) Lew 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 avalable for small mammal Sver and serum was smaller than w expected. Spiking at the predetermined 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-determined 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 win cover the analyte concentrations expected hi the water samples. _ ,_ , _ IMPACT.ONSTUBI The LOQ is 100 ng/g fora 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 w ll ensure that more QC recovery data can be used. LIBRARYIO: W 000122M` Exygen Research Exygen QAU Review / ,15 M.iilltT ADMINISTRATIVE FORM Page 130 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231 -1580 Amendment Num ber Effective Date: Exygen Study Number PROTOCOL AMENDMENT 2 03/07/05 P0001131 Client Study Num ber Pagai of 1 None DESCRIPTION OF AMENDED SECTION 1) Report, page 11 of 65 2) Test Materials, page 6 of 65: PFOS transition monitored 499 -> 99. 1) Instead of one final report, Interim reports will be Issued. 2) PFOS transition monitored may also be 499 -> 80. RATIONALE 1) Due to the excessiva sizes of the data sets, interim reports w ll be issued to allow the d en t to receive data in a timelier manen a w eifa/tj- - / 2) The API 4000 LG/MS/MS systems detect the 499 > 80 PFOS transition with greater sensitivity than the 499-> 99 transition. IMPACT ON STUDY 1) The d en t win be able to receive and review the date more quickly. 2) The 499 -> 80 transition can be detected with greater sensitivity; therefore, giving bettor chromatography. 'Z / n d U S Jb'LHWuAJNmUI mImVuMASlnQaMAOnrt aftQtr,rMiMO,,,n MA: Saidy Director Management Signatura B t P MarwgafinMMum SponwrStgnfiur(IfraquM) s h /* ? Date ' , Dali .......... Data i/it/o f Dali ' Exvpan QAU Review LU OjJosliS UBRARYID: V000122S4- ADMINISTRATTVE FORM Exygen Research Page 131 o f 136 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0001131 CHEM EHSR 236 i t 651 733 1958 87-11*2001 03:Sfpa 9rHISTBI WLOTIOKS 07/11 '06 1 4 :09 N0.142 02/02 T-KI P.01W12 WM : ! ' i . * SMtaMte*C*ot#lc*fe#, FA*H*K1 -. V * V ; * . " Ful 11,4-231-15W research .. t . 6*- . . A rt^n*w it Number AMENDMENT 7 ^ . 1 <*1 ^ S n w S 'Nuirtber TOQ0i~l'3l P le n t M y Number. i M - r Aribndto I, Analytod Method ^0001782. rr be le e e tb dad uah* the WlQwIngmethod. W--^VO' "IjJnifW*--M iv n Mamed: Beforre the aaniMaa aR^flhed.*qr B arteello R . m aM .raouBntiyybpydgocouayaatW ^ t h e p o r r t ^ . A o h e ^ r tm p o ^ o n w r _ lefoelBhed Intea ittwHniililttWnntfwor cofeinnCMi tuMtuba.frthe arib c ^ aoatlc ad In mathanoifa added* jm i eaoga. The earapttethen a M fflf r hand, vorteaad, ahd eenieatad forWry mtndea. The e a f n p l^ t hah cwtrt^ W -WmlnuMaM-apoOrern. Eacheampfe.isanalyzedbyLC/MS/MBefoetToapray. .... --'" ........ : .. ' UtMHpBT' MoreueebtedateMilboobtained}*using,analternatemedwd. uawwv'iftvoooiajM RECEIVED TICE JUL.ll. 4:27PM -. PRINT TIME v * > * A D M dw nidrn>M N JUL.ll. 4:28PM Exygen Research Page 132 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 Waiting on Amendment 10 signatures Exygen Research Page 133 o f 136 Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study N o.: P0001131 Ezysen \ \ / fJRESEARCH 3058 Research Drive Phone: 814-272-1039 State Collese, PA 16801 Fa x: 814-231-1580 DEVIATION FORM _______ General: X Project Specific Deviation ____Facility Deviation ______________ Pag 1 of2 Date of Occurrence: 04/26/06 Exygen Project # : P76Q/P1131 Deviation#: _____5 Client Project#: NA R e fere n ce# : 064)76 Regulatory Driver: X _____ _____ GMP GLP Other None Samle Description: Deviation Typg: (Include W fo r m ethods a n d S O P s) X Protocol Method SOP V #:N A Notebook reference: NA Looin#: L000819 Container#: NA L o t# : NA Summary o f Deviation: The three sediment samples in L8191 (C0172892 - C0172894) were originally extracted using the sediment method V0001782. Poor recoveries were obtained for PFOS, PFOA and ,3C PFOA. Because of this, the study sponsor requested the use of an alternative extraction for these compounds, as follows: Direct Injection Method: Before the samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A one-gram portion of sediment was weighed into a 15-minaiter centrifuge tube for the extraction. Ten milliliters of 1% acetic add in methanol was added to each sample. The samples were then shaken by hand, vortexed, and sonicated for thirty minutes. The samples were then centrifuged for -1 0 minutes at -3000 rpm. Each sample was analyzed by LC/MS/MS electrospray. Using this method acceptable data was obtained for PFOS, but the recoveries for PFOA and ,SC PFOA were stlH poor. Another alternative method was then used for PFOA and WC PFOA, as follows: Alternative SPE Method: The samples that were prepared In 1% acetic acid for the direct injection method were used for this extraction. Five milliliters of each sample was aliquoted into a 50-mL polypropylene centrifuge tube and the volume was taken to 40 mL with water. The samples were then centrifuged for -1 0 minutes at -3000 rpm. The supernatant was then loaded onto a C SPE cartridge conditionedwith10m Lofm ethanoiand5m Lofwater. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five m illters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. Cause: ____Preparation_____Analysis____ Instrument_____: Client Request X Other Impact: More usable data was obtained. LIBRARY IO: V0001640-6 Exygen Research ADMINISTRATIVE FORM Page 134 o f 13b Interim Report #24 - Analysis o f Water, Sludge, and Sediment Exygen Study No.: P0001131 CHEM EHSR 236 1B 651 733 1958 IM M M IM I FrtHKTW SOLUTIMS 05/01 '06 13:46 N0.106 03/03 t-m p.m /m m u SIA*Ch *. . u n w w rifc v u o iM M Exygen Research s , v< r y . yi . . *V :*.v !.' ; / " >. . . ' ,v * s/K , - f , s \ ; . ' ' i'V .j " , : ,, ' '' ................... . '-. V ;r . ! *h ' * * t, ' ,'* V *,f t* #', * , fi . ' \A %' ' . i* ' ' * * " >**: * ' ' ! /` s.** ,V Sv Page 135 o f 136 Interim Report #24 - Analysis of Water, Sludge, and Sediment Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB $ 651 778 4226 07/11/06 11:46 0 :0 7 /0 7 NO:681 '* ; ^RESEARCH 3058 Retearch Drive Phone: 8144^-1039 State College, PA16801 Fax: 814-231-1580 .' . DEVIATION FORM X " Pralaet Specific Deletion _ ___ Facility Deviation Data of Oocurranoa: ^ tttw on P rotect*: . P 7eo /P ll3t ' " DawiaMlon#: j u i cileitt P re s e t# : ______ SgB SSfiffii . OMP X OLP ... Other __ _ None Voo: ftncfadS V# h r m athods end S O P ) X ' Protocol ' ____Method ___ .;$bP' V#: NA Notebook reference: Login#: U 0ei21 C orM her#: 'LOdfliBOai . / . . . .. " T fr[rn T T T Devlellon: Samples were filled to 250 mL Instead o f200 mL. 00109347 CO1.0M54 eo m b ae:. L o t# : - . :RA 6a#ee:' X Preparation . ' Analysts Instrument t_ _ d e n t Request tmeaet! '^ " !T 3ampies could not be extracted according to the protocol, O tter Corraottva ActtonoT SpDdftglavale were adjusted to eccommodels the alternative volume. BI*f*t-n-op--Mimii. . rtlgator 7 * 8tudy.---------duality Aeeunince / ------ tik i D ate. . "Pete.. Exygen Research Page 136 o f 136