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INTERIM REPORT #11 Analysis of Public Water. Wastewater, and Sludge Samples Amendment Number 1 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 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 07/07/06 and 10/05/06 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: 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples 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. Principal Investigator Exygen Research Jaisimha Study Director Weston Solutions, Inc. Michael A. San Sponsor Repres 3M Company Exygen Research Amendment Number 1 Date Page 2 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No. : 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 Date Inspected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Studv Director 21. Raw Data Review and Interim Analytical Report Review 09/21-22/05 05/01/06 05/04/06 05/09/06 31. Raw Data Review and Final Interim Analytical Report Review 07/06/06 07/07/06 07/07/06 07/07/06 35. Amended Raw Data Review and Amended Final Interim Report Review 09/28/06 10/05/06 10/05/06 10/05/06 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion of the study. This QA statement involves only the review of the interim report and associated raw data. Exygen Research Amendment Number 1 Page 3 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 CERTIFICATION OF AUTHENTICITY This interim report, for Exygen Study Number P0001131, is a true and complete representation of the raw data. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Principal Investigator, Exygen: ( ii/v tu ^ohn Flaherty Vice President Exygen Research V Exygen Research Facility Management: S ~ O l Date Weston Solutions, Inc. Sponsor Representative, 3M Company: Michael A. Santoro/' Director of Regulatory Affairs Exygen Research Amendment Number 1 Page 4 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 STUDY IDENTIFICATION 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 PROTOCOL NUMBER: P0001131 EXYGEN STUDY NUMBER: P0001131 TYPE OF STUDY: Residue SAMPLE MATRIX: Public Water, Wastewater, and Sludge 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: 07/01/05 Interim Analytical Termination Date: 05/25/06 Interim Report Completion Date: 07/07/06 10/05/06 Exygen Research Amendment Number 1 Page 5 o f 112 Interim Report #11- Analysis o f Water, Wastewater, and Sludge Samples 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 of this interim portion of the study: Name John Flaherty Karen Risha Paul Connolly Mark Ammerman Amy Sheehan Eric Edwards Brittany Kravets Frances Crespi Scott Crain Krista Gallant Mindy Cressley Kim Hall Abby Caporuscio Title Vice President, Principal Investigator Scientist Technical Lead-LC/MS Sample Custodian Associate Scientist Sample Custodian Technician Technician Technician Technician Technician Technician Intern Technician Exygen Research Amendment Number 1 Page 6 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 TABLE OF CONTENTS Page TITLE PAGE.......................................................................................................................1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................. 2 QUALITY ASSURANCE STATEMENT......................................................................... 3 CERTIFICATION OF AUTHENTICITY.......................................................................... 4 STUDY IDENTIFICATION............................................................................................... 5 PROJECT PERSONNEL.................................................................................................... 6 TABLE OF CONTENTS.................................................................................................... 7 LIST OF TABLES.............................................................................................................. 8 LIST OF FIGURES............................................................................................................. 9 LIST OF APPENDICES....................................................................................................10 SUMMARY OF CHANGES AND REASON FOR AMENDMENT...............................11 1.0 SUMMARY................................................................................................................11 2.0 OBJECTIVE...............................................................................................................11 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 Percent Solids Procedure For Sludge.......................................................................14 6.4 Preparation of Standards and Fortification Solutions...............................................15 6.5 Chromatography.......................................................................................................16 6.6 Instrument Sensitivity...............................................................................................16 6.7 Description of LC/MS/MS Instrument and Operating Conditions...........................16 6.8 Quantitation and Example Calculation.....................................................................17 7.0 EXPERIMENTAL DESIGN......................................................................................18 8.0 RESULTS...................................................................................................................19 9.0 CONCLUSIONS.................................. 19 10.0 RETENTION OF DATA AND SAMPLES.............................................................19 Exygen Research Amendment Number 1 Page 7 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Table I. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Water Samples.................................21 Table II. Summary of PFBS, PFHS and PFOS in Sludge Samples................................22 Table III. Summary of PFOS in Re-extracted Sludge Samples..................................... 23 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Water Samples.......... 24 Table V. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sludge Samples..........25 Table VI. Matrix Spike Recovery of PFOS in Re-extracted Sludge Samples.............. 26 Table VII. Total Percent Solids for Sludge Samples...................................................... 27 Amendment Number 1 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFBS in Reagent Water................................. 29 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 30 Figure 3. PFBS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................................... 31 Figure 4. Chromatogram Representing a Water Sample Analyzed for PFBS (Exygen ID: C0070099, Data Set: 070105A)............................................... 32 Figure 5. Chromatogram Representing a Sludge Sample Analyzed for PFBS (Exygen ID: C0071211, Data Set: 070505G)................................................ 33 Figure 6. Typical Calibration Curve for PFHS in Reagent Water................................ 34 Figure 7. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 35 Figure 8. PFHS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................................... 36 Figure 9. Chromatogram Representing a Water Sample Analyzed for PFHS (Exygen ID: C0070099, Data Set: 070105A).................................................37 Figure 10. Chromatogram Representing a Sludge Sample Analyzed for PFHS (Exygen ID: C0071211, Data Set: 070505G)................................................ 38 Figure 11. Typical Calibration Curve for PFOS in Reagent Water................................ 39 Figure 12. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 40 Figure 13. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................................... 41 Figure 14. Chromatogram Representing a Water Sample Analyzed for PFOS (Exygen ID: C0070099, Data Set: 070105A)................................................ 42 Figure 15. Chromatogram Representing a Sludge Sample Analyzed for PFOS (Exygen ID: C0071211, Data Set: 052206B)................................................ 43 Exygen Research Amendment Number 1 Page 9 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 LIST OF APPENDICES Page Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods, and Protocol Amendments 1, 2 and 3...................... 44 Exygen Research Amendment Number 1 Page 10 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 SUMMARY OF CHANGES AND REASON FOR AMENDMENT The headings on the columns in Table I were incorrect. The headings were for soil samples but the table listed water samples. The column headings were changed to reflect the proper units for water samples. The incorrect column headings had no affect on the overall results reported. 1.0 SUMMARY Exygen Research extracted and analyzed public water and wastewater samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001780 and sludge samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001781 (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 was 0.2 ng/g (wet weight). Analytical results for the analysis of PFBS, PFHS and PFOS found in public water and wastewater samples are summarized in Table I. Analytical results for the analysis of PFBS, PFHS and PFOS found in sludge samples are summarized in Table II. Analytical results for the analysis for PFOS found in re-extracted sludge samples are summarized in Table III. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in water samples are summarized in Table IV. The overall average percent recoveries standard deviations for field and laboratory spikes for PFBS, PFHS, and PFOS in water samples were 108 + 16%, 109 12%, and 121 23%, respectively. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in sludge samples are summarized in Table V. The overall average percent recoveries standard deviations for field and laboratory spikes for PFBS and PFHS in sludge samples were 90 4% and 86 3%, respectively. Fortification recoveries for the analysis of PFOS in re-extracted sludge samples are summarized in Table VI. The overall average percent recovery standard deviation for laboratory spikes for PFOS in re-extracted sludge samples was 101 1%. 2.0 OBJECTIVE The objective of the analytical part of this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and Exygen Research Amendment Number 1 Page 11 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 perfluorooctanesulfonate (PFOS) in public water, wastewater, and sludge according to Protocol P0001131 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFBS, PFHS and PFOS in water and wastewater using the analytical method entitled, "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" and in sludge using the analytical method entitled, "V0001781: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS." 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 July 1, 2005, and the analytical termination date for this interim report was May 25,2006. 4.0 ANALYTICAL TEST SAMPLES Seven public water supply samples (Exygen ID C0070099-C0070105) representing one public water treatment site, along with field blanks, were received on wet ice on April 27, 2005 from Tim Frinak at Weston Solutions, Inc. Seven public wastewater samples (Exygen ID C0071212-C0071218) representing one public wastewater treatment site, along with field blanks, and two sludge samples (Exygen ID C0071210-C0071211) were received on wet ice on May 4, 2005 from Tim Frinak at Weston Solutions, Inc. Seven public water supply samples (Exygen ID C0076774-C0076780) representing one public water supply site, along with field blanks, were received on wet ice on July 1, 2005 from Tim Frinak at Weston Solutions, Inc. The samples were logged in by Exygen personnel and placed in refrigerated storage. Sample ID number DPWS-PW-DWTP-0-050425 and DPWS-PW-FINW-0-050629 are Decatur Public Water System finished water. Sample ID number DPWS-WW-DCTP010-050503 is Decatur Dry Creek WWTP effluent. Sample log-in and chain of custody information is located in the raw data package associated with this interim report. Storage records will be kept at Exygen Research. 5.0 REFERENCE MATERIAL The analytical standards, PFBS and PFHS, were supplied by 3M. PFBS was received from 3M at Exygen on July 6, 2000, and 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. Exygen Research Amendment Number 1 Page 12 o f 112 Interim Report #11 - Analysis of Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated. Comnound PFBS PFBS PFHS PFOS Exveen Inventory No. SP0000252 SP0005726 SP0002401 SP0002694 Lot # 101 101 SE036 430180-1 Puritv (%) 96.7 96.7 98.6 101.2 Expiration Date 12/04/06 12/04/06 10/18/06 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 (C4F9SC>3'K+) Transitions Monitored: 299 -> 99 Structure: FF FF F FF 3 PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (C6Fi3S0 3 'K+) Transitions Monitored: 399 - 80 Structure: so, PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFi7S0 3 'K+) Transitions Monitored: 499 - 80 Structure: FFF FFFF F SO3 FFFF F Exygen Research Amendment Number 1 Page 13 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 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" and "V0001781: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" were used for this study. 6.1 Extraction Procedure for Water A 40 mL aliquot of the water sample was used for the extraction procedure. After fortification of appropriate samples, the samples were loaded onto a 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. 6.2 Extraction Procedure For Sludge Before the samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the sample container. A 5-gram portion of sludge was weighed into a fifty-milliliter centrifuge tube for the extraction. After fortification of appropriate samples, 5 mL of methanol was added to the samples. The samples were allowed to shake on a wrist action shaker for ~15 minutes and were then sonicated in an ultrasonic bath for -15 minutes. The volume was taken to 40 mL with water and the samples were then centrifuged for -10 minutes at -3000 rpm. The supernatant was then loaded onto a C]8 SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.3 Percent Solids Procedure For Sludge Percent solids were determined using the procedure indicated in Exygen method V0000427-3. 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 1 C. Then the samples were transferred to a dessicator and allowed to cool for -15 minutes. Each sample was then weighed again, including the weight of the pan. The percent solid for each sample was then calculated. See Table V for percent solids results. Exygen Research Amendment Number 1 Page 14 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 6.4 Preparation of Standards and Fortification Solutions Individual stock standard solutions of PFBS, PFHS and PFOS were prepared. The stock standard solutions were prepared at a concentration of 100 pg/mL by dissolving 10 mg of each of the standards (corrected for purity and salt content) in methanol. From these solutions, mixed 1.0 pg/mL fortification standard solutions were prepared by taking 1 mL of each of the appropriate stock solutions and bringing the volume up to 100 mL with methanol. By taking 10 mL of the mixed 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the mixed 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.01 pg/mL fortification standard was prepared. A mixed stock standard solution of PFBS, PFHS, and PFOS was also prepared. The stock standard solution was prepared at a concentration of 1000 pg/mL by dissolving 100 mg of each of the standards (corrected for purity and salt content, if necessary) in methanol. From this solutions, a 100 pg/mL fortification standard solution was prepared by taking 10 mL of the stock and bringing the volume up to 100 mL with methanol. By taking 10 mL of the 100 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 10 pg/mL fortification standard was prepared. By taking 10 mL of the 10 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 1.0 pg/mL fortification standard was prepared. By taking 10 mL of the 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 0.01 pg/mL fortification standard was prepared A set of standards containing PFBS, PFHS and PFOS was prepared in water and processed through the extraction procedure, identical to samples. The following concentrations were prepared: Cone, of Fort Fort Solution Volume (ng/mL)1 (pL) 00 10 100 10 200 10 400 100 100 100 200 100 400 1of PFBS, PFHS and PFOS Volume of Fortified Sample (mL) 40 40 40 40 40 40 40 Final Cone, of Calibration Std. (ng/L) 0 25 50 100 250 500 1000 Exygen Research Amendment Number 1 Page 15 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.5 Chromatography Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~ 0.6 mins, ~ 10.5 mins, and ~ 12.8 mins, respectively. Peaks above the LOD were not detected in any of the reagent blank samples corresponding to the analyte retention time. 6.6 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 25 ng/L of PFBS, PFHS and PFOS. 6.7 Description of LC/MS/MS Instrument and Operating Conditions Instrument: Interface: Computer: Software: HPLC: API 4000 Biomolecular Mass Analyzer Turbo Ion Spray Liquid Introduction Interface DELL OptiPlex GX400 Windows NT, Analyst 1.4.1 Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: 30 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time (mini 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: ~18 min Flow Rate: 0.3 mL/min %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Exygen Research Amendment Number 1 Page 16 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Ions monitored: Analvte PFBS PFHS PFOS Mode negative negative negative Exygen Study No.: P0001131 Transition Monitored 299 99 399 -> 80 499 -> 80 Approximate Retention Time (min) -0.6 min. -10.5 min. -12.8 min. 6.8 Quantitation and Example Calculation Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations of standards. The concentration was determined from the equations below. Equation 1 calculated the amount of analyte found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/L) = (Peak area - intercept! x DF Slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. For samples fortified with known amounts of 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 corresponding sample (ng/L)) xl00% amount added (ng/L) Equation 3 was used to convert the amount of analyte found in ng/L to ng/g (ppb) for the sludge samples. Equation 3: Analyte found (ppb, wet weight) = iAnalvte found (ng/L! x volume extracted (0.04L)1 sample weight (5 g) Exygen Research Amendment Number 1 Page 17 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Equation 4 was then used to calculate the amount of analyte found in ppb based on dry weight for the sludge samples. Equation 4: Analyte found (ppb) dry weight = Analyte found (ppb, wet) x [100% / total solids(%)] An example of a calculation using an actual sample follows (calculation is for PFOS only): Water sample Exygen ID: C0070099 Spk D (Set: 070105A), fortified at 1000 ng/L with PFHS where: peak area = 25035 intercept = 814 slope = 248 dilution factor = 10 ng/L PFOA added (fort level) = 1000 amt in corresponding sample = ND (Not Detected) From equation 1: Analyte found (ng/L) = [25035 - 8141 x 10 248 From equation 2: % Recovery 977 ng/L (977 ne/L-Ong/L) x 100% 1000 ng/L = 98% 7.0 EXPERIMENTAL DESIGN For water samples designated as field matrix spikes, PFBS, PFHS and PFOS were added at a known concentration to the bottles in the laboratory before being shipped to the field. The samples were filled to a 200 mL volumetric fill line in the field. The water samples were extracted in three sets. Each set included one reagent blank and two reagent blanks fortified at known concentrations. Each water set contained one sample site along with the field blank and field blank spikes collected for the ground water samples. For each site, a sample, a field duplicate and two matrix field spikes were collected and extracted. For each site, a laboratory duplicate was extracted and two laboratory matrix spikes were also extracted. Two sludge samples, consisting of a field sample and a field duplicate, were received representing one sample site. The sludge Exygen Research Amendment Number 1 Page 18 o f 112 Interim Report #11- Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 samples were extracted in two sets, one of which was a re-extraction set. Each set included one reagent blank and two reagent blanks fortified at known concentrations. For each sludge sample, a laboratory duplicate and two laboratory matrix spikes were also extracted. In the re-extraction set only one spike and one aliquot of sample was analyzed due to low sample volume. 8.0 RESULTS Analytical results for the analysis of PFBS, PFHS and PFOS found in public water and wastewater samples are summarized in Table I. Analytical results for the analysis of PFBS, PFHS and PFOS found in sludge samples are summarized in Table II. Analytical results for the analysis for PFOS found in re-extracted sludge samples are summarized in Table III. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in water samples are summarized in Table IV. The overall average percent recoveries + standard deviations for field and laboratory spikes for PFBS, PFHS, and PFOS in water samples were 108 16%, 109 12%, and 121 23%, respectively. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in sludge samples are summarized in Table V. The overall average percent recoveries standard deviations for field and laboratory spikes for PFBS and PFHS in sludge samples were 90 4% and 86 3%, respectively. Fortification recoveries for the analysis of PFOS in re-extracted sludge samples are summarized in Table VI. The overall average percent recovery standard deviation for laboratory spikes for PFOS in re-extracted sludge samples was 101 1%. 9.0 CONCLUSIONS The public water, wastewater, and sludge samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical methods V0001780 and V0001781, respectively. 10.0 RETENTION OF DATA AND SAMPLES All original paper data generated by Exygen Research that pertains to this interim report will be shipped to the study director. This does not include facility-specific raw data such as instrument or temperature logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. Exygen Research Amendment Number 1 Page 19 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 TABLES Exygen Research Amendment Number 1 Page 20 o f 112 Interim Report #11- Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Water Samples ExygenID C lie nt Sam ple ID C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS P erfluorobutineK ulfonata_______ Perftuorohexenesulfonete________ P w fluorooctanesulfonate Analyte Found (ng/L> Assessed Accuracy </-%) Analyte Found (ng/L) Assessed Accuracy <+/-%) Analyte Found <ng/L) Assessed Accuracy (+/-% ) C0070099 D P W S -P W -D W TP -0-050425 ND 30 ND C0070099 Rep DPWS-PW-DWTP-0-050425* ND 30 ND C0070100 DPWS-PW-DWT P-DP-050425 ND 30 ND 30 ND 30 ND 30 ND 30 30 30 C0070103 BLANK ND 30 ND 30 ND 30 C0071212 DPWS-WW-DCTP01 -0-050503 95.6 30 296 C0071212 Rep DPW S-WW-DCTP01-0-050503* 105 30 297 C0071213 D PW S-W W -D C T P01 -DP-050503 104 30 297 30 2710 30 2840 30 3010 50 50 50 C0071216 D P W S -W -T R IP -0 -0 5 05 0 3 ND 30 ND 30 ND 30 C0076774 DPWS-PW- FINW-0-050629 ND 30 ND C0076774 Rep DPWS-PW-FINW-0-050629* ND 30 ND C0076775 D P W S -P W -F IN W -D B -050629 ND 30 ND 30 ND 30 ND 30 ND 30 30 30 C0076778 DPWS-PW-T RIP-0-050629 ND 30 ND 30 ND 30 ` Laboratory Duplicate ND s Not detected at or above 25 ng/L. Exygen Research Amendment Number 1 Page 21 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Table II. Summary of PFBS, PFHS and PFOS in Sludge Samples ExygenID Client Sample ID C4 Sulfonate PFBS P e rfluo rob utan esulfonite Analyte Found Assessed (ppb. ng/g) Dry Weight Accuracy <+/- %) C0071210 DPWS-S L-DCTP01-0-050503 C0071210 Rep DPWS-S L-DCTP01-0-050503* C0071211 DPWS-S L-DCTP01-DP-O50503 C0071211 Rep DPWS-SL-DCTP01-DP-050503* ND ND 1.59 ND 30 30 30 30 C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorohexarm sulfonate_______ P arfluorooctaneaulfonata Analyte Found Assessed Analyte Found Assessed (ppb, ng/fl) Dry Weight Accuracy <+/- %) (ppb, ng/g) Dry Weight Accuracy <'-*> 13.8 15.5 30 30 NR NA NR NA 12.7 15.2 30 30 NR NA NR NA 'Laboratory Duplicate ND c Not detected at or above 0.2 ng/B (wet weight). NR = Not reported due to quality control failure. For re-analysis see Table III. Exygen Research Amendment Number 1 Page 22 o f 112 Interim Report # 1 1 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Table III. Summary of PFOS in Re-extracted Sludge Samples E xygenID C 0071210 C0071211 C lie n t S am p le ID D PW S -S L-D C TP 01 -0-050503 D PW S -S L-D C TP 01 -D P -050503 C8 Sulfonate PFOS Perfluorooctanesulfonate Analyte Found (PPb, ng/g) Dry W eight Assessed Accuracy (+/-% ) 2110 1930 30 30 Exygen Research Amendment Number 1 Page 23 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Water Samples Sample Description DPWS-PW-DWTP-0-050425 (C00700M Spk C, 100 ngfl. Lab Spike) DPWS-PW-DWTP-0-050425 (C0070099 Spk D, 1000 n^L Lab Spike) DPWS-PW-DWTP-LS-050425 (C0070101, 100 ng/L Field Spike) DPWS-PW-DWTP-HS-Q50425 (C0070102,1000 ng/L Field Spike) C4 Sulfonate PFBS____________ C6 Sulfonate PFHS____________C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Semple Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/L) (nfl/L) (ng/L) l* l (ng/L) (ng/L) (ng/L) (ng/L) <%) 100 ND 113 113 ND 99.4 99 ND 106 106 1000 ND 1090 109 ND 979 98 ND 1060 106 100 ND 112 112 ND 105 105 ND 91.9 92 1000 ND 1100 110 ND 1020 102 ND 932 93 BLANK-LS (C0070104, 100 ng/L Field Spike) 100 ND 103 103 ND 109 109 ND 122 122 BLANK-HS (C0070108,1000 ng/L Field Spike) 1000 ND 1010 101 ND 1140 114 ND 1180 118 DPWS-WW-DCTP01 -0-050503 (C0071212 Spk C, 1000 ng/L Lab Spike) DPWS-WW-DCTPOt -0-050503 (C0071212 Spk D, 10000 ng/L Leb Spike) DPWS-WW-DCTP01 -LS-050503 (C0071214,1000 ng/L Field Spike) DPWS-WW-DCTP01-HS-05Q503 (C0071216,10000 ng/L Field Spike) 1000 10000 1000 10000 95.6 95.6 95.6 95.6 1200 11100 1370 13600 110 110 127 135 296 1340 104 2710 4360 165 296 10500 102 2710 14300 116 296 1570 127 2710 4220 151 296 14700 144 2710 20100 174 DPWS-W-TRIP-LS-050503 (C0071217,100 ng/L Field Spike) 100 ND 73.0 73 ND 106 106 ND 125 125 DPWS-W-TRIP-HS-050503 (C0071211,1000ng/L Field Spike) 1000 ND 733 73 ND 1120 112 ND 1210 121 DPWS-PW-FINW-0-050629 (C007S774 Spk C, 1000 ng/L Lab Spike) DPWS-PW-FINW-0-050629 (C007I774 Spk D, 10000 ng/L Lab Spike) DPWS-PW-FINW-LS-050629 (C0078778,100 ng/L Field Spike) DPWS-PW-FINW-HS-050629 (C007S777,1000 ng/L Field Spike) 1000 10000 100 1000 ND ND ND ND 1230 11000 124 1010 123 110 124 101 ND 1100 110 ND 10400 104 ND 117 117 ND 919 92 ND 1180 118 ND 11000 110 ND 135 135 ND 952 95 DPWS-PW-TRIP-LS-050629 (C007I779,100 ng/L Field Spike) DPWS-PW-TRIP-HS-050629 (C0074780,1000 ng/L Field Spike) 100 1000 ND 89.3 89 ND 1110 Average: Standard Deviation: in 108 16 ND 100 100 ND 1080 Average: Standard Deviation: 108 12 ND 115 115 ND 1240 Average; Standard Deviation: 124 1 23 ' Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated ND * Not detected at or above 25 ng/L (ppt). Note: Sinee this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. Exygen Research Amendment Number 1 Page 24 o f 112 Interim Report #11- Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Table V. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sludge Samples Sample Description DPWS-SL-DCTP01 -0-050503 (C0071210 Spk C, 4 ppb Spike) DPWS-SL-DCTP01-0-050503 (C0071210 Spk 0 ,40 ppb Spike) Amount Spiked (no/g) C4 Sulfonate PFBS Vtet Weight Amt Found Amount in Sample Recovered Recovery (ng/g) (ng/g) (%) C6 Sulfonate PFHS Wet Weight Amt Found Amount in Sample Recovered Recovery (ng/g) (ng/g) (%) C8 Sulfonate PFOS Wet Weight Amt Found in Sample (ng/g) Amount Recovered Recovery (ng/g) (%) 4 ND 3.85 96 1.90 5.21 83 NR NR NR 40 ND 35.3 88 1.90 37.6 89 NR NR NR DPWS-SL-DCTP01 -DP-050503 (C0071211 Spk E, 4 ppb Spke) DPWS-SL-DCTP01 -DP-050503 (C0071211 Spk F, 40 ppb Spike) 4 40 0.229 3.74 88 0.229 35.1 Average: Standard Deviation: 87 90 4 1.82 5.20 85 1.82 36.6 Average: Standard Deviation: 87 86 3 NR NR NR NR NR Average: Standard Deviation: NR NA NA ND Not detected at o r above 0.2 ng/g (wet weight). NR = Not reported due to quality control failure. For re-analysis see Table VI. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in d ie raw data. Exygen Research Amendment Number 1 Page 25 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Table VI. Matrix Spike Recovery of PFOS in Re-extracted Sludge Samples Sample Description DPWS-SL-DCTP01 -0-050503 (C0071210 Spk E, 400 ppb Spike) DPWS-SL-DCTP01 -DP-050503 (C0071211 Spk G, 400 ppb Spike) Amount Spiked (ng/g) 400 400 Amt Found in Sample (ng/g) C8 Sulfonate PFOS Wet Weight Amount Recovered (ng/g) Recovery (%) 290 694 101 278 685 102 Average: Standard Deviation: 101 1 Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Amendment Number 1 Page 26 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Table VII. Total Percent Solids for Sludge Samples Exygen ID____________ Client Sample ID_________ Total Percent Solids (%) C0071210 C0071211 DPWS-SL-DCTP01-0-050503 DPWS-SL-DCTP01-DP-050503 13.76 14.41 Exygen Research Amendment Number 1 Page 27 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 FIGURES Exygen Research Amendment Number 1 Page 28 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 1. Typical Calibration Curve for PFBS in Reagent Water 070105A Water.rdb CPFBS* "Lineal" Regression weighting* y - 118 * 0.00180 (r* 0.0067) Area, counts Exygen Research Amendment Number 1 Page 29 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively I XCP71505-9 - PFBS (Stuudurd) 29%V99.0om u -vurp/ 1 o f 27 from Q7$1Q5A.wtff p ak not found) 10.11 Tim , min 1 X 00 7 13 00 -1 PFBS (Standard)2flQ.Q/OQ.O am u sa m p le 2 o f 27 from 0 7 0 1 0 5 A .w lff A rea: 3 00 0 oounto H eight: 0 7 .4 ops R T :3 .B 2 m in 3.B2 Exygen Research Amendment Number 1 Page 30 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 3. PFBS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively I f f i f f M t Control - PFBS (Unknown) 29%9f9%0nmn m pf* 9 o f 27 from 979105A.wfff ponk not found) Intensity, cps Intensity, cps R eagent Spk A - PFBS (Q C )20Q .Q A 0.0 am u sa m p le 10 o f 2 7 fro m Q70105A.iNiff A r t a: 0 3 3 5 counts H eight: 8 5 .0 cps RT: 3 .7 4 m in 3 .7 4 /S 1 D 0.00 7.40 O K k 10/J5f1D-48 5 0 7 8 0 10 Tim e, min | R a ag a nt Spk B - PFBS ( 0 0 ) 2 0 0 .0 ^ 0 .0 am u - u m p l c 11 o t 27 from O7O1O0A.ulff Area: 5027B counts H e ig h t 040. cps R T :3 .0 1 min 3.61 13.30 1 4 .8 8 ^_ t6 3 0 ^,1 5 .7 0 ,1 0 .7 0 -Q - ^7**- r Tim e, min Intensity, cps Exygen Research Amendment Number 1 Page 31 o f 112 Interim Report # 11- Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Water Sample Analyzed for PFBS (Exygen ID: C0070099, Data Set: 070105A) C N 7N N PFflS (UHlutowH) 2 M IM S M il -ta m p ff t o f 7} from *f*1 *5 A ytfff A n a : m ie o a a ta HtigOt: 3 S .ic p t RT: 9.5*4mia 0.98 Intensity, cps Exygen Research Amendment Number l Page 32 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 5. Chromatogram Representing a Sludge Sample Analyzed for PFBS (Exygen ID: C0071211, Data Set: 070505G) I C0071211 PFBS (UmtKOWH) 29910/99.0 amu -tamp! 22 o f 27 from 070505G.wiff A na: 4880 counts HOigltt: 33.3 epa RT: 0.535mia 11.81 Intensity, cps Time, min ,1 2 .0 0 ^14.03 Exygen Research Amendment Number 1 Page 33 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 6. Typical Calibration Curve for PFHS in Reagent Water 070108A W jtr.rdt> (PFHS): " Line a l" R t g m lo n ('1 / m ig h tin g ): y - 248 x + 8 14 (r 0.0880) Area, counts Exygen Research Amendment Number 1 Page 34 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 7. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively XC$715$5+ - PFHS (Standard) 399L0/NL0 ama sam pk 1 o f 27 from 079105A.WM A r*a : ZU coaat fSL2cpt RT: 11.3 m ia 11.20 Intensity, cps Intensity, cps Tim , min XC 071505-1 - PFHS (S tan d ard) 3Q0.EMBO.O m u - sam ple 2 o f 27 Iro m 0701Q 5A .w iff A rea: 7 0 7 6 counts H eight: 3 01 . ops RT: 10.5 m in 10.54 Tim e, min _ 13JB2 13 14 15 10 17 Exygen Research Amendment Number 1 Page 35 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 8. PFHS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively Rnngont C ontrol PFHS (Unknown) 3 9 I M M omo -nnm plo 9 o f 27 from 97919SA.wiff A roo: i M c w i k H nigkt: 19.1 opo RT; 11.9 m in Tim , min R t jg t n i Spk A - PFHS (0 0 )3 0 0 .0 /0 0 .0 m u - w m p lt 10 of 27 from 070105Auwff A r j: 1 14 04 counts H eight: 5 26 . eps RT: 10.5 m in 400 l 200 10.52 ij^ " 1 2 3 4 5 B 7 8 0 10 11 12 13 14 15 10 17 Tim , min I R t a g t n t Spk B - PFHS (Q C )3Q 0.0/B 0.0 im u w m p lt 11 of 27 from 0 7 0 1Q 5 A w fr A ra: 1 13008 eounts H ig h f:5 1 S 0 . eps RT: 10.5 min 10.53 Exygen Research Amendment Number 1 Page 36 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 9. Chromatogram Representing a Water Sample Analyzed for PFHS (Exygen ID: C0070099, Data Set: 070105A) C007W 99 PFHS ffM Am wa) 399l/V4Lf ama n m p tt 76 o f 27 from 7076SA.W777 A n a : H3Teoaat* HtlgHt: 1%3tp* RT: 1*.5mm 10.81 Intensity, cps Exygen Research Amendment Number 1 Page 37 o f 112 Interim Report #11- Analysis o f Water, Wastewater, and Sludge Samples Exygen Study N o.: P0001131 Figure 10. Chromatogram Representing a Sludge Sample Analyzed for PFHS (Exygen ID: C0071211, Data Set: 070505G) C0071211 -PFHS (Unknown)39910/S&0 M ir -sam ple 22o f 27 from 070S0SG.wifT Area: 49227count H eight: 3979. cpa HT: 70.6m in 10.03 Intensity, cps Exygen Research Amendment Number 1 Page 38 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 11. Typical Calibration Curve for PFOS in Reagent Water 070109AW tr.rdb (PFOS): "L in te l" R e g re w io n fl / * " weighting): y 142 x + 0.00173 ( r - 0.0070) Area, counts Exygen Research Amendment Number 1 Page 39 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 12. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively XCO77505-0 - PFOS (Standard) 499.9/99.9 amu -sumpf* 1 o f 27 from 070103A.wifT paaft aot found) 12.00 Exygen Research Amendment Number 1 Page 40 o f 112 Interim Report #11- Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No. : P0001131 Figure 13. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively I f f H f H t Control - PFOS (IMMmowh) 49%C/ML9 ama - aampt* 9 o f 27 from $7Q15A.wifT p ia tt aot found) 14.17 .,14.87 Tim , min R ia g tn t Spk A - PFOS (QC) 4 0 0 .0 *0 .0 amu - sample 10 of 27 from 07Q105A.wiff A rea: 7 1B 4 counts H a ig h t 2 20 . ops RT: 12.8 m in iaun. 200- |0) 1000-I..1.03 2.13-r^r --1-i--" -- 1 -.i-*O--- 8.41. .8.8I2.8.3411^D_S11 1237\1 , ,\k5.04 -- i----*-- i - i ~ -- r - ........i ' ------1-- ^ i ---- i 1 . - . --i----- ! 17.30, 1 2 3 4 5 0 7 8 0 10 11 12 13 14 15 10 17 Tim , min I R agnt Spk B . PFOS(QC )4M .O/BO.O a m u -s a m p le 11 of 27 from 070105A.w iff Ara: 02005 counis Hight: 2010. cps RT: 12.8 min in S* 1800- f 1000- aC s o d - 01 12.70 34 50 7 10.52 0 10 11 12 13 14 15 10 17 Tim , min Exygen Research Amendment Number 1 Page 41 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 14. Chromatogram Representing a Water Sample Analyzed for PFOS (Exygen ID: C0070099, Data Set: 070105A) C0970M 9 - PFOS (IMtKOWH) 499.0*0.0 t m t -M im pfr H o t 27 from OTOIOSA.vritf A n t: yN 2ccw t> H oigtt: S0l7cp AT: IZ tm it 12.81 Intensity, cps Exygen Research Amendment Number 1 Page 42 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Figure 15. Chromatogram Representing a Sludge Sample Analyzed for PFOS (Exygen ID: C0071211, Data Set: 052206B) I C0971211 - PFOS (UktMOWMj 49**919 Mma -Murp/e 21 o f 23 from 05Z296B.wiff A rtt: 116997 count Height: 7.22+053 cp# HT: 9,97 min 7000 0.07 0500 0000 5500- aooo- 4500 4000 3500 3000 2500 2000 1500 - 1000 500 0 1 i ' ----------- 1 ------------,----- ------,-----------123 45 07 T im *, min 12 13 14 15 10 17 Intensity, cps Exygen Research Amendment Number 1 Page 43 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods, and Protocol Amendments 1,2 and 3 Exygen Research Amendment Number 1 Page 44 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in W ater, Soil, Sedim ent, Fish, Clam s, Vegetation, Small M am m al Liver and Small M am m al Serum Using LC/M S/M S for the 3M Decatur M onitoring Program Exygen Protocol Number: P0001131 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 Sponsor Representative: M ichael A. Santoro Director o f Regulatory Affairs 3M Building 0 2 3 6 -0 1-B -10 St. Paul, M N 55144 Phone: (651) 733-6374 Page / of 65 Exygen Research Amendment Number 1 Page 45 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 0 0 0 1131 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Amendment Number 1 Page 2 o f 65 Page 46 of 112 Interim Report #11- Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: POOO1131 PROTOCOL APPROVAL Study Title: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 APPROVALS JaisimhalKesan, S' Weston Solutions Michael A. 3M Comp: Sponsor Representative m Date 7q-acr-xz^ Date ad, Quality Assurance Unit Date Page J oj 65 Exygen Research Amendment Number 1 Page 47 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 TABLE OF CONTENTS TITLE PA G E ............................................................................................................................................................................. 1 D ISTRIBUTION.......................................................................................................................................................................2 PROTOCOL A PPROVAL..................................................................................................................................................... 3 TABLE OF C O N TEN TS........................................................................................................................................................4 I N T R O D U C T IO N .....................................................................................................................................................................S TEST M A TER IA LS................................................................................................................................................................ 5 O BJEC TIV E.............................................................................................................................................................................. 6 TESTING FA CILITY..............................................................................................................................................................t> STU D Y DIRECTOR................................................................................................................................................................ 2 SPONSOR REPRESENTATIVE............................................................................................................................................7 PRINCIPAL INVESTIGATOR.............................................................................................................................................. 7 PROPOSED EXPERIM ENTAL START A N D TERMINATION D A T E S ............................................................... 7 IDENTIFICATION A N D JUSTIFICATION OF THE TEST S Y S T E M ..................................................................8 SAM PLE PROCUREM ENT, RECEIPT A ND R ETENTIO N .....................................................................................8 SAM PLE IDENTIFICATION.............................................................................................................................................. 9 A NA LYTICAL PROCEDURE SUM M A R Y ................................................................................................................... 9 VERIFICATION OF ANALYTICAL PROCEDURE....................................................................................................9 METHOD FOR CONTROL OF B I A S ................................................................................................................................. 11 STATISTICAL M E T H O D S.................................................................................................................................................... 11 GLP S T A T E M E N T ................................................................................................................................................................. 11 R EPO R T..................................................................................................................................................................................... U SAFETY A N D H E A L T H .......................................................................................................................................................12 AM EN DM ENTS TO PR OTO CO L.....................................................................................................................................13 D ATA RECORD K E E P IN G .................................................................................................................................................13 Q U ALITY A S S U R A N C E ..................................................................................................................................................... 14 RETENTION OF D ATA A N D A R C H IV IN G .................................................................................................................14 APPENDIX I, ANALYTICAL M E TH O D S......................................................................................................................15 Page 4 o f 65 Exygen Research Amendment Number 1 Page 48 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 INTRODUCTION The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum using LC/MS/MS for the 3M Decatur Monitoring Program. The study will be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit o f Exygen Research. TEST MATERIALS The test materials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M. PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4FgSOj'K+) Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 -* 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CiFuSt^TC*) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 - 80 Structure: Page 5 o f 65 Exygen Research Amendment Number 1 Page 49 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number. P 0 0 0 1 131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CsFirSO TO Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 - * 99 Structure: OBJECTIVE The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions o f the following Exygen analytical methods: V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Page 6 o f 65 Exygen Research Amendment Number 1 Page 50 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 1 3 1 STUDY DIRECTOR Jaisimha (Cesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax:(610)701-7401 j.k esa ri@ w esto n so lu tio n s.co m SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M. Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 john.flaherty@exygen.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion o f this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and termination dates will be included in the final report. Page 7 o f 65 Exygen Research Amendment Number 1 Page 51 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: Water (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study. The test systems were chosen to access the environmental impact o f PFBS, PFHS and PFOS in the Decatur, Alabama area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples will be received at Exygen directly from Weston Solutions. The details o f sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types o f samples collected will vary depending availability in the field. The total number o f samples received and analyzed for each matrix will be documented in the final report associated with this study. Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at 2C-8C. Fish, clam, vegetation and small mammal liver samples will be processed according to the appropriate analytical method (see Appendix I)' These sa m p les w ill b e stored frozen at 5 -10C. S m a ll m a m m al w h o le b lood samples will be centrifuged in the field at the time o f collection and the serum fraction w ill be used for the study. Small mammal se r u m will b e sto red frozen at -10"C. The receipt and processing o f the samples will be documented in the final report and raw data associated with the study. Page 8 o f 65 Exygen Research Amendment Number 1 Page 52 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 SAMPLE IDENTIFICATION Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Sample storage conditions and locations will be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" The above methods use analytical conditions capable o f separating the isomers o f PFBS, PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sample will be used for the preparation o f fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen will supply one bottle per sample collected. The bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 9 o f 65 Exygen Research Amendment Number 1 Page 53 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 /" > Exygen Protocol Number; POOO1131 collection at the testing facility and have been shown to be free o f PFBS, PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike o f each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid ( 13C PFOA). PFOA and l3C PFOA are included in the solutions used to spike the samples. The results for PFOA and l3C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containers/bags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and 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 determine method accuracy and to check for bias. For small mammal liver, Exygen will supply a 50 mL polypropylene centrifuge tube. For small mammal serum, Exygen w ill supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. Low and high spiking levels for each matrix are defined below: M atrix Low Spiking Level High Spiking Level Water 500'ng/L 5000 ng/L Soil 4ng/g 40 ng/g Sediment 4 ng/g 4 0 n g/g Fish lOng/g 100 ng/g Clams 10 ng/g 100 ng/g Vegetation 10ng/g 100 ng/g Small Mammal Liver 10 ng/g 100 ng/g Small Mammal Serum 10 ng/mL 100 ng/mL Page 10 o f 65 Exygen Research Amendment Number 1 Page 54 o f 112 Interim Report #11- Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: POOOl 131 Recoveries are anticipated to be between 70% and 130% o f the fortified levels; however, the exact precision and accuracy w ill be determined by the analysis o f the quality control samples described above. A statement o f accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control o f bias will be addressed by taking representative sub-samples from a homogeneous mixture o f each matrix from untreated control samples, and by analyzing at least two levels o f fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation o f average recoveries, as applicable. GLP STATEMENT All aspects o f this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative. REPORT A final report will be prepared by the principal investigator or their designee at the conclusion o f the study. The report will include, but will not be limited to, the following: The name and address o f the Study Director, Sponsor Representative, and o f the testing facility. A statement o f GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). Page II o f 6j Exygen Research Amendment Number 1 Page 55 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates o f study inspections and dates findings were reported to the Study Director and Management. A description o f the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy o f the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page. Description o f the instrumentation used and operating conditions. All results from all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level. Representative chromatograms for each analyte in each matrix, including chromatograms o f a standard and a control sample, and a chromatogram at a fortification level. The location o f the analyte peaks w ill be clearly identified in all chromatograms. All circumstances that may 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 the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Representative. All applicable requirements for reporting o f study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory person nel w ill practice g ood sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environment to the test or reference substance(s). Page 12 o f 65 Exygen Research Amendment Number 1 Page 56 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000 U31 AMENDMENTS TO PROTOCOL All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation o f study plan change. However, when a change is required without sufficient time for the issue o f a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date o f the written amendment will be the date o f the documented change. Copies o f the signed amendments will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description o f any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence Any other documentation necessary for the reconstruction o f the study Chromatograms- All chromatograms will contain the following: Sample identification, injection date, arrow or other indication o f the area o f interest, and injection number corresponding to the run. Additionally, fortifications will include the amount o f analyte added and the sample number o f the sample that was fortified. Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL). Page 13 o f 65 Exygen Research Amendment Number 1 Page 57 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 As part o f the documentation the following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITY ASSURANCE The QA Unit o f Exygen Research w ill inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings o f audits to the Study Director, Exygen Management, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period o f time specified in 40 CFR 792.195. An exact copy o f the materials submitted to the study director will also be kept at Exygen Research. Exygen w ill obtain permission from the study director before discarding or returning samples. Exygen Research Amendment Number 1 Page 14 o f 65 Page 58 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 APPENDIX I ANALYTICAL METHODS V0001780 vooomi V0001782 V0001783 V0001784 V0001785 V0001786 "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" 'Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC M S/M S" Exygen Research Amendment Number 1 Page 15 o f 65 Page 59 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 ANALYTICAL METHOD Method Number: V0001780 M ethod o f Analysis fo r the DetermlnaUoa o f Perfluerooctanolc A d d (PFO A) in W ater byLC /M S /M S Analytical Testing Facility: Exygen Research 3058 Research D rive State College, PA 16801 Approved By; C-- _________ Paul Connolly 1 Technical Leader, LC-M S, Bxygen Research mltuAW Date A-1 Exygen Research Amendment Number 1 Total Pages: 7 Page 6 of 65 Page 60 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 Exyea Rwoarch Method Number V0QQI7W A N A LY T IC A L m e t h o d Method o f Analysis for the Determination ofPcrfluoroocU noic A cid (PFOA) in Water by LC /M S/M S 1.0 Scope This method it to be employed fo r the ieolation and quantitation o f p e riluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/M S) in water. 2.0 Safety 2.1 A tw ayi observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 40 m L o f test sample fo r extraction. 3.2 No sample processing is needed fo r water samplee. 3.3 Simples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 A il samples must be thoroughly mixed before being sampled for extraction 3.5 Any samples conttining particles should be centrifuged at '300 0 rpm for '5 minutes and the supernatant used for the extraction. 3.6 Sample collection procedures w ill be specified in the sampling plan for this p ro je c t 4.0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigm a-Aklrich 5.0 Instrument and Equipment 5.1 A high performance liq u id chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume yector capable o f injecting $-200 |iL connected to a tandem Mass Spectrometer (LC/M S/M S). 3.2 A device to collect raw data fo r peak integration and quantitation. $.3 Analytical balance capable o freading to 0.00001 g. $.4 SOm L disposable polypropylene centriftige tube#. 5.5 15 m Ldi^osable polypropylene centrifoge tube*. 5.6 Disposable m icropipets (50*100qL, 100-200uL). 5.7 12S-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC via l k it. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), w ith disposable tips. 5.11 Waters Sep Pak Vac 6 cc(lg)tC 18S P E cartridges. Page2v i1 Page / 7 o f 65 Exygen Research Amendment Number 1 Page 61 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygraXauarcb MethodNumber VQ00178Q ANALYTICAL method Method o f Analysis fo r the Determination ofPeriluoroocU noic A cid (PFOA) in Water by LC /M S/M S 3.12 SPE vacuum manifold. 3.13 C entriftge capable o f spinning 30 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.) Analytical Column: Fluophaae RP (Keystone S cientific), 2.1 m m x50m m . 5h (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ): 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ): Methanol 6.5 Gradient Program: Tim e fm in l 0.0 1.0 8.0 20.0 22.5 2LA 65 65 23 25 63 Flow Rate % B fm L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions ere intended as a guide and may be changed in order to optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative M RM mode, m onitoring 413 369 nvz. The above conditions are intended as a guide and may be changed m order to optim ize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water it prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. Page * Page 18 o f 65 Exygen Research Amendment Number 1 Page 62 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 Exygea Research Method Number V0001780 ANALYTICAL METHOD Method o f Analyse for the Detemunation o f Perfluorooctanoic A cid (PFOA) in Water by LC /M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a tock solution o f MOO pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-m LLDPE bottle. 9.1.2 A 10 pg/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 100 pg/m L solution to a fin a l volume o f 100 w ith methanol in 125 m L LDPE bottle. 9.1.3 A 1.0 p^m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f die 10 pg/m L solution to a fin a l volume o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.4 A0.1 pg/m L fortifica tion solution ofPFO A is prepared by bringing 10 m L o fthe 1.0 pg/m L solution to a fin a l volume o f 100 w ith methanol in a 125 m L LOPE bottle. 9.1.5 A 0.01 pg/m L fortifica tion solution ofP FO A is prepared by bringing 10 m L o f the 0.1 pg/m L solution to a final volume o f 100 with methanol in a 125 m L LOPE bottle. 9.1.6 The stock and fortifica tion solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o fpreparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The follow ing is a typical example: additional concentrations may be prepared u needed. Concentration o fFortification Solution (nob) 0 10 to to 100 100 100 Fortification Volume o f Volume Fortified Control (PL) Smele (mL) 0 40 100 40 200 40 400 40 100 40 200 40 400 40 Final Concentration of Calibration Standard (pet)* 0 23 SO 100 230 300 1000 Calibration Standard ID (exanrnlel XCmmddyy-0 XCmmddyy*] XCmmddyy*2 XCmmddyy*) XCmmddyy-4 XCmmddyy-5 XCmmddyy-6 * The extracted concentration o f the calibration standard is equal to 8x its in itia l concentration, due to the concentra!ton o fthe standard during the extraction (SPE). XC " extracted calibration standard. Pige 4 ol' ' Page 19 o f 65 Exygen Research Amendment Number 1 Page 63 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 Exygon JUm rch Method Number V0001780 ANALYTICAL METHOD Method o f Analysis fo r the Determination o f Perfluorooctanoic A cid (PFOA) in Water by LC /M S/M S 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) m u it be prepared w ith each set o f standards extracted. Store a ll extracted calibration standards in 15-mL polypropylene tubes at 2*C to 6*C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one reagent control (method blank using HPLC water) and two reagent controls fortifie d st known concentrations (lab control spike) to verily procedural recovery for the batch. 10.2 Requirements fo r field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Measure 40 mL o f am ple or a portion o f sample diluted to 40 m L w ith water into 50 mL polypropylene centrifoge tubes (fo rtify as needed, replace lid and m ix w ell). 11.2 Condition the C u SPE cartridges (1 g, 6 m L) by passing 10 m L methanol followed by 5 m L o fHPLC water (~ 2 drop/eec). Do not let column run dry 11.3 Load sample on conditioned C u SPE cartridge. Discard etuate. 11.4 Elute w ith ~5 m L 100% methanol. C ollect 5 m L o f eluate into graduated 15 m L polypropylene centrifoge tubes (fin a l volume * 5 mL). 11.5 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sim ple and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set 12.3 An entire set o f extracted calibration standards must be included st the beginning and st the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards may be injected at the beginning o f a sei followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the firs t and last injection in a sample sei. 12.4 Use linear standard curves fo r quantitation. Linear standani curves are generated for the analyte by linear regression using 1/x weighting o f peak area Pagei ol i Page 20 o f 65 Exygen Research Amendment Number 1 Page 64 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 0 0 0 1 131 ExygtnIU m reh Method Number V00017S0 | ........ A N A LY T IC A L M ETH O D ........... | Method o f Analysts fo r the Determination o fPerfluorooctanoic A cid (PFOA) in Water by LC /M S/M S versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.3 Sample response should not exceed standard responses. Any samples that exceed standard responses should be farther diluted and resnsljoed. 13.0 Acceptance C riteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. II * blank contains PFOA at levels greater than 50 ng/L, then s new blank sample must be obuinod and the entire set must be re-extracted. 13.3 Recoveries o f control spikec and m atrix spikes must be between 70-130V o f their known values. I f control spike fells outside the acceptable lim its, the entire set o f samples should be re-extracted. Any m atrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is 13.4 Any calibration standard found to be it statical ou tlie r by using the Huge Error Test, may be excluded from foe calculation o f the calibration curve However, foe total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. 13.5 The em ulation coefficient (R) fo r calibration curves generated must be 20.992 (R2 20.985). I f calibration results fo il outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than i 4 % w ithin an analytical run. I f retention tim e d rift exceeds this lim it within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in ng/L. baaed on peak area) using the standard curve (linear regression parameters! generated by foe Mass Lynx software program: PFOA found (ng/L) - /Peak area - intercept) x DF slope DF factor by which the final volume was diluted, if necessary. Pag*6oi1 Page 2 / o f 65 Exygen Research Amendment Number 1 Page 65 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 Exygfo Rcwvcb Metfeod Number V00017W | A1SALVTICAL M ETH O D ~1 Method o f Analyne for the Determination o fPerfluorooctanoic A cid (PFOA) in Water by LC /M S/M S 14.2 For um plaa fo rtifie d w ith known amounts o f PFOA prior to extraction, use the follow ing equation to calculate the percent recovery. Recovery (% ) [ total analyte found (n g fl.) analyte found in control (ng/L)] ^ ^^ analyte added (ng/L) Exygen Research Amendment Number 1 Page 7 of 7 Page 22 o f 65 Page 66 o f 112 Interim Report # 1 1 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 ANALYTICAL METHOD Method Num ber VOOOI78! M ethod o fA n e ly iit to r the D eterm ination e f Perthioreoctanolc A d d (PFO A) Id Soil by LC /M S/M S Analytical Teetin g Facility; Exygen Research 3058 Reaearch D rive State College, PA 16801 Approved By: t u m ,__ Paul Connolly ' Technical Leader, LC-M S, Exygen Reaearch io ln /< W Date n Flaherty f Vice Prsidait, Operations, Exygen Research Date Exygen Research Amendment Number 1 Total Pages: 7 Page 23 o f 65 Page 67 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 ExygtaJUanrck Me&od Nwaber V00017g | I A N A LY T IC A L M ETH O D M hod o f A n a ly tii for the Detemunatlon ofPerfluoroocUmoic Acid (PFOA) in Soil by LC /M S/M S 1.0 Scope This method i i to be employed for the eolation and quantitation o f perfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/M S/M S) in to il. 2.0 Safety 2.1 A iw ayi observe safe laboratory practice!. 2.2 Conault the appropriate MSDS before handling any chemical for proper safety precaution!. 3.0 Sample Requirement 3.1 A t least 13 g o f ted sample fo r extraction. 3.2 No sample processing ii needed fer Mil samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 A ll samples must be thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedures w ill be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P LC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma*A ldrich 5.0 Instrument and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith variable volume hyectoT capable o f injecting 5-200 pL connected to a tandem M an Spectrometer (LC/M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene ccntrifogs tubes. 15 m L disposable polypropylene centrifoge tubes. Disposable m icropipets (50-100uL, 100-200uL). 125-mL LDPE nanow-mouth bottles. 2 mL clear HPLC via l k it. Disposable pipettes. Autopipettes (100*1000 pL and 10-100 pL), w ith disposable tips. Waters Sep P ik Vac 6 cc (Ig ) tC18 SPE cartridges. SPE vacuum manifold. Ultrasonic bath. Page 2 o f ' Page 24 o f 65 Exygen Research Amendment Number 1 Page 68 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 Exypn Ramrcb ANALYTICAL METHOD MethodNumber VOOOI781 1 Method o fAnalysis for the Determination o fPerfluocooctanoic A cid (PFOA) in Soil by LC /M S/M S 5.14 W rift-a ctio ii shaker. 5.15 Centrifuge capable o f spinning 50 m L polypropylene tube* at 5000 rpm, 6.0 Chromatographic System 6.1 Analytical Column: Fluophase R? (Keystone S cientific}, 2.1 mm x 50 mm. 5p (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ) : Methanol 6.5 Gradient Program: Tim e faiiiri 0.0 1.0 8.0 20.0 22.S 65 65 25 25 65 Flow Rate % B (m l/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Q uantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and may be changed in order to optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospmy Negative M RM mode, m onitoring 4 1 3 - 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optim ize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volum e! may be prepared. P a fcJo n Page 25 o f 65 Exygen Research Amendment Number 1 Page 69 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 Exyjc* Battateli Method Number VQOOPIl ANALYTICAL METHOD Method o f Analysis fo r the Determination o f Perihiorooetanoic A cid (PFOA) in Soil by IC/M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortiflcation Solution 9.1.1 Prepare a stock aolution o f-100 pg/raL o f PFOA by weighing 10 mg o f analytical atandard (corrected fo r p u rity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pgfrnL fortifica tion solution o f PFOA it prepared by bringing Hi m L o f the 100 p j/m L solution to a fin a l volume o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 1.0 pg/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 10 pg/m L solution to a fin a l volume o f 100 w ith methanol m a 125 m L LDPE bottle. 9.1.4 A 0.1 pg/m L fortifica tion aolution o f PFOA ie prepared by bringing 10 m L o ffh e 1.0 pgfm L solution to a fin a l volume o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.5 A 0.01 pgftnL fortifica tion solution o f PFOA ia prepared by bringing 10 m L o f foe 0.1 pg/m L solution to a fin a l volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.6 The stock and fortifica tion solutions are to be stored in a refrigerator at approximately 4"C and are stable fo r a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to sample. The follow ing is a typical example: additional concentrations may be prepared as needed. Final Concentration o f Fortification Solution (nob) 0 10 10 Fortification Volume o f Volume Fortified Control 0L) Sam oli (mL) 0 40 100 40 200 40 Concentration of Calibration Standard (not)* 0 25 50 Calibration Standard ID (examole) XCmmddyy-0 X C m m ddyy'l XCmmddyy-2 10 400 100 100 40 40 100 XCmmddyyO 250 XCnunddyy*4 100 200 too 400 40 40 500 1000 XCmmddyy-5 XCmmddyy*6 * The extracted concentration o f the calibration standard is equal to 8 * its in itia l concentration, due to the concentration o f the standard during the extraction (SPE). XC extracted calibration standard. Pag 4 o f7 Page 26 o f 65 Exygen Research Amendment Number 1 Page 70 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xyg e n P ro to c o l N u m be r: P 0 001131 Exypo Ramrck Mttbod Number V000I78I ANALYTICAL METHOD Method o f Analysis fo r the Determination o f Perfhiorooctanoic A cid (PFOA) in Soil by LC /M S/M S 9.2.3 9.2.4 9.2.5 A zero itandard eohition (reagent blank) m in i be prepared w ith each set o f standards extracted. Store a ll extracted calibration standards in 15-mL polypropylene lubes at 2*C to 6C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one reagent control (method blank using 5 m L o f methanol) and two reagent controls fortifie d at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assurance plan fo r this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f sample into 50 m L polypropylene ccntrifoge tubes (fo rtify as needed, replace lid and m ix w ell). 11.2 Add 5 m L o fmethanol and shake on a w rist action shaker for **15 minutes. 11.3 Transfer the tubes to an ultrasonic bath rod sonicate for -15 minutes. 11.4 Bring the volume up to 40 m L w ith water in the 50 m L polypropylene ccntrifoge tube. 11.5 Centrifoge for -1 0 minutes at -3000 rpm. 11.6 Condition the C n SPE cartridges (1 g, 6 m L) by passing 10 m L methanol followed by 5 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.7 Load (decant) the sample on the conditioned C n SPE cartridge. Discard eluate. 11.8 Elute w ith -5 m L 100% methanol. Collect 5 m L o f eluate into graduated 15 m L polypropylene centrifoge tubes (fin a l volume - 5 mL). 11.9 Analyze sim ples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Iqject the same amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included st the beginning and at the end o f a sample set Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f Pair 5 o ff Page 27 o f 65 Exygen Research Amendment Number 1 Page 71 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 BxygtaRMMBch Method Numtwr VQ0017B1 I ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by LC /M S/M S extracted calibration itandards may be injected at the beginning o f a act followed by extracted calibration itandards interspersed every 5*10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the firs t and lu t injection in a sample set 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using l/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.3 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance C riteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (309 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. I f a blank contains PFOA at levels greater than 30 ng/L, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes must be between 70*130% o f their known values. I f a control strike foils outside the acceptable lim its, the entire set o f samples should be re-extracted. Any m atrix spike outside 70 130% should be evaluated by the analyst to determine if re-extrection is 13.4 Any calibration standard found to be a statstica] outlier by using the Huge Error Test, may be excluded from the calculation o f die calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. 13.3 The correlation coefficient (R) for calibration curves generated must he 20.992 (R3 20.983). I f calibration results fa ll outside these lim ns, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than 4 % w ithin an w ly tic a l run. I f retention tim e d rift exceeds this lim it within in analytical run then die set must be reanalyzed. Page 6 o f 7 Page 28 o f 65 Exygen Research Amendment Number 1 Page 72 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 0 0 0 1 131 BxyitB IUnmcIi Mvthed Nwnbtr V000178I _______________________ ANALYTICAL METHOD_______________________ Method o f Analysis fo r the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by LC /M S/M S 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in n g /l, baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/L) - (Pm K TO IH tfflgpO * OF slope DF factor by which foe fin a l volume was diluted, i f necessary. 14.2 For samples fortifie d w ith known amounts o f PFOA pror io extraction, use th follow ing quation to calcolate th percent recovery. Recovery (% ) - [to ta l analyte found (ng/L) analyte found in control(ng/L)] analyte added (ng/L) 14.3 Use foe follow ing equation to convert the amount o f PFOA found in ng/L to ng/g(ppb). PFOA found (ppb) - (PFOA found <n/Ll x volume extracted f0.04L)l ample weight (5 g) 14.4 Use foe follow ing equation to calculate foe amount o f PFOA found in ppb based on dry weight. PFOA found (ppb) dry w eight" PFOA found (ppb) x [ 100% / total iolids(% )} Pag 7 of7 Page 29 o f 65 Exygen Research Amendment Number 1 Page 73 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: POOO1131 ANALYTICAL METHOD Method Number V0001782 M ethod o f A a alytis fo r the Determ ination o f Perffeerooetanolc A cid (PPO A) in Sediment by LC /M S/M S Analytical Testing Fecility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: c jL Paul Connolly 1 Technical Leader, LC-M S, Exygen Research io k h /a < r Date a J/K > / h i / /John Flaherty ' / Vice President, Operations, Exygen Research Date Exygen Research Amendment Number 1 Total Pages: 7 Page 30 o f 65 Page 74 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number; P 0 0 0 1 131 ExypaRawarch Method Number VOOOI782 I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perftuorooctanoic Acid (PFOA) in Sediment by LC /M S/M S 1.0 Scope This method is to be employed fo r the isolation and quantitation o f perfluorooclanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectromtrie Detector (LC/M S/M S) in aedhnent. 2.0 Safety 2.1 Always obeerve safe laboratory practice!. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety 3.0 Sample Requirement 3.1 A t least 30 g o ftest sample for extraction. 3.2 No sample processing is needed for sediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 A lt samples must be thoroughly m ixed before being sampled for extraction. 3.3 Sample collection procedures w ill be specified in the sampling plan for tins p ro je c t 4.0 Reagents and Standards 4.1 Water -H P L C grade 4.2 Methanol - HPLC grade 4.3 Acetic A cid - Reagent grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic A cid - Sigm a-Aldrich 3.0 Instrument and Equipment 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume injector capable o f injecting 3-200 jiL connected to a tandem Maas Spectrometer (LC/M S/M S). 3.2 A device to collect raw data for peak integration and quantitation. 3.3 Analytical balance capable o f reading to 0.00001 g. 3.4 $0 m L disposable polypropylene centriftige tubes. 5.3 15 m L disposable polypropylene centrifoge tubes. 5.6 Disposable m icropipets (50-100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC via l idt. 5.9 Disposable pipettes. 5.10 Autopipettei (100-1000 pL and 10-100 pL), w ith disposable tips. 5.11 Waters Sep Pak Vac 6 oc (Ig ) tC18 SPE cartridges. 5.12 SPE vacuum m anifold. Pag* 2 of 7 Page 31 o f 65 Exygen Research Amendment Number 1 Page 75 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 0 0 0 1 131 ExyfmRumrcb Method Number V0001782 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment bv LC /M S/M S ' 3.13 Vmtexer. $.14 W rist-action shaker. $.13 C entriftige capable o f (pinning 30 m L polypropylene tube at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone S cientific), 2.1 mm x 30 mm, 5p (P/N: 82303-032130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ): Methanol 6.3 Gradient Program: Time (m in) 0.0 1.0 8.0 20.0 22.3 ftA 63 63 23 23 63 Flow Rate (m L/m in) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: IS pL (can be increased to as much as 30 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative M RM mode, m onitoring 413 - * 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optim ize die MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.134 g o f ammonium acetate to 1000 m L o f water. Page 3 of 7 Page 32 o f 65 Exygen Research Amendment Number 1 Page 76 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No. : P0001131 E xygen Protocol Number: P 0 0 0 1131 Bxyfan Ri--icfc Mttkod Number V00017S2 I A N A LY T IC A L M ETH O D | Method o fAnalysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Sedimern by LC /M S/M S 8.2 Extraction Solution 8.2.1 1H acetic acid in water is prepared by adding 10 m L o f acetic acid to 1000 m L o fwater- Alternate volume may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f-1 0 0 jig /ra L o f PFOA by weighing 10 mg o f analytical standard (corrected fo r p u rity) and dilute to 100 mL with methanol in a 125-raL LDPE bottle. 9.1.2 A 10 p m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 100 ug/m L solution to a fin a l volume o f 100 w ith methanol in 123 m L LDPE bottle. 9.1.3 A 1.0 MgfaiL fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 10 pg/raL solution to a fin a l volume o f 100 w ith methanol in a 12$ m L LDPE bottle. 9.1.4 A 0.1 gg/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o fth e 1.0 p ^m L solution to a fin a l volume o f l 00 w ith methanol in a 12$ m L LDPE bottle. 9.1.5 A 0.01 pg/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 0.1 pgftnL solution to s final volume o f 100 with methanol in a 12$ m L LDPE bottle. 9.1.6 The stock end fortifica tion solutions are to be stored in e refrigerator at approximately 4*C and are stable fo r a maximum period o f 6 months from the date o fpreparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o ffoe 0.1 yg/m L fo rtifica tio n solution. 9.2.2 The follow ing is a typical example; addition] concentrations may be Concentration o f Fortification Solution (na/mL) 100 too 100 10 $ 2 Volume (ml.) 10 $ 2 10 10 10 Diluted to (mL) 100 100 too too 100 too Final Concentration (ne/m U 10.0 $0 20 10 0$ 02 Page * of 7 Page 33 o f 65 Exygen Research Amendment Number 1 Page 77 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xyg e n P ro to c o l N u m be r: P 0 001131 Exygea Raeparch Mfthed Number V000PI2 | A N A LY T IC A L M ETH O D | Method o fAnalysis fo r the Determination ofPerfluorooctanoic Acid (PFOA) in Sediment by LC /M S/M S 9.2.3 9.2.4 Store a ll calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to d*C* up to six months. Alternate volume# and concentration* o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one untreated control and two untreated controls fo rtifie d at known concentrations (lab control spike) to ve rify procedural recovery fo r the batch 10.2 Requirements for field and laboratory duplicate# end spike* w ill be specified in the quality aaeunneeplan fo r this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f sample into $0 m L polypropylene centrifuge tubes (fo rtify us needed, replace lid and m ix w ell). 11.2 Add 35 mLo f 1% acetic acid, cap, vortex and shake on a w rist action shaker fo r -6 0 minute#. 11.3 Centrifuge the tubes at -3000 rpm for -2 0 minutes. 11.4 Condition the C u SPE cartridges ( I g. 6 m L) by passing 10 m L methanol followed by 20 mLo f HPLC water ( - 2 drop/aec). Do not let column run dry 11.5 Load (decant) the sample on the conditioned C u SPE cartridge. Discard eiuate. 11.6 Add 20 m L o f methanol to the sediment le ft in the bottom o f the 50 mL ccntrifiige tube. Cap, vortex and shake on a w rist action shaker for -30 minutes. 11.7 CentrifUge the tubes at -3000 rpm for -2 0 minutes. 11.8 Decant the methanol onto the same SPE cartridge. C ollect the eiuate. 11.9 Wash the column w ith 4 m L o f mothanoL C ollect the eiuate and add it to the chute collected in step 11.8. 11.10 Condition a second C u SPE cartridge (1 g. 6 m L) by passing 10 m L methanol followed by 20 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.11 Add the methanol to -200 m L o f water and load on the second conditioned SPE cartridge. 11.12 Elute w ith -5 m L 100% m ethanol C ollect 5 m L o f eiuate into graduated 15 m L polypropylene centrifoge tube* (fin a l volume * 5 mL). 11.13 Analyze sunples using electroepray LC/MS/MS. Page 3 o i 7 Page 34 o f <S3 Exygen Research Amendment Number 1 Page 78 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 Exygen ReMarch Motfcod Number V00017 | A N A LY T IC A L M ETH O D [ Method o f Analysis fo r the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment bv LC /M S/M S 12.0 Chromatography 12.1 Inject the lim e amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 122 Standards o f PFOA corresponding to at least five o r more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included st the beginning and at the end o f a temple set. Standards must be interspersed between every 3*10 sample. As an alternative, an entire set o f calibration standards may be injected at foe beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account fo r a second set o f standards). In tid ie r case, calibration standard! must be the first and last injection in a sample set. 12.4 Use linear standard curvet for quantitation. Linear standard curves arc generated fo r the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3 3 (or equivalent) software system. 12.5 Sim ple response should not exceed standard responses. Any samples that exceed standard responses should be foither diluted snd reanalyzed. 13.0 Acceptance C riteria 13.1 Chromatogram must show a peak o f a daughter km at 369 amu from a parent o f 413 amu. The 413 arau parent corresponds to the PFOA anion, w hile the daughter km (369 amu) represents die loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. I f a blank contains PFOA at levels greater than 0.2 ng/m L, then a new blank sample must be obtained and the entire eel must be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes must be between 70-130% o f their known values. I f a control spike folia outside the acceptable lim its, the entire set o f samples should be re-extracted. Any m atrix spike outside 70 130% should be evaluated by the analyst to determine if ro-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards iiyected. 13.5 The correlation coefficient (R ) for calibration curves generated must be 20.992 (R2 20.925). I f calibration results fa ll outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. Page ef 7 P age 35 o f 65 Exygen Research Amendment Number 1 Page 79 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xyg e n P ro to c o l N um ber: P00011 i I Exyfta JlwMrch Motbod Nimbar VOOOI782 A N A LY T IC A L M ETH O D | Method o f Analysis fo r the Detenninatian o fPerfluorooctanoic A cid (PFOA) in Sediment by LC /M S/M S 13.6 Retention times between standards and samples must not d rift more than 4 H w ithin an analytical run. I f retention tim e d rift exceeds this lim it w ithin an analytical ran then the set must be reanalyzed. 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in ng/mL, baaed on peak area) uaing the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/m L) - ( T a k in - intercrop x DF slope DF " factor by which the final volume was diluted, i f necessary. 14.2 For saraples fortifie d w ith known amounts o f PFOA p rio r to exlraction, use th follow ing equation to calculate th percent recovery. Recovery (% ) - f total analyte found (ng/m L) analyte found in control (ng/m L)] ^ analyte added (ng/m L) 14.3 Use the follow ing equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). p f o a found (ppb) * [PFQA found ( n tta l) * fin d *olw ng (S m ill sample weight (3 g) 14.4 Use die follow ing equation ( If tteceasary) to calculate the amount o f PFOA found in ppb based on dry weight. PFO A found (ppb) dry w eight PFO A found (ppb) x [ 100% / total solids!*/.)) Ptge 7 of 7 P a g e 36 o f 65 Exygen Research Amendment Number 1 Page 80 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number; P 0 0 0 1 131 ANALYTICAL METHOD Method Number VOOOI783 M ethod o f A nalysis fo r th e D eten n i stio n o f P erfluoroocteoolc A cid (P F O A ) in Fish a id C lam i by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research D rive State College PA 16801 Approved By: v U f - J t ___ Paul Connolly I Technical Leader, LC-M S, Exygen Research Du fohn Flaherty Vice President, Operations, Exygen Research Date Exygen Research Amendment Number 1 Total Pages: 8 Page 37 o f 65 Page 81 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 B xy|n lU nueb Method Number VOOOI7I) A N A LY T IC A L m e t h o d ] Method o f Analysis fo r the Determination o fPerfluorooctanoic A cid (PFOA) in Fish and C lam i by LC/MS/MS 1.0 Scope Tins method is to be employed for the isolation and quantitation o f pertluorooclanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/M S) in fish and clams. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safely precautions. 3.0 Sample Requirement 3.1 A t least 20 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize w ith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublim ation. Seal and place the samples in frozen storage u n til time o f analysis. 3.3 Sample collection procedures w ill be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 4.2 4.3 4.4 4.3 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 W ater-H P LC grade Acetonitrile - HPLC grade Carbon (120-400 mesh) - Reagent grade Methanol - HPLC grade S ilica get (60-200 mesh) - Reagent grade F lo ris il (60-100 mesh) - Reagent grade Superclean LC -N H j - Reagent grade 1-Octanol - HPLC grade L-Ascotbic acid - Reagent grade Dim ethyldichlororilane - Reagent grade Toluene - Reagent grade Ammonium Acetate - A.C.S. Reagent Grade Perfluorooctanoic Acid - Sigm a-Aldrich 3.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume Injector capable o f injecting S-20O ML connected to a tandem M ast Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Page 2 o f8 Page 38 of 65 Exygen Research Amendment Number 1 Page 82 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 0 0 0 1 131 ExygcoReacwdi Mctfeod Number V0001783 ANALYTICAL METHOD Method o f A n a ly tii for the Determination o fPerfluorooctanoic Acid (PPOA) in Fish and Claras by LO M S/M S $.4 5.5 5.6 5.7 5.8 5.9 5.10 511 5.12 5.13 5.14 5.15 5.16 Rotary evaporator. Tissumizer. 125 m L pear-shaped flasks. 50 m L diapotable polypropylene centrifuge tubes. 15 m L disposable polypropylene centrifuge tubes. Disposable micropipets (50100uL, 100-200uL). 12S-mL LDPB narrow-mouth bottles. 2 mL clear HPLC via l k it. Disposable pipettes. Autopipettes <100*1000 pL and 10-100 pL), w ith disposable tips. SPE tubes (20m L) (Supelco cat. no. N057177). W rist action shaker. Centrifuge capable o f spimsng 50 m L polypropylene tube* at 2000 tpm. 6.0 Chromatographic System 6.1 Analytics] Column: Fluophasc RP (Keystone S cientific), 2.1 mm x 50 mm, 5m (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 a M Ammonium Acetate in Water 6.4 M obile Phase (B ): Methanol 6.5 Gradient Program: Time 0.0 Flow Rate * 4 % B fm L/m in) 65 35 0.3 1.0 65 35 0.3 8.0 25 75 0.3 20.0 25 75 0.3 22.5 65 35 0.3 6.6 Injection Volume: I S pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: <-23 minutes. The above conditions are intended as a guide and may be changed in order to optim ize the HPLC system. P it*) rs Page 39 o f 65 Exygen Research Amendment Number 1 Page 83 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen P ro to c o l N u m be r: P 0 001131 Bxygtn R w r c h Method Nunbfr VOOOI71} ANALYTICAL METHOD Method o fA nalytic for tbe Determination o fPerfhiorooctenoic A cid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: E lectrotpny Negative M RM mode, m onitoring 413 -> 369 mi? for PFOA. The above condition! are intended at a guide and may be changed in order to optim ize the MSMS syetem. 8.0 Preparation o f Solution! 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0.IS4 g o f ammonium acetate to 1000 m L o fwater. 8.2 Extraction Solution* 8.2.1 8.2.2 2% ascorbic acid in methanol U prepared by dissolving 2 g o f ascorbic add in 100 m L o f methanol. 30% O im etityldichlorDtitane in toluene it prepared by dingin g 3 mL o f dim efoyldichlorostlane to a Anal volume o f 10 m L w ith toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.3 Prepare a dock solution o f-100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected fo r purity) and dilute to 100 mL with methanol in a 125-raL LDPB bottle. A 1.0 jig fa iL fortifica tion solution o f PFOA ia prepared by bringing I m L o f foe 100 pg/raL solution to a final volume o f 100 w ith methanol in a 123 m L LDPE bottle. A 0.1 pg/m L fortifica tion solution ofP P O A is prepared by bringing Id m L o ftbe 1.0 pg/m L solution to a fin a l volume o f 100 w ith methanol in 125 mL LDPB bottle. A 0.01 pg/m L fortifica tion solution o f PFOA it prepared by bringing 10 mL o f the 0.1 jig /m L solution to a fin a l volume o f 100 with methanol In a 123 m L LDPE bottle. The stock and fortifica tion solutions are to be stored in a refrigerator at approximately 4"C and are stable for a maximum period o f 6 months from the date o f preparation. Page 4 ofC P a g e 40 o f 65 Exygen Research Amendment Number 1 Page 84 of 112 Interim Report #11- Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 0 0 0 1 131 Exygen Research Method Number VOOO1783 ANAL! T1CAL METHOD Method o fAnalysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Fish and Clams by LC/MS/MS 9.2 Standard Calibration Solution 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/m L fortifica tion solution. The follow ing is a typical example: additional concentrations may be prepared at needed. Concentration Final o f Fortification Volume Diluted to Concentration Solution (ut/raL ) (mL) (mL) Gl/mL) 1.0 9.0 100 0.05 1.0 2.5 1.0 lA 0.05 10 100 too 100 0.025 0.01 0.005 0.025 10 100 0.0025 0.1 10 100 0.005 to 100 0.001 0.0005 9.2.3 Store a ll calibration standards in 125-mL LDPE ninow -m outh bottles it 2*C to 6*C, up to six months. 9.2.4 Alternate volumes snd concentrations o f standards may be prepared as 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or le ts) must include at least one untreated control and two untreated controls fo rtifie d at known concentrations (lab control q iik o ) to ve rify procedural recovery fo r the batch. 10.2 Requirements fo r field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 Weigh 5 g o f frozen sample into 50 mL polypropylene centrifuge tubes (fo rtify as needed, replace lid and m ix w ell). 11.2 Add 30 m L o f acetonitrile and shake on a w rist action shaker for --IS minutes 11.3 Place the tubes in a freezer for -1 hour. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack die 20 m L SPE tubes in sequence w ith 2 g flo ris il, 2 g silica gel, 2 g carbon, and 1 g LC -N H j. Condition die columns w ith 20 m L o f methanol, then 20 m L o f acetonitrile. Discard a ll washes. Do not allow the column to dry. 11.6 Silanize the 125 m L pear-shaped flasks by rinsing w ith the 30% dim ethyldicblorosiline in toluene solution. Rinse the flask w ith toluene once, followed by methanol (three tim e*). D ry die flasks com pletely before use. either by air-drying or w ith a stream o f nitrogen. Page * ufH Page 4! o f 65 Exygen Research Amendment Number 1 Page 85 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 Exyfea Itmarch Motbod Number V0001783 | A N A LV T IC 'A l. M K TH O P [ Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 11.7 Centrifuge the 50 raL polypropylene tubes containing sample at -2000 rpm fo r-1 0 minutes. 11.8 Decani the extract on to a conditioned SPE column fitted inside the mouth o f the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape flask. 11.9 Add 10 m L o f acetonitrile to the sample in the SO m L centrifuge tube. Homogenize the frozen fat phase using a tiaaumizer for -3 0 seconds and rinse the tumuaizer w ith -1 0 m L o f acetonitrile into the tube. 11.10 Shake the sample again fo r-1 0 minutes on a w rist-action shaker. 11.11 Place the tubes in a freezer fo r- 1 hour more. 11.12 Centrifrige the 50 m L polypropylene tubes containing sample at -2000 rpm fo r-1 0 minutes. 11.13 Decant the extract onto the same SPE column. C ollect the eluate into the same pev-ahaped flask and combine w ith the eluent from the in itia l extraction. 11.14 Pass 20 m L o f acetonitrile through the SPE column and combine the eluate in the same pear-shaped flask. 11.15 Add 3-4 drops o f 1-octanol to die extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < *0C). 11.16 Make the Anal volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and sw irl to mix/diaaolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets, 11.18 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and ai the end o f a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5*10 samples (to account for a second set o f standards). In either case, calibration standards must be die firs t and last nveetton in a sample set 12.4 Use linear standard curves fo r quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. PtgebofS Page 42 o f 65 Exygen Research Amendment Number 1 Page 86 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 B xyy Rsmrch Mrtwd Number V0001783 [ ANALYTICAL METHOD ] Method o fAnalysis fo r the Determination o fPcrfhiorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted and reanalyzed 13.0 Acceptance C riteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA tnion , w hile the daughter ioo (369 amu) represents the lo ts o f carbon dioxide. 13.2 Method blanks must not oontain PFOA at levels greater than the LOO I f a blank contains PFOA at levels greater than 0.5 ppb, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f oootrol spikes and m atrix spikes must be between 70-130% o f their known values. I f a control qrike falls outside the acceptable lim its, the entire set o f samples should be re-extraded. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f die total number o f standards injected. 13.5 The correlation coefficient (R ) fo r calibration curves generated must be 0.992 (R2 0.985). I f calibration results fo il outside these lim its, then appropriate steps must be taken to adjust instrum ent operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention tunes between standards and samples must not d rift more than 4 % w ithin an analytical run. I f retention tim e d rift excoeds this lim it within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ngftnL) - (Peak area - intercept) slope 14.2 Use the follow ing equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) * IPPQA found fne/m L) x fin a l volume fm L) x DF1 sample weight (g) DF " factor by which the final volume was diluted, if necessary Pig* 7 of S Page 43 o f 65 Exygen Research Amendment Number 1 Page 87 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 Exygia Rtteafch Method Number VOOOl A N A L Y T IC A L m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For samples fortifie d w ith known amounts o f PFOA prior to extraction, use the follow ing equation to calculate the percent recovery. Recovery (% )- [ total analyte found (o g /g ) analyte found in control (ng/g)l ^ analyte added (ng/g) Exygen Research Amendment Number 1 Page I of 8 Page 44 o f 65 Page 88 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 ANALYTICAL METHOD Method Number: V0001784 M ethod o f Analysis fo r the Determ ination o f PerRuorooctauoic A cid (PFO A) in Vegetation by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research D rive State College, PA 16801 Approved By: ________ c--tL* Paul Connolly ' Technical Leader, LC-M S, Exygen Research ___iq J M Dale q / m f / * 4 Y ----------------------------------J o h n Flaherty ^ Vice President, Operations, Exygen Research ' J r Date Exygen Research Amendment Number 1 Total Page: 7 Page 45 o f 65 Page 89 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 Exyyea Rm m ic Ii Method Number V000178 A N A LY T IC A L m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope This method is lo b e employed fo r the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/M S) in vegetation. 2.0 Safety 2.1 Always obeerve ta le laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 20 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize w ith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow fo r carbon dioxide sublim ation. Seal and place the samples in frozen storage until time o f analysis. 3.3 Sample collection procedures w ill be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P LC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120*400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.3 Silica g e l(60*200mesh) -Reagent grade 4.6 F lo risll (60-100 mesh) -Reagent grade 4.7 Superclean LC -N H i - Reagent grade 4.8 l*O ctanol** HPLC grade 4.9 L*Aseorbic acid - Reagent grade 4.10 Dim ethyldichloroailane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic A cid - Sigma*Aldrich 5.0 Instrument and Equipment 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume injector capable o f injecting 3*200 pL connected to a tandem Maas Spectrometer (LC/M S/M S). 3.2 A device to collect raw data fo r peak integration and quantitation. 3.3 Analytical balance capable o f reading to 0.00001 g. Pag 2 of 7 Page 46 o f 65 Exygen Research Amendment Number 1 Page 90 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xyg e n P ro to c o l N u m b e r POOO1131 ExyynJUaNKli Mffbod Number V0001784 A N A LY T IC A L M ETH O D ....... I Method o f Analysis fo r the Determination ofPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5. 1 5.12 5.13 5.14 5.15 Rotary evqmrator. 12S m L pev-sbqm d flask. 50 m L disposable polypropylene centrifuge tubes. 15 m L disposable polypropylene centriluge tubes. Dupoaable micropipets (S0-100uL, 100-200uL). 125-roL LDPE narrow-mouth bottles. 2 m L clear KPLC v ia l k it. Dispoiabie pipettes. Autopipettes (100-1000 pL and 10-100 pL), w ith disposable tips. SPE tubes (20mL) (Supelco cat no. N0S7177). W rist action shaker. Centriftige capable o fspinning 50 m L polypropylene tubes st 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Ftuophase RP {Keystone S cientific), 2.1 nun x 50 mm. 5}i (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile P b iw (A ): 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ): Methanol 6.5 Gradient Program: Time (m ini 00 1.0 8.0 20.0 22.5 &A 65 65 25 25 65 Flow Rate X J (mL/min> 35 03 33 0.3 75 03 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak A rea-external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: BlectroeprayNegativeM R M m ode.m onitoring4 1 3 -*3 6 9 m /rfo r PFOA. Pige 3 of 7 Page 47 o f 6S Exygen Research Amendment Number 1 Page 91 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: POOO1131 Exypn Rwawch Method Number VOOU1784 I A N A LY T IC A L m e t h o d Method o f Analysis for the Determination o f P eriluoroocunoic A cid (PFOA) in Vegetation by LC/MS/MS The above conditions are intended aa a guide and may be changed in order to optim ize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol i t prepared by dissolving 2 g o f ascorbic add in 100 m L ofmethanoL 30% Dim ethyidichlorosilane in toluene ie prepared by bringing 3 mL o fdimethyldichlorosUane to a fin a l volume o f 10 m L w ith toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f ^100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected fix ' purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/m L fortifica tion solution o f PFOA is prepared by bringing I m L o f the 100 jig ta L solution to a fin a l volume o f 100 w ith methanol in a 125 m L LDPE bottle. A 0.1 pg/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o fth c 1.0 pg/raL solution to a fin a l volume o f 100 w ith methanol in a 125 m L LDPE bottle. A 0.01 ng/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 0.1 pg/m L solution to a fin a l volume o f 100 with methanol in a 125 mL LDPE bottle. The stock and fortifica tion aolutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/m L fortifica tion solution. Pa 4 1 ' Page 48 o f 65 Exygen Research Amendment Number 1 Page 92 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen P ro to c o l N u m be r: P 0 001131 Exy|tn Rmarch Mffbod Number V0QQ1784 [ a is a l y u c a l m e t h o d Method o f Analyse for the Determination ofPerfluoroocianoic Acid (PFOA) in Vegetation by LC/MS/MS 9.2.2 The follow ing ie a typical example: additional concentrations may be prepared as needed. Concentration Final ofFcrtifioation Volume Diluted to Concentration Solution (ua/mL) (mL) (mL) (M /m L) 1.0 3.0 too 0.03 1.0 2.5 100 0.02$ 1.0 1.0 100 0.01 0.05 10 100 0.005 0.025 10 100 0.0025 0.1 10 100 0.005 10 too 0.001 0.0003 9.2.3 Suae a ll calibration standards in 125-mL LDPE narrow-mouth bonks it 2*C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Bach batch o f samples extracted (typically 20 or le u ) must include et least one untreated control and two untreated controls fo rtifie d et known concentrations (lab control spike) to v e rily procedural recovery for the batch. 10.2 Requirements fin: field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into 50 m L polypropylene centrifuge tubes (fo rtify u needed, replace lid and m ix w ell). 11.2 Add 30 m L o f acetonitrile and shake on a w rist action shaker for -15 minutes. 11.3 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm fo r-1 0 minutes. 11.4 Pack and condition the SPE tubes and silanizc the pear-shaped flasks. 11.5 Pack the 20 m L SPE tubes in sequence w ith 2 g flo ris il, 2 g silica gel. 2 g carbon, and 1 g LC -N Hj. Condition the columns w ith 20 m L o f methanol, then 20 m L o f acetonitrile. Discard a ll washes. Do not allow the column to dry. 11.6 Silanize the 125 m L pear-shaped flask* by nnsing w ith the 30% dimethyldich)on>silane in toluene solution. Rinse the flask w ith toluene once, followed by methanol (three tim es). D ry the flasks com pletely before use. either by air-drying or w ith a stream o f nitrogen. 11.7 Decant the extract on to a conditioned SPE column fitted inside the mouth o f the pear-shaped flask. Collect the eluste in the 125 m L silanized pear-shape flask. Page J of ' Page 49 o f 65 Exygen Research Amendment Number 1 Page 93 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 ExyicaResearch Method Number V000I7*4 ANALYTICAL m ethod Method o f Analyst for the Determination o f P eriluorooctanoic A dd (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 m L o f acetonitrile to the sample in the SOm L centrifuge tube. 11.9 Shake the sample again fo r -1 0 minutes on a w rist-action shaker. 11.10 C entriftige the SO m L polypropylene tubes containing sample at -2000 ipm for -5 minutes. 11.11 Decant the extract onto the same SPE column. C ollect the eluate into the same pear-shaped flask and combine w ith the eluent from the in itia l extraction. 11.12 Repeat steps 11.8 through 11.11 again. 11.13 Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.14 Make the fin a l volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and sw irl to m ix/dissolve. 11.15 Transfer the extracts to HPLC vials using disposable pipets. 11.16 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed sample. 12.2 Standards o fPFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 3-10 samples (to account for a second set o f extracted standards). In either esse, extracted calibration standards must be the firs t and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves arc generated fo r tire analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.3 Sample response should not exceed standard responses. Any samples that exceed standard response* should be fiuther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from e parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, w hile the daughter ion (369 amu) represents the loss o f earbon dioxide. P**e 6 ofv Page 50 o f 65 Exygen Research Amendment Number 1 Page 94 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: POOO1131 Exy|w>R iiwreh Method Number V0Q0I7M | ........ A fS 'A LV T lC A i. M ETH O D | Method o f Analysis for the Determination o f Perfluoroocttnoic Acid (PFOA) in Vegetation by LC/MS/MS 13.2 Method blank* must not contain PFOA at levels greater thsn the LOQ. If a blank contains PFOA at levels greater than O.S ppb, then a new blank sample must be obtained and the entire aet must be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes must be between 7<M30% o f their known vsluos. I f a control spike falls outride the acceptable lim its, the entire set o f samples should be re-extracted. 13.4 Any calibration standard found to be a statistical o utlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards it\jected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R2 20.985). I f calibration results fa ll outside these lim its, then appropriate steps must be taken to adjust Instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than 4 % w ith in an analytical ran. I f retention tim e d rift exceeds this lim it within an analytical ran d im the set must be reanalyzed. 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in ng/mL, based on peak ares) using the standard curve (linear regression parameters) generated by the Masa Lynx software program: PFOA found (ngfaiL) - fPeak area - intercept) slope 14.2 Use the follow ing equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found (ppb) - fPFOA found (na/m U x Anal volume fm U x DF1 sample weight (g) DF - lector by which the final volume was diluted, i f necessary. 14J For samples fortifie d w ith known amounts o f PFOA prior to extraction, use die follow ing equation to calculate die percent recovery. Recovery (% ) * [to ta l analyte found (ng/g) - analyte found in control (ng/g)] analyte added (ng/g) Page 7 ><7 Page 5/ o f 65 Exygen Research Amendment Number 1 Page 95 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOti 1131 ANALYTICAL METHOD Method Number: VOOOI785 M ethod o f Analysis fo r the Determ ination o f Perfloorooeteoolc A d d (PFO A) In Smell Mamm a! L iv e r by LC /M S/M S Anelytleal Tearing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C -JiL ____ Paul Connolly I Technical Leader, LC-MS, Exygen Research ___ iQ 'i- i M Date M John Flaherty L / Vice President, Operations, Exygen Research Due Exygen Research Amendment Number 1 T o o l Paget: 7 Page 52 o f 65 Page 96 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 EsygenR m eeb Method Number V0001785 I AiSALYTlCAJL M E T H O D Method o fAnalysis for the Determination ofPerfluorooctanoic A cid (PFOA) in Small Mammal Liver by LC/MS/MS 1.0 Soope This method is to be employed fo r the isolation and quantitation ofperfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/M S/M S) in sm all mammal liver. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least Sg o f test sample for extraction. 3.2 Sample should he processed before extraction. Place the frozen sample in a food processor and homogenize w ith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow fo r carbon dioxide sublimation- Seal and place the samples m frozen storage u n til time o f analysis. Alternately i f there ie in insufficient amount o f sample (-less than S g), then no processing is necessary and the sample can be used as supplied. 3.3 Sample collection procedures w ill be specified in the sampling plan for this p ro je c t 4.0 Reagenta and Standards 4.1 Water-H P L C grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooetanoie A cid - Sigm a-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 3 solvents equipped w ith a variable volume injector capable o f injecting 5-2(Xj pL connected to a tandem M an Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 m L disposable polypropylene centrifoge tubes. 5.5 15 m L disposable polypropylene centrifoge tubes. 5.6 Disposable micropipets (SO-)OOuL, 100-200uL). 5.7 125-raL LDPB narrow-mouth bottles. 5.8 2 m L clear HPLC v ia lk it. Page 2 o r Page 53 o f 65 Exygen Research Amendment Number 1 Page 97 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 Exygea RMMitk Method Number VOOC)78$ | A N A LY T IC A L M ETH O D ..... Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Small Mammal Liver by LC/MS/MS 5.9 Disposable pipettes. 5.10 Autopipettes (100*1000 pL and 10*100 p L ), w ith disposable tips. 5.11 Waters Sep Pak Vac 6 c c (lg )tC )8 S P E cartridges. 5.12 SPE vacuum m anifold. 5.13 Tissuemizer. 5.14 W rist'Sction shaker. 5.15 Centrifuge capable o f spinning 15 m L polypropylene tubes at 3000 rpm. 5.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone S cientific), 2 .1 mm %$0 mm, 5p (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ) : Methanol 6.5 Gradient Program: Thne {m ini 0.0 1.0 8.0 20.0 22.5 SLA 65 65 25 25 65 Flow Rate 2 k fm L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: ~ 2 3 minutes. The above conditions a rt intended as a guide and may be changed in order to optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative M RM mode, m onitoring 4 )3 - 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optim ize the MSMS system. Page 3 o f ' Page 54 o f 65 Exygen Research Amendment Number 1 Page 98 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: POOO1131 Exygra Research Method Number V0Q0|7|j I AN,'ATICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PPOA) in Small Mammal Liver by LC/MS/MS 8.0 Preparation o f Solution! 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volume may be prepared. 9.0 Standard Preparation 9 .1 Standard Stock/Fortificalion Solution 9.1.1 Prepare a atock solution o f-100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/m L fortifica tion solution o f PFOA is prepared by bringing I m L o f the 100 pg/m L solution to a final volume o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 0.1 jig/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f foe 1.0 p ^m L solution to a fin a l volume o f 100 w ith methanol in a 123 m L LDPE bottle. 9.1.4 The stock and fortifica tion solutions are to be stored in a refrigerator at approximately 4*C and are stable fo r a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 922 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 pgfaiL fortifica tion solution. The follow ing is a typical example: additional concentrations may he prepared as needed. Concentration Final o f Fortification Volume Diluted to Concentration Solution (ni/m L) 100 100 too iroU 5.0 2.0 t.0 (m L ) 100 100 100 (na/mL) 50 2.0 10 5.0 to 100 2.0 10 100 1.0 10 100 0.5 0.2 0.1 9.2.3 Store a ll calibration standards in )25-m L LDPE narrow-mouth bottles at 2*C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. Plge 4 o f1 P a g e 55 o f 65 Exygen Research Amendment Number 1 Page 99 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 ExyjealtMMrefc Method Number VOOOI785 I A N A LY T IC A L M ETH O D Method o f Analysis fo r the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 10.0 Batch Set Up 10.1 Each batch o f aamplea extracted (typically 20 o r lees) must include at least one untreated control and two untreated controls fortifie d at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirements fo r field and laboratory duplicates and spikes w ill be specified in the quality assurance plan fo r this project. 11.0 Sample Extraction 11.1 Weigh 1 g o f sample into a 50 m L polypropylene centrifUge tubes (fo rtify as needed, replace lid and m ix w ell). Note that alternate weights o f liver may be measured depending on the sample sire available fo r use. 11.2 Add water to the sample for s fin a l volume o f 10 m L 11.3 Homogenize sample using a tissuemizer fo r-1 minute. 11.4 Transfer 1 m L o f die sample using a disposable pipette into a 15 mL disposable centrifUge tube. 11.5 Add 5 m L o f acetonitrile and shake fo r 20 minutes on a w rist-aclion shaker 11.6 Centrifuge die tubes at 3000 rpm for 5 minutes. 11.7 Decant die supernatant into a 50 m L disposable centrifUge tube and add 35 m L o f water. 11. Condition the C n SPE cartridges (1 g, 6 m L) by passing 10 mL methanol followed by 5 m L o f HPLC water ( 2 drop/soc). Do not let column run dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eluate. 11.10 Elute w ith 2 m L o f methanol. C ollect 2 m L o f eluate into a graduated 15 m L polypropylene centrifUge tube (fin a l volume 2 m L). . 11.11 Analyze samples using eloctrospray LC/MS/MS. 12.0 Chromatography 12.1 Iqject the same amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS syatem. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o fPFOA corresponding to at least five or more concentration levels must be included in in analytical set. 12.3 An entire set o fcalibration standards m u be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5- lu samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set follow ed by calibration standards interspersed every 5*10 samples (to account for seoond set o f standards), in either case, calibration standards must be the firs t and last injection in a sample set 12.4 Use linear standard curvet for quantitation. Linear standard curves are generated fo r the analyte by linear regression using 1/x weighting o fpeak area Page 5 of 7 Page 56 of 65 Exygen Research Amendment Number 1 Page 100 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1131 Exytm RmskcIi Mttfaod Number V00017tS | A N A LY T IC A L M ETH O D Method o f Analysis for the Determination ofPorfluorooctanoic A cid (PFOA) in Small Mammal Liver by LC/MS/MS versus calibration standard concentration using MassLynx 3.3 (or equivalent! software system. 12.3 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Amber diluted and reanalyzed 13.0 Acceptance C riteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. I f a blank contains PFOA at levels great dun 10 ng/g, then a new blank sample must be obtained and the entire set must be reextracted. 13.3 Recoveries o f control spikes in d m atrix spikes must be between 70* 130% o f their known valuei. I f a control spike Calls outside the acceptable lim its, the entire set o f samples should be re-extracted. Any m atrix spike outside 70 130% should be evaluated by the analyst to determine i f re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exoeed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R ) fo r calibration curves generated must be 20.992 (R2 20.983). I f calibration results ib ti outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards rad samples must not d rift more than 1 4 % w ithin an analytical run. I f retention tim e d rift exceeds this lim it within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters] generated by the Mass Lynx software program: PFOA foim d (ng ta L ) * fPeak area - intercept! x DF x aliquot factor slope DF " factor by which the final volume was diluted, i f necessary. A liquot fa c to r-10 Page 6 of 7 Page 57 o f 65 Exygen Research Amendment Number 1 Page 101 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: POOOI131 Bxygffd RoM rch Mtlhod Number VOOOP8J I a n a l y t ic a l m e t h o d Method o f A nalytic fo r the Determination o f Perfluorooctanoic A cid (PFOA) in Smalt Mammal Liver by LC/MS/MS M 2 For aamplee fortifie d w ith known amount* o f PFOA prior to extraction, use die follow ing equation to calculate the percent recovery. Recovery (% ) [to ta l analyte found (ngfoiL) - analyte found in control (ng/m L)] ^ analyte added (ng/m L) 14.3 Use the follow ing equation to convert the amount o f PFOA found in ng/m l 10 n tfg ip p b ). PFOA found (ppb) - [ffQA (toad (n ^rn L ) \ fin a l volume fm U l ample weight (g) Exygen Research Amendment Number 1 Pa 7 o f7 Page 58 o f 65 Page 102 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 ANALYTICAL METHOD Method Number VOOOI786 Method of Analysis for the Determination of Perttiiorooctanolc AeM (PFOA) to Smelt Mammal Serum by LC/MS/MS Analytical T o tin g Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: w t J , c j UL Paul Connolly 1 Technical Leader, LC-M S, Exygen Research Date JohnaFFllaahherty / ' VVice Preosidid<ent, Operations, Exygen Research Date Exygen Research Amendment Number 1 Total Pages: 7 Page 59 o f 65 Page 103 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: POOO1131 Exyitfl RMMTCh Method Number VOOG1796 I AW AX.V11CAL M &TM OP Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA ) in Small Mamma) Serum by LC/MS/MS 1.0 Scope This method ia to be employed fo r the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/M S) in email mammal serum. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical fo r proper safety precautions. 3.0 Sample Requirement 3.) A t least I m L o f test sample fo r tra ction. 3.2 No sample processing is needed for senun samplee. However, frozen serum samples mum to allowed to com pletely thaw to room temperature before use. 3.3 Sample collection procedures w ill be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Methanol - HPLC grade 4 3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.3 Perfluorooctanoic Acid - Sigm a-Aldrich 3.0 Instrument and Equipment 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/M S/M S). 3.2 A device to collect raw data for peak integration and quantitation. 3.3 Analytical balance capable o f reading to 0.00001 g. 5-4 30 m L disposable polypropylene centrifiige tubes. 3.3 13 m L disposable polypropylene centrifiige tube*. 3.6 Disposable m icropipets (50-lOOuL, 100-200uL). 3.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC via l kit, 3.9 Disposable pipettes. 3.10 Autopipettes (100-1000 pL and 10-100 pL), w ith disposable tips. 3.11 Waters Sep Pak Vac 6 cc(lg)tC 18S P E cartridges. 3.12 SPE vacuum manifold. 5.13 Vortexer. Pge2t>n Page 60 o f 65 Exygen Research Amendment Number 1 Page 104 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xyg en P ro to c o l N u m be r: P 0 001131 Biygra RmmikIi Method Number V000I786 I A iW V T lC A L M ETH O D Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 5.14 W rist-action ahiker. 5.15 C entriftige capable o f spinning 15 m L polypropylene tubes at 3000 rpm 6.0 Chromatographic System 6.1 Analytical Column: FluophaseRP (Keystone S cientific), 2.1 mm x SOmm. 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ): 2 mM Ammonium Acetate in W ster M obile Phase (B ) : Methanol Gradient Program: Flow Rate (m L/m rn^ 0.0 65 35 0.3 1.0 65 35 0.3 8.0 25 75 0.3 20.0 25 75 0.3 22.5 65 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL) 6.7 Quantitation: Peak A rea-external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order io optim ize foe HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative M RM mode, m onitoring 4 1 3 - 369 nv? for PFOA. The above conditions are intended as a guide and may be changed in order to optim ize the MSMS system. 8.0 Preparation o fSolutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f waiter. Alternate volumes may be prepared. Fag*3 of? Page 61 o f 63 Exygen Research Amendment Number Page 105 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xyg en P ro to c o l N um ber: POOO1131 E x y in Research Mtlbod Number VOOO1786 I ANALYTICAL METHOD Method o f Analysis for the Determination o fPerfluorooctanoic A cid (PFOA) in Small Mamma) Serum by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f-100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in 125-mL LDPE bottle. 9.1.2 A 1.0 pg/m L fortifica tion solution o f PFOA is prepared by bringing I m L o f the 100 pg/raL solution to a fin a l volume o f 100 w ith methanol in a 12S m L LDPE bottle. 9.1.3 A0.1 pg/m L fortifica tion solution ofPFO A is prepared by bringing 10 m L o f the 1.0 pg/m L solution to a fin a l volume o f 100 w ith methanol in 125 m L LDPE bottle. 9.1.4 The stock rad fortifica tion solutions are to be stored in a refrigerator it approximately 4*C and are stable fo r a maximum period o f 6 months from the thrte o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o fthe 0.1 pg/m L fortifica tion solution. The follow ing is a typical example: additional concentrations may be prepared as needed. Concentration Final o f Fortification Volume Diluted to Concentration Solution faa/mL) (mL) (n i) (n/mL) 100 5.0 100 5.0 too 2.0 100 2.0 100 1.0 100 1.0 S.O 10 100 0.5 2.0 10 100 0.2 1.0 10 100 01 9.2.3 Store a ll calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each b itch o f samples extracted (typically 20 or less) must include at least one untreated oontrol and two untreated controls fo rtifie d at known concentrations (lab control spike) to ve rify procedural recovery for the batch 10.2 Requirements fo r field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. Page 4 o f7 Page 62 o f 65 Exygen Research Amendment Number 1 Page 106 of 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P0001 ] 31 Exygeo Kmarcb Mtthod Number V0OOI786 I ANALYTICAL m e t h o d Method o f Analysis fo r the Determination o f Perfluoroocunoic A cid (PFOA) in Small Mammal Serum by LC/MS/MS 1 1.0 Sample Extraction 11.1 Meaeure 1 mL o f sample into a 50 mL polypropylene centrifuge tubes (fo n ify as needed, replace lid and m ix w ell). Note that alternate volumes o f serum may be measured depending on the sample size available for use. 11.2 Add water to the sample for a fin a l volurao o f 20 m L Cap tig h tly 11.3 Vortex fo r minute. 11.4 Transfer 1 m L o f the sample using a disposable pipette into s 13 mL disposable eentrifage tube. 11.5 Add 5 m L o facetonitrile and shake fo r -2 0 minutes on a w risl-action shaker. 11.6 CenariAige the tubes at -3000 rpm fo r -5 minutes. 11.7 Decant the supernatant into a 50 m L disposable centrifuge tube and add 35 m L o f water. U .ft Condition the C n SPE cartridges (1 g, 6 m L) by passing 10 m L methanol followed by 5 m L o f HPLC water ( - 2 drop/soc). Do not let column run dry 11.9 Load the sample on conditioned Cis SPE cartridge- Discard eluate. 11.10 Elute w ith -2 m L o f methanol. C ollect 2 m L o f eluate into a graduated 15 m L polypropylene eentrifage tube (fin a l volume - 2 m L). 11.11 Analyze samples using eloctrosprsy LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o fPFOA corresponding to at least five or more concentration levels must be included in an analytics) sat. 12.3 An entire set o f calibration standards must be included at the beginning and ai the end o f a sample set. Standards must be interspersed between every 5-1<) besamples. As an alternative, an entire set o f calibration standards may injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account fo r a second set o f standards)- In either ease, calibration standards must be the firs t and last injection in a sample set 12.4 Use linear standard curvea for quantitation. Unear standard curves are generated for the analyte by linear regression using l/x weighting o f peak area versus calibration standard concentration using MaasLynx 3.3 (or equivalen!) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be farther diluted and reanalyzed. Page5 o f? Page 63 o f 65 Exygen Research Amendment Number 1 Page 107 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: P 0 0 0 1 131 ExygM Rm ich Mvihod Number V000I786 I AWALVTlCALMirrHOP Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Small Mammal Serum by LC/MS/MS 13.0 Acceptance C riteria 13.1 Chromatogram must show a peak o f a daughter ion it 369 amu from a parent o f 413 amu. The 413 amu parent correspond* to the PFOA anion, while the daughter ion (369 amu) represents the Iocs o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. i f a blank contains PFOA at levels greater than 10 ng/m L, then a new blunk sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes must be between 70-130% o f their known values. I f a control spike fails outside the acceptable lim its, the entire aet o f samples should be re-extracted. Any m atrix pike outside 70 130% riio u ld be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o fthe total number o f standards injected. 13.3 The correlation coefficient (R) fo r calibration curves generated must he 20.992 (R* 20.983). I f calibration results fa ll outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant sot o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more ihun 1 4 % w ithin in analytical run. I f retention tim e d rift exceeds this lim it within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in ng/mL. baaed on peak area) using foe standard curve (linear regression parameters) generated by foe Mass Lynx software program: PFOA found fng/m L) (Peak area - intercept) x DF x aliquot factor elope DF " A ctor by which foe final volume was diluted, i f necessary. A liquot factor 20 14.2 For samples fortifie d w ith known amounts o f PFOA prior to extraction, use the follow ing equation to calculate foe percent recovery. Recovery (% ) * [ total analyte found (ng/m L) - analyte found in control (ng/m L)] m^^ analyte added (ng/m L) Pg{ 6 of 7 Page 64 o f 65 Exygen Research Amendment Number 1 Page 108 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 E xygen Protocol Number: POOO1131 Erew h m attll_____________________ __________ _____________Method Number VQOO1716 I a n a L v t ic a i. m e t h o d Method o f Analyaia fa t the Dweemlnatlon o fFerfluorooctanole Acid (PFOA ) in Small Mammal Seram by LC/MS/MS 14.3 Uee the follow ing equation to convert the amount o f PFOA found in ngmtL to PPb. PFOA found foObl - fPFOA found fnafm U i fin a l volume Im L l) (ample volume (m L) Exygen Research Amendment Number 1 Page 7 ot 7 Page 65 uj 65 Page 109 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment Num ber 1 Effective Date: 01/19/05 Exygen Study Number P0001131 Client Study Num ber. Page 1 of 1 None D ES C R IPTIO N O F AM ENDED S EC TIO N 1) Analytical Procedure Summary V0001780:Section 9.1 2 ) Verification o f Analytical Procedure AMENDED TO 1) Add to Section 9.1: Section 9 .1 .6 , Alternate weights of standards m ay be used to prepare alternate concentrations o f stock solutions as necessary. Alternate levels of fortification solutions may also be prepared. 2 ) Low and high spiking levels o f the analytes fo r each matrix m ay be altered depending on sam ple size available for extraction and/or to cover analyte concentrations expected in the sam ples. BATIQNALE 1 ) Higher concentrations of standards need to be prepared in order to spike the sam ple bottles at higher levels. 2 ) The sam ple size available for sm all mammal liver and serum w as sm aller than expected. Spiking at the pre-determ ined levels in the protocol puts the spiked concentration low er than the detection lim it Also, the analyte levels in the ground w ater sam ples are expected to greatly exceed the pre-determ ined spiking levels listed In the protocol. W hen the levels in the sam ples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the Q C sam ple. Higher spiking levels in the bottles will cover the analyte concentrations expected in the w ater sam ples. IM PA C T O N STU D Y T h e LO Q is 100 ng/g for a 0.1 g sam ple of small mam mal liver and is 1000 n g /m l for a 0.01 mL sam ple o f small mammal serum. Higher levels o f spiking for the w ater samples w ll ensure that more Q C recovery data can be used. LIBRARY ID: W0001226d- . Exygen Research Amendment Number 1 ADMI NI STRATI VE FORM Page 110 of 112 Interim Report #11 - Analysis o f Water, . Wastewater, and Sludge Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231 -1580 Am endm ent Num ber E ffective D ate: E xygen S tudy N um ber PROTOCOL AMENDMENT 2 0 3 /0 7 /0 5 P0001131 C lie n t S tu d y N um ber. Page 1 of 1 None D ESC RIPTIO N O F AM END ED S EC TIO N 1) Report, page 11 o f 65 2 ) T est M aterials, page 6 o f 65: PFO S transition monitored 499 -> 99. AMENPEPTQ 1) Instead o f one Anal report, Interim reports will be issued. 2 ) PFO S transition monitored may also be 499 > 80. RATIO NALE 1) Due to the excessive sizes o f the data sets, interim reports w ill be issued to allow the client to receive data in a tim elier mener-. <eur 2 ) The A PI 4 000 LC/M S/M S systems detect the 4 9 9 > 80 PFO S transition with greater sensitivity than the 4 9 9 -> 99 transition. IM PACT O N STU D Y 1) The cfient will be able to receive and review the data more quickly. 2 ) The 4 9 9 -> 80 transition can be detected with greater sensitivity; therefore, giving better chromatography. SponsorSlgn^ura (Ifrequired) LIBRARYID: VD00122M- Data Exygen QAU Review L l5 P ilo titi ADMINISTRATIYE FORM Exygen Research Amendment Number 1 Page 111 o f 112 Interim Report #11 - Analysis o f Water, Wastewater, and Sludge Samples '-2006 04:09pm From-WESTON SOLUTIONS Exygen Study No.: P0001131 T-152 P.0oj/001 F-l/8 RES Amendment Nu- iben Effective Date: xygen Study Number 3058 ResearchDrive Phone: 614-Z7Z-1039 StaneCollege, PA16801 Fa 814-231*1580 RCM T'-OCOL AMENDMENT p a g e l e r.1 12/01/05 P0011S1 Client Study Number _ _NA P^lF!T lO N O A M iPteL SE glM ` TestMaterials, page & of Protocol. 'ApembEp W An alternate lot ofPFOS m y usedtorthisstudy. ..... ftfiffNAlE The P F 0 8 used previously in this study (W number 217 horn SM ) ran out and #i was necessary to use a new lo t IMPACTbSsTUOT No negativa impact on study. 1 r 1114 " " " -*'** -- -- Q ^ / tJ .L' !S3y__________f / L / i b b ii hrfhclpel Utwis&galD.' Sigrraai, v o sr-- 1 * t s L RT D a- " jr-A jf^ J S S dSET*--------- UBRARYID: V003" 'BffMI r u . rM RECEIVED TIME 1RR. fi '28PM Bygen QAUInll/Dte T -I J jk lsS s. , ADMINISTRATIVE FORM PRINT TIME I MflR.21. 5:29PM