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INTERIM REPORT #26 - Analysis of Groundwater Samples 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 January 9, 2007 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: 118 Interim Report #26 - Analysis o f Groundwater 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. Ch Pri . ~ Exygen Research Jaisimha ,E Study Director Weston Solutions, Inc. M: Sp--------x --------3M Company Exygen Research Date Page 2 o f 118 Interim Report #26 - Analysis o f Groundwater 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 Study Director 45. Interim Report and Raw Data Review 12/28,29/06 01/09/07 01/09/07 01/09/07 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion of the study. This QA statement involves only the review of the interim report and associated raw data. Exygen Research Page 3 o f 118 Interim Report #26 - Analysis of Groundwater 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: Chas Simons Operations Manager Exygen Research Exygen Research Facility Management: Date Richard A. Gr Executive Directdr Analytical Sciences Exygen Research Study Director, Weston Solutions, Inc. Jaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: Michael A. Santpfo Director of Regulatory Affairs Date Date' Dat H ! n Exygen Research Page 4 o f 118 Interim Report #26 - Analysis o f Groundwater 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: Ground Water 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: 11/24/06 Interim Analytical Termination Date: 12/06/06 Interim Report Completion Date: 01/09/07 Exygen Research Page 5 of 118 Interim Report #26 - Analysis o f Groundwater 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 Chas Simons John Flaherty Karen Risha Amy Sheehan Mark Ammerman Mindy Cressley Ellen Dashem Chrissy Edwards Krista Gallant Title Operations Manager Vice President Laboratory Supervisor Associate Scientist Sample Custodian Technician Technician Technician Technician Exygen Research Page 6 of 118 Interim Report #26 - Analysis o f Groundwater 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 1.0 SUMMARY................................................................................................................11 2.0 OBJECTIVE...............................................................................................................11 3.0 INTRODUCTION.......................................................................................................11 4.0 ANALYTICAL TEST SAMPLES..............................................................................11 5.0 REFERENCE MATERIAL........................................................................................12 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................13 6.1 Extraction Procedure................................................................................................13 6.2 Preparation of Standards and Fortification Solutions...............................................13 6.3 Chromatography.......................................................................................................14 6.4 Instrument Sensitivity...............................................................................................14 6.5 Description of LC/MS/MS Instrument and Operating Conditions...........................14 6.6 Quantitation and Example Calculation.....................................................................15 7.0 EXPERIMENTAL DESIGN......................................................................................16 8.0 RESULTS...................................................................................................................17 9.0 CONCLUSIONS.........................................................................................................17 10.0 RETENTION OF DATA AND SAMPLES.............................................................17 Exygen Research Page 7 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Table I. Table n. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Groundwater Samples..................... 19 Matrix Spike Recovery of PFBS, PFHS and PFOS in Groundwater Samples.......................................................................................................... 20 Exygen Research Page 8 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 1. LIST OF FIGURES Page Typical Extracted Calibration Curve for PFBS in Reagent Water................ 22 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 23 Figure 3. PFBS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................... 24 Figure 4. Chromatogram Representing a Groundwater Sample Analyzed for PFBS (Exygen ID: C0222027, Data Set: 112406C)................................................ 25 Figure 5. Typical Extracted Calibration Curve for PFHS in Reagent Water................ 26 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 27 Figure 7. PFHS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................... 28 Figure 8. Chromatogram Representing a Groundwater Sample Analyzed for PFHS (Exygen ID: C0222027, Data Set: 112406C).................................................29 Figure 9. Typical Extracted Calibration Curve for PFOS in Reagent Water................ 30 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 31 Figure 11. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................... 32 Figure 12. Chromatogram Representing a Groundwater Sample Analyzed for PFOS (Exygen ID: C0222023, Data Set: 112406C)..................................................... 33 Exygen Research Page 9 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 LIST OF APPENDICES Page Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method V0001780, Protocol Amendments, and Deviations................................................................................................. 34 Exygen Research Page 10 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 1.0 SUMMARY Exygen Research extracted and analyzed groundwater samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001780 (Appendix A). The limit of quantitation (LOQ) for PFBS, PFHS and PFOS in ground water was 25 ng/L. Analytical results for the analysis of PFBS, PFHS and PFOS found in groundwater samples are summarized in Table I. Due to quality control failures, PFBS results for two samples are not reported (NR). Fortification recoveries for PFBS, PFHS and PFOS in the groundwater samples are detailed in Table II. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in the groundwater samples were 102 4%, 100 10%, and 103 7%, respectively. 2.0 OBJECTIVE The objective of the analytical part of this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) in ground water 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 groundwater using the analytical methods entitled, "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water 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 November 24, 2006, and the analytical termination date for this interim report was December 6,2006. 4.0 ANALYTICAL TEST SAMPLES Eleven groundwater samples (Exygen ID C0222023 - C0222033) were received on wet ice on November 21, 2006 from Tim Frinak at Weston Solutions, Inc. Eight groundwater samples represented two groundwater sampling sites and associated field quality control samples. Three water samples represented one trip blank Mid two trip blank spikes. All samples were logged in by Exygen personnel and placed in refrigerated storage. Exygen Research Page 11 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 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 (SP0008058) and PFHS (SP0008057), were supplied by 3M. PFBS (SP0008058) was received from 3M at Exygen on September 6,2006. PFHS (SP0008057) was received from 3M at Exygen on September 5, 2006. The analytical standard PFOS (SP0002694) was purchased from Fluka Corporation and was received at Exygen on April 23, 2003. The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated. Compound Exygen Inventory No. Lot# Purity (%) PFBS SP0008058 2 97.3 PFHS SP0008057 NB-120067-69 98.6 PFOS SP0002694 430180-1 101.2 The molecular structures of PFBS, PFHS and PFOS are given below: Expiration Date 01/17/07 10/18/07 10/31/07 PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4F9S0 3 'K+) Transitions Monitored: 299 -> 99 Structure: FF FF F SO 3 FF FF PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CeFnSOsTC*) Transitions Monitored: 399 -> 80 Structure: FF F F F F F SO3 FFF FFF Exygen Research Page 12 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CsFnSOs'K*) Transitions Monitored: 499 -> 80 Structure: FFFF FFFF FFFF FFFF 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical method "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" was used for the groundwater samples in this study. 6.1. Extraction Procedure 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 Preparation of Standards and Fortification Solutions A mixed stock standard solution of PFBS, PFHS and PFOS was prepared as specified in Exygen method V0001780. The stock standard solution was prepared at a concentration of 10,000 pg/mL by dissolving 1.0 g of each of the standards (corrected for purity and salt content, if necessary) in methanol. From these solutions, a 1000 pg/mL fortification standard solution was prepared by taking 10 mL of each stock and bringing the volume up to 100 mL with acetonitrile. By taking 10 mL of the 1000 pg/mL fortification standard and bringing the volume up to 100 mL with acetonitrile, a 100 pg/mL fortification standard was prepared. By taking 10 mL of the 100 pg/mL fortification standard and bringing the volume up to 100 mL with acetonitrile, 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 acetonitrile, 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 acetonitrile, a 0.1 pg/mL Exygen Research Page 13 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 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 acetonitrile, a 0.01 pg/mL fortification standard was prepared. A set of standards containing PFBS, PFHS and PFOS were 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. Calibration ! (ng/L) 0 25 50 100 250 500 1000 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.3 Chromatography Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~0.5 mins, -8.5 mins, and -11.1 mins, respectively. Peaks above the LOQ were not detected in any of the reagent blank samples corresponding to the analyte retention time. 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 25 ng/L of PFBS, PFHS and PFOS. 6.5 Description of LC/MS/MS Instrument and Operating Conditions Instrument: Interface: Computer: Software: API 4000 Biomolecular Mass Analyzer Turbo Ion Spray Liquid Introduction Interface DELL OptiPlex GX400 Windows NT, Analyst 1.4.1 Exygen Research Page 14 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: -30 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time (mini 0.0 1.0 8.0 10.0 11.0 18.0 %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Total run time: ~18min Flow Rate: 0.3 mL/min Ions monitored: Analvte PFBS PFHS PFOS Mode negative negative negative Transition Monitored 299 -> 99 399 - 80 499 -> 80 Approximate Retention Time (min) -0.5 min. ~8.5 min. -11.1 min. 6.6 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 seven concentrations of standards. The concentration was determined from the following equations. 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 Exygen Research Page 15 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessaiy. For samples fortified with known amounts of PFBS, PFHS and PFOS prior to extraction, Equation 2 was used to calculate the percent recovery. Equation 2: Recovery (%) = (analyte found (ng/L) - analyte in control (ng/L)) xl00% amount added (ng/L) An example of a calculation using an actual sample follows (calculation is for PFHS only): Ground water sample Exygen ID: C0222023 Spk C (Set: 112406C), fortified at 250 ng/L with PFHS where: peak area intercept slope dilution factor ng/L PFHS added (fort level) amt in corresponding sample = = = = = = 51377 0.000296 154 1 250 75.5 (Set: 112406C) From equation 1: Analyte found (ng/L) f5 1 3 7 7 -0.0002961 x 1 154 334 ng/L From equation 2: % Recovery (334 ng/L - 75.5 ng/L) x 100% 250 ng/L = 103 % NOTE: Numbers may differ slightly from raw data due to rounding. 7.0 EXPERIMENTAL DESIGN For 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. Exygen Research Page 16 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 The samples were extracted in one set. The set included one reagent blank, two reagent blanks fortified at known concentrations. The set also contained two sample sites and the field blank and the field blank spikes collected for the groundwater samples. For each site, a sample, a field duplicate and two-matrix field spikes were collected. For each site, a laboratory duplicate and two laboratory matrix spikes were also extracted. 8.0 RESULTS Analytical results and assessed accuracies for the analysis of PFBS, PFHS and PFOS found in groundwater samples are summarized in Table I. Accuracies were assessed for each sample by reviewing the individual quality control results obtained for each sample site. In most cases, there were two laboratory and two field spike recovery results available for each sample site that were used to assess the accuracy. In instances of failed laboratory or field spikes, recoveries associated with other spikes were used to assess sample accuracy. Quantitative results were obtained for all samples and analytes except for PFBS in two groundwater samples that are not reported (NR) due to quality control failures. Fortification recoveries for PFBS, PFHS, and PFOS in groundwater samples are detailed in Table II. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in ground water samples were 1024%, 10010%, and 1037%, respectively. 9.0 CONCLUSIONS Except as noted above, the groundwater samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001780. 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 Page 17 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 TABLES Exygen Research Page 18 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Groundwater Samples Exyqen ID Client Sample ID C0222023 C0222023 Rep C0222024 D A L -G W -R E S W 0 1-0-061117 DAL-GW -RESW 01-0-061117* D A L-G W -R ESW 01-D B -061117 C0222027 C0222027Rep C0222028 DAL-GW -RESW 02-0-061117 D AL-GW -RESW 02-0-061117* DAL-G W -R ESW 02-DB -061117 C0222031 DAL-G W -TR IP-0-061117 C4 Sulfonate PFBS P e rfluo rob utan esulfonate Analyte Found (ng/L) Assessed Accuracy (+/- %) NR . NR NR - NR NR NR ' ND 30 C6 Sulfonate PFHS P erfluQ rohB xanesulfonate Analyte Found (ng/L) Assessed Accuracy (+/- %) 75.5 83.3 83.3 30 30 30 764 30 754 30 778 30 ND 30 C8 Sulfonate PFOS P a rfluorooctanesu lfonata Analyte Found (ng/L) Assessed Accuracy (+/- %) 602 30 604 30 611 30 9270 7540 10800 30 30 30 ND 30 'Laboratory Duplicate ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures. Exygen Research Page 19 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Groundwater Samples Sample Description C4 Sulfonate PFBS____________ C6 Sulfonate PFHS____________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked In Sample Recovered Recovery In Sample Recovered Recovery In Sample Recovered Recovery (no/L) (ng/L.) (ng/L) (%) (nn/L) __ ("g'L)_____ (* ) (na/L) (na/L)_____ (%) DAL-GW -RSW 01-0-061117 (C0222023 Spk C, 2M ng/L Lab Spike) DAL-GW-RESW01-0-061117 (CQ222Q23 Spk D, S000 ng/L Lab Spike) DAL-GW-RESW01 -LS-061117 (C0222025,0.25 ppb Field Splice) DAL-GW -RESW 01-HS-061117 (C0222026,5 ppb Field Spike) 250 5000 250 5000 NR NR NR NR NR NR 75.5 335 104 NR NR NR NR NR 75.5 4940 97 602 5850 105 NR NR 75.5 374 119 NR NR NR NR NR 75.5 4790 94 602 6040 109 DAL-GW -RESW 02-0-061117 (C0222027 Spk E, 250 ng/L Lab Spike) DAL-GW -RESW 02-0-061117 (C0222027 Spk F, 5000 ng/L Lab Spike) D A L -G W -R E S W 0 2 -L S -0 6 1 117 (C0222020,0JS ppb Field Spike) D A L-G W -R E S W 02-H S -061117 (C02220M, 5 ppb Field Spike) 250 5000 250 5000 NR NR NR NR NR NR 764 920 . 9270 10900 NR NR 764 5240 90 9270 14300 101 NR NR 764 969 * 9270 7660 * NR NR 764 5130 87 9270 13800 91 DAL-GW-TRIP-LS-061117 (C0222032,0.25 ppb Field Spike) 250 NO 247 99 ND 264 106 ND 260 104 D A L -G W -T R IP -H S -0 6 1 117 (C0222033, 5 ppb Field Spike) 5000 ND 5210 104 ND 5040 101 ND 5410 108 Average: Standard D eviation: 102 4 Average: Standard D eviation: 100 10 Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated NO * Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR Not reported due to quality control failures. Note: Since th is sum m ary table show s rounded results, recovery values may vary s lig h tly from the values in the raw data. Average: Standard D eviation: 103 7 Exygen Research Page 20 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 21 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 1. Typical Extracted Calibration Curve for PFBS in Reagent Water 112400CR P700-1131 W jte r R rev2.idb (PFBS): "U n ji" Regression H I * weighting): y - 86.1 x + 357 ( i - 0 .9925) Area, counts Exygen Research Page 22 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively XC112406`0 PFBS (Standard) 299.0/99.bam* 6>9a* n ot found) 1 o f 34from 112406C.wiff 11.81 Intensity, cps Area: 253 5 counts H eigh t 1 .7 7 t+ 0 0 2 cps RT: 0.542 min intensity', cps Exygen Research Page 23 o f 118 Interim Report #26 - Analysis o f Groundwater 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 Intensity, cps : Intensity, cps I Reagent Control -PFBS (Unknown) 299.0/99,0anta -sample 9 o f 34from 112406C.wiff peak not foand) 220 1Hon.A4n0 2.50 ,, ,, .___ ft .0 8 0.57 Q.43vA 10A.30A,10^.82* 1- a3-A1VJ \ A 8 - ,,3o 2r . ,,15.77 10.71 1 2 3 4 5 0 7 B 0 10 11 12 13 14 15 10 17 Time, min Reagent Spk A - PFBS (QC) 290.0/00.0 am u-sam ple 10 o f3 4 fio m 112400C.miff Area: 5307 counts Height: 3.07e+002 cps RT: 0.550 min 0.50 .lx 2.48 5.00 0 l7--.8"0,0-r.1-5" ,I8...8--0 --10.`4--5 T 12.22 13.2^,13.50 ,,14.02 8 0 10 11 12 13 14 15 10 ____ _____ Time, min ____ Reagent Spk B - PFBS (QC) 200.0/00.0 amu - sample 11 of M fro m i l 2400CjAiiff Area: 54410 counis Height: 4.08e+003 cps RT: 0.547 min --I-- 17 0.55 I 1.53 I I I 1 I 8 0 10 11 12 13 14 15 10 17 Time, min Intensity, cps Exygen Research Page 24 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Groundwater Sample Analyzed for PFBS (Exygen ID: C0222027, Data Set: 112406C) C0222027 - PFBS (Uhhitowu) 299.0/99.0 HU ample 24o f 34 from 112496C.wiff A n a ; 37915 count Height: 2.47e+003c> RT: 9.549 min 0.55 Intensity, cps --t- --1-- **1----1" ~~i~ --r-- 4 0 10 11 12 13 14 15 18 17 Time, min Exygen Research Page 25 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 5. Typical Extracted Calibration Curve for PFHS in Reagent Water H i 12400 C P70O-1131 Water Res.rdb (PFHS): "Lineai" Regression f i /'^w )e iV h tinB ):y1 S 4x+ '^0.000208 ( r - 6:0004) Area, counts Exygen Research Page 26 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively | XC112400-0 - PFHS (Standard) 300.0/80.0 amu - sample 1 of 20 from 112408CR.miff (peak not found) 0.00 10.21 Time, min | XC1124M-1 -PFHS {Standard)399.0/90.0 amu -samit plie 23 o f 29 from 112406CR.wiff Area: 3924counts H eight f.63e+002cpr RT: 8L5#mia 150 uUQ). & 100c 2c so- 0 - -Q-- t- 8.S8 Exygen Research Page 27 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Figure 7. PFHS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively Intensity, cps Reagent Control - PFHS (Unknown) 39LMML0 ama -ample 9 o f 34 from 1124MC.wiff peak not found) i0.40 0.0 2.23 3.17 ia Aa /4 >1.30 0 -M- 7.04 A A 8 12.17 13Q1 0.12,0.44 11.02v 14.87 f/\A A p \ jU JV i l a/U 0 10 11 12 13 14 15 Time, min Reagent Spk A - PFHS (QC)300.0/BO.O emu - sample 10 of 34 from 112406C.iwiff Area: 7505 counts Height: 2.00e+002 cps RT:B.61 min V 16 17 8.61 Intensity, cps Intensity, cps Time, mm__ Reagent Spk B - PFHS(QC) 300.0/80.0 am u-sam ple 11 of 34 from 112406C.miff.... Area: 75576 counts Height: 2.Q2e+Q03 cps RT: 8.61 min 2000 0- 8.61 Time, min Exygen Research Page 28 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Groundwater Sample Analyzed for PFHS (Exygen ID: C0222027, Data Set: 112406C) Intensity, cps | CQ222027 - PFHS (Unknown) 39ft<V8aO ama -ample 24o f 34 from 112406C.wiff Atom: 117390count Heigt: 5.350+063 ep* RT: 9.63 mia 0.03 5000 4600 4000 3500 3000 2500 2000 1500 1000 500- Q- 12 3 45 07 1-- -- i-- -- r i I i 0 0 10 11 12 13 14 15 10 17 Time, min Exygen Research Page 29 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Figure 9. Typical Extracted Calibration Curve for PFOS in Reagent Water 1124O0C P7BQ-1131 Water Res.rdb (PFOS): "Linear11Regression f'1 /x? weighting): y = 122 x + -4.83e-005 ( r - 0.0007) Area, counts Exygen Research Page 30 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively I XC112406-0 - PFOS (Standard) 499-0/90,0 ama - sample 1 o f 34 from 112406C-wiff frealt aot foaad) 11.07 Intensity, cps Intensity, cps Time, min XC112400-1 - PFOS (Standard)4QQ 0/SG.0 amu - sampIe 2 of 34 from 112400C.roiff... Area: 2818 counts Height: 1.3Be+002 cps RT: 11.1 min 11.11 100 00 0 t- i 12 !-- --- i-- " " !---- - T~ -- r 345 07 Time, min Exygen Research Page 31 o f 118 l Intensity, cps Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Figure 11. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively i Reagent Control -PFOS (Unknown)499.tiMl.ilamu -ample 9 o f 34from ttZ496C.wiff peak not found) 100- 0.37 -- t-- --i -- e o io 11 12 13 14 15 15 17 Time, min Reagent Spk A - PFOS (QC)4OQ.0riBO.O amu - sample 10 of 34 from 112400C.wiff Area: 0030 counts Height: 3.21 e+002 cps RT: 11.1 min 10.02 --i-- -- i-- -- i" '1 8 0 10 11 12 13 14 15 10 17 Time, min Reagent Spk B - PFOS (00)400.0/80.0 am u-sam ple 11 of 34 from 112400C.miff Area: 01200 counts Height: 3.28e+003 cps RT: 11.1 min 11.10 Time, min I Intensity, cps Intensity, cps Exygen Research Page 32 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Groundwater Sample Analyzed for PFOS (Exygen ID: C0222023, Data Set: 112406C) I CO222023 - PFOS (Uakaowa) 499.0/90.0 aam -tam p! T6 o f 24 from 112406C,wiff A n a : 73346 coaat* Height: 3.65?+003 cp t RT: 11.1 atia 3500 11.11 3000 2500- Intensity, cps T im *, min Exygen Research Page 33 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods, Protocol Amendments, and Deviations Exygen Research Page 34 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soli, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 Perform ing Laboratory: Exygen Research 3058 R esearch Drive State College, PA 16801 Phone: (814) 272-1039 Sponsor Representative: M ichael A . Santoro D irector o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, M N 55144 Phone: (651) 733-6374 Page I J 6S Exygen Research Page 35 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 DISTRIBUTION: 1) Jaisim ha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 o f 65 Page 36 o f 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol NunVber: P0001131 PROTOCOL APPROVAL Study T itle: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small M ammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 APPROVALS JaisirohatKesan, S' Weston Solutions M ichael A. Sgntoro, Sponsor Representative 3M Comparfy lohn M. Flaherty, Prnincciippaal Investigator Exygen Research A /A mJ /. y ' Richard A. Gi Exygen Resi ident, Facility Management ad, Quality Assurance Unit Date Date PageJ o f 65 Exygen Research Page 37 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 TABLE OF CONTENTS TITLE PA G E .....................................................................................................................................................................1 D IS T R IB U T IO N ...............................................................................................................................................................2 PROTOCOL APPROVAL..............................................................................................................................................3 TABLE OF CONTENTS................................................................................................................................................4 INTRODUCTION............................................................................................................................................................ 5 TEST M A TERIA LS........................................................................................................................................................5 OBJECTIV E..................................................................................................................................................................... 6 TESTING FACILITY......................................................................................................................................................i> STUDY DIRECTOR........................................................................................................................................................7 SPONSOR REPRESENTATIVE................................................................................................................................... 7 PRINCIPAL INVESTIGATOR..................................................................................................................................... 7 PROPOSED EXPERIMENTAL START AND TERMINATION D A TE S........................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SY ST EM ................................................................8 SAMPLE PROCUREMENT, RECEIPT AND RETENTION..................................................................................8 SAMPLE IDENTIFICATION......................................................................................................................................9 ANALYTICAL PROCEDURE SUMMARY.............................................................................................................. 9 VERIFICATION OF ANALYTICAL PROCEDURE................................................................................................9 METHOD FOR CONTROL OF B IA S..........................................................................................................................11 STATISTICAL M ETH O D S........................................................................................................................................... 11 GLP STA TEM EN T..........................................................................................................................................................H R EPO R T.............................................................................................................................................................................11 SAFETY AND H EA LTH ................................................................................................................................................12 AMENDMENTS TO PROTOCOL............................................................................................................................... 13 DATA RECORD K EE PIN G .......................................................................................................................................... 13 QUALITY A SSURANCE............................................................................................................................................... RETENTION OF DATA AND ARCHIVING.............................................................................................................M APPENDIX I, ANALYTICAL M ETHODS.................................................................................................................15 Page 4 of 65 Exygen Research Page 38 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000113I INTRODUCTION The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), periluorohexanesulfonate (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), periluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M. PFBS Chemical Name: Perfluorobutanesulfonate M olecular W eight: 338 supplied as the potassium salt (CgFgSCVK*) Lot Number: 101 Purity: 96.7% Transitions Monitored: 2 9 9 -* 99 Structure: PFHS Chemical Name: Periluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFuSOrTC*) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 -> 80 Structure: Page 5 o f 65 Exygen Research Page 39 o f 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate M olecular Weight: 538 supplied as the potassium salt (C 8F |7S03'K +) Lot N um ber 217 Purity: 86.9% Transitions Monitored: 499 99 Structure: FFF FFFF F S03" 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 M onitoring 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 Page 40 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDY DIRECTOR Jaisimha Kesari P.E., DEE W eston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax:(610)701-7401 j .kesari@ westonsolutions.com SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, M N 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M. Flaherty Exygen Research 3058 Research Drive State 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 Page 41 o f 118- Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 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 W eston 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 W ork Plan for Sampling Environmental M edia." 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 sam ples will be stored frozen at : -10C. Sm all m am m al w hole blood samples will be centrifuged in the field at the time o f collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at S -10C. 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 Page 42 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol Number: POOOI131 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 W ater 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" V000I784: "M ethod 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 w ater 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 of65 Exygen Research Page 43 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 collection at the testing facility and have been shown to be free o f PFBS, PFHS and PFOS. Samples w ill be added to each container to a volum etric fill line at 200 mL. A field duplicate, a low field spike and a high field spike o f each 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 (l3C PFOA). PFO A and l3C PFOA are included in the solutions used to spike the samples. The results for PFOA and l3C PFO A will not be reported in this study. Exygen w ill supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. A t 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 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. For small mammal liver, Exygen will supply a 50 mL polypropylene centrifuge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with 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 W ater 500ng/L 5000 ng/L Soil 4ng/g 40 ng/g Sediment Fish 4 ne/a lO ng/g 40 na/a 100 ng/g Clams lO ng/g 100 ng/g Vegetation 10 ng/g 100 ng/g Small Mammal Liver 10ng/g 100 ng/g Small Mammal Serum lO ng/m L lOOng/mL Page Oaf6$ Exygen Research Page 44 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 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 of 63 Exygen Research Page 45 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 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. I f the method is photo-reduced, the project number and page number must be included on each page. Description o f the instrumentation used and operating conditions. All results from all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level. Representative chromatograms for each analyte in each matrix, including chromatograms o f a standard and a control sample, and a chromatogram at a fortification level. The location o f the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity o f the data will be documented in the report. Locations where raw data and the final report are to be archived. Additions or corrections to the final report shall be in the form o f an amendment signed by the Study Director. The amendment shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Representative. All applicable requirements for reporting o f study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel will practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environment to the test or reference substancefs). Page 12 o f 65 Exygen Research Page 46 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 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- A ll chromatograms w ill contain the following: Sample identification, injection date, arrow o r 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 Page 47 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 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 will inspect the study at intervals adequate to assure compliance w ith G LP's, and w ill 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 w ith the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period o f time specified in 40 CFR 792.195. An exact copy o f the materials submitted to the study director will also be kept at Exygen Research. Exygen will obtain permission from the study director before discarding or returning samples. Exygen Research Page 4 o f 65 Page 48 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 APPENDIX I ANALYTICAL METHODS V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in W ater by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: `M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: `M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V 0001784: `M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V000178S: `M ethod o f Analysis for the D eterm ination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" Exygen Research Page IS of 6S Page 49 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol Number; P0001131 ANALYTICAL METHOD Method Number: V0001780 Method of Analysis for the Determination o f Perflaorooctanoic Acid (PFOA) in Woter by LC/MS/MS Analytical Totting Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: i - l L ______ Paul Connolly 1 Technical Leader, LC-MS, Exygen Research 3L y /orJohhnnFFlalahhierty / VVicMe PDmresiident, Operations, Exygen Research toltwAW Date Exygen Research Total Pages: 7 Page 6 o f 65 Page 50 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 S "* ExygcsRMMKfe Method Number VOOOPBO ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid <PFOA) in W ater by L C /M S/M S 1.0 Scope This method is to be employed for the iaolation and quantitation o f perfluorooctanoic a d d by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in water. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult die appropriate MSDS before handling any chemical for proper safely precauti(ms. 3.0 Sample Requirement 3.1 At least 40 m L o f test sample for extraction. 3.2 No sample processing is needed for water samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction. 3.3 Any samples containing particles should be centrifuged at -3 0 0 0 rpm for -5 minutes and the supernatant used for the extraction. 3.6 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich S.O Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5*200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene eentriflige tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50*100uL, 100-200uL). 5.7 12S*mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettaa (100*1000 pL and 10-100 pL), w ith disposable tips. 5.11 Waters Sep Pak Vac 6 c c (lg )tC 1 8 S P E cartridges. Page 2 o n Page 17 of65 Exygen Research Page 51 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygn&nMicb Mbod Numb V00017*0 L ]ANALYTICAL m e t h o d ________________________ Method o f A ntlyiie for the Dtermination o f Perfluorooctmoic Acid (PFOA) in W ater by L C /M S/M S 5.12 SPB vacuum manifold. 5.13 Ceotriftigo capable o f spuming 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5^ (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ): 2 mM Ammonium Acetate in W ater 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time iminl 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate fmL/minl 3$ 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 p L (c m be increased to as much as 50 pL). 6.7 Quantitation: Peak A n a -e x te rn a l standard calibration curve. 6.8 RunTim e: * 2 3 minutes. The above conditions ere intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Etcctrosprsy Negative MRM m ode, monitoring 413 -> 369 nv^z. The above are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared b y adding 0.154 g o f ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. Fayeol Page IS o/ 65 Exygen Research Page 52 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E xygR *m rck Method Number V0001780 ANALYTICAL M ETH O D ___________________________ Method o f Analysis far the Determination o f Periluorooctanoic Acid (PFOA) in Water by L C /M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f - 1 0 0 p g /m L o f PFOA by weighing 10 mg o f analytical atandard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPB bottle. 9.1.2 A 10 pt/m L fortification solution o f PFOA if prepared by bringing 10 m L o f the 100 pgrinL solution to a final volume o f 100 with methanol in a 12S mL LDPB bottle. 9.1.3 A 1.0 p ^ m L fortification solution o f PFOA ta prepared by bringing 10 m L o f the 10 pg/mL solution to a final volume o f 100 with methanol in * 125 m L LDPB bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA i t prepared by bringing 10 m L o fth e 1.0 pg/raL solution to a final volume o f 100 with methanol in a 125 m L LDPB bottle. 9.1.5 A 0.01 pgfaiL fortification solution o f PFOA is prepared by bringing 10 m L o f tite 0.1 pg/m L solution to a final volume o f 100 with methanol in a 125 mL LDPB bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. 92 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 die extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared aa needed. Final Concentration Fortification Volume of Concentration of Calibration o f Fortification Volume Fortified Control Calibration Standard ID Solution (nob) fuL) Samolo (mL) Standard (tnx>* (examolel 0 0 40 0 XCnsoddyy-0 10 100 40 10 200 40 10 400 40 100 too 40 25 XCnunddyyl 50 XCnunddyy2 100 XCnunddyy-3 250 XCmmddyy-4 100 200 40 500 XCmmddyy-5 100 400 40 1000 XCmmddw-6 * The extracted concentration o f the calibration standard is equal to 8 k its initial concentration, due to the concentration o f the standard during the extraction (SPC). XC extracted calibration standard. PtS 4 o f' Page 19 o f 65 Exygen Research Page 53 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 BxygMRNMicfc M*tb4 Number VOOOI7SO ANALYTICAL METHOD Method o f A n a ly st for the Determination o f Pcrihiorooctanotc Acid (PFOA) in Water by L C /M S /M S 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent b lin k ) m u it be prepared with each aet o f standards extracted. Stare all 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 m ay be prepared as 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 HPLC water) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in foe quality assurance plan for this project. 11.0 Sample Extraction 11.1 Measure 40 m L o f sample o r a portion o f sample diluted to 40 m L with water into 50 m L polypropylene ecntriftige tubes (fortify as needed, replace lid and mix well). 11.2 Condition foe C SPE cartridge (I g, 6 mL) by passing 10 mL methanol followed by 5 m L o f HPLC water (~ 2 drop/sec). Do not let column run dry 113 Load sample on conditioned C n SPE cartridge. Discard eluate. 11.4 Elute with - 5 m L 100% m ethanol Collect 5 m L o f eluate into graduated 15 m L polypropylene centrifoge tubes (final volume - 5 mL). 11.5 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the fam e amount o f each standard, sample and fortified sample into the LO M S/M S system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels must be included in an analytical set. 12.3 A n entire aet o f extracted calibration standards m ust be included at the beginning and at the end o f a sample aet. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the first and last injection in s sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for foe analyte by linear regression using 1/x weighting o f peak area Pag* 5 of 7 Page 20 o f 65 Exygen Research Page 54 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 ExypaB w irrh Method Number V0001780 | ANALYTICAL M ETHOD .... | Method o f Analysis fix'the Determination ofPerfluorooctsnoic Acid (PFOA) in Water by L C /M S/M S versus calibration standard concentration using MassLynx 3.3 (or equivalcni) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be lUrther diluted and reanalyzed. 13.0 Acceptance Criteria 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 (he daughter ion (369 amu) representa the lota o f carbon dioxide. 13.2 Method blank must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank am p le must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130V o f their known values. I f a control spike fiUls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraclion is warranted. 13.4 Any calibration standard found to be a statistical outlier 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 die total num ber o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (Ra 20.985). I f calibration results foil outside these limits, 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 and samples must not drift more than 1 4 % within an analytical run. I f retention time drift exceeds this limit within an analytical run then foe set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate foe amount o f PFOA found (in ng/L, based on peak area) using foe standard curve (linear regression parameters) generated by the M an Lynx software program: PFOA found (ng/L) - (Peak area - intercept) x DF slope DF - Actor by which foe final volume was diluted, if necessary. Paat6on Page 21 of65 Exygen Research Page 55 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 Exygas RttNKfe H td to d Nicobar VOOOI710 I ANALVT1CAL M ETHOD I Method o f A nalyni for the Determination o f Perfluorooctanoic Acid (PFOA) in W ater by L C /M S/M S \4 2 For samplea fortified with known amount* o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [ total analyte found (ng/L) analyte found in control (ng/L)] i[|Q^ analyte added (ng/L) Exygen Research Page 7 o f7 Page 22 of 65 Page 56 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method N um ber V0001781 Method of Aaalyaia forth Determination o f Ferflaorooctnneic Acid (PFOA) lit Soil by LC/MS/MS Analytic! Tooting Facility: Exygen Research 3058 Reaearch Drive Stato College, PA 16801 Approved By: T tA . C J i .____ Paul Connolly Technical Leader, LC-MS, Exygen Reaearch Due J6bti Flaherty ' / Vice President, O perations Exygen Reaearch Date Exygen Research Total Pagea: 7 Page 23 o f6$ Page 57 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Eiyj Ramtck Method Number V0001781 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctaooic Acid (PFOA) in Soil by LC/MS/MS ] 1.0 Scope ThU method is to be employed for the isolation and quantitation o f perfluorooctinaie a d d by High Performance liq u id Chromatography coupled to a tandem Mass Spettrometrie D e te ct (LC/MS/MS) in soil. 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 At leest IS g o f test sample for extraction. 3.2 N o sample processing is needed for soil sample*. 3.3 Samples stored refrigerated should h e allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluoroottenoie A dd - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high perfotmance liquid chromatograph capable o f pumping up to 2 solvente equipped w ith a variable volume injector capable o f injecting 5-200 pL connected to a tandem Maas Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centriftige tubes. 5.6 Dispostble micropipets (SO-lOOuL, 10G-200uL). $.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L d e a r HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep P tk Vac 6 cc (ig ) tC IS SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. HfC2 of 7 Page 24 o f 65 Exygen Research Page 58 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygoa Rmurch Mttfcod Nwobtr V0001781 ANALYTICAL MKTHOP Method o f Analysis for the Determinstion ofPerfluorooctinotc Acid (PFOA) in Soil by L C /M S/M S 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 50 m L polypropylene tubes al 5000rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x SO mm. 5m (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A ): 2 mM Ammonium Acetate in W ater 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Tima fminl 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate 2LB fmL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 |iL (can be increased to as m uch 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 a id may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Blectrospray Negative MRM mode, monitoring 413 -+ 369 m/z for PFOA. The above conditions are intended a t a guide and m ay be changed in order to optimize die MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water i< prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. PapJon Page 25 o f 65 Exygen Research Page 59 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol Number: POOOl 131 Exygea Rnaaich Method N um ber VOOO1781 ANALYTICAL METHOD Method o f Analysis for the Determination o f Pcrfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/raL 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 10 p g fa L fortification solution o f PFOA is prepared by bringing 1() mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.3 A 1.0 pg/mL fortification eolutionofPFO A is prepared by bringing 10 mL o f tiie 10 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.4 A 0 .1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f |h e 1.0 p g ta L solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from tiie 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 proceeeed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Concentration ofFoitifioation Solution (nob) 0 10 10 10 100 100 100 Fortification Volume of Volume Fortified Control (pL) SamdefmL) 0 40 100 40 200 40 400 40 100 40 200 40 400 40 Final Concentration o f Calibration Standard loot)* 0 25 50 100 250 500 1000 Calibration Standard ID fexamole) XCmmddyy-0 XCjTunddyy-l XCmmddyy-2 XCmmddyy-3 XCmmddyy-4 XCmmddyy-5 XCmmddvvO * The extracted concentration o f the calibration standard i t equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC extracted calibration standard. Pag4 o f7 Page 26 o f 65 Exygen Research Page 60 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Mttbod N i n t e VOOO1711 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) m ust be prepared with each set o f standards extracted. Store all extracted calibration standards in 13-mL polypropylene lubes at 2*C to 6*C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r leas) must include at least ooc reagent control (method blank using S m L o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to verity procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 3 g o f sample into 50 m L polypropylene centrifuge tubes (fortify as 1U needed, replace lid and mix well). Add 3 m L o f methanol and shake on a wrist action shaker for -1 5 minutes. 11J Transfer the tubes to an ultrasonic hath and sonicate for ~ l 5 minutes. 11.4 Bring the volume up to 40 m L with water in the SO m L polypropylene centrifbge tube. ^ 11.3 Ceotrifoge fo r- 1 0 minutes at -3 0 0 0 ipm. 11.6 Condition the Cis SPE cartridges (1 g. 6 m L) b y passing 10 mL 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 u SPE cartridge. Discard 11.8 Elute with - 5 mL 100% m ethanol Collect 5 m L o f eluate into graduated 15 mL polypropylene centrifUge tubes (final volume " 5 mL). 11.9 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Iqject the -- n e amount o f each standard, sample and fortified 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 m ore concentration levels must be Included in an analytics] set. 12.3 An entire set o f extracted calibration standards must b e 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 Pag5 of7 Page 27 o f 65 Exygen Research Page 61 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyges RMearch M ethod Number V0001711 ANALYTICAL METHOD ] Method o f Analytic for the Determination o f Perfluorooctanoie Aoid (PFOA) in Soil by L C /M S/M S extracted calibration standards m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5*10 samples (to account for a second set o f extracted standards), h i either case, extracted calibration standards must be die tin t and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standaid curves are generated for die analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MaaaLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram m ust 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. If a blank contains PFOA at levels greater than SO ng/L, then a new blank sample must be obtained and the entire set must be re-extraeted. 13.3 Recoveries o f control spikes and matrix spikes must b e between 70-130% o f their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine i f re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier b y using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total num ber o f extracted calibration standards that could be excluded must not exceed 20% o f the total num ber o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R2 20.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples m ust not drift more than 1 4 % within an analytical nm . I f retention tim e drift exceeds this limit within an analytical run then the eet must be reanalyzed. p*t* Page 28 of 65 Exygen Research Page 62 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 Exygea RMMfch Method N u a b VOOO17I ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 14.0 Calculation! 1A 1 Use the following equation to calculate the amount o f PFOA found (in ng/L, baaed on peak a n a ) using the standard curve (linear regression parameters) generated by foe M ail Lynx software program: PFOA found (ng/L) slope xDF DF - factor by which foe final volume w as diluted, i f necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use foe following equation to calculate foe percent recovery. Recovery (% ) [ total analyte found (ng/L) - analyte found in control (ng/L)] analyte added (n ^L ) 14.3 Use foe following equation to convert foe amount o f PFOA found in ng/L to ttg /| (ppb). PFOA found (ppb) - (PFOA found fna/L) x volume extracted (0.04D1 sample weight (5 g) 14.4 Use foe following equation to calculate the amount o f PFOA found in ppb based on dry weight PFOA found (ppb) dry weight PFOA found (ppb) x [100% / total iolids(% Pag*7or7 Page 29 o f65 Exygen Research Page 63 of 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method N um ber V0001782 Method o f Analysis for the D eferlaitlon of Perflaoreoctaaoic Add (PFOA) lo Sediment by LC/MS/MS Analytical Testing Facility: Exygen Research 3038 Research Drive State College, PA 1680! Approved By: c j L ____ Paul Connolly I Technical Leader, LC-MS, Exygen Research &tAa(ohn Flaherty / Vice President, Operations, Exygen R esearch __iSkbM. Date Due Exygen Research Total Page: 7 Page 30 o f 65 Page 64 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 B*yan Rnewch Method Number VOQO1782 j ANALYTICAL M ETHOD | Method o f Analyst for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 1.0 Scope This method is to be employed for die isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometrie Deteetor (LC/MS/MS) in sedim ent 2.0 Safety 2.1 Always observe sale laboratory practices. 2 2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 30 g o f test sample for extraction. 3 2 N o sample processing ia needed for sediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly m ixed before b a n g sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - HPLC grade 4.3 Acetic A d d -R eag en t grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic A d d - Sigma-Aldrich $.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 iqjeetor capable o f injecting 5*200 pL connected to a tandem Maas Spectrometer (LC/MS/MS). 5.2 A device to co lled raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifiige tubes. 5.5 15 m L disposable polypropylene centrifiige tubes. 5.6 Disposable micropipets (50-lOOuL, 100-200uL). 5.7 125*mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipette. 5.10 Autopipcttes (100-1000 pL and 10*100 pL), with disposable tips. 5.11 Waters Sep Pak Vsc 6 cc ( lg ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. Pag 2 o f7 Page 31 o f 65 Exygen Research Page 65 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E iyinldM cb Method Number VOOOI7I2 ANALYTICAL M ETHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Sediment bv L C /M S/M S 5.13 Vortexer. 5.14 Wrist-action shaker. 5.15 CentriAige capable o f spinning 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase HP (Keystone Scientific), 2.1 nun x 50m m . 5p (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water Mobile Phase (B) : Methanol ( M i n t Program: Tima fminl 0.0 1.0 8.0 20.0 22.5 2 tA 65 65 25 25 65 Flow Rate fmL/minl 35 0.3 33 0.3 75 0.3 75 0.3 35 0.3 6.6 iqjectioo Volume: 15 p L (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as t guide and m ay b e changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: EtectrOsprayNegativeMRM m ode, monitoring 413 369 m/z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o f water. Pigs 3 Of7 Page 32 o f65 Exygen Research Page 66 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygcn Protocol Number: P0001131 Exygca Rai--rofa Method Number VOOO1712 I A N A LY TICA L M ETH O D ~| Method o f Analysis for the Determination o f Pcrfluorooctanoic Acid (PFOA) in Sediment by L C /M S /M S 8.2 Extraction Solutions 8.2.1 I S acetic acid in water is prepared by adding 10 m L o f acetic acid to 1000 m L o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f MOO pgfaiL 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 10 pg/mL fortification solution o f PFOA is prepared by bringing to m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA ia prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a m aximum period o f 6 months from tbs 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 foe 0.1 pg/mL fortification solution. 9.Z2 The following it a typical example: additional concentrations may be CwtiCTirtmtiffli o f Fortification Solution fal/mL) 100 100 100 10 5 2 Vohune (mU 10 5 2 10 10 10 Diluted to (mL) 100 100 100 too 100 100 Final Concentration ins/mU 10.0 5.0 2.0 1.0 0.5 0.2 Page 4 of 7 Page 33 o f 65 Exygen Research Page 67 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 IxyinX iM Rk Matfaod Num ber V0001782 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanolc Acid (PFOA) in Sediment by L C /M S/M S 9.2.3 9.2.4 Store all calibration atandirde in 12S-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six montha. Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) m ust include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verity procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assuranoe plan for tins project 11.0 Sample Extraction 11.1 Weigh 5 g o f sample into 50 m L polypropylene centrifoge tubes (fortify as needed, replace Hd and m ix well). 11.2 Add 35 m L o f 1% acetic acid, cap, vortex end shake on a wrist action shaker fo r- 6 0 minutes. 11.3 Centrifoge the tubee at -3 0 0 0 rpm for - 2 0 minutes. 11.4 Condition the C u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed b y 20 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.5 Load (decant) the sample on the conditioned C u SPE cartridge. Discard eluate. 11.6 Add 20 m L o f methanol to the aodimcnt left in the bottom o f the 50 mL centrifoge tube. Cap, vortex and shake o n a wrist action shaker for -3 0 minute. 11.7 Cantrifoge foe tubes at -3 0 0 0 rpm for - 2 0 minutes. 11.8 Decant the methanol onto th s same SPE cartridge. Collect the eluate. U .9 Wash the column with 4 m L o f methanol. Collect the eluate end add it to the eluate collected in step 11.8. 11.10 Condition a second C u SPE cartridge (1 g, 6 m L) by passing 10 mL methanol followed by 20 m L o fH P L C water ( - 2 drop/sec). Do not let column run dry 11.11 Add the methanol to - 2 0 0 m L o f water and load on the second conditioned SPE cartridge. 11.12 Elute with - 5 m L 100% m ethanol Collect 5 m L o f eluate into graduated 15 mL polypropylene centrifoge tube* (final volume " 5 mL). 11.13 Analyze samples using electrospray LC/MS/MS. PastS of 7 Page 34 o f65 Exygen Research Page 68 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E iy |a R w c h M tthodN tabcr V00017I2 A N A LY TICA L M ETH O D ~ I Method o f Analysis for the Detannination o f Perfluorooctmoic Acid (PFOA) in Sediment by L C /M S/M S 12.0 Chromatography 12.1 Ii\ject the n o w amount o f each standard, sample and fortified 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 i t least five o r m ore concentration levels m int be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning sod at the end o f a sample s e t Standards must be interspersed between every 3-10 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 of standards). In either case, calibration standards must be the first and Iasi injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves arc generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using M aisLynx 3.3 (or equivalent) software system. 12.3 Sim ple response should not exceed standard responses. Any samples that exceed standard responses should be Author diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 anui parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents foe lots o f carbon dioxide. 33.2 Method blanks m ust no t contain PFOA at levels greater than the LOQ. If a blank PFOA at levels greater than 0.2 ng/mL, then a new blank sample must be obtained and foe entire set must b e re-exIracted. 13.3 Recoveries o f control spikes and matrix spikes m ust be between 70-130% o f their known values. I f a control spike foils outside foe acceptable limits, the entire set o f t 1-- should be re-extracted. Any matrix spike outside 70 130% should be evaluated by foe analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from foe calculation o f foe calibration curve However, foe total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total num ber o f extracted standards injected. 13.3 The correlation coefficient (IQ for calibration curves generated must be 0.992 (R* 0.983). I f calibntion results foil outside these limits, (hen appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should be reanalyzed. Pag* 6 of 7 Page 35 o f 65 Exygen Research Page 69 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 E x y g n Research Method Num ber VOOOI7I2 I AftALVTlCAI. M ETHOD | Method o f Analysis for the Determination o f Perfloorooctanoic Acid (PFOA) in Sediment by L C /M S /M S 19.6 Retention ttm ei between standards and samples must not drift more then 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical tun then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate foe amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the M u s Lynx software program: PFOA found (ng/mL) - (Peak area - intercept) x DF slope DF factor by which the final volume w u diluted, i f necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (% ) f total analyte found (ng/mL) analyte found in control (ng/mL)] ^ ^ analyte added (ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng'mL to ng/gfppb). PFOA found (ppb) - (PFOA found fn j/m L l x final volume (S mLtl sample weight (5 g) 14.4 U se foe following equation ( if necessary) to calculate the amount o f PFOA found in ppb based on dry weight. PFOA found (ppb) dry weight PFOA found (ppb) x (100% / total sohdi(%>] S*fC 7 of 7 Page 36 of65 Exygen Research Page 70 o f 118 Interim Report #26 -- Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method H um ber V0001783 Method o f A iib ih fertile Detcnniaatloa ofPerflaorooctaoolc Add (PPOA) io Fish and Clams by LC/MS/MS Analytical Testing Facility: Exygen Research 3038 Research Drive State College, PA 16801 Approved By. y>~l C-j L Paul ComaoUy I Technical L o d a r, LC-MS, Exygen Reeearch H IU .M Date i Flaherty r Vice President, Operations, Exygen Research Date Exygen Research Total Pages: 8 Page 37 o f 65 Page 71 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 / "* Exygen Protocol Number: P0001131 ExygeoltMMieb Method Number VOOOI7SJ | ANALYTICAL M ETHOD ~| M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Pish and Clama t y LC/MS/MS 1.0 Scope This method ia to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Perfoimance Liquid Chromatography coupled to a tandem M ass Spectromtrie Dctector (LC/MS/MS) in fiah and clama. 2.0 Safety 2.1 Always obaerve aafe laboratory practices, 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g o f test sample for extraction. 3.2 Samples should be proceswd before extraction. Place the frozen sample in a food processor ami homogenize w ith (fry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation Seal and place the samples in frozen storage until time o f analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagenta and Standards 4.1 W ater-H P L C grade 4.2 A cetonitrile-HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.3 Silica gel (60-200 mesh) Reagent grade 4.6 Ftorisil (60-100 m esh) Reagent grade 4.7 Superclean LC-NHj Reagent grade 4.8 1-Octanol- HPLC grade 4.9 L-Ascorbic a d d - Reagent grade 4.10 Dimethyldichlorosilane - Reagent grade 4.11 Toluene Reagent grade 4.12 Ammonium Acetate A.C.S. Reagent Crade 4.13 Perfluorooctanoic A d d - Sigma-Aldrich 5.0 Instrument and Equipment ' 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped witti a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to oollect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Page 2 o f S Page 38 of 65 Exygen Research Page 72 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol N um ber P0001131 EiyittHm iK b Method Number V00I78) ANALYTICAL 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 3.4 Rotaiy evaporator. 3.3 Tissumizer. 3.6 123 raL pear-shaped flasks. 3.7 50 m L disposable polypropylene centrifuge tubes. 5.8 1S m L dispoeable polypropylene centrifuge tubes. 5.9 Disposable micropipets (SO-lOOuL, 100-200uL). 5.10 125-mL LDPE narrow-mouth bottles. 5.11 2 m L clear HPLC vial Idt. 5.12 Disposable pipettes. 5.13 Autopipettes (100-1000 pL and 10-100 >iL), w ith disposable tips. 5.14 SPE tubes (20mL) (Supelco cat. no. N057I77). 5.15 Wrist action taker. 5.16 Centrifuge capable o f spuming 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x SO mm. 5m (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in W ater 6.4 Mobile Pbaae (B) : Methanol 6.5 Gradient Program: Tnwa fmhri 0.0 1.0 8.0 20.0 22.5 5LA 65 65 25 25 65 Flow Rate & j(2qUDU&) 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 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions am intended as a guide and may be changed in order to optimize tiw HPLC system. Pass 3 oTS Page 39 of 65 Exygen Research Page 73 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Raaearch . Method N ste r V00017S3 1 ANALYTICAL M fiTHOD | Method o f Analysis for the D eterm initi) ofPerfluorooetanoic Acid (PFOA) in Fish and Clama by LOMS/MS 7.0 MS/MS System 7.1 Mode: Electrospny Negative MRM mode monitoring 413 -> 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water i t 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 a d d in methanol i t prepared by dissolving 2 g o f ascorbic acid in 100 m L o f methanol. 30% Dimethyldichlorosilane in toluene is prepared by bringing 3 mL ofdimethyldichlorotUane to a final volume o f 10 mL with toluene. Alternate volumes m ay b e prepared. 9.0 Standard Preparation 9.1 Standard Stock/Foitifieation Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f - 1 0 0 p ffa iL o f PFOA by w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LOPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 1 m L o f foe 100 pg/mL solution to a final volume o f 100 with methanol in a 123 m L LOPE bottle. A 0.1 pgfaiL fortification solution o f PFOA is prepared by bringing 10 m L o fth a 1.0 p ^ m L solution to a final volume o f 100 with methanol in a 125 m LLDPE bottle. A 0.01 pgfaiL fortification solution o f PFOA is prepared by bringing 10 m L o f the 0.1 jigfaiL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a m aximum period o f 6 months from the date o f prepvation. Page 4 ofS Page 40 o f 65 Exygen Research Page 74 o f 118 M erini Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 Bxygea JUaurch Method Number V0001783 ANALYTICAL METHOD ] Method o f Analysis for the Detominatioo o f Pcrfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 9.2 Standard Calibration Solution 9.2.1 9.22 LC/MS/MS ealftfation standard! are prepared in methanol via dilution o f die 1.0 |ig/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentration o f Fortification Solution fuErinL) 1.0 Volume (mL) S.0 Diluted to tinU 100 Final Concentrati) <AnL) 0.05 IX) 2.5 100 IX) 1.0 100 0.05 10 too 0.025 0.01 0.005 0.025 10 100 0.002S 0.1 0.005 10 10 100 100 0.001 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to d*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) m ust include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in foe quality aaaurance plan for this project. 11.0 Sample Extraction 11.1 W eigh 5 g o f frozen sample into 50 m L polypropylene centrifuge tubes (fortify ae needed, replace lid and mix well). 11.2 Add 30 m L o f acetonitrile and shake on a wrist action shaker for -1 5 minutes. 11.3 Place the tubes in a freezer for -1 hour. 11.4 Pack and condition the SPE tubes and ailanize the pear-shaped flasks. 11.5 Pack the 20 m L SPE tubes in sequence w ith 2 g florisil, 2 g silica gel, 2 g carbon, and 1 g LC-NHj. Condition the columns with 20 mL o f methanol, then 20 m L o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanizc fo e 125 m L pear-shaped flasks b y rinsing with the 30% dimethykiichtoroailane in toluene solution. Rinse foe flask with toluene once, followed by methanol (three times). Dry foe flasks completely before use. either by air-drying o r with a stream o f nitrogen. Page 9 of X Page 4i o f 65 Exygen Research Page 75 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 B tyintiinch Matbod Number VOOOH83 ANALYTICAL MJETHOP 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 mL polypropylene lubes containing sample at -2 0 0 0 rpm tor - 1 0 minutes. 11.8 Decant the extract on to a conditioned SPE column fitted inside the mouth o f the pear-shaped flask. Collect the eluate in the 12S m L tilanized pear-shape flask. 11.9 Add 10 m L o f acetonitrile to the sample in the 50 m L centrifuge tube. Homogenize die frozen fht phase using a tiiaumizer for - 3 0 seconds and rinse the tiaaumizer w ith -lO m L o f acetonitrile into the tube. 11.10 Shake the sample again fo r- 1 0 minutes on a wrist-action ahaker. 11.11 Place the tubes in a freezer for - 1 hour more. 11.12 Centrifuge the 50 m L polypropylene tubes containing sample at -2 0 0 0 rpm fo r- 1 0 minutes. 11.13 Decant the extract onto the same SPE column. Collect the eluate into the same pear-chaped flask and combine w ith the eluent from the initial extraction. 11.14 Pass 20 m L o f acetonitrile through the SPE column and combine the eluate in the same pear-chaped flask. I M S Add 3-4 drops o f l-octaool to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40*C). 11.16 M ake the final volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/disolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze sample using clectrospray LC/MS/MS. 12.0 Chromatography 12.1 Iqject the same amount o f each standard, sam ple and fortified sample into (he LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA oonw ponding to at least five o r more concentration levels must be included in an analytical set 12.3 An entire set o f calibration standards must be included i t the beginning and at die end o f a sample set. Standards must be interspersed between every 5-10 samples. A s an alternative, an entire set o f calibration standards may be injected at the beginning o f 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 the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte b y linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. P*S6of8 Page 42 o f 65 Exygen Research Page 76 of 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 ExyfwiR m trch Method Nmbr V0001713 I ANALYTICAL METHOD I Method o f Analysis for the Determination o f Perftuorooctinoic Acid (PFOA) in Fish and Clams by LG/MS/MS * 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should he Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show t peak o f a daughter ion at 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 loss o f carbon dioxide. 13.2 Method blanks m ust not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.5 ppb, then a new blank sample must be obtained and foe entire set m ust be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike foils outside foe acceptable limits, the entire set o f samples should b e re-extracted. 13.4 Any calibration standard found to be s statistical outlier by using the Huge Error Test may be excluded from the calculation o f the calibration curve. However, foe total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R2 20.985). I f calibration results All outside these limits, then appropriate stepe must b e 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 and samples must not drift more than 4 % within an analytical run. I f retention time drift exceeds this limit within an analytical run then foe set must b e reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peek a n t) using foe standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) (Peak area - interemi) slope 14.2 Use the following equation to convert the amount o f PFOA found in ng'mL to n0/g(ppb). PFOA found (ppb) - (PFOA ftxmd fng/m l.! * final volume ImL) x DF1 sample weight (g) DF factor by which the final volume was diluted, if necessary. Page 43 o f 65 Exygen Research Page 77 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 ExygeoRiiw ch MMbod Humber V00017$) ANa LVTIC.U* METHOD Method o f Analysis for the Determinati! o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (% )- [ total analyte found (ng/g) - analyte found in control (ng/g)l j[]0Q analyte added (ng/g) Exygen Research Pag I of S Page 44 o f 65 Page 78 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOt 131 ANALYTICAL METHOD Method Number: V0I784 M ethod oTA aalyrls fo r the D eterm ination o f Perflnorooctnaolc Acid (PFO A ) In V egetation fey LC/M S/M S Analytical Totting Facility: Exygen Beatateli 3038 Reaeatch Drive State College. PA 16801 Approved By: T t-1 C JiL Puil Connolly ' Technical Leader, LC-MS, Exygen Research Q // n / i U /________ Jbhn Flaherty ' Vice Prsidait, Operations, Exygen Research ___ l O f r t M Date Date Exygen Research Total Pages: 7 Page 45 o f 65 Page 79 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 Exygta Rr n i r ch Method Number VOOtfl 7B4 ANALYTICAL MTHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope This method 1 to be employed for the isolation and quantitation o f perfluorooctanoic a d d b y High Perfonnance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in vegetation. 2.0 Safety 2.1 Always obeerve safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g o f tact sample for extraction. 3.2 Samples should be processed before extraction. Place die frozen sample in a food processor and homogenize with d ry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time o f analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 A cetonitrile-HPLC grade 4.3 Carbon (120-400 mesh) -R eegent grade 4.4 Methanol - HPLC grade 4.3 Silica gel (60-200 mesh) - Reagent grade 4.6 Floriiil (60-100 mesh) - Reagent grade 4.7 Superclean LC-NHj - Reagent grade 4.5 1-Octanol-HPLC grade 4.9 L-Ascorbic a d d - Reagent grade 4.10 Diroethyldichlorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 3.0 Instrument sad Equipment 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume izyector capable o f injecting 3*200 l*L connected to a tandem Maas Spectrometer (LC/MS/MS). 3.2 A device to collect raw data for peak integration and quantitation. S J Analytical balance capable o f reading to 0.00001 g. Page 2 o f 7 Page 46 o f65 Exygen Research Page 80 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyiuJUwarch Mftbod Niunber V00017M ANALYTICAL M ETH O D _________________________ Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 5.4 Rotary evaporator. 5.5 123 m Lpesr-shipod flasks. 5.6 50 m L disposable polypropylene centri& ge tithes. 5.7 15 m L disposable polypropylene ceotrifoge tubes. 5.8 Disposable mfcropipeis (SO-lQOuL, 10Q-200uL). 5.9 125-raL LDPE narrow-mouth bottles. 5.10 2 m L clear HPLC vis] kit. 5.11 Disposable pipettes. 5.12 Autopipcttee (100-1000 p L and 10-100 pL), w ith disposable tipe. 5.13 SPE tubas (20mL) (Supelco cat. no. N057177). 5.14 Wrist action shaker. 5.15 Ceotrifoge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophsse RP (Keystone Scientific), 2.1 mm x 50 mm, 5^ (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A ): 2 mM Ammonium Acetate in W ater 6.4 Mobile Phase ( B ) : Methanol 6.5 Gradient Program: T im a ftwinl 0.0 1.0 8.0 20.0 22.5 SkA 65 65 25 25 65 Flow Rate fm L /m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 hgeetion Volume: 15 pL (can b e 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 are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electroepray Negative MRM mode, monitoring 413 --369 m/z for PFOA. Page 3 of 7 Page 47 o f 6J Exygen Research Page 81 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: POOOl 131 Exygen RcsMieh Method Number V00I7S4 ANALYTICAL M ETHOD Method o f Analysis for the Determination ofPcrfluorooctanoic A d d (PFOA) in Vegetation by LC/MS/MS The above conditions are imended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% asoobac acid in methanol is prepared b y dissolving 2 g o f ascorbic acid in 100 mL o f methanol 30H Oimefoyidichiofosilane in toluene is prepared by bringing 3 mL ofdimcthyldichlorosilane to a final volum e o f 10 mL with toluene. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Stodc/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock sohit)on o f ~100 p ^ 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. A 1.0 pg/mL fortification solution o f PFOA i i prepared b y bringing 1 mL o f the 100 p g fa L solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. A 0.1 p ^ n L fortification solution o f PFOA i t prepared by bringing 10 m L o f the t .0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 p g ta L solution to a final volume o f 100 with methanol in a 125 m L LDPB bottle. The stock and fortification solutions a n to be stored in a refrigerator at approximately 4*C and m e 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/mL fortification solution. Page 4 ol Page 48 of 65 Exygen Research Page 82 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number P0001131 ExygmlMMKh Method Number V0001784 ANA LY TICA L M E T H O D | Method o f Analyse for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation byL C /M S/M S 92,2 H ie following ii a typical example: additional concentration* may be prepared a t needed. Coocenkatian Final ofFortifioatioa Volume Diluted to Concentration Solution fus/mL) <L) (mL) (ni/mL) 1.0 5.0 100 1.0 2.5 100 0.05 0.025 1.0 1.0 100 0.01 0.05 10 100 0.005 0.025 10 100 0.0025 0.1 0.005 10 10 100 100 0.001 0.0005 9.2.3 Store aU calibration nandanU in 125-mL LDPE narrow-mouth bonles at 2*C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one untreated control and two untreated controls fortified st known concentrations (lab control spike) to verify procedural recovery for the batch. 1 0 J Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sim ple Extraction 11.1 Weigh 5 g o f frozen sample into 50 mL polypropylene centrifuge tubes (fortifyas needed, replace lid and mix well). 11.2 Add 30 m L o f acetonitrile and ihake on a wrist action shaker f o r -1 5 minutes. 11.3 Centrifuge the 50 mL polypropylene tubes containing sample at -2 0 0 0 rpm fo r- 1 0 minutes. 11.4 Pack and condition the SPE tubes and ailanize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in sequence with 2 g florisil, 2 g silica gel, 2 g carbon, and 1 g LC-NHj. Condition the columns w ith 20 m L o f methanol, d u n 20 m L o f acetonitrile. Discard all washes. D o not allow the column to dry. 11.6 Silanize the 12S mL pear-shaped flasks by rinsing with the 30% dimetbyldichlorosUane in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). D ry the flasks completely before use. either by air-drying or with a stream o f nitrogen. 11.7 Decant the attract on to a conditioned SPE column fitted inside the mouth of the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape Pag* 5 of ' Page 49 o f 65 Exygen Research Page 83 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 ExygraRMMtch Mtfcod Number V000I7I4 ANALYTICAL m e t h o d Method o f Analysis for the Determination ofPerfluoroocunoic Acid (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 m L o f acetonitrile to the sample in the SO m L centrifUge tube. 11.9 Shake the a m p le again for - 1 0 minutes on a wrist-actioa shaker. 11.10 CentrifUge the SO mL polypropylene tubes containing sample at -2 0 0 0 rpm fo r- 3 minute*. 11.11 Decant the extract onto the same SPE column. Collect the eluate into the same pear-slu^ed flask and combine with the eluent from the initial 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 pcar-diaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40*C). 11.14 M ake die final volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/disaolve. 11.15 Transfer the tra c ts to HPLC v isit using disposable pipets. 11.16 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 12.2 12.3 12.4 12.5 Inject die same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. Standards o f PFOA corresponding to at least five o r m ore concentration levels must b e included in an analytical set. An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample s e t Extracted standards must be interspersed between every 3-10 samples. A t an alternative, an entire set o f extracted calibration standards m ay b e injected i t the beginning o f a sei followed by extracted calibration standards interspersed every 3-10 samples (to account for a second set o f tr a d e d standards). In either case, extracted calibration standards must be the first and last injection in a sample set. Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using \fx weighting o f peak ares versus cstibration standard concentration using M sssLynx 3.3 (or equivalent) software system. Sample response should not exceed standard responses. Any samples ilui exceed standard rcaponm should be further diluted and reanalyzed. 13.0 Acceptance Criteria 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 ihe daughter ion (369 amu) represents the loss o f carbon dioxide. P a | 6 o n Page SOo f65 Exygen Research Page 84 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOI131 ExyguR--i r h MMhodNuaber V00017M j A ftA lV nC A L METHOD | Method o f Analysis ih r the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation b yL C /M S/M S 13.2 Method blanks m ust not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than O.S ppb, d u n a sew blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70*130% o f their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should b e re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error T e st may b e 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 injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R* 20.985). I f calibration results fall outride these limits, 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 between standards and samples must not drift m ore than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run titan the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/m L. based on peek area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ngfmL) - Peak area * in terred ) slope 14.2 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found fnph1 - (PFOA found <aa/mL) x final volume (mL) x DF1 sample weight (g) DP - ftetor by which the final volume was diluted, if necessary. 143 For sanples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [ total analyte found (n ^ g ) analyte found in control (ng/g)J analyteadded (ng/g) Page 7o i7 Page SI o f 65 Exygen Research Page 85 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 E x y g e n P r o to c o l N u m b e r: POOO1131 ANALYTICAL METHOD Method N um ber V0001785 Method o f Aaalyala for the D eterm lultoB o f Perfinero o cu o atc Acid (PVOA) hi Smell M emm el Liver by LC/MS/MS Anelyticel T a tin g Facility: Exygen Research 3058 Research Drive Stete College, PA 1801 Approved By: 'B J s _--------------- Paul Connolly I Technical Leader, LC-MS, Exygen Research a/ w ________ Jhn Flaherty / Vice President, Operations, Exygen Research Date Dote Exygen Research Total P eg a: 7 Page 52 o f65 Page 86 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 Exyira Rrnarch Method Nwaber VQ0QI7IJ | ANALYTICAL M ETHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f pcrfhioiooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in email 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 At least S g o f test sample for extraction. 3.2 Samples should b e processed before extraction. Place Ok frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time o f analysis. Alternately, i f there it an insufficient amount o f sample ('less than S g), then no processing is necessary aod the sample can be used as supplied. 3.3 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - HPLC p a d s 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A .C S. Reagent Grade 4.5 Perfluorooctanoic A d d - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5*200 jiL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifoge tubes. 5.5 15 m L djspottble polypropylene centrifoge tubes. 5.6 Disposable micropipets (50-lOOuL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottle*. 5.8 2 m L clear HPLC vini kit. P*ge2oH Page S3 o f63 Exygen Research Page 87 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen P r o to c o l Number P 0 0 0 1 1 3 1 ExygcaJUmrch Matkod Number V000I7IS J ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perftuorooctanoic A d d (PFOA) in Small Mamma] Liver by LC/MS/MS 5.9 5.10 5.11 5.12 5.13 5.14 5.15 Diflponbleptpetlee. Autopipettes (100*1000 |iL and 10-100 pL), with disposable tips. W aten Sep Pak Vac 6 oc (lg ) tC18 SPE cartridges. SPE vacuum manifold. Tiewtemizer. Wrist-action shaker. Centrifitge capable o f spinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5^ (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase ( A ) : 2 raM Ammonium Acetate in Water Mobile Ph sse(B ): Methanol Gradient Program: Tima (mini 0.0 1.0 8.0 20.0 22.5 5kA 65 65 25 25 65 Flow Rate fm L /m inl 35 0 J 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: IS pL (can b e increased to u m uch as 50 *iL). 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 m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Blectrospray Negative MRM m ode, monitoring 413 - a 369 m/z for PFOA. The shove conditions are intended as s guide end m ay be changed in order to optimize the MSMS system. Pafe3 of? Page 54 o f 65 Exygen Research Page 88 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExypaRMMnh MethodNumber V0001783 I ... 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 Small Mammal Liver by LC/MS/MS ; 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volumee may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Foctification Solution 9.1.1 Prepare a etock solution ofMOO pg/mL 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 yg/mL fortification solution o f PFOA is prepared by bringing 1 m L o f the 100 yg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.3 A 0.1 MgfaL fortification solution o f PFOA is prepared by bringing 10 m L o fth e 1.0 yg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.4 The stock and foitifieation solutions are to b e stored in e refrigerator at approximately 4*C and a n stable for maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solution 9.2.1 92 2 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 yg/mL fortification solution. The following is a typical example: additional concentrations may be prepared aa needed. Concentration Final of Fortification Volume Diluted to Concentration Solution (na/mL) (mL) (mL) (na/mL) 100 5.0 100 5.0 100 2.0 100 2.0 100 1.0 100 1.0 $.0 10 100 0.5 2.0 10 100 1.0 to 100 OJ 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, u p to a ix month. 9.2.4 Alternate volumes and concentrations o f standards m ay be prepared as tage 4 of? Page 55 o f 65 Exygen Research Page 89 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyiealtMMick Mwbod Number V0001785 | ANALYTICAL METHOD Method o f A nelytii for the Determination o f Perfluorooctanoic A d d (PFOA) in Smell Minimal Liver by LC/MS/MS 10.0 Batch Set Up 10.1 Each batch o f sample* extracted (typically 20 o r lees) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the betch. 10.2 Requirements for field and laboratory duplicate* and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 1 g o f sample into a 50 raL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate weights o f liver may be measured depending on the sample rise available for use. 11.2 Add water to the sample for a final volume o f 10 mL. 11.3 Homogemw sample using a tissuemizer for ~1 minute. 11.4 Transfer 1 mL o f foe sample uaing a disposable pipette into a IS mL disposable centrifuge tube. 11.5 Add 5 m L o f acetonitrile and shake fo r ~20 minutes on a wrist-action shaker. 11.6 Centrifuge foe tubes at >3000 rpm for - 3 minutes. 11.7 Decant the supernatant into a SO m L disposable centrifoge tube and add 35 m L o f water. 11.8 Condition the C ti SPE cartridges ( l g, 6 mL) by passing 10 mL methanol followed b y 5 m L o f HPLC water (~ 2 drop/soc). Do not let column nin dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eluate. 1 M 0 Elute with ~2 mL o f mrihanoL Collect 2 mL o f eluate into a graduated 1$ m L polypropylene centrifoge tube (final volume 2 mL). 11.11 Analyse samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Iryect the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or m ore concentration levels must b e included in an analytical set. 12.3 An entire eat o f calibration standards m ust be included at the beginning and at foe end o f a sample s e t Standards m ust be interspersed between every 5-IU 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 3-10 sim ples (to account for a second set o f standards). In either case, calibration standards must be foe first and last 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 1/x weighting o f peak area Page 5 of? Page 56 o f 65 Exygen Research Page 90 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyganftaMifcb M*Siod N uotor V000178S | ANALYTICAL M ETHOD M ethod o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS | venue catibradoc etandaid concentration using MaeeLynx 3.3 (or equivalent) software system. 12.3 Sample response should not exceed standard responses. Any samples that exceed standard responses should be fluther diluted and reanalyzed. 13.0 Acceptance Criteria 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 lose o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. if a blank contains PFOA at levels greeter then 10 ng/g, then a new blank sample must ba obtained and foe entire set must be (extracted. 13.3 Recoveries o f control spikes and matrix spikes m ust be between 70-130% o f their known values. I f e control spike foils outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to b e a statistical outlier by using the Huge Error Tori, may be excluded from foe calculation o f foe calibration curve However, foe total number o f calibration standards that could be excluded must not exeeed 20% o f foe total number o f standards injected. 13.3 The correlation coefficient (R) for calibration curves generated must be 20.992 (RJ 20.983). I f calibration results foil outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or foe relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical tun. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using foe standard curve (linear regression parameters) generated by foe Mam Lynx software program: PFOA found (ng/mL) - (Peak area - intercept) x D F x aliquot factor DF " factor by which foe final volume was diluted, i f necessary. Aliquot factor - 10 Pa*6 of 7 Page 57 o f 65 Exygen Research Page 91 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 ExygmiUMHck Mathod Number VOOOI785 I ANALYTICAL M ETHOD .............. Method o f Analysis for the Determination ofPerfluorooctinoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 14.2 For sample fortified with known amounts o f PFOA prior to extraction, use die following equation to calculate the percent recovery. Recovery (H )* [total analyte found (ng^nL) analyte found in control (n g /m t)] |1QQ analyte added (ngfaL ) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL io ng^gippb). PFOA found fppbl - fPFOA found fay/m M x final volume fmLll sample weight (g) Exygen Research Pate 7 of7 Page 58 o f 65 Page 92 of 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Num ber V0001786 Method of Analysis for the Determination of Porfluorooctooolc Add (PFOA) to Small Mammal Serum by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: p t-l CJtL, Paul Connolly I Technical Leader, LC-MS, Exygen Research 4 2 /#> / / Jw m Flaherty / Vice President, Operations, Exygen Research Date Date Exygen Research Total Paget: 7 P age 59 o f 65 Page 93 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number P0001131 Exygen R w arch Method N u n * V000!716 _________________________ AWALVT1CAL m e t h o d M ethod o f Analysis for the Determination o f Perfloorooctanoie Acid (PFOA) in Small Manunal S o w n by LC/MS/MS 1.0 Scope This method is to be alloyed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectroroetric Detector (LC/MS/MS) in small mammal serum. 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 At least l m L o f test sample for extraction. 3.2 N o sample processing is needed for serum samples. However, frozen scrum samples must to allowed to completely thaw to room temperature before use. 3 J Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater - HPLC grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume iqjector capable o f injecting 5*200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL duposable polypropylene centrifoge tubes. 5.5 15 m L disposable polypropylene centrifUge tubes. 5.6 Disposable micropipets (50*100uL, 100-200uL). 5.7 125-raL LDPB narrow-mouth bottles. 5.8 2 m L clear HPLC vial IdL 5.9 Disposable pipettes. 5.10 Autopipettes (HXMOOOpL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 c c (lg )tC l8 S P E cartridges. 5.12 SPB vacuum manifold. 5.13 Vortexer. Page 2 of' Page 60 o f 65 Exygen Research Page 94 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyfBBKcmrch Method Number V00UI7S6 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 Small Mammal Serum by LC^MS/MS 5.14 Wrist-action shaker. S. 15 Centrifuge capable o f spinning 15 mL polypropylene tubes at 3000 rpm 6.0 Chromatographic System 6.1 Analytical Column: Fluophise RP (Keystone Scientific). 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Tamparature: 30*C 6.3 Mobile Phaae (A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phaae (B ): Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate % B fmL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increaaed to as m uch aa 50 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 m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM m ode, monitoring 4 1 3 - * 369 m/z for PFOA. The above conditions are intended as e guide and m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium scutate in water is prepared by adding 0.154 g o f nmfliiium acetate to 1000 mL o f water. Alternate volumes may be prepared. Page 3 of 7 P age 61 o f 65 Exygen Research Page 95 o f 118 Interim Report #26 -- Analysis o f Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 EiypaJUM ueb Method Nunber VOOOI7S6 | ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluoiooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortificatioo Solution 9.1.1 Prepare a stock solution o f~ 1 0 0 p g /m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mU with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 ng/mL fortification solution o f PFOA is prepared by bringing I mL o f the 100 pg/raL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o f PFOA it prepared b y bringing 10 m L o f the 1.0 jig/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottk. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C end are stable for a m aximum 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 methanol via dilution o f foe 0.1 ftg/mL fortification solution. Tbs following is a typical example: additional concentrations may be prepared as needed. Concentration ofFortificstion Solution (oa/mL) 100 Volume (m l) 5.0 Diluted to (mL) too Final Concentration (na/mL) 5.0 100 2.0 100 J.0 too too 2.0 1.0 5.0 10 100 OS 2.0 10 100 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6 "C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r lass) m ust include at least one untreated control and two untreated controls fortified at known concentrations (lib control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. Page 4 of 7 Page 62 o f 65 Exygen Research Page 96 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 Exyya R w reh Method Number VOOO1716 ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 11.0 Sample Extraction 11.1 M eanire 1 m L o f a m p le into a SO m L polypropylene centrifuge lube (fortify u needed, replace lid and mix well). Note that alternate volumes o f serum may be measured depending on die a m p le size available for use. 11.2 Add water to the sample for final volume o f 20 mL. Cap tightly 11.3 Vortex for - 1 minute. 11.4 Transfer 1 m L o f the sample using a diapoeabte pipette into a 13 mL disposable eentri&ge tube. 11.3 Add 3 mL o f acetonitrile and shake for - 2 0 m inutes on wrist-action shaker. 11.6 Centrifuge the tubes at -3 0 0 0 rpm for - 3 minutes. 11.7 Decant the supernatant into a 30 m L disposable centrifuge tube and add 35 m L o f water. 11.8 Condition the C u SPE cartridge# (1 g, 6 mL) by passing 10 mL methanol followed by 5 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eluate. 11.10 Elute with - 2 mL o f methanol. Collect 2 m L o f eluate into a graduated 15 m L polypropylene eentrifoge tube (final volume 2 mL). 11.11 Analyze samples using electrocpray LC/MS/MS. 12.0 Chromatography 12.1 Iqject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards ofPFO A corresponding to at least five o r m ore 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 at the end o f a sample set. Standards must be interspersed between every 5 -1n 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 ease, cal&ration standards must be the first and last injection in a sample set 12.4 Use linear standard curves for quantitation. Linear standard curves sre generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MaasLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. Page 3 of 7 Page 63 o f65 Exygen Research Page 97 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000U 31 Exygen JL ttU fth M ethod Number VOOO1786 I A NA LYTICAL M K TH O P Method o f Analysis for the Determination o f Perfluorooctaaoic Acid (PFOA) in Small Mammal Seram by LC/MS/MS 13.0 Acceptance Criteria 13.1 Chromatogram m u tt how a peak o f a daughter on at 369 amu from a parent o f 413 amu. The 413 amu parent corroapondc to the PFOA anion, whtle die daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blank! muat not contain PFOA at levela greater than the LOQ. If a blank contains PFOA at levela greater than 10 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known vahtee. I f a control spike falls outside the acceptable limits, the entire act o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by die analyst to determine if re-extraction is warranted. 13.4 Any calibration 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 the total number o f standards injected. 13.3 The correlation coefficient (R) for calibration curves generated must be 20.992 (RJ 20.985). I f calibration results fall outside these limits, 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 and samples must not drift more than 4 % within an analytical nm . If retention tim e drift exceeds this limit within an analytical ran then the set must Ire reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak ares) using tire standard curve (linear regression parameters) generated by tire M a n Lynx software program: PFOA found (ng/mL) (Peak area - intercept) x DF x aliquot factor slope DF firetor by which tire final volume w u diluted, i f necessary. Aliquol factor - 20 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use tire following equation to calculate tire percent recovery. Recovery ( % ) [ total analyte found (ng/mL) - analyte found in control (ng/mL)] analyte added (na/mL) Page 6 o f7 Page 64 o f 65 Exygen Research Page 98 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Bxygen Protocol Number: P0001131 ExygttltM M rcfa M ethod Number V 00017S6 I A N A L rnC A t METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 14.3 Use the following equation to convert the amount o f PFOA found in ngfmL to ppb. PFOA found (ppb) - fPFQA found fticftnlA * Bm I volume frnl tl sample volume (mL) Exygen Research Pafs 7 of "f Page 65 o f65 Page 99 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 PROTOCOL AM ENDM ENT Amendment N um berM Effective Date: 01/19/05 Exygen Study Num ber: P0001131 Client Study Number: Page 1 of 1 D E S C R IP T IO N O F A M E N D E D S E C T IO N 1) Analytical Procedure Sum m ary V0001780:Section 9.1 2) Verification of Analytical Procedure None AM ENDED TO 1) Add to Section 9.1: Section 9.1.6, Alternate w eights o f standards m ay be used to prepare alternate concentrations of stock solutions a s necessary. Alternate levels of fortification solutions m ay also be prepared. ' 2) Low and high spiking levels of the analytes for 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. R A T IO N A L E 1) H igher 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 m am m al liver and serum w as sm aller than expected. Spiking at the pre-determined levels in the protocol puts the spiked concentration lower than the detection limit. A lso, the analyte levels in the ground water sam ples are expected to greatly exceed the pre-determined 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. H igher spiking levels in the bottles will cover the analyte concentrations expected in the water sam ples. IM P A C T O N ST U D Y The L O Q is 100 ng/g for a 0.1 g sam ple of sm all m am mal liver and is 1000 ng/m L for a 0.01 m L sam ple of sm all m am mal serum. Higher levels of spiking for the water sam ples will ensure that' m ore Q C recovery data can be used. LIBRARY ID: W0001226-6 ' . ADMINISTRATIVE FORM Exygen Research Page 100 o f 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 3058 R esearch D rive Phone: 814-272-1039 State C o lle ge , PA 16801 Fax: 814-231-1580 Am endm ent Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 2 03/07/05 P0001131 Client Study N um ber Page 1 of 1 None D E S C R IP T IO N O F A M E N D E D S E C T IO N 1) Report, p a g e 11 o f 65 2) T e st M aterials, p age 6 o f 65: P F O S transition m onitored 499 -> 99. AM ENDED TO 1) In ste ad o f o ne final report, interim reports will be issu ed . 2) P F O S transition m onitored m ay a lso be 4 9 9 -> 80. R A T IO N A L E 1) D u e to the e x ce ssiv e siz e s o f the data se ts, interim reports w ill be issu e d to allow the client to receive d a ta in a tim elier maner-. 'wo1' osfalu- 4 * * 2 ) T h e A P I 400 0 L C /M S /M S sy ste m s detect the 499 -> 80 P F O S transition with gre ate r sensitivity than the 49 9 -> 99 transition. IM P A C T O N S T U D Y 1) T h e client will be ab le to receive an d review the data m ore quickly. 2) T h e 499 -> 80 transition can be detected with gre ate r sensitivity; therefore, givin g better chrom atography. Study Director Signature / X2u' f t fJ U jr Pnnnccipi al Investigator Signature ili Study Director Managiement Signature Exygen Manager" " '*" W v k jj 1. Sponsor Signature (if required) Date Date 8 -MW--os Date i/n /o f Date Exyge n Q A U R eview _ L u u io s it s LIBRARY ID: V0Q01226-8 ADMINISTRATIVE FORM Exygen Research Page 101 o f 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 JUL.25.005 0:58*1 EXYGEN RESEARCH H 0 .7 T 4 P .3 nr RESEARCH 3058 Research Drive Phone: 814-272-1039 SU te College, PA 16801 Fax: 814-231-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 3 07/18/06 P1131 Client Study Number: Pagai o ri NA DESCRIPTION OF AMENDED SECTION Verification ofAnalytical Procedura, page 10 of protocoL AMENDED TO The fWd duplicate can be used for the tsboratoiy spikes and replicate when the primary sample volume is limited. B A T IQ N & E The sample size fora watersample Is 200 ml- Ha samplesite requires nwwtredton for any reason, there would not be enough of the primary sample to repeat two laboratory spikes and a replicata. The field duplicate Is technicallythe same sample as the primary sample and therefore, can be used for laboratory spikes and replicates as needed. ---- - jm ea ct.o n s t v p y No negative impact on the study. Using the duplicate sample allows for the futi QC of the sample site to be completed. QMaL StudyDJnrMrstonati " ?,/ / n o/ciX ' 'itn d p ir m v e s s g s io f S ig n a y 1 ^- Date Date U ~ jm - t s Dale th '.o s Exygen QAU Review LIBRARYID: V000122M ADMINMTRATTVE FORM Exygen Research Page 102 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 'T 1lfJJ-2005 04:22m FraHESTOH SOLUTIONS ; NOVt22.Z005 CSBPM EXYGEN RESEARCH T-677 F.003/003 F-713 ru .2 1* r .a n& g g ?R E S E A R C H 30 Research Ori Phonal 014-272-1039 State College, PA 16801 Fax: 814-231-1380 AmendmentNumber: EffectiveDate: Exygen Study Number PROTOCOLAMENDMENT 4 T1/228 ~ Ptt31 aientStudyNumber. Pag* 1 of 1 NA__ D ESCRIPTIO N OP A M EN D ED SECTDBN Analytical Procedure Summary: V00(M7W:'Method of Analysis fe rita Detennination of PerftuoreaoctehoicAcid (PPOAitn Water by LC1M 8/M S' Section 11.0 rftfwm atnod. T* M ffr Q E P T g ~ .. _ Secton 11.0. Sample* may be diluted before going through It a eoctratfon procedure. E&I M B IS S - If a 40 mL portion of tampte wfl not loot onto the Ci S P E csrtride, a pre-dilution can te prepared and extracted. l 1M PA CLW ST VE . , No negative impact on Ite study. Mora usable data may be obtained. vXM L study mature lpoikwafSgator! atuteD teO B r Exygan Maria S p e n terb ig n ttu rep freq teB ^ - D *t* U * + n v-4 J! Da dH T V -v-r USRARVIO: V00012ZM RECEIVED TIME NOV.23. 5:40PM administrative form PRINT TIME NOV.23. 5 :41PM Exygen Research Page 103 o f 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 fCHEM EHSR 236 1B 651 733 1958 JU L > - 2 0 0 3 0 5 : 1 2 F P O M sS I B W . L flB 6 5 1 7 7 8 6 1 7 6 r, 2 2 .2 0 0 5 4 )5 B P n EXYSCN R E S E flK 11/23 '05 14:12 N0.979 03/03 . 711:66517331930 NO.31 4 p .3 3058 Retard! Drive Miao 4-272-10 StateCottele, PA16601 Fax: 814-23T-1MO RESEARCH AmendmentNumber. EffectiveDate: ExygenStudyNumber PROTOCOLAMENDMENT 4 naaoB P1131 ~ ClientStudyNumber: PogoioM NA PSfcRUmONOFAMEMDEDldlffS Analytical Procedure Summary: VC0t)t78OTMmi ofAnalysis for the Determinata! of Perfluwoooetanolc Acid (PFOA) In Water by IQ M S/M S," section H O oTtN method. A M fe N D EP T O Section 11.0. Sample may be diluted betre pomp throughtna sxtraeSan procedura. --------------------------------s s m iM -------------------------------- Ifa d O m L portion or sample will not toad onto the C m S P E c e rM s . a pneKflution can b a prep ared arto extracted. IMBACT ON STUDY <\ No negators impact ontne study. Mare usaMe data may be obtained. S tu d y B M n f s g n a u i -- S a > w tr4 r 5 Ma i. l i . w>S ExvcenQALl Review uanAM Yim voooiaaaa AOMINWTTVdlVEFORM RECEIVED TIME N0V.23. 3:32PM PRINT TIME N0V.23 . 3:33PM * N Exygen Research Page 104 o f 118 Interim Report #26 - Analysis of Groundwater Samples -20DS 04:09m Frot-KJTOH SOtUTION * * Exygen Study No.: P0001131 H 51 P.00^/D0^ P IN J lt I 3 *R e *8 f!S FW w Ph5 !!! E H ' 2 Siam O M f , PA 16*01 P te *14-231*13*0 ^ R :,.1SCH .Am andinm i Nu Aer: Eflecttw D cte ExygBfi Study Number T 'O C O t AM ENDM ENT J ST" Client Study N um ter 1 B T .1 naT TaatMateriato, page M of Pratoeal. V a 'T * ['d 7 * An Hamate toto'PFO S/n 'y j;-usedfortwsafudy. .........~ ^ k r i N A L E D w PF09 uaad pnw)oubr in this study (MnuniOar 217 ftn*M > m o u ta n d iw a a naoaaaniyte w a new lot No negativ impact on study. E& r t - r r r i T ^ see: _________ v J w n L j s ^ j 'L L ' * A / l t s e ----------------------- WM. B * y g iQ A U M U D 4 e e ^ ^ _ U L 5 fc fe$ i. UBRMWTttVWKM'S* RECEIVED TIME *HR. 1 28PM . a bm n o trm n epcrm PRINT TIME MRR.21. 5:28PM Exygen Research Page 105 o f 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231 -1580 Amendment Number. Effective Date: Exygen Study Number PROTOCOL AMENDMENT 6 05/17/06 ' P0001131 Client Study Number Pag l o f i NA D E S C R IP T IO N O F A M E N D E D SE C T IO N Appendix I, Analytical Method V0001762 AMENDED TO . *4 ' A s per client request sam ples in login L4026 that have been tagged for re-extraction by the sponsor will be re-extracted using the following method: D irect Injection M ethod: Before the sam ples are w eighed for the extraction, they are mixed thoroughly by vigorously shaking the container. A one-gram portion o f sedim ent is weighed into a 15-m illliter centrifuge tube for the extraction. Ten milliliters o f 1 % acetic ad d in methanol is added to each sam ple. The sam ples are then shaken by hand, vortexed, and sonicated for thirty minutes. The sam ples are then centrifuged for -1 0 m inutes at -3 0 0 0 rpm. Each sam ple is analyzed by L C /M S/M S electrospray. BAH0M .A LE M ore usable data will be obtained by using an alternate method. No negativeim pact on study. IM P A C T O N ST U D Y )kkJJy StudyoueAerSignaaire Date ^ n d p e l Investigator Signature 1 Data Sponsor Signature (Uequlred) Date 34-AM** Date id /ill i Dal E x yge n Q A U Init/Date 'f t - / Sfa/of LIBRARY ID: V0001226-S ' ADMINISTRATIVE FORM Exygen Research Page 106 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 3058 Research D rive Phone: 814-272-1039 State C ollege, PA 16801 Fax: 814-231-1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 8 07/06/06 P0001131 Client Study N um ber Page 1 of 1 NA D E S C R IP T IO N O F A M E N D E D S E C T IO N A p p en d ix I, A n alytical M ethod V0001782. AM ENDED TO A s per client request, sedim ent sam p le s re-tagge d for re-extraction b y the sp o n so r w ill be re-extracted u sin g the follow ing m ethod: D ire c t In je c tio n M e th o d : Before the sa m p le s are w eighed fo r the extraction, they are m ixed thoroughly b y vigorou sly sh ak in g the container. A o n e -gram portion o f sedim en t is w eighed into a 15-m illiliter centrifuge tube for the extraction. T e n m illiliters o f 1 % acetic acid in m ethanol is added to e ach sam ple. T h e sa m p le s are then sh ak e n by hand, vortexed, and sonicated for thirty m inutes. T h e sa m p le s are then centrifuged for - 1 0 m inutes at -3 0 0 0 rpm. E a c h sam p le is analyzed b y L C /M S /M S electrospray. R A T IO N A L E M ore u sab le data will be obtained by u sin g an alternate m ethod. IM P A C T O N S T U D Y N o negative im pact on study. Study Director Signature / A x '________ ^nncipal Investigator Signature Date _ Date Study Director Management Signature Date Exygen Management Signature Date Sponsor Signature (if required) Date Exyge n Q A U In it/D a te k X - / lil/lofe LIBRARY ID: V0001226-9 ADMINISTRATIVE FORM Exygen Research Page 107 of 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 CHEM EHSR 236 lfe 651 733 1958 OM W OK 03:S7pa FroHSTW SOLUTIOMS 07/11 '0 6 14:09 N0.142 02/02 + T-5I P.OM/OM H 30?aIteMrh0nit lino State Colles. PA *H 01 Pwc ^RESEARCH . '.j ' . ^ T O C O U AM ENDM ENT Amendment Num ber E fflo c ve D a te S S S m m m trn am ***** ::p o a :. v f i 3T7 J - V Appendix I, Analytical Method V0KJ17S2. ba no-rteeted uaing the frtciwlng-method: le weighed Into a 16-mlRmtwrenMUS Bioa iorino areacegn. Ton w-- aorte acid in methanol le added W Ncheernpie. The m r t s w e h g m j r hand, vortaxed, and eonteated for.thirfc mtnutee. The e a m ^ am ^ h W -TO minuta at *3Q00 rpm. Each sample is analyzed by L C M S /M 6 el^ctftapray. ... . -- ------------------ - : fwnsfrttE .. . ' i. '. . 'i More useWe data Will be obtainedttyuelng anpltametemfthod. ffifPftgt'.ON8TUS~ No negrtye impact on study. UBWAHVIP: VD0012284 RECEIVED TIME J U L .U . 4:27PM ' PRINT TIME ju jM iW w m iiT W t W sh u J U L .ll. 4:28PM Exygen Research Page 108 of 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 01-01-2007 03:58p Froa-NESTON SOLUTIONS T-021 P.002/005 P-050 Amendment Number Effective Date: RCH 3058Research Drive Phont: 814-272-10 State College, PA 16801 Fax: 814-231-1580 PROTOCOLAMENDMENT 11 12721/08 to M ---------------------------- DESCRIPTION O F AMENDE^} SECTION Protocol Distribution Section: 2) John M. Flaherty, Principal Investigator, Exygen Research '* -A M g N p a ia _ 2) Chartas Simons, Principal Investigator, Exygen Research nationale John Flahsrty retired and Chas Simons lies M a n over John's reta as Principal InvestiBaterfbr the study. No negativa Impact IMPACT ON STUDY 4 MQ a u d y o 4 ciirlv H O m c 0l _ A . Mn nucLpmiI m_W-!-,--jy-M--wCJ*Mn--i-i-i-w- iif UA syoach>Mw iggDB*sen*v --1111111 "" // JDat1feS /D '+7 Dais '-- om s -J tJ 'ia r i urn SponsorS bwM (ffoquM) OM OAU WL/Oate U S / fll/W d? uaw um nvaooiass RECEIVED TIM E JflN . 8 . 4:02P M AOMMiarmmveraAM PR IN T TIM E JAN. 8 . 4:04P M Exygen Research Page 109 o f 118 Interim Report #26 -- Analysis o f Groundwater Samples Exygen Study No.: P0001131 SEP.20.2035 152PM EXYGEN RESEfiRCH NO.377 P .2 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 D EV IA T IO N FO R M ________________ ' Penerai: X Project Specific Deviation ____ Facility Deviation _______ Pao 1 of 1 Date o f Occurrence: 03/16/05 Exygen Project#: P760/P1131 D eviation#: 1/1 Client Project # : NA R e fe re n ce # : 05-122 R sau latorv Driver: Deviation TVoe: (Include V for m ethods end SO PsJ X _____ _____ GMP OLP Other None Sam le D escrlntion: Protocol V#: 0001658-3 Notebook reference: N A M ethod X SOP Loo in #: N A Container#: N A L o t# : NA Sum m ary o f Deviation: T ills deviation pertains to all soil and sediment sam ples analyzed for percent solids before 07/07/05. a. No blanks or duplicates were run a s required by section 9.3. b. Som e sam ple weights exceed the allowable range f> 10g). C ause: ____Preparation_____A n a ly sis____ Instrum ent_____ d e n t Request X Other Irmsset: 1 There has bean no negative impact on the study. Ail of the percent solid values that were determined during the time period In question are considered valid, although the S O P w as not followed. In the newly revised version of the S O P blanks and duplicates are no longer required. Also, In the new SO P , the alow able amount of sam ple to be used is < 20 g. All of the sam ples In question in this deviation weighed less than 20 g. The technician analyzing the sam ples for percent so lid s w as following the new procedure before it w as formally approved. Corrective Action: A new version of the S O P has been Issued and approved (VOOO0427-3). LIBRARY ID: V0001640-8 Exygen Research ADMINISTRATIVE FORM Page 110 o f 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 SEP.20.2005 i:52PM EXYGEN RESERRCH n RESEARCH 3058 Research Drive State College, PA 16801 NO.377 P .3 Phone; 814-272-1039 Fax: 814-231-1580 d e iw il; X Project Specific Deviation D EV IA T IO N FO R M Facility Deviation P s a e lo fl Date of Occurrence: 06/29/05 Exygen Project#: P760/PU31 Deviation # ; 2/2 Client Project#: NA Reference#: O - / 3 ^ Regulatory Driver: Deviation Type: (Include V# formethods and SOPs) GMP GLP Other None Sample Daecrtatlon: X Protocol _____ Method V#:NA Notebook reference: NA _____ SOP Login#: L4264/L4256 Container#: C0056480-88 L o t# : _________ NA Summary of Deviation: The protocol states that control and fortified control samples of each matrix will be analyzed; however, control dam was not obtained. Fish was used as the control for the analysis of these six dam samples. Cause; ____Preparation_____Analysis____ Instrument_____Client Request X Other No negative impact on this study. Corrective Actions: Deviation Issued. I Investigator idy Director Quality Assurance f/sLo/tS" Date Sponsor Representative Jl Sponsor Management Date LIBRARY ID: V000164M ADMINISTRATIVE FORM Exygen Research Page 111 o f 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 SEP.20.2005 1!52PM EXYGEN RESEARCH n RESEARCH 3058 Research Drive State College, PA 16801 NO.377 P. 4 Phone: 814-272*1039 Fax: 814-231-1580 General: X Project Specific Deviation D EV IA T IO N FO R M Facility Deviation Paaal of 1 Date o f Occurrence: 12/27/04 Exygen Project # : P760/P1131 Deviation # : 3/3 Client Project#: NA Reference # : O S '-(5 Y ^ R eauH torv D rive r Deviation Tvne: (Include W formethods end SO Ps) QMP GLP Other None Sam ple Description: ____ Protocol V#: 202-20 Section 5.2.3 Notebook reference: N A Method X SOP Lo gin #: _______ NA C o n ta in e r# : NA Lot#: NA Sum m ary o f D eviation: SL2114 (30% Dimethyldichlorosilane in Toluene) w as given the expiration date of 02/13/05, but RE544 (Toluene) used to make SL2114 expired on 03/28/04. SL 2 1 14 wee used to eilanlzed glassw are prepared for the fish extraction from 12/27/04 through 01/07/05. C au se: ____Preparation_____A n a ly sis_____Instrum ent_____ Client Request X Other Im pact: No negative impact on the study. Toluene w as used only a s a solvent for the glassw are preparation. Dimethyldichlorosilane, which is the coating agent, w as not expired. Corrective A ctions: Deviation Issued. S ig n a tu ra : J a //$ d r U ^Principajinvetgatbr Study Director OyCa. S k d L t'Z Quality Assurance Date -------- ' Exygen Kfnpgdnent mm D atif UN Sponsor Representative Date klN Sponsor Managem ent A u tW Date Date Date LIBRARY ID: V0001640-6 ADMINISTRATIVE FORM Exygen Research Page 112 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 D E V IA T IO N F O R M ________________________________________ __ ___________________ Pag 1of2 G enew i: X Prefect Specific Deviation ___ Facility Deviation Date of Occurrence: 04126/06 Exygen Prefect # : P760IP1131 Deviation#: S Client Project#: ______NA_____ Referance#: 06-076 Regulatory Driver Deviation Type: /Indude VttformethodsandSOPs) ____ X ____ ____ GMP GLP Other None Sample Description: X Protocol ____ Method V#:NA Notebook reference: NA ____ SOP Login#: L0008191 Container#: NA Lot#: ________ NA Summary of Dovlstlon: The three sediment samples in L8191 (C0172892 - C0172894) ware originally extracted using the sediment method V0001782. Poor recoveries were obtained for PFOS, PFOA and nC PFOA. Because of this, the study sponsorrequested the use of an alternative extraction for these compounds, as follows: Direct Injection Method: Before the samples ware weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A one-gram portion of sediment was weighed into a 15-m31liter centrifuge tube for the extraction. Ten milliliters of 1% acetic acid in methanol was added to each sample. The samples were then shaken by hand, vortexed, and sonicated for thirty minutes. The samples ware then centrifuged for -10 minutes at -3000 ipm. Each sample was analyzed by LC/MS/MS electrospray. Using this method acceptable data was obtained for PFOS, but the recoveries for PFOA and UC PFOA were stilt poor. Another alternative method was then used for PFOA and ,JC PFOA, as follows: Alternative SPE Method: The samples that were prepared In 1%acetic add for the direct injection method were used for this extraction. Five m ilte rs of each sample was aliquoted into a 50-mL polypropylene centrifuge tube and the volume was taken to 40 mL with water. The samples were then centrifuged for -10 minutes at -3000 rpm. The supernatant was then loaded onto a C ,, SPE cartridge conditioned with 10 mL of methanol and 5 mL ofwater. The eluate was discarded. Approximately five nlliliters of methanol was added to the cartridge. Five m ilte rs of eluate was collected into a graduated 18 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS alactrospray. ' Causa: ___ Preparation____ Analysis____ Instrument____ Client Request X Other Impact: More usable data was obtained. LIBRARYID. V0001640-6 ADMINISTRATIVE FORM Exygen Research Page 113 of 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 CHEH EHSR 236 1B 651 733 1958 IM fM N OI:llN F rarSim SUITIORS 05/01 '06 13:46 NO.106 03/03 + m m r.Mvm m ii ` ` n M ta a rttM w ' P h o h r'ft 'in j . . SC A N C H . 0|VITIOWFOWi.. K a a "1 4 '." v faaflaiiu-- ' \ . Cbitm fiet: . . *< r jv> !.* t*<. .. * . **> : 'f$) , 7/ U U M tV IQ rV M U to t , ' Q U 4 'V p o n p p /, sV /.C . / * . * : . * . . 4< .5 ' * ' ./ , * ..V . * .' / v . . \.y . V , : ' \ '" < S >k ; ; > ; . . *, *. . 4 4 i*`\f .f .4 'rW.4444'4*}4' . . < . ...M i , . 4 "V ' .. 4 44 ' *.. . j\ ,, .. ' v '- 'i ; :i i t. : . i . ' i ' 4.:, 4 . v.4 /; . 1 . i' . 4 4 : . . - 4' i ; - "`V- ' '. i.\V ; & 4 .* 4V i ' . 4 t.` . ' 4. !>' ." . 4 ' 4 ' ( , , ' f , I i , , 4 , \ \ ;\s\ J V : * v** 4 ' 'lWA Mi B MN jMi v iljSniwJ Mi nM'Q ijHp il. .* . t i , , .* * " w 1> * . Exygen Research Page 114 o f 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB ' 651 778 4226 07/11/06 11:46 0 :04/07 NO:681 RESEARCH 3058 Research Prive Phone: 4-272-1039. State College, PA 16801 Fax: 814-231-1580 O B V IA T IO N F O R M sh e rfl: ^ '. X Project Specific Deviation ' Faculty 'Deviation Ml D ale of Occurrence: (W 2M B6 Exygen Project # : P780ijP1131 Deviation # :' ' .8 I-" . Client Project # : MA' ' P efirtn oe # : V V . 'f f i i f r " . Rmilitflfv PriYtr ' OMP ~ r~ olp ___;__ Other __ None Siimi PwplaflML L o gin # : NA D evliH pn fy n e : (Indudi VX formeffiotfa and SO P a) .. _____ _ Protocol ' X Method ' _____' 0 P : V*V00 0427-3 Notebook reference; NA C o n ta in e r# : 000189446' Lot#: M. Summery of Deviation: ": .. One eoi ampia (CO0158446) was weighed at -8g for percent solid analyaie, rather than at -20g which Is stated In the method. ' " : . ' C au sa: ____Preparation_____A n a ly sis_____Instrument Cileni Request Olhisr Im pact; ; ' .. . . N o negative Im pact An accurate percent solid number wee obtained by allghlly altering the caleulallonused. \ ' ... ' . g g n tc W sA c flo rw i Deviation Issued. `Untumi I , . a T r o fL L f Ipveetlgator ' Date / Exygen d r a g a m i ` Studyualm ctor itiM Ma Sponsor JthkL^r _,^iA Sponsor Managem ent Exygen Research Page 115 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB @ 651 778 4226 07/11/06 11:46 0 :06/07 NO.-681 RESEARCH 3058 Reiearch Drive Phone;' 814-272*1039 State College, P16801 Fair: 814^31-1580 DEVIATION FORM X . Project Specific Deviation __ _ Facility Deviation S L L Dita ofOccurranrii: 66/28/08 ExygenProject#; P760/P1131 Deviation#: k jt Client Pnjfleet # : NA , ' ' ; ' fteferenoe # : ' ReotilatOr Driver: Deviation TVna: (Includa V formathodt and SOP) \ ,, OMP X GLP ____ Other . ' . None lemrtePtecrletlgnz L o gin # : _______N A _____ Protocol ____ _ Method ' Vtfc 1 8 0 0 Notebook reference: - X - ' S F ;- C o n ta in e r# : NA Loft#:' L dip tim arire fD e viation: ' "" ' " : 1" Peer review of raw date w aa not documented per S O P V1800 prior td 8/28/06.. Cavee: ___ Preparation_____ Anfyle____ betrumont Im pact: . Client Regueet Raw data w ll be more thoroughly evaluated and reviewed before Q A Inepectlon. ' 5E i> Corrective Actione: A note to fie wW be leiudd to a i eubtequent reports i tifino that overall eummariea have bden pear reviewed. f Exygen Research Page 116 o f 118 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB 9 651 778 4226 0 :07/11/06 11:46 07/07 N0:681 n RESEARCH 3058 Research Drive Phone: t4-#2-1039 State Collie, PA 16801 Fax: 814-231-1580 .' ' . .; DEVIATION FORM Q u a rt: .. X Project Specific Deviation _ Facility Deviation Exygen Project#: P76/P131 PaP<^i D ita of Occurrence: 6/28AW D eviation#: U fr Client Project # : A. lloBulatdnr P rivar: D SvteM onTypa: ntuOa v for methods and SO P il ___ _ .X ... ___ ' OMP OLP Other Nona X Protocol _____ M eihod v a n a ;.. Notebook reference: ___ 8 flW l ftiB fta iM i L o g in *: L00Q8121 LO08O61 C o n ta in e r# : . llIffIBMry Bf BW lltfW fi ! Sam ples ware filiad to 250 mL Insteadof 200 mL. C 0 6 9 3 4 7 L o t# : C0169354 Ct71 6a, . A Catase: X Preparation __ ;A hoyis ;___ Instrument Im pact: .; . .; Sam ples could not ba extracted aecbrdlrrg to the protocol. Client Request Obrer S o tT K lh a t o l a i r SptklngTavela ware adjusted to accommodate the atfernetlvo ylme. pel Investigator / Study iuad^r Assurance (il2Sl(Xe D ate. Sponsor Managem ent -? o > / la tT Data Exygen Research Page 117 o f 118 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 0 1-0 8 -2 0 0 7 03:01m Froa-NESTOH SOLUTIONS T-021 P.005/005 F-0SS feyseir 3058 Research Drive Phonr 814-272-1039 State College, M1WM Fax: 814-231-15 y j ^ I research DEVIATIO N FO RM L ist i. X ProjectSpecificDeviation F a d % Deviation Exygen Project#: pooomi ta li ofOccurrence: Nov.2M6- jsu seL . D e viatio n # : 10 Client Pl^ect#: P0001131 Reference#: (\ l~ O Q L RwgWonrPrhrg" J & r tr fa T m P K ^ W /b r m a O x x k iin d S a P * ) OMP GLP Other Sarete beecmmon: X Metiod Vft V0001780 Notebook natetene*: SOP Login#: L0010155 UMICI65 Container#: Lot#: L0010254 Summary of Deviation: w d gk s u n w r a t o o w nw cpD pfi standardsused Inthe extracticn terthese logine wore prepared in aeelanMe Insteadof mohanol. X Preparation___ Analytic _ _ _ twtnenent____ Client Request _ Other No negative Impact. Compounds araeem pleW ysofublainaoaionMeasIheyarehm olhanoL Corrective AcUoiS T DeviationIssued. No action taken. 4 LIBRARYID: 1*0816*0-7 RECEIVED T IM E JAN. 8 . 4 :0 2 P M ADMINSTRATIV8 FORM PR IN T TIM E JAN. 8 . 4:04P M '. I Exygen Research Page 118 o f 118