Document MJMqQ5qDjo1NQpxbqVmD2L9wz

INTERIM REPORT # 4 - Analysis of Surface Soil Samples STUDY TITLE Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone:610-701-3761 . INTERIM REPORT COMPLETION DATE October 6, 2006 PERFORMING LABORATORY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR 3M Company 3M Building 0236-01-B -10 St. Paul, MN 55144 Phone: 651-733-6374 PROJECT Protocol Number: P0001131 Exygen Study Number: P0001131 Total Pages: 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number P0001131, entitled "Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program," conducted for 3M Company, is being performed in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by Exygen Research. Exygen Research Jaisimha , Study Director Weston Solutions, Inc. Michael A. Santoro' Sponsor Represenfittaa.tive 3M Company Exygen Research Date Page 2 of 115 Interim Report #4 - Analysis o f Surface Soil 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 phases' were inspected for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Exygen Principal Investigator and Management and to the Study Director. Phase Date Inspected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Studv Director lO.Raw Data Review and Interim Analytical Report Review 05/19-20/05, 05/23/05 06/30/05 08/19/05 09/13/05 37.Raw Data Review and Revised Interim Analytical Report Review 10/05,06/06 10/06/06 10/06/06 10/06/06 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion o f the study. This QA statement involves only the review o f the interim report and associated raw data. Exygen Research Page 3 o f 115 Interim Report #4 - Analysis of Surface Soil Samples Exygen Study No.: P0001131 CERTIFICATION OF AUTHENTICITY This interim report, for Exygen Study Number P0001131, is a true and complete representation o f the raw data. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Principal Investigator, Exygen: Date Exygen Research Facility Management: Richard A. Grazzini President Exygen Research (cfcl G m U n j Study Director, .Weston Solutions, Inc. Q& kL Jaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: Michael A. Santoro Director o f Regulatory Affairs Exygen Research h-Q C X -tyfp Date Date Page 4 o f 115 Interim Report #4 - Analysis o f Surface Soil 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: P 0 0 0 1131 EXYGEN STUDY NUMBER: P0001131 TYPE OF STUDY: Residue SAMPLE MATRIX: Soil and 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: 02/24/05 Interim Analytical Termination Date:06/13/06 Interim Report Completion Date: 10/06/06 Exygen Research Page 5 of 115 Interim Report #4 - Analysis o f Surface Soil 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 o f the study: Name John Flaherty Karen Risha Paul Connolly Christine Edwards Mark Ammerman Amy Sheehan Mark Neeley James Miller Brittany Kravets Mindy Cressley Kimberly Hall Frances Crespi Krista Gallant Ed Cams Title Vice President Scientist Technical Lead-LC/MS Technician Sample Custodian Associate Scientist Scientist Scientist Technician Technician Technician Technician Technician Sample Custodian Exygen Research Page 6 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 TABLE OF CONTENTS Page TITLE PA G E................................................................................................................................. 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEM ENT................................ 2 QUALITY ASSURANCE STATEM ENT................................................................................ 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 SUM MARY..........................................................................................................................11 2.0 OBJECTIVE.........................................................................................................................11 3.0 INTRODUCTION................................................................................................................ 11 4.0 ANALYTICAL TEST SAMPLES.....................................................................................12 5.0 REFERENCE M ATERIAL................................................................................................13 6.0 DESCRIPTION OF ANALYTICAL METHOD.............................................................14 6.1. Extraction Procedure For S o il........................................................................................ 14 6.2 Percent Solids Procedure For Soil...................................................................................14 6.3 Extraction Procedure For W ater......................................................................................15 6.4 Preparation o f Standards and Fortification Solutions................................................... 15 6.5 Chromatography................................................................................................................ 16 6.6 Instrument Sensitivity....................................................................................................... 16 6.7 Description o f LC/MS/MS Instrument and Operating Conditions............................. 16 6.8 Quantitation and Example Calculation........................................................................... 17 7.0 EXPERIMENTAL D ESIG N ............................................................................................. 19 8.0 R ESU LTS............................................................................................................................. 19 9.0 CONCLUSIONS..................................................................................................................20 10.0 RETENTION OF DATA AND SA M PLES..................................................................20 Exygen Research Page 7 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table I. LIST OF TABLES Page Summary o f PFBS, PFHS and PFOS in Soil Samples....................................22 Table II. Summary o f PFHS and PFOS in Re-extracted Soil Sam ples..........................24 Table III. Summary o f PFBS, PFHS and PFOS in Water Samples................................. 25 Table IV. Matrix Spike Recovery o f PFBS, PFHS and PFOS in Soil Sam ples............ 26 Table V. Matrix Spike Recovery o f PFHS and PFOS in Re-extracted SoilSamples ...31 Table VI. Summary o f Percent Solids in Surface Soil Sam ples.......................................33 f Exygen Research Page 8 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFBS in Reagent Water.................................... 35 Figure 2. Extracted Standards o f PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively............................................................................................................ 36 Figure 3. PFBS in Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively..............................................37 Figure 4. Chromatogram Representing a Soil Sample Analyzed for PFBS (Exygen ID: C0059474, Data Set: 022405C).....................................................38 Figure 5. Typical Calibration Curve for PFHS in Reagent W ater.....................................39 Figure 6. Extracted Standards o f PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively............................................................................................................ 40 Figure 7. PFHS in Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.............................................. 41 Figure 8. Chromatogram Representing a Soil Sample Analyzed for PFHS (Exygen ID: C0059474, Data Set: 022405C).....................................................42 Figure 9. Typical Calibration Curve for PFOS in Reagent W ater.....................................43 Figure 10. Extracted Standards o f PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively............................................................................................................ 44 Figure 11. PFOS in Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively...............................................45 Figure 12. Chromatogram Representing a Soil Sample Analyzed for PFOS (Exygen ID: C00059484, Data Set: 022405DR2)............................................. 46 Exygen Research Page 9 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 LIST OF APPENDICES Page A ppendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods and Protocol Am endm ents...........................................47 Exygen Research Page 10 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 1.0 SUMMARY Exygen Research extracted and analyzed surface soil samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001781 and analyzed water samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001780 (A ppendix A). The limit o f quantitation for PFBS, PFHS and PFOS in soil (wet weight) was 0.2 ng/g. The limit o f quantitation for PFBS, PFHS and PFOS in water was 25 ng/L. Analytical results for the analysis of PFBS, PFHS and PFOS found in soil samples and assessed accuracy for each sample is summarized in T able I. Samples designated as not reportable (NR) due to quality control failures were re-extracted and reanalyzed in an attempt to obtain quantitative results. Analytical results for the analysis o f PFHS and PFOS found in re-extracted soil samples and assessed accuracy for each sample are summarized in Table II. Analytical results for the analysis of PFBS, PFHS and PFOS found in water samples are summarized in Table III. Quantitative results were obtained for all samples and analytes except for PFOS in one sample, which is not reported due to quality control failures. Fortification recoveries for PFBS, PFHS and PFOS in the surface soil samples are detailed in Table IV. The average percent recoveries standard deviations for PFBS, PFHS and PFOS in the surface soil samples were 132 30%, 89 13% and 84 17%, respectively. Fortification recoveries for PFHS and PFOS in the re-extracted surface soil samples are detailed in Table V. The average percent recoveries standard deviations for PFHS and PFOS in the re-extracted surface soil samples were 101 24% and 99 25%, respectively. The total percent solids for soil samples are detailed in Table VI. 2.0 OBJECTIVE The objective o f the analytical part of this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) in soil and water according to Protocol P0001131 (A ppendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFBS, PFHS, and PFOS in water and soil using the analytical methods entitled, "V0001780: Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by Exygen Research Page 11 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 LC/MS/MS," and "V0001781: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS." The study was initiated on November 05, 2004, when the study director signed protocol number P0001131. The analytical start date for this interim report was February 24, 2005, and the analytical termination date for this interim report was June 13, 2006. 4.0 ANALYTICAL TEST SAMPLES Forty-six soil samples (Exygen ID C0059474-C0059519) and two water samples (Exygen ID C0059520 and C0059521) were received on wet ice on January 29, 2005 from Tim Frinak at Weston Solutions, Inc. The samples were logged in by Exygen personnel and placed in refrigerated storage. The soil sample identification (ID) codes for the samples analyzed and reported in Interim Report #22 are in the following format: Dxxx-xx-xxxxxx-x(x)-xxxx For soil samples, the first character D represents Decatur, Alabama samples and the xxx defines the sampling location within the study area where: DF06 = Field 6 DF8b = Field 8b DF09 = Field 9 DF14 = Field 14 (reference field) DBKG = Northwest comer reference area The second string defines the type o f sample where: SS = surface and shallow subsurface soil sample For soil samples the third string defines the soil sampling location. The fourth string additionally defines the sample type where: 0 Primary field sample 2 = Equipment rinseate blank sample The fifth string defines the collection depth o f the soil samples where: 0000 = 0 to 3 inches 0003 = 3 to 6 inches Sample log-in and chain o f custody information is located in the raw data package associated with this interim report. Storage records will be kept at Exygen Research. Exygen Research Page 12 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 5.0 REFERENCE MATERIAL The analytical standards, PFBS (SP252) and PFHS, were supplied by 3M. PFBS was received from 3M at Exygen on July 6, 2000. PFHS was received from 3M at Exygen on January 20, 2003. The analytical standards, PFBS (SP5726) was supplied by 3M and was received from 3M at Exygen on May 13, 2005. PFOS was purchased from Fluka Corporation and was received at Exygen on April 23,2003. The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated. Compound PFBS PFBS PFHS PFOS Exygen Inventory No. SP0000252 SP0005726 SP0002401 SP0002694 Lot # 101 101 SE036 430180-1 Puritv (%) 96.7 96.7 98.6 101.2 Expiration D 12/04/06 12/04/06 10/18/06 10/31/07 The molecular structures o f PFBS, PFHS and PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4F9S0 3 'K+) Transitions Monitored: 299 --> 99 Structure: F F SO3 FF FF PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CeFuSOsTC*) Transitions Monitored: 399 - 80 Structure: FF F F F F F FFF FFF O3 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFi7S0 3 'K+) Transitions Monitored: 499 --> 80 Exygen Research Page 13 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Structure: Exygen Study No.: P0001131 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical method "V0001781: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" was used for the soil samples in this study. The analytical method "V0001780: Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" was used for the water samples in this study. 6.1. Extraction Procedure For Soil Before the samples were weighed for the extraction, they were placed into a new, clean Ziploc bag and mixed thoroughly. The samples were then transferred back to the sampling container. A 5-gram portion o f soil was weighed into a fifty-milliliter centrifuge tube for the extraction. After fortification of appropriate samples, 5 mL of methanol was added to the samples. The samples were allowed to shake on a wrist action shaker for -15 minutes and were then sonicated in an ultrasonic bath for -15 minutes. The volume was taken to 40 mL with water in the same fifty-milliliter centrifuge tube and the samples were then centrifuged for -1 0 minutes at -3000 rpm. The supernatant was then loaded onto a Ci8 SPE cartridge conditioned with 10 mL o f methanol and 5 mL o f water. The eluate was discarded. Approximately five milliliters o f methanol was added to the cartridge. Five milliliters o f eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.2 Percent Solids Procedure For Soil Percent solids were determined using the procedure indicated in Exygen method V0000427. Approximately 20 grams of sample was weighed into a pan. The weight of the sample plus the pan was recorded. The sample was then dried in an oven overnight at 104 2 C. Then the sample was transferred to a dessicator and allowed to cool for -1 5 minutes. Each sample was then weighed again, including the weight o f the pan. The percent solid for each sample was then calculated. For percent solids data see Table VI. Exygen Research Page 14 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 6.3 Extraction Procedure For Water A 40 mL aliquot of the water sample was used for the extraction procedure. The samples were loaded onto a Cis SPE cartridge conditioned with 10 mL o f methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters o f methanol was added to the cartridge. Five milliliters o f eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.4 Preparation of Standards and Fortification Solutions Individual stock standard solutions of PFBS, PFHS and PFOS were prepared as specified in Exygen method V0001780. The stock standard solutions were prepared at a concentration o f 100 pg/mL by dissolving 10 mg o f each o f the standards (corrected for purity and salt content) in methanol. From these solutions, mixed 1.0 pg/mL fortification standard solutions were prepared by taking 1 mL of each of the appropriate stock solutions and bringing the volume up to 100 mL with methanol. By taking 10 mL o f the mixed 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.1 pg/mL fortification standard was prepared. By taking 10 mL o f the mixed 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.01 pg/mL fortification standard was prepared. A set of standards containing PFBS, PFHS and PFOS was prepared in water and processed through the extraction procedure, identical to samples. The following concentrations were prepared: Cone, of Fort Fort Solution Volume (ng/mL)1 0 (PL) 0 10 100 10 200 10 400 100 100 100 200 100 400 ' of PFBS, PFHS and PFOS Volume of Fortified Sample (mL) 40 40 40 40 40 40 40 Final Cone, of Calibration Std. (ng/L) 0 25 50 100 250 500 1000 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation o f standard preparation is located in the raw data package associated with this interim report. Exygen Research Page 15 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 6.5 Chromatography Quantification o f PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times o f PFBS, PFHS and PFOS were ~ 3.1 mins, ~ 10.1 mins, and ~ 12.8 mins, respectively. Peaks above the LOD were not detected in any o f the reagent blank samples corresponding to the analyte retention time. 6.6 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration o f 25 ng/L o f PFBS, PFHS and PFOS. 6.7 Description of LC/MS/MS Instrument and Operating Conditions Instrument: Interface: Comuter: Software: HPLC: API 4000 Biomolecular Mass Analyzer Turbo Ion Spray Liquid Introduction Interface DELL OptiPlex GX400 Windows NT, Analyst 1.4.1 Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: -30 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time ("min') 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: ~18min Flow Rate: 0.3 mL/min %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Exygen Research Page 16 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Ions monitored: Analvte PFBS PFHS PFOS Mode negative negative negative Transition Monitored 299 -> 99 399 80 499 80 Approximate Retention Time (min) ~3.1 min -10.1 min. -12.8 min. 6.8 Quantitation and Example Calculation Fifteen microliters o f sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations o f standards. The concentration was determined from the equations below. Equation 1 calculated the amount o f analyte found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/L) = (Peak area - intercept) x DF Slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. For samples fortified with known amounts of PFBS, PFHS, and PFOS prior to extraction, Equation 2 was used to calculate the percent recovery. Equation 2: Recovery (%) = (analyte found (ng/L-) - analyte in control (ng/LV) xl00% amount added (ng/L) Note: For the analyte recovery calculation, the "control" is the unspiked aliquot of the primary field sample. Equation 3 was used to convert the amount o f analyte found in ng/L to ng/g (ppb). Equation 3: analyte found (ppb) = fPFOA found (ng/L) x volume extracted (0.04L11 sample weight (5 g) Exygen Research Page 17 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Equation 4 was then used to calculate the amount o f analyte found in ppb based on dry weight. Equation 4: analyte found (ppb) dry weight = analyte found (ppb) x [100% / total solids(%)] NOTE: Total solids (%) = [wet weight (g) / dry weight (g)] x 100% An example o f a calculation using an actual sample follows: Soil sample Exygen ID C0059476 Spk G (Set: 022405CR), fortified at 500 ng/L with PFBS where: peak area = 15163 intercept = 0.000922 slope 140 dilution factor = 10 ng/L PFBS added (fort level) = 500 ng/L in corresponding sample (ng/L) = 553 volume extracted (L) = 0.04 sample weight (g) =5 total solids (%) = 82.23 From equation 1: Analyte found (ng/L) = T15163 - 0.0009221 x 10 140 = 1080 ng/L From equation 2: % Recovery = H080 ne/L - 533 ne/Ll x 100% 500 ng/L = 109% From equation 3: PFBS found (ppb) = H 080 ne/L x 0.04L1 5g = 8.64 ppb From equation 4: PFBS found (ppb) dry weight = 8.64 ppb x (100% / 82.23%) = 10.5 ppb Note: Numbers may vary slightly due to rounding. Exygen Research Page 18 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 7.0 EXPERIMENTAL DESIGN For soil samples designated as laboratory matrix spikes, PFBS, PFHS, and PFOS were added at a known concentration to the samples in the laboratory after the samples were weighed for extraction. For the water samples, bottles were shipped to the field, and filled to a 200 mL volumetric fill line. The soil samples were extracted in ten sets, five o f which contained re-extractions. Each set consisted o f one reagent blank and two reagent blanks fortified at known concentrations. Two sets contained ten sample sites, two sets contained eleven sample sites, and one set contained four sample sites. Two re-extraction sets contained five sample sites, one re-extraction set contained ten sample sites, one re-extraction set contained eight sample sites, and one re-extraction set contained three sample sites. One sample was collected for each sample site. For each site, two laboratory matrix spikes and a duplicate were extracted. The two rinse water samples from two sample sites were extracted together in their own set. No laboratory matrix spiking or duplicating was done on the rinse water samples. 8.0 RESULTS Analytical results for the analysis o f PFBS, PFHS and PFOS found in soil samples and assessed accuracy for each sample are summarized in Table I. Samples designated as not reportable (NR) due to quality control failures were re-extracted and reanalyzed in an attempt to obtain quantitative results. Analytical results for the analysis o f PFHS and PFOS found in re-extracted soil samples and assessed accuracy for each sample are summarized in Table II. Analytical results for the analysis o f PFBS, PFHS and PFOS found in water samples are summarized in Table III. Quantitative results were obtained for all samples and analytes except for PFOS in one sample, which is not reported due to quality control failures. To assess the accuracy o f the results, the matrix spikes were used. Matrix spikes that were in the appropriate concentration range (primary concentration has to be than or equal to three times the spiking level) were used to calculate the assessed accuracies. When the field matrix spikes were not available or failed to meet recovery criteria, the laboratory spikes were used. Note that several recovery percentages are marked as an asterisk. These samples were found to have analyte concentrations greater than three times that of the given spiking level. Fortification recoveries for PFBS, PFHS and PFOS in the surface soil samples are detailed in Table IV. The average percent recoveries standard deviations for PFBS, PFHS and PFOS in the surface soil samples were 132 30%, 89 13% and 84 17%, respectively. Fortification recoveries for PFHS and PFOS in the re-extracted surface soil Exygen Research Page 19 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 samples are detailed in Table V. The average percent recoveries standard deviations for PFHS and PFOS in the re-extracted surface soil samples were 101 24% and 99 25%, respectively. The total percent solids for soil samples are detailed in Table VI. 9.0 CONCLUSIONS Except as noted above, the soil samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001781. The water samples were also successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001780. 10.0 RETENTION OF DATA AND SAMPLES When the final analytical report is complete, all original paper data generated by Exygen Research will be shipped to the sponsor. This does not include facility-specific raw data such as instrument or temperature logs. Exact copies o f all raw data, as well as a signed copy o f the final analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period o f time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. Exygen Research Page 20 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 21 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Soil Samples Exygen ID C0059474 C0059474 Rep C0059475 C0059475 Rep C0059476 C0059476 Rep C0059477 C0059477Rep C0059478 C0059478 Rep C0059479 C0059479 Rep C0059480 C0059480 Rep C0059481 C0059481 Rep C0059482 C0059482 Rep C0059483 C0059483 Rep C0059484 C0059464 Rep C0059485 C0059485 Rep C0059486 C0059486 Rep C0059487 C0059487 Rep C0059488 C0059488 Rep C0059489 C0059489 Rep C0059490 C0059490 Rep C0059491 C0059491 Rep C0059492 C0059492 Rep C0059493 C0059493 Rep C0059494 C0059494 Rep C0059495 C0059495 Rep Client Sample ID DF06-SS-0001-0-0000 DF06-SS-0001-0-0000* DF06-SS-0001-0-0003 DF06-SS-0001-0-0003* DF06-SS-0002-0-0000 DF06-SS-0002-0-0000* DF06-SS-0002-0-0003 DF06-SS-0002-0-0003* DF06-SS-0003-0-0000 DF06-SS-0003-0-0000* DF06-SS-0003-0-0003 DF06-SS-0003-0-0003* DF06-SS-0004-0-0000 DF06-SS-0004-0-0000* DF06-SS-0004-0-0003 DF06-SS-0004-0-0003* DF06-SS-0005-0-0000 DF06-SS-0005-0-0000* DF06-SS-0005-0-0003 DF06-SS-0005-0-0003* DF09-SS-0001-0-0000 DF09-SS-0001-0-0000* DF09-SS-0001-0-0003 DF09-SS-0001-0-0003* DF09-SS-0002-0-0000 DF09-SS-0002-0-0000* DF09-SS-0002-0-0003 DF09-SS-0002-0-0003* DF09-SS-0003-0-0000 DF09-SS-0003-0-0000* DF09-SS-0003-0-0003 DF09-SS-0003-0-0003* DF09-SS-0004-0-0000 DF09-SS-0004-0-0000* DF09-SS-0004-0-0003 DF09-SS-0004-0-0003* DF09-SS-0005-0-0000 DF09-SS-0005-0-0000* DF09-SS-0005-0-0003 DF09-SS-0005-0-0003* DF8b-SS-0001-0-0000 DF8b-SS-0001-0-0000* DF8b-SS-0001-0-0003 DF8b-SS-0001-0-0003* C4 Sulfonate PFBS C6 Sulfonate PFHS Psrfluorobutsntsulfonste_______ PsrflMorohexsnesulfonale Analyte Found Assessed Analyte Found Assessed (ppb, n g ig ) Dry Weight Accuracy <+/-%> (ppb, ntfg) Dry Weight Accuracy (+/-%> 0.510 0.583 50 50 3.62 4.63 30 30 C8 Sulfonate PFOS PsrfluorooctartMulfonats Analyte Found (ppb, ngTg) Assessed Accuracy Dry Weight (+/- %) NR NR NR NR 0.551 0.720 50 50 5.19 10.1 30 30 NR NR NR NR 5.38 50 31.6 30 NR 3.09 50 24.0 30 NR NR NR 3.09 50 29.6 30 NR 3.39 50 25.7 30 NR NR NR 3.49 50 31.7 30 NR 3.73 50 31.3 30 NR NR NR 4.60 50 40.7 30 NR 3.12 50 38.2 30 NR NR NR 1.81 50 16.7 30 NR 2.39 50 17.6 30 NR NR NR 5.86 50 26.4 30 NR 2.36 50 21.6 30 NR NR NR 2.47 50 22.6 30 NR 2.78 50 21.6 30 NR NR NR 5.07 50 40.6 30 NR 3.28 50 32.7 30 NR NR NR 1.15 50 7.18 30 NR 1.19 50 7.90 30 NR NR NR 0.808 0.923 50 50 8.20 7.43 30 30 NR NR NR NR 1.34 50 8.60 30 NR 1.24 50 8.37 30 NR 1.21 50 9.47 30 NR 1.94 50 10.4 30 NR NR NR NR NR 3.64 50 37.0 30 NR 2.94 50 32.5 30 NR NR NR 2.60 50 54.9 30 NR 2.49 50 53.9 30 NR 5.59 50 83.9 40 NR 5.00 so 72.4 40 NR NR NR NR NR 4.58 50 59.4 30 NR 5.56 50 72.7 30 NR NR NR 4.39 50 47.9 30 NR 4.99 50 48.8 30 NR NR NR 5.35 50 80.7 40 NR 4.84 50 79.5 40 NR NR NR 13.6 9.72 50 50 NR NR NR NR NR NR NR NR 15.4 16.2 50 50 NR NR NR NR NR NR NR NR Laboratory Duplicate ND = Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). NR = Not reported due to quality control failure. For re-analysis see Table II. Exygen Research Page 22 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Soil Samples Continued Exygen ID C0059496 C0059496 Rep C0059497 C0059497 Rep C0059498 C0059498 Rep C0059499 C0059499 Rep C0059500 C0059500 Rep C0059501 C0059501 Rep C0059502 C0059502 Rep C0059503 C0059503 Rep C0059504 C0059504 Rep C0059505 C0059505 Rep C0059506 C0059506 Rep C0059507 C0059507 Rep C0059508 C0059508 Rep C0059509 C0059509 Rep C0059510 C0059510Rep C0059511 C0059511 Rep C0059512 C0059512 Rep C0059513 C0059513 Rep C0059514 C0059514 Rep C0059515 C0059515 Rep C0059516 C0059516 Rep C0059517 C0059517 Rep C0059518 C0059518 Rep C0059519 C0059519 Rep Client Sample ID DF8D-SS-0002-0-0000 DF8b-SS-0002-0-0000* DF8P-SS-0002-0-0003 DF8b-SS-0002-0-0003* DF8P-SS-0003-0-0000 DF8b-SS-0003-0-0000* DF8b-SS-0003-0-0003 DF8b-SS-0003-0-0003* DF8b-SS-0004-0-0000 DF8b-SS-0004-0-0000* DF8P-SS-0004-0-0003 DF8b-SS-0004-0-0003* DF8b-SS-0005-0-0000 DF8b-SS-0005-0-0000* DF8b-SS-0005-0-0003 DF8b-SS-0005-0-0003* DF14-SS-0001-0-0000 DF14-SS-0001-0-0000* D F14-SS-0001-0-0003 DF14-SS-0001-0-0003* DF 14-SS-0002-0-0000 DF14-SS-0002-0-0000* DF 14-SS-0002-0-0003 DF14-SS-0002-0-0003* DF 14-SS-0003-0-0000 DF14-SS-0003-0-0000* DF 14-SS-0003-0-0003 DF14-SS-0003-0-0003* DF14-SS-0004-0-0000 DF14-SS-0004-0-0000* DF 14-SS-0004-0-0003 DF14-SS-0004-0-0003* DF14-SS-0005-0-0000 DF14-SS-0005-0-0000* DF 14-SS-0008-0-0003 DF14-SS-0005-0-0003* DBKG-SS-0001-0-0000 DBKG-SS-0001-0-0000* DBKG-SS-0001-0-0003 DBKG-SS-0001-0-0003* DBKG-SS-0002-0-0000 DBKG-SS-0002-0-0000* DBKG-SS-0002-0-0003 DBKG-SS-0002-0-0003* DBKG-SS-0003-0-0000 DBKG-SS-0003-0-0000* DBKG-SS-0003-0-0003 DBKG-SS-0003-0-0003* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Pertluorobutanesulfonsto_______ P e rflu o ro h ix s n ts u tfo n its ______ Perfluorooctanesulfonite Analyte Found (ppb, ng/g) Dry Weight Assessed Accuracy (+/- %) Analyte Found (ppb, ng/g) Dry Weight Assessed Accuracy <'-*> Analyte Found (PPb, ng/g) Dry Weight Assessed Accuracy <+/-% ) 17.5 9.00 50 50 NR NR NR NR NR NR NR NR 15.4 11.0 50 50 NR NR NR NR NR NR NR NR 8.93 9.57 50 50 152 40 139 40 NR NR NR NR 8.48 8.25 50 50 189 50 184 50 NR NR NR NR 6.33 50 65.6 30 NR 7.00 50 68.0 30 NR NR NR 4.64 50 59.3 50 NR 5.35 50 63.6 50 NR NR NR 4.11 50 59.8 30 NR 6.96 50 78.8 30 NR NR NR 1.18 50 19.2 30 NR 1.40 50 22.9 30 NR NR NR 0.308 30 0.872 30 210 0.385 30 0.862 30 205 30 30 0.314 0.291 30 0.841 30 30 0.864 30 NR NR NR NR 0.437 0.288 30 30 ND 30 15.6 ND 30 15.7 30 30 ND 30 ND 30 30.5 30 ND 30 ND 30 30.2 30 0.253 ND 30 30 ND 30 11.8 ND 30 11.1 30 30 ND 30 ND 30 24.9 30 ND 30 ND 30 29.3 30 0.348 0.330 30 30 ND 30 28.6 ND 30 30.9 30 30 0.265 0.269 30 30 ND 30 36.7 ND 30 33.8 30 30 0.586 0.449 30 30 ND 30 69.7 ND 30 74.3 30 30 0.254 0.255 30 30 ND 30 126 ND 30 130 30 30 1.59 1.64 30 0.315 30 44.9 30 0.348 30 50.4 30 30 1.03 1.09 30 0.252 30 36.0 30 0.290 30 42.0 30 30 0.735 0.885 30 30 ND 30 23.1 ND 30 25.2 50 50 0.702 0.524 30 30 ND 30 23.7 ND 30 24.5 40 40 0.850 0.919 30 30 ND 30 61.4 ND 30 62.1 40 40 0.484 0.446 30 30 ND 30 151 ND 30 153 30 30 laboratory Duplicate ND = Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight) NR = Not reported due to quality control failure. For re-analysis see Table II. Exygen Research Page 23 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table II. Summary of PFHS and PFOS in Re-extracted Soil Samples C6 Sulfonate PFHS PerfluorohexsnesUfonste C8 Sulfonate PFOS P e riluorooctxn e sulfo n ste ExygenID Client Sample ID Analyte Found (ppb, ng/g) Dry W eight Assessed Accuracy (+/- %) Analyte Found (ppb, ng/g) Dry W eight Assessed Accuracy (+/.%) C0059474 DF06-SS-0001-0-0000 - - 1320 40 C0059475 C0059476 C0059477 C0059478 C0059479 C0059480 C0059481 C00S9482 C0059483 C0059484 C005948S C0059486 C0059487 C0059488 DF06-SS-0001-0-0003 DF06-SS-0002-0-0000 DF06-SS-0002-0-0003 DF06-SS-0003-0-0000 DF06-SS-0003-0-0003 DF06-SS-0004-0-0000 DF06-SS-0004-0-0003 DF06-SS-0005-0-0000 DF06-SS-0005-0-0003 DF09-SS-0001-0-0000 DF09-SS-0001 -0-0003 DF09-SS-0002-0-0000 DF09-SS-0002-0-0003 DF09-SS-0003-0-0000 - - - -' - - - - - 2790 5760 12300 12800 22700 3330 14400 5300 10800 1610 2490 2470 2330 21600 30 40 30 30 30 50 30 40 50 30 30 30 30 30 C00S9489 C0059490 C00S9491 C0059492 DF09-SS-0003-0-0003 DF09-SS-0004-0-0000 DF09-SS-0004-0-0003 DF09-SS-0005-0-0000 - - 59900 30 - 53500 30 - 65000 50 - 17300 30 C0059493 C00S9494 C0059495 DF09-SS-0005-0-0003 DF8b-SS-0001-0-0000 DF8b-SS-0001-0-0003 15.6 368 - NR 30 418000 30 668000 30 30 C00S9496 DF8b-SS-0002-0-0000 15.2 30 117000 30 C0059497 DF8b-SS-0002-0-0003 27.7 30 433000 50 C0059498 C0059499 C0059S00 C0059501 C0059502 C0059503 C0059505 DF8b-SS-0003-0-0000 DF8b-SS-0003-0-0003 DF8b-SS-0004-0-0000 DF8b-SS-0004-0-0003 DF8b-SS-0005-0-0000 DF8b-SS-0005-0-0003 DF14-SS-0001 -0-0003 - - 273000 30 - 273000 30 - 166000 30 - 156000 30 - 131000 30 - 52800 30 - 278 30 Laboratory Duplicate ND = Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). NR = Not Reported due to Quality Control failures. Exygen Research Page 24 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table III. Summary of PFBS, PFHS and PFOS in Water Samples Exygen ID C0059520 C0059521 Client Sample ID DF09-SS-0003-2-0000 DF8b-SS-0004-2-0000 Analyte Found (ppt, ng/L) ' C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesutfonate Perfluorohexaneaulfonate Perfluorooctanesulfonate ND ND 112 ND ND 27.4 ` Laboratory Duplicate ND = Not detected at or above the Lim it of Quantitation (LOQ) of 25 ng/L. Exygen Research Page 25 of 115 Interim Report #4 - Analysis of Surface Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Sample Description Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (na/g) (ng/g) wet wt. (no/g) wet wt. <%) (no/o) wet wt. (ng/g) wet wt. <%) (na/g) wet wt. (ng/g) wet wt. <%> DF06-SS-0001-0-0000 (C0QS9474SpkC, 4 ppb Spike) DF06-SS-0001-0-0000 (C0089474 Spk D, 40 ppb Spike) DF06-SS-0001-0-0003 (COO5*471 Spk E, 4 ppbSpike) DF06-SS-0001-0-0003 (COO99476 SpkF, 40 ppb Spike) 4 40 4 40 0.445 0.445 0.484 0.484 7.48 62.3 6.65 62.8 176 3.15 155 3.15 154 4.57 156 4.57 7.55 110 NR 39.8 92 NR 8.96 110 NR 41.3 92 NR NR NR NR NR NR NR NR NR DF06-SS-0002-0-0000 (COOSS47SSpk 0 ,4 ppb Spike) DF06-SS-0002-0-0000 (C0009470 Spk H, 40 ppb Spike) DF06-SS-0002-0-0003 (COO99477 Spk 1,4 ppb Spike) DF06-SS-0002-0-0003 (C0099477 Spk J, 40 ppbSpike) 4 40 4 40 4.43 4.43 2 66 2.66 8.66 106 26.0 65.1 152 26.0 7.72 127 25.5 61.2 146 25.5 24.8 62.8 92 24.6 57.7 81 NR NR NR NR NR NR NR NR NR NR NR NR DF06-SS-0003-0-0000 (COO69478 Spk K, 4 ppb Spike) DF06-SS-0003-0-0000 (COO99478 Spk L, 40 ppb Spike) 4 40 2.96 2.96 9.08 153 26.9 64.6 154 26.9 29.2 62.1 86 NR NR NR NR NR NR DF06-SS-0003-0-0003 (C00S9479 Spk M, 4 ppb Spike) DF06-SS-0003-0-0003 (COO99479 Spk N, 40 ppb Spike) 4 40 3.91 391 10.5 165 34.6 599 140 34.6 47.1 67.3 82 NR NR NR NR NR NR DF06-SS-0004-Q-0000 (C00S9480 Spk 0 ,4 ppb Spike) DF06-SS-0004-0-0000 (COO99480 Spk P, 40 ppb Spike) 4 40 1.55 1 55 7.54 150 14.3 59.1 144 14.3 20.3 46.2 60 NR NR NR NR NR NR DF06-SS-0004-Q-0003 (C0099481 Spk Q, 4 ppb Spike) DF06-SS-0004-0-0003 (C0099481 Spk R, 40 ppbSpike) 4 40 4.99 4.99 7.58 65 22.5 67.1 155 22.5 19.9 57.0 86 NR NR NR NR NR NR DF06-SS-0005-0-0000 (COO99482Spk S, 4 ppb Spike) DF06-SS-0005-0-0000 (COO99482SpkT. 40 ppb Spike) 4 40 2.10 2.10 7.94 146 19.2 61.6 149 19.2 26.3 60.6 104 NR NR NR NR NR NR DF06-SS-0005-0-0003 (C0069483SpkU, 4 ppbSpike) DF06-SS-0005-0-0003 (COO99483 SpkV, 40 ppb Spike) 4 40 4.12 4.12 9.82 143 33.0 61.0 142 33.0 33.1 63.2 76 NR NR NR NR NR NR Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND Not detected at or above the Limit of Quantitation (LOQ) o f 0.2 nj^g {wet weight). NR * Not reported due to quality control failure. For re-analysis see Table V. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. Exygen Research Page 26 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Amount Spiked (ng/g) C4Sulfoiiby*FBSWsWelght Amt Found Amount in Sample Recovered Recovery (ng/g) wet wt. (ng/g) wet wt. (%) Amt Found Amount in Sample Recovered (ng/g) wet wt. (ng/g) wet wt. Recovery (%> Amt Found Amount in Sample Recovered (no/a) wet wt. (ng/a) wet wt. Recovery (%> DF09-SS-0001-0-0000 (COOSMM8pk C, 4 ppb Spike) DF09-SS-0001-0-0000 (COOSMM Sph D, 40 ppb Spike) 4 40 0.976 0.976 7,16 155 6.10 60.0 148 6.10 9.81 93 43.5 94 NR NR NR NR NR NR DF09-SS-0001-0-0003 (COOSMSSSpk E, 4 ppb Spike) DF09-SS-0001-0-0000 (COOSMSSSpk F, 40 ppb Spike) 4 40 0.694 0.694 6.90 155 7.03 57.9 143 7.03 11.9 122 44.3 93 NR NR NR NR NR NR DF09-SS-0002-0-0000 (COOSMSSSpk0 ,4 ppb Spike) DF09-SS-Q002-0-0000 (COOSMSSSpk H, 40 ppb Spike) 4 40 1.13 1.13 7.06 148 7.28 57.0 140 7.28 11.2 98 44.7 94 NR NR NR NR NR NR DF09-SS-0002-0-0003 (C00SMS7 Spk 1,4 ppb Spike) DF09-SS-0002-0-0003 (C005MS7 Spk J, 40 ppbSpike) 4 40 1.05 1.05 6.75 143 8.17 56.8 139 8.17 13.0 121 45.8 94 NR NR NR NR NR NR DF09-SS-0003-0-0000 (COOSMMSpkK, 4 ppb Spike) DF09-SS-0003-0-0000 (C00SS4M Spk L, 40 ppb Spike) 4 40 3.03 3.03 8.05 126 30.8 59.2 140 30.6 31.3 * 65.4 87 NR NR NR NR NR NR DF09-SS-0003-0-0003 (COOSMSSSpk M, 4 ppb Spike) DF09-SS-0003-0-0003 (COOSMSSSpk N, 40 ppb Spike) 4 40 2.22 2.22 8.03 145 46.8 58.1 140 46.8 47.1 . 77.4 77 NR NR NR NR NR NR DF09-SS-0004-0-0000 (C00SS4S0Spk0 ,4 ppbSpike) DF09-SS-0004-0-0000 (COOSMSOSpk P, 40 ppb Spike) 4 40 4.42 4.42 10.6 155 66,4 59.2 137 66.4 68.7 91.1 62 NR NR NR NR NR NR DF09-SS-0004-0-0003 (C00S94S1 Spk 0 ,4 ppb Spike) DF09-SS-0004-0-0003 (C006S491 Spk R, 40 ppb Spike) 4 40 3.87 3.87 9.60 143 50.1 60.3 141 50.1 57.4 . 86.4 91 NR NR NR NR NR NR DF09-SS-0005-0-0000 (C00SM92 SpkS, 4 ppb Spike) DF09-SS-0005-0-0000 (COOSS4S2SpkT, 40 ppb Spike) 4 40 3.44 344 8.93 137 37.5 61.9 146 37.5 40.8 * 754 95 NR NR NR NR NR NR DF09-SS-0005-0-0003 (COOSMSSSpk U, 4 ppb Spike) DF09-SS-0005-0-0003 (COOSMSSSpkV. 40 ppb Spike) 4 40 4.40 4.40 10.3 148 66.4 61.4 143 66.4 746 . 92.6 66 NR NR NR NR NR NR ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND * Not detected at or above the Limit of Quantitation (LOQ) o f 0.2 ng/g (wet weight). NR * Not reported due to quality control failure. For re-analysis see Table V. Note: Since thle summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 27 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Description C4 Sulfonate PFBS Wet Weight C6 Sulfonate PFHS Wet Weight C8 Sulfonate PFOS Wet Weight Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/a) (no/a) wet wt. (no/a) wet wt. i%) (nofQ) wet w t (na/o) wet wt. (%> (nata) wetwt. (no/a) wet wt. <%) DF8b-SS-0001-0-0000 (C00SS4S4SpkC, 4 ppbSpike) DF8b-SS-0001-0-0000 (C00SMS4 Spk D, 40 ppbSpike) 4 40 10.1 10.1 14.4 108 NR 68.8 147 NR NR NR NR NR NR NR NR NR NR NR DF8b-SS-0001-0-0003 (C00SS4SSSpk E, 4 ppbSpike) DF8b-SS-0001-0-0003 (C00SS4SSSpkF, 40 ppb Spike) 4 40 12.3 12.3 13.5 NR 68.6 141 NR NR NR NR NR NR NR NR NR NR NR DF8b-$S-0002-0-0000 (C00SS4M Spk 0 ,4 ppb Spike) DF8b-SS-0002-0-0000 (C00SS4M Spk H, 40 ppb Spike) 4 40 12.2 12.2 12.7 NR 62.6 126 NR NR NR NR NR NR NR NR NR NR NR DF8b-SS-0002-0-0003 1C009S497Spk 1,4 ppb Spike) DF8b-SS-0002-0-0003 (COOSS497 Spk J. 40 ppbSpike) 4 40 11.2 11.2 14.8 90 NR 681 142 NR NR NR NR NR NR NR NR NR NR NR DF8b-SS-0003-0-0000 (C005S4M SpkK. 4 ppb Spike) DF8b-SS-0003-0-0000 (C00S04S8 Spk L, 40 ppb Spike) 4 40 6.46 6.46 13.5 176 110 63.6 143 110 105 NR 134 60 NR NR NR NR NR DF8b-SS-0003-0-0003 (COOSS499Spk M, 4 ppb Spike) DF8b-SS-0003-0-0003 (COOSS4M Spk N, 40 ppb Spike) 4 40 6.62 6.62 13.8 180 147 64.8 145 147 162 . NR 204 143 NR NR NR NR NR DF8b-SS-0004-0-0000 (C00SM00Spk 0 .4 ppb Spike) DF8b-SS-0004-0-0000 (COOS9SOOSpk P, 40 ppb Spike) 4 40 4.81 4.81 11.0 155 49.9 61.7 142 49.9 56.1 * 83 3 84 NR NR NR NR NR NR DF8b-SS-0004-0-0003 (CQOM501 spk Q, 4 ppb Spike) DF8b-SS-0004-0-0003 (C00S0S01 Spk R, 40 ppb Spike) 4 40 3.85 3.85 10.4 164 49.3 58.1 136 49.3 52.2 * 69.7 51 NR NR NR NR NR NR DF8b-SS-0005-0-0000 (C006M02 spk 8 .4 ppb Spike) DF8b-SS-0005-0-0000 (C00SNQ2SpkT, 40 ppb Spike) 4 40 3,21 3.21 15.2 300 46.7 61.3 145 46.7 94.1 * NR 78.1 79 NR NR NR NR NR DF8b-SS-0005-0-0003 (CO0B95O3SpkU. 4 ppb Spike) DF8b-SS-0005-0-0003 (C00S9903 SpkV. 40 ppb Spike) 4 40 1.02 1.02 6,67 141 16.7 58.7 144 16.7 27.7 . 55.0 96 NR NR NR NR NR NR DF14-SS-0001-0-0000 (C0049504 Spk W. 4 ppbSpike) DF14-SS-0001-0-0000 (C00MS04 Spk X. 40 ppb Spike) 4 40 0.250 0.250 4.54 107 0.708 40.9 102 0.708 4.09 65 36.6 90 170 170 166 198 70 'Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND * Not detected at or above the Limit of Quantitation (LOQ) o f 0.2 ngfg (wet weight). NR * Not reported due to quality control failure. For re-analysis see Table V. Note: 8ince this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. / Exygen Research Page 28 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample C4 Sulfonate PFBS Wet Weight C6 Sulfonate PFHS Wet W ight C8 Sulfonate PFOS Wat Weight Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/g) (ng/g) wet wt. (ng/g) wet wt. <%> (no/Q) wet wt. (ng/g) wet wt. (%) (na/g) wet wt. (ng/g) wet wt. <%> DF14-SS-0001-0-0003 (COOSSSOSSpkC, 4 ppb spike) DF14-SS-0001-0-0003 (COOSSSOSSpk D, 40 ppb Spike) 4 40 0.257 0.257 4.77 113 0.688 46.0 114 0.688 4.45 94 36.3 89 NR NR NR NR NR NR DF14-SS-0002-0-0000 (COOSSSOSSpk E, 4 ppbSpike) DF14-SS-0002-0-0000 (COOSSSOSSpk F, 40 ppb Spike) 4 40 0.367 0.367 4.19 96 ND 42.0 104 ND 3.78 95 13.1 35.9 90 13.1 19.3 51.2 95 DF14-SS-0002-0-0003 (C00SSS07SpkOt 4 ppbSpike) DF14-SS-0002-0-0003 (C00SSS07Spk H, 40 ppb Spike) 4 40 ND ND 3.95 99 ND 43.6 109 ND 3.56 89 26.0 36.2 91 26.0 27.8 62.6 92 DF14-SS-0003-0-0000 (COOSSSOSSpk 1,4 ppb Spike) DF14-SS-0003-0-0000 (COOSSSOSSpk J, 40 ppbSpike) 4 40 0.212 0.212 4.53 108 ND 53.3 133 ND 3.59 90 9.87 36.2 91 9.87 14.1 106 45.5 89 DF14-SS-0003-0-0003 (COOSSSOSSpk K, 4 ppb Spike) DF14-SS-0003-0-0003 (COOSSSOSSpk L, 40 ppb Spike) 4 40 ND ND 4.83 121 ND 46.8 117 ND 3.60 90 20.2 34.9 87 20.2 17.8 . 47.6 69 DF14-SS-0004-0-0000 (COOSSSIOSpk M, 4 ppb Spike) DF14-SS-0004-0-0000 (C00SSS10Spk N, 40 ppb Spike) 4 40 0.284 0.284 4.44 104 ND 40.0 99 ND 3 75 94 23.3 35.1 88 23.3 30.4 . 61.3 95 DF14-SS-0004-0-0003 (C00SSS11Spk 0 ,4 ppb Spike) DF14-SS-0004-0-0003 (C005SS11 Spk P, 40 ppbSpike) 4 40 0.225 0.225 4.50 107 ND 38.7 96 ND 3.70 93 31.2 34.7 87 31.2 32.3 . 64.6 84 DF14-SS-0005-0-0000 (C00SSS12Spk Q, 4 ppb Spike) DF14-SS-0005-0-0000 (C00SSS12Spk R, 40 ppb Spike) 4 40 0.492 0.492 4.96 112 ND 41.7 103 ND 3.62 91 58.5 36.4 91 58.5 63.1 . 93.6 88 DF14-SS-0005-0-0Q03 (COOSHIS Spk S. 4 ppb Spike) 4 0.216 2.97 69 ND DF14-SS-0005-0-0003 (C00SH1S SpkT, 40 ppb Spike) 40 0.216 33.6 83 ND 3.63 91 107 33.4 84 107 114 . 135 70 DBKG-SS-0001 -0-0000 (C00SH14 Spk U, 4 ppb Spike) DBKG-SS-0001-0-0000 (C00SSS14SpkV, 40 ppb Spike) 4 40 1.32 1.32 623 123 0.262 52.2 127 0.262 3.66 85 37.3 34.8 66 37.3 44.6 73.8 91 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND * Not detected at or above the Limit of Quantitation (LOQ) o f 0.2 ngfg (wet weight). NR = Not reported due to quality control failure. For re-analysis see Table V. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 29 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Description C4 Sulfonate PFBS Wet Weight C6 Sulfonate PFHS Wet Weight C8 Sulfonate PFOS Wet Weight Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/a) (no/a) wet wt. (na/a) wet wt. (%) (ng/g) wet wt. (ng/a) wet wt. <%) (ngig)wetwt. fno/Q) wet wt. <%) DBKG-SS-0001-0-0003 (C0069S1SSpkW, 4 ppbSpike) DBKG-SS-Q001-0-0003 (COOStHSSpk X, 40 ppb Spike) 4 40 0.853 0.853 6.05 130 0.209 51.7 127 0.209 3.70 87 29.9 32.7 81 29.9 32.5 71.3 104 DBKG-SS-0002-0-0000 (C005SS1SSpkC. 4 ppbSpike) DBKG-SS-0002-0-0000 (C00MS1S Spk D, 40 ppbSpike) 4 40 0.612 0.612 4.55 98 39.8 98 ND ND 3.34 84 19.2 32.6 82 19.2 21.9 41.5 56 DBKG-SS-0002-0-0003 (COOSSS17Spk E, 4 ppbSpike) 4 0.599 4.08 87 ND DBKG-SS-0002-0-0003 (C00SM17 Spk F, 40 ppb Spike) 40 0.599 38.6 95 ND 3.38 85 20.2 34.2 86 20.2 22.8 45.9 64 DBKG-SS-0003-0-0000 (C00SK18 Spk 0 .4 ppbSpike) DBKG-SS-0003-0-0000 (C005*S18 Spk H, 40 ppbSpike) 4 40 0.699 0.699 4.92 106 ND 44.6 110 ND 3.63 91 50.5 35.5 89 50.5 52.3 75.0 61 DBKG-SS-0003-0-0003 (COOffS1SSpk 1,4 ppb Spike) DBKG-SS-0003-0-0003 icoonsie Spk J, 40 ppbSpike) 4 40 0.405 0.405 4.22 95 ND 40.3 100 ND 3.59 90 35.5 89 127 127 121 170 108 Average: 132 Standard Deviation: 30 Average: Standard Deviation: 89 13 ' Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NO * Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). NR * Not reported due to quality control failure. For re-analysis see Table V. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Average: Standard Deviation: 84 17 Exygen Research Page 30 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table V. Matrix Spike Recovery of PFHS and PFOS in Re-extracted Soil Samples Sample Description DF06-SS-0001-0-000G (C0059474 Spk E, 4000 ppb Spike) DF06-SS-0001-0-0003 (C0059475 Spk G, 8000 ppb Spike) DF06-SS-0002-0-0000 (C0059476 Spk 1, 20000 ppb Spike) DF06-SS-0002-0-0003 (C0059477 Spk K. 20000 ppb Spike) DF06-SS-0003-0-0000 (COOS9478 Spk M, 20000 ppb Spike) DF06-SS-0003-0-0003 (C0059479 Spk O, 80000 ppb Spike) DF06-SS-0004-0-0000 (C0059480 Spk Q, 8000 ppb Spike) DF06-SS-0004-0-0003 (C0059481 Spk S, 20000 ppb Spike) DF06-SS-0005-0-0000 (C0059482 Spk U, 20000 ppb Spike) DF06-SS-0005-0-0003 (C0059483 Spk W, 20000 ppb Spike) DF09-SS-0001-0-0000 (COOS9484 Spk E, 4000 ppb Spike) DF09-SS-0001-0-0003 (COOS9485 Spk G, 4000 ppb Spike) DF09-SS-0002-0-0000 (C00S9486 Spk I, 4000 ppb Spike) DF09-SS-0002-0-0003 (C00S94B7 Spk K. 8000 ppb Spike) DF09-SS-0003-0-0000 (C0059488 Spk M , 40000 ppb Spike) DF09-SS-0003-0-0003 (C0059489 Spk O, 80000 ppb Spike) DF09-SS-0004-0-0000 (C0059490 Spk Q, 60000 ppb Spike) DF09-SS-0004-0-0003 (COOS9491 Spk S, 60000 ppb Spike) DF09-SS-0005-0-0000 (C0059492 Spk U, 40000 ppb Spike) DF09-SS-0005-0-0003 (C0059493 Spk W, 60000 ppb Spike) Amount Spiked (nfl/a) C6 Sulfonate PFHS Wet Weight C8 Sulfonate PFOS Wet Weight Amt Found Amount in Sample Recovered (ng/g) wet wt. (ng/g) wet w t Am t Found Amount Recovery in Sample Recovered <%) (ng/g) w et wt. (ng/g) w et wt. Recovery (%) 4000 - 1150 6390 131 8000 - 2420 10100 96 20000 - 4730 31200 132 20000 - 10500 29000 93 20000 - 10900 27600 84 80000 - 19400 11600012 1 8000 - - 2850 14100 141 20000 - - - 12300 32200 100 20000 - - 4500 32500 140 20000 - 8760 19600 54 4000 - - 1370 4940 89 4000 - 2140 4950 70 4000 - - 2090 5310 81 8000 40000 - - 2010 9840 98 - - 17900 52700 87 80000 - - 51000 14700012 0 80000 - - - 42300 116000 92 80000 40000 80000 - - 54900 99900 56 - 13500 58800 113 - - NR NR NR Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated ND = Not detected at or above 0.2 ng/g (wet weight) NR * N ot reported due to Quality Control failures. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 31 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table V. Matrix Spike Recovery of PFHS and PFOS in Re-extracted Soil Samples Continued Sample Description DF8b-SS-0001-0-0000 (C00SS4M Spk E, 40 ppb Spike) DF8b-SS-0001-0-0000 (C0059494 Spk F, 400000 ppb Spike) DF8b-SS-0001-0-0003 (C0059496 Spk G, 800 ppb Spike) DF8b-SS-0001-0-0003 (C0069495 Spk H, 800000 ppb Spike) DF8b-SS-0002-0-0000 (C0069496 Spk 1, 40 ppb Spike) DF8b-SS-0002-0-0000 (C0069496 Spk J, 400000 ppb Spike) DF8b-SS-0002-0-0003 (C00S9497 Spk K, 800 ppb Spike) DF8b-SS-0002-0-0003 (C0059497 Spk L, 800000 ppb Spike) DF8b-SS-0003-0-0000 (C00S9498 Spk M, 400000 ppb Spike) DF8b-SS-0003-0-0003 (C0069499 Spk 0 ,800000 ppb Spike) DF8b-SS-0004-0-0000 (C0069500 Spk Q, 400000 ppb Spike) DF8b-SS-0004-0-0003 (C0059601 Spk S, 400000 ppb Spike) DF8b-SS-0005-0-0000 (C0089S02 Spk U, 400000 ppb Spike) DF8b-SS-0005-0-0003 (C0069503 Spk W, 400000 ppb Spike) DF14-SS-0001-0-0003 (C0069506 Spk E, 800 ppb Spike) Amount Spiked (ng/g) C6 Sulfonate PFHS Wet Weight C8 Sulfonate PFOS Wet Weight Amt Found Amount Amt Found Amount in Sample Recovered Recovery in Sample Recovered Recovery (ng/g) wet wt. (ng/g) wet wt. (%) (ng/g) wet wt. (ng/g) wet wt. (%) 40 400000 800 800000 40 400000 800 800000 11.6 - 292 10.6 - 20.2 - 60.5 122 - - 310000 604000 74 941 81 . 530000 1110000 73 42.4 80 .. - - 81500 523000 110 986 121 .. - - 315000 1470000 144 400000 - - 197000 586000 97 800000 - - - 213000 1080000 108 400000 - - - 126000 520000 99 400000 - - - 129000 649000 130 400000 - - - 102000 439000 84 400000 - - - 45800 291000 61 800 - - - 227 1070 105 Average: Standard Deviation: 101 24 Average: Standard Deviation: 'Sam ple residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated ND = Not detected at or above 0.2 ng/g (wet weight) Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. 99 25 Exygen Research Page 32 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Table VI. Summary of Percent Solids in Surface Soil Samples ExygenID C0059474 C0059475 C0059476 C0059477 C0059478 C0059479 C0059480 C0059481 C0059482 C0059483 C0059484 C0059485 C0059486 C0059487 C0059488 C0059489 C0059490 C0059491 C0059492 C0059493 C0059494 C0059495 C0059496 C0059497 C0059498 C0059499 C0059500 C0059501 C0059502 C0059503 C0059504 C0059505 C0059506 C0059507 C0059508 C0059509 C0059510 C0059511 C0059512 C0059513 C0059514 C0059515 C0059516 C0059517 C0059518 C0059519 Client Sample ID DF06-SS-0001-0-0000 DF06-SS-0001-0-0003 DF06-SS-0002-0-0000 DF06-SS-0002-0-0003 DF06-SS-0003-0-0000 DF06-SS-0003-0-0003 DF06-SS-0004-0-0000 DF06-SS-0004-0-0003 DF06-SS-0005-0-0000 DF06-SS-0005-0-0003 DF09-SS-0001-0-0000 DF09-SS-0001-0-0003 DF09-SS-0002-0-0000 DF09-SS-0002-0-0003 DF09-SS-0003-0-0000 DF09-SS-0003-0-0003 DF09-SS-0004-0-0000 DF09-SS-0004-0-0003 DF09-SS-0005-0-0000 DF09-SS-0005-0-0003 DF8b-SS-0001-0-0000 DF8b-SS-0001-0-0003 DF8b-SS-0002-0-0000 DF8b-SS-0002-0-0003 DF8b-SS-0003-0-0000 DF8b-SS-0003-0-0003 DF8b-SS-0004-0-0000 DF8b-SS-0004-0-0003 DF8b-SS-0005-0-0000 DF8b-SS-0005-0-0003 DF14-SS-0001-0-0000 DF14-SS-0001-0-0003 DF14-SS-0002-0-0000 DF14-SS-0002-0-0003 DF14-SS-0003-0-0000 DF14-SS-0003-0-0003 DF14-SS-0004-0-0000 DF14-SS-0004-0-0003 DF14-SS-0005-0-0000 DF14-SS-0005-0-0003 DBKG-SS-0001-0-0000 DBKG-SS-0001-0-0003 DBKG-SS-0002-0-0000 DBKG-SS-0002-0-0003 DBKG-SS-0003-0-0000 DBKG-SS-0003-0-0003 Percent Solid 87.10 87.94 82.23 85.96 84.84 85.15 85.60 85.19 84.84 81.26 84.97 85.82 84.61 86.30 83.19 85.26 79.16 84.45 78.29 82.36 74.21 79.38 69.94 72.78 72.27 78.10 76.06 83.09 78.04 86.71 81.24 81.83 84.04 84.95 83.62 81.05 81.64 85.01 83.99 85.12 83.09 82.88 83.21 85.40 82.33 83.65 Exygen Research Page 33 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 34 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 1. Typical Calibration Curve for PFBS in Reagent Water 0224060.rdb (PFBS): " U n t il" R tg r n io n ("1 / * ' lig h tin g ): y - 144 x + -0.000147 ( i= 0.0060) Area, counts Exygen Research Page 35 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively XC022295-4 9 ng/L Standard PFBS (Standard) pa n* not found) -aampt* 1 o f 57 from $22405tXwiff Intensity, cps Tim , min XC 022203-1, 2 0 n g/L S tandard - PFBS (S ta n d a rd )2 00 0 /0 0 0 im u - sam ple 2 of 0 7 fio m O224O0D.wiff Area: 3800 counts Height: 1D8. cps RT: 3.17 min 3.17 Intensity, cps Exygen Research Page 36 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 3. PFBS in Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively Rtagaut Controf A ' PFBS (Unknown) 299H/M0m -aampfa 9 o f 57 from 0224Q5C*wiff p a a t aot found) 11.34 ,,1 1 .0 4 1* ? 8 >14.00 Intensity, cps 0 10 Tim a, min g R e agent Spike A . SO n g /L . PFBS COC) 2 00 .0/0 0.0 im u - sam ple 10 e l 5 7 from 0 2 2 4 0 9 C.miff A re e :4 8 7 B counts Height: 130. ops RT: 3 .1 3 m in 11 3.13 100 12 13 14 15 Intensity, cps 0 10 11 12 13 14 Tim e, min | R t a g t n t Spile* B, 9 00 n g/L - PFBS (QC)20Q.OA)Q.O am u s jm p li 11 o f5 7 <rom022<KKC.*ifl A rea: 4 4 0 0 0 counts Height: 1050. ops RT: 3 .1 4 m in Intensity, cps Tim e, min Exygen Research Page 37 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Soil Sample Analyzed for PFBS (Exygen ID: C0059474, Data Set: 022405C) CMS9474 PFBS ftM Ammm) 29&W910 tm u - im m p lt 1S o f 37 from 9224MC.WHT m A rm : 7835 .*<* H tig tl: 1S3. c p t RT: 3.1Sm in 3.15 Exygen Research Page 38 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 5. Typical Calibration Curve for PFHS in Reagent Water 0 22 4 0 3 C rtv2 .rd b (PFHS): "L in a a i" R . g r . M o n f l / x " lig h tin g ): y = 567 x + 130 0 .0 0 73 ) Area, counts Exygen Research Page 39 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively I XC02229S-4 ag/L Standard - PFHS (Standard) 399.0/1010 am a -aam p/a 1 o f 57 from Q224Q5C>wifT A n a : 13$eo aat HaigHt: 9.30 cpa RT: 19.1 min 10.13 Tim , min XC22205-1, 23 ng/L Standard - PFHS (S tandard)30fl.0ffl0.0 mu sample 2 of 37 from G22403C.wiff Area: 1 44 74 counts H eight: 1200. cps RT: 10.0 min Intensity, cps Intensity, cps Exygen Research Page 40 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 7. PFHS in Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively I R tu g tu t Control A -PFHS (UnitMown) 399*0/90.0 am * - ta m p ft 9 o f 57 from 922405C,wiff G>*a9 n o t found) 12.32 Intensity, cps Intensity, cps Li,It . llMV?*i0.70, ,, 2 .0 0 I -3 .7 2 . __ 0.27 7.78. | R eagent Spike A . SO n g /L - PFHS (Q C)3QQ.0/B0.0 im u - sam ple 10 o f 57 fro m 0 2 2 4 0 5 C.wiff A rea: 2Q530 oounts H e ig h t 2 30 0. ops RT: 10.0 m in 2000 10.01 1000 0 e.37se^ 0 10 Tim e, min I R e agent Spike B. 5 0 0 n g /L - PFHS (Q C )3Q 0.0/B0.0 amu sam ple 11 o f5 7 from 02240 5 C .w iff Area: 200 07 0 counts Height: 24000. cps RT: 10.0 min s --15.42 7.0 Q 14 15 10 17 14 15 15 17 Intensity, cps Exygen Research Page 41 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Soil Sample Analyzed for PFHS (Exygen ID: C0059474, Data Set: 022405C) C00SW74 - PFHS (UkUkowm) 3t9lMHlLt am a - m m p ie IS o fS T fr o m 022405C.twff A n a ; 23*205 c e n rte H tigkt: 500. cpa RT; 1*.ttmia 10.04 Intensity, cps Exygen Research Page 42 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 9. Typical Calibration Curve for PFOS in Reagent Water 022409 DR2 Surface S oil Rev4.rdb (PFOS): "L in e a i" Regression ("1 w eighting): y 390 x + 0.13e+003 C r - 0 .0008) Area, counts Exygen Research Page 43 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively XC032195-% 0 *?(L Stadrd PFOS (Stmndard) 49910/1* 0 m u -sam pi* 1 o f 57 from 022495DR2.wifT A n ; 9134cot Haight: 1159. cp RJ; 12.%m i 12.00 A rea: 1 74 34 counts H eight: 1000. ops RT: 12.0 min 12.70 Intensity, cps Intensity, cps Exygen Research Page 44 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 11. PFOS in Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively Rangent Control A - PFOS (Unknown) now -nam pte 9 o f 37 from k224kSDRZwiff 1 A n n : 31641 c o n n * Hkfgkt: 426k. cpa RT: 12.%m in uUaJ 4 0 0 0 U> 2 0 0 0 a> 1 2 3 4 5 5 7 8 0 10 11 Tim , min I Raagant Spika A, 50 n g/L * PFOS (QC) 4 M .0/80.0 am u* sample 10 of 07 from 022405DR2.wifi A rt : 3 12 10 eounts H aight: 3 30 0. eps RT: 12 8 min 12.80 I 1 2 .3 2 1 At A 12 13 I R a a g a n tS p ika B. 5 0 0 n g /L * PFOS (Q C) 4 M .0/80.0 a m u -s a m p le 11 of 5 7 fr o m 0 2 2 4 0 5 DR2.miff A la a : 2 1 2 7 5 3 counfe H aight: 2 3 3 0 0 . ops RT: 12.6 m in Exygen Research Page 45 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Soil Sample Analyzed for PFOS (Exygen ID: C00059484, Data Set: 022405DR2) C$059494, DF-1090 PFOS (UtAttown) 499.0/80.0 s m s ss m p ls 15 o f 57 from 0224050RZwiff A n s : 59337 count H eight: 7939. cp s RT: 12.9 m in 12.00 Intensify, cps Exygen Research Page 46 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods and Protocol Amendments Exygen Research Page 47 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in W ater, Soil, Sedim ent, Fish, Clam s, Vegetation, Small M am m al Liver and Small M am mal Serum Using LC/M S/M S for the 3M Decatur M onitoring Program E xygen P rotocol N um ber: P 0 0 0 1 131 Perform ing Laboratory: E xygen Research 3058 R esearch D rive State C ollege, PA 16801 Phone: (814)272-1039 Sponsor R epresentative: M ichael A. Santoro D irector o f R egulatory A ffairs 3M B uilding 0 2 3 6 -0 1-B -10 St. Paul, M N 55144 Phone: (651) 733-6374 Page I o f 63 Exygen Research Page 48 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 DISTRIBUTION: 1) Jaisim ha Kesari, Study Director, W eston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) M ichael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 o f 65 Page 49 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PROTOCOL APPROVAL S tu d y T itle: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohcxanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in W ater, Soil, Sediment, Fish, Clams, Vegetation, Small M am mal Livers and Sm all M am m al Serum U sing LC/M S/M S for the 3M Decatur M onitoring Program E xygen Protocol N um ber: P 00 0 1 131 APPROVALS Jaisim h atK esan , S' W eston Solutions M ichael A. S toro, Sponsor Representative 3M Comparo' Date LydfifShaffer, Tec E xigen Research * 1 ____________ :ad, Q uality A ssurance U nit D ate A Page 3 oj 05 Exygen Research Page 50 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 TABLE OF CONTENTS TITLE PAGE...................................................................................................................................................... 1 DISTRIBUTION.................................................................................................................................................2 PROTOCOL APPROVAL..................................................................................................................................3 TABLE OF CONTENTS................................................................................................................................... 4 INTRODUCTION...............................................................................................................................................5 TEST MATERIALS...........................................................................................................................................5 OBJECTIVE.......................................................................................................................................................6 TESTING FACILITY.........................................................................................................................................6 STUDY DIRECTOR...........................................................................................................................................7 SPONSOR REPRESENTATIVE....................................................................................................................... 7 PRINCIPAL INVESTIGATOR......................................................................................................................... 7 PROPOSED EXPERIMENTAL START AND TERMINATION DATES...................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM..........................................................8 SAMPLE PROCUREMENT, RECEIPT AND RETENTION.......................................................................... 8 SAMPLE IDENTIFICATION........................................................................................................................... 9 ANALYTICAL PROCEDURE SUMMARY.................................................................................................... 9 VERIFICATION OF ANALYTICAL PROCEDURE....................................................................................... 9 METHOD FOR CONTROL OF BIAS...............................................................................................................11 STATISTICAL METHODS............................................................................................................................... 11 GLP STATEMENT............................................................................................................................................ 11 REPORT............................................................................................................................................................. 11 SAFETY AND HEALTH................................................................................................................................... 12 AMENDMENTS TO PROTOCOL....................................................................................................................13 DATA RECORD KEEPING.............................................................................................................................. 13 QUALITY ASSURANCE.................................................................................................................................. 14 RETENTION OF DATA AND ARCHIVING...................................................................................................14 APPENDIX I, ANALYTICAL METHODS.......................................................................................................15 Page 4 o f 65 Exygen Research Page 51 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 INTRODUCTION The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFO S) in water, soil, sedim ent, fish, clam s, vegetation, sm all m am m al livers and sm all mam m al serum using LC/M S/M S for the 3M Decatur M onitoring Program . The study w ill be audited for compliance with EPA TSCA G ood Laboratory Practice Standards 40 CFR 792 by the Quality A ssurance U nit o f Exygen Research. TEST MATERIALS The test m aterials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M. PFBS Chemical Nam e: Perfluorobutanesulfonate M o lecu lar W eight: 338 supplied as the p o tassium salt (C 4F9SO 3I O L ot N um ber: 101 Purity: 96.7% Transitions M onitored: 299 -> 99 Structure: PFHS Chem ical Name: Perfluorohexanesulfonate M olecular W eight: 438 supplied as the potassium salt (CeFuSOjTC*) Lot Number: SE036 Purity: 98.6% Transitions M onitored: 399 -* 80 Structure: Page 5 o f 65 Exygen Research Page 52 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Prolocol Number: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate M olecular W eight: 538 supplied as the potassium salt (C sFnSO j'K *) Lot Number: 217 Purity: 86.9% Transitions M onitored: 499 - 99 Structure: F S03 OBJECTIVE The purpose o f this study is to perform analysis for perfluorobutanesulfonaie (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFO S) in w ater, soil, sedim ent, fish, clam s, vegetation, sm all m am m al livers and sm all m am m al serum for the 3M D ecatur M onitoring Program using the current versions o f the following Exygen analytical methods: V0001780: V0001781: V0001782: V0001783: V0001784: V0001785: V0001786: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in W ater by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Fish and Clam s by LC/M S/M S" "M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Small M am mal Liver by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Small M am mal Serum by LC/M S/M S" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 Page 6 o f 65 Exygen Research Page 53 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000113 1 STUDY DIRECTOR Jaisim ha Kesari P.E., DEE W eston Solutions, Inc. 1400 W eston W ay W est Chester, PA 19380 Phone: (610) 701-3761 Fax: (610) 701-7401 j .kesari@ w estonsolutions.com SPONSOR REPRESENTATIVE M ichael 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 Decem ber 31, 2005. The actual experimental start and termination dates will be included in the final report. Page 7 o f 65 Exygen Research Page 54 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The follow ing are the test system s for this study: W ater (groundwater and surface water) Soil Sediment Fish Clams Vegetation Sm all M am m al Liver Small M am m al Serum The sam ples will be collected by W eston Solutions. The control sam ples will be purchased and prepared by the testing facility. Purchase and processing details for the control sam ples will be included in the final report associated w ith this study. The test system s were chosen to access the environm ental impact o f PFBS, PFHS and PFOS in the Decatur, Alabam a area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION W ater, soil, sediment, fish, clam, vegetation, sm all m am m al liver and small m am m al serum sam ples w ill be received at Exygen directly from W eston Solutions. The details o f sam ple procurem ent for this study are outlined in the 3M w ork plan entitled "Phase 2 W ork Plan for Sam pling Environm ental M edia." The num ber and types o f sam ples collected will vary depending availability in the field. The total num ber o f sam ples received and analyzed for each m atrix w ill be docum ented in the final report associated with this study. W ater, 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 m am m al liver sam ples will be processed acco rd in g to the ap p ropriate analytical m eth o d (see A ppendix I). T hese samples will be stored frozen at -10"C. Small mammal whole blood sam ples will be centrifuged in the field at the tim e o f collection and the serum fraction w ill be used for the study. Sm all m am m al serum will be stored frozen at S -10C. The receipt and processing o f the sam ples w ill be docum ented in the final report and raw data associated with the study. Page 8 o f 65 Exygen Research Page 55 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 <*" * Exygen Protocol Number P0001131 SAMPLE IDENTIFICATION Prior to analysis, each sam ple will be assigned a laboratory sam ple reference number. T he reference num ber will be unique and will distinguish each laboratory sam ple that is processed throughout the analytical procedure. Chrom atographic data w ill be identified by the laboratory sam ple reference num ber. Sam ple storage conditions and locations w ill be docum ented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: V0001780: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in W ater by LC/M S/M S" V0001781: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/M S/M S" V0001782: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/M S/M S" V0001783: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Fish and C lam s by LC/M S/M S" V0001784: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/M S/M S" V0001785: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Small M ammal Liver by LC/M S/M S" V0001786: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Small M am mal Serum by LC/M S/M S" The above m ethods use analytical conditions capable o f separating the isom ers o f PFBS, PFHS and PFOS. The final report will include the isomers sum m ed into total PFBS, total PFHS, and total PFO S found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sam ple will be used for the preparation o f fortified control samples. The test substance w ill be m ade into solutions as per the m ethod, and added to the m atrices via a micropipette. For w ater sampling, Exygen w ill supply one bottle per sam ple collected. The bottles w ill be 500 m L precleaned Sci/Spec Prem ier wide m outh HDPE bottles. These bottles have been routinely used for fluorochem ical sample Page 9 o) 65 Exygen Research Page 56 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 r**' collection at the testing facility and have been show n to be free o f PFBS, P F H S and P F O S . Sam ples w ill b e added to each co n tain er to a v o lu m etric fill line at 200 mL. A field duplicate, a low field spike and a high field spike o f each sam ple will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (13C P F O A ). P F O A and 13C P F O A are included in the solutions used to spike the samples. The results for PFOA and l3C P F O A w ill n ot be reported in th is study. E xygen w ill su p p ly on e field blank (control water) and two field blank spikes (control w ater fortified with PFBS, PFHS and PFOS at a low and high level) for every tw enty sam ples collected. At the testing facility, each w ater sam ple (excluding field duplicates and field spikes) w ill be extracted in duplicate and w ill also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determ ine method accuracy and to check for bias. For soil, sedim ent, clam s, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Prem ier wide m outh HD PE bottle per sam ple collected or a zip-seal bag. All containers/bags used for sam ple collection will be shipped to the sam ple location. Sam ples will be added to each container or bag in the field. At the testing facility, each sam ple will be extracted in duplicate and w ill also be fortified at a know n concentration w ith PFB S, PFH S and PFO S at ^ both a low and high level and processed through the described procedure to determ ine method accuracy and to check for bias. For sm all m am m al liver, Exygen will supply a 50 m L polypropylene centrifuge tube. For sm all m ammal serum, Exygen will supply a collection kit for each sam ple containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sam ple w ill be extracted in duplicate and w ill also be fortified at a known concentration w ith PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determ ine m ethod accuracy and to check for bias. Low and high spiking levels for each matrix are defined below: M atrix Low Spiking Level High Spiking Level W ater 500 ng/L 5000 ng/L Soil S edim ent 4 ng/g 4 n g /g 40 ng/g 4 0 n g /g Fish 10 ng/g 100 ng/g C lam s 10 ng/g 100 ng/g V eg etatio n 10 ng/g 100 ng/g Small M ammal Liver 10 ng/g 100 ng/g Small M am mal Serum lO ng/m L 100 ng/mL Page 10 o f 65 Exygen Research Page 57 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Recoveries are anticipated to be betw een 70% and 130% o f the fortified levels; however, the exact precision and accuracy w ill be determ ined by the analysis o f the quality control sam ples described above. A statem ent o f accuracy w ill be included in the final report. METHOD FOR CONTROL OF B U S Control o f bias will be addressed by taking representative sub-sam ples from a hom ogeneous m ixture o f each matrix from untreated control sam ples, and by analyzing at least two levels o f fortifications. STATISTICAL METHODS Statistics will be lim ited to those specified in the subject m ethods and to the calculation o f average recoveries, as applicable. GLP STATEMENT All aspects o f this study shall be perform ed and reported in com pliance with EPA T SC A G ood Laboratory Practice Standards 40 C FR 792. T he final report or data package (supplied to the Sponsor) shall contain a statem ent that the study was conducted in compliance with current and applicable GLP standards and w ill outline any deviations in the study from those standards. This statem ent will be signed by the Study Director and Sponsor R epresentative. REPORT A final report will be prepared by the principal investigator or their designee at the conclusion o f the study. T he report will include, but will not be limited to, the following: The nam e and address o f the Study Director, Sponsor Representative, and o f the testing facility. A statem ent o f GLP com pliance (any related docum entation, such as chain-of-custody records, m ust be in the study records). Page II o f 65 Exygen Research Page 58 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 The signed and dated statem ent by the Exygen Research Quality A ssurance Unit regarding dates o f study inspections and dates findings were reported to the Study Director and M anagement. A description o f the exact analytical conditions em ployed in the study. If the subject m ethod w as follow ed exactly, it is necessary to include only a copy o f the analytical method. A ny m odifications to this m ethod will be incorporated into the report. If the m ethod is photo-reduced, the project num ber and page num ber m ust be included on each page. Description o f the instrumentation used and operating conditions. All results from all sets analyzed. Control and fortified sam ples will be identified and the data table w ill include sam ple num ber and fortification level. Representative chrom atogram s for each analyte in each m atrix, including chrom atogram s o f a standard and a control sam ple, and a chrom atogram at a fortification level. The location o f the analyte peaks will be clearly identified in all chrom atogram s. All circumstances that m ay have affected the quality or integrity o f the data will be docum ented in the report. Locations where raw data and the final report are to be archived. A dditions or corrections to the final report shall be in the form o f an am endm ent signed by the Study Director. The am endm ent shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendm ent will be signed and dated by the Study D irector and the Sponsor Representative. All applicable requirem ents 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 environm ent to the test or reference substance(s). Page /2 o f 65 Exygen Research Page 59 of 115 Interim Report #4 - Analysis o f Surface Soil 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 D irector and Sponsor Representative. Am endments usually will be issued prior to initiation o f study plan change. H ow ever, w hen a change is required w ithout sufficient tim e for the issue o f a w ritten amendm ent, that change m ay be effected verbally with supporting docum entation signed and dated by the Study Director and follow ed w ith a w ritten am endm ent as soon as possible. In this case, the effective date o f the written am endm ent w ill be the date o f the documented change. Copies o f the signed amendm ents w ill b e appended to all distributed study plan copies. T he original amendm ent w ill be m aintained with the original study plan. A ny deviations from the study plan or from the analytical m ethod as provided will be docum ented and reported prom ptly to the Sponsor Representative. DATA RECORD KEEPING Records to be m aintained include the follow ing (as appropriate): Sam ple tracking sheet(s) Sam ple receipt records, storage history, and chains o f custody H istory and preparation o f standards (stock, fortification, calibration) Description o f any m odifications to the m ethod Instrum ent run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sam ple extraction and analysis dates A com plete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence A ny other documentation necessary for the reconstruction o f the study Chromatograms- A ll chrom atogram s w ill contain the follow ing: Sam ple identification, injection date, arrow or other indication o f the area o f interest, and injection num ber corresponding to the run. Additionally, fortifications w ill include the am ount o f analyte added and the sam ple num ber o f the sam ple that w as fortified. Analytical standard chrom atogram s will additionally include the concentration (e.g., pg/mL). Page 13 o f 65 Exygen Research Page 60 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 A s p at o f the docum entation the follow ing sheets w ill be included in each analytical set: a run sheet listing the sam ples to be run in the set, and an instrum ent conditions sheet describing the instrum ent type and operating conditions. QUALITY ASSURANCE The Q A Unit o f Exygen Research w ill inspect the study at intervals adequate to assure com pliance with G L P 's, and will report the findings o f audits to the Study Director, Exygen M anagement, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING All hard copy raw data, including, but not lim ited to, the original chromatogram s, worksheets, correspondence, and results shall be included with the data package subm itted to the Study Director. These will be archived with the original study plan, amendm ents, final report, and all pertinent inform ation from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipm ent, and storage logs for the period o f tim e specified in 40 CFR 792.195. An exact copy o f the m aterials subm itted to the study director will also be kept at Exygen Research. Exygen w ill obtain perm ission from the study director before discarding or returning samples. Exygen Research Page 14 o/'6S Page 61 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 APPENDIX I ANALYTICAL METHODS V0001780: V0001781: V0001782: V0001783: V0001784: V000178S: V0001786: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in W ater by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Sedim ent by LC/M S/M S" "M ethod o f Analysis for the D eterm ination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/M S/M S" `M e th o d o f A n alysis for the D eterm ination o f P erfluorooctanoic A cid (PFOA) in Vegetation by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Sm all M am mal Liver by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Small M am mal Serum by LC /M S/M S" Exygen Research Page IS o f 65 Page 62 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOI131 ANALYTICAL METHOD Method Number: V000I780 M ethod o f A a alyiia fo r the Determ laadoa o f Perfluorooctaoolc A d d (PFD A) ia W ater byLC /M S /M S Analytical Teeting Facility: Eaygen Reeearch 3058 Reeearch Drive State College, PA 16801 Approvod By: Paul ConnoHy 1 Technical Leader, LC-M S, Exygen Research 2/ ^ /hAzf /J o h n Flaherty 7 ' VV iiccee PD rmesi iidUennlt, Of Vpaeeraattiifotni s, Exygen Research w in.AW Date Date Exygen Research Total Pages: 7 Page /6 o f 65 Page 63 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reewrci Meibod Number VOOOPgO L A N A LY T IC A L M ETHO D Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC /M S/M S 1.0 Scope This method ia to be employed fo r the isolation and quantitation o f perfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectromethc Detector (LC/MS/M S) in water. 2.0 Safety 2.1 Atwaya observe aafe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical fo r proper safely precaution. 3.0 Sample Requirement 3.1 A t least 40 o iL 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 A ll samples must be thoroughly mixed before being sampled for extraction. 3.$ Any samples containing particles should be centrifuged at *3000 rpm for -5 minutes and the supernatant used for the extraction. 3.6 Sample collection procedures w ill be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P LC grade 4.2 Methanol - HPLC grade 4.3 Ammonium A cetate-A .C .S . Reagent Grade 4.4 Perfluorooctanoic Acid - Sigm a-Aldrich 3.0 Instrument and Equipment 3.1 A high performance liq u id chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/M S/M S). 3.2 A device to collect raw data fo r peak integration and quantitation. 3.3 Analytical balance capable o freading to 0.00001 g. 5.4 SOm L disposable polypropylene eentrifiige tubes. 3.3 13 m L disposable polypropylene centrifuge tubes. 3.6 Disposable micropipets (SO-lOOuL, 100-200uL). 3.7 125-roL LDPE narrow-mouth bottles. 5.8 2 m L cle a rH P L C via lkit. 5.9 Disposable pipettes. 5.10 Autopipectes (100-1000 pL and 10-100 pL), w ith disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tC IS SPE cartridges. Page2 o f' Page 7 o f 65 Exygen Research Page 64 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000M31 Exygtn R auvcb Method Number V00017M ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC /M S/M S $.12 SPB vacuum manifold. S.13 Centrifoge capable o f spinning $0 m b polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: FluophaseRP (Keystone S cientific), 2.1 mm x 50 mm, 5jj (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase< A ): 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ): Methanol 6.5 Gradient Program: Time (m ini 0.0 1.0 8.0 20.0 22.5 21A 65 65 25 25 65 Flow Rate % B fm L/m in) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as SO pL). 6.7 Q uantitation: Peak A rea-external standard calibration curve6.8 RunTim e: - 2 3 minutes. The above conditions sre intended as a guide and may be changed in order to optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospny Negative M RM mode, m onitoring 413 369 m/z. The above conditions are intended as a guide and may be changed in order to optim ize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. Exygen Research P age 18 o f 65 Page 65 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO131 Exygen Research Method Number VOW1780 ANALYTICAL METHOD Method o f A n a lyst fo r the Determination o f Perfloorooctanoic A cid (PFOA) in Water by LC /M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f ~100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected fo r purity) and dilute to l DO m l with methanol in a 125-mL LDPB bottle. 9.1.2 A 10 pg/m L fortifica tion solution o f PFOA is prepared by bringing ID m L o f the 100 pg/m L solution to a final volume o f 100 w ith methanol in a 125 m l LDPB bottle. 9.1.3 A 1.0 pgDnL fortifica tion solution o f PFOA is prepared by bringing ID m L o fth e 10 pg/m L solution to a final volume o f 100 w ith methanol m a 125 m L LDPE bottle. 9.1.4 A 0.1 pg/m L fortifica tion solution ofPFO A is prepared by bringing ID m L o fth e 1.0 pg/m L solution to a fin a l volume o f 100 w ith methanol in a 125 m L LDPB bottle. 9.1.5 A 0.01 p g /m l fortifica tion solution ofP FO A is prepared by bringing 10 m L o f the 0.1 pg/tnL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.6 The stock and fortifica tion solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The follow ing is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortificati) Volume o f Concentration of Calibration o f Fortificati! Volume Fortified Control Calibration Standard ID Solution (nob) (PL) Semole (mL) Standard (not)* (example) 0 0 40 0 XCmmddyy-0 to 100 40 25 XCmmddyy-1 10 200 10 400 100 100 too 200 100 400 40 40 40 40 40 50 100 250 500 1000 XCmmddyy-2 XCmmddyyO XCmmddyy-4 XCmmddyy-5 XCmnxWyy-6 * The extracted concentration o f the calibration standard is equal to Bx its in itia l concentration, due to the concentration o fthe standard during the extraction (SPE). XC - extracted calibration standard. Page 4 o f "* Page 19 o f 65 Exygen Research Page 66 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bxygm ReMuch Method Number VQOOI7SO L ANALYTICAL METHOD Method o fAnalysis fo r the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LCVMS/MS 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) m m t be prepared w ith each set o f standards extracted. Store a ll extracted calibration standards in 13-mL polypropylene tubes 2*C to 6CC. up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f sample! extracted (typically 20 or less) must include at least one reagent control (method blank uaing HPLC water) and two reagcm controls fortifie d st known concentrations (lab control spike) to verify procedural recovery fo r the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Measure 40 m L o f sample or a portion o f sample diluted to 40 m L w ith water into 50 m L polypropylene centrifiige tubes (fo rtify as needed, replace lid and m ix w ell). 11.2 Condition the C u SPE cartridges (1 g, 6 m L) by passing 10 mL methanol followed by 5 m L o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.3 Load sample on conditioned C u SPE cartridge. Discard eluate. 11.4 Elute w ith -5 m L 100% methanol. C ollect 5 mL o f eluate into graduated IS raL polypropylene centrifiige tubes (fin a l volume - 5 mL). 11.5 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards may be injected * the beginning o f a sei followed by extracted calibration standards interspersed every 5*10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the firs t and last injection in a sample set. 12.4 Use linear standard curves fo r quantitation. Linear standard curves are generated for the analyte by linear regression using l/x weighting o f peak area Psgi Ol 7 Page 20 o f 65 Exygen Research Page 67 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Eem rch Method Number V0001780 ANALYTICAL METHOD Method o f Analysis fo r the Determination o fPerfluorooctanoic A cid (PFOA) in Water by LC /M S/M S venue calibration standard ooneantration using MassLynx 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 C riteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA snion, while the daughter ion (369 amu) represents the lo u 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 50 ng/L, then a new blank sample must be obtained and the entire act must be re-extracted. 13.3 Recoveries o f control spikes and m atrix ipikes must be between 70*130% o f their known values. I f a control spike falls outside the acceptable lim its, the entire set o f samples should be re-extracted. Any m atrix spike outside 70* 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Teat, may be excluded from the calculation o f the calibration curve However, die total number o f extracted calibration standards that could be excluded must not exceed 20% o f die total number o f extracted standards injected. The correlation coefficient (R ) for calibration curve generated must be 0.992 (R2 0.985). I f calibration results fa ll outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than 4 % w ithin an analytical nm. I f retention tim e d rift exceeds this lim it within an analytical nm then the set must be reanalyzed. 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Maas Lynx software program: PFOA found (ng/L) * (Peak area - intercept) x DF slope DF Actor by which the final volume was diluted, if necessary. Page 4 el 7 Page 21 o f 65 Exygen Research Page 68 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Rcwarch Method Number V000I780 I ANALYTIC AL METHOD | Method o f Analysis for the Determination o fPerfluorooctanoic A cid (PPOA) in Water by LC /M S/M S 14.2 For samples fo rtifie d w ith known amounts o f PFOA prior to extraction, use the follow ing equation to calculate the percent recovery. Recovery (% ) " [ totalanalyte found (ng/L) - analyte found in control (ng/L)[ ^ 1QQ analyte added (ng/'L) Exygen Research Page 7 of? Page 22 o f 65 Page 69 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 ANALYTICAL METHOD Method Num ber V0001781 M ethod o f Analysis fo r the D eterm lnstloB o f P eruorooctuotc A d d (PFO A) Id Soil by LC/M S/M S Analytical Teating Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: "R A CAL Paul Connolly ' Technical Leader, LC-M S, Exygen Research lOlnAW Date Date Exygen Research Total Pages: 7 Page 23 o f 65 Page 70 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen JUeeirch Method tomber V000l7g| ANALYTICAL M ETHOD MethodofAnalysis fortheDeterminationofPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 1.0 Scope T b it method it to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/M S/M S) in soil. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS W ore handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 15 g o fte s t sample fo r extraction. 3.2 No sample processing is needed fo r soil samples. 3.3 Samplee stored refrigerated should be allowed to equilibrate to room temperature. 3.4 A ll samples must be thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedures w ill be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P LC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate -A C .S . Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-AJdrich 5.0 Instrument and Equipment 5.1 A high performance Hquid chromatograph capable o f pumping up to 2 solvents equipped w ith i variable volume hyector capable o f injecting 5 -2 0 0 pL connected to a tandem Maes Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 m L disposable polypropylene centrifuge tubes. 5.5 15 m L disposable polypropylene ccntriftige tubes. 5.6 Disposable m icropipets (50-lO0uL, 100-200uL). 5.7 l2S-m LLDP8nanow*m outh bottles. 5.8 2 m L clear HPLC via l kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10*100 pL), w ith disposable rips. 5.11 Waters Sep Pak Vac 6 c c (lg )tC l8 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. Page 2 o f7 Page 24 o f 65 Exygen Research Page 71 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Rotarch Method Number VOOO1781 ANALYTICAL METHOD J Method o f Analysis fo r the Determination o f Perfluorooctanoic A cid (PFOA) in Soil by LG/MS/MS 5.14 W rist-action shaker. 5.1 $ Centrifuge capable o f spinning 50 m L polypropylene lubes at 5000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: FluophaseRP (Keystone S cientific), 2.1 mm x 50 mm. 5p (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ): 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ): Methanol 6.5 Gradient Program: Time mini 0.0 1.0 8.0 20.0 22.5 L 65 65 25 25 65 Flow Rale i U to V m iP ) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6 6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and may be changed in order to optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: Blectrospray Negative M RM mode, m onitoring 413 -* 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optim ize the MSMS system. 8.0 Preparation o f Solutions 81 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.1S4 g o f ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. Page 3 of? Page 25 of 65 Exygen Research Page 72 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen RMMrcb Mstbod Number VOOOP8I ANALYTICAL METHOD Method o f Analysis fo r the Determination o fPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.0 Standard Preparation 9.1 Standard S tock/Foitiflcstion Solution 9.1.1 Prepare a stock solution o f-100 pg/roL o f PFOA by weighing 10 mg o f analytical standard (corrected for p u rity) and dilute to 100 mL with methanol in 125*mL LDPE bottle. 9.1.2 A 10 pg/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 100 pgfaiL solution to a final volume o f 100 w ith methanol in a I2S m L LDPE bottle. 9.1.3 A 1.0 pg/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 10 pg/m L solution to a fin a l volume o f 100 w ith methanol m a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f foe 1.0 pg/raL solution to a fin a l volume o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 0.01 pgfaiL fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 0.1 pgfaiL solution to a final volume o f 100 with methanol in 123 m L LDPE bottle. 9,1.6 The stock and fortifica tion solutions are to be stored in a refrigerator at approximately 4*C and are stable fo r a maximum period o f 6 months from the date o f preparation. 9 2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards ire processed through the extraction procedure, identical to samples. The follow ing is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Volume o f Concentration of Calibration o fFortification Solution (nob) 0 Volume (pL) 0 Fortified Control Calibration Sample (mL) Standard (doO* 40 0 Standard ID (example) XCmmddyy-0 10 100 10 200 10 400 100 100 100 200 100 400 40 40 40 40 40 40 23 XCmmddyy-1 30 XCmmddyy-2 100 XCmmddyy-3 230 XCmmddyy-4 500 XCmmddyy-5 1000 XCmmddvY-6 * The extracted concentration o f the calibration standard is equal to 8x its in itia l concentration, due to the concentration o f the standard during the extraction (SPE). XC extracted calibration standard. Pag 4 of 7 P age 26 o f 65 Exygen Research Page 73 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygra Rfwaivh MvthodNumber VOOOP8I I ANALYTICAL METHOD Method o fAnalysis fo r the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) must be prepared w ith each set o f standards extracted. Store a ll extracted calibration standards in 15-mL polypropylene lubes at 2*C to 6*C, up to two weeks. Alternate volumes and concentrations o f standards m ty be prepared as 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one reagent control (method blank using 5 raL o f methanol) and two reagent controls fortifie d at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assurance plan fo r this project. 11.0 Sample Extraction 1l. l Weigh 5 g o f sample into 50 m L polypropylene centrifuge tubes (fo rtify as needed, replace lid and m ix well). 11.2 Add 5 m L o f methanol and shake on a w rist action shaker for -15 minutes 11.3 T ruefor the tubes to an ultrasonic bath and sonicate for ->15 minutes. 11.4 Bring the volume up to 40 m L w ith water in the 50 m L polypropylene centrifuge tube. 11.5 Centrifoge fo r-1 0 minutes at -3000 rpro. 11.6 Condition the Cj SPE cartridges (1 g, 6 m L) by passing 10 mL methanol followed by 5 m L o f HPLC water ( - 2 drop/iec). Do not let column run dry 11.7 Load (decant) the sample on the conditioned C u SPE cartridge. Discard eiuate. 11.8 Elute w ith -5 m L 100% methanol. C olled 5 m L o f eiuate into graduated 1Sm L polypropylene centrifoge tubes (fin a l volume - 5 mL). 11.9 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the lame amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 A n entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set Extracted standards must be interspersed between eveiy 5-10 samples. A i an alternative, an entire set o f PigeJoH Page 27 o f 65 Exygen Research Page 74 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen I w n h M nhodNum b V00CI7S1 I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Soil by LC/MS/MS extracted calibration standards may be iqjected 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 esse, extracted calibration standards m utt be the firs t and last injection in a sample set 12.4 Use linear standard curves for quantitation. Linear standard curves are generated fo r the analyte by linear regression using t/x weighting o f peak area versus calibration standard concentration using MassLynx 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 C riteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, w hile the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks m utt not contain PFOA at levels greater than the LOQ. I f a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set m utt be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikee m utt be between 70-130% o f their known values. I f a control spike foils outside the acceptable lim its, the entire act o f samples should be re-extractod. Any m atrix spike outside 7Q130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f extracted calibration standards that could be excluded m utt not exceed 20% o f the total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated mus be 20.992 (RJ 20.985). I f calibration results fa ll outside these lim its, then appropriate tteps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than 4 % w ithin an analytical run. I f retention tim e d rift exceeds this lim it within an analytical run then the set must be reanalyzed. P tse o n Page 28 of 65 Exygen Research Page 75 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 BxygenRmareh Mfthod Number VOOOI7S1 I A N A LY T IC A L M ETH O D Method o fAnalysis fo r the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC /M S/M S 14.0 Calculations 14.1 Uee the follow ing equation to calculate the amount o f PFOA found {in ng/L, baled on peak area) using the standard curve (linear regression parameters) generated by the Maes Lynx software program: PFOA found (n g /L ) (Pm K I f f - intercept! x DF elope DF " factor by which the final volume waa diluted, i f necessary. 14.2 For samples fortifie d w ith known amounts o f PFOA prior to extraction, use the follow ing equation to calculate the percent recovery. Recovery (H ) [ total analyte found (ng/L) analyte found in control (ng/L)] ^ analyte added (og/L) 14.3 Use the foUowing equation to convert the amount o f PFOA found in ng/L to ng/g (ppb). PFOA found (ppb) - [PFOA found in u /L l x volume extracted f0.04L)l ample weight (5 g) 14.4 Use foe follow ing equation to calculate the amount o f PFOA found in ppb baaed on dry w eight PFOA found (ppb) (fry weight * PFOA found (ppb) x [ 100% / total io lid i(% )] Pag*7 of? P age 29 o j 65 Exygen Research Page 76 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001782 Method of Aoalyiie for the DettrmloOtJoD of Perfluorooctesole Acid (PFOA) io Sediment by LC/MS/MS Analytic] Testing Facility: Exygen Research 30S8 Research D rive State College, PA 16801 Approved By: C_j L Paul Connolly ( Technical Leader, IC -M S , Exygen Research / jy 'ohn Flaherty Vice President, Operations, Exygen Research ___ ta b -M Date Date Exygen Research Total Pages: 7 Page 30 o f 65 Page 77 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 B xypa kesaarcb Method Number VOOOI782 I ANALYTICAL METHOD Method ofAnalysis forthe DeterminationofPerfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 1.0 Scope This method it to be employed fo r the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/M S/M S) in sediment. 2.0 Safety 2.1 Always obaerve safe laboratory practices. 2 2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 30 g o f teat sample fo r extraction. 3.2 No sample processing is needed for sediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 A ll samples must be thoroughly m ixed before being sampled for extraction. 3.3 Sample collection procedures w ill be specified in the sampling plan for this p ro je c t 4.0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Methanol - HPLC grade 4.3 Acetic A dd - Reagent grade 4.4 Ammonium A cetate-A .C .S . Reagent Grade 4.5 Perfluorooctanoic Acid - Sigm a-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 iqjector capable o f injecting 5-200 p L connected to a tandem Maas Spectrometer (LC/M S/M S). 5.2 A device to collect raw data fo r peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 m L disposable polypropylene eentriftige tubea. 5.5 15 m L diepoaable polypropylene centrifuge tubes. 5.6 Disposable m icropipets {50-100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC via l kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), w ith disposable tips. 5.11 Waters Sep Pak Vac 6 cc(lg)tC 18S P E cartridges. 5.12 SPE vacuum manifold. Pag* 2 of 7 Page 3/ o f 65 Exygen Research Page 78 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 E xygen P ro to c o l N u m be r: P 0 001131 Exygen RMWth Method Number VQOOI782 | ANALYTICAL METHOD [ Method o fAnalysis for the Determination o f Perftuorooctanoic Acid (PFOA) in Sediment bv LC/MS/MS 3.13 Voitexer. 3.14 W rist-action shaker. 3.13 Centrifbge capable o f spuming 30 m L polypropylene tube at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone S cientific), 2.1 mm x SOmm, 5n (P/N: 823034)32130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ): 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ): Methanol 6.5 Gradient Program: Time (m in) 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate J8 (m L/m in) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume; IS pL(can be increased to as much as 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 optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative M RM mode, m onitoring 413 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optim ize die MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Page 3 of 7 Page 32 o f 65 Exygen Research Page 79 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Ex/gm Rntarch Method Number V0001712 | ANALYTICAL METHOD | Method o fAnalysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Sedimem by LC /M S/M S 8.2 Extraction Solutions 8.2.1 1H acetic add in water is prepared by adding 10 m L o f acetic acid to 1000 m L o fwater. Alternate volumea may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/FortificatioQ Solution 9.1.1 Prepare a stock solution o f--100 pg/raL o f PFOA by weighing 10 mg o f analytical standard (corrected fo r purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/raL fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 100 pg/raL solution to a fin a l volume o f 100 w ith methanol in a 123 m L LDPE bottle. 9.1.3 A 1.0 pg/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 10 pg/m L solution to a fin a l volume o f 100 w ith methanol in a 123 m L LDPE bottle. 9.1.4 A 0.1 pg/mL fortifica tion solution o f PFOA is prepared by bringing 11> m L o f the 1.0 pg/m L solution to a fin a l volume o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.5 A 0.01 p ^m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 0.1 pg/m L solution to s final volume o f 100 with methanol in a 123 m L LDPE bottle. 9.1.6 The stock and fortifica tion solutions are to be stored in a refrigerator it approximately 4*C and are stable fo r a maximum period o f 6 months from the date o fpreparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o fthe 0.1 pg/m L fortifica tion solution. 9.2.2 The follow ing it a typical example: additional concentrations may be Concentration o f Fortification Solution (na/mL) 100 too too 10 3 2 Volume (mL) 10 5 2 10 10 10 Diluted to (id .) too too too 100 100 too Final Concentration (nirtiU .) 10.0 50 20 10 03 02 Page 4 of 7 Page 33 o f 65 Exygen Research Page 80 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number P0001131 Exyfffl RwMTCfe Method Number VOOOPI2 | ANALYTICAL METHOD | Method o f Analyns fo r the Determination o fPerfluorooctanoic Acid (PFOA) in Sediment by LC /M S/M S 9.2.3 9.2.4 Store a ll caUbration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, uptosbt months. Alternate voluntas and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Bach batch o f aamplea extracted (typically 20 or less) must include at least one untreated oonirol and two untreated controls fo rtifie d at known concentrations (lab control spike) to ve rify procedural recovery fo r the batch 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project 11.0 Sample Extraction 11.1 Weigh $ g o f ample into 50 m L polypropylene centrifoge tubes (fo rtify as needed, replace lid and m ix w ell). 11.2 Add 3$ m L o f 1% acetic acid, cap, vortex and shake on a w rist action shaker fo r -6 0 minutes. 11.3 Centrifuge the tube at -3000 rpm for -2 0 minutes. 11.4 Condition the C n SPB cartridges (1 g. 6 m L) by passing 10 m L methanol followed by 20 m L o fHPLC water (> 2 drop/sec). Do not let column run dry 11.5 Load (decani) the sample on the conditioned C u SPE cartridge. Discard eluate. 11.6 Add 20 m L o f methanol to the sediment le ft in the bottom o f the 50 mL centrifoge tube. Cap, vortex B id shake on a w rist action shaker for ~30 minutes. 11.7 Centrifoge the tubes at >3000 rpm fo r >20 minutes. 11.8 Decant the methanol onto the same SPE cartridge. C ollect the eluate. 11.9 Wash the column w ith 4 m L o f m ethanol C ollect the eluate and add it to the elusle collected in step ] ] .8. 11.10 Condition a second C u SPE cartridge ( l g. 6 m L) by passing 10 m L methanol followed by 20 m L o f HPLC water (> 2 drop/sec). Do not let column run dry 11.11 Add the methanol to >200 m L o f water and load on the second conditioned SPE cartridge. 11.12 Elute w ith >5 m L 100% m ethanol Collect 5 m L o f eluate into graduated 15 m L polypropylene centrifoge tube (fin a l volume " S m L). 11.13 Analyze saraplee using electrospray LC/MS/MS. Page 5 Of 7 Page 34 o f 65 Exygen Research Page 81 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 BxypnRuMrcb Method Number V00017 | A N A LY T IC A L M ETH O D 1 Method o f Analysis fo r the Determination o f Perfluorooctanoic A cid (PFOA) in Sediment by LC /M S/M S 12.0 Chromatography 12.1 Inject the tame amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5*10 sample. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5*10 samples (to account for a second set o f standards). In either case, calibration standards must be the first and last iq jaction in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves arc generated fo r the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3 3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance C riteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, w hile the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blades must not contain PFOA at levels greater than the LOQ. I f a blank contains PFOA at levels greater than 0.2 og/m L, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes must be between 70*130% o f their known values. I f a control spike foils outside the acceptable lim its, the entire set o f samples should be re-extrected. Any m atrix spike outside ?0* 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards uycctod. 13.5 The correlation coefficient (R ) for calibration curves generated must be 20.992 (R* 20.985). I f calibration results fa ll outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed Psic6 of? Page 35 o f 65 Exygen Research Page 82 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyfuR ttM idi Method Number V0001782 I A N A LY T IC A L M ETH O D | Method o f Analysis fo r the Determination ofPorftuorooctanotc A cid (PFOA) in Sedimem by LC /M S/M S 13.6 Retention times between standards and samples must not d rift more than 4 H w ithin an analytical run. I f retention tim e d rift exceeds this lim it within an analytical ran then the set must be reanalyzed. 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in ng/mL, baaed on peak area) using the standard curve (linear regression parameters) generated by the Maes Lynx software program: PFOA found (ng/m L) * (Peak area - intercept) x DF slope DF factor by which the final volume was diluted, i f necessary. 14.2 For samples fortifie d w ith known amounts o f PFOA prior to extraction, use the follow ing equation to calculate the percent recovery. Recovery (% ) [to ta l analyte found (pg/m L) - analyte found in oontrol (ng/m L)] analyte added (ng/m L) 14.3 Use the follow ing equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found (ppb) - (PFOA found (ng/m L) x fin a l volume (3 mLH sample weight (3 g) 14.4 Use the follow ing 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 [100H / total solids(V)] Page 7 of 7 Page 36 o f 65 Exygen Research Page 83 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V000I783 Method of Aialyili for the Detsrmlaatioo of Perflvorooctanolc Add (PFOA) in Fish and Clam by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research D rive State College, PA 16801 Approved By. v L l C-Jt Paul Connolly 1 Technical Leader, LC-MS, Exygen Research a Jolohhnn Flaherty ' Vice President, Operations, Exygen Reseaicb Date Date Exygen Research Total Pages: 8 Page 37 o f 65 Page 84 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Sxygta lUcatrcfe Method Number VOO0I7S3 ANALYTICAL METHOD Method o f Analysis for the Determination o fPerfluorooctanoic A cid (PFOA) in Fish and Clams by LC/MS/MS 1.0 Scope T b ii method it to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/M S) in fUrb and clams. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safely precautions. 3.0 Sample Requirement 3.1 A t least 20 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize w ith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage u n til tim e o f analysis. 3.3 Sample collection procedures w ill be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P LC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120<400 mesh) - Reagent grade 4.4 Methanol - HPLC grade S ilica gel (60-200 mesh) - Reagent grade F loriail (60*100 mesh) - Reagent gride Superclean LC-NH - Reagent grade l*O ctanol - HPLC grade 4.9 L-Aacorbic acid - Reagent grade 4.10 Dim ethyldkhlorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma*A ldrich 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 Injector capable o f injecting 5*200 iL connected to a tandem Mass Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Pag* 2 ofS P age 38 of 65 Exygen Research Page 85 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyges fUttsrch Method Number V00017SJ ANALYTICAL METHOD Method o f Analysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Claras byLC/M S/M S 5.4 Rotary evqtorator. 5.5 Tisaumizer. 5.6 125 m L pear-shaped flasks. 5.7 50 m L disposable polypropylene centrifuge tubes. 5.8 15 raL disposable polypropylene centrifuge tubes. 5.9 Disposable micropipets (50-lOOuL, 100-200uL). 5.10 125-mL LDPB narrow-mouth bottles. 5.11 2 m L clear HPLC v ia l kit. 5.12 Disposable pipettes. 5.13 Autopipettes (100*1000 pL and 10-100 pL), w ith disposable tips. 5.14 SPB tubes (20m L) (Snpelco cat. no. N057177). 5.15 W rist action shaker. 5.16 Centrifuge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone S cientific), 2.1 mm x 50 mm. $m (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase ( A ) : 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ): Methanol 6.5 Gradient Program: Time (m ini 0.0 1.0 8.0 20.0 22.5 2kA 65 65 25 25 65 Flow Rate % B fm L/m uri 35 0 J 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optim ize (he HPLC system. Pi*J of< Page 39 o f 65 Exygen Research Page 86 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bxygttl fcMMRh MethodNumber VOOOI713 ANALYTICAL METHOD Method o fAnalysis fo r the Determination o f Perfiuorooctanoic A cid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: Electrocpvay Negative MRM mode, m onitoring 413 --e 369 rrvz for PFOA. The above condition* are intended as a guide and may be changed in order to optim ize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0.IS4 g o f ammonium acetate to 1000 m L o fwater. 8.2 Extraction S olution 8.2.1 8.2.2 2% ascorbic acid in methanol ia prepared by diaaolving 2 g o f ascorbic add in 100 m L o fmethanol. 30H Dim ethyldichlorosilane in toluene is prepared by bringing 3 mL o f dimetbyldichlorosilane to a Anal volume o f 10 m L w ith toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 9.12 9.1.3 9.1.4 9.1.3 Prepare stock solution o f-100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected Ex purity) and dilute to 100 mL with methanol in a 125-raL LDPB bottle. A 1.0 pgfatL fortifica tion solution o f PFOA is prepared by bringing 1 m L o f the 100 pgftnL solution to a final volume o f 100 w ith methanol in a 123 m L LDPE bottle. A0.1 pg^mL fortifica tion solution ofPFO A is prepared by bringing 10 m L o f the 1.0 pg/m L solution to a fin a l volume o f 100 w ith methanol in a 123 m l LOPE bottle. A 0.01 pg/m L fortifica tion solution ofP FO A is prepared by bringing 10 mL o f the 0.1 pg/m L solution to a final volume o f 100 with methanol in a 123 m L LDPE bottle. The stock and fortifica tion solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. Pages sfS P a g e 40 o f 65 Exygen Research Page 87 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 E xyg e n P ro to c o l N u m be r: P 0 001131 Exygea Research Method Numb? V0001783 ANALYTICAL METHOD Method o f Analysis for the Detemnnatum o f Perfluorooctanoic A cid (PFOA) in Fish and Clams by LC/MS/MS 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/m L fortifica tion solution. The follow ing it a typical example: additional concentrations may be prepared as needed. Concentration Final o f Fortification Volume Diluted to Concentration Solution (ua/mL) (mL) (mL) (ua/mL) 1.0 S.0 100 0.05 1.0 2.5 100 0.025 1.0 \A 100 0.01 0.05 10 100 0.005 0.025 10 100 0.002S 0.1 10 100 0001 0.005 10 100 0.0005 9.2.3 Store a ll calibration standards in 125-mL LDPE narrow-mouth bottles at 29C 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 Bad) batch o f samples extracted (typically 20 o r less) must include at least one untreated control and two untreated controls fortifie d at known concentrations ( lib control spike) to v e rily procedural recovery for the batch. 10.2 Requirements fo r field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into SO m L polypropylene centrifuge tubes (fortify as needed, replace lid and m ix w ell). 11.2 Add 30 m L o f acetonitrile and shake on a w rist action shaker for -1 Sminutes U .3 Place the tubes in a freezer for - l hour. 11.4 Pack and condition the SPE tubes and eilanize the pear-shaped flasks. 11.5 Pack foe 20 m L SPE tubes in sequence w ith 2 g flo ris il, 2 g silica gel, 2 g carbon, and 1 g LC -N H j. Condition foe columns w ith 20 m L o f methanol, then 20 m L o f acetonitrile. Discard a ll washes. Do not allow the column to dry. 11.6 Silanize foe 123 m L pear-shaped flasks by rinsing w ith the 30% diroethyldichlororilane in toluene solution. Rinse foe flask w ith toluene once, followed by methanol (three tim es). D ry the flasks com pletely before use. either by air-drying or w ith a stream o f nitrogen. Page ' a<X Page 41 o f 65 Exygen Research Page 88 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 E xyg en P ro to c o l N u m be r: P 0 001131 ExygenResearch Method Number V00017S3 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluoiooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 11.7 Centrifuge the 50 m l polypropylene tubes containing sample at -2000 rptn fo r-1 0 minutes. 11.8 Decant the extract on to a conditioned SPE column fitted in tide the mouth o f the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape flask. 11.9 Add 10 m L o f acetonitrile to the sample in the 50 m L centrifuge tube. Homogenize the frozen fat phase using tissum izer for -3 0 seconds and rinse the tissum izer w ith -1 0 m L o f acetonitrile into the tube. 11.10 Shake the sample again fo r -1 0 minutes on s w rist-action shaker. 11.11 Place tho tubes in a freezer for - 1 hour more. 11.12 CentrifUge the SO m L polypropylene tubes containing sample st -2000 rpm fo r-1 0 minutes. 11.13 Decant the extract onto the same SPE column. Collect the eluate into ihe same pear-shaped flask and combine w ith the eluent from the in itia l extraction. 11.14 P us 20 m L o f acetonitrile through the SPE column and combine the eluate in the same pear-shaped flask. 11.15 Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.16 Make the final volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and sw irl to m ix/dissolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze sim ples using electrosprsy LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow ail analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in in analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and ai the end o f a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, in 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 fo r a second set o f standards). In either case, calibration standards must be the firs t and last injection in a sample set 12.4 Use linear standard curves fo r quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. P ap 6 f s P age 42 o f 65 Exygen Research Page 89 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VQ0017J3 ANALYTICAL METHOD Method o f Analysis fo r the Determination o f Perftuorooctanoic A cid (PFOA) in Fish end Clems by LC7MS/MS 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be flirth e r diluted and reanalyzed 13.0 Acceptance C riteria 13.1 Chromatogram must show a peak o f daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, w hile the daughter ioo (369 amu) represents the lose o fcarbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ I f a blank contains PFOA at levels greater then 0.5 ppb, then a new blank sample must be obtained end the entire set must be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes must be between 70-130% o f their known values. I f a control spike falls outside the Keeptable lim its, the entire set o f samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using (he 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 foe total number o f standards injected. 13.5 The correlation coefficient (R ) fo r calibration curves generated must be 20.992 (R3 20.985). I f calibration results fo il outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or foe relevant set o fsamples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than 1 4 % w ithin an analytical ran. I f m ention tim e d rift excoeds this lim n within in analytical ran then foe set must be reanalyzed. 14.0 Calculations 14.1 Use foe follow ing 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 Maes Lynx software program: PFOA found (ngftnL) - (Peak area - intercept) elope 14.2 Use foe follow ing equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found (ppb) (PFOA found fne/m L) x fin a l volume (m Ll x DF1 sample weight (g) DF factor by which foe final volume was diluted, if necessary. Peg* ? o f K Page 43 o f 65 Exygen Research Page 90 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOl ANa L Y T IC .U , m e t h o d Method o f Analysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Clam* by LC/MS/MS U .3 For samples fortifie d w ith known amounts o f PFOA prior to extraction, use the follow ing equation to calculate the percent recovery. Recovery (% ) - [ totalanalyte found (ng/g) analyte found in control (ng/g)l t ^ analyte added (ng/g) Exygen Research Page 8of8 Page 44 o f 65 Page 91 of 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001784 Method of AaoJyste for the Determination of Perflsorooctaaolc Acid (PFOA) io Vegetation by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research D rive State College, PA 16801 Approved By: 'VLX C JU Paul Connolly Technical Leader, LC-MS, Exygen Research n / z n / i U / ________ J o h n Flaherty ^ Vice President, Operations, Exygen Research Date Exygen Research ToU l Pages: 7 Page 45 o f 65 Page 92 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygcp fUMirch Method Number V000!7M ANALYTICAL METHOD Method o f Analysis for the Detennination o f Periluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope 1111 meebod is to be employed for tbe isolation end quantiration o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled lo a tandem Mass Spectromtrie Detector (LC/MS/M S) in vgtation. 2.0 Safety 2.1 Always obeerve safe laboratory practices. 2.2 Consult die appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 20 g o ftest sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize w ith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage u n til time o f analysis. 3.3 Sample collection procedures w ill be specified in tbe sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P LC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.3 S ilica gel (60-200 mesh) - Reagent grade 4.6 F lo riril (60-100 mesh) - Reagent grade 4.7 Supcrclesn LC-NH* - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L-Ascorbic add - Reagent grade 4.10 Dim ethykticblorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigm a-Aldrich S.O Instrument tnd Equipment 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith variable volume injector capable o f injecting 3-200 pL connected to a tandem Maes Spectrometer (LC/M S/M S). 3.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Page 2 i>i ? Page 46 o f 65 Exygen Research Page 93 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygra RcMMck Mhod Number V00017S4 A N A LY T IC A L M KTH O P ....... ............ | Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 5.4 Rotary ev^xirator. 5.5 125 mL pear-shaped flasks. 5.6 50 m L disposable polypropylene centrifuge tube. 5.7 15 m L diapoaable polypropylene centrifuge tubes. 5.8 D i^aable m icropipet* (S0-100uL, 100-200uL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 m L clear HPLC v ia l kit. 5.11 Diapoaable pipettes. 5.12 Autopipettes (100-1000 pL and 10-100 pL), w ith disposable tips. $.13 SPE tubes (20m L) (Supelco cat. no.N057177). 5.14 W rist action shaker. 5.15 C entriftge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: PluophaseRP (Keystone S cientific), 2.1 m m x 50 mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ): 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ) : Methanol 6.5 Gradient Program: Tim e (m ini 0.0 1.0 8.0 20.0 22.5 5LA 65 65 25 25 65 Flow Rate &J8 (m l/1TM) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can bo increased to as much as 50 pL). 6.7 Q uantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: Blectrospray Negative MRM mode, m onitoring 413 - 369 m /z for PFOA. PeJul'7. Page 47 o f 65 Exygen Research Page 94 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bxypn lUsMfcb Method Number VOOOI784 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Vegetation by LC/MS/MS The above condition! are intended at a guide and may be changed in order to optim ize the MSMS system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water ie prepared by adding 0.154 g o f ammonium acetate to 1000 tnL o f water. 8.2 Extraction Solution! 8.2.1 8.2.2 2% ascorbic acid in methanol ia prepared by diasolving 2 g o f ascorbic acid in 100 mL o fmethanol. 30% Dim othyidichloioailane in toluene ie prepared by bringing 3 mL o fdim ethyktichlorofilane to a fin a l volume o f 10 m L w ith toluene. Alternate volume# may be prepared. 9.0 Standard Preparation 9 .1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f-100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected fo r purity) and dilute to 100 mL with methanol in a 12S-mL LDPE bottle. A 1.0 pg/m L fortifica tion solution o f PFOA is prepared by bringing I m L o f the 100 pg/m L solution to a fin a l volume o f 100 w ith methanol in n 125 m L U )PE bottle. A0.1 p g ta L fortifica tion solution ofPFO A is prepared by bringing 10 m L o f die 1.0 pg/m L solution to a final volume o f 100 w ith methanol in a 125 raL LDPE bottle. A 0.01 pg/m L fortifica tion solution o f PFOA is prepared by bringing 10 m L o f the 0.1 pg/m L solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. The stock and fortifica tion solutions are to be stored in a refrigerator si approximately 4*C and are stable fo r a maximum period o f 6 months from die date o fpreparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/m L fortifica tion solution. Piyc 4 I ' Page 48 o f 65 Exygen Research Page 95 o f 115 Interim Report #4 - Analysis of Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyia fUaewch Method Number V000I7S4 | A N A LY T IC A L m TH O P Method o f Analysis fo r the Determination o f Perfluoroocunoic Acid (PFOA) in Vegetation by LC/MS/MS 9.2.2 The follow ing is a typical example: additional concentrations may be prepared as needed. Concentration ofFortifiostioa Solution (ua/raL) 1.0 1.0 1.0 Volume (mL) 5.0 2.5 1.0 Diluted to (mL) too 100 100 Final Concentration (m rtnL ) 0.05 0.025 0.01 0.05 10 100 0.005 0.025 10 100 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 Store a ll calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Bach batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortifie d at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirements fo r field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 11.2 11.3 11.4 11.5 11.6 11.7 Weigh 5 g o f frozen sample into 50 m L polypropylene centrifuge tubes (fo rtify as needed, replace lid and m ix w ell). Add 30 m l, o f acetonitrile and shake on a w rist action shaker for -15 minutes Centrifuge the 50 m L polypropylene tubes containing sample at -2000 rpm fo r-1 0 minutes. Pack and condition the SPE tubes and silanize the pear-shaped flasks. Pack the 20 m L SPE tubes in sequence w ith 2 g flo ris il, 2 g silica gel. 2 g carbon, and 1 g LC-NH*. Condition the columns w ith 20 m L o f methanol, then 20 m L o f acetonitrile. Discard a ll washes. Do not allow the column to dry. Silanize the 125 m L pear-shaped flasks by nnsing w ith the 30% dimctbykUchlorosUane in toluene solution. Rinse the flask w ith toluene once, followed by methanol (three tim es). D ry the flasks com pletely before use, either by air-drying or w ith a stream o f nitrogen. Decant the extract on to a conditioned SPE column fitted inside the mouth o f the pear-shaped flu k . Collect the eluate in the 125 m L silanized pear-shape flask. Page 5 of ' Page 49 o f 65 Exygen Research Page 96 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001784 A N A LY T IC A L m s t h o p Method o f Analysts fo r the Determination o f Perfluoroooanoic Acid (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 m L o f acetonitrile to the sample in the 50 m L centriftge tube. 11.9 Shake the sample again for-lO m inutee on a w rist-action shaker. 11.10 Centrifuge the SO m L polypropylene tubes containing cample at -*2000 rpm for 5 uiimitea. 11.11 Decant the extract onto the came SPE column- C ollect the eluate into the same pear-shaped flask and combine w ith the eluent from the in itia l extraction. 11.12 Repeatctepf 11.8through 11.11 again. 11.13 Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.14 Make the final volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and sw irl to m ix/dissolve. IM S Transfer the extracts to HPLC vials using disposable pipets. 11.16 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire eet o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards may be iiyoctod at the beginning o f a sei followed by extracted calibration standards interspersed every 5-10 simples (to account fo r a second set o f extracted standards). In either case, extracted calibration standards must be the firs t and last injection in a sample set. 12.4 Use linear standard curves fo r quantitation. Linear standard curves are generated fo r the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Semple response should not exceed standard responses. Any samples (hat exceed standard responses should be farther diluted and reanalyzed. 13.0 Acceptance C riteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. T1m 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. Page 6 o f* Page SOo f 65 Exygen Research Page 97 o f 115 Interim Report #4 - Analysis of Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygmRMMrcb Mho<INiubiVOOOI7l4 | A N A LY T IC A L m e t h o d I Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 13.2 Method blanks moat 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 the entire set must be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes must be between 70-130% o f their known vslues. I f a control spike falls outside the acceptable lim its, the entire set o f samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Teat, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must oot exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R1 20.985). I f calibration results fk ll outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than 4 % w ithin an analytical nm. I f retention tim e d rift exceeds this lim it within an analytical ran then the act must be reanalyzed. 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in ng/mL. baaed on peak area) using the standard curve (linear regression parameters) generated by the Masa Lynx software program: PFOA found (ng/m L) - (Peak area - intercept! slope 142 Use the follow ing equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found /ppM [PFOA found fag/m Ll x fin a l volume fm L l x DF1 sample weight (g) DF factor by which the final volume was diluted, if necessary. 14J For samples fortifie d w ith known amounts o f PFOA prior to extraction, use die follow ing equation to calculate the percent recovery. Recovery (% ) * ( total analyte fotmd (ng/g) analyte found in control (ng/g)] analyte added (ng/g) Pag 7 o i 7 P age 51 o f 65 Exygen Research Page 98 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 ANALYTICAL METHOD Method Number: V0001785 Method of Analysis for the Determination of Perflnorooctanolc Acid (PFOA) in Smell M anu) Liver by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research D rive State College PA 16801 Approved By: ___ c -- iX u _________ Paul Connolly I Technical Leader LC-M S, Exygen Research o //H d-M- John Flaherty / Vice President,. Operations Exygen Research ___ lM M Date Dato Exygen Research Total Figoi: 7 P a g e 52 o f 65 Page 99 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Eaygaa Research Mtihod Number VOOOI78S I A N A LY T IC A L M ETH O D Method o fAnalysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Small Mammal Liver by LC/MS/MS 1 1.0 Scope This method is to be employed fo r the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Maas Spectrometric Detector (LC/M S/M S) in sm all mammal liver. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 5 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize w ith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublim ation. Seal and place the samples in frozen storage until time o f analysis. Alternately i f there is an insufficient amount o f sample (-less than 5 g). then no processing is necessary and the sample can be used as supplied 3 3 Sample collection procedures w ilt be specified in the sampling plan for this project. 4.0 Reagents and Standard! 4.1 Water-H P L C grade 4.2 M ethanol-H PLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate -A .C .S . Reagent Grade 4.5 Perfluorooctanoic A cid - Sigm a-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 injector capable o f injecting 5-2(Xj pL connected to a tandem Maas Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5 4 50 m L disposable polypropylene centrifoge tubes 5.5 15 m L disposable polypropylene centrifuge tubes. 5.6 Disposable m icropipets (50-lOOuL, 100-200uL). 5.7 125-mLLDPG narrow-mouth bottles. S.S 2 m L clear HPLC via l kit. Page 2 o f Page 53 o f 65 Exygen Research Page 100 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P001131 Exygra RMnrck Method Number VOOO179$ | A N A LYTICAL m e t h o d Method o fAnalysis fo r the Detenninstion o f Perfluorooctanoic A cid (PFOA) in Small Mammal Liver by LC/MS/MS 5.9 5.10 5.11 5.12 5.13 5.14 5 .15 Disposable pipettes. Autopipettes (100*1000 |iL and 10-100 pL), w ith disposable tips. Waters Sep Pak Vac 6 cc(lg)tC 18S P E cartridges. SPE vacuum manifold. Tissaemizer. W rist-action shaker. Centrifuge capable o f spinning 15 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographie System 6.1 Analytical Column: FIuophaseRP (Keystone S cientific), 2.1 mm x 50 mm, 5n (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ) : Methanol 6.5 Gradient Program: Time {m ini 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate & fm lV m ini 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative M RM mode, m onitoring 413 - 369 m ft for PFOA. The above condition are intended es a guide and may be changed in order to optim ize the MSMS system. Page 3 of 7 Page 54 o f 65 Exygen Research Page 101 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygw RsMveh Mrhod Number VOOOl' SJ 1 AN/wL\TICAl `m ETHOD Method o f Analysis for the Determination o fPerftuorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS B.O Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 tnM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volum e! may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f-100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pgAnL fortifica tion solution o f PFOA is prepared by bringing I m L o f the 100 pg/raL solution to final volume o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 0.1 pgftnL fortifica tion solution o f PFOA is prepared by bringing 10 m L o f foe 1.0 pg/inL solution to a fin a l volume o f 100 w ith methanol in a 123 m L LDPE bottle. 9.1.4 The stock and fortifica tion solutions are to be stored in a refrigerator at approximately 4*C and are stable fo r a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 92J LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 pg/m L fortifica tion solution. The follow ing is a typical example: additional concentrations may he prepared as needed. Concentration o f Fortification Volume Solution (ni/m L) (mL) Diluted to (mL) Final Concentration (nc/mL) 100 5.0 100 2.0 100 1.0 100 100 10Q 5.0 20 10 5.0 to too 2.0 10 100 1.0 10 100 0.5 0.2 0.1 9.2.3 Store a ll calibration standards in 123-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as pgc 4 o f y P age 55 o f 65 Exygen Research Page 102 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyfraJtMMRli Method Number V0001785 | A N A LY T IC A L M ETHO D Method o f Analysis for the Determination ofPerfluorooctsnoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or lose) must include at least m e untreated control and two untreated controls fortifie d at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirements fo r field and iaboratoiy duplicates and spikes w ill be specified in the quality assurance plan fo r this project. 11.0 Sample Extraction 11.1 Weigh 1 g o f sample into a 50 m L polypropylene centrifuge tubes (fo rtify as needed, replace lid and m ix w ell). Note that alternate weights o f liver may be measured depending on the sample size available fo r use. 11.2 Add water to the sample for a fin a l volume o f 10 mL. 11.3 Homogenize sample using a tiasuenuzer for -1 minute. 11.4 Transfer 1 m L o f die sample using a disposable pipette into a 15 mL disposable centriftge tube. 11.5 Add 5 m L o f acetonitrile and shake fo r 20 minutes on w rist-action shaker 11.6 Centrifuge the tubes at 3000 rpm for - 5 minutes. 11.7 Decant the supernatant into a 50 m L disposable centrifuge tube and add 35 m L o f water. 11.8 Condition the C u SPE cartridge (1 g, 6 m L) by passing 10 m l methanol followed by 5 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eluate. 11.10 Elute w ith 2 m L o f methanol. C ollect 2 m L o f eluate into a graduated 15 m L polypropylene centrifoge tube (fin a l volume - 2 m L). 11.11 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject die same amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at (he beginning and at the end o f a sample set. Standards must be interspersed between every 5-K> samples. As in alternative, an entire set o f calibration standards may be injected at the beginning o f a set follow ed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must be the firs t and last injection m a sample set. 12.4 Use linear standard curves fo r quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area Pag 5 or? P age S6 o f 65 Exygen Research Page 103 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyfto R*Much Mflfaod Number VOOOtTIS I A N A LY T IC A L M ETHO D Method o f Analysis for the Determination o fPeriluorooctanoic A d d (PFOA) in Small Mammal Liver by LC/MS/MS venus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.3 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed 13.0 Acceptance C riteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponda to the PFOA anion, w hile 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 10 ng/g, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes must be between 70-130% o f their known values. I f a control spike foils outside the acceptable lim its, the entire sat o f samples should be re-extracted. Any m atrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is 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. 13.5 The correlation coefficient (R ) fo r calibration curves generated must be 0.992 (R1 0.91$). I f calibration results fa ll outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention tunee between standards and samples must not d rift more than 4 % w ithin an analytical run. I f retention tim e d rift exceeds this lim it w ithin an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters] generated by the Mass Lynx software program: PFOA found (ng/m L) - (Peak area - intercept x DF x aliquot factor slope DP - factor by which the final volume was diluted, i f necessary. A liquot A ctor -1 0 Pageof 7 P age 57 o f 65 Exygen Research Page 104 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOI131 Exypo R*mrcfa Mtlbod Number VQ00P8S I a n a l y t ic a l m e t h o d Method o f A n lriy iii for the Determination o f Perfluorooctanoic Acid (PFOA) in SmatF Mammal Liver by LC/MS/MS 142 For samples fortifie d w ith known amount* o f PFOA prior to extraction, use die follow ing equation to calculate the percent recovery. Recovery (% ) [ total analyte found (ngftnL) - analyte found in control (ng/m L)l k } ^ analyte added(ng/m L) 14.3 U ie the follow ing equation to convert tho amount o f PFOA found in ng/mL 10 ntfg(ppb ). PFOA found (ppb) - ffP O A found (ng/m L) x fin a l volume fm D I sample weight (g) Exygen Research P*fe 7 of 7 P age 58 o f 65 Page 105 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 ANALYTICAL METHOD Method Number V0001786 Method of Analyst! for the DetermiaetSon of PerfUioroocUoolc Add (PFOA) la Small Mammal Seram by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research D rive State College, PA 16801 Approved By: Paul Connolly 1 Technical Leader, LC-M S, Exygen Research J/D / / ihn Flaherty Vice President, Operations, Keygen Research ___ is k W g 'l Dim Date Exygen Research Total Pages: 7 Page 59 o f 65 Page 106 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Enyjm Rrnaich Method N utter VOOOI786 I AWAJLVT1CAL M ETH O D Method o f A n ly n t for the Detetminetion o f Perfloorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 1.0 Scope T b ii method is to be employed for the isolation and quantitation o f porfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/M S/M S) in small mammal serum. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical fo r proper safety precautions. 3.0 Sample Requirement 3.1 A t In s t 1m L o f test sample fo r extraction. 3.2 No sample processing is needed for serum samples. However, frozen serum sam ples must to allowed to com pletely thaw to room temperature before use. 3.3 Sample collection procedures w ill be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P LC grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigm a-Aldrich 5.0 Instrument and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume injector capable o f injecting $ -2 0 0 pL connected to a tandem Mass Spectrometer (LC/M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene centriftjge tubes 1S m L disposable polypropylene centrifuge tubes. Disposable m icropipets (50-100uL, 100-200uL). 125-mL LDPB narrow-mouth bottles. 2 m L clear HPLC v ia l kit. Disposable pipettes. Autopipettes (100-1000 uL and 10-100 pL), w ith disposable tips. Waters Sep Pak Vac 6 cc (Ig ) tC l8 SPE cartridges. SPE vacuum m anifold. Vortexcr. PSe2oP P age 60 o j 65 Exygen Research Page 107 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bxyfca ReMveb Method Number V000I7K6 I ...... A iW V IIC A L M ETHOD Method o fAnalysis for the Deteimination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Swum by LC^MS/MS 5. 14 W rist-action shaker. S.1S Centrifuge capable o f spinning 15 m L polypropylene lubes ai 3000 rpm 6.0 Chromatographic System 6.1 Analytical Column: Fluophaae RP (Keystone S cientific), 2 .1mm x SOmm. 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ): 2 mM Ammonium Acetate in Water 6.4 M obile Phase (B ) : Methanol 6.S Gradient Program: Time /m in i 0.0 1.0 8.0 20.0 22.S 2LA 65 65 25 25 65 Flow Rate fm L/m in) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: IS pL (can be increased to as much as SQ pL) 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optim ize the HPLC system. 7.0 MS/MS System 7.1 Mode: Eleetroepray Negative MRM mode, m onitoring 413 - * 369 mn for PFOA. The above oonditions are intended as a guide and may be changed in order to optim ize the MSMS system. 8.0 Preparation o fSolutions 8.1 M obile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. Page 3 of 7 P a g e 61 o f 65 Exygen Research Page 108 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 E xypnR cm rch Method Number VOOO1786 I ANALYTICAL METHOD Method o f Analysis fo r the Determination o fPerfiuorooctanotc A cid (PFOA) in Small Mammal Scrum by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Foitification Solution 9.1.1 Prepare a stock solution o f 100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to ! 00 mL with methanol in 125-raL LDPE bottle. 9.1.2 A 1.0 jt m L fortifica tion solution o f PPOA is prepared by bringing I m L o f the 100 pg/raL solution to a fin a l volume o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 0.1 pg/m L fortifica tion solution o f PPOA is prepared by bringing 10 m L o ftb c 1.0 pg/m L solution to a fin a l volume o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.4 The stock and fortifica tion solutions are to be stored in a refrigerator it approximately 4*C and are stable fo r a maximum period o f 6 months from the date o fpreparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o fthe 0.1 pg/m L fortifica tion solution. The follow ing ia a typical example: additional concentrations may be prepared as needed. Concentration ofFoctificstion Volume Diluted to Pinal Concentration Solution faa/iuL) (mL) (mL) (na/inL) 100 5.0 100 5.0 100 2.0 100 100 1.0 100 2.0 1.0 5.0 10 100 0.5 2.0 10 100 1.0 10 100 0.2 0.1 9.2.3 Store a ll calibration standards in 125-mL LDPE nam>w*mouth bottles at 2*C to 6"C1up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r lose) must include at least one untreated control and two untreated controls fortifie d ai known concentrations (lab control spike) to ve rify procedural recovery fo r the batch 10.2 Requirements fo r fie ld and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. page 4 o f7 Page 62 o f 65 Exygen Research Page 109 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exy|Cfl Research Method Number V0001786 1..... ..... ...... A N A LY T IC A L m e t h o d " Method o fAnalysis for the Determination o f Perfiuorooctanoic A cid (PFOA) in Small Mammal Serum by LC/MS/MS 11.0 Sample Extraction 11.1 Measure 1 mL o f sample into a 50 m L polypropylene centrifuge tubes (fo rtify as needed, replace Ud and m ix w ell). Note that alternate volumes o f serum may be measured depending on the sample sue available for use. 11.2 Add water to the sample for a final volume o f 20 m L Cap tig h tly 11.3 Vortex for-I minute. 11.4 Transfer 1 raL o f the sample using a disposable pipette into a 15 mL disposable centrifuge tube. 11.5 Add S m L o facetonitrile and abske fo r -2 0 minutes on a wrist-action shaker. 11.6 Centrifoge the tubes at -3000 rpm fo r -5 minutes. 11.7 Decant the supernatant into a 50 m L disposable centrifoge tube and add 35 m L o f water. 11.8 Condition the Ci SPE cartridges ( I g, 6 m L) by passing 10 mL methanol followed by Sm 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 w ith ~2 m L o f methanol. C ollect 2 m L o f eluate into a graduated 15 m L polypropylene centrifoge tube (fin a l volume * 2 m L). 11.11 Analyze samples using electrocpray LC/MS/MS- 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fo rtifie d sample into the LC/MS/MS system. A calibration standard must precede and follow ail analyzed samples. 12.2 Standards o fPFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and ai the end o f a sample set. Standards must be interspersed between every S-1<> sample. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a act followed by calibration standards interspersed every 5-10 samples (to account for second set o f standards) in either case, calibration standards must be the firs t and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be fortber diluted and reanalyzed. Page 5 of" Page 63 o f 65 Exygen Research Page 110 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygmRMMicb Method Number V000!?86 | A N A LY T IC A L M ETH O D Method o f Analysis for the Determination o f Perfhiorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 13.0 Acceptance C riteria 13. t Chromatogram m uit ahow a peak o f a daughter ion it 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represent the loes o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contain! PFOA at levels greater than 10 ng/m L, then a new blunk sample must be obtained and the entire set must be ro-extracted. 13.3 Recoveries o f control spikes and m atrix spikes must be between 70-130% o f tbeir known values. I f a control spike falls outside the acceptable lim its, the entire set o f samples should be re-extracted. Any m atrix spike outside 70 130% rfiould be evaluated by the analyst to determine if re-extraction is warranted. 13.4 A ny calibration standard found to be a statistical outlier by using the Huge Error Teat, may be excluded from the calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o fthe total number o f standards injected. 13.5 The correlation coefficient (R) fo r calibration curves generated must he 20.992 (R* 20.985). I f calibration results fa ll outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than 1 4 % w ithin an analytical run. I f retention tim e d rift exceeds this lim it within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the follow ing equation to calculate the amount o f PFOA found (in ng/m L based on peak area) using foe standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/raL) (Peak area - intercept) x DF x aliquot factor slope DF * A ctor by which foe final volume w u diluted, i f necessary. A liquot factor " 20 14.2 For samples fortifie d w ith known amounts o f PFOA prior to extraction, use foe follow ing equation to calculate the percent recovery. Recovery (% ) * [to ta l analyte found (ng/m L) - analyte found in control (ng/m L)] analyte added (ng/m L)___________________________ Page 6 of ? Page 64 o f 65 Exygen Research Page 111 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 Exygea Protocol Number: POOOI13] E l y l w R a c m t _____________________ _________ ______________ Method Numb VOOOP86 I ANaI vHCAI. m e t h o d _____________________ Method o f A ndys for the Dotem tinetion o fPerfUiorooctanoic Acid (PFOA) in Smell M sm m il Serum by LC/MS/MS 14.3 Use the follow ing equation u>convert the amount o f PFOA found in ng'rnL to ppb. PFOA found (uobt fPFQA found (na/m Li x fin a l volume (m L li ample volume (m l) Exygen Research Page 1 of Page 65 o f 65 Page 112 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Am endm ent Num ber_1__ Effective Date: 0 1 /1 9 /0 5 Exygen Study Num ber P0001131 Client Study N um ber Paga 1 of 1 D ES C R IPTIO N O F AM ENDED SEC TIO N 1) Analytical Procedure Sum m ary V0001780:Section 9.1 2 ) Verification o f Analytical Procedure None AMENPEPTP 1) Add to Section 9.1: Section 9 .1 .6, Alternate weights of standards m ay be used to prepare alternate concentrations of stock solutions as necessary. A lternate levels of fortification solutions m ay also be prepared. 2} Low and high spiking levels o f the analytes fo r each matrix m ay be altered depending on sam ple size available for extraction and/or to cover analyte concentrations expected in the sam ples. R A T IO N A LE 1 ) Higher concentrations of standards need to be prepared In order to spike the sample bottles at higher levels. 2 ) The sam ple size available for sm all m am mal liver and serum w as sm aller than expected. Spiking at the pre-determ ined levels in the protocol puts the spiked concentration lower than the detection lim it Also, the analyte levels in the ground w ater sam ples are expected to greatly exceed the pre-determ ined spiking levels listed in the protocol. W hen the levels in the sam ples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the Q C sam ple. Higher spiking levels in the bottles will cover the analyte concentrations expected in the w ater sam ples. IM PACT O N STU D Y T he LOQ is 100 ng/g for a 0.1 g sam ple o f small m am mal liver and is 1000 ng/m L for a 0.01 mL sam ple o f sm all mam mal serum. Higher levels of spiking for the w ater sam ples w ll ensure that m ore Q C recovery data can be used. LIBRARY ID: W0001226-6` - . Exygen Research ADMINISTRATIVE FORM Page 113 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 305B Research D rive Phone: 814-272-1039 S tate College, PA 16801 Fax: 814-231-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 2 03/07/05 P0001131 Client Study Number Pase 1of 1 None D ESC R IPTIO N O F AM ENDED SEC TIO N 1) Report, page 11 o f 65 2 ) T est M aterials, page 6 o f 65: PFO S transition monitored 4 96 -> 99. AMENDED TQ 1) Instead o f one final report, interim reports will be issued. 2 ) PFO S transition monitored m ay also be 4 99 > 80. RATIO NALE 1) Due to the excessive sizes o f the data sets, interim reports will be issued to allow the client to receive data in a tim elier re a r *. e*h!*r 2 ) The A PI 4 000 LC /M S/M S systems detect the 4 9 9 > 80 PFO S transition with greater sensitivity than the 4 9 9 -> 99 transition. IM PACT O N S TU D Y 1) T he client will be able to receive and review the data more quickly. 2 ) T he 4 6 9 -> 80 transition can be detected w ith greater sensitivity; therefore, giving better chromatography. o LIBRARY ID: V000122S-6- Exygen QAU R eview . tJJ odosits ADMINISTRATVE FORM Exygen Research Page 114 o f 115 Interim Report #4 - Analysis o f Surface Soil Samples Exygen Study No.: P0001131 J U L .2 S .2 0 0 S 8:56AM EXYGEN RESEARCH NO.T?4 P . 3 3058 Research Drive Phone: 814*272-1039 State College, PA 16801 Fax: 814-231*1580 Amendment Number E ffective D ate: Exygen Study Number PROTOCOL AMENDMENT 3 0 7 /1 8 /0 5 P1131 Client Study Number: Pas 1 f 1 NA ' DESCRIPTION OF AMENDED SECTIO N Verification of Analytical Procedure, page 10 of protocol AMBtBECLT The field duplicate can be used for the laboratory spikes and replicate when the primary sample volume is limited- B A IlQ NALg The sample size for a w ater sample is 200 m L If a sample site requires re-extraction for any reason, there would not be enough of the primary sample to repeat two laboratory spikes and a replicate. The field duplicate Is technically the sam e sam ple as the primary sample and therefore, can be used for laboratory spikes and replicates as needed. IMPACT ON STUDY No negative Im pact on the study. Using the duplicate sample allows for the full QC of the sample site to be completed. SI L/.. Study Director Signatum n .L / V ' / a U C f principal investigator Slgnaly/e y Exygn M argen !A A 6 ' * $ 2 ___________ Sponsor S lgriiivr*/! required) PaW / O ste D ate U - j t / i -S Date 7/is/o s Date E xygen QAU R ev iew 7 /W r LIBRARY IO: V000122S-8 ADMINISTRATIVE FORM Exygen Research Page 115 o f 115