Document MM2pLrg3zE0BDx3QVZ4X3aORM

INTERIM REPORT #22 - Analysis of Soil Samples STUDY TITLE Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: 610-701-3761 INTERIM REPORT COMPLETION DATE October 2,2006 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: 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number P0001131, entitled "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program," conducted for 3M Company, is being performed in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by Exygen Research. -John Flaherty Principal Investigator Exygen Research Jaisimha Kesari P.E., DEE Study Director Weston Solutions, Inc. Mici Sponsor Representative 3M Company Exygen Research Date Page 2 o f 134 Interim Report #22 - Analysis o f 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 phases1 were inspected for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Exygen Principal Investigator and Management and to the Study Director. Phase 28. Raw Data and Interim Report Review 36. Raw Data and Interim Report Review Date Inspected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Studv Director 06/20-23/06 06/28/06 07/05/06 10/02/06 09/28,29/06 10/02/06 10/02/06 10/02/06 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion of the study. This QA statement involves only the review of the interim report and associated raw data. Exygen Research Page 3 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 CERTIFICATION OF AUTHENTICITY This interim report, for Exygen Study Number P0001131, is a true and complete representation of the raw data. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Principal Investigator, Exygen: Date Exygen Research Facility Management: 2 ~OCl-70Ql> Date Study Director,iWeston Solutions, Inc. Jaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: i/lM ijd Michael A. Santoro Director of Regulatory Affairs Exygen Research Page 4 of 134 Interim Report #22 - Analysis o f 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: P0001131 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: 04/12/06 Interim Analytical Termination Date: 07/24/06 Interim Report Completion Date: 10/02/06 Exygen Research Page 5 o f 134 Interim Report #22 - Analysis o f 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 of the study: Name John Flaherty Karen Risha Eric Edwards Christine Edwards Mark Ammerman Amy Sheehan Mindy Cressley Brittany Kravets Krista Gallant Scott Crain Ling Ling Liu Cammy Graybill Frances Crespi Title Vice President Scientist Sample Custodian Technician Sample Custodian Associate Scientist Technician Technician Technician Technician Technician Technician Technician Exygen Research Page 6 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 TABLE OF CONTENTS Page TITLE PAGE....................................................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................. 2 QUALITY ASSURANCE STATEMENT..........................................................................3 CERTIFICATION OF AUTHENTICITY...........................................................................4 STUDY IDENTIFICATION................................................................................................5 PROJECT PERSONNEL.....................................................................................................6 TABLE OF CONTENTS.....................................................................................................7 LIST OF TABLES...............................................................................................................8 LIST OF FIGURES..............................................................................................................9 LIST OF APPENDICES....................................................................................................10 1.0 SUMMARY................................................................................................................ 11 2.0 OBJECTIVE............................................................................................................... 12 3.0 INTRODUCTION....................................................................................................... 12 4.0 ANALYTICAL TEST SAMPLES.............. >..............................................................12 5.0 REFERENCE MATERIAL........................................................................................ 13 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................ 15 6.1. Extraction Procedure For Soil................................................................................. 15 6.2 Percent Solids Procedure For Soil............................................................................ 15 6.3 Extraction Procedure for Water................................................................................16 6.4 Preparation of Standards and Fortification Solutions............................................... 16 6.5 Chromatography....................................................................................................... 17 6.6 Instrument Sensitivity...............................................................................................17 6.7 Description of LC/MS/MS Instrument and Operating Conditions...........................17 6.8 Quantitation and Example Calculation..................................................................... 18 7.0 EXPERIMENTAL DESIGN......................................................................................20 8.0 RESULTS...................................................................................................................20 9.0 CONCLUSIONS.........................................................................................................21 10.0 RETENTION OF DATA AND SAMPLES.............................................................21 Exygen Research Page 7 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table I. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Soil Samples.................................. 23 Table H Summary of PFOS in Re-extracted Soil Samples......................................... 29 Table HI. Summary of PFBS, PFHS and PFOS in Associated Rinse Blanks and Trip Blanks.....................................................................................................32 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples............33 Table V. Matrix Spike Recovery of PFOS in Re-extracted Soil Samples................... 45 Table VI. Matrix Spike Recovery of PFBS, PFHS and PFOS in Associated Trip Blanks.............................................................................................................48 Table VH. Total Percent Solids in Soil Samples...................................................... 49 Exygen Research Page 8 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFBS in Methanol......................................... 55 Figure 2. Extracted Standards of PFBS in Methanol, 25 ng/L and 50 ng/L, Respectively....................................................................................................56 Figure 3. PFBS in Methanol Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.................................... 57 Figure 4. Chromatogram Representing a Soil Sample Analyzed for PFBS (Exygen ID: C0156370, Data Set: 041306B).................................................58 Figure 5. Typical Calibration Curve for PFHS in Methanol......................................... 59 Figure 6. Extracted Standards of PFHS in Methanol, 25 ng/L and 50 ng/L, Respectively.....................................................................................60 Figure 7. PFHS in Methanol Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.....................................61 Figure 8. Chromatogram Representing a Soil Sample Analyzed for PFHS (Exygen ID: C0156368, Data Set: 041306BR)...............................................62 Figure 9. Typical Calibration Curve for PFOS in Methanol......................................... 63 Figure 10. Extracted Standards of PFOS in Methanol, 25 ng/L and 50 ng/L, Respectively....................................................................................................64 Figure 11. PFOS in Methanol Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.................................... 65 Figure 12. Chromatogram Representing a Soil Sample Analyzed for PFOS (Exygen ID: C0156344, D ata Set: 0 4 1 2 0 6 A R ).....................................................66 Exygen Research Page 9 of 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 LIST OF APPENDICES Page Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods and Protocol Amendments 1 and 2........................... 67 Exygen Research Page 10 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 1.0 SUMMARY Exygen Research extracted and analyzed soil samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001781 and water samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001780 (Appendix A). Seventeen of the analyzed soil samples were not reported (NR) for PFBS and six of the analyzed soil samples were not reported (NR) for PFHS due to quality control failure. These samples failed for unknown reasons. Fifty-two of the analyzed soil samples were not reported (NR) for PFOS due to quality control failure. Forty-five of the fifty-two samples failed because the spikes were too low relative to the endogenous level in the sample to allow assessment of matrix interference. Seven of the fifty-two samples failed for unknown reasons. Additional analytical work was performed to provide quantitative results for these fifty-two samples. The limit of quantitation for PFBS, PFHS, and PFOS in soil was 0.2 ng/g. The LOQ provided is assuming wet weight values and the results for the actual samples are reported based on dry weight. The limit of quantitation for PFBS, PFHS, and PFOS in water 25 ng/L. Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS in soil samples based on dry weight are summarized in Table I. Analytical results and assessed accuracies for the analysis of PFOS in re-extracted soil samples based on dry weight are summarized in Table II. Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS in water samples are summarized in Table III. To assess the accuracy of 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 three times or greater than that of the given spiking level. Quantitative results were obtained for all samples and analytes except for PFBS in seventeen samples, PFHS for six samples, and PFOS in seven samples that are not reported due to quality control failures. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in soil samples based on wet weight are summarized in Table IV. The overall average percent recovery standard deviation for PFBS, PFHS, and PFOS in soil samples based on wet weight were 111 25%, 86 8%, and 88 16%, respectively. Fortification recoveries for the analysis of PFOS in re-extracted soil samples based on wet weight are summarized in Exygen Research Page 11 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Table V. The overall average percent recovery standard deviation for PFOS in re extracted soil samples based on wet weight was 94 23%. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in water samples are summarized in Table VI. The overall average percent recovery standard deviation for PFBS, PFHS, and PFOS in water samples were 97 6%, 94 4%, and 86 + 4%, respectively. The total percent solids for soil samples are detailed in Table VII. 2.0 OBJECTIVE The objective of the analytical part of this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) in soil and water according to Protocol P0001131 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFBS, PFHS, and PFOS in soil using the analytical methods entitled, "V0001781: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" and for the determination of PFBS, PFHS, and PFOS in water using the analytical method entitled, "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS." The study was initiated on November 05, 2004, when the study director signed protocol number P0001131. The analytical start date for this interim report was April 12, 2006 and the analytical termination date for this interim report was July 24,2006. 4.0 ANALYTICAL TEST SAMPLES Five water samples, representing one trip blank and its associated spikes and two rinse blanks (RB), and forty-seven soil samples (Exygen ID C0156344 - C0156395) were received on wet ice on March 2, 2006 from Tim Frinak at Weston Solutions, Inc. Six water samples, representing one trip blank and its associated spikes and three rinse blanks (RB), and twenty-three soil samples (Exygen ID CO159443 - CO159471) were received on wet ice on March 10, 2006 from Tim Frinak at Weston Solutions, Inc. Four water samples, representing one trip blank and its associated spikes and one rinse blank (RB), and thirty-four soil samples (Exygen ID CO160867 - CO160904) were received on wet ice on March 15, 2006 from Tim Frinak at Weston Solutions, Inc. Four water samples, representing one trip blank and its associated spikes and one rinse blank (RB), and thirteen soil samples (Exygen ID CO164222 - CO164260) were received on wet ice on Exygen Research Page 12 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 March 24, 2006 from Tim Frinak at Weston Solutions, Inc. Please note that rinse blank (RB) samples do not have associated field spikes or laboratory spikes. The samples were logged in by Exygen personnel and placed in refrigerated storage. The soil samples collected represent both soil boring samples and surface soil samples. Sample ID coding for the soil samples consisted of five sections or strings in the following format: Dxxx-xx-xxxxxx-x(x)-xxxx. The first string of code, for the soil boring samples, contains DAL, which represents Decatur, Alabama. For the surface soil samples, the first string begins with D for Decatur, Alabama and the xxx defines the sampling location within the study area where: DF05 = Field 5 DF11 = Field 11 DF13= Field 13 DF10 = Field 10 DF12 = Field 12 The second string defines the type of sample where: SB = soil boring sample SS = surface soil sample The third string for the soil boring samples defines the soil boring identification. For the surface soil sample, the third string defines the location within the sampling area. The fourth string more specifically defines the sample type where: 0 = primary field sample RB = equipment rinseate blank sample DB = field duplicate sample The fifth string defines the collection depth o f the soil samples where: 0000 = 0 to 3 inches 0005 = 6 inches to 1 foot 0050 = 5 to 5.5 feet 0003 = 3 to 6 inches 0010 = 1 to 1.5 feet 0100 = 10 to 11 feet Sample login and chain of custody information is located in the raw data package associated with this interim report. Storage records will be kept at Exygen Research. 5.0 REFERENCE MATERIAL The analytical standard PFBS (SP0005726) was received from 3M at Exygen on May, 13, 2005. The analytical standard PFBS (SP0007771) was received from 3M at Exygen on June 13, 2006. The analytical standard PFHS (SP0002401) was received from 3M at Exygen Research Page 13 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Exygen on January 20, 2003. The analytical standard PFHS (SP0007770) was received from 3M at Exygen on June 13, 2006. The analytical standard PFOS (SP0002694) was purchased from Fluka Corporation and was received at Exygen on April 23, 2003. The analytical standard PFOS (SP0007769) was received from 3M at Exygen on June 13, 2006. The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated. Comnound PFBS PFBS PFHS PFHS PFOS PFOS Exveen Inventorv No. SP0005726 SP0007771 SP0002401 SP0007770 SP0002694 SP0007769 Lot# 101 101 SE036 NB 120067-69 430180-1 2795-53-93 Puritv (%) Expiration Date 96.7 12/04/06 96.7 10/17/06 98.6 10/18/06 98.6 10/18/06 101.2 10/31/07 86.9 08/31/06 The molecular structures of PFBS, PFHS and PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt ^FgSOaTC*) Transitions Monitored: 299 - 99 Structure: FF FF F SO 3 FF FF PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFuSOs'K*) Transitions Monitored: 399 - 80 Structure: FFF FF F F SO3 FFF FFF Exygen Research Page 14 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CsFnSOa'K4) Transitions Monitored: 499 80 Structure: F F F FF FFF SO3 FFFF FFFF 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical methods "V0001781: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" and "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" were used for 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 of 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 and the samples were then centrifuged for --10 m inutes at --3000 rpm. The supernatant w as then loaded onto a Cig SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.2 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 samples was then dried in an oven overnight at 104 2 C. Then the samples were transferred to a dessicator and allowed to cool for -15 minutes. Each sample was then weighed again, including the weight of the pan. The percent solid for each sample was then calculated. See Table VII for percent solids results. Exygen Research Page 15 o f 134 Interim Report #22 - Analysis o f 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. After fortification of appropriate samples, the samples were loaded onto a Cig SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.4 Preparation of Standards and Fortification Solutions A mixed stock standard solution of PFBS, PFHS, and PFOS was prepared at a concentration of 1000 pg/mL by dissolving 100 mg of each of the standards (corrected for purity and salt content) in 100 mL of methanol. From this solution, the following fortification standards were prepared: Cone, of Fort Stock or Fort. Volume Solution (mL) (pg/mL)1 1000 10 100 10 10 10 1.0 10 0.1 10 1of PFBS, PFHS, and PFOS Final Volume (mL) 100 100 100 100 100 Final Cone, of Fortification Std. (pg/mL) 100 10 1.0 0.1 0.01 A set of extracted calibration standards containing PFBS, PFHS, and PFOS were also made. The following concentrations were prepared before extraction: Cone, of Fort Fort Volume of Solution Volume Fortified Sample (ng/mL)1 (mL) (mL) - - 40 10 0.1 40 10 0.2 40 10 0.4 40 100 0.1 40 100 0.2 40 100 0.4 40 1of PFBS, PFHS, and PFOS Final Cone, of Calibration Std. (ng/mL) 0 25 50 100 250 500 1000 Exygen Research Page 16 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.5 Chromatography Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~0.5 mins, ~9.0 mins, and ~11.5 mins, respectively. Peaks above the LOQ were not detected in any of the reagent blank samples corresponding to the analyte retention time. 6.6 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 0.2 ng/mL of PFBS, PFHS and PFOS. 6.7 Description of LC/MS/MS Instrument and Operating Conditions Instrument: API 4000 Biomolecular Mass Analyzer Interface: Turbo Ion Spray Liquid Introduction Interface Computer: DELL OptiPlex GX400 Software: Windows NT, Analyst 1.4.1 HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: ~30 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time (mini 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: ~18 min %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Exygen Research Page 17 o f 134 Interim Report #22 - Analysis o f Soil Samples Flow Rate: 0.3 mL/min Ions monitored: Analvte PFBS PFHS PFOS Mode negative negative negative Exygen Study No.: P0001131 Transition Monitored 299 -> 99 399 -> 80 499 -80 Approximate Retention Time (min) ~0.5 min. -9.0 min. -11.5 min. 6.8 Quantitation and Example Calculation Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations of standards, except in a few cases of PFBS, where only five were used; as the 500 ng/L standard results were rejected as outliers. The concentration was determined from the following equations. Equation 1 calculated the amount of analyte found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/L) = (Peak area - intercept) x DF Slope 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 (ne/L)) xl00% amount added (ng/L) Equation 3 was used to convert the amount of PFBS, PFHS and PFOS found in ng/L to ng/g (ppb). Equation 3: Analyte found (ppb) = fAnalyte found (ng/Lj x volume extracted (0.04L)1 sample weight (5 g) Exygen Research Page 18 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Equation 4 was then used to calculate the amount of PFBS, PFHS and PFOS 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 of a calculation using an actual sample follows (calculation is for PFBS only): Soil sample Exygen ID: C0156360 Spk G (Set: 041206E), fortified at 500 ng/L with where: peak area = 28588 intercept = 0.000832 slope = 55.1 dilution factor =1 ng/L PFOA added (fort level) = 500 ng/L in corresponding sample = 28.8 volume extracted (L) = 0.04 sample weight (g) =5 total solids (%) = 81.74 From equation 1: Analyte found (ng/L) = F28588 - 0.0008321 x 1 55.1 From equation 2: % Recovery From equation 3: Analyte found (ppb) 519 ng/L (519 ne/L-2 8 .8 ne/L) x 100% 500 ng/L : 98% = (519 ng/L x 0.04L) 5g 4.15 ppb From equation 4: Analyte found (ppb) dry weight = 4.15 ppb x (100% / 81.74%) = 5.08 ppb Note: Numbers may vary slightly due to rounding. Exygen Research Page 19 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 7.0 EXPERIMENTAL DESIGN For water samples designated as field matrix spikes, PFBS, PFHS, and PFOS was added at a known concentration to the bottles in the laboratory before being shipped to the field. The water sample bottles were filled to a 200 mL volumetric fill line in the field. 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. The soil samples were extracted in thirty-two sets. Each set included one reagent blank and two reagent blanks fortified at known concentrations. Twenty-three of the sets contained five samples sites and one set contained two sample sites. One sample was collected for each sample site. For each site, two laboratory matrix spikes and a laboratory duplicate were extracted. The last eight sets were re-extraction sets. Four sets contained eight samples, three sets contained seven samples, and one set contained six samples. Each re-extraction set consisted of one sample and one laboratory matrix spike. The water samples were extracted in two sets. Each set included one reagent blank and two reagent blanks fortified at known concentrations. Set one contained three rinse blanks, two trip blanks with the associated spikes. Set two contained four rinse blanks and two trip blanks with the associated spikes. 8.0 RESULTS Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS in soil samples based on dry weight are summarized in Table I. Analytical results and assessed accuracies for the analysis of PFOS in re-extracted soil samples based on dry weight are summarized in Table II. Analytical results and assessed accuracies for the analysis o f PFBS, PFHS, and PFOS in water samples are summarized in Table III. To assess the accuracy of the results, the matrix spikes were used. Matrix spikes that were in die 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 three times or greater than that of the given spiking level. Quantitative results were obtained for all samples and analytes except for PFBS in seventeen samples, PFHS for six samples, and PFOS in seven samples that are not reported due to quality control failures. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in soil samples based on wet weight are summarized in Table IV. The overall average percent recovery standard deviation for PFBS, PFHS, and PFOS in soil samples based on wet weight were 111 25%, 86 8%, and 88 16%, respectively. Fortification recoveries for the analysis of PFOS in re-extracted soil samples based on wet weight are summarized in Exygen Research Page 20 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table V. The overall average percent recovery standard deviation for PFOS in re extracted soil samples based on wet weight was 94 23%. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in water samples are summarized in Table VI. The overall average percent recovery standard deviation for PFBS, PFHS, and PFOS in water samples were 97 6%, 94 4%, and 86 4%, respectively. The total percent solids for soil samples are detailed in Table VII. 9.0 CONCLUSIONS Except as noted above, the soil and water samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical methods V0001781 and V0001780, respectively. There were no circumstances that would have affected the quality or integrity of the data. 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 study director. This does not include facility-specific raw data such as instrument or temperature logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. Exygen Research Page 21 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 22 of 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study N o.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Soil Samples Exvnen ID C0156344 C0156344 Rep C0156345 C0156345 Rep CO156346 C0156346 Rep C0156347 CO156347 Rep CO156351 C0156351 Rep CO156352 C0156352 Rep CO156353 C0156353 Rep C0156354 C0156354 Rep C0156355 CO156355 Rep C0156356 C0156356 Rep C0156357 CO156357 Rep C0156358 CO156358 Rep C0156360 CO156360 R ep C0156361 C0158361 Rep C0156362 C0156362 Rep CO156363 CO156363 Rep CO156364 C0156364 Rep C0156365 C0 1 56365 Rep C lient Sam ple ID D F 1 3-S S -S S 0 3-0-000 0 D F 1 3-S S -S S 0 3-0-000 0* D F 1 3-S S -S S 0 3-0-000 3 D F 1 3-S S -S S 0 3-0-000 3* D F 1 3-S S -S S 0 4-0-000 0 D F 1 3-S S -S S 0 4-0-000 0* D F 1 3-S S -S S 0 4-0-000 3 D F 1 3-S S -S S 0 4-0-000 3* D F 1 3-S S -S S 0 5-0-000 0 D F 1 3-S S -S S 0 5-0-000 0* D F 1 3-S S -S S 0 5-0-000 3 D F 1 3-S S -S S 0 5-0-000 3* D F 13-S S -S S 05-D B -0003 D F 1 3-S S -S S 0 5-D B -00 03* D F 1 0-S S -S S 0 1-0 -0 0 0 0 D F 1 0-S S -S S 0 1-0-000 0* D F 1 0-S S -S S 0 1-0 -0 0 0 3 D F 1 0-S S -S S 0 1-0-000 3* D F 1 0-S S -S S 0 2-0-000 0 D F 1 0 -S S -S S 0 2 -0 -0 0 0 0 * D F 10-S S -S S 02-D B -0000 D F 1 0-S S -S S 0 2-D B -00 00* D F 1 0-S S -S S 0 2-0-000 3 D F 1 0-S S -S S 0 2-0-000 3* D F 1 0-S S -S S 0 3-0-000 0 D F 1 0 -S S -S S 0 3 -0 -0 0 0 0 * D F 1 0-S S -S S 0 3-0-000 3 D F 10-S S -S S 03-0-0003* D F 1 0-S S -S S 0 4-0-000 0 D F 1 0 -S S -S S 0 4 -0 -0 0 0 0 * D F 1 0-S S -S S 0 4-0-000 3 D F 10-S S -S S 04-0-0003* D F 1 0-S S -S S 0 5-0-000 0 D F 10-S S -S S 05-0-0000* D F 1 0-S S -S S 0 5-0-000 3 D F 1 0-S S -S S 0 5-0-000 3* C4 Sulfonate PFBS PorfluorobutaiMSuHonita Analyte Found (PPb, ng/g) Dry Weight Assessed Accuracy (/-% ) C6 Sulfonate PFHS Parfluorohexanaiulfoflata Analyte Found Assessed (ppb, ng/g) Dry Weight Accuracy (+/- %) C8 Sulfonate PFOS Parfluorooctanatulfonate Analyte Found Assessed (ppb, ng/g) Dry Weight Accuracy (+/- %) NR 44.9 30 NR NR - 35.2 30 NR * 3.22 30 66.4 30 1.77 30 37.1 30 NR NR _ * 2.07 30 3.33 30 332 50 1.93 30 3.12 30 350 50 3.14 30 27.6 30 620 30 3.23 30 24.3 30 561 30 3.06 30 90.8 30 2.51 30 72.5 30 NR NR _ - 2.03 30 63.8 30 2.33 30 48.7 30 NR NR . 4.00 40 89.3 30 2.17 40 61.7 30 1.15 30 9.37 30 1.08 30 7.97 30 NR NR NR NR - _ - 1.79 30 9.22 30 1.11 30 9.05 30 NR NR 0.883 30 NR . 824 40 0.698 30 NR - 771 40 1.06 30 5.99 30 746 40 1.15 30 8.14 30 859 40 0.603 0.970 40 40 4.45 8.06 30 30 NR NR _ - 0.282 30 0.791 30 0 .3 5 2 30 0 .5 7 9 3 0 0.339 30 0.686 30 0.247 30 0.633 30 190 161 199 213 30 30 30 30 4.02 50 31.3 30 4.34 50 35.8 30 NR NR . - 2.09 40 37.1 30 1.79 40 40.4 30 NR NR _ - 0.760 0.723 30 30 17.6 17.4 30 30 NR NR - 1.10 30 26.2 30 0.731 30 21.2 30 NR NR - * ` Laboratory Duplicate NR = Not Reported due to Quality Control failures, See Table II for PFOS re-extract data. Exygen Research Page 23 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Soil Samples Continued Exygen ID C0156366 C0156366 Rep C0156367 C0156367 Rep C0156368 C0156368 Rep C0156370 C0156370 Rep C0156371 C0156371 Rep C0156372 C0156372 Rep C 0156373 C0156373 Rep C 0156374 C0156374 Rep CO156375 C0156375 Rep CO156376 CO156376 Rep C0156377 CO156377 Rep C0156378 C0156378 Rep CO156379 C0156379 Rep C0156380 C0156380 Rep CO156381 C0156381 Rep C0156382 C0156382 Rep C0156383 C0156383 Rep C0156384 CO156384 Rep C lient Sam ple ID D F 1 1-S S -S S 0 1-0-000 0 D F 1 1-S S -S S 0 1-0-000 0* D F 1 1-S S -S S 0 1-0-000 3 D F 1 1-S S -S S 0 1-0-000 3* D F 1 1-S S -S S 0 2-0-000 0 D F 1 1-S S -S S 0 2-0-000 0* D F 1 1-S S -S S 0 2-0 -000 3 D F 1 1-S S -S S 0 2-0-000 3* D F 1 1-S S -S S 0 3-0-000 0 D F 1 1-S S -S S 0 3-0-000 0* D F 1 1-S S -S S 0 3-D B -00 00 D F 1 1-S S -S S 0 3-D B -00 00* D F 1 1 -S S -S S 0 3 -0 -0 0 0 3 D F 1 1-S S -S S 0 3-0-000 3* D F 1 1-S S -S S 0 4-0-000 0 D F11-SS-SS04-0-0000* D F11-SS-SS04-0-0003 D F 1 1-S S -S S 0 4-0-000 3* D F 1 1-S S -S S 0 5 -0 -0 0 0 0 D F 1 1-S S -S S 0 5 -0 -0 0 0 0 * D F 1 1-S S -S S 0 5 -0 -0 0 0 3 D F 1 1-S S -S S 0 5-0-000 3* D F 0 5-S S -S S 0 1-0 -0 0 0 0 D F 0 5-S S -S S 0 1-0 -0 0 0 0 * DF05-SS-SS01 -0-0003 DF05-SS-SS01-0-0003* D F 05-S S -S S 02-0-0000 D F 05-S S -S S 02-0-0000* D F 05-S S -S S 02-0-0003 D F 05-S S -S S 02-0-0003* D F 1 2-S S -S S 0 1-0-000 0 DF12-SS-SS01-0-0000* D F 1 2-S S -S S 0 1-0 -0 0 0 3 D F 1 2-S S -S S 01-0-0003* D F 1 2-S S -S S 0 2-0-000 0 D F 12-S S -S S 02-0-0000* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Parfluorobutanasulfonate_____ Parfluorohexanaiulfonata_____ ParttuorooctanasuHonata Analyte Found Assessed Analyte Found Assessed Analyte Found Assessed (ppb, ng/g) Dry Weight Accuracy (+/-% ) (ppb, ng/g) Dry Weight Accuracy <+/-%) (ppb, ng/g) Dry Weight Accuracy </-%) 1.14 30 78.6 30 1.21 30 42.8 30 NR NR 0.984 1.42 30 30 21.3 30.8 30 30 NR NR 20.5 30 197 30 22.2 30 205 30 NR NR NR 69.8 30 NR - 62.1 30 NR _ 14.1 30 NR - 15.7 30 2.16 30 15.7 30 1.93 30 19.1 30 NR NR NR NR NR NR 1.09 0.837 30 30 16.3 10.3 30 30 NR NR ND 40 1.15 30 219 30 ND 40 1.22 30 248 30 0.383 30 NR . 516 30 0.307 30 NR 510 30 0.527 30 2.40 30 496 30 0.515 30 2.11 30 475 30 0.448 30 2.70 30 494 30 0.494 30 2.55 30 574 30 0.791 0.783 0.370 0.252 30 30 50 50 7.68 9.49 2.43 1.19 30 30 30 30 NR NR NR NR . - - - NR 56.2 30 NR - NR * 28.9 30 NR * 2.25 2.27 0.569 0.840 30 30 30 30 33.7 31.0 NR NR 30 30 . - NR NR NR NR * - - 1.05 40 21.6 30 1.18 40 20.5 30 NR NR - NR . 58.0 30 NR . NR 57.6 30 NR " ' Laboratory Duplicate ND = Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). N R = Not Reported due to Quality Control failures, S ee Table II for PFO S re-extract data. Exygen Research Page 24 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Table I. Summary of PFBS, PFHS and PFOS in Soil Samples Continued Exvaen ID C0156385 C0156385 Rep C0156386 C0156386 Rep C0156387 C0156387 Rep C0156388 CO156388 Rep C0156389 C0156389 Rep C0156390 C0156390 Rep C0156391 CO156391 Rep C0156392 C0156392 Rep C0156393 CO156393 Rep C0156394 C0156394 Rep C0156395 CO156395 Rep C0159446 C0159446 Rep CO159447 C0159447 Rep C0159448 CO159448 Rep C0159449 C0159449 Rep C0159450 CO159450 Rep C 0159451 C0159451 Rep C0159453 C0159453 Rep C lient Sam ple ID D F 1 2 -S S -S S 0 2 -0 -0 0 0 3 D F 1 2-S S -S S 0 2-0-000 3* D F 1 2-S S -S S 0 3-0-000 0 D F 1 2-S S -S S 0 3-0-000 0* D F 1 2-S S -S S 0 3-0-000 3 D F 1 2-S S -S S 0 3-0-000 3* D F 1 2-S S -S S 0 4-0-000 0 D F 1 2-S S -S S 0 4-0-000 0* D F 1 2-S S -S S 0 4-0-000 3 D F 1 2-S S -S S 0 4-0-000 3* D F 1 2-S S -S S 0 5-0-000 0 D F 1 2-S S -S S 0 5-0-000 0* D F 1 2-S S -S S 0 5-0-000 3 D F 1 2-S S -S S 0 5-0-000 3* D F 1 3-S S -S S 0 1-0 -0 0 0 0 D F 1 3-S S -S S 0 1-0 -0 0 0 0 * D F 1 3-S S -S S 0 1-0 -0 0 0 3 D F 1 3-S S -S S 0 1-0-000 3* D F 1 3-S S -S S 0 2-0-000 0 D F 1 3-S S -S S 0 2-0-000 0* D F13-SS-SS02-0-0003 D F 1 3-S S -S S 0 2-0-000 3* D A L-S B -603 R -0-0000 D A L -S B -6 0 3 R -0 -0 0 0 0 * D A L-S B -603R -0-0005 D A L-S B -603 R -0-0005 * D A L-S B -603R -0-0010 D A L-S B -603 R -0-0010* D A L-S B -603R -0-0050 D A L-S B -603R -0-0050* D A L -S B -6 0 3 R -0 -0 1 00 D A L -S B -6 0 3 R -0 -0 1 00 * D A L-S B -603 R -D B -010 0 D A L-S B -603 R -D B -0100 * D A L-S B -604R -0-0000 D A L-S B -604R -0-0000* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS PsrtluorobutanesuM onsli______PstlhiorohexantsuW onsts______PsrtluorooctenesuHOnsts Analyte Found (PPb, ng/g) Drv Weiaht Assessed Accuracy (+/- %) Analyte Found (PPb, ng/g) Dry Weight Assessed Accuracy </-%) Analyte Found (PPb, ng/g) Dry Weight Assessed Accuracy (/-% > 5.47 30 92.8 30 5.95 30 92.6 30 NR NR - 0.349 0.281 30 30 1.34 1.09 30 30 NR NR _ - ND 30 1.00 30 388 30 ND 30 1.73 30 394 30 3.11 30 107 30 4.09 30 107 30 NR _ 210 30 NR - 202 30 1.91 50 3.58 30 2.28 50 5.04 30 NR NR NR NR NR NR - _ - _ - 2.45 40 13.9 50 2.42 40 14.2 50 NR NR - 1.87 50 29.9 30 2.17 50 23.8 30 2.13 30 33.7 30 2.02 30 33.1 30 NR NR NR NR * _ * 3.38 40 98.1 30 2.97 40 77.0 30 NR NR - 4.96 30 140 30 2.36 30 96.5 30 NR NR . * 0.277 30 ND 30 13.5 30 0.244 30 ND 30 14.4 30 ND 40 ND 30 21.8 30 ND 40 ND 30 23.6 30 ND 30 ND 30 15.5 30 ND 30 ND 30 13.2 30 0.396 30 0.406 30 0.358 30 0.356 30 19.8 8.74 30 30 ND 50 ND 30 0.298 30 ND 50 ND 30 ND 30 ND 50 ND 30 ND 30 ND 50 ND 30 ND 30 ND 30 ND 30 34.9 30 ND 30 ND 30 33.8 30 ` Laboratory Duplicate ND - N ot detected a t o r above the L im it o f Q uantitation (LOQ) o f 0.2 ng/g (w et w eight). NR = N ot Reported due to Q uality C ontrol b ilu re s , See Table II fo r PFOS re-extract data. Exygen Research Page 25 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Table I. Summary of PFBS, PFHS and PFOS in Soil Samples Continued Exvaen ID C0159454 C0159454 Rep CO159455 C0159455 Rep C0159456 C0159456 Rep C0159457 C0159457 Rep CO159458 C0159458 Rep CO159459 C0159459 Rep C0159460 C0159460 Rep C 0159461 C0159461 Rep CO159462 C0159462 Rep CO159463 C0159463 Rep C0159464 C0159464 Rep C0159466 C0159466 Rep CO159467 CO159467 Rep CO159468 CO159468 Rep C0159470 CO159470 Rep C 0159471 C0159471 Rep C0160871 C0160871 Rep CO160872 CO160872 Rep C lient Sam ple ID D A L-S B -604 R -0-0005 D A L-S B -604 R -0-0005 * D A L -S B -6 0 4 R -0 -0 0 1 0 D A L -S B -6 0 4 R -0 -0 0 1 0 * D A L-S B -604R -0-0050 D A L-S B -604 R -0-0050 * D A L -S B -6 0 4 R -0 -0 1 00 D A L -S B -6 0 4 R -0 -0 1 00 * D A L-S B -605 R -0-0000 D A L-S B -605 R -0-0000 * D A L-S B -605 R -D B -000 0 D A L-S B -605 R -D B -000 0* D A L-S B -605 R -0-0005 D A L-S B -605 R -0-0005 * D AL-SB-605R-0-0010 D A L -S B -6 0 5 R -0 -0 0 1 0* D A L-S B -605 R -0-0050 D A L-S B -605 R -0-0050 * D A L -S B -6 0 5 R -0 -0 1 00 DAL-SB-605R -0-0100* D A L-S B -1 3 8 R -0 -0 0 0 0 D A L -S B -1 3 8 R -0 -0 0 0 0 * D A L -S B -1 3 8 R -0 -0 0 0 5 D A L-S B -138 R -0-0005 * D A L-S B -1 3 8 R -0 -0 0 1 0 D A L-S B -138 R -0-0010 * D A L-S B -138 R -0-0050 D A L-S B -138 R -0-0050 * D A L -S B -1 3 8 R -0 -0 1 00 D A L-S B -138 R -0-0100* D A L-S B -138 R -D B -010 0 D A L-S B -138 R -D B -0100* D F 0 5-S S -S S 0 3-0-000 0 D F 05-S S -S S 03-0-0000* D F 0 5-S S -S S 0 3-0-000 3 D F 05-S S -S S 03-0-0003* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Parfluorobutanesulfonato_____ P arfluorohaxanaiulfonatt______ParfluorooctaneauHonata 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 (+/- %) 0.262 30 ND 30 19.9 30 ND 30 ND 30 17.9 30 ND 30 ND 30 NR ND 30 ND 30 NR . - ND 30 ND 30 34.1 30 0.244 30 ND 30 31.3 30 NR _ ND 30 ND 30 NR - ND 30 ND 30 0.859 40 ND 30 49.9 30 0.876 40 ND 30 52.8 30 0.803 30 ND 30 48.6 30 0.869 30 ND 30 50.3 30 0.644 30 ND 30 51.8 30 0.633 30 ND 30 48.4 30 ND 30 ND 30 NR ND 30 ND 30 NR _ * ND 50 ND 30 1.04 30 ND 50 ND 30 1.69 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 1.35 30 272 30 ND 30 1.50 30 276 30 0.247 30 2.30 30 454 30 0.257 30 2.86 30 585 30 0.449 30 2.65 30 264 30 0.368 30 2.69 30 388 30 9.86 50 28.3 30 223 50 7.58 50 22.5 30 279 50 3.82 50 17.8 30 238 30 2.87 50 14.2 30 175 30 2.83 30 NR . 216 30 3.56 30 NR 173 30 NR _ 40.1 30 NR - NR 35.6 30 NR " 20.7 40 87.5 30 4.22 40 42.1 30 NR NR - "Laboratory D uplicate ND - N ot detected at o r above the Lim it o f Q uantitation (LOQ) o f 0.2 ng/g (w et weight). NR = N ot Reported due to Q uality C ontrol failures, See Table II fo r PFOS re-extract data. Exygen Research Page 26 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Soil Samples Continued Exygen ID C 0160873 C0160873 Rep CO160874 CO160874 Rep C0160875 CO160875 Rep C0160876 CO160876 Rep CO160877 C0160877 Rep C0160878 CO160878 Rep C0160879 CO160879 Rep C0160880 CO160880 Rep C0160881 C0160881 Rep C0160882 CO160882 Rep C0160883 CO160883 Rep C0160884 C0160884 Rep CO160885 C0160885 Rep CO160886 CO160886 Rep C0160887 CO160887 Rep C0160888 CO160888 Rep C0160889 C0160889 Rep CO160890 C0160890 Rep C lient Sam ple ID C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Pw tluorobutsneiulfonats_____ Pfluorohxan--uHonat_____ PerfluofOOCtanwuHbruts 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 (+/- %> D F 0 5-S S -S S 0 4-0-000 0 0.353 30 ND 30 25.6 30 D F 0 5-S S -S S 0 4-0-000 0* 0.351 30 ND 30 20.7 30 D F 0 5-S S -S S 0 4-0-000 3 ND 30 ND 30 28.4 30 D F 0 5-S S -S S 0 4-0-000 3* ND 30 ND 30 32.2 30 D F 05-S S -S S 05-0-0000 ND 30 ND 30 38.1 30 D F 0 5-S S -S S 0 5-0-000 0* ND 30 ND 30 44.1 30 D F 0 5-S S -S S 0 5-0-000 3 ND 30 ND 30 60.4 30 D F 0 5-S S -S S 0 5-0-000 3* ND 30 ND 30 62.6 30 D F 0 5-S S -S S 0 5-D B -00 03 ND 30 ND 30 72.6 30 D F 0 5-S S -S S 0 5-D B -00 03* ND 30 ND 30 58.7 30 D F 0 9 -S S -0 0 0 6 -0 -0 0 0 0 0.321 30 0.615 30 206 30 D F 0 9-S S -000 6-0 -00 00* 0.373 30 0.697 30 216 30 D F 0 9 -S S -0 0 0 6 -0 -0 0 0 3 0.323 40 0.669 30 206 30 D F 0 9-S S -000 6-0 -00 03* 0.318 40 0.606 30 197 30 D F 0 9-S S -000 6-0 -00 10 D F 0 9-S S -000 6-0 -00 10* ND ND 40 0.308 30 40 0.284 30 DF09-SS-0007-0-0000 1.59 30 15.5 30 D F 0 9-S S -000 7-0 -00 00* 1.41 30 13.1 30 D F 0 9 -S S -0 0 0 7 -0 -0 0 0 3 D F 0 9-S S -000 7-0 -00 03* 0.662 0.732 30 30 NR NR _ - D F 0 9-S S -000 7-0 -00 10 D F 0 9 -S S -0 0 0 7 -0 -0 0 1 0 * ND ND 30 1.19 30 30 1.26 30 198 200 NR NR 1260 1190 379 389 30 30 . - 40 40 30 30 D F 0 9-S S -000 7-D B -0 010 0.248 30 1.21 30 291 30 D F 0 9-S S -000 7-D B -0 010* ND 30 1.04 30 392 30 D F 0 9 -S S -0 0 0 8 -0 -0 0 0 0 0.782 30 NR - NR D F 0 9 -S S -0 0 0 8 -0 -0 0 0 0 * 0.590 30 NR - NR D F 0 9 -S S -0 0 0 8 -0 -0 0 0 3 D F 0 9 -S S -0 0 0 8 -0 -0 0 0 3 * 0.943 0.625 30 30 8.36 8.56 40 40 NR NR D F 0 9 -S S -0 0 0 8 -0 -0 0 1 0 D F 0 9-S S -000 8-0 -00 10* 0.454 0.323 30 30 4.00 2.99 40 40 NR NR D F 0 9 -S S -0 0 0 9 -0 -0 0 0 0 0.377 30 0.473 30 201 30 D F 0 9 -S S -0 0 0 9 -0 -0 0 0 0 * 0.356 30 0.491 30 216 30 D F 0 9 -S S -0 0 0 9 -0 -0 0 0 3 ND 30 0.401 30 254 30 D F 0 9 -S S -0 0 0 9 -0 -0 0 0 3 * ND 30 0.523 30 353 30 D F 0 9-S S -00 09-D B -0 003 ND 30 0.417 30 255 30 D F 0 9-S S -000 9-D B -0 003* ND 30 0.478 30 294 30 ` Laboratory Duplicate ND = N ot detected a t o r above the L im it o f Q uantitation (LOQ) o f 0.2 ng/g (w et w eight). NR = N ot Reported due to Q uality C ontrol failures, See Table II fo r PFOS re-extract data. Exygen Research Page 27 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Soil Samples Continued Exygen ID C0160891 C0160891 Rep CO160892 C0160892 Rep CO160893 C0160893 Rep CO160894 C0160894 Rep C016089S C0160895 Rep C0160896 CO160896 Rep C0160897 CO160897 Rep C0160898 CO160898 Rep C0160899 CO160899 Rep C0160900 C0160900 Rep C0160901 C0160901 Rep CO160902 CO160902 Rep CO160903 CO160903 Rep C0160904 CO160904 Rep C0164247 CO164247 Rep C0164248 C0164248 Rep C0164249 C0164249 Rep CO164250 C0164250 Rep C lient Sam ple ID D F 0 9 -S S -0 0 0 9 -0 -0 0 1 0 D F 0 9-S S -000 9-0 -00 10* D F 0 9-S S -0010 -0-00 00 D F 0 9-S S -0010 -0 -0 0 0 0 * D F 0 9 -S S -0 0 1 0-0 -0 0 0 3 D F 0 9-S S -001 0-0 -00 03* D F 0 9-S S -0010 -0-00 10 D F 0 9-S S -0010 -0 -0 0 1 0 * D F09-SS-0011-0-0000 D F09-SS-0011-0-0000* D F09-SS-0011-0-0003 D F09-SS-0011-0-0003* D F09-SS-0011-0-0010 D F09-SS-0011-0-0010* D F 0 9-S S -0012 -0-00 00 D F 0 9 -S S -0 0 1 2 -0 -0 0 0 0 * D F09-SS-0012-DB-0000 D F 0 9-S S -001 2-D B -0 000* D F 0 9 -S S -0 0 1 2 -0 -0 0 0 3 D F 0 9 -S S -0 0 1 2 -0 -0 0 0 3 * D F09-SS-0012-0-0010 D F 0 9-S S -0012-0-00 10* D F 0 9-S S -0013-0-00 00 D F 0 9 -S S -0 0 1 3 -0 -0 0 0 0 * D F 0 9 -S S -0 0 1 3 -0 -0 0 0 3 D F 0 9-S S -0013 -0 -0 0 0 3 * D F 0 9 -S S -0 0 1 3-0 -0 0 1 0 D F 0 9 -S S -0 0 1 3 -0 -0 0 1 0 * D A L-S B -602R -0-0000 D A L-S B -602 R -0-0000 * D A L-S B -6 0 2 R -0 -0 0 0 5 D A L-S B -602 R -0-0005 * D A L-S B -602R -0-0010 D A L -S B -6 0 2 R -0 -0 0 1 0* D A L-S B -6 0 2 R -0 -0 0 5 0 D A L-S B -602 R -0-0050 * C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanmlfonate_____ PetWuorohaxanasuHbnat_____ Pwfluofooctan--ulfonato 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 (/-% ) ND 30 0.582 30 521 50 ND 30 0.595 30 624 50 NR 61.8 30 NR _ NR - 62.4 30 NR * 7.10 30 36.1 30 1580 30 5.99 30 36.9 30 1590 30 NR _ 30.8 30 1180 30 NR - 22.6 30 759 30 3.89 30 19.1 30 2.91 30 20.6 30 NR NR _ - 3.34 30 18.9 30 3.44 30 24.4 30 NR NR _ - NR 26.4 30 NR _ NR 33.6 30 NR - 5.23 50 27.5 30 5.73 50 35.0 30 NR NR . - NR _ 25.7 30 1310 30 NR - 23.0 30 1190 30 NR _ 39.0 30 NR . NR - 36.8 30 NR * 4.59 50 23.2 30 4.96 50 24.6 30 NR NR ,, ' 0.262 30 0.691 30 250 30 ND 30 0.705 30 236 30 ND 30 0.566 30 255 30 ND 30 0.831 30 305 30 ND 30 1.03 30 402 30 ND 30 0.719 30 344 30 0.593 30 ND 30 31.6 30 0.578 30 ND 30 33.6 30 0.535 30 ND 30 40.2 30 0.450 30 ND 30 41.2 30 ND 40 ND 30 45.0 30 ND 40 ND 30 33.5 30 ND 40 ND 30 ND 30 ND 40 ND 30 ND 30 'Laboratory Duplicate ND = N ot detected a t o r above the L im it o f Q uantitation (LOQ) o f 0.2 ng/g (w et w eight). NR = N ot Reported due to Q uality C ontrol failures, See Table II fo r PFOS re-extract data. Exygen Research Page 28 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Soil Samples Continued Exygen ID C0164251 CO164251 Rep C0164252 CO164252 Rep C0164253 C0164253 Rep C0164254 C0164254 Rep CO164255 C0164255 Rep C0164256 C0164256 Rep C0164257 CO164257 Rep CO164259 C0164259 Rep CO164260 CO164260 Rep C lient Sam ple ID D A L -S B -6 0 2 R -0 -0 1 00 D A L-S B -6 0 2 R -0 -0 100* D A L-S B -601 R -0-0000 DAL-SB-601R -0-0000' DAL-SB-601 R-DB-0000 DAL-SB-601 R-DB-0000 DAL-SB-601 R-0-0005 DAL-SB-601 R-0-0005 DAL-SB-601 R-0-0010 DAL-SB-601 R-0-0010 DAL-SB-601 R-DB-0010 DAL-SB-601 R-DB-0010 DAL-SB-601 R-0-0050 DAL-SB-601 R-0-0050 DAL-SB-601 R-0-0100 D A L -S B -6 0 1R -0-0100 * DAL-SB-601 R-DB-0100 DAL-SB-601 R-DB-0100* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Pertluorobutanesulfonate_____ Parfluorohexaneaulfonala_____ Parfluorooctanaiulfonata 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 <+/-%> NR ND 30 0.496 30 NR - ND 30 0.422 30 ND 30 ND 30 5.04 30 ND 30 ND 30 5.82 30 NR ND 30 5.61 30 NR - ND 30 5.28 30 NR ND 30 5.27 30 NR - ND 30 5.77 30 NR ND 30 NR NR - ND 30 NR - 0.350 50 ND 30 5.08 30 ND 50 ND 30 7.00 30 ND 30 ND 30 ND 30 ND 30 ND 30 0.315 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 'Laboratory Duplicate ND = Not detected at or above the Limit o f Quantitation (LOQ) of 0.2 rtg/g (wet weight). NR = Not Reported due to Quality Control failures, See Table II for PFOS re-extract data. Exygen Research Page 29 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study N o.: P0001131 Table II. E xyp e n ID Summary of PFOS in Re-extracted Soil Samples C8 Sulfonate PFOS P w flu o ro o cta n a tu lfo rn t C lie n t S am ple ID Analyte Found (ppb, ng/g) Dry W eight Assessed A ccura cy (+ /-% ) C0156344 D F13-S S -S S 03-0-0000 91900 40 C0156345 D F13-S S -S S03-0-0003 68900 30 C 0156351 D F13-S S -S S 05-0-0000 97900 30 C0156352 D F13-S S -S S 05-0-0003 43300 30 C0156353 D F13-S S -S S 05-D B -0003 78800 30 C0156354 D F10-SS-SS01 -0 -0000 1290 30 C0156355 DF 10-SS-SS01 -0 -0003 2400 30 C0156358 D F10-S S -S S02-0-0003 591 30 C0156362 D F10-S S -S S04-0-0000 4720 50 C0156363 D F10-S S -S S04-0-0003 9800 30 C0156364 D F10-S S -S S05-0-0000 3980 30 C0156365 D F10-S S -S S05-0-0003 9980 30 C0156366 D F11-S S -S S01-0 -000 0 NR - C0156367 D F11-S S -S S 01-0-0003 6240 30 C0156368 D F11-S S -S S 02-0-0000 59700 30 C0156370 D F11-S S -S S 02-0-0003 48140 50 C 0156371 D F11-S S -S S 03-0-0000 4500 30 C0156372 D F11-S S -S S 03-D B -0000 5110 30 C0156373 C0156378 D F11-S S -S S 03-0-0003 D F05-SS-SS01 -0 -0000 36600 2160 30 30 C0156379 D F05-S S -S S 01-0 -0003 572 30 C0156380 D F05-S S -S S 02-0-0000 14500 30 C 0156381 D F05-S S -S S 02-0-0003 20300 30 C0156382 DF 12-SS-SS01 -0 -0000 3100 40 C0156383 D F12-S S -S S 01-0-0003 68500 50 C0156384 C0156385 D F12-S S -S S02-0-0000 D F12-S S -S S 02-0-0003 NR 316000 30 NR = N ot R eported due to Q u a lity C ontrol fa ilu re s. Exygen Research Page 30 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table II. Summary of PFOS in Re-extracted Soil Samples Continued Exygen ID Client Sample ID C8 S ulfonate PFOS PerfluorooctanesuHbnate A nalyte Found Assessed (PPb, ng/g) D ry W eight Accuracy (+/-%) C0156386 DF12-SS-SS03-0-0000 263 30 C0156388 C0156389 D F 12 -S S -S S 04 -0 -0 0 0 0 DF12-SS-SS04-0-0003 NR 367000 30 C0156390 DF 12-SS-SS05-0-0000 8090 30 C0156391 DF 12-SS-SS05-0-0003 NR - C0156392 DF 13-SS-SS01-0-0000 64200 30 C0156393 DF 13-SS-SS01-0-0003 49500 50 CO156394 DF 13-SS-SS02-0-0000 NR - C0156395 C0159455 DF13-SS-SS02-0-0003 DAL-SB-604R-0-0010 211000 15.4 40 40 C0159461 C0160871 DAL-SB-605R-0-0010 DF05-SS-SS03-0-0000 NR 8460 30 C0160872 DF05-SS-SS03-0-0003 13200 30 C0160881 DF09-SS-0007-0-0000 NR - C0160885 DF09-SS-0008-0-0000 2700 30 C0160886 DF09-SS-0008-0-0003 4370 40 C0160887 DF09-SS-0008-0-0010 2110 30 C0160892 C 0160893 DF09-SS-0010-0-0000 D F09-S S -0010-0-00 03 3910 1550 30 NA C0160895 DF09-SS-0011-0-0000 1710 30 C0160896 DF09-SS-0011-0-0003 2030 30 C0160897 D F 0 9 - S S -0 0 1 1-0 -0010 1700 30 C0160898 DF09-SS-0012-0-0000 1110 30 C0160900 DF09-SS-0012-0-0003 2620 30 C0160901 DF09-SS-0012-0-0010 1540 30 C0164255 DAL-SB-601R-0-0010 7.35 40 NR = Not Reported due to Quality Control failures. Exygen Research Page 31 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table III. Summary of PFBS, PFHS and PFOS in Associated Rinse Blanks and Trip Blanks Exygen ID C0156348 C0156359 C01S6369 C0159443 C0159452 C0159465 C0159469 C0160867 C0160870 C0164222 C0164258 C lie n t S am ple ID D F 13-S S -T R IP 1-0-060221A DF10-SS-SS03-RB-0000 DF11-SS-SS02-RB-0003 DAL-SB-TRIP1 -0-060302A D A L-S B -603 R -R B -0100 DAL-SB-138R-RB-0000 DAL-SB-138R-RB-0100 D F 0 5-S S -T R IP 2-0-06 030 8A D F 05-S S -S S 03-R B -0000 D A L-S B -TR I P -0 -060316A D A L-S B -601R -R B -0100 C4 S ulfonate PFBS PorfluorobutanMulfonato ND ND ND ND ND ND ND ND ND ND ND Analyte Found (ppt, ng/L) C6 S ulfon ate PFHS PorfluorohaxanMulfonate ND ND ND ND ND ND ND ND ND ND ND C8 S ulfon ate PFOS PufluorooctamsuHonatt ND ND ND ND ND ND ND ND ND ND ND ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. * The assessed accuracy for all analytes in the sample is +/- 30%. Exygen Research Page 32 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Sample Descriotion C4 Sulfonate PFBS Wet Weight C6 Sulfonate PFHS Wet Weight C8 Sulfonate PFOS Wot 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) (nato) wet wt. (nato) wet wt. (%) (nato) wet wt. (noto) wet w t <%) (no/a) wet wt. (ng/g) wet wl. (%) DF13-SS-SS03-0-0000 (C0196344 SpkC,4 ppb Spille) DF13-SS-SS03-0-0000 (COI56544Spk 0,400 ppb Splke) 4 400 NR NR NR NR 29.4 NR NR 29.4 32.0 380 88 NR NR NR NR NR NR DF13-SS-SS03-0-0003 (COI50345 SpkE, 4ppb Splke) DF13-SS-SS03-0-0003 (C0150345Spk F,400 ppbSplke) 4 400 2.47 2.47 5.86 85 51.0 429 107 51.0 42.3 352 75 NR NR NR NR NR NR DF13-SS-SS04-0-0000 (Cd56346Spk 6,4 ppb Splke) DF13-SS-SS04-0-0000 (COI56346Spk H,400 ppb Spike) 4 400 1.73 1.73 5.38 91 2.78 416 104 2.78 6.49 93 310 345 86 310 325 545 59 DF13-SS-SS04-0-0003 (Cd56347Spk1,4 ppb Splke) DF13-SS-SS04-0-0003 (C0156347 SpkJ, 400ppb Splke) 4 400 2.74 2.74 5.71 74 24.1 316 78 24.1 27.8 541 399 94 541 536 856 79 DF13-SS-SS05-0-0000 (Cd56351 Spk K4 ppb Spike) DF13-SS-SS05-0-0000 (C0156351 SpkL,400 ppbSpila) 4 400 2.39 2.39 6.95 114 71.0 466 116 71.0 72.7 429 90 NR NR NR NR NR NR DF13-SS-SS05-0-0003 (Cd56352 SpkC,4 ppbSplke) DF13-SS-SS05-0-0003 (Cd56352Spk 0,400 ppb Spike) 4 400 172 1.72 6.27 114 54.1 547 136 54.1 54.1 378 81 NR NR NR NR NR NR DF13-SS-SS05-DB-0003 (C0156353 Spk E,4 ppb Splke) DF13-SS-SS05-DB-0003 (C0156353Spk F,400 ppb Spike) 4 400 3.34 3.34 5.61 62 74.4 471 117 74.4 49.8 356 70 NR NR NR NR NR NR DF10-SS-SS01-0-0000 (Cd56354Spk G,4 ppb Spike) DF10-SS-SS01-0-0000 (Cd56354Spk H,400 ppb Splke) DF10-SS-SS01-0-0003 (C0196359Spk1,4 ppb Splke) DF10-SS-SS01-0-0003 (C0156355SpkJ, 400ppb Splke) 4 400 4 400 0.968 0.968 1.55 1.55 5.51 114 7.90 474 118 7.90 5.67 103 8.00 537 134 8.00 11.6 93 327 80 11.8 95 358 88 NR NR NR NR NR NR NR NR NR NR NR NR DF10-SS-SS02-0-0000 (Cd56356Spk K,4 ppb Splke) DF10-SS-SS02-0-0000 (C0156356SpkL. 400 ppbSpike) 4 400 0.762 0.762 5.07 108 NR 450 112 NR NR NR 711 NR NR 711 758 952 60 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NR * Not Reported due to Quality Control failures, See Table V for PFOS re-extract data. Note: Since this summarytable shows rounded results, recoveryvalues may vary slightly from the values In the raw data. Exygen Research Page 33 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study N o.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Description C4 Sulfonate PFBS Wat Weight C6 Sulfonate PFHS WetWeight 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 (na/a) wet wt. (na/a) wet wt. (%) (na/a) wet wt. (na/a) wet wt. (%) (na/a) wet wt. (na/a) wet wt. <%) DF10-SS-SS02-DB-0000 (CM56357SpkC,4 ppbSpike) DF10-SS-SS02-DB-0000 (CM56357Spk D.400 ppb Spike) 4 400 0.920 0.820 5.30 110 5.18 518 129 5.18 8.88 93 645 354 87 645 614 920 69 DF10-SS-SS02-0-0003 (C015S355 SpkE, 4ppb Spike) DF10-SS-SS02-0-0003 (C0156353Spk F, 400ppbSpike) 4 400 0.522 0.622 5.93 135 3.86 530 132 3.86 7.02 79 NR 358 89 NR NR NR NR NR DF10-SS-SSO3-0-0000 (C0156360Spk G, 4ppb Spike) DF10-SS-SS03-0-0000 (C0156360Spk H,400 ppb Spike) DF10-SS-SS03-0-0003 (C0156361 Spk1.4 ppb Spike) DF10-SS-SS03-0-0003 (C0156361 SpkJ, 400ppb Spike) 4 400 4 400 0.230 0.230 0.286 0.286 4.15 98 0.646 390 97 0.646 3.54 81 0.570 378 94 0.578 3.93 361 4.34 379 82 90 94 95 155 155 167 167 129 441 72 154 512 86 DF10-SS-SS04-0-0000 (CM56362Spk K, 4 ppb Spike) DF10-SS-SS04-0-0000 (C0156362SpkL, 400ppb Spike) 4 400 2.88 2.88 8.72 146 22.4 536 133 22.4 20.9 NR 338 79 NR NR NR NR NR DF10-SS-SS04-0-0003 (CM56363SpkC.4 ppb Spike) DF10-SS-SS04-0-0003 (CC0156363SpkD, 400ppb Spike) 4 400 1.58 1.58 6.82 131 28.1 599 149 28.1 34.6 . 393 91 NR NR NR NR NR NR DF10-SS-SS05-0-0000 (C0156364Spk E.4 ppb Spike) DF10-SS-SS05-0-0000 (C0156364SpkF, 400ppb Spike) 4 400 0.625 0.625 4.53 98 14.5 464 116 14.5 22.6 374 90 NR NR NR NR NR NR DF10-SS-SS06-0-0003 (CM56365Spk 0,4 ppb Spike) DF10-SS-SS05-0-0003 (CM56366 Spk H,400ppb Spike) 4 400 0.904 0.904 5.77 122 21.6 576 144 21.6 27.4 382 90 NR NR NR NR NR NR DF11-SS-SS01-0-0000 (C015S3SSSpk 1,4 ppb Spike) DF11-SS-SS01-0-0000 (C0156366SpkJ, 400ppb Spike) 4 400 0.928 0.928 5.42 112 83.8 525 131 63.8 41.1 NR 390 82 NR NR NR NR NR DF11-SS-SS01-0-0003 (CMS6367SpkK. 4 ppb Spike) DF11-SS-SS01-0-0003 (C01S6367SpkL, 400ppb Spike) 4 400 0.824 NR 5.85 126 17.8 NR NR 17.8 30.6 359 85 NR NR NR NR NR NR ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NR Not Reported due to Quality Control failures, See Table V for PFOS re-extract data. Note: Since this summarytable shows rounded results, recoveryvalues mayvary slightly from the values In the raw data. Exygen Research Page 34 o f 134 Interim Report #22 - Analysis of Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Description Amount Amt Found Spiked in Sample fna/a) wet wt. Amount Recovered Amt Found Amount Amt Found Amount Recovery in Sample Recovered Recovery in Sample Recovered Recovery (% i (no/a) wet wt. (na/a) wet wt. (%) (no/a) wet wt. (nata) wet wt. (%) DF11-SS-SS02-0-0000 (C019S3SSSpk C.4 ppb Spike) DF11-SS-SS02-0-0000 (COISS3SI Spk D,400ppb Spike) 4 400 13.9 13.9 24.1 134 465 110 134 157 * NR 440 77 NR NR NR NR NR DF11-SS-SS02-0-0003 (C01S6370 SpkE,4 ppb Spike) DF11-SS-SS02-0-0003 (C0156370Spk P,400ppb Spike) 4 400 NR NR NR NR 56.4 NR NR 56.4 69.7 362 76 NR NR NR NR NR NR DF11-SS-SS03-0-0000 (C0156371 Spk 6,4 ppb Spike) DF11-SS-SS03-0-0000 (COI50371 SpkH,400 ppb Spike) 4 400 NR NR NR NR 11.3 NR NR 11.3 15.9 115 NR 326 79 NR NR NR NR NR DF11-SS-SS03-DB-0000 (COI56372Spk 1,4ppb Spike) DF11-SS-SS03-DB-0000 (C01S6372SpkJ, 400ppb Spike) 4 400 1.73 1.73 6.82 127 12.6 576 144 12.6 16.4 373 90 NR NR NR NR NR NR DF11-SS-SS03-0-0003 (COI56373 SpkK,4 ppbSpike) DF11-SS-SS03-0-0003 (C0156373Spk L, 400ppb Spike) 4 400 0.928 0.828 5.78 121 13.9 536 134 13.9 23.5 340 82 NR NR NR NR NR NR DF11-SS-SS04-0-0000 (C0150374Spk C,4 ppb Spike) DF11-SS-SS04-0-0000 (C0180374 Spk D.400ppb Spike) 4 400 ND ND 5.62 141 1.00 438 110 1.00 4.83 96 190 373 93 190 218 590 100 DF11-SS-SS04-0-0003 (C0150375Spk E,4 ppb Spike) DF11-SS-SS04-0-0003 (C0150375Spk F, 400ppb Spike) 4 400 0.342 0.342 4.83 112 NR 440 110 NR NR NR 461 NR NR 461 880 888 107 DF11-SS-SS05-0-0000 (C0150376Spk 0,4 ppb Spike) DF11-SS-SS05-0-0000 (COI50370SpkH, 400ppb Spike) 4 400 0.441 0.441 5.13 117 2.01 461 115 2.01 5.46 86 415 382 95 415 307 715 75 DF11-SS-SS05-0-0003 (COI00377 Spk1,4 ppb Spike) DF11-SS-SS06-0-0003 (C0150377SpkJ, 400ppb Spike) 4 400 0.382 0.382 5.11 118 2.30 482 120 2.30 5.50 80 421 347 86 421 454 732 78 DF05-SS-SS01-0-0000 (COI50371SpkK, 4 ppb Spike) DF05-SS-SS01-0-0000 (C0156378Spk L, 400ppb Spite) 4 400 0.646 0.646 5.77 128 6.26 469 117 6.26 10.6 109 NR 337 83 NR NR NR NR NR Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NR = Not Reported due to Quality Control failures. See Table V for PFOS re-extract data. ND = Not detected at or above the lim it of Quantitation (LOQ) of 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. Exygen Research Page 35 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study N o.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample DescriDtion C4 Sulfonate PFBS Wet W eight C6 Sulfonate PFHS Wat W eight C8 Sultanato PFOS W tt W eight Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery fno/a)wetwt. fnafa) wet wt. <%> (ng/g) wet wt. fno/a) wet wt. <%> fna/a) wet wt. (na/g) wet wt. <%) DF05-SS-SS01-0-0003 (COI56379SpkC,4 ppbSpike) DF05-SS-SS01-0-0003 (C0156379Spk D,400 ppb Spike) 4 400 0.311 0.311 6.28 149 2.04 595 149 2.04 5.57 88 380 94 NR NR NR NR NR NR DF05-SS-SS02-0-0000 (C0156390SpkE, 4 ppb Spike) DF05-SS-SS02-0-0000 (C0156390Spk F,400 ppb Spike) DF05-SS-SS02-0-0003 (COI56361 Spk 6,4 ppb Spike) DF05-SS-SS02-0-0003 (COI56391 SpkH, 400ppb Spike) DF12-SS-SS01-00000 (COI56392Spk 1,4ppb Spike) DF12-SS-SS01-0-0000 (COI56392SpkJ. 400ppb Spike) 4 400 4 400 4 400 NR NR 1.90 1.90 0.470 0.470 NR NR 46.3 NR NR 46.3 5.77 97 28.4 474 118 28.4 5.34 122 NR 522 130 NR 106 374 82 37.8 346 79 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR DF12-SS-SS01-00003 (COI56393Spk K, 4 ppb Spike) OF12-SS-SS01-0-0003 (C0156393Spk L,400 ppb Spike) 4 400 0.656 0.856 6.15 132 17.5 470 117 17.5 29.1 NR 334 79 NR NR NR NR NR DF12-SS-SS02-0-0000 (COI56394SpkC,4 ppbSpike) DF12-SS-SS02-0-0000 (COI56394Spk D,400ppb Spike) 4 400 NR NR NR NR 39.8 NR NR 39.8 42.6 . 383 86 NR NR NR NR NR NR DF12-SS-SS02-0-0003 (C0156396spk E,4 ppb Spike) DF12-SS-SS02-0-0003 (C0156395SpkF, 400ppb Spike) 4 400 4.03 4.03 9.04 125 68.4 562 137 68.4 65.4 . 366 74 NR NR NR NR NR NR DF12-SS-SS03-0-0000 (CM56396SpkG4ppb Spike) DF12-SS-SS03-0-0000 (CM56396SpkH, 400ppb Spike) 4 400 0.287 0.287 4.81 113 1.10 477 119 1.10 4.57 87 NR 336 84 NR NR NR NR NR DF12-SS-SS03-0-0003 (00106397 Spk 1,4ppb Spike) DF12-SS-SS03-0-0003 (CM56397SpkJ, 400ppb Spike) 4 400 ND ND 4.03 101 0.880 4.36 87 340 342 86 0.860 341 65 340 227 . 668 80 DF12-SS-SS04-0-0000 (CM56399SpkK, 4 ppb Spike) DF12-SS-SS04-0-0000 (C0156399Spk L,400ppb Spike) 4 400 2.31 2.31 7.67 134 79.4 572 142 79.4 93.6 . NR 434 89 NR NR NR NR NR ` Sample residue exceeds die spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NR * Not Reported due to Quality Control failures, See Table V for PFOS re-extract data. ND * Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). Note: Since tills summarytable shows rounded results, recovery values mayvary slightly from the values in the raw data. Exygen Research Page 36 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Descrlotion C4 Sutfonata PFBS Wet Weight C6 Sulfonate PFH3 Wet Weight C8 Sulfonate PFQ3 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 fna/g) wet wt. ino/a) wet wt. <%> fna/a) wet wt. (na/o) wet wt. <%> (ng/g) wet wt. (ng/g) wet wt. <%> DF12-SS-SS04-0-0003 (COI56389Spk C,4 ppb Spile*) DF12-SS-SS04-0-0003 (COI56389Spk D,400 ppb Spik*) 4 400 NR NR NR NR 164 NR NR 164 153 NR 472 77 NR NR NR NR NR DF12-SS-SS05-0-0000 (C0156390SpkE, 4 ppb Spik*) DF12-SS-SS05-0-0000 (C0156390Spk F,400 ppb Spik*) 4 400 1.50 1.50 7.19 142 2.80 582 145 2.80 6.38 90 360 87 NR NR NR NR NR NR DF12-SS-SS05-0-0003 (COI56391 Spk6 4 ppb Spik*) DF12-SS-SSO5-0-0003 (COI56391 Spk H.400 ppb Spik*) 4 400 1.99 1.99 7.53 139 11.3 566 141 11.3 13.3 50 338 82 NR NR NR NR NR NR DF13-SS-SS01-0-0000 (COI56392Spk 1,4 ppb Spik*) DF13-SS-SS01-0-0000 (C0156382SpkJ. 400ppb Spik*) 4 400 1.45 1.45 7.23 145 23.1 543 135 23.1 28.4 NR 358 84 NR NR NR NR NR DF13-SS-SS01-0-0003 (COI56393Spk K, 4 ppb Spik*) DF13-SS-SS01-0-0003 (C0156393SpkL, 400ppb Spik*) 4 400 1.70 1.70 6.B2 128 27.0 581 145 27.0 28.2 * NR 374 87 NR NR NR NR NR DF13-SS-SS02-0-0000 (COI56394Spk C,4 ppb Spik*) DF13-SS-SS02-0-0000 (COI56394SpkD,400ppb Spik*) 4 400 2.56 2.56 8.08 138 74.2 572 142 74.2 57.7 * NR 435 90 NR NR NR NR NR DF13-SS-SS02-0-0003 (C0159395Spk E,4 ppb Spik*) DF13-SS-SS02-0-0003 (COI56395Spk F, 400ppb Spik*) 4 400 3.98 3.98 7.53 89 113 510 127 113 96.0 391 70 NR NR NR NR NR NR DAL-SB-603R-0-0000 (C01S9446Spk E,4 ppb Spik*) DAL-SB-603R-0-0000 (COI59446Spk F, 400ppb Spik*) DAL-SB-603R-0-0005 (0018*447 Spk G , 4 ppb Spitw) DAL-SB-603R-0-0005 (Cd59447Spk H,400ppb Spik*) 4 400 4 400 0.251 0.251 ND ND 4.50 106 ND 407 102 ND 2.59 65 ND 323 81 ND 3.07 77 12.2 324 81 12.2 2.95 74 18.7 350 88 18.7 17.4 314 21.4 366 . 75 . 87 DAL-SB-603R-0-0010 (Cd59448Spk1,4 ppbSpik*) DAL-SB-603R-0-0010 (C01S9448Spk J,400ppb Spik*) 4 400 ND ND 2.85 71 ND 274 69 ND 3.12 78 12.6 344 86 12.6 16.2 . 359 87 'Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NR * Not Reported due to Quality Control failures. See Table V for PFOS re-extract data. ND * Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). Note: Since this summarytable shows rounded results, recovery values mayvary slightly from the values In the raw data. Exygen Research Page 37 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample OescriDtion C4 Sulfonate PFBS Wet W ight C6 Sulfonate PFHS WotWeight Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery (nofa) wet wt. (%) (rm/s) wet wt. (ng/g) wet wt. (%) C8 Sulfonate PFOS Wet Weight Amt Found Amount in Sample Recovered Recovery fnota) wet wt. <%> DAL-SB-603R-0-0050 (C0159449Spk K, 4 ppb Spike) DAL-SB-603R-0-0050 (C018M49SpkL, 400ppb Spike) 4 400 0.328 0.328 3.66 83 0.336 3.23 72 16.4 440 110 0.336 343 86 16.4 19.3 334 70 DAL-SB-603R-0-0100 (C019M50SpkC, 4 ppb Splks) DAL-SB-603R-0-0100 (C01S9490Spk D,400 ppb Spike) 4 400 ND ND 2.07 52 ND 205 51 ND 3.18 80 0.249 3.42 79 366 92 0.249 334 83 DAL-SB-603R-DB-0100 (C01SS451 SpkE,4 ppb Spike) DAI-SB-603R-DB-0100 (C0159451 SpkF,400 ppb Spike) 4 400 ND ND 2.07 52 ND 172 43 ND 3.36 84 332 83 ND ND 3.34 84 340 85 DAL-SB-604R-0-0000 (C019MS3Spk 6,4 ppb Spike) DAL-SB-604R-0-0000 (00199459Spk H,400 ppb Spike) 4 400 ND ND 4.30 108 ND 502 126 ND 3.22 81 31.3 330 83 31.3 29.8 358 82 DAL-SB-604R-0-0005 (C0159454Spkt, 4 ppb Spike) DAL-SB-604R-0-0005 (00189454Spk J, 400ppb Spike) 4 400 0.215 0.215 5.04 121 ND 542 135 ND 3.25 81 16.3 355 89 16.3 22.6 399 96 DAL-SB-604R-0-0010 (00159488SpkK, 4 ppb Spike) DAL-SB-604R-0-0010 (C0189489Spk L, 400ppb Spike) 4 400 ND ND 4.97 124 ND 584 146 ND 3.18 80 376 04 NR NR NR NR NR NR DAL-SB-604R-Q-0050 (C01S9458SpkC, 4 ppb Spike) DAL-SB-604R-0-0050 (C0189458Spk0,400 ppb Spike) 4 400 ND ND 4.42 111 ND 402 101 ND 3.60 90 30.1 332 83 30.1 33.4 . 358 82 DAL-SB-604R-0-0100 (00189487Spk E, 4 ppb Spike) DAL-SB-604R-0-0100 (Cd59457SpkF,400 ppbSpike) 4 400 NR NR NR NR ND NR NR ND 2.98 75 ND 342 86 ND 2.94 74 329 82 DAL-SB-605R-0-0000 (C01SS4SSSpkO4ppb Spite) DAL-SB-605R-0-0000 (C0189451 Spk H,400ppb Spike) 4 400 0.600 0.699 6.06 134 ND 486 121 ND 3.14 79 40.6 328 82 40.6 48.2 358 79 DAL-SB-605R-DB-0000 (C0189489Spk1,4ppb Spike) DAL-SB-605R-DB-0000 (C0189459SpkJ, 400ppb Spike) 4 400 0.657 0.657 5.69 126 ND 526 131 ND 3.04 76 39.8 336 84 39.8 49.7 346 77 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NR Not Reported due to Quality Control failures, See Table V for PFOS re-extract data. ND * Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). Note: Since this summary table shows rounded results, recovery values may vary slightly from die values In the raw data. Exygen Research Page 38 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study N o.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Description Amount Spiked ino/a) C4 Sulfonate PFBS Wat Weight C6 Sulfonate PFHS WotWeight C8 Sulfonate PFQ8 WotWeight Amt Found Amount Amt Found Amount Amt Found Amount in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (%) (na/g)wetwt. (no/g) wet wt. (%> (ng/g) wet wt. (ng/g) wet wt. <%) DAL-SB-605R-0-0005 (C0159490SpkK, 4 ppbSpike) DAL-SB-605R-0-0005 (C015S460SpkL, 400ppb Spike) 4 400 0.546 0.546 4.69 104 ND 435 109 ND 3.18 80 43.8 335 64 43.8 41.4 370 82 DAL-SB-605R-0-0010 (COI59461 Spk C,4 ppb Spike) DAL-SB-605R-0-0010 (C0159461 Spk D,400 ppb Spike) 4 400 ND ND 4.50 113 ND 458 115 ND 3.32 83 353 88 NR NR NR NR NR NR DAL-SB-605R-0-0050 (C0159462Spk E.4 ppb Spike) DAL-SB-605R-0-0050 (C01S9462Spk F,400 ppbSpike) 4 400 ND ND 2.33 68 ND 220 55 ND 3.58 90 0.832 4.74 98 362 91 0.832 358 89 DAL-SB-605R-0-0100 (COI59463Spk0 ,4 ppb Spike) DAL-SB-605R-0-0100 (C0159463 Spk H,400 ppb Spike) 4 400 ND ND 2.78 70 ND 245 61 ND 3.28 82 335 64 ND ND 3.34 84 346 87 DAL-SB-138R-0-0000 (C0159464Spk1,4 ppb Spike) DAL-SB-138R-0-0000 (C0189464Spk J,400 ppbSpike) 4 400 ND ND 5.15 129 1.16 564 141 1.16 4.80 91 234 390 97 234 247 717 121 DAL-SB-138R-0-0005 (COI59466Spk K, 4 ppb Spike) DAL-SB-138R-0-0005 (C01S9466Spk L 400 ppbSpike) 4 400 0.218 0.218 4.46 106 2.02 462 120 2.02 5.82 95 399 364 90 399 424 880 120 DAL-SB-138R-0-0010 (COI59467SpkC,4 ppb Spike) DAL-SB-138R-0-0010 (COI59467Spk D,400 ppb Spike) 4 400 0.386 0.386 6.62 156 2.28 584 146 2.28 6.70 111 227 375 93 227 262 . 654 107 DAL-SB-138R-0-0050 (C0159466 SpkE, 4ppb Spike) DAL-SB-138R-0-005Q (COI59469 Spk F, 400 ppb Spike) DAL-SB-138R-0-0100 (00159470SpkQ,4 ppb Spike) DAL-SB-138R-0-0100 (COI59470Spk H,400 ppbSpike) 4 400 4 400 8.08 8.08 3.00 3.00 10.3 56 23.2 470 115 23.2 8.80 145 14.0 305 76 14.0 20.5 182 314 73 182 23.9 . 187 331 79 187 211 398 64 195 . 518 83 DAL-SB-136R-DB-0100 (COI59471 Spk1,4 ppb Spike) DAL-SB-138R-DB-0100 (C0159471 SpkJ,400ppb Spike) 4 400 2.27 2.27 7.13 122 NR 273 68 NR NR NR 174 NR NR 174 398 . 506 83 DF05-SS-SS03-0-0000 (COI60871 Spk6,4 ppb Spike) DF06-SS-SS03-0-0000 (COI60871 Spk H,400ppb Spike) 4 400 NR NR NR NR 32.4 NR NR 32.4 31.4 . NR 361 82 NR NR NR NR NR ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NR Not Reported due to Quality Control failures. See Table V for PFOS re-extract data. NO * Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). Note: Since this summary table shows rounded results, recovery values mayvary slightly from the values in the raw data. Exygen Research Page 39 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Description C4 Sulfonate PFB8 Wet Weight C6 Sulfonate PFHS WetWeight CS Sulfonate PFQ8 Wot W ig h t Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (no/nlw etw t. (np/fl) wet wt. (%) (no/Q) wet wt. fno/g) wet wt. (%) (ng/p) wet wt. (nata) wet wt. (%) DF05-SS-SS03-0-0003 (C01MI72 Spk 1,4ppb Spike) DF05-SS-SSO3-0-0003 (C01S0t72 Spk J. 40Qppb Spike) 4 400 17.2 17.2 12.2 72.8 551 133 72.8 35.5 386 78 NR NR NR NR NR NR DF05-SS-SSO4-0-0000 (C01S0I73Spk K, 4 ppb Spike) DF05-SS-SS04-0-0000 (C0160S73SpkL, 400ppb Spike) 4 400 0.298 0.298 3.72 86 ND 412 103 ND 3.13 78 21.6 339 85 21.6 21.9 362 85 DF05-SS-SS04-0-0003 (00160674SpkC,4 ppb Spike) DF05-SS-SS04-0-0003 (00160674Spk 0,400 ppb Spike) 4 400 ND ND 3.46 87 ND 386 97 ND 3.85 96 24.8 383 96 24.8 33.0 406 95 DF05-SS-SS05-0-0000 (00160678Spk E, 4ppb Spike) DF05-SS-SS05-0-0000 (C0160S75Spk F, 400ppb Spike) 4 400 ND ND 3.20 80 ND 359 90 ND 3.75 94 33.0 369 92 33.0 43.3 390 89 DF05-SS-SSO5-0-0003 (C0160676Spk G. 4ppb Spike) DF05-SS-SS05-0-0003 (C0160676Spk H, 400ppb Spike) 4 400 ND ND 3.64 91 ND 343 86 ND 3.39 85 52.3 358 90 52.3 58.2 418 91 DF05-SS-SS05-DB-0003 (C0160877Spk1,4 ppb Spike) DF05-SS-SS05-DB-0003 (C0160677SpkJ. 400ppb Spike) 4 400 ND ND 3.20 80 ND 392 98 ND 3.77 94 63.1 354 89 63.1 49.9 * 416 89 DF09-SS-0006-0-0000 (00160676Spk K.4 ppb Spike) DF09-SS-0006-0-0000 (C0160678Spk L.400 ppbSpike) 4 400 0.286 0.286 3.92 91 0.615 427 107 0.615 3.92 83 330 82 183 183 146 * 485 76 DFOO-SS-0006-0-0003 (00160670SpkC,4 ppb Spike) DF09-SS-0006-0-0003 (00160679SpkD,400ppb Spike) 4 400 0.282 0.282 5.54 131 0.584 506 126 0.584 4.24 91 358 89 180 180 202 618 85 DF09-SS-0006-0-0010 (C0160660SpkE, 4ppb Spike) DF09-SS-0006-0-0010 (C0160660Spk F, 400ppbSpike) 4 400 ND ND 6.89 142 0.266 3.72 86 170 472 118 0.266 353 88 170 182 528 90 DF09-SS-0007-0-0000 (00160661 SpkG,4 ppb Spike) DF09-SS-0007-0-0000 (00160661 SpkH,400 ppb Spike) 4 400 1.38 1.38 8.27 122 13.4 529 132 13.4 11.4 * 355 85 NR NR NR NR NR NR ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NR = Not Reported due to Quality Control failures, See Table V for PFOS re-extract data. ND * Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ngfg (wet weight). Note: Since tele summary table shows rounded results, recovery values mayvary slightly from the values in the raw data. Exygen Research Page 40 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study N o.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Descriotion Amount Spiked C43u!fonats PFBS WatWeight C6 Sulfonate PFHS WetWeight C Sultanati PFOS WotWeight Amt Found Amount Amt Found Amount Amt Found Amount in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (nota) wet wt. (%> fna/Q) wet wt. (na/a)wetwt. <%) (ng/g) wet wt. (nfl/H) wet wt. (%) DF09-SS-0007-0-0003 (C0160M2Spk1,4 ppb Spike) DFOO-SS-0007-0-0003 (C0160M2 SpkJ, 400ppb Spila) 4 400 0.593 0.593 5.21 115 NR 507 127 NR NR NR 1130 1010 NR NR 1130 1380 63 DF09-SS-0007-0-0010 (COI60993SpkK, 4 ppb Spike) DFO0-SS-OOO7-O-OO1O (C0160SS3Spk L,400 ppbSpike) 4 400 ND ND 4.27 107 1.06 502 126 1.06 4.14 77 338 346 86 338 322 647 77 DF09-SS-0007-DB-0010 (COI90994SpkC,4 ppbSpike) DF09-SS-0007-DB-0010 (COI60984Spk0,400 ppb Spike) 4 400 0.222 0.222 4.91 117 1.08 477 119 1.08 4.66 90 260 380 95 260 273 625 91 DF09-SS-0008-0-0000 (C0100088 Spk e, 4ppb Spike) DF09-SS-0008-0-0000 (C0160SS6Spk F,400 ppbSpila) 4 400 0.676 0.676 5.51 121 NR 478 119 NR NR NR NR NR NR NR NR NR NR NR DF09-SS-0008-0-0003 (COI600MSpk 0 ,4ppb Spila) DF09-SS-0008-0-0003 (COI00000SpkH. 400ppb Spike) 4 400 0.832 0.832 5.38 114 7.38 510 127 7.38 9.76 60 NR 314 77 NR NR NR NR NR DF09-SS-0008-0-0010 (COI60007Spk 1,4ppb Spike) DF09-SS-0008-0-0010 (C0100007SpkJ, 400ppb Spike) 4 400 0.402 0.402 5.32 123 3.54 537 134 3.54 6.10 64 362 90 NR NR NR NR NR NR DF09-SS-0009-0-0000 (COI60000Spk K, 4 ppb Spila) DF09-SS-0009-0-0000 (C0100000Spk L,400 ppbSpike) 4 400 0.331 0.331 4.78 111 0.416 455 114 0.416 3.49 77 322 80 177 177 192 * 507 83 DF09-SS-0009-0-0003 (COI00009SpkC.4 ppbSpike) DF09-SS-0009-0-0003 (COI00009Spk 0,400 ppb Spike) 4 400 ND ND 4.14 104 0.354 614 154 0.354 3.78 86 224 411 103 224 261 714 123 DF09-SS-0009-DB-0003 (C0100000Spk E,4 ppb Spito) DF09-SS-0009-DB-0003 (C0100090Spk F,400ppb Spike) 4 400 ND ND 4.65 116 0.370 438 110 0.370 4.05 92 369 92 226 226 247 595 92 DF09-SS-0009-0-0010 (COI00091 Spk6,4 ppb Spila) DF09-SS-0009-0-0010 (COI00091 SpkH,400ppb Spila) 4 400 ND ND 4.21 105 0.511 554 139 0.511 3.78 82 458 367 92 458 420 1050 . 148 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NR = Not Reported due to Quality Control failures, See Table V for PFOS re-extract data. NO * Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). Note: Since this summary table shows rounded results, recoveryvalues mayvary slightly from the values In the raw (M s. Exygen Research Page 41 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Descriotion C4 Sulfonate PFBS W tt W ig h t C6 Sulfonate PFH8 Wot 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 (no/a) (na/a) wet wt. (na/a) wet wt. (% ) (no/a) wet wt. (na/a) wet wt. (%> (na/a) wet wt. (ntfa)w etw t. <%> DF09-SS-0010-0-0000 (C01MM2 Spk 1,4 pptoSpike) DF09-SS-0010-0-0000 (C0160S92SpkJ, 400ppb Spite) 4 400 NR NR NR NR 44.6 NR NR 44.6 56.4 442 09 NR NR NR NR NR NR DFOO-SS-O010-0-0003 (C01S0M3Spk K.4 ppb Spite) DF09-SS-0010-0-0003 (C0160683Spk U 400 ppb Spite) 4 400 5.37 5.37 8.72 84 27.3 434 107 27.3 29.6 . 1100 1280 374 87 1100 1710 130 DF09-SS-0010-0-0010 (C01SOSS4SpkC,4 ppb Spite) DF09-SS-0010-0-0010 (CMMOM Spk D,400 ppb Spike) 4 400 NR NR NR NR 23.6 NR NR 23.6 30.2 904 397 93 004 1140 1380 . 119 DF09-SS-0011-0-0000 (C01B0896Spk E.4 ppb Spite) DF09-SS-0011-0-0000 (C0160M6SpkF, 400ppb Spite) 4 400 3.41 3.41 7.20 05 16.7 430 100 16.7 20.6 . 402 06 NR NR NR NR NR NR DF09-SS-0011-0-0003 (CM60006Spk6.4 ppb Spike) DF09-SS-0011-0-0003 (CM60666Spk H,400 ppb Spite) 4 400 2.00 2.00 6.22 83 16.4 302 97 16.4 21.6 . 370 88 NR NR NR NR NR NR DF09-SS-0011-0-0010 (C0160M7Spk1,4 ppb Spite) DF09-SS-0011-0-0010 (C0160M7 SpkJ, 400ppb Spite) 4 400 NR NR NR NR 22.7 NR NR 22.7 33.3 * 410 00 NR NR NR NR NR NR DFOO-SS-0012-0-0000 (CM60666Spk K.4 ppb Spite) DF09-SS-0012-0-0000 (CM60666Spk L, 400ppb Spike) 4 400 4.73 4.73 6.84 53 24.9 354 87 24.0 30.1 * NR 374 87 NR NR NR NR NR DFOQ-SS-0012-DB-0000 (CM60666Spk C,4 ppb Spite) DF09-SS-0012-DB-0000 (CM60668SpkD,400 ppb Spite) 4 400 NR NR NR NR 23.3 NR NR 23.3 26.0 1180 1020 . 406 06 1180 1460 70 DF09-SS-0012-0-0003 (C0160600SpkE,4 ppb Spite) DF09-SS-0012-0-0003 (C0160600Spk F,400 ppbSpite) 4 400 NR NR NR NR 34.3 NR NR 34.3 23 0 NR 402 92 NR NR NR NR NR DF09-SS-0012-0-0010 (CM60601 Spk Q,4 ppb Spite) DF09-SS-0012-0-0010 (CM606M Spk H,400 ppb Spike) 4 400 3.99 3.99 10.0 150 20.2 550 137 20.2 23.2 . NR 423 101 NR NR NR NR NR 'Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NR * Not Reported due to Quality Control failures, See Table V for PFOS re-extract data. Note: Since this summary table shows rounded results, recoveryvalues mayvary slightly from the values in the raw data. Exygen Research Page 42 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study N o.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Descriotion Amount Spiked C4&ulfonat0PFBSW rtW eigh^_^aJ!M f6nateP FH 8|M ^ Amt Found Amount Amt Found Amount in Sample Recovered Recovery in Sample Recovered fna/a) wet wt. (%> irra/a) wet wt. Amt Found Amount Recovery in Sample Recovered Recovery f%) (ng/g) wet wt. (ng/g) wet wt. (%> DF09-SS-0013-0-0000 (C0160S02Spk 1,4ppb Spike) DFOO-SS-0013-0-0000 (C01S0902SpkJ, 400ppb Spike) 4 400 0.230 0.230 3.82 90 0.605 407 102 0.605 4.03 86 219 371 93 219 230 553 84 DF0S-SS-O013-0-0003 (00180003Spk K,4 ppb Spike) DF09-SS-0013-0-0003 <00180903SpkL. 400 ppb Spike) 4 400 ND ND 3.60 90 0.405 416 105 0.495 3.88 65 223 414 103 223 210 626 101 DF09-SS-0013-0-0010 (00160004SpkC,4 ppbSpike) DF09-SS-0013-0*0010 (CQ160904Spk D,400 ppb Spike) 4 400 ND ND 3.02 96 0.880 4.14 82 345 356 89 0.880 358 89 345 354 701 86 DAL-SB-602R-0-0000 (00164247SpkK.4 ppbSpike) DAL-SB-602R-0-0000 (C0184247Spk u 400ppb Spike) 4 400 0.462 0.462 5.03 114 ND 452 113 ND 3.10 78 24.6 329 82 24.6 29.4 341 79 DAL-SB-602R-0-0005 (C0164248Spk C,4 ppb Spike) DAL-S8-602R-0-0005 (00184248 SpkD, 400ppb Spike) 4 400 0.440 0.440 4.14 93 ND 465 116 ND 3.50 88 33.1 347 87 33.1 36.2 . 366 83 DAL-SB-802R-0-0010 (C0184249Spk E.4 ppb Spike) DAL-SB-602R-0-0010 (C0184248SpkF, 400ppb Spike) 4 400 ND ND 5.26 132 ND 543 136 ND 3.52 88 37.9 386 97 37.9 27.0 438 100 DAL-SB-602R-0-0050 (C01842S0Spk G,4 ppb Spike) DAL-S6-602R-0-0050 (00184280 SpkH,400 ppb Spike) 4 400 ND ND 3.55 69 ND 357 89 ND 3.14 79 376 94 ND ND 3.45 86 386 97 DAL-SB-602R-0-0100 (C0184281 Spk1,4 ppb Spike) DAL-SB-802R-0-0100 (C0184281 SpkJ, 400ppb Spike) 4 400 NR NR NR NR ND NR NR ND 3.11 78 0.414 3.34 73 353 88 0.414 308 77 DAL-S8-601R-0-0000 (C0184252SpkK. 4 ppb Spike) DAL-SB-601R-0-0000 (00184282Spk L,400 ppbSpike) 4 400 ND ND 5.04 126 ND 614 154 ND 3.26 82 4.50 366 92 4.50 8.96 112 360 89 DAL-SB-601R-DB-0000 (00184283SpkC,4 ppb Spike) DAL-SB-601 R-DB-0000 (C01642S3SpkD.400 ppb Spike) 4 400 NR NR NR NR ND NR NR ND 3.64 91 4.94 383 96 4.94 8.40 87 396 98 ` Sample residue exceeds the spiking level significantly (3x spiking level): therefore, an accurate recovery value cannot be calculated NR * Not Reported due to Quality Control failures, See Table V for PFOS re-extract data. ND = Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). Note: Since this summarytable shows rounded results, recovery values mayvary slightly from tha values In the raw data. Exygen Research Page 43 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil Samples Continued Sample Description C4 Sulfonate PFB8 Wet W eight C6 Sulfonate PFHS W ot W eight C8 Sulfonate PFQ3 Wet W eight Amount Amt Found Spiked in Sample (na/a) wet wt. Amount Recovered Amt Found Amount Amt Found Amount Recovery in Sample Recovered Recovery in Sample Recovered Recovery <%) (ng/g) wet wt. (na/a) wet wt. <%> (na/a) wet wt. (na/a) wet wt. (%) DAL-SB-601R-0-0005 (C01S42S4Spk E, 4 ppb Spite) DAL-SB-601 R-0-0005 (C0164284SpkF,400ppb Spite) 4 400 NR NR NR NR ND NR NR ND 3.23 81 4.68 354 89 4.68 8.64 99 374 92 DAL-SB-601 R-0-0010 (CM642S5Spk 6,4 ppb Spite) DAL-SB-601 R-0-0010 (CM042S5 Spk H,400ppb Spike) 4 400 NR NR NR NR ND NR NR ND 3.86 97 NR 367 92 NR NR NR NR NR DAL-SB-601R-DB-0010 (C0104256Spk 1,4 ppb Spite) DAL-SB-601 R-DB-0010 (C01042S6SpkJ, 400ppb Spite) 4 400 0.306 0.308 6.32 150 ND 714 178 ND 3.86 92 4.47 379 95 4.47 6.65 55 368 91 DAL-SB-601 R-0-0050 (00104257Spk K, 4 ppb Spite) DAL-SB-601R-0-0050 (00164257Spk L.400 ppbSpite) 4 400 ND ND 3.17 79 ND 430 108 ND 3.14 79 NO 341 85 ND 3.48 87 344 86 DAL-SB-601 R-0-0100 (CM04250Spk0,4 ppb Spite) DAL-SB-601 R-0-0100 (CM04250Spk0 ,400ppb Spike) 4 400 ND ND 3.04 76 ND 298 75 ND 3.62 91 385 96 ND ND 3.47 87 390 100 DAL-SB-601 R-DB-0100 (CM04200Spk E,4 ppb Spite) DAL-SB-601 R-DB-0100 (C0164260Spk F,400 ppbSpike) 4 400 ND ND 2.78 70 ND 261 65 ND 3.44 86 378 95 ND ND 3.53 88 378 95 Average; 111 Standard Deviation: 25 Average: Standard Deviation: 86 8 NR 1 Not Reported due to Quality Control failures. See Table V for PFOS re-extractdata. ND * Not detected at or above the LimitofQuantitation (LOQ)of 0.2 ng/g (wetweight). Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Average: 68 Standard Deviation: 16 Exygen Research Page 44 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table V. Matrix Spike Recovery of PFOS in Re-extracted Soil Samples Sample Description D F 1 3-SS-SS03-0-0000 (C0159344 Spk F, 80,000 ppb Splka) D F 13-SS-SS03-0-0003 (C0158345 Spk H, 80,000 ppb Splka) DF13-SS-SS05-0-0000 (C0158351 Spk N, 80,000 ppb Splka) DF13-SS-SS05-0-0003 (C0158352 Spk F, 80,000 ppb Splka) DF13-SS-SS05-DB-0003 (C0158353 Spk H, 200,000 ppb Splka) D F 10-SS-SS01 -0-0000 (C0158354 Spk J, 2000 ppb Splka) DF 10-SS-SS01-0-0003 (C0158355 Spk K, 4000 ppb Splka) DF10-SS-SS02-0-0003 (C0156358 Spk G, 400 ppb Spike) DF 10-SS-SS04-0-0000 (C0158362 Spk M, 4000 ppb Splka) D F 10-SS-SS04-0-0003 (CC0158383 Spk E, 20,000 ppb Splka) D F 10-SS-SS05-0-0000 (C0158384 Spk G, 4000 ppb Splka) DF 10-SS-SS05-0-0003 (C0156305 Spk 1,20,000 ppb Spike) DF 11-SS-SS01 -0-0000 (C0158386 Spk K, 20,000 ppb Spike) D F 1 1-SS-SS01 -0-0003 (C0158387 Spk 0 , 20,000 ppb Splka) DF11-SS-SS02-0-0000 (C0158388 Spk F, 80,000 ppb Splka) DF 11-SS-SS02-0-0003 (C0158370 Spk H, 80,000 ppb Splka) DF11-SS-SS03-0-0000 (C0158371 Spk J, 4000 ppb Splka) DF11-SS-SS03-DB-0000 (C0158372 Spk K, 4000 ppb Splka) D F 1 1-SS-S S0 3-0 -000 3 (C0158373 Spk N, 40,000 ppb Splka) Amount Spiked (ng/g) 80000 80000 80000 80000 200000 2000 4000 400 4000 20000 4000 20000 20000 20000 80000 80000 4000 4000 40000 111 1C8 Sulfonate PFOS Wet W eight -- ... " -- i Amt Found Amount in Sample Recovered (ng/g) wet wt. (ng/g) w et wt. Recovery (%) 60200 111000 64 52000 130000 96 76600 145000 86 36600 134000 122 65700 223000 79 1090 2740 83 2080 5160 77 511 952 110 3380 5620 56 7410 25000 88 3270 7020 94 8240 NR 26800 NR 93 NR 5220 23000 89 40500 107000 83 34100 76400 53 3580 7260 92 4080 9040 124 31200 75300 110 NR = Not Reported due to Quality Control failures. Note: Since this summarytable shows rounded results, recovery values may vary slightly from the values In the raw data. Exygen Research Page 45 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table V. Matrix Spike Recovery of PFOS in Re-extracted Soil Samples Continued Sample Description DF05-SS-SS01-0-0000 (COI56378 Spk M, 2000 ppb Spike) DF05-SS-SS01 -0-0003 (C015637 Spk E, 400 ppb Spike) DF05-SS-SS02-0-0000 (C0156380 Spk G. 20,000 ppb Spike) DF05-SS-SS02-0-0003 (C0156361 Spk J, 40,000 ppb Spike) DF12-SS-SS01-0-0000 (C0156382 Spk K, 4000 ppb Spike) D F 1 2 -S S -S S 0 1-0 -000 3 (C0156363 Spk N, 80,000 ppb Spike) DF12-SS-SS02-0-0000 (C0156384 Spk F, (0,000 ppb Spike) DF12-SS-SS02-0-0003 (C0156385 Spk H, 200,000 ppb Spike) DF12-SS-SS03-0-0000 (C0156366 Spk J, 400 ppb Spike) DF12-SS-SS04-0-0000 (C0156368 Spk N, 200,000 ppb Spike) DF12-SS-SS04-0-0003 (C0156380 Spk F, 800,000 ppb Spike) DF12-SS-SS05-0-0000 (C0156390 Spk G, 20,000 ppb Spike) DF12-SS-SS05-0-0003 (C0156391 Spk I, 8000 ppb Spike) DF13-SS-SS01-0-0000 (COI56392 Spk K, 80,000 ppb Spike) DF13-SS-SS01-0-0003 (C0158393 Spk N, 40,000 ppb Spike) DF13-SS-SS02-0-0000 (C0156394 Spk F, 200,000 ppb Spike) DF13-SS-SS02-0-0003 (C0156395 Spk H, 200,000 ppb Spike) DAL-SB-604R-0-0010 (C0159455 Spk M, 8 ppb Spike) DAL-S B-605R-0-0010 (C0150461 Spk E, 4 ppb Spike) Amount Spiked (ng/g) 2000 400 20000 40000 4000 80000 80000 200000 400 200000 800,000 20000 8000 80000 40000 200000 200000 8 4 C8 Sulfonate PFOS Wet Weight Amt Found Amount in Sample (ngla ) wet wt. Recovered (ng/g) wet wt. Recovery (%) 1760 3320 78 481 832 88 11900 36700 124 17100 51000 85 2560 8160 140 55700 176000 150 NR NR NR 233000 432000 100 216 575 90 NR NR NR 286000 952000 83 6330 25400 95 NR 49700 NR 136000 NR 108 39700 97600 145 NR NR NR 170000 449000 140 12.6 17.6 63 NR NR NR NR = Not Reported due to Quality Control failures. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. Exygen Research Page 46 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table V. Matrix Spike Recovery of PFOS in Re-extracted Soil Samples Continued Sample Description DF05-SS-SS03-0-0000 (C0160S71 Spk 1,8000 ppb Spike) DF05-SS-SS03-0-0003 (C01S0872 Spk L, 20,000 ppb Spike) DF09-SS-0007-0-0000 (C0160881 Spk 1,2000 ppb Spike) DF09-SS-0008-0-0000 (C0160885 spk G, 2000 ppb Spike) DF09-SS-0008-0-0003 (00160886 Spk 1,4000 ppb Spike) DF09-SS-0008-0-0010 (C0160887 Spk K, 2000 ppb Spike) DF09-SS-0010-0-0000 (C0160892 Spk K, 4000 ppb Spike) DF09-SS-0011-0-0000 (C016080S Spk G, 2000 ppb Spike) DF09-SS-0011-0-0003 (C0160896 Spk 1,2000 ppb Spike) DF09-SS-0011-0-0010 (C01608S7 Spk L, 2000 ppb Spike) DF09-SS-0012-0-0000 (C0160898 Spk N, 2000 ppb Spike) DF09-SS-0012-0-0003 (C0160900 Spk H, 4000 ppb Spike) DF09-SS-0012-0-0010 (C0160901 Spk l. 2000 ppb Spike) DAL-SB-601R-0-0010 (C0164255 Spk 1,4 ppb Spike) Amount Spiked (ng/g) 8000 20000 2000 2000 4000 2000 4000 2000 2000 2000 2000 4000 2000 4 C8 Sulfonate PFOS Wot Weight Amt Found Amount in Sample (ng/g) wet wt. Recovered (ng/g) w et wt. Recovery (%) 6830 13500 83 11000 29000 90 NR NR NR 2340 4100 88 3860 9360 138 1870 3860 100 2820 6170 84 1500 2990 75 1760 3330 79 1460 3590 107 1010 2620 81 2300 5930 91 1340 6.53 2930 9.12 80 65 Average: Standard Deviation: NR - Not Reported due to Quality Control failures. Note: Since this summary table shows rounded results, recoveryvalues may vary slightly from the values In the raw data. 94 23 Exygen Research Page 47 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table VI. Matrix Spike Recovery of PFBS, PFHS and PFOS in Associated Trip Blanks Sample Description Amount Spiked (nan.) C4 Sulfonate PFBS________________ C$ Sulfonate PFHS__________________ CS Sulfonate PFOS Amt Found in Sampls (no/L> Amount Amt Found Recovered Recovery in Sample (ng/L) (%> (ng/L) Amount Recovered (ng/L) Recovery (S ) Amt Found in Sample (ng/L) Amount Recovered (ng/L) Recovery (%) DF13-SS-TRIP1-LS-060221 (C015S34S,1.0ppb FieldSpike) 1000 ND 1010 101 ND 892 89 DF13-SS-TR1P1-HS-060221 (C015S350,100ppb Field Spike) 100000 ND 99200 99 ND 91200 91 ND 851 65 ND 87900 88 DAL-SB-TRIP1-LS-060302 (COI90444,1.0ppbField Spike) 1000 ND 978 96 ND 970 97 ND 877 68 DAL-SB-TRIP1-HS-060302 (C019S445,100ppbField Spike) 100000 ND 91300 91 ND 99800 100 ND 91500 92 DF05-SS-TRIP2-LS-060308 {COIS08SS,1.0ppbField Spike) DF05-SS-TRIP2-HS-060308 (C01S00S9,100ppbField Spike) 1000 . 100000 ND ND 1030 97700 103 98 ND ND 928 69300 93 89 ND 660 86 ND 85300 85 DAL-SB-TRIP-LS-060316 (C0104223,1.0ppb Field Spike) 1000 ND 862 86 ND 991 99 ND 875 88 DAL-SB-TRIP-HS-060316 (COI#4224,10 ppb Field Spike) 10000 ND 9930 99 ND 9250 93 ND 7710 77 Average: Standard Deviation: 97 6 Average: Standard Deviation: 94 4 NO * Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. Note: Since this summary table shows rounded results, recoveryvalues mayvary slightly from the values In the raw data. Average: Standard Deviation: 89 4 Exygen Research Page 48 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table VII. Total Percent Solids in Soil Samples Exygen IO C0156344 C0156345 CO156346 C0156347 C0156351 C0156352 CO156353 C0156354 CO156355 C0156356 CO156357 C0156358 CO156360 C 0 1 56361 CO156362 C0156363 CO156364 C0156365 CO156366 C0156367 C0156368 C0156370 C0156371 C0156372 C0156373 CO156374 C0156375 C0156376 C 0156377 S am ple D escription D F 1 3-S S -S S 0 3-0-000 0 DF13-SS-SS03-0-0003 D F 1 3-S S -S S 0 4-0-000 0 DF13-SS-SS04-0-0003 DF 13-SS-SS05-0-0000 DF13-SS-SS05-0-0003 DF13-SS-SS05-DB-0003 D F 10-S S -S S 01-0-0000 DF 10 -S S -S S 0 1-0-0003 DF10-SS-SS02-0-0000 DF10-SS-SS02-DB-0000 DF10-SS-SS02-0-0003 D F 1 0-S S -S S 0 3-0-000 0 DF10-SS-SS03-0-0003 DF10-SS-SS04-0-0000 DF 10-S S -S S 04-0-0003 DF 10-SS-SS05-0-0000 D F 10-S S -S S 05-0-0003 D F 1 1-SS-SS01 -0-0000 D F 1 1-S S -S S 0 1-0-0003 D F 1 1-S S -S S 02-0-0000 D F 1 1-S S -S S 0 2-0-000 3 DF11-SS-SS03-0-0000 D F 1 1-S S -S S 03-D B -0000 D F 1 1-S S -S S 03-0-0003 DF 11-SS-SS04-0-0000 DF11-SS-SS04-0-0003 D F 1 1-S S -S S 05-0-0000 DF11-SS-SS05-0-0003 T o tal S o lid s (%) 65.54 76.73 83.41 87.27 78.17 84.70 83.33 84.27 86.73 86.29 86.48 86.56 81.74 84.16 71.57 75.62 82.18 82.54 81.11 83.77 67.83 70.80 79.72 79.82 85.23 86.67 89.38 83.65 85.17 Exygen Research Page 49 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table VII. Total Percent Solids in Soil Samples Continued Exygen ID C0156378 C0156379 CO156380 C 0 1 56381 CO156382 C0156383 C0156384 C0156385 C0156386 C0156387 C0156388 C0156389 CO156390 C 0 1 56391 C0156392 CO156393 C0156394 C0156395 C0159446 C0159447 C0159448 C0159449 C0159450 C0159451 C0159453 C0159454 C0159455 C0159456 S am ple D escription DF05-SS-SS01-0-0000 DF05-SS-SS01-0-0003 DF05-SS-SS02-0-0000 DF05-SS-SS02-0-0003 D F 12-S S -S S 01-0-0000 D F 12-SS -S S 01-0-0003 DF12-SS-SS02-0-0000 DF 12-SS -S S 02-0-0003 D F 12-SS -S S 03-0-0000 DF 12-SS -S S 03-0-0003 DF 12-SS -S S 04-0-0000 DF 12-SS -S S 04-0-0003 DF 12-SS -S S 05-0-0000 DF 12-SS -S S 05-0-0003 DF 13-SS-SS01-0-0000 D F 13-SS -S S 01-0-0003 DF13-SS-SS02-0-0000 DF 13-SS-SS02-0-0003 DAL-SB-603R-0-0000 DAL-SB-603R-0-0005 D A L -S B -6 0 3 R -0 -0 0 10 DAL-SB-603R-0-0050 D A L -S B -6 0 3 R -0 -0 100 D A L -S B -6 0 3 R -D B -0 1 00 DAL-SB-604R-0-0000 DAL-SB-604R-0-0005 DAL-SB-604R-0-0010 DAL-SB-604R-0-0050 T o ta l S o lid s (%) 81.57 84.09 82.35 84.38 82.65 81.26 68.74 73.68 82.19 87.57 74.29 78.13 78.20 81.22 77.44 80.09 75.65 80.31 90.63 85.84 81.62 82.85 83.40 78.33 89.54 82.13 81.93 88.21 Exygen Research Page 50 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table VII. Total Percent Solids in Soil Samples Continued Exygn ID C0159457 C0159458 C0159459 C0159460 C 0 1 59461 C0159462 C0159463 C0159464 CO159466 C0159467 C0159468 C0159470 C 0 1 59471 C0160871 C0160872 CO160873 C0160874 C0160875 C0160876 CO160877 C0160878 C0160879 C 0160880 C0160881 C 0160882 CO160883 C0160884 C0160885 S am ple D escription DAL-SB-604R-0-0100 DAL-SB-605R-0-0000 D A L-S B -605 R -D B -000 0 DAL-SB-605R-0-0005 D A L-S B -605 R -0-0010 DAL-SB-605R-0-0050 D A L -S B -6 0 5 R -0 -0 100 DAL-SB-138R-0-0000 DAL-SB-138R-0-0005 DAL-SB-138R-0-0010 D A L-S B -138 R -0-0050 D A L-S B -138 R -0-0100 DAL-SB-138R-DB-0100 DF05-SS-SS03-0-0000 DF05-SS-SS03-0-0003 DF05-SS-SS04-0-0000 D F 0 5-S S -S S 0 4-0-000 3 DF05-SS-SS05-0-0000 DF05-SS-SS05-0-0003 D F 0 5-S S -S S 0 5-D B -00 03 DF09-SS-0006-0-0000 DF09-SS-0006-0-0003 D F09-S S -0006-0-0010 DF09-SS-0007-0-0000 DF09-SS-0007-0-0003 DF09-SS-0007-0-0010 DF09-SS-0007-DB-0010 DF09-SS-0008-0-0000 T o ta l S o lid s (%) 77.17 81.42 81.76 84.78 82.01 79.83 73.86 86.23 87.94 86.15 81.93 78.61 80.36 80.74 83.20 84.22 87.24 86.54 86.68 86.89 89.09 87.28 86.20 86.39 89.57 89.36 89.39 86.47 Exygen Research Page 51 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Table VII. Total Percent Solids in Soil Samples Continued Exygen ID C0160886 C 0160887 CO160888 C0160889 CO160890 C0160891 CO160892 C0160893 CO160894 C0160895 CO160896 C0160897 CO160898 C0160899 C0160900 C0160901 C0160902 C0160903 C0160904 C0164247 C0164248 C0164249 CO164250 C0164251 C0164252 CO164253 C0164254 C0164255 S am ple D escription DF09-SS-0008-0-0003 D F 0 9-S S -008 -0-0010 DF09-SS-0009-0-0000 DF09-SS-0009-0-0003 D F 0 9 -S S -0 0 0 9 -D B -0 0 0 3 D F 0 9-S S -000 9-0 -00 10 D F 0 9-S S -0010-0-00 00 D F 0 9 -S S -0 0 10-0-0003 D F 0 9-S S -0010-0-00 10 D F 0 9 -S S -0 0 1 1-0-00 00 D F 0 9 -S S -0 0 1 1-0-0003 D F 0 9 -S S -0 0 1 1 -0-00 10 DF09-SS-0012-0-0000 D F 0 9 -S S -0 0 1 2 -D B -0 0 0 0 D F 0 9-S S -0012-0-00 03 DF09-SS-0012-0-0010 DF09-SS-0013-0-0000 D F 0 9-S S -0013-0-0003 D F 0 9-S S -0013-0-00 10 DAL-SB-602R-0-0000 DAL-SB-602R-0-0005 DAL-SB-602R-0-0010 DAL-SB-602R-0-0050 DAL-SB-602R-0-0100 D A L -S B -6 0 1R -0-000 0 DAL-SB-601R-DB-0000 DAL-SB-601R-0-0005 DAL-SB-601 R-0-0010 Total S olids 88.27 88.54 87.96 88.35 88.75 87.79 72.09 75.60 76.57 87.53 86.92 86.19 90.45 90.43 88.07 87.02 87.52 87.52 85.76 77.82 82.30 84.24 82.18 83.52 89.43 87.98 88.75 88.87 Exygen Research Page 52 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Table VII. Total Percent Solids in Soil Samples Continued Exygen ID C0164256 C0164257 C0164259 C0164260 S am ple D escription D A L -S B -6 0 1 R -D B -0 0 1 0 DAL-SB-601R-0-0050 DAL-SB-601 R-0-0100 DAL-SB-601 R-DB-0100 T o ta l S olids 87.99 81.36 86.85 83.56 Exygen Research Page 53 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 54 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Figure 1. Typical Calibration Curve for PFBS in Methanol 041300B P760-1131 Surfjo Soil.rdb (PFBS): "Linear" Regression C'1 / * * weighting): y * 00.8 x + 0.00103 (r= 0.0071) Area, counts Exygen Research Page 55 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 2. Extracted Standards of PFBS in Methanol, 25 ng/L and 50 ng/L, Respectively XC941396-1 - PFBS (Standard) 299.W 99.9ama-ta m p lt 2 o f 3 from 941396B.wiff Araa: 3442coaatt H tig kt: 1.99t+002 cp KT: 0.S92 mia 0.90 3.34,3.92 eoe 9.38 7.23 ,7.75 8.15 11J4 ^12.77^13.00 11.32^ ft . s k ,,14.80 10.00 10.80 8 0 10 11 12 13 14 10 10 17 Tim, min XC041308-2 - PFBS (Standard) 209.0A9.0 amu sampl 3 of 38 from 041300B.wiff Ara:5222 counts Haight: 1.55+002 ops RT: 1.70 min 1.70 Exygen Research Page 56 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 3. PFBS in Methanol Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively R ta g a at C o n tro l-P F B S (U ataow a) 299.a w 0 a -sam pI t 9 o f 3 from 0413@iB.win 6> ta* u o t fo u nd ) Intensity, cps Intensity, cps Area: 480 1 oounts Haight: 1 .5 5 *+ 0 0 2 cps RT: 1.7 4 min 190 0.60 100- 8 0 10 T im a, min I R eagent Spk B PFBS (QC ) 2 0 0 .0 /0 0 .0 amu sample 11 of 3 8 from 041306B .w iff Area: 5 0 7 6 7 oounts Haight: 1 .53e+ 003 cps RT: 0 .5 0 6 min 0.60 1.74 3.36 10.8 15.16 8 0 10 11 12 13 1 4 15 16 17 T im a, min Intensif, cps Exygen Research Page 57 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Soil Sample Analyzed for PFBS (Exygen ID: C0156370, Data Set: 041306B) I C$1SS3 T t P F B S ffM Im ow ii) 29M / M l f w -ta m p lt 29 o f 39 from 04130SB.w fff A m : 84735 count* H ight: S.5* + 003c p t KT: 9.540 m in intensity, cps Exygen Research Page 58 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 5. Typical Calibration Curve for PFHS in Methanol 041306B P760-1131 Surface Soil.rdb (PFHS): "Lineai" Regression ("1 weighting): y 405 x + 0.00020 ( r * 0.0004) Area, counts Exygen Research Page 59 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 6. Extracted Standards of PFHS in Methanol, 25 ng/L and 50 ng/L, Respectively I XC041306-1 PFHS (S tandard) 3S 9.trtO .tam u -a a a tp lt 2 o f 38 from 04130B.wiff A raa: 10527 co u n t* H aight: 8.57+002 cps RT: 9.18 m in 0 .1 8 Intensity, cps Tim e, m in XC 041300-2 - PFHS (S tjn d e rd ) 300.0/80.0 jm u - s im p le 3 o f 38 from 041300B .w iff A m : 22172 counts H eight: 1.81e+003 ops R T .0 .1 7 m in 0.17 Intensity, cps Exygen Research Page 60 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 7. PFHS in Methanol Reagent Blank, 0 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively R a a g tat C o n tro l PFHS (Unknown) 399.9/99.0 a aia -ta m p ft 9 o f 39 from 041306B.wiff (peak n o t found) 020 Tim , min R eagent Spk A - PFHS (QC ) 3 0 9 .0 /8 0 .0 j m u - s im p le 10 of 3 8 from 041308B.*iff A im : 2 0 8 0 8 count: Holght: 1.7 4 e + 0 0 3 cp: RT: 0.1S min T im e, min I R e jg e n t S p k B - PFHS (Q C )300.0/80.0 e m u -s im p le 11 of 3 8 fio m 0413088.)! A i m : 2 0 7 0 7 4 counts Height: 1.7S e+ 004 ops RT: 0 .1 5 min Exygen Research Page 61 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Soil Sample Analyzed for PFHS (Exygen ID: C0156368, Data Set: 041306BR) Intensity, cps I C M SC3C* - PFHS (Jakaow a) 3 L M 0 ama aam pla 13 o f 78 from 94130BR.wfff A n a : tfflc o a a t* H afgkt: 5L5*+802cp* RT: 9.12 m ia A5 5 0 0.12 500 450 400 350 300 250 \2 0 0 150 100 50 0 T im e, min Exygen Research Page 62 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 9. Typical Calibration Curve for PFOS in Methanol 0 4 1 2 0 6 AR P760-1131 Surface Soil R E V I.rd b (PFOS): "L in * j i" Regression ? 1 /x " weighting): y 2 7 8 x + 1 .0 3 *+ 0 0 3 (r 0 .0 0 8 6 ) Area, counts Exygen Research Page 63 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 10. Extracted Standards of PFOS in Methanol, 25 ng/L and 50 ng/L, Respectively XO4130C-1 -PFOS (S tandard)499.AWl$ ama -sam pfa 2 o f 49 from Q41206AR.wiff A n a : 7032 counts H aig&t: 4.71*+092 cps RT: 11.5 m in 11.50 intensity, cps Area: 1 4 5 0 4 counts Haight: 0 .3 0 t+ 0 0 2 cps RT: 11.5 min 11.40 Intensity, cps Exygen Research Page 64 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 11. PFOS in Methanol Reagent Blank, 0 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively I ffM fM t C o n tro l PFOS (Unknown) 499.00.9 amu -tam p ! 9 o f 49 from 041206AR.wifT (ponk n o t fo u nd ) 1S.60 I R . i g . n t Spk A - PFOS (Q C ) 4 0 0 .0 /8 0 .0 a m u - u m p l t 10 of 4 0 from 041206A R .w iff A lta : 134 03 count! Height: 8 .3 8 .+ 0 0 2 ops RT: 11.5 min 11.40 * Tim a, min R jg * n t Spk D PFOS (Q 0 ) 4 0 0 .0 /8 0 .0 jm u s im p le 11 of 4 0 from 04120CAR.wiff Area: 1 2 0 3 2 4 oounts Height: 8 .0 0 e+003 ops RT: 11.5 min 11.48 Exygen Research Page 65 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Soil Sample Analyzed for PFOS (Exygen ID: C0156344, Data Set: 041206AR) I CH5S344 - PFOS (Uhkaowa) 419.5/90.0 amu -ta m p !* o f 49 from M12MAR.W M A n a : 212721 coalite H tig A b 1.92e+004cp* RT: 11.5 m in 11.47 intensity, cps Exygen Research Page 66 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods and Protocol Amendments Exygen Research Page 67 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number P0001131 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Sponsor Representative: M ichael A . Santoro Director o f Regulatory Affairs 3M Building 0236-01-B -10 St. Paul, M N 55144 Phone: (651) 733-6374 Page I oj 65 Exygen Research Page 68 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 o f65 Page 69 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PROTOCOL APPROVAL Study Title: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 APPROVALS Jaisimhaufesan, S' Weston Solutions Michael AL.. Sgflitoro, Sponsor Representative 3M Comp:aify (Shaffer, Technij gen Research u .__________ ad, Quality Assurance Unit Date 7 ^ -c c r-tc ^ Date & Page 3 o f 65 Exygen Research Page 70 o f 134 Interim Report #22 - Analysis o f 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 B U S ...................................................................................................... 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............................................................................................... IS Page 4 of 65 Exygen Research Page 71 o f 134 Interim Report #22 - Analysis o f 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 (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum using LC/MS/MS for the 3M Decatur Monitoring Program. The study w ill be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit o f Exygen Research. TEST MATERIALS The test materials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M. PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4F9SOj"K+) Lot Number: 101 Purity: 96.7% Transitions Monitored: 2 9 9 -* 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CtFuSOjIC*) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 - 8 0 Structure: Page 5 o f 65 Exygen Research Page 72 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFnSOj'K*) Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 --> 99 Structure: OBJECTIVE The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions o f the following Exygen analytical methods: V0001780 V0001781 V0001782 V0001783 V0001784 V0001785 V0001786 "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Page 6 o f65 Exygen Research Page 73 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax:(610)701-7401 j .kesari@westonsolutions.com SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, M N 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M. Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 john.flaherty@exygen.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion o f this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and termination dates w ill be included in the final report. Page 7o f 65 Exygen Research Page 74 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 IDENTIFICATION AP JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: Water (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study. The test systems were chosen to access the environmental impact o f PFBS, PFHS and PFOS in the Decatur, Alabama area. ^ SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples w ill be received at Exygen directly from Weston Solutions. The details o f sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types o f samples collected will vary depending availability in the field. The total number o f samples received and analyzed for each matrix will be documented in the final report associated with this study. Water, soil, and sediment samples w ill be used as received without further processing at Exygen. These samples will be stored refrigerated at 2C-8C. Fish, clam, vegetation and small mammal liver samples w ill be processed according to the appropriate analytical method (see Appendix I). These samples will be stored frozen at S -10"C. Small mammal whole blood samples will be centrifuged in the field at the time o f collection and the serum fraction w ill be used for the study. Small mammal serum will be stored frozen at 5 -10C. The receipt and processing o f the samples w ill be documented in the final report and raw data associated with the study. Page 8 o f 65 Exygen Research Page 75 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 SAMPLE IDENTIFICATION Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Sample storage conditions and locations will be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" The above methods use analytical conditions capable o f separating the isomers o f PFBS, PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sample will be used for the preparation o f fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen w ill supply one bottle per sample collected. The bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 9 o f 65 Exygen Research Page 76 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 />--- collection at the testing facility and have been shown to be free o f PFBS, PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike o f each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid ( 13C PFOA). PFOA and l3C PFOA are included in the solutions used to spike the samples. The results for PFOA and l3C PFOA w ill not be reported in this study. Exygen w ill supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containers/bags used for sample collection w ill be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and w ill also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. For small mammal liver, Exygen will supply a 50 mL polypropylene centrifuge tube. For small mammal serum, Exygen w ill supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and w ill also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. Low and high spiking levels for each matrix are defined below: M atrix Low Spiking Level High Spiking Level Water 500 ng/L 5000 ng/L Soil Sediment 4ng/g 4ng/g 40ng/g 40ng/g Fish lOngfg 100ng/g Clams lO n g /g lOOng/g Vegetation lOng/g 100 ng/g Small Mammal Liver 10ng/g 100ng/g Small Mammal Serum lOng/mL 100 ng/mL Page !0 of 65 Exygen Research Page 77 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 /v. Exygen Protocol Number: POOOl 131 Recoveries are anticipated to be between 70% and 130% o f the fortified levels; however, the exact precision and accuracy w ill be determined by the analysis o f the quality control samples described above. A statement o f accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control o f bias will be addressed by taking representative sub-samples from a homogeneous mixture o f each matrix from untreated control samples, and by analyzing at least two levels o f fortifications. STATISTICAL METHODS Statistics w ill be limited to those specified in the subject methods and to the calculation o f average recoveries, as applicable. GLP STATEMENT A ll aspects o f this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and w ill outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative. REPORT A final report w ill be prepared by the principal investigator or their designee at the conclusion o f the study. The report will include, but w ill not be limited to, the following: The name and address o f die Study Director, Sponsor Representative, and o f the testing facility. A statement o f GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). Page II of 65 Exygen Research Page 78 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number P0001131 The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates o f study inspections and dates findings were reported to the Study Director and Management. A description o f the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy o f the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page. Description o f the instrumentation used and operating conditions. A ll results from all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level. Representative chromatograms for each analyte in each matrix, including chromatograms o f a standard and a control sample, and a chromatogram at a fortification level. The location o f the analyte peaks will be clearly identified in all chromatograms. A ll circumstances that may have affected the quality or integrity o f the data w ill be documented in the report. Locations where raw data and the final report are to be archived. Additions or corrections to the final report shall be in the form o f an amendment signed by the Study Director. The amendment shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendment w ill be signed and dated by the Study Director and the Sponsor Representative. A ll applicable requirements for reporting o f study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel w ill practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environment to the test or reference substance(s). Page 12 o f 65 Exygen Research Page 79 o f 134 Interim Report #22 - Analysis o f 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 Director and Sponsor Representative. Amendments usually will be issued prior to initiation o f study plan change. However, when a change is required without sufficient time for the issue o f a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date o f the written amendment will be die date o f the documented change. Copies o f the signed amendments will be appended to all distributed study plan copies. The original amendment w ill be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description o f any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence Any other documentation necessary for the reconstruction o f the study Chromatograms- A ll chromatograms w ill contain the following: Sample identification, injection date, arrow or other indication o f the area o f interest, and injection number corresponding to the run. Additionally, fortifications w ill include the amount o f analyte added and the sample number o f the sample that was fortified. Analytical standard chromatograms w ill additionally include the concentration (e.g., pg/mL). Page 13 o f65 Exygen Research Page 80 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 A s part o f the documentation the following sheets w ill be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITY ASSURANCE The QA Unit o f Exygen Research w ill inspect the study at intervals adequate to assure compliance with GLP's, and w ill report the findings o f audits to the Study Director, Exygen Management, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING A ll hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period o f time specified in 40 CFR 792.195. An exact copy o f the materials submitted to the study director will also be kept at Exygen Research. Exygen w ill obtain permission from the study director before discarding or returning samples. Exygen Research Page 4 o f 65 Page 81 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 APPENDIX I ANALYTICAL METHODS V0001780: "Method o f Analysis for die Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: `M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: `M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: `M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" Exygen Research Page 15 of 65 Page 82 of 134 Interim Report #22 - Analysis of Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001780 Method o fAnalysis fo r the DetermtoatJoa o f Perfloorooctanolc Add (PFOA) to Water byLO M S/M S Analytical Testing Facility: Exygen Roeareh 3058 Research Drive State College, PA 16801 Approved By: ______ Paul Coonolly ' Technical Leader, LC*MS, Exygen Research tokwAW Date ./J o to Flaherty / ^ Vice President, Operations, Exygen Research Exygen Research Total Pages: 7 Page 16 o f 65 Page 83 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygsaRin n ch MethodNumber V000P80 a n a l y t ic a l m e t h o d Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 1.0 Scope This method iito b e employed for the eolation and quantitation o f perfhiorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in water. 2.0 Safety 2.1 Always observe safe laboratorypractices. 22 Consultfoe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least40 m L o ftest 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 samplesmust be thoroughly mixed before being sampled for extraction 3.3 Any samples containing particles should be centrifuged at `*3000 rpm for -5 minutes andthe supernatantused 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 Water-HPLC grade 4.2 Methanol-HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfhiorooctanoic Acid - Sigma-Aldrich 3.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 3.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 50 mL disposable polypropylene centriftigo tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-1OOuL, 100-200uL). 5.7 123-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waten SepPak Vac 6 cc (lg ) tCISSPE cartridge. Pagt?or? Page 17 o f65 Exygen Research Page 84 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number P0001131 ExyfHftfMreb MethodNumb VOOOlttO ANALYTICAL m e t h o d Method o fAnalysis for the Determination o fParfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 5.12 SPEvacuum manifold. 5.13 Centrifugo capableo fapiiming 50 raL polypropylene tubee at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluopha*eRP(]Ceystooe Scientific), 2.1 mm x SOmm, V (P/N: 62505*052130) 6.2 Temperature: 30*C 6.3 Mobile Phase(A ) : 2 mM Ammonium Acetate in Water 6.4 Mobi!ePbase(B): Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22J 65 65 25 25 65 Flow Rate 2LB (mL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (canbe increased to as much ai SOjiL ). 6.7 Quantitation: PeakArea - external standard calibration curve. 6.8 RunTime: -2 3 minuter The above conditions are intended aaa guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Blecuoepray Negative MRM mode, monitoring 413 - 369 m/z. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Pisparatioo o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.i54 g o f ammonium acetateto 1000 mL o fwater. Alternate volumea may be prepared. Page wi * Page 18 of 65 Exygen Research Page 85 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygcalUm ich MethodNwnbcr V0001710 ANALYTICAL METHOD Method o fAnalyst fix the Determination o fPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution of~IOO jig/m L ofPFOA by weighing 10 mg o f analytical standard (corrected fix purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification aolutioa o f PFOA is prepared by bringing 10 mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/ttiL fortification aolution ofPFOA is prepared by bringing 10 mL o fthe 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pgftnL fortification aolution ofPFOA is prepared by bringing 10 mL o fthe 1.0 pg/raL solution to a final volume o f 100 w ith methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pgftnL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the dateo fpreparation. 92 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are pcoceased through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared asneeded. Final Concentration Fortification Volume of Concentration of Calibration o f Fortification Votums Fortified Commi Calibration Sttndsrd ID Solution (mb) (PL) Santoleim Ll Standard fumi* (examole) 0 0 40 0 XCmmddyy-0 10 100 40 25 XCmraddyy-1 10 200 40 50 XCramddyy-2 10 400 40 100 XCnunddyyO 100 100 40 250 XCmmddyy-4 100 200 40 500 XCmmddyy-3 100 400 40 1000 XCmmddw-6 * The extracted concentration o f foe calibration standard is equal to 8 its initial concentration, due to the concentration o fthe standard during foe extraction (SPE). XC extracted calibration standard. PaarSol"' Page 19 o f 65 Exygen Research Page 86 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 BxygMRMMrch Mrthod Number V00017S0 I ........ ANALYTICAL METHOD [ Method o fAnalysis for the Determination ofPerfiuorooctanoic Acid (PFOA) in Water by LC/MS/MS 9.13 9.2.4 9.2.5 A un> standard solution (reagent blank) mum be prepared with each seto fstandards extracted. Store a ll extracted calibration standards in 15-raL polypropylene tubes at 2"C to 6C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or leas) must include at least one reagent control (method blank using HPLC water) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural reoovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified In the quality assurance plan for this project 11.0 SampleExtraction 11.1 Measure 40 mL o fsample or a portion o f sample diluted to 40 mL with water into 50 mL polypropylene eentriflige tubes (fo rtify as needed, replace lid and mix well). 11.2 Condition the Cu SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL o fHPLC water ( - 2 drop/sec). Do not let column run dry 11.3 Load sampleon conditioned Cii SPE cartridge. Discard eluate. 11.4 Elute with -5 mL 100% methanol Collect 5 mL o f eluate into graduated 15 mL polypropylene centrifiage tubes (final volume 5 mL). 11.5 Analyze samplesusing electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to tt 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 he interspersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards may be rejected at the beginning o f a set followed by extracted calibration standard interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the first and last injection in aample 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 fpeak area Page) of? Page 20 o f 65 Exygen Research Page 87 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygiaRaNKli MatfaodNumber V00017S0 C ANALYTICAL MBTHOP________________________ Method o fAnalysis for foe Determination o fPerfluorooctanoie Acid (PFOA) in Water by LC/MS/MS versus calibration etandaid cooeentration using MawLynx 3.3 (or equivalent} software system. 12.5 Sample response should not exceed standard responses. Any sample* that exceed standard responses should be flirther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o fa daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to foe PFOA anion, while the daughter ion (369 amu) represent! the low o fcarbon dioxide. 13.2 Method blanks must not contain PFOA at level greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must beobtained and the entire set must be re-extracted. 13J Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike falls outride the acceptable lim its, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by tire analyst to determine i f re-extraction is 13.4 Any caUbration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from tire calculation o f the calibration curve. However, foe total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curvet generated must be 0.992 (R* 0.985). I f calibration resulta fo il outride these lim its, then appropriate stops must be taken to adjust instrument operation, and the standards or the relevant seto f samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than 4 % within an analytical nm. I f retention time d rift exceeds this lim it within an analytical run then the set mustbe reanalyzed. 14.0 Calculations 14.1 Use the following aquation to calculate the amount o f PFOA found (in ng/L. baaed on peak area) using foe standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found fne/Ll - ( t a i re, inlncem l DF slope DF Actor by which the final volume was diluted, if necessary. Pge6or7 Page 21 o f 65 Exygen Research Page 88 o f 134 Interim Report #22 - Analysis of Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bxypa Sawareh Method Number V0001780 I a n a l v t ic a l m e t h o d Method o f Analydi for the Determination o fPerfluoroocttnoic Acid (PFOA) in Water by LC/MS/MS 14.2 For aamplea fortified w ith known amount! o f PFOA prior to extraction, uie the following equation to calculate the percent recovery. Recovery(H ) [ total analyte found(ng/L) - analyte found in control (ng/L)] qq analyte added (ng/L) Exygen Research Page 22 o f 6$ Page 89 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Exygen Protocol Number: PQ001131 ANALYTICAL METHOD Method Number V0001781 Method o fA aalyib fo r the Detenniemtioe o f Perlleorooetupie AcM (PFOA) In Soil by LC/MS/MS Analytical Tenting Facility: Exygen Research 3058 Research Drive State College. PA 16801 Approved By: l a c-xL Paul Connolly ' Technical Leader, LC-MS, Exygen Research Date fohn Flaherty f Vice President, Operations, Exygen Research Date Exygen Research Total Pages: 7 Page 23 o f63 Page 90 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E iy f ti Kaaearcb Method Number V0001781 ANALYTICAL METHOD Method o fA n a lyiii for the Determination o fPerfluorooctaooic Acid (PFOA) in Soil by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o fperfluorooctenoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectromctric Detector (LC/MS/MS) in aotl. 2.0 Saftty 2.1 Alwayi observe safe laboratory practicea. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t leaet 15g o ftest sample for extraction. 3.2 No sampleprocessing is needed fo r soil samples. 3.3 Samples fiored refrigerated should be allowed to equilibrate to room temperature. 3.4 A ll samplesmustbe 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-HPLC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfiuorooctaooic Add - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvent! equipped w ith a variable volume iqjector capable o f injecting 5-200 pL connected to atandem Maes Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 50 mL disposable polypropylene ccntrifoge tubes. 5.5 l SmL disposablepolypropylene centriflige tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial Id t 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 ( Ig) tC 18SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. Page 2 of 7 Page 24 o f 65 Exygen Research Page 91 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exyiea Rwwrrh MethodNumberV0001711 I ANALYTICAL METHOD Method o fAnalysis for die Determination ofPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 5.14 Wrist-action shaker. 5.1$ Centrifiige capable ofspim ing 50 mL polypropylene tubes at 5000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 62 Temperature: 30*C 6.3 Mobile Phase(A) : 2 mM Ammonium Acetate in Water Mobile Phase(B) : Mediano! Gradion Program: Flow Rate 3LA 2 i faiL/m in) 0.0 65 35 0.3 1.0 65 35 0.3 8.0 25 75 0.3 20.0 25 75 0.3 22.5 65 35 0.3 6.6 Iqjection Volume: 15 pL (can be increased to u much as 50 p.L). 6.7 Quantitation: PeakA rea- external standard calibration curve. 6.8 Run Time: - 23 minutea. The above conditions are intended aaa guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - f 369 m/z for PFOA. The above conditions an intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM emmonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o fwater. Alternate volumes maybe prepared. Pag3of 7 Page 25 o f 65 Exygen Research Page 92 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number P0001131 Exygea Um weh Motbod KnnberVOOOt711 ANALYTICAL METHOD Method o fAnalysts for the Determination o f Pcrftuorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.0 Standard Prepantioo 9.1 StandardStock/Fortification 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 for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LOPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o fthe 10 pg/mL solution to a final volume oflOO with methanol in 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortificatim i solution o f PFOA is prepared by bringing 10 mL o fpie 1.0 pgfaL aolutioo to a final volume o f 100 w ith methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f die 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions sretobe stored in refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from die dateo fpreparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standard are prepared in HPLC water The calibration standards are processed through the extraction procedure, identical to samples. 9.2.2 The following is a typical example: additional concentrations may be prepared asneeded. Final Concentration Fortification V olum e o f Concentration o f C alib ratio n o f Fortification V olum e Fortified C ontrol C alibration S tandard ID Solution (neb) 0 ( U 0 Santole im L l 40 Standard (ooO* 0 (extm plc) X C m m ddyy-0 10 100 40 23 X Cm m ddyy-l 10 200 4 0 SO X C m m ddyy-2 10 400 40 100 X Cm mddyyO 100 100 40 250 XCmmddyy-4 100 200 40 500 XCmmddyy-5 100 400 40 1000 XCm m ddw -4 * The extracted concentrationo fdie calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC extracted calibration standard. Pag 4 of7 Page 26 o f 65 Exygen Research Page 93 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P01131 ExyfM RaaMrck MattedNuotar V000178) I ANALYTICAL METHOD Method o fAnalysis for the Determination o fPerfhwroocunolc Acid (PFOA) in Soil by LC/MS/MS 9.2.3 A zero standard solution (reegent blank) m int be nrepared with each set o fstandards extracted. 92.4 Store a ll extracted calibration standards in 15-rnL polypropylene tubes at 2*C to 6"C, up to two weeks. 9.2.5 Alternate volumes and concentration o f standards may be prepared as 10.0 Batch SetUp 10.1 Each batch o f sample extracted (typically 20 or less) must include at least one reagent control (method blank using S mL o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes win be specified in the quality assuranceplan for due project. 11.0 Sample Extraction 11.1 Weigh 5 g o f sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 5 mL o fmethanol and shakeon a w rist action shaker fo r-13 minutes 11J Transfer the tubes to an ultraaonic bath and sonicate fo r-1 5 minutes. 11.4 Bring the volume up to 40 mL w ith water in the 50 mL polypropylene centri&ge tube. 11.5 Centrifage fo r-10 minutes at-3000 rpm. 11.6 Condition the Cu SPB cartridges ( I g 6 m L) by passing 10 mL methanol followed by 5 mL o fHPLC water ( - 2 drop/sec). Do not let column run dry 11.7 Load (decent) the sample on the conditioned Cu SPE cartridge. Discard etuiie. 11.8 Elute with 5 mL 100% methanol. Collect 5 mL o f eluate into graduated 15 mL polypropylene centriAtgetubes (final volume 5 mL). 11.9 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Iq'ect the aime amount o f each standard, sample and fortified sample into (he LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Stendardi o fPFOA conwponrimg to at least five or more concentration levels must beincluded 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 Pat*S of7 Page 27 o f65 Exygen Research Page 94 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000U31 Exygen JU m id i Method Number VOOO1781 ANALYTICAL METHOD Method o fAnalyrii for the Determination o fPerftaorooctanoic Aoid (PFOA) in Soil by LC/MS/MS extracted calibration standards may be injected at the beginning o f set followed by extracted calibration standards interspersed every 5*10 samples (to account for a second seco f extracted standards). In either case, extracted calibration standards must be the first and last iiyeetion in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using l/x weighting o fpeak 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 furtherdiluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the 1m s o fcarbon dioxide. 132 Method blanks must not oontain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater titan 50 ng/L, then a new blank sample mustbe obtained and the entire set roust be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. If a control spike falls outride the acceptable lim its, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine i f re-extraction is 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Tost, may bo 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 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R1 20.985). I f calibration results f ill outride these limns, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant seto fsamples should be reanalyzed. 13.6 Retention timee between standards and aamplca must not d rift more than 4 % within ananalytical run. I f retention time d rift exceeds this lim it within an analytical run then the set mustbe reanalyzed. P steS on Page 28 o f 65 Exygen Research Page 95 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Eayya Iwtswh MtthodNaabv V00017S1 ANALYTICAL METHOD ] Method o fAnalysts for the Determination o fPtrfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/L, based on peak ana) using die standard curve (linear regression parameters) generated by die Mass Lynx software program: PFOA found fae/D -(P eak area .intercept) x DF slope DF - factor by which die final volume was diluted, i f necessary. 14.2 For samples fortified w ith known amounts o f PFOA prior to extraction, use the following equation to calculate the percentrecovery. Recovery (%) - [ totalanalyte found(ng/L) - analyte found in control(ng/L)] ^ analyte added (ng/L) 14.3 (Jae die following equation to convert the amount o f PFOA found in ng/L to n g /g to b ). pfoa found(ppb)- [PFOAfoundfaa/U\ wham ttttictod flMHUl sample weight (5 g) 14.4 Use die following equation to calculate die amount o f PFOA found in ppb basedon dry weight PFOA found (ppb) dry weight - PFOA found (ppb) x (100% / total solids(%)] Fa7 of? Page 29 o f 65 Exygen Research Page 96 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number P0001131 A N A LY T IC A L M ETH O D Method Number V0001782 Method o f Analysis fo r the Determination o f Perfiaorooctaaolc Add (PFOA) in Sediment by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C J t L _____________ Paul Connolly { Technical Leader, LC-MS, Exygen Research a/fl? t f U / __________ ydohn Flaherty t Vice President, Operations, Exygen Reaearch ___ io h .b h i Date Date Exygen Research Total Pages: 7 Page 30 o f65 Page 97 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 M ^tod Number VOOOI7J2 I ANALYTICAL MBTHOD j Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 1.0 Scope This method iito b e employed for the isolation and quantitation o fperfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in sediment 2.0 Safety 2.1 Always observeeafo laboratorypractioes. 2.2 Consultthe appropr iate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 30 g o ftest sample for extraction. 3.2 No sample procenutg is needed for sediment samples. 3.3 Samples stored reftigersted 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 win be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 Water-HPLC grade 4.2 Methanol- HPLC grade 4.3 Acetic Add - Reagent grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.3 Perfluorooctanoic A dd - Sigma-Aldrich 5.0 Instrument and Equipment 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume injector capable o f injecting 5-2QQ pL connected to atandem Maas Spectrometer(LC/MS/MS). 3.2 A device to collectraw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 50 mL disposable polypropylene centriiUge tubes. 5.5 13 mL duposablo polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-lOOuL, 100-200uL). 5.7 125-mL LDPB narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 3.9 Disposable pipettes. 3.10 Autopipette* (10 0 -10 0 0 pL and 10 -10 0 ^L ), w ith di sposable tips. S.l 1 Waters SepPak Vac 6 cc (lg ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. Pas* 2 of 7 Page 31 o f 65 Exygen Research Page 98 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 Exypaftsseveb Method Number V0QO1712 I ANALYTICAL METHOD f Method o fAnalysis for the Determination o fPerfluorooctaooic Acid (PFOA) in Sediment bv LC/MS/MS * 5.13 Vortexer. 5.14 Wrist-action shaker. 5.1$ CentriAige capable o fplaning 50 mL polypropylene tubes at 3000 rpm. 6.0 Chromstopsphic System 6.1 Analytical Column: FtuophaseRP (Keystone Scientific), 2.1 mm x SOmm. 5m (P/N: 82305-032130) 6.2 Temperature: 30*C 6.3 Mobile Phase(A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phase(B ): Methanol 6.5 Gradient Program: Tim a/m int 0 .0 1.0 8 .0 20 .0 22.5 2tA 65 63 25 25 65 Flow Rate & (mL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 injection Volume: 15 pL (can be increased to as much ss 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions an intended asa guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 -* 369 m/z for PFOA. The sbove conditions are intended is a guide and may be changed in order to optimize die MSMS system. 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetateto 1000 mL o fwater. Pac^)o f7 Page 32 o f65 Exygen Research Page 99 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Snyi Ksw ch Method Number V0Q017I2 | ANALYTICAL METHOD | Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 8.2 Extraction Solutions 8.2 .1 lH aceticacid in water is prepared by adding 10 mL o f acetic seid to 1000 mL o fwater. Alternate volumes maybeprepared. 9.0 Standard Preparation 9.1 Standard Stock/Farti6cation Solution 9.1.1 Prepare a stock solution o f-100 p y ta L o f PFOA by weighing 10 mg o f analytical standard (corrected fix purity) and dilute to 10 0 mL with methanolin a 125-oL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 100 pg/mL solution to a final volume o f 100 w ith methanol in a 12SmL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o fthe 10 pg/mL solution to a final volume o f 100 with methsnol in a 125mL LDPE bottle. 9.1.4 A0.1 pg/mL fortification solution o f PFOA it prepared by bringing 10 mL o fthe 1.0 pg/mL solution to a final volume o f 100 with methanol in a 123mL LDPE bottle. 9.1.3 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o fpreparation. 92 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution oftheO .l pg/mL fortification solution. 9.2.2 The following is a typical example: additional concentrations may be Concentration o fFortification Solution fartsL) 100 100 too 10 3 2 Vohirat (mL) 10 5 2 10 10 10 Diluted to (mL) 10 0 100 100 100 100 100 Final Concentration infl/mL) 10.0 5.0 20 1.0 0.3 0 .2 Pag4of 7 Page 33 o f 65 Exygen Research Page 100 of 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Num ber P0001131 Exypu KaanreS ANALYTICAL METHOD Method o fAnalysis for foe Determination ofPerfiuorooctanoic Aeid (PFOA) in Sediment by LC/MS/MS 9.2.3 Store a ll calibration standards in 125-raL LDPE narrow-mouth bottles at 2*C to 6*C, up to six months. 9.2 A Alternate volumes id concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or le u ) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verity procedural recovery for foe batch. 10.2 Requirements for field id laboratory duplicates end spikes w ill be specified in foe quality assuranceplan for this projert. 11.0 Sample Extraction 11.1 Weigh 3 g o f sample into 50 mL polypropylene centrifoge tubes (fortify as needed, replace lid and mix well). 11.2 Add 35 mL o f 1% acetic acid, cap, vortex end shake on a wrist action shaker fo r-40 minutes. 11.3 CentrifUge foe tubes i t -3000 rpm for -20 minutes. 11.4 Condition foe Cu SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 20 mL o fHPLC water ( - 2 drop/sec). Do not let column run dry 11.5 Load (decant) foe sample on foe conditioned Cu SP6 cartridge. Discard eluate. 11.6 Add 20 mL o f methanol to the aediment left in the bottom o f foe 50 mL centrifoge tube. Cap, vortex and shake on e wrist action shaker for -30 minulea. 11.7 Centrifoge foe tubes at-3000 rpm for -2 0 minutes. 11.8 Decantthe aefoapol onto foe sameSPE cartridge. Collect the eluate. 11.9 Wash the column w ith 4 mL o fmethanoL Collect the eluate and add it to the eluate collected in step 1 1 .8. 11.10 Condition a second Cu SPE cartridge (1 g, 6 mL) by passing 10 mL methanol followed by 20 mL o fHPLC water ( - 2 foop/scc). Do not let column run dry 11.11 Add foe methanol to -200 mL o f water and load on foe second conditioned SPE cartridge. 11.12 Elute with -5 mL 100H methanoL Collect 5 mL o f eluate into graduated 15 mL polypropylene ceatrifoge tubes (final volume 5 mL). 11.13 Analyse samples using electroepray LC/MS/MS. Pagf 5 o f? Page 34 o f 65 Exygen Research Page 101 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen P rotocol N um ber: P0001131 BxygsaRsMweh MethodNumberVOOO1782 I ANALYTICAL METHOD I Method o f Analysis for the Determination o fParfluorooetanoic Add (PFOA) in Sediment by LC/MS/MS 12.0 Chromatography 12.1 Inject the tame amount o feach standard, M aple end fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o fPFOA corr esponding to at least five or more concentration levels must be included in an analytical act. 12.3 An entiie set o f extracted calibration standards must be included at the beginning and at foe end o f a sample eel. Standard! must be interspersed between every 3-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 3-10 samples (to account for a second set of standards). In either case, calibration standards must be the first and last injection in a sample sec. 12.4 Use linear standard oaves for qusntitatioa. Linear standard curves arc generated for foe analyte by linear regressioa using l/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.3 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted end reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to foe PFOA anion, while the HtigM w on (359 amu) represents the loss o fcarbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.2 ng/mL, then a new blank sample must be obtained and the entire set must be re-oxtraded. 133 Recoveries o f control spikes end matrix spikes must be between 70-130% o f their known values. I f a control spike foils outside the acceptable lim its, the entire eel o f temple* abould be re-extracted. Any matrix spike outside 70' 130% should be evaluated by foe analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from foe calculation o f the calibration curve However, foe total number o f extracted calibration standards that could be excluded must not exceed 20% o f foe total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R* 0.985). I f calibration result* fo il outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standardsor foe relevant set o f samples should be reanalyzed. Pag6of 7 Page 35 o f 65 Exygen Research Page 102 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen P rotocol N um ber: P0001131 MethodNimber V0001782 ANALYTICAL METHOD Method o fAnalytic for the Detannination o fPerfluorooeunoic A dd (PFOA) in Sediment by LC/MS/MS 13.6 Retention times between standards snd samples must not d rift more than 4 % within an analytical run. I f retention time d rift exceeds this lim it within an analytical run then the setmust be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generatedby the Mass Lynx software program: PFOA found (ngftnL) - (Peak area - intercept) x DF lope DF factor by which the final volume waa diluted, i f neceeaary. 142 For maples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculatethe percent recovery. Recovery(% )- f total analyte found (ng/mL) - analyte foundin control(ng/mL)] ^ 1QQ analyte added (ng/m L) 14.3 Uethe following equation to convert the amount o f PFOA found in ng/mL to n*/g(ppt>). PFOA found (im bl - fPFQA found (iW m U I m l vnlnni. n m l.11 cample weight (5 g) 14.4 Use the following equation ( if nocesiary) to calculate the amount o f PFOA found in ppb based on dry weight. PFOA found (ppb) dry weight " PFOA found (ppb) x {100% / total sohds(%)] Paf*7 of 7 Page 36 o f 65 Exygen Research Page 103 o f 134 Interim Report #22 - Analysis of Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V00017S3 Method o fAnalysis fo r the Determination ofFerftnorooctanotc AcM (PFOA) io Fisb u d C lem by LC/MS/MS Analytic] Testing Facility: Exygen Research 305$ Research Drive State College, PA 16801 Approved By. y t-l c- J l Peunli Connolly I Technical Leader, LC-MS, Exygen Reeearch n iw M Date Flaherty Vice President, Operations, Exygen Research Dele Exygen Research Total Pagee: 8 Page 37 o f 65 Page 104 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygenItowucli MethodNumberV00017I3 [ ANALYTICAL m e t h o d Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Clara* by LC/MS/MS 1.0 Scope Tide method it to be employed for the isolation and quantitation o fperfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric E>etector(LC/MS/MS) in fish and dam*. 2.0 Safety 2.1 Always observe safe laboratory practice*. 2.2 Consultdie appropriate MSDS before handling any chemical for proper safety precautions. 3.0 SampleRequirement 3.1 A t least20 g o ftest sample for extraction 3.2 Samples dwuld be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place die samples in frozen storage until time o f analysis. 3.3 Sample collection procedures w ill be specified in the sampling plan for this project 4.0 Reagents and Standards ^ 4.1 W ater-HPLC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60-200 mesh)- Reagent grade 4.4 Flocisil (60*100 mesh)- Reagent grade 4.7 Superclean LC*NHj - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L*Aecotbic add - Reagentgrade 4.10 DimethyldichloroaUane-Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Add - Sigma-Aldricb 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up <o 2 solvents equipped with a variable volume injector capable o f injecting 5*200 pL connected to a tandem Mast Spectrometer(LC/MS/MS). 5.2 A device to oollect raw data for peak integration and quantitation 5.3 Analytical balance capable o freading to 0.00001 g. Page 2 o f8 Page 38 of 65 Exygen Research Page 105 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 BxyfeaRsscseh M sto d Number VQ00P8J analytical m etho d Method o fAnalysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 5.4 Rotary evaporator. 5.5 Tiscumizer. 5.6 125 mL pear-shaped flasks. 5.7 50 mL disposable polypropylene centriftige tubes. 5.8 15mL disposable polypropylene centrifuge tubes. 5.9 Disposable micropipets (50-100uL, 100-200uL). 5.10 125-mL LDPB narrow-mouth bottles. 5.11 2 mL clearHPLC vial kit. 5.12 Disposable pipettes. 5.13 Autopipettes (100-1000 pL and 10-100 jxL), w ith disposable tips. 5.14 SPEtubes (20mL) (Supelco cat no. N057I77). 5.15 W rist action shaker. 5.16 Centrifuge capable o fspuming 50m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5m (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase(A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phaae(B ): Methanol 6.5 GradientProgram: Tim orm ini 0 .0 1.0 8 .0 20 .0 22.5 65 65 25 25 65 Flow Rate & fm L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to aa much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The aboveconditions me intended as guide and may be changed in order to optimize the HPLC system. pi* i ora Page 39 of 65 Exygen Research Page 106 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygenK-- weh M ethod N um ber VQQ017I3 L ANALYTICAL MXTHOD ] Method o fAnalysis for the Determination o fPerfluorooetntoic Acid (PPOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - 369 m/z for PFOA. The above conditions am intended u a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0.134 g o f ammonium acetateto 1000 mL o fwater. 8.2 Extraction Solution* 8.2 .1 8.2.2 2% aaeorbic add in methanol is prepared by dissolving 2 g o fascorbic add in 100 mL o fmethanol. 30% DimethyldicWorosilane in toluene is prepared by twinging 3 mL o fdimethyldichlorosilane to a final volume o f 10 mL with toluene. Alternate volume* may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 1 mL o ftile 100 pgrinL solution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.3 A 0.1 pgfaL fortification solution o fPFOA prepared by bringing 10 m Lofthe 1.0 pg/mL solution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.4 A 0.01 pgfaiL fortification solution o f PFOA ia prepared by bringing 10 mL o f the 0.1 pgtoiL solution to a final volume o f 100 with mediano) in a 123 mL LDPE bottle. 9.1.5 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the dale o fpreparation. p t|e 4 o fI Page 40 o f 65 Exygen Research Page 107 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyycaRateateli MethodNumberV001783 ANALYTICAL METHOD Method o fAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Fish and Gams by LC/MS/MS 9.2 Standard Calibratioa Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f the l .0 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared at needed. Concentration Final efFertifieatien Votai Solution (ua/mL) (mL) Diluted to (mL) Concentration (Mg/mL) IX) 5.0 100 0.05 1.0 1JO 0.05 0.025 2.5 1.0 10 10 too 100 10 0 10 0 0.025 0.0 1 0.005 0.002S 0 .1 0.005 10 10 100 100 0 .0 0 1 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six montiti. 9.2.4 Alternate volumes and concentrations o fstandards may be prepared as 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or leas) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the bitch. 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 5 g o f frozen sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid sod mix well). 11.2 Add 30 mL o facetonitrile and shaleson a wrist actum shaker for -13 minutes 11.3 Place the tubes in i freezer for ~1 hour. 11.4 Pack andcondition the SPE tubes and silsnize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in sequence w ith 2 g flo riril, 2 g silica gel, 2 g carbon, and l g LC-NHj. Condition the columns with 20 mL o f methanol, than 20 mL o f acetonitrile. Discard a ll washes. Do not allow the column to dry. 11.6 Silsnize the 125 mL pear-shaped flasks by rinsing w ith the 30% dimethyldichloroailsne in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use, either by amdrying or with a stream o fnitrogen. h | t * of K Page 4 ! o f6$ Exygen Research Page 108 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 B xygsaRM--rc h Mflthod N um ber VOOOPSJ [ ANALYTICAL METHOD Method o f Asalymi for the Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 11.7 Centriflige the SOmL polypropylene tuba containing sample at -2000 rpm fo r-1 0 minutes. 11.8 Decant the extract on to a conditioned SPE column fitted inside the mouth o f the pear-ahaped flask. Collect the eluate in the 125 mL silanized pear-shape flask. 11.9 Add 10 mL o f acetonitrile to the sample in the SO mL centrifuge tube. Homogenize die frozen frt phase using a tiaaumizer for -30 seconds and rinse the riiernnirer with -10 mL o facetonitrile into the tube. 11.10 Shakethe sample again fo r-10 minutes on a wrist-action shaker. 11.11 Place the tubes in a freezer for -1 hour more. 11.12 Centrifoge die SOmL polypropylene tubes containing sample at -2000 rpm fo r-1 0 minutes. 11.13 Decant the extract onto the same SPB column. Collect the eluate into the same pear-shaped flask and combine w ith die eluent from the initial extraction. 11.14 Pais 20 mL o f acetonitrile through the SPE column and combine the eluate in the samepear-shaped flask. 11.15 Add 3-4 (hops o f l-octanol to the extract in the pear-shaped flask and evaporate at reducedpressure using a rotary evaporator (at < 40C). 11.16 Make the final volume, by adding 2 mL o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. 11.17 Transfer the extracts to HPLC vials using disposable pipetj. 11.18 Analyze samplesusing electroepray LC/MS/MS. 12.0 Chromatography 12.1 Inject die same amount o f each standard, sample and fortified 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 seL 12.3 An entire set o fcalibration standards must be included at the beginning and at die end o f a sample set. Standards must be interspersed between every 5-10 samples. A t an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either ease, calibration standards must be the first and last injection in a sample set 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o fpeak area versus calibration standard coocerantion using MassLynx 3.3 (or equivalent) software system. PSe6ofS Page 42 o f 65 Exygen Research Page 109 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number POOOJ131 Bxyya Wm--wh MtdwdNnrtbcr V000I793 L ANALYTICAL METHOD Method o fAnslyiis for the Determination o fPerfluorooetanoic Acid (PFOA) in Fish and Clime by LG/MS/MS 12.5 Sample reeponse ehould not exceed atandaid recponeee. Any samples that exceed standard responses should be Author diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show apeak o f a daughter ion at 369 nu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughterion (369 amu) ispiusents the Iocs o fcaibon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. I f a blank contains PFOA at levels greater than 0.5 ppb, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f ooatrol spikes and matrix spikes must be between 70-130% of their known values. I f a control spike foils outside the acceptable lim its, the entire seto fsamples should be re-extracted. 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 ffoe total numbero fstandards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R2 20.985). I f calibration results fo il outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standardsor the relevant set o fsamplesshould be reanalyzed. 13.6 Retention times between standards a d samples mutt not d rift more than 4 % within an analytical nm. I f retention time d rift exceeds this lim it within an analytical run then foe set must be reenalyzed. 14.0 Calculations 14.1 Use foe following equation to calculate foe amount o f PFOA found (in ng/mL, based on peek srsa) using foe standard curve (linear regression parameters) generated by foeMaas Lynx software program: PFOA found (ng/raL) fPeak area - intm ep^ slope 14.2 Use foe following equation to convert the amount o fPFOA found in ng/mL to n^g(ppb). PFOA found fontA fPFQAfoundfng/mlA . fira l vnlum gfm l ^ . fiF I sample weight (g) DF factor by which foe final volume was diluted, i f necessary. psi*7ofS Page 43 o f63 Exygen Research Page 110 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E x y y Rucwd t Motbod Number V000178J ANALYTICAL METHOD Method o fAnalysis for tbe Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Clami by LC/MS/MS 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use fits following equation to calculate foe percent recovery. Recovery (%) - [ totalanalytefound (ngfr) analytefound in control(ng/g)] analyte added(n ^ g ) Exygen Research Papsofs Page 44 o f65 Page 111 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001784 Method o fAnalysis fo r the Determination e f Perflnorooctanoic Add (PFOA) In Vegetation by LC/MS/M5 Analysed Testing Facility: Exygen Research 3058 ReeearchDrive State College, PA 16801 Approved By: C JiL ___ Put Cotmolly 1 Technical Leader, LC-MS, ExygenResearch o / r t f / U / __________ Jw oi Flaherty ^ Vice President, Operations, Exygen Research iQ fr tlH Date Date Exygen Research Total Pages: 7 Page 45 o f65 Page 112 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygcn Protocol Number: P0001131 E x y y lU m reh Method Number V00VI7M ANAL V'TICAL METHOD j Method o fAnalysis for the Detenninadon o fPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope Thi method ie to be employed for the {eolation and quantitation o f perfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Maas Spectrometric Detector (LC/MS/MS) in vegetation. 2.0 Safety 2.1 Always obeerve safe laboratory practices. 2.2 Consult foe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t leeat 20 g o fte it sample for extraction. 3.2 Samples should be processed before extraction. Place die 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 until time o f analysis. 3.3 Sample collection procedures w ill be spedfied in foe sampling plan for this project 4.0 Reagentsand Standards 4.1 Water -HPLC grade 4.2 Acetonitrile-HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.3 Silica gel (60-200 mesh) - Reagent grade 4.6 Florinl (60-100 mash) - Reagent grade 4.7 Supcicletn LC-NH* - Reagent grade 4.8 1 -OctanoI-HPLC grade 4.9 L-Ascorbic add - Reagent grade 4.10 Dimcthyldichloroiilane - Reagent grade 4.11 Toluene - Reegent grade 4.12 Ammonium Acetate - A.C.S. ReegentOtade 4.13 Perfluorooctanoic Acid - Sigma-Aidrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume iqjector capable o f injecting 5-200 jiL connected to atandem Mass Spectrometer (LC/MS/MS). 5-2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. Psgf 2ttl'7 Page 46 o f 65 Exygen Research Page 113 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygca Protocol Number: P0001131 ExypnlU iM rcti Method Number V00017M ANALYTICAL METHOD Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 5.4 Rotaryevaporator. 55 125 mL pear-shaped flasks. 56 50 mL duposable polypropylene centrifrge tabes. 5.7 15 mL disposable polypropylene centrilbge tubes. Disposable micropipets (50-100uL, 100-200uL). 12S-mL LDPE nairow*mouth bottles. 5.10 2 mL clear HPLC vial kit. 5.11 Disposable pipettes. 5.12 Autopipettes (100*1000 pL and 10*100 pL), w ith disposable tips. 5.13 SPEtubes (20mL) (Supeleo cat no. N057177). 5.14 Wrist aetion shaker. 5.15 Csntrifiige capable o fspinning 50 mL polypropylene tubes at 2000 tpm. 6.0 Chromatographic System 6.1 Analytical Column: FluophaieRP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 32505*052130) 6.2 Temperature: 30*C 6.3 Mobile Phase(A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phase(B ): Methanol 6.5 Ondisot Program: Tima fm irri 0 .0 1.0 8 .0 20 .0 22.5 S tA 65 65 25 25 65 Flow Rate fm L/m ini 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 RunTime: -2 3 minutes. The above conditions are intended asa guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - * 369 m/z for PFOA. Page 3 of? Page 47 o f65 Exygen Research Page 114 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Bxyisa Rauick Muhod Nunter V00017M I ANALYTICAL METHOD Method o fA m ly iii for the Detenninetion o fPerfluorooctenoic A dd (PFOA) in Vegetation by LC/MS/MS The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o fSolutions 8 .1 Mobile Phase 8.1.1 2 mM ammonium ecetttc in water is prepared by adding 0.1S4 g o f ammonium acetate to 1000 mL o fwater. 8.2 Extraction Solutions 8.2 .1 8.2.2 2% asooittc arid in methanol is prepared by dissolving 2 g o f ascorbic acid in 100 mL o fmethanoL 30% Dhnethyidichlorosilane in toluene is prepned by bringing 3 mL o fdimsthyldichlorosiline to a final volume o f 10 mL w ith toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 ug/mL fortification eohition o f PFOA ie prepared by bringing 1 mL o f the 100 fig/mL solution to a final volume o f 100 with methanol in a 125 mL LOPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o fPFOA is prepared by bringing 10 m Lofthe 1.0 pgtaL solution to a final volume o f 100 with methanol in a 125 mL LOPE bottle. 9.1.4 A 0.01 pg/knL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 ug/mL solution to a final volume o f 10 0 with methanol In a 125 mL LOPE bottla. 9.1.5 The stock ind fortification solutions are to be stored in a refrigerator at approximately 4*C and are liable for 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 1.0 pgtoL fortification solution. Page 4 ui ` Page 48 o f 65 Exygen Research Page 115 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number POOOl 131 ExyinKMMich Method Number VOOOl7S4 I ANA in ilA L METHOD Method o fAnalysis for the Determination o fPerfluorooctsnoic Acid (PFOA) in Vegetation by LC/MS/MS 92.2 The following if a typical example: additional concentrations may be prepared atneeded. Concentration ofFortifkatiOD Solution fuftfaL) 1.0 1.0 1.0 O.OS 0.025 0.1 0.005 Volume faL ) 5.0 2.5 1.0 10 10 10 10 Diluted to (mL) 1O0 100 100 100 100 100 100 Final Concentration (ufl/mL) 0.05 0.025 0.01 0.00$ 0.0025 0.001 0.0005 at 2*C to 6*C, up to nx months. 92.4 Alternate volumes and concentrations o f standards may be prepared as 10.0 Batch Setlip 10.1 Each batch o f sample# extracted (typically 20 or leas) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 102 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 5 g o f frozen sample into 50 mL polypropylene centrifuge tubes (fortify t t needed, replace lid end mix well). 11.2 Add 30 mL o facetonitrile end shake on awrist action shaker fo r-15 minutes. 11.3 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm fo r-1 0 minutes. 11.4 Pack and condition the SPEtubes and ailanize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tidies in sequence w ith 2 g flo risil, 2 g silica gel. 2 g carbon, and 1 g LC-NHj. Condition the columns w ith 20 mL o f methanol, then 20 mL o f acetonitrile. Discard a ll washes. Do not allow the column to dry. 11.6 Silaaize the 125 mL pear-shaped flasks by nosing with the 30% dimethyldichlORMUsae in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use. eitherby air-drying or w ith astream o fnitrogen. 11.7 Decant the extract on to a conditioned SPE column fitted inside the mouth o f the peer-shaped flask. Collect the eluate in the 125 mL silanized pear-shape flask. PageS o f Page 49 o f 65 Exygen Research Page 116 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyfm Research MethodNumber VOOOI7S4 a n a l y t ic a l m e t h o d Method o fAnalysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 mL o facetonitrile to the sample in the 50 mL centriftjge tube. 11.9 Shake the sample again for ~10 minutes cma wrist-action shaker. 11.10 Centriftige die SO mL polypropylene tubes containing sample at 2000 rpm for 5 minutes. 11.11 Decant the extract onto die same $PE column. Collect the eluate into the same pear-shaped flask ind combine w ith the eluent from the initial extraction. 1 1 .1 2 Repeal steps 11.8 through 1 1 .1 1 again. 11.13 Add 3*4 drops o f l-octanol to the extract in the pear-ehaped flask and evaporate at reduced prem ie using arotary evaporator (at < 40*0). 11.14 Malm die final volume, by adding 2 mL o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/disaolve. 11.15 Transfer the extracts to HPLC vials using disposable pipeii. 11.16 Analyze samples using eiectrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed sample. 12.2 Standards o fPFOA corrsaponding 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 end at the end o f a am ple set Extracted standards must be interspersed between every 3-10 samples. As an alternative, an entire set o f extracted calibration standards may be injected at the beginning o f a set followed by attracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards mustbe die first and last injection in a sample set. 12.4 Use linear stanched curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o fpeak 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 exceol standardraponata should be fhrtbcr diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 aau from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughterkm (369 amu) represents the loss o fcarbon dioxide. PaS*6of7 Page SOo f65 Exygen Research Page 117 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExypeXweeh MethodNuatorVOOOI7S4 ANALYTICAL m e t h o d 1 Method o fAnalysts for the Determination o fPcrfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 13.2 Method blanks must not oontain PFOA at levels greater than the LOQ. If a black contains PFOA at levels greater Otan 0.5 ppb, then a new blank sample must be obtained and the entire ie t muat be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes muat be between 70>130*/ o f their known values. I f control spike falls outside the acceptable lim its, the entire seto fsamples shouldbe re-extracted. 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 staadvda that could be excluded must not exceed 20% o fthe total numbero fstandards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R* 20.985). If calibration resulta fo il outside these lim its, then appropriate steps must be talma to adjust instrument operation, and the standards or the relevant set o fsamples should be reanalyzed. 13.6 Retention between standards and samples must not d rift more than 1 4 % within an analytical run. I f retention time d rift exceeds this lim it within an analytical run then die actmust be reanalyzed. 14.0 Calculations 14.1 Use foe following equation to calculate foe amount o f PFOA found (in ng/mL, baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) - /p ~ v * " - intercept) slope 14.2 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found (ppbl- fPFOA found fna/fcnLlx final volume fm Ll x DF1 sample weight (g) DF - factor by which the final volume was diluted, i f necessary. 14J For samples fortified w ith known amounts o f PFOA prior to extraction, use die following equation to calculate the percent recovery. Recovery (%) - f totalanalytefound (ng/g) analyte found in control (ng/g)] ][10p analyte added (ng/g) Pie ?ui ` Page 5! o f65 Exygen Research Page 118 o f 134 Interim Report #22 - Analysis of Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD MethodNumber V0001785 Method of Analysis for the Petermleerten of Ferfleerooctaeoic AeM (PFOA) hi Smell Mammal U var by LG/MS/MS Analytic] Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: ''v^--V-_C--iL ____ Paul Connolly I Technical Leader, LC-MS, Exygen Research a/ # JobhnFFlalahheerty ' ' VicePPrreessuidsnt, Operations, Exygeo Research Date Date Exygen Research Total Pages: 7 Page 52 o f 65 Page 119 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exysra RNMch Method N u o ta VOOOI7S5 I ANALYTICAL MTHOD .... Method o fAnalysis fbr the Detennination o fPerfluorooctanoic Acid (PFOA) in Smell Mammal Liver by LC/MS/MS 1.0 Scope This method is 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/MS) in small mammal liver. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult toe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t lead Sg o fteat sample for extraction. 3.2 Sample* should be proceteed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place toe samples in frozen storage until time of analysis. Alternately, i f there is an insufficient amotan o f sample Hess than 5 g), then no processing is necessary and the sample can be used as supplied. 3.3 Simple collection procedures w ill be specified in toe sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-HPLC grade 4.2 Methanol-HPLC pads 4.3 Acetonitrile-HPLC grade 4.4 Ammonium Acetate- A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich $.0 Instrument and Equipment 5.1 A Ugh performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer(LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capableo freading to 0.00001 g. 5.4 SOmL disposable polypropylene centriftige tubes. 5.5 15 mL disposable polypropylene centrifUge tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouthbottles. 5.8 2 mL clear HPLC vial kit. P sf2oP Page 53 of 65 Exygen Research Page 120 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E a y a l U i--ich Method Number V000PB5 I ANALYTICAL METHOD Method o fAnalysis for die Determination o fPerfluorooctanoic Acid (PFOA) in Small Mammal liv e r by LC/MS/MS 3.9 Disposable pipettes. 3.10 Autopipette (100*1000 pL and 10-100 pL), with disposable tips. 3.11 Waten Sep Pak Vac 6 ec (lg ) tC18 SPE cartridge*. 3.12 SPE vacuum manifold. 3.13 Tiiauemizer. 3.14 Wrist-action ahakcr. 3.15 Centrifuge capable o fspinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chroraatognphic System 6.1 Analytical Column: FluophaseRP (Keystone Scientific), 2.1 mm x$0 mm, 5h (P/N: 82503-032130) 62 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water Mobile Phase(B) : Methanol Gradient Program: Time (mint 0 .0 1.0 8.0 20 .0 22.3 63 65 25 25 65 Flow Rate SLfi (m lAnin) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 nL (can be increased to as much as 50 >iL). 6.7 Quantitation: PeakArea - external standard calibration curve. 6.8 RunTime: -2 3 minutes. The above conditions are intended as a guide and may be changed in order to optimize die HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 -* 369 m/z for PFOA. The above conditions ate intended as a guide and may be changed in order to optimize the MSMS system. P a tii of? Page 54 o f65 Exygen Research Page 121 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 BnypaRwuwh______________________________________ MettodNanftcr V00Q|7|j f ANAL\TICALMgTHOD .... Method o fAnalysis for the Determination o fPerfluomoctanoic Acid (PFOA) in Smell Mammal Liver by LC/MS/MS 8.0 Pnpacatioo o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in wato ia prepared by adding 0 154 g o f ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Foctification Solution 9.1.1 Prepare a nock solution o f 100 pg/mL o f PFOA by weighing 10 mg o fanalytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125*mL LDPE bottle. 9.1.2 A 1.0 jig/m L fortification solution o f PFOA is prepared by bringing I mL o f the 10 0 |ig/mL solution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.3 A 0.1 pgtoiL fortification solution o f PFOA is prepared by bringing 10 m Lofthe 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125mL LDPE bottle. 9.1.4 Tbe stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o fpreparation. 91 Standard Calibration Solutiona 91.1 LC/MS/MS calibration standards are prepared in methanol via dilution o fthe 0.1 MgfaiL fortification solution. 9 1 1 The following is a typical example: additional concentrations prepared asneeded. CooeeMmtidn Finti ofFortification Votone Diluted to Concentration Solution fa^mL) (mL) (mL) (nafaiL) too 5.0 100 5.0 100 2.0 too 2.0 100 1X1 100 1.0 3.0 10 100 01 2.0 10 100 IA 10 too 01 0.) 9 1 1 Store all calibration standards in 12S*mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six months. 91.4 Alternate volumes and concentrations o fstandards may be prepared as Pages of? Page 55 o f 65 Exygen Research Page 122 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exypafemich MethodNiunber V00Q17U I ANALYTICAL METHOD Method o fAnalyse for the Determination ofPeriluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 10.0 Batch Set Up 10.1 Each batch o f camples extracted (typically 20 or teas) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verily procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assuranceplan for this project. 1 1 .0 Sample Extraction 11.1 Weigh 1 g o f sample into a SOrnL polypropylene centrifuge tubes (fo rtify as needed, replace lid and mix well). Note that alternate weights o f liver may be measured depending on the sample siae available for use. 11.2 Add water to the sample for afinal volume o f 10 mL. 11.3 Homogenize sample using atiaiuemiaer for ~1 minute. 11.4 Transfer 1 mL o f the sample using a disposable pipette into a IS mL disposable centriftige tube. 11.5 Add S mL o facetonitrile and shake for ~20 minutes on a wrist-action shaker 11.6 Centrifuge fee tubes at -3000 rpm for ~3 minutes. 11.7 Decant the supernatant into a SOmL disposable centriftige tube and add 3S mL o fwater. ll.S Condition the Cis SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mLofHPLC water (~ 2 drop/sec). Do not let column run dry 11.9 Load the sampleon conditioned Cn SPE cartridge. Discard eluate. 1 1.10 Elute with ~2 mL o f methanol Collect 2 mL o f eluate into a graduated IS mL polypropylene centriftige tube (final volume - 2 mL). 11.11 Analyse samples usingelectrospray LC/MS/MS. 12.0 Chromstofftphy 12.1 Iqject tiie same amount o f each standard, am ple and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analysed samples. 122 Standards o fPFOA corresponding to at least five or more concentration levels mustbe included in <n analytical set 12.3 An entire act o fcalibration standards must be included at the beginning snd at the end o f a sample set. Standards must be interspersed between every 5-IO samples. As an alternative, an entire set o f calibration standards may be tiyected at the beginning o f a set followed by calibration standards interspersed every 3-10 samples (to account for a second set o f standards). In either ease, calibration standards must be foe first and last injection in a sample set 12.4 Uae linear standard curvet for quantitation. Linear standard curves are generated for the analyte by linearregression using l/x weighting o fpeak area PageJof? Page 56 of 65 Exygen Research Page 123 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Eaygenftmaidi MrtwdNwnbw V0Q07I3 I .. ANALYTICAL METHOD Method o fAnalysis for tbs Determination ofPerfluorooctanoic Acid (PFOA) in Small MammalLiver by LC/MS/MS 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 Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parem corresponds to the PFOA anion, while the daughter it (369 amu) represents the loss o fcarbon dioxide. Method blanks must not contain PFOA at levels greater than the LOQ. i f a blank contains PFOA at levels greater than 10 ng/g, then a new blank sample must beobtained and the eotire setmust be re-extracted. Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. I f a control spike foils outside the acceptable lim its, the entice eel o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine i f re-extraction is warranted. Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded ftom the calculation o f the calibration curve However, foe total number o f calibration standards that could be excluded must not exceed20% o fthe total number o fstandards tqjected. th e condition coefficient (R) for calibration curves generated must be 20.992 (R1 20.985). I f cal&ration results foil outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevantset o fsamples should be reanalyzed. Retortion tiinee between standards and samples must not d rift more than 4 % within in analytical run. I f retortion time d rift exceeds this lim it within an analytical run then the setmust be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o fPFOA found (in ng/mL. based on peak area) uring the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) - (Peak area - intercept x DF x aliquot factor lope DF * factorby which the final volume was diluted, i f neoesiary. Aliquot factor* 10 Pag6of7 Page 57 o f 65 Exygen Research Page 124 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bxyttt Reacvcfc Method Number V00017I5 | ANALYTICAL METHOD Method o fAnalysis for the Determination o fPerfluorooctmoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use die following equation to calculate the percent recovery. Recovery (%) [ total analyte found (ng/mL) - analytefound in control (ng/mL)l [1Q0 analyte added (ng/taL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to n rt(p p b ). PFOA found (pobl - fPFOA found fne/m D x final volume (mLM sample weight (g) Exygen Research Page 7 o f7 Page 58 o f 65 Page 125 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V00017M Method o fAnalysis fo r the DetermtaeHoa o f Perfluorooetanolc Acid (PFOA) io Smalt Mammal Serum by LC/MS/MS A nilytkai Testing Facility: Exygen Research 3058 Research Drive State College, FA 16801 Approved By: _C-- ___ Paul Connolly I Technical Leader, LC-MS, Exygen Research _________ John Flaherty / Vice President, Operations, Exygen Research Date /'A a Dete Exygen Research Total Pago: 7 Page 59 o f 65 Page 126 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bona Xawcb Mtthod Numbtr VOOO17M I ANALYTICAL METHOD Method o fAnelyne (hr the Determination o f Perfluorooctanoic Acid (PFOA) m Smell Mammal Serum by LC/MS/MS 1.0 Scope Thie method ia to be employed for foe {eolation and quantitation o fperfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Maas Spectnxnetric Detector(LC/MS/MS) in email mammal serum. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consultfoe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 1mL o fteat sample for extraction. 3.2 No simple processing is needed for serum simples. However, frozen swum mmples mustto allowed to completelythaw to room temperature before use. 3.3 Sample collection procedures w ill be specified in foe sampling plan for this project 4.0 Reagents and Standards 4.1 Water-HPLC grade 4.2 Methanol - HPLC grade 4 J Acetonitrile-HPLC grade 4.4 Ammonium Acetate-A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sipna-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 50 mL duposable polypropylene centrifogc tubes. 5.5 15 mL disposable polypropylene centriftige tubes. 5.6 Disposable micropipets (50-lOOuL. 100>200uL). 5.7 125-mL LDPE narrow-mouthbottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (HXMOOOpL and 10-100 pL), w ith disposable tips. 5.11 Waters SepPak Vac 6 cc (lg ) tC U SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Voitexer. Page 2 o P Page 60 o f 65 Exygen Research Page 127 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000U31 ExyteateMuch Method NiMfccrVOOO17*6 I A iW Y T lC A L m e t h o d Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Small Mammal Seram by LC^MS/MS 9.14 Wrist-action shaker. 5.15 Centrifuge capable o fspinning 15 mL polypropylene tubes at 5000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Tenpenture: 30*C 6.3 Mobile Phase(A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase(B ): Methanol 6.5 GradientProgram: Tune (m ini 0 .0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate 51S (mUmin) 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 aa 50 pL). 6.7 Quantitation: PeakArea- external standard calibration curve. 6.8 RunTime: -2 3 minutes. The above conditions are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrosprty Negative MRM mode, monitoring 4! 3 - 369 m/z for PFOA. The above conditions are intended a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate In water is prepared by adding 0.154 g o f nmanium acetate to 1000 mL o fwater. Alternate volumes maybe prepared. P a p ) of 7 Page 6/ o f 65 Exygen Research Page 128 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 EaygseR-- arch Mwbod Number VQ00I78& 1 ANALYTICAL METHOD Method o fAnalysis for foe Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS | 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f MOO pg/m Lof PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125*mL LDPE bottle. 9.1.2 A 1.0 pg/mL fortification rotation o f PFOA is prepared by bringing \ mL o f foe 100 pg/mL solution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m Loftbe l .0 pg/mL solution to a final volume o f 100 w ith methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C end are stable for a maximum period o f 6 months from tire data o fpreparation. 9.2 StandardCalibration Solutions 9.2.1 92.2 LC/MS/MS calibration standard! are prepared in methanol via dilution o ffoe 0.1 pg/mL fortification solution. Tbe following is a typical example: additional concentrations may be prepared as needod. Concentration ofFortification Votum Solution ( a lb i) (mL) Diluted to (mL) Final Concentration (nt/mL) 100 3.0 100 3.0 100 2.0 100 2.0 100 1.0 100 S.0 10 100 1.0 0.3 2.0 10 100 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6"C, up to six months. 9.2.4 Alternate voltanes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or leas) must include et least one untreated control and two untreated controls fortified at known concentration* (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 assuranceplan for this project. PifcSofT Page 62 o f 65 Exygen Research Page 129 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number. P0001131 Exygoi RtfM icb Mfthod Nwnbtr V00017$ | ANALYTICAL METHOD ~ ........ Method o fAnalysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Senun by LC/MS/MS 11.0 Sample Extraction 11.1 Measure l mL o fsample into a SOmL polypropylene centrifrge tubes (fortify as needed, replace lid and mix well). Note diet alternate volumes o f serum maybe measured depending on foe sample size available for use. 11.2 Add water to foe sample for a final volume o f20 m L Cap tightly 11.3 Vortex for-1 minute. 11.4 Transfer 1 mL o f foe sample using a disposable pipette into a 15 mL disposable entrifltge tube. U.5 Add SmL o facetonitrile and shake for -20 minutes on s wrist*action shaker. 11.6 Centrifuge the tubes at -3000 rpm for -S minutes. 11.7 Decant the supernatant into a 50 mL disposable centriftige tube and add 35 mL o fwater. 11.8 Condition foe Cis SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL ofHPLC water (~ 2 drop/sec). Do not let column run dry 11.9 Load foe sampleon conditioned Cu SPE cartridge. Discard eluate. U.10 Elute with -2 mL o f methanol. Collect 2 mL o f eluate into a graduated 15 mL polypropylene centriftige tube (final volume * 2 mL). 11.11 Analyze samples using electrocpray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o feach stndard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards ofPFOA corresponding to at least five or more concentration levels mustbe included in m analytical set, 12.3 An entire set o fcalibration standards must be included tt the beginning and at foe end o f a sample set Standards must be interspersed between every 5' l <> samples. As an alternative, an entire act o f calibration standards may be iqjectod 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, calforation standards must be foe first and last injection in a sample set 12.4 Use lin e * standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o fpeak 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 ftuther diluted and reanalyzed. Page 5 o f 7 Page 63 o f 65 Exygen Research Page 130 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Rtaeaitb Method Numbtr VOM 716 I ANALYTICALMKTHOD Method o f Analysis for the Detemiination o fPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 13.0 Acceptance Criteria 13.1 Cbromatognun must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) representsthe lose o fcarbon 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/mL, then a new blank sample mustbe obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike falls outside the acceptable lim its, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o fthe total numbero f standards injected. 13.3 The correlation coefficient (R) for calibration curves generated must be 0.992 (Ra 0.985). I f calibration results fo il outside these lim its, then sppropriste steps must be tsken to adjust instrument operation, and the standards or the relevant set o fsamples should bs reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical ran. I f retention time d rift exceeds this lim it within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Usethe following equation to calculate the amount o fPFOA found (in ng/mL. based on peak area) using the standard curve (linear repression parameters) generated by the M an Lynx software program: PFOA found (nfl'm L) " (Peakarea - intercept) x DF x aliquot factor slope DF " foctor by which the final volume was diluted, i f necessary. Aliquot factor * 20 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [total analytefound(ng/mL) - analytefound in control (ng/mL)] k |Qij analyte added(na/roLl Pag6 of7 Page 64 o f 65 Exygen Research Page 131 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study N o .: P0001131 Exygen Protocol Number: P0001131 E xyfM jtM M rcb M ftbod Nuobcr V0001716 I .... A N A tV T IC A L METHOD Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 14.3 Ute the fallowing equation to oouvert the amount o f PFOA found in ngrinL to ppb. PFOA found Cppb) - fPFOA found fagftnU X final volume ftn ll) am ple volume (mL) Exygen Research P Page 65 o f 65 Page 132 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 S tate College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment Num ber 1 Effective Date: 0 1/19/05 Exygen Study Number P0001131 Client Study N um ber Page 1 of 1 BESG BIPT IQ N .P F AM EN P E P 8E C T IQ N 1) Analytical Procedure Sum m ary V0001780:Section 9.1 2 ) Verification o f Analytical Procedure None ~ AMENDED TO 1) Add to Section 9.1:S ectton 9 .1 .6 , Alternate weights o f standards m ay be used to prepare alternate concentrations o f stock solutions as necessary. A tterriate levels o f fortification solutions m ay also be prepared. 2 } Low and high spiking levels o f the analytes for each matrix m ay be altered depending on sam ple size available for extraction and/or to cover analyte concentrations expected in the sam ples. RATIO NALE 1 ) Higher concentrations o f standards need to be prepared in order to spike the sam ple bottles at higher levels. 2 ) T he sam ple size avalab le fo r sm all mammal Iv e r and serum w as sm aller than expected. Spiking at the pre-determ ined levels in the protocol puts the spiked concentration low er than the detection lim it Also, the analyte levels in the ground w ater sam ples a re 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 w ill cover the analyte concentrations expected in the w ater sam ples. IM PACT O N S TU D Y T he LOQ is 100 r> g /g fb ra0.1 g sam ple o f small m am m al liver and is 1000 n g /m L fo ra 0.01 mL sam ple o f sm all mammal serum. Higher levels of spiking fo r the w ater sam ples w ill ensure that m ore Q C recovery data can be used. LIBRARY ID: W 00012264` ' ADMINISTRATIVE FORM Exygen Research Page 133 o f 134 Interim Report #22 - Analysis o f Soil Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 S tate College, PA 16801 Fax: 814-231-1580 Am endm ent N um ber E ffec tiv e D a te: Exygen S tu dy N um ber PROTOCOL AMENDMENT 2 0 3 /0 7 /0 5 P0001131 C lie n t S tu dy N u m b e r Page 1 o f 1 None D ESC R IPTIO N O F AM ENDED SE C TIO N 1) Report, page 11 o f 65 2 ) T est M aterials, page 6 o f 65: PFO S transition monitored 499 -> 99. AMENDED TO 1 ) Instead o f one final report, Interim reports will be issued. 2 ) P FO S transition monitored may also be 4 9 9 -> 80. RATIO NALE 1) Due to th e excessive sizes of the data sets, interim reports w ill be issued to allow the client to receive data In a tim elier m a rn e ' '* * * ' e t f a f w 2 ) The A P I 4 0 0 0 LC /M S /M S systems (M e e t 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 d e n t will be able to receive and review the data m ore quickly. 2 ) T he 4 9 9 -> 80 transition can be detected with greater sensitivity; therefore, giving b etter chrom atography. LIBRARY ID: VD00122M-' - Exygen Q AU R eview . L u M t s ADMINISTRATIVE FORM Exygen Research Page 134 o f 134