Document ymeg18EzMbagq81o0qGQBpBD3
INTERIM REPORT # 8 - Analysis of Sediment 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 07/13/06
PERFORMING LABORATORY Exygen Research
3058 Research Drive State College, PA 16801
Phone: 814-272-1039
STUDY SPONSOR 3M Company
3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: 651-733-6374
PROJECT Protocol Number: P0001131 Exygen Study Number: P0001131
Total Pages: 121
Interim Report #8-Analysis of Sediment 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.
Exygen Research
Jaisimha K
,
Study Director
Weston Solutions, Inc.
1 /l/U lJy &
Michael A. Santoro Sponsor Representative 3M Company
Exygen Research
Date Page 2 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
QUALITY ASSURANCE STATEMENT
Exygen Research's Quality Assurance Unit reviewed Exygen Study Number P0001131, entitled, "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program". All reviewed phases1 were inspected for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Exygen Principal Investigator and Management and to the Study Director.
Phase
Date Inspected
Date Reported to Date Reported to
Principal
Exygen
Date Reported to
Investigator Management Study Director
15. Raw Data Review and Interim Analytical Report Review
07/11-13/05
08/24/05
09/02/05
09/13/05
30. Raw Data Review and Final Interim Analytical Report Review
07/03-04/06
07/13/06
07/13/06
07/13/06
Lydia S, Technical Lead, Quality As^forance Unit
Date
'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.
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Interim Report #8-Analysis of Sediment 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:
Exygen Research Exygen Research Facility Management:
Study Director, Weston Solutions, Inc
Jaisimha Kesan P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company:
1/hw LJj O*.
Michael A. Santoro Director of Regulatory Affairs
Exygen Research
Date
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Interim Report #8-Analysis of Sediment 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:
Sediment
TEST SUBSTANCE:
Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS)
SPONSOR:
3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144
STUDY DIRECTOR:
Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380
STUDY MONITOR:
Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144
PERFORMING LABORATORY: Exygen Research 3058 Research Drive State College, PA 16801
ANALYTICAL PHASE TIMETABLE:
Study Initiation Date:
11/05/04
Interim Analytical Start Date:
05/23/05
Interim Analytical Termination Date: 05/30/06
Interim Report Completion Date: 07/13/06
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PROJECT PERSONNEL
The Study Director for this project is Jaisimha Kesari at Weston Solutions, Inc. The following personnel from Exygen Research were associated with various phases of this interim portion of the study:
Name John Flaherty Karen Risha Paul Connolly Christine Edwards Brittany Kravets Mindy Cressley Krista Gallant
Scott Crain Mark Ammerman
Title Vice President
Scientist Technical Lead-LC/MS
Technician Technician Technician Technician Technician Sample Custodian
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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.................................................................................................... 11 1.0 SUMMARY................................................................................................................ 12 2.0 OBJECTIVE............................................................................................................... 12 3.0 INTRODUCTION....................................................................................................... 13 4.0 ANALYTICAL TEST SAMPLES.............................................................................. 13 5.0 REFERENCE MATERIAL........................................................................................ 13 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................ 14
6.1. Extraction Procedure For Sediment......................................................................... 15 6.2 Solvent Extraction Procedure For Sediment............................................................ 15 6.3 Percent Solids Procedure For Sediment................................................................... 15 6.4 Preparation of Standards and Fortification Solutions............................................... 16 6.5 Chromatography....................................................................................................... 16 6.6 Instrument Sensitivity............................................................................................... 17 6.7 Description of LC/MS/MS Instrument and Operating Conditions...........................17 6.8 Quantitation and Example Calculation..................................................................... 17 7.0 EXPERIMENTAL DESIGN...................................................................................... 19 8.0 RESULTS...................................................................................................................20 9.0 CONCLUSIONS.........................................................................................................20 10.0 RETENTION OF DATA AND SAMPLES.............................................................21
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Table I.
LIST OF TABLES
Page Summary of PFBS, PFHS and PFOS in Sediment Samples............................23
Table D. Summary of PFBS, PFHS and PFOS in Re-extracted Sediment Samples..... 25
Table ID. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sediment Samples..... 26
Table IV Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Sediment Samples.......................................................................................... 29
Table V Total Percent Solids in Sediment Samples.................................................... 32
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Figure 1.
LIST OF FIGURES
Page Typical Calibration Curve for PFBS in Methanol......................................... 34
Figure 2. Non-Extracted Standards of PFBS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively................................................................................ 35
Figure 3. PFBS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively........................................ 36
Figure 4. PFBS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B by Solvent Extraction, Respectively..... 37
Figure 5. Chromatogram Representing a Sediment Sample Analyzed for PFBS (Exygen ID: C0053119, Data Set:052405D)..................................................38
Figure 6. Chromatogram Representing a Sediment Sample Analyzed for PFBS by Solvent Extraction, (Exygen ID: C0053132, Data Set: 051906D)........................................................................................................ 39
Figure 7. Typical Calibration Curve for PFHS in Methanol.......................................... 40
Figure 8. Non-Extracted Standards of PFHS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively.................................................................................41
Figure 9. PFHS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively.........................................42
Figure 10. PFHS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B by Solvent Extraction, Respectively..... 43
Figure 11. Chromatogram Representing a Sediment Sample Analyzed for PFHS (Exygen ID: C0053119, Data Set: 052405D).................................................44
Figure 12. Chromatogram Representing a Sediment Sample Analyzed for PFHS by Solvent Extraction, (Exygen ID: C0053132, Data Set: 051906D)........................................................................................................45
Figure 13. Typical Calibration Curve for PFOS in Methanol......................................... 46
Figure 14. Non-Extracted Standards of PFOS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively.................................................................................47
Figure 15. PFOS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively.........................................48
Figure 16. PFOS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B by Solvent Extraction, Respectively..... 49
Figure 17. Chromatogram Representing a Sediment Sample Analyzed for PFOS (Exygen ID: C0053119, Data Set:052405D)..................................................50
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LIST OF FIGURES
Page
Figure 18. Chromatogram Representing a Sediment Sample Analyzed for PFOS by Solvent Extraction, (Exygen ID: C0053132, Data Set: 051906D)....................................................................................................... 51
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LIST OF APPENDICES
AppendixA Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method V0001782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS", Protocol Amendments 1,2 and 8 and Protocol Deviation 1..................................................................................
Page 52
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1.0 SUMMARY
Exygen Research extracted and analyzed Sediment samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001782 (Appendix A).
The limit of detection for PFBS, PFHS and PFOS in sediment was 0.2 ng/g and the limit of detection for PFBS, PFHS and PFOS in water was 25 ng/L.
The standard sediment method (V0001782) was originally used to extract all of the samples. Twenty-two of the twenty-four analyzed samples were not reported due to quality control failure for at least one analyte. The QC failures were observed for samples with both apparent low endogenous levels and high endogenous levels. Due to this reason, the samples that were spiked too low relative to endogenous were not re analyzed using V0001782, but were solvent extracted and then analyzed, without further purification, through direct injection (Section 6.2).
Analytical results for the analysis of PFBS, PFHS and PFOS found in sediment samples are summarized in Table I. Analytical results for the analysis of PFBS, PFHS and PFOS found in the re-extracted sediment samples are summarized in Table II.
Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in sediment samples extracted with the original method are summarized in Table III. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in sediment samples were 71 8%, 95 15%, and 70 8%, respectively. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in sediment samples extracted with the solvent direct injection method are summarized in Table IV. The average percent recoveries standard deviations for PFBS, PFHS, and PFOS in the re-extracted sediment samples were 83 20%, 94 7%, and 95 25%, respectively.
The total percent solids for sediment samples are detailed in Table V.
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 sediment according to Protocol P0001131 (Appendix A).
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3.0 INTRODUCTION
This report details the results of the analysis for the determination of PFBS, PFHS, and PFOS in sediment using the analytical method entitled, "V0001782: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" and a solvent extraction method, detailed in Section 6.2.
The study was initiated on November 05, 2004, when the study director signed protocol number P0001131. The analytical start date for this interim report was May 23, 2005, and the analytical termination date for this interim report was May 30,2006.
4.0 ANALYTICAL TEST SAMPLES
Twenty-four sediment samples (Exygen ID C0053113-C0053136) were received on wet ice on December 10, 2004 from Charles Young at Weston Solutions, Inc. The samples were logged in by Exygen personnel and placed in refrigerated storage.
Sample log-in and chain of custody information is located in the raw data package associated with this interim report. Storage records will be kept at Exygen Research.
5.0 REFERENCE MATERIAL
The analytical standards PFBS and PFHS were supplied by 3M. PFBS was received from 3M at Exygen on May 13, 2005. PFHS was received from 3M at Exygen on January 20, 2003. PFOS was purchased from Fluka Corporation and was received at Exygen on April
23,2003.
The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated.
Compound PFBS PFHS PFOS
Exygen Inventory No. SP0005726 SP0002401 SP0002694
Lot # 101 SE036 430180-1
Purity (%) 96.7 98.6 101.2
Expiration Date 12/04/06 10/18/06 10/31/07
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The molecular structures of PFBS, PFHS and PFOS are given below:
PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (T^FgSCh'K^) Transitions Monitored: 299 - 99 Structure:
F F
F SO3
FF FF
PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CeFnSOaTC*)
Transitions Monitored: 399 -* 80 Structure:
FFF F
F
F S03
FFF FFF
PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFnSOaTC*) Transitions Monitored: 499 - 80 Structure:
FFF F SO3
FFFF FFFF
6.0 DESCRIPTION OF ANALYTICAL METHOD
The analytical method "V0001782: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" and a direct injection method (Section 6.2) were used for the sediment samples in this study.
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6.1. Extraction Procedure For Sediment
Before the samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A 5 gram portion of sediment was weighed into a fifty milliliter centrifuge tube for the extraction. After fortification of appropriate samples, 35 mL of 1% acetic acid in water was added to the samples. The samples were vortexed and allowed to shake on a wrist action shaker for ~60 minutes. The samples were centrifuged for -20 minutes at -3000 rpm. The supernatant was then loaded onto a Cis SPE cartridge conditioned with 10 mL of methanol and 20 mL of water. The eluate was discarded. Twenty milliliters of methanol was added to the sediment samples left in the centrifuge tube. The samples were vortexed and allowed to shake on a wrist action shaker for another 30 minutes. The samples were centrifuged again for ~20 minutes at ~3000 rpm. The supernatant was then loaded onto the same Cis SPE cartridge. The eluate was collected into a 500 mL Nalgene Bottle. The column was washed with 4 mL of methanol. The wash was collected in the same bottle as the eluate. Approximately two hundred milliliters of water was added to the bottles. The samples were mixed by shaking and loaded onto another Cis SPE cartridge conditioned with 10 mL of methanol and 20 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 Solvent Extraction Procedure For Sediment
Before the samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A one-gram portion of sediment was weighed into a 15-milliliter centrifuge tube for the extraction. Ten milliliters of 1% acetic acid in methanol was added to each sample. The samples were then shaken by hand, vortexed, and sonicated for thirty minutes. The samples were then centrifuged for ~10 minutes at -3000 rpm. Each sample was analyzed by LC/MS/MS electrospray.
6.3 Percent Solids Procedure For Sediment
Percent solids were determined using the procedure indicated in Exygen method V0000427. Approximately 20 grams of sample was weighed into a pan. The weight of the sample plus the pan was recorded. The samples were 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 V for percent solids results.
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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 non-extracted calibration standards containing PFBS, PFHS, and PFOS was prepared in methanol, as specified in Exygen method V0001782. The following concentrations were prepared:
Cone, of Fort Fort
or Calibration Volume
Solution
(mL)
(ng/mL)1
100 1.0
100 0.5
100 0.2
10 1.0
5.0 1.0
2.0 1.0
1of PFBS, PFHS, and PFOS
Final Volume
(mL)
10 10 10 10 10 10
Final Cone, of Calibration Std.
(ng/mL)
10 5.0 2.0 1.0 0.5 0.2
The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report.
6.5 Chromatography
Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~ 0.6 mins, ~ 9.2 mins, and -1 1 .7 mins, respectively. Peaks above the LOD were not detected in any of the reagent blank samples corresponding to the analyte retention time.
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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 ('min) 0.0 1.0 8.0 10.0 11.0 18.0
Total run time: ~18min Flow Rate: 0.3 mL/min Ions monitored:
%A 65 65 25 25 65 65
Analvte
Mode
PFBS PFHS PFOS
negative negative negative
%B 35 35 75 75 35 35
Transition Monitored 299 - 99 399 -80 499 -> 80
Approximate Retention Time
(min) ~0.6 min. ~9.2 min. -11.7 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
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linear regression) by Analyst software using six concentrations of standards. The concentration was determined from the following equations. Equation 1 calculated the amount of analyte found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program.
Equation 1:
Analyte found (ng/mL) = (Peak area - intercept) x DF Slope
Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary.
Equation 2 was used to convert the amount of PFBS, PFHS and PFOS found in ng/mL to ng/g (ppb) on a wet weight basis.
Equation 2:
Analyte found (ppb) = fAnalyte found (ng/mL) x final volume (5 mDl sample weight (5 g)
or
Analyte found Direct Inject (ppb) = fAnalyte found (ng/mL) x final volume (10 mDl sample weight (1 g)
For samples fortified with known amounts of PFBS, PFHS and PFOS prior to extraction, Equation 3 was used to calculate the percent recovery.
Equation 3: Recovery (%) =
(analyte found (ng/g) - analyte in control (ng/eV) xl00% amount added (ng/g)
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 (%)]
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An example of a calculation using an actual sample follows (calculation is for PFBS
only):
Sediment sample Exygen ID: C0053113 Spk C (Set: 052305F), fortified at 4 ng/mL
with where:
peak area
= 57889
intercept
= 2590
slope
= 14100
dilution factor
=1
ng/g PFBS added (fort level) = 4
amt in corresponding sample = 0
final volume (mL)
=5
sample weight (g)
=5
total solids (%)
= 59.43
From equation 1: Analyte found (ng/L)
= l~57889- 25901 x 1 14100
= 3.92 ng/mL
From equation 2: Analyte found (ppb)
= (3.92 ng/mL x 5 mL) 5g
= 3.92 ppb
From equation 3: % Recovery
= (3.92 ppb - Qppb) x 100% 4 ppb
= 98%
From equation 4: Analyte found (ppb) dry weight = 3.92 ppb x (100% / 59.43%)
= 6.60 ppb
NOTE: This value may be slightly different than that of the raw data due to rounding.
7.0 EXPERIMENTAL DESIGN
For sediment 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.
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The sediment samples were originally extracted in eight sets. Two sets contained six samples each, one set contained five samples, three sets contained two samples each, and one set contained one sample. The eighth set contained three samples designated for re extraction with the original method. For each sample, two laboratory matrix spikes and a duplicate were extracted. The re-extract sediment samples were extracted in five sets. Three sets contained five samples each, one set contained four samples, and one set contained three samples. For each sample, a duplicate (except for C0053135) and variable matrix spikes of adequate concentration were extracted. Each set included one reagent blank and two reagent blanks fortified at known concentrations.
8.0 RESULTS
The standard sediment method (V0001782) was originally used to extract all of the samples. Twenty-two of the twenty-four analyzed samples were not reported due to quality control failure for at least one analyte. The QC failures were observed for samples with both apparent low endogenous levels and high endogenous levels. Due to this reason, the samples that were spiked too low relative to endogenous were not re analyzed using V0001782, but were solvent extracted and then analyzed, without further purification, through direct injection (Section 6.2).
Analytical results for the analysis of PFBS, PFHS and PFOS found in sediment samples are summarized in Table I. Analytical results for the analysis of PFBS, PFHS and PFOS found in the re-extracted sediment samples are summarized in Table II.
Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in sediment samples extracted with the original method are summarized in Table III. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in sediment samples were 71 8%, 95 15%, and 70 8%, respectively. Fortification recoveries for the analysis o f PFBS, PFHS, and PFOS in sediment samples extracted with the solvent direct injection method are summarized in Table IV. The average percent recoveries standard deviations for PFBS, PFHS, and PFOS in the re-extracted sediment samples were 83 20%, 94 7%, and 95 25%, respectively.
The total percent solids for sediment samples are detailed in Table V.
9.0 CONCLUSIONS
The sediment samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001782 and using a solvent direct injection method, detailed in Section 6.2.
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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.
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TABLES
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Table I. Summary of PFBS, PFHS and PFOS in Sediment Samples
E xvnen ID
C lie n t S a m p le ID
C 4 S u lfo n a te P F B S
C 6 S u lfo n a te P F H S
C 8 S u lfo n a te P F O S
PetltuorobutanesuHonate______ Psrfluorohexsnesulfonats_______Perfluorooctsnssulfonate
A nalyte Found (ppb. ng/g) Drv W eight
Assessed A ccuracy
<+/-%>
Analyte Found (ppb, ng/g) D ry W eight
Assessed A ccuracy
<+/-% )
A nalyte Found (ppb, ng/g) D rv W eight
Assessed A c c u ra c y
(+/-% >
C 0053113
D L 3-S D -LO C 001 -0-041 201
ND
30
ND
30
ND 30
C 0 053113 R ep D L 3 -S D -LO C 001 -0-041 201*
ND
30
ND
30
ND 30
C 0053114
D L3-S D -LO C 002 -0-041 201
ND
30
ND
30
ND 40
C 0053114 R ep D L 3 -S D -LO C 002 -0-041 201*
ND
30
ND
30
ND 40
C0053115
D L3-S D -LO C 003 -0-041 201
ND
40
ND
30
NR NR
C 0 053115 R ep D L 3 -S D -L O C 0 0 3 -0 -0 4 1 2 0 1 *
ND
40
ND
30
NR NR
C 0053116
D L2-S D -LO C 001 -0-041 201
ND
40
ND
30
NR NR
C 0053116 R ep D L 2 -S D -L O C 0 0 1 -0 -0 4 1 2 0 1 *
ND
40
ND
30
NR NR
C 0053117
D L 2 -S D -L O C 0 0 2 -0 -0 4 1201
ND
40
ND
30
NR NR
e0053117 R ep D L 2 -S D -L O C 0 0 2 -0 -0 4 1 2 0 1 *
ND
40
ND
30
NR NR
C0053118
D L2-S D -LO C 003 -0-041 201
ND
40
ND
30
NR NR
C 0053118 R ep D L 2 -S D -L O C 0 0 3 -0 -0 4 1 2 0 1 *
ND
40
ND
30
NR NR
C0053119 C0053119 Rep
D B C -S D -LO C 0 01-0-0 41202 D B C -S D -LO C 0 01-0-0 41202 *
ND ND
40 0 .6 1 2 40 0 .6 5 7
30 30
NR NR NR NR
C0053120 C0053120 Rep
D B C -S D -L O C 0 0 2 -0 -0 4 1202 D B C -S D -LO C 0 02-0-0 41202 *
ND ND
30 0 .6 1 5 30 0 .6 0 6
30 30
NR NR NR NR
C 0053121
D BC-SD -LO CO O 3 -0 -0 4 1 2 0 2
ND
40
ND
30
NR NR
C 0053121 R ep D B C -S D -LO C 0 03-0-0 41202 *
ND
40
ND
30
NR NR
C0053122
D O U -S D -LO C 001-0-041202
3 .7 7
30
5 .6 8
C 0053122 R ep D O U -S D -LO C 001 -0 -0 4 1 2 0 2 *
3 .7 9
30
5 .1 3
30 30
NR NR NR NR
C0053123
D O U -S D -LO C 002-0-041202
6 .5 9
50
NR
NR
C 0 053123 R ep D O U -S D -LO C 002-0-041202*
7 .3 7
50
NR
NR
NR NR NR NR
C 0053124 C0053124 Rep
D O U -S D -LO C 003-0-041202 D O U -S D -LO C 003-0-041202*
0 .5 4 5 0 .5 9 1
30 30
2 .8 9 2 .1 0
30 30
NR NR NR NR
C0053125
D L 1 -S D -LO C 001 -0-041 202
ND
30
ND
30
NR NR
C 0 053125 R ep D L 1 -S D -L O C 0 0 1 -0 -0 4 1 2 0 2 *
ND
30
ND
30
NR NR
C0053126
D L 1 -S D -L O C 0 0 2 -0 -0 4 1202
ND
30
ND
C 0 053126 R ep D L 1 -S D -L O C 0 0 2 -0 -0 4 1 2 0 2 *
ND
30
ND
30 30
NR NR NR NR
COO5 3 1 2 7
DL1 -SD-LO CO O 3 -0 -0 4 1 2 0 2
NO
30
ND
30
NR NR
C 0 053127 R ep D L 1 -S D -LO C 003 -0-041 202*
ND
30
ND
30
NR NR
C 0053128
D M C -S D -LO C 001-0 -0 4 1 2 0 2
ND
30
ND
30
NR NR
C 0 053128 R ep D M C -S D -LO C 001-0-04120 2*
ND
30
ND
30
NR NR
COO5 3 1 2 9
D M C -S D -LO C 002-0-04120 2
ND
40
ND
30
NR NR
C 0053129 R ep D M C -S D -LO C 002-0-04120 2*
ND
40
ND
30
NR NR
COO5 3 1 3 0
D M C -S D -LO C 003-0-04120 2
ND
40
ND
30
NR NR
C 0 053130 R ep D M C -S D -LO C 003-0-041202*
ND
40
ND
30
NR NR
COO5 3 1 3 1
D A A -S D -L O C 0 0 6 -0 -0 4 1202
8 .2 5
40
NR
NR
NR NR
COO53131 R ep D A A -S D -LO C 0 06-0-0 41202*
6 .6 0
40
NR
NR
NR NR
COO531 32
D A A -S D -L O C 0 0 5 -0 -0 4 1202
NR
NR
NR
C 0 053132 R ep D A A -S D -L O C 0 0 5 -0 -0 4 1202*
NR
NR
NR
NR NR
NR NR NR NR
C0053133
D A A -S D -LO C 0 04-0-0 41202
NR
NR
NR
NR
NR
NR
C 0053133 R ep D A A -S D -L O C 0 0 4 -0 -0 4 1202*
NR
NR
NR
NR
NR NR
` Laboratory D uplicate ND - N ot detected a t o r above the Lim it o f Q uantitation (LO Q ) o f 0.2 ng/g (w et w eight). NR * N ot reported due to qua lity control failure . For re-analysis see Table II.
Exygen Research
Page 23 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Table I. Summary of PFBS, PFHS and PFOS in Sediment Samples Continued
E x y g e n ID
C lie n t S a m p le ID
C4 Sulfonate PFBS
C8 Sulfonate PFHS
C8 Sulfonate PFOS
Psrfluorobutanesulfonats______ Pertluorohsxaiieiutfonats______ Perfluorooctanssulfonats
A n a lyte Found (ppb, ng/g) D ry W e ig h t
Assessed A ccu racy
(+ /-% )
A n a lyte Found (ppb, ng/g) D ry W e ig h t
Assessed A ccu racy
< + /-% )
A n a lyte Found (p p b , ng/g) D ry W e ig h t
Assessed A ccuracy
( /-% )
C0053134 C 0053134 Rep
D A A -S D -L O C 0 0 3 -0 -0 4 1 2 0 2 D A A -S D -L O C 0 0 3 -0 -0 4 1 2 0 2 *
NR NR
N R 5 4 .1 N R 59.1
30 30
NR NR NR NR
C 0053135 C 0053135 Rep
D A A -S D -L O C 0 0 2 -0 -0 4 1 202 D A A -S D -L O C 0 0 2 -0 -0 4 1 2 0 2 *
NR NR
N R 0 .6 6 6 N R 1 .1 5
30 30
3 7 .7 4 2 .3
40 40
C0053136 C0053136 Rep
D A A -S D -L O C 0 0 1 -0 -0 4 1 2 0 2 D A A -S D -L O C 0 0 1 -0 -0 4 1 2 0 2 *
ND ND
3 0 0 .3 3 6 3 0 0 .3 4 1
40 40
NR NR NR NR
` L a b o ra to ry D u plicate N D = N o t d etected a t o r above th e L im it o f Q u a n tita tio n (LO Q ) o f 0 .2 n g /g (w e t w e ight). N R = N ot reporte d due to q u a lity co n tro l fa ilu re . F o r re -a n a lysis se e T able II.
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Page 24 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Table II. Summary of PFBS, PFHS and PFOS in Re-extracted Sediment Samples
E xyg e n ID
C0053115 C0053115 Rep
C lie n t S am ple ID
D L3-S D -LO C 003-0-041201 D L3-S D -LO C 003-0-041201*
C 4 S u lfo n a te P FBS
C 6 S u lfo n a te PFH S
C 8 S u lfo n a te PFOS
PtrtluorotoutanssuHonate_______ Pw1luofOtwxnwuHonH________Pw tluofO O CtinM ulfom t
Analyte Found (ppb. ng/g) Dry W eight
Assessed Accuracy
(*/-%)
Analyte Found (ppb, ng)g) Drv W eight
Assessed Accuracy
(*/-%)
Analyte Found (ppb. ng/g) Drv W eight
Assessed Accuracy
<+/-% )
. ND 30
- ND 30
C0053116 C0053116 Rep
D L2-S D -LO C 001-0-041201 D L 2-S D -LO C 001-0-041201*
ND 30 ND 30
C0053117 C0053117 Rep
D L2-S D -LO C 002-0-041201 D L2-S D -LO C 002-0-041201*
ND 30 ND 30
C 0053118 C 0053118R ep
D L2-S D -LO C 003-0-041201 D L2-S D -LO C 003-0-041201 *
ND 30 ND 30
C0053119 C0053119 Rep
D B C -S D -LO C 001-0-041202 D B C -S D -LO C 001-0-041202*
7 3 .0 7 1 .0
50 50
C0053120 C0053120 Rep
D B C -S D -LO C 002-0-041202 D B C -S D -LO C 002-0-041202*
3 7 .0 3 7 .2
50 50
C0053121
D B C -S D -LO C 003-0-041202
C 0053121 R ep D B C -S D -LO C 003-0-041202*
3 3 .8 3 2 .6
40 40
C0053122
D O U -S D -LO C 001-0-041202
C 0053122 R ep D O U -S D -LO C 001-0-041202*
500 30 460 30
C0053123
D O U -S D -LO C 002-0-041202
C 0053123 R ep D O U -S D -LO C 002-0-041202*
16.1 30 215 30
15.6
30
209
30
C0053124
D O U -S D -LO C 003-0-041202
C 0053124 R ep D O U -S D -LO C 003-0-041202*
390 30 324 30
C00S312S C0053125 Rep
DL1 -S D -LO C 0014)4)41202 D L 1-S D -LO C 001-0-041202*
9 .5 9 6.61
30 30
C0053126 C0053126 Rep
D L 1-S D -LO C 002-0-041202 D L1-S D -LO C 002-0-041202*
7 .7 6 8 .6 5
40 40
C0053127 C0053127Rep
D L1-S D -LO C 003-0-041202 D L 1-S D -LO C 003-0-041202*
9 .3 6 7 .9 3
30 30
C00S3128
D M C -S D -LO C 001-04)41202
C 0053128 R ep D M C -S D -LO C 001-0-0 41202*
C0053129
D M C -S D -LO C 002-0-0 41202
C 0 053129 R ep D M C -S D -LO C 002-0-041202*
3.51 3 .6 3
6 .4 1 8 .1 3
30 30
30 30
C 0053130
D M C -S D -LO C 003-0-041202
C 00S3130 R ep D M C -S D -LO C 003-0-041202*
6 .9 4 7 .7 4
30 30
COOS3131 C0053131Rep
D A A -S D -LO C 006-0-041202 D A A -S D -LO C 006-0-041202*
-
84.1
30
1700
50
*
117
30
1960
50
C 0053132
D A A -S D -LO C 005-0-041202
1 7 .8
50
41.1
50
616
30
C 0053132 R ep D A A -S D -LO C 005-0-041202*
17.7
50
4 0 .4
50
649
30
C 0053133
D A A -S D -LO C 004-0-041202
8 5 .9
30
709
C 0053133 R ep D A A -S D -LO C 004-0-041202*
102
30
665
30 4730 30 30 4550 30
C00S3134 C0053134 Rep
D A A -S D -LO C 003-0-041202 D A A -S D -LO C 003-0-041202*
ND ND
40 40
-
2010
30
-
2230
30
C 0053135
D A A -S D -LO C 002-0-041202
ND
30
-
-
--
C 0053136 C0053136 Rep
D A A -S D -LO C 001-0-041202 D A A -S D -LO C 001-0-0 41202*
. '
-
-
3 8 .5
30
4 5 .0
30
Laboratory Duplicate ND * Not detected at o r above the Lim it o f Q uantitation (LOQ) o f 0.2 ngig (w et weight).
Exygen Research
Page 25 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sediment Samples
S am ple D e scription
C 4iS u lfo n a t P F B S J W U W lj|h t_ ^ S S u jfo n a t i P F H S J W e U W e lg h ^ _ C 2 ^ u tfo n a t^ F O S W e tW ij[trt
Am ount S piked
(n o lg )
A m t Found in S am ple
(n fl/g )
Am ount R ecovered
<ng/0 )
R ecovery <%>
Am t Found in S am ple
(n g /g )
Am ount R ecovered
(n g /g )
R ecovery (*>
A m t Found in S am ple
Am ount R ecovered
(n g /g )
R ecovery <*>
D L3-S D -LO C 001-0-041201
(C0053113 Spk C, 4 ppb Spike)
4
ND
3 .9 2
98
ND
4.21
105
ND
DL3-SD-LO CO 01-0-041201 (C0053113 Spk D, 40 ppb Spike)
40
ND
3 1 .3
78
ND
3 4 .9
87
ND
3 .2 6 2 9 .4
82 74
D L3-S D -LO C 002-0-041201
(C0053114 Spk E. 4 ppb Spike)
4
ND
2 .9 3
73
ND
3 .2 4
81
ND
2.41
60
D L3-S D -LO C 002-0-041201
(C0053114 Spk F, 40 ppb Spike)
40
ND
30.1
75
ND
3 4 .2
86
ND
2 7 .7
69
D L3-S D -LO C 003-0-041201
(C0053115 Spk G. 4 ppb Spike)
4
ND
2 .5 6
64
ND
4 .2 8
107
NR
DL3-S D -LO C 003-0-Q 41201
(C0053115 Spk H. 40 ppb Spike)
40
ND
2 9 .0
73
ND
4 1 .4
104
NR
NR NR NR NR
D L2-S D -LO C 001-0-041201 (C0053116 Spk i, 4 ppb Spike)
D L2-SD-LO CO 01-0-041201 (C0053116 Spk J, 40 ppb Spike)
4 40
ND ND
2.71 2 6 .5
68 66
ND
4 .3 0
108
NR
ND
4 2 .5
106
NR
NR NR NR NR
D L2-S D -LO C 002-0-041201
(C0053117 Spk K, 4 ppb Spike)
4
ND
2 .7 0
68
D L2-S D -LO C 002-0-041201
(C0053117 Spk 1,40 ppb Spike)
40
ND
27.1
68
ND
4 .3 7
109
NR
ND
4 2 .8
107
NR
NR NR NR NR
D L2-S D -LO C 003-0-041201
(C0053118 Spk C. 4 ppb Spike)
4
ND
2 .7 7
69
D L2-S D -LO C 003-0-041201
(C0053118 Spk D, 40 ppb Spke)
40
ND
2 8 .4
71
ND
4.31
108
NR
ND
4 3 .8
110
NR
NR NR NR NR
D BC-SD -LO CQ 01-0-041202
(C0053119 Spk E. 4 ppb Spike)
4
ND
2 .6 0
65
0 .2 5 9
4 .4 2
104
NR
D B C -S D -L0 0001-0-0412 02
{00053119 Spk F. 40 ppb Spfce)
40
ND
30.1
75
0 .2 5 9
4 1 .7
104
NR
NR NR NR NR
D B C -S D -LO C 002-0-041202 (C0053120 Spk G. 4 ppb Spike)
D B C -S D -LO C 002-0-041202 (C0053120 Spk H, 40 ppb Spike)
4 40
ND ND
2 .9 5
74
0 .2 5 8
4.31
101
28.1
70
0 .2 5 8
4 0 .0
99
NR NR
NR NR NR NR
D B C -S D -LO C O O 3 -0 -0 4 1 2 0 2 (C0063121 Spk 1.4 ppb Spike)
4
ND
2.61
65
ND
2 .9 0
73
NR
D B C -S D -LO C 003-0-041202 (C0053121 Spk J, 40 ppb Spike)
40
ND
2 4 .4
61
ND
3 4 .5
86
NR
NR NR NR NR
DO U-SD -LO C O 01-0-041202 (C0053122 Spk K. 4 ppb Spike)
DO U-SD -LO C O 01-0-041202 (C00S3122 Spk L, 40 ppb Spike)
4 40
2 .3 4 2 .3 4
5 .6 8 2 9 .6
84 68
3 .5 2 3 .5 2
6 .5 0 4 1 .6
75 95
NR NR
NR NR NR NR
` Sample residue exceeds the spiking level significantly (3x spiking level): therefore, an accurate recovery value cannot be calculated ND * Not detected at o r above the Lim it o f Q uantitation (LOQ) o f 0.2 ng/g (wet weight). NR * Not reported due to quality control failure For reanalysis see Table IV. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data.
Exygen Research
Page 26 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sediment Samples Continued
S am ple D e scription
Am ount S piked (n g /g )
C 4 S u lfo n ate PFBS W et W eig h t
Am t Found In S am ple
(n g /g )
Am ount
R ecovered R ecovery
(n g /g )
(% )
C 6 S u lfo n ate PFHS W ot W eig h t
Am t Found in S am ple
(n g /g )
Am ount
R ecovered R ecovery
(n g /g )
(% )
C 8 S u lfo n ate PFO S W ot W eig h t
A m t Found In S am ple
(n g /g )
Am ount R ecovered
(n g /g )
R ecovery <%)
D O U -S D -LO C 002-0-041202
(C0053123 Spk C, 4 ppb Spike)
4
4 .6 0
6.91
58
NR
NR NR
NR
D O U -S D -LO C 002-0-041202
(C0053123 Spk 0.40 ppb Spfce)
40
4 .6 0
2 4 .2
49
NR
NR NR
NR
NR NR NR NR
D O U -S D -LO C 003-0-041202 (C0053124 Spk E, 4 ppb Spike)
D O U -S D -LO C 003-0-041202 (C0053124 Spk F, 40 ppb Spike)
4 40
0 .3 5 9 0 .3 5 9
3 .5 7 2 5 .2
80 62
1.90 1.90
6 .8 3 3 2 .9
123 78
NR NR
NR NR NR NR
DL1 -SD-LO CO 01-0-041202 (C0053125 Spk G. 4 ppb Spfce)
D L1-S D -LO C 001-0-041202 (C006312S Spk H. 40 ppb Spfce)
4 40
ND ND
2 .9 4 3 0 .0
74 75
ND
2 .9 6
74
ND
2 8 .2
71
NR NR
NR NR NR NR
D L1-S D -LO C 002-0-041202 (C0053126 Spk 1,4 ppb Spike)
D L 1-S D -LO C 002-0-041202 (COO53120 Spk J, 40 ppb Spike)
4 40
ND ND
3 .0 0 2 8 .8
75 72
ND
3.51
88
ND
3 0 .8
77
NR NR
NR NR NR NR
D L1-S D -LO C 003-0-041202 (C0053127 Spk K, 4 ppb Spike)
D L 1-S D -LO C 003-0-041202 (C0053127 Spk L, 40 ppb Spfce)
4 40
ND ND
3 .0 8 2 9 .6
77 74
ND
4 .1 3
103
NR
ND
3 9 .2
98
NR
NR NR NR NR
DM C -SD-LO C O 01-0-041202
(C00S3128 Spk C. 4 ppb Spike)
4
ND
2 .9 7
74
ND
4 .2 4
106
NR
D M C -S D -LO C 001-0-041202
(C0053128 Spk D, 40 ppb Spfce)
40
ND
2 9 .2
73
ND
4 1 .5
104
NR
NR NR NR NR
D M C -S D -L 0 0 0 0 2 -0 -0 4 1202 (C00S3129 Spk E, 4 ppb Spike)
D M C -S D -LO C 002-0-041202 (C0053129 Spk F, 40 ppb Spfce)
4 40
ND ND
2 .7 6 2 8 .8
69 72
ND
3 .6 5
91
ND
3 9 .2
98
NR NR
NR NR NR NR
D M C -S D -LO C 003-0-041202 (C0063130 Spk G, 4 ppb Spike)
D M C -S D -LO C 003-0-041202 (C0053130 Spk H, 40 ppb Spfce)
4 40
ND ND
2 .7 2 2 7 .2
68 68
ND
3 .8 6
97
ND
3 6 .0
90
NR NR
NR NR NR NR
D A A -S D -LO C 006-0-041202
(C0053131 Spk 1,4 ppb Spike)
4
4 .3 0
6 .8 4
64
NR
NR NR
NR
D A A -S D -LO C 006-0-041202
(C0053131 Spk J. 40 ppb Spike)
40
4 .3 0
31.1
67
NR
NR NR
NR
NR NR NR NR
D A A -S D -LO C 005-0-041202 (00053132 spk K, 4 ppb Spike)
D A A -S D -LO C 005-0-041202 (C0053132 Spk L. 40 ppb Spike)
4 40
NR NR
NR NR NR NR
NR NR
NR NR NR NR
NR NR
NR NR NR NR
Sam ple residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NO * Not detected a t or above the Lim it o f Q uantitation (LOQ) o f 0.2 ng/g (wet weight). NR * Not reported due to quality control failure. For re-analysis see Table IV. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data.
Exygen Research
Page 27 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sediment Samples Continued
S am ple D e scription
Am ount S piked (n g /g )
C 4 S u lfo n a te PFBS W et W e ig h t
A m t Found In S am ple
(n g /g )
Am ount
R ecovered R ecovery
(n g /g )
(% )
C 6 S u lfo n a te PFHS W e t W a lg h t
Am t Found in S am ple
(n g /g )
Am ount
R ecovered R ecovery
(n g /g )
(% )
C 8 S u lfo n a te PFO S W e t W e ig h t
Am t Found Am ount In S am ple R ecovered R ecovery
(n g /g )
(n g /g )
<%>
D A A -S D -LO C 004-0-041202 (C0053133 Spk C. 4 ppb Spike)
D A A -S D -LO C 004-0-041202 (C0053133 Spk D. 4 ppb Spike)
4 40
NR NR
NR NR NR NR
NR NR
NR NR NR NR
NR NR
NR NR NR NR
D A A -S D -LO C O O 3 -0 -0 4 1 2 0 2 (C0053134 Spk E. 4 ppb Spike)
D A A -S D -LO C 003-0-041202 (C0053134 Spk F. 40 ppb Spike)
4 40
NR NR
NR NR NR NR
17.2 17.2
2 0 .3 62.1
* 112
NR NR
NR NR NR NR
D A A -S D -LO C 002-0-041202 (C0053135 Spk 6 .4 ppb Spfce)
D A A -S D -LO C 002-0-041202 (C00S3135 Spk H, 40 ppb Spike)
4 40
NR NR
NR NR 0.373 4.91 113 21.1 NR NR 0.373 43.9 109 21.1
2 3 .3 4 7 .3
* 66
D A A -S D -LO C 001-0-041202
(C0053136 Spk 1,4 ppb Spike)
4
ND
3 .2 8
82 0.203 2.61
60
D A A-SD -LO C 001-0-041202
(C0053136 Spk J, 40 ppb Spike)
40
ND
2 9 .6
74
0 .2 0 3
2 6 .4
65
NR NR
NR NR NR NR
A ve ra g e : S ta n d a rd D e v ia tio n :
71 8
A ve ra g e : S ta n d a rd D e v ia tio n :
95 15
A ve ra g e : S ta n d a rd D e v ia tio n :
` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND > Not detected a t or above the Lim it o f Q uantitation (LOQ) o f 0.2 ng/g (wet weight). NR * Not reported due to quality control b ilu re . For re-analysis see Table IV. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data.
70 8
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Page 28 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Table IV Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Sediment Samples
Sam ple D escription
C4 S ulfonate PFBS W et W eight
Am ount Am t Found
Spiked in Sam ple
(n g /g )
(n g /g )
Am ount
Recovered Recovery
(n g /g )
(%>
C 6 S ulfonate PFHS W et W eight
Am t Found in Sam ple
(n g /g )
Am ount
R ecovered R ecovery
(n g /g )
(% )
C 8 S u lfonate PFOS W et W eight
Am t Found in Sam ple
Am ount R e co ve re d
R e covery
(n g /g )
(n g /g )
(% )
D L3-SD -LO C 003-0-041201 (COO53115 Spk 1,4 ppb Spike)
DL3-SD-LO C003-0-041201 (C00S3115 Spk J. 40 ppb Spike)
4 40
-
--
-
ND 3.73 93 - - ND 32.2 81
DL2-SD-LO C001-0-041201 (C0053116 Spk K, 4 ppb Spike)
DL2-SD-LOCOO1-0-041201 (C0053116 Spk L, 40 ppb Spike)
4 40
-
.
--
-
. ND 3.64 96
ND
39.3
98
DL2-SD-LO C002-0-041201 (C0053117 Spk M, 4 ppb Spite)
DL2-S D-LO C 002-0-041201 (C0Q53117 Spk N, 40 ppb Spfce)
4 40
-
.
--
-
.
ND
4.14
104
- - ND 34.1 85
DL2-S D -LO C 003-0-041201 (C00531 I t Spk E, 4 ppb Spite)
DL2-SD-LO C003-0-041201 (C005311I Spk F, 40 ppb Spike)
4 40
-
.
--
-
. ND 3.67 92 - - ND 33.2 83
DBC-SD-LOC001 -0-041202 (C005311I Spk 6 ,2 0 ppb Spfce)
DBC-SD-LO C001-0-041202 (C0053119 Spk H, 200 ppb Spike)
20 200
-
.
-
-
.
30.9
4 2 .3
57
- 30.9 204 87
D B C -S D -LO C 002-0-041202 (C0053120 Spk 1,20 ppb Spite)
DBC-SD-LO C002-0-041202 (C0053120 Spk J. 200 ppb Spite)
20 200
-
.
--
-
.
15.5
4 3 .9
142
- - 15.5 198 91
DBC-SD-LO C003-0-041202 (COO53121 Spk K, 20 ppb Spike)
D BC-SD -LO C003-0-041202 (C0053121 Spk L, 200 ppb Spike)
20 200
-
.
-
. 14.3 42.1 139
-
- 14.3 212
99
DO U-SD-LO C001-0-041202 (C0053122 Spk M , 400 ppb Spite)
DO U-SD-LO C001-0-041202 (C00S3122 Spk N, 4000 ppb Spite)
400 4000
-
,,
--
-
. 310 684 94 - - 310 3720 85
ND * Not detected at or above the Lim it of Quantitation (LOQ) o f 0.2 ng/g (wet weight}. N ote: S in e * th is sum m ary tab le show s rounded results, recovery values m ay vary s lig h tly from the values In the raw data.
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Exygen Study No.: P0001131
Table IV Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Sediment Samples Continued
Sam ple D escriotion
Am ount S p ike d (n tffl)
C 4 S ulfonate PFBS W et W eight
Am t Found Am ount
in Sam ple Recovered Recovery
(n g /fl)
(n g /g )
(% )
C 6 S ulfonate PFHS W et W eight
Am t Found Am ount
in Sam ple R ecovered R ecovery
(n o /fl)
(n g /g )
(%>
C8 S u lfonate PFOS W et W eight
A m t Found in Sam ple
(n g /g )
Am ount R ecovered
(n g /g )
R e covery (% )
DO U-SD-LOC002-0-041202 (COO5)123 Spk E. 4 ppb Spike)
D O U-SD -LO C 002-0-041202 (C0053123 Spk F, 500 ppb Spike)
D O U-SD -LO C 002-0-041202 (C005312) Spk 6,4 0 00 ppb Spike)
4 800 4000
. -
11.2 708 87
150
737
73
-
-
11.2
3920
98
150
3810
92
DO U-SD -LO C 003-0-041202 (C0053124 Spk 0 .4000 ppb Spike)
D O U -SD -LO C 003-0-041202 (C0053124 Spk H, 40000 ppb Spike)
4000 40000
_
-
--
.
-
257 3800 89 - - 257 40900 102
DL1-SD-LO C001-0-041202 (C0053125 Spk 1.4 ppb Spike)
DL1-SD-LO C001-0-0 41202 (C0053125 Spk J, 40 ppb Spike)
4 40
-
.
--
. 3.93 7.17 81
-
-
3.93
41.7
94
D L1-S D -LO C 002-0-041202 (C005S120 Spk K, 4 ppb Spike)
D L1-S D -LO C 002-0-041202 (C0053124 Spk L. 40 ppb Spike)
4 40
-
.
--
. -
3.42
6 .1 3
68
-
-
3.42
4 1 .2
94
DL1-SD-LO C003-0-041202 (C005S127 Spk M, 4 ppb Spike)
D L1-S D -L00003-0-041202 (C00S3127 Spk N, 40 ppb Spike)
4 40
-
--
-
4 .0 6
8.04
100
-
-
4 .0 6
41.5
94
DM C-SO-LOC001-0-041202 (C0053120 Spk E, 4 ppb Spfte)
D M C -S D -LO C O 01-0 -0 4 1 2 0 2 (C005312S Spk F, 40 ppb Spike)
4 40
-
. --
-
2 .0 6
6 .1 0
101
- 2.06 37.4 88
D M C -SD-LO C 002-0-041202 (C00S3120 Spk 0 ,4 ppb Spike)
D M C -SD-LO C 002-0-041202 (C0053129 Spk H. 40 ppb Spike)
4 40
-
2 .5 3
5.82
82
-
-
-
- 2.53 39.1
91
D M C -SD-LO C 003-0-041202 (COO531)0 Spk 1.4 ppb Spike)
DM C -SD-LO C 003-0-041202 (C0053130 Spk J. 40 ppb Spike)
DAA-SD -LO C006-0-041202 (C0053131 Spk K, too ppb Spike)
DAA-SO -LO C 006-0-041202 (C00S3131 Spk L, 4000 ppb Spike)
4 40 800 4000
-
. . 2.61 6.18 89
- - 2.61 40.5 95
.
43.8 809 96
914
2580
208
-
-
4 3 .8
3660
90
914
5200
107
D AA-SD -LO C005-0-041202 (C0053132 Spk N, 40 ppb Spike)
D AA-SD -LO C005-0-041202 (C0053132 Spk 0 ,4 0 0 ppb Spike)
DAA-SD-LOCOO5-0-041202 (C00S3132 Spk P, 4000 ppb Spike)
40 400 4000
12.9 12.9 12.9
33.4 322 3570
51 77 89
2 9 .8 2 9 .8
,, 464 3800
. 109 94
. 447 447
. 620 3970
. 43 88
ND = Not detected at or above the Lim it of Quantitation (LOQ) o f 0.2 ng/g (wet weight). Note: S ince th is sum m ary tab le show s rounded resu lts, recovery values m ay vary s lig h tly from the values In the raw data.
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Table IV Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Sediment Samples Continued
Sam ple D escription
Am ount Spiked (nglg )
C4 Sulfonate PFBS W ot W ig h t
Am t Found Am ount
in Sam ple Recovered R ecovery
(n g /g )
(n g /g )
<%)
C6 S ulfonate PFHS W et W eight
Am t Found Am ount
in Sam ple R ecovered R ecovery
(n g /g )
(n g /g )
(% )
C 8 S u lfonate PFOS W et W eight
Am t Found in Sam ple
Am ount R e co ve re d
R e covery
(n g /g )
(n g /g )
(% i
D AA-SD -LO C004-0-041202 (C00S3133 Spk E, 4 ppb Spike)
4
10.7
13.9
80
D AA-SD -LO C004-0-041202
(COO53133 Spk F, 400 ppb Spike)
400
10.7
301
73
115 491 94
767
1260
123
D AA-SD -LO C004-0-041202
(C0033133 Spk G, 4000 ppb Spike)
4000
10.7
3130
78
115
3640
88
767
4220
86
DAA-SD -LO C003-0-041202 (C0053134 Spk G, 4 ppb Spike)
DAA-SD -LO C003-0-041202 (C0053134 Spk H. 40 ppb Spike)
D A A -S D -LO C 003-0-0412Q 2 (C0063134 Spk L 400 ppb Spike)
DAA-SD-LO C003-0-C41202 (C0053134 Spk J. 4000 ppb Spike)
4 40 400 4000
ND NO ND ND
5.37 31.7 313 3280
134 79 78 82
-
. ..
. ..
. 640 990 88 - - 640 4120 87
D AA-SD -LO C002-0-041202
(C0053135 spk 1,4 ppb Spike)
4
ND
3.47
87
--
-*
DAA-SD-LOC001 -0-041202 (C0053131 Spk K, 20 ppb Spike)
DAA-SD-LOC001 -0-041202 (C0053134 Spk L, 200 ppb SpBte)
20 200
-
..
--
-
.
2 3 .3
4 9 .3
130
- - 23.3 192 84
A verage: Standard D eviation:
83 20
A verage: Standard D eviation:
94 7
ND * Not detected at or above the Lim it of Quantitation (LOQ) o f 0.2 ng/g (wet weight). Note: S ince th is sum m ary tab le show s rounded resu lts, recovery values m ay vary s lig h tly from th e values In the raw data.
A verage: Standard D eviation:
95 25
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Exygen Study No.: P0001131
Table V Total Percent Solids in Sediment Samples
Exygen ID C0053113 C0053114 C0053115 C0053116 C0053117 C 0053118 C0053119 C0053120 C0053121 C0053122 C0053123 C0053124 C0053125 C0053126 C0053127 C0053128 C0053129 C0053130 C0053131 C0053132 C0053133 C0053134 C0053135 C 0 0 5 3 1 36
Client Sample ID DL3-SD-LOC001-0-041201 D L 3 -S D -L O C 0 0 2 -0 -0 4 1 201 D L 3 -S D -L O C 0 0 3 -0 -0 4 1 201 DL2-SD-LOC001 -0-041201 D L 2 -S D -L O C 0 0 2 -0 -0 4 1 201 D L 2 -S D -L O C 0 0 3 -0 -0 4 1201 DBC-SD-LOC001 -0-041202 DBC-SD-LOC002-0-041202 DBC-SD-LOC003-0-041202 DOU-SD-LOC001 -0-041202 DOU-SD-LOC002-0-041202 DOU-SD-LOC003-0-041202 D U -SD-LOC001-0-041202 DL1-SD-LOC002-0-041202 DL1-SD-LOC003-0-041202 DMC-SD-LOC001-0-041202 DMC-SD-LOC002-0-041202 DMC-SD-LOC003-0-041202 DAA-SD-LOC006-0-041202 DAA-SD-LOC005-0-041202 DAA-SD-LOC004-0-041202 DAA-SD-LOC003-0-041202 DAA-SD-LOC002-0-041202 DAA-SD-LOC001 -0-041202
Total Percent Solids (%) 59.43 39.60 41.28 42.03 45.53 40.07 42.34 41.94 42.30 62.01 69.77 65.82 40.98 44.06 43.36 58.71 39.48 37.60 52.09 72.52 16.23 31.80 56.03 60.49
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Exygen Study No.: P0001131
FIGURES
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Exygen Study No. : P0001131
Figure 1. Typical Calibration Curve for PFBS in Methanol
0524050 Sediment Revl.idb (PFBS): "Lineai11Regression (M /xT weighting): y 3.6e+004x + 1.25e+003 ( r * 0.0000)
Area, counts
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Exygen Study No.: P0001131
Figure 2. Non-Extracted Standards of PFBS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively
SS7134 - P f f lS (Standard) 299.V99.0amu -aam ph 1 o f 42 from 052405O.wifT A n a : 9342 count* Height: $29, cps RT: 0.591 mi*
Intensity, cps
Intensity, cps
Area: 10727 oounts Height: 1100. ops RT: 0.600 min 0.57
1000 800 000 400
200 2.02
0
8 0 10 Time, min
17
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Exygen Study No.: P0001131
Figure 3. PFBS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively
I f l u jH f Control - PFBS (Unknown) 299. 0/93.Qama -tnm plt t o t 41from 0524Q5D.wiff A na: 9tte o a a ta H tg it 2 a Sep* KT: .Sttm ia
iL--
| Reagent Spk A- PFBS (00)200.0/00.0 amu -sample 0 of42 from 052405D.wiff Arta: 13401 count! Height: 544.opt RT: 0.605 min
0.01
Tima, min
I Rtagtnt SpkB- PFBS (QC)200.0/00.0 amu* sample 10 of4 2 from 052405D.twiff Arta: 121074 eounts Height 4790.ops RT: 0.584 min
u>
c
12
5 T0 7
8 ' 0 ' 10 11 ' 12 ' 13 14 19 10 7 Time, min
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Exygen Study No.: P0001131
Figure 4. PFBS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B by Solvent Extraction, Respectively
Roagomt Comtrot PFBS (Btmmk) 29SL*99.0 amm sam pia 9 o f 35 from 05190D.wiff fra a * mot found)
12-28
13.46 A -13.76
Tim, min
I R agntS pkA - PFBS (QC)2QQ.Q/Q0.0 amu sampl 0 of 36 from Q61006D.wiff Area: 6770 counts Haight: 3.34+002 ops RT: 0.627 min
to 300-I j ao .
? 200- I I c a>
"L
1.46
i
6.73
Tim, min
I Ragnt Spk B PFBS (00)200.0/0 0.0 amu sampl 10 of 36 from 0610060.miff Ara: 60108 counts H eight 3.80+O03 ops RT: 0.613 min
0.61
11.01 12.81 ^13.42 - ri ^ n
17.73^.
12 13 14 15 16 17
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Exygen Study No.: P0001131
Figure 5. Chromatogram Representing a Sediment Sample Analyzed for PFBS (Exygen ID: C0053119, Data Set:052405D)
0*33119 - PFBS (Uakaoam) 29a.09S.tMmi/ .ta m p ia 34o f 42 from 0S24SlXwiff A n a : 3 *4 3 c o a a tt H a ig tt *914 c p * RT: a 3 4 * M iff 0.04
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Exygen Study No.: P0001131
Figure 6. Chromatogram Representing a Sediment Sample Analyzed for PFBS by Solvent Extraction, (Exygen ID: C0053132, Data Set:051906D)
CM S3T32 PFBS (U ataow a) 29t.W 9i.tam M -aam pla I t o f 35 from ts n ttD M ttf A n a : f9 M c o ifirte H a ig tt: 5.55+001cpt R7: 293m ia
Intensity, ops
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Exygen Study No.: P0001131
Figure 7. Typical Calibration Curve for PFHS in Methanol
0 3 2 4 0 5 D S e d im e n t R e v l.rd b (PFH S): " Lineai" Regression ('1 / x" w eighting): y = 5 .7 9 e + 0 0 4 x + 2 .0 6 e + 0 0 3 (r = 0 .9 9 9 5 )
Area, counts
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Figure 8. Non-Extracted Standards of PFHS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively
SS7134 - PFHS (Standsrd ) 399 .0*010 w wir -sam p/s 1 o f 42 from 0S2495Oiwiff A n a : 12319 coaat* H sig S t 437. cps RT: 9.23 m in 0.23
Intensity, cps
387133 PFHS (Standard) 3 0 0 .0 *0 .0 im u sam plt 2 of 42 from 052405D.wiff Area: 32229 oounti H oight 1010. ops RT: 0.26 min
Intensity, cps
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Exygen Study No. : P0001131
Figure 9. PFHS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively
(LMD *)393. t R *g t*t C ontrol PFHS
kow
0 /M .am * -* *m p lt o f 42 from M 24M D .w itf
tro m t mot fommd)
0.51
f 20'
100-k- AA-/V ,in^wic . 1.60^2.1 2 3 1 3 4.17 f i n 0.63
12.Q1
10^16.08
I n A rw y ., n
1 2 3 4 0 e 7 e 0 10 11 12 13 14 19 16 17
Tim, min
Ragnt Spk A - PFHS (QC) 300.0/80.0 im u sample 0 of 42 from 062406D.wifl Arj : 27837 counts Height: 1100. ops RT:8.23 min
Tim, min
Reagent Spk B PFHS (QC) 3Q8.0/B0.0 amu - sample 10 of 42 from 002406 Djulff Area: 271862 oounts H eight 10600. ops RT: 0.26 min
025 (oo0. 1XM)4-
2?
'55 5000.00 a> c
0.00J-- r 1 2 3 4 6 6 7 8 0 10 11 12 13 14 19 16 17 Tim, min
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Exygen Study No.: P0001131
Figure 10. PFHS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B by Solvent Extraction, Respectively
I A i f f M t CoMtrof PFHS Hamk) 399L0/8OL# ama -ta rn p it $ o f 35 from iS fM O lL w ff met foM ird)
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Exygen Study No. : P0001131
Figure 11. Chromatogram Representing a Sediment Sample Analyzed for PFHS (Exygen ID: C0053119, Data Set: 052405D)
- PFHS (Uakaowm) ^SLO/M lO ama *a m p i* 34 o f 42 from 0S 2^S fllm Y f A n a : 17H 7 counts H aight: 757. cp* R 7 :9.2g m ia
Intensity, cps
T im t, min
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Figure 12. Chromatogram Representing a Sediment Sample Analyzed for PFHS by Solvent Extraction, (Exygen ID: C0053132, Data Set: 051906D)
(U*** )C9953132 - PFHS
owm 39910*0, 9 amn -aam pta 2$ o f 35 from tSfMfiOLwOT
Aroa: 954 3count* H a ig h t 479+092cp # RT: U lm in
460
Intensity, cps
150
100
7 8 0 10 11 12 13 14 16 16 17 Time, mir
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Figure 13. Typical Calibration Curve for PFOS in Methanol
0 5 2 4 0 5 D S e d im e n t R e v l.rd b (P FO S ): "Lineai" Regression (" 1 / * ' w eighting): y = 6 .2 0 e + 0 0 4 x + 9 .8 7 e + 0 0 4 (r = 0 .9 0 8 6 )
Area, counts
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Exygen Study No.: P0001131
Figure 14. Non-Extracted Standards of PFOS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively
I SS7134 - PFOS (Standard) 499. OTA $ ama f t w p /i 1 o f 42 from 9S24$5D.wrff A r t : t f 2 5 M coant* H aigkt: m i cps RT: 11.7 m in
11.00
Intensity, cps
1 2 3 4 6 6 7 8 0 10 11 12 13 14 10 10 17 Tim *, min
887 133* PFOS (Standard) 400.0/80JD amu sample 2 of 42 from D524O0[>.iff Aroa: 133740 oounts Haight: 1000. ops RT: 11.0 min
11.05
Intensity, cps
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Exygen Study No.: P0001131
Figure 15. PFOS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively
Rongont C ontrol - PFOS (Unknown) 499. $ am * -sam pfo 9 o f 42 from 9S24M ikm ff A n a : 95399 count H aight: 791. cp* RT: 12.3 m in
T im t, min
I R ta g tn t Spk A PFOS (QC) 409.0/80.0 amu sam plt 0 of 42 from 052405D.wiff A rta : 110752 oounts H tig h t 1780. cps RT: 11.7 min
11.09
T im t, min
I R ta g tn t Spk 9 PFOS (QC)4800/80.0 amu sam plt 10 of 42 from 052405D.wiff A rta : 327528 counts H tig h t1 1 8 0 0 .e p s RT: 11.7 min
11.57
T im t, min
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Exygen Study No.: P0001131
Figure 16. PFOS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B by Solvent Extraction, Respectively
f e w * C ontrol - PFOS (B lank) 4991*90.1 amn -sam ple 9 o f 35 from $S19$D.wiff p n a k n o t found)
1477
Intensity, cps
Area: 0878 oourts Height: 3.51e+002 ops RT: 11.2 min
11.24
Intensity, cps
Area: 70041 oountf H e ight 34e+003 ops RT: 11.2 min
11.21
Intensity, cps
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Exygen Study No.: P0001131
Figure 17. Chromatogram Representing a Sediment Sample Analyzed for PFOS (Exygen ID: C0053119, Data Set:052405D)
Intensity, cps
(U O )C # 0 5 3 ff9 - PFOS b bowb 499KOT0L0 f a i -ta m p l* 34 o f 42 from 052405D.witf
A r t : 533403 c o a irt* HoigkV 25200. cp* RT: 11.7 m in
2.4*4
22*4
2.0*4 1.8*4 1.0*41.4*4 1.2*4 1.0*4* 8 0 0 0 .0
0000.0
4000.0 2000.0 *
10 11 12 13 14 15 10 17 Tim *, min
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Exygen Study No.: P0001131
Figure 18. Chromatogram Representing a Sediment Sample Analyzed for PFOS by Solvent Extraction, (Exygen ID: C0053132, Data Set:051906D)
I C0033T32 - PFOS (U n te o w i) 49910*0. m s -sam ple 26 o f 3S from t5 n 0 6 D .w iff A n a : 753 *0 coasts H eight: 4 2 9 .+03 cps RT: 11.2 m ia
11.22
Intensity, cps
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APPENDIX A
Study Protocol P0001131
(Exygen Study No. P0001131) with Analytical Method,
Protocol Amendments 1,2, and 8, and Protocol Deviation 1
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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
Perform ing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039
Sponsor R epresentative: M ichael A . Santoro D irector o f R egulatory A ffairs 3M Building 0236-01-B-10 St. Paul, M N 55144 Phone: (651) 733-6374
Page I oj 65
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Exygen Protocol Number: POOO1131
DISTRIBUTION:
1) Jaisim ha Kesari, Study Director, W eston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit
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PROTOCOL APPROVAL
Study T itle: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small M ammal Livers and Small M ammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program
Exygen Protocol Number: P0001131
APPROVALS
Jaisimhaucesan, Study Director Weston Solutions
a // /L i/
J o h n M. Flaherty, Principal Investigator / Exygen Research
. / R i c h a r d A. Gi Exygen Resi
ident, Facility Management
2-
L y d Shaffer, Technij Exygen Research
z
Quality Assurance Unit
Date
Date
/ fa l )/
Date
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TABLE OF CONTENTS
TITLE P A G E ..................................................................................................................................................................... 1 DISTRIBUTION............................................................................................................................................................... 2 PROTOCOL APPROVAL.............................................................................................................................................. 3 TABLE OF CONTENTS................................................................................................................................................ 4 IN T R O D U C T IO N ............................................................................................................................................................. 5 TEST M ATERIA LS........................................................................................................................................................ 5 OBJECTIV E......................................................................................................................................................................6 TESTING FACILITY--................................................................................................................................................... 6 STUDY DIRECTOR.........................................................................................................................................................7 SPONSOR REPRESENTATIVE....................................................................................................................................7 PRINCIPAL INVESTIGATOR......................................................................................................................................7 PROPOSED EXPERIMENTAL START AND TERMINATION D A TE S............................................................7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SY ST EM ................................................................ S SAMPLE PROCUREMENT, RECEIPT AND R ETEN TION ..................................................................................8 SAMPLE IDENTIFICATION........................................................................................................................................9 ANALYTICAL PROCEDURE SUMMARY...............................................................................................................9 VERIFICATION OF ANALYTICAL PROCEDURE................................................................................................ 9 METHOD FOR CONTROL OF B U S ..........................................................................................................................11 STATISTICAL M ETHODS............................................................................................................................................11 GLP STA TEM EN T.......................................................................................................................................................... 11 R EPO R T............................................................................................................................................................................. 11 SAFETY AND H EA LTH ................................................................................................................................................ 12 AMENDMENTS TO PROTO CO L................................................................................................................................13 DATA RECORD K EE PIN G ...........................................................................................................................................13 QUALITY ASSU RA N CE............................................................................................................................................... 14 RETENTION OF DATA AND A RCHIVING.............................................................................................................14 APPENDIX I, ANALYTICAL M ETHODS................................................................................................................. IS
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INTRODUCTION
The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum using LC/MS/MS for the 3M Decatur Monitoring Program.
The study will be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit o f Exygen Research.
TEST MATERIALS
The test materials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M.
PFBS Chemical Name: Perfluorobutanesulfonate
M olecular W eight: 338 supplied as the potassium salt (C4FS03"K+)
Lot Number: 101 Purity: 96.7% Transitions Monitored: 2 9 9 -+ 9 9 Structure:
PFHS
Chemical Name: Perfluorohexanesulfonate
M olecular Weight: 438 supplied as the potassium salt (C&uSOflC)
Lot Number: SE036 Purity: 98.6%
Transitions M onitored: 399 -- 80
Structure:
FFF
FF F
S03
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PFOS Chemical Name: Perfluorooctanesulfonate M olecular Weight: 538 supplied as the potassium salt (CsFnSOj'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 M onitoring Program using the current versions o f the following Exygen analytical methods:
V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS"
V 0001781: "M ethod o f Analysis for the D eterm ination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS"
V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS"
V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS"
V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS"
V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS"
V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS"
TESTING FACILITY
Exygen Research 3058 Research Drive State College, P A 16801 Phone:(814)272-1039
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STUDY DIRECTOR
Jaisimha Kesari P.E., DEE W eston Solutions, Inc. 1400 Weston Way W est 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.
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IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM
The following are the test systems for this study: Water (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum
The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study.
The test systems were chosen to access the environmental impact o f PFBS, PFHS and PFOS in the Decatur, Alabama area.
SAMPLE PROCUREMENT, RECEIPT AND RETENTION
Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples will be received at Exygen directly from Weston Solutions. The details o f sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 W ork 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 m atrix will be documented in the final report associated with this study.
Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at 2C-8C. Fish, clam, vegetation and small mammal liver samples will be processed according to the appropriate analytical method (see Appendix I). These samples will be stored frozen at S -10C. Small mammal w h o le b lo o d samples will be centrifuged in the field at the time o f collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at S -10C.
The receipt and processing o f the samples will be documented in the final report and raw data associated with the study.
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^ SAMPLE IDENTIFICATION
Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number.
Sample storage conditions and locations will be documented throughout the study.
ANALYTICAL PROCEDURE SUMMARY
References: V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in W ater by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for die Determination o f Perfluorooctanoic
Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for die Determination o f Perfluorooctanoic
Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in Small Mammal Serum by LC/MS/MS"
The above methods use analytical conditions capable o f separating the isomers o f PFBS, PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found.
VERIFICATION OF ANALYTICAL PROCEDURE
A laboratory control sample will be used for the preparation o f fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette.
For water sampling, Exygen will supply one bottle per sample collected. The bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample
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collection at the testing facility and have been shown to be free o f PFBS, PFHS and PFOS. Samples w ill be added to each container to a volum etric fill line at 200 mL. A field duplicate, a low field spike and a high field spike o f each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (13C PFOA). PFO A and l3C PFO A are included in the solutions used to spike the samples. The results for PFOA and 13C PFOA will not be reported in this study. Exygen w ill supply one field blank (control water) and two field blank spikes (control w ater fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias.
For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containers/bags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration w ith PFBS, PFHS and PFOS at
^ both a low and high level and processed through the described procedure to
determine method accuracy and to check for bias.
For small mammal liver, Exygen will supply a 50 m L polypropylene centrifuge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias.
Low and high spiking levels for each matrix are defined below:
M atrix
Low Spiking Level
High Spiking Level
W ater
500ng/L
5000 ng/L
Soil
4ng/g
40ng/g
Sediment
4ng/g
40ng/g
Fish
lO ng/g
lOOng/g
C lam s
10ng/g
lOOng/g
Vegetation
10ng/g
100 ng/g
Small Mammal Liver
10ng/g
lOOng/g
Small Mammal Serum
lO ng/m L
lOOng/mL
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Recoveries are anticipated to be between 70% and 130% o f the fortified levels; however, the exact precision and accuracy w ill be determined by the analysis o f the quality control samples described above. A statement o f accuracy will be included in the final report.
METHOD FOR CONTROL OF BIAS
Control o f bias will be addressed by taking representative sub-samples from a homogeneous mixture o f each matrix from untreated control samples, and by analyzing at least two levels o f fortifications.
STATISTICAL METHODS
Statistics will be limited to those specified in the subject methods and to the calculation o f average recoveries, as applicable.
GLP STATEMENT
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 will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative.
REPORT
A final report will be prepared by the principal investigator or their designee at the conclusion o f the study. The report will include, but will not be limited to, the following: The name and address o f the Study Director, Sponsor Representative, and
o f the testing facility.
A statement o f GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records).
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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. A ny modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page.
Description o f the instrumentation used and operating conditions.
All results from all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level.
Representative chromatograms for each analyte in each matrix, including chromatograms o f a standard and a control sample, and a chromatogram at a fortification level. The location o f the analyte peaks will be clearly identified in all chromatograms.
All circumstances that may have affected the quality or integrity o f the data will be documented in the report.
Locations where raw data and the final report are to be archived.
Additions or corrections to the final report shall be in the form o f an amendment signed by the Study Director. The amendment shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Representative.
All applicable requirements for reporting o f study results as per 40 CFR 792.185.
SAFETY AND HEALTH
Laboratory personnel will practice good sanitation and health habits.
Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environment to the test or reference substance(s).
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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 m ay 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 will be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative.
DATA RECORD KEEPING
Records to be maintained include the following (as appropriate):
Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description o f any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence Any other documentation necessary for the reconstruction o f the study
Chromatograms- A ll chromatograms w ill contain the following:
Sample identification, injection date, arrow or other indication o f the area o f interest, and injection number corresponding to the run.
Additionally, fortifications will include the amount o f analyte added and the sample number o f the sample that was fortified.
Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL).
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As part o f the documentation the following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions.
QUALITY ASSURANCE
The QA Unit o f Exygen Research will inspect the study at intervals adequate to assure compliance w ith G LP's, and w ill report the findings o f audits to the Study Director, Exygen Management, and the Sponsor Representative.
RETENTION OF DATA AND ARCHIVING
All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived 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 tim e specified in 40 CFR 792.195. A n exact copy o f the materials submitted to the study director will also be kept at Exygen Research.
Exygen will obtain permission from the study director before discarding or returning samples.
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APPENDIX I
ANALYTICAL M ETHODS
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"
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ANALYTICAL METHOD Method Num ber V0001780
Method e fAnalyste lier the Determination o f Perfloerooctaaok Add (PFOA) in Water byLC /M S/M S
Analytical Testing Facility:
Exygen Research 3058 Research Drive State College. PA 16801
Approved By:
Paul Connolly
*
Technical Leader, LC-MS, Exygen Research
iL /dtfC .-------------
/J oJhohnn Flaherty 7 / VV5ic^eeProud!, O pm doiii, Exj^oo Research
Date Dus
Exygen Research
Total Ptga: 7
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Exyya Rtnwch
Mrtbod Number VOOOI7SO
| ANALYTICAL METHOP
I
Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS
1.0 Scope
Thu method ia to be employed for foe iaolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spcctrometric Detector(LC/MS/MS) in water.
2.0 Safety
222.1 Always obeerve safe laboratory practices. Consultthe appropriate MSDS before handling any chemical for proper safety precautions.
3.0 Sample Requirement
3.1 A t least 40 mL o ftestsample for extraction. 3.2 No sample processing is needed for water samples. 3J Samples stored refrigerated should be allowed to equilibrate to room
temperature. 3.4 A ll sanqriesmust be thoroughly mixed before being sampled for extraction 3.3 Any samples containing particles should be centrifuged at -3000 rpm for '5
minutes and foe supernatant used for the extraction. 3.6 Sample collection procedures w ill be specified in the sampling plan for this
project
4.0 Reagents and Standards
4.1 W ater-HPLC grade 4.2 Methanol-HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich
5.0 Instrument and Equipment
5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume 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.
of5.3 Analytical balancecapable raiding to 0.00001 g.
5.4 50 mL disposable polypropylene ccntrifUge tubes. 5.5 15 mL disposable polypropylene oentrifoge tubes. 5.6 Disponble micropipets (50-lOOuL, 100-200uL). 5.7 125>mL LDPE narrow-mouth bottles. 5.8 2 mL dear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettee (100*1000 pL and 10-100 pL), w ith disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tC U SPE cartridges.
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y *"
Exygen Protocol N um ber P0001131
ExygmltM Nreb
Mftbod Number V00017M
ANALYTICAL m e t h o d
Method o fAnalytic for the Determination o fPerfluorooctsnoic Acid (PFOA) in Water by LC/MS/MS
5.12 SPBvacuum manifold. 5.13 Ceotriitige capable o f ipinoing 50 mL polypropylene tubes at 3000 rpm.
6.0 Chromatographic Syrian
6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x SOmm. 5m (P/N: 82S05-052130)
6.2 Temperature: 30*C 6.3 Mobile Phase(A ): 2 mM Ammonium Acetate in Water
Mobile Phase(B ): Methanol Gradient Program:
Tima (mint
0.0 L0 8.0 20.0 22.5
&A 65
65
25
25 65
Flow Rate SkJB fmL/min) 35 0.3
35 0.3
75 0.3 75 0.3 35 0.3
6.6 Injection Volume: 15pL (can be increased to as much aaSOpL). 6.7 Quantitation: PeakArea - external standard calibration curve. 6.8 Run Time: - 23 minutes.
The above conditions are intended at a guide and may be changed in order to optimize the HPLC system.
7.0 MS/MS System
H.7.1 Mode: Elcctroapray Negative MRM mode, monitoring 413 - 369 m
The above conditions are intended aa a guide and may be changed in order to optimize die MSMS system.
8.0 Preparation o fSolutions 8.1 Mobile Phan
8.1.1 2 mM ammonium acetate in water ie prepared by adding 0.154 g of ammonium acetateto 1000 mL o fwater.
Alternate volumes maybe prepared.
PintJul'
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ExyjnRaMtidi
Method NwabefVOOO 1710
ANALYTICAL METHOD
Method o fAnalyiifl for the Determination o fPeriluorooctanoic Acid (PFOA) in Water by LC/MS/MS
9.0 Standard Preparation
9.1 Standard Stock/Fortificatian Solution 9.1.1 Prepare a tock solution o f-100 ny'm L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA ia prepared by bringing 10 mb o fthe 100 jtgftnL solution to a final volume o f 100 with methanol in a 125mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o fPFOA is prepared by bringing Id mL o f tire 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 pgtaL fortification solution o fPFOA i i prepared by bringing 10 mL o fthe 1.0 pgtaL eolution to a final volume o f 100 w ith methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pgfaiL fortification aolution o f PFOA is prepared by bringing 10 mL o f the 0.1 pgfaL solution to a final volume o f 100 with methanol in a 125mL LDPE bottle.
of69.1.6 The Mock tad fortification solutions are to be stored in a refrigerator at approximately 4*C and ere stable for a maximum period months from tire date o fpreparation.
9.2 Standard Calibration Solutions
9.2.1 LC/MS/MS calibration standards are prepared in HPLC water. The caUbntioo standards are processed through the extraction procedure, identical lo samples.
229 The following is a typical example: additional concentrations may be
Final
Concentration Fertificatioo Volumsof Concentration of Calibration
o fFortification Votane Fortified Control Calibration
Standard ID
Solutionfoot) (PL) Samsle(mL) Standard (dm)* (example)
0 0 40
0 XCramddyy-0
10 100 40
25 XCmmddyy'l
to 200 40
50 XCnunddyy2
10 400 40
100 XCmmddyy-3
too too 40
250 XCmmddyy*4
100 200 40
500 XCmraddyy-3
100 400
40
1000 XCmmddw-6
* The extracted concentration o fthe calibration standard is equal to 8x its initial
concentration, dueto the concentration o fthe standard during the extraction (SPE).
XC extracted calibration standard.
Pg* 4 o f '
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Bxygm km areh
Method Number V000I7K)
|
ANALYTICAL METHOD
.........
"]
Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS
9.23 A zero standard eotutioa (reagent blank) mum be prepared with each
set o fatandarda extracted.
9.2.4 Store a ll extracted calibration standards in 15-raL polypropylene tubes at 2*C to 6CC, up to two weeks.
9.2.5 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 Sample Extraction
11.1 Measure 40 mL o fsample or a portion o f sample diluted to 40 mL with water into 50 mL polypropylene centtifiige 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/soc). Do not let column run dry
11.3 Load sample on conditioned Ci i SPE cartridge. Discard eluate. 11.4 Elute with -5 mL 100% methanol Collect 5 mL o f eluate into graduated
15mL polypropylene centrifbge tubes (final volume * SmL). 11.5 Analyze sanlesusing electrospriy LC/MS/MS.
12.0 Cteomatopiphy
12.1 Iqject the same amounto feach standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples.
12.2 Standard! o fPPOA corraponding to at leeet five or more concentration levels must be included fatan analytical set.
12.3 An entire set o f extracted calibration standards must be included si (be bfgitym g and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 sample. As an alternative, an entire set o f extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a seoond set o f extracted standards). In either case, extracted calibration standards mustbe 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
Page 5of7
Page20of65
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ExyfM Ramrch
Method Number V00017U
|
..... ANALYTICAL METHOD
...
|
Acid(PFOA)Method o fAnalysis fo rthe Determination ofPerfluorooctanoic
in Waterby
LC/MS/MS
venui calibration standard concentration using MsssLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed.
13.0 Acceptance Criteria
13.1 Chromatogram must show t 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 lost 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 50 ng/L, then a new blank sample must beobtained and the entire set mustbe reextracted.
133 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 entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by die analyst to determine i f re-extraction is warranted.
13.4 Any calibration stmdard found to be a atatiatical outlier by using the Huge Error Teat, may be excluded from the calculation o f the calibration curve. However, die total number o f extracted calibration standards that could be excluded must not exceed 20% o f die total number o f extracted standards injected.
13.5 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, (hen appropriate slept must be taken to adjust instrument operation, and the standards or the relevant sal o f samples should be reanalyzed.
13.6 Retention times between standards and samples must not d rift more than 4 % within in 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
24.1 Use the following equation to calculate the amount o f PFOA found (in ng/L. based on peak area) using die standard curve (linear regression parameters) generated by the Mass Lynx software program:
PFOA found (ng/L) (Peak area - intercept) x DF slope
DF - factorby which die final volume was diluted, i f necessary.
P i|t 6 of 7
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Exygen Study No.: P0001131
Exygea Protocol N u m be r: P0Q01131
BxysMkMMKb
I
Method NuaberVOOO 1710
ANALYTICALMtTBOD
I
Method o fAnalysis for the Determination o fPeritaorooctenoic Acid (PFOA) in Water by
LC/MS/MS
\A2 For samples fortified with known amounts o f PFOA prior to extraction, use
the following equation to calculate the percent recovery.
Recovery(H)
[totalanalytefound(ng/L) - analyte foundin control(ng/L)] ^
analyteadded (ng/L)
Exygen Research
Ha*7of7
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number; P0001131
ANALYTICAL METHOD Method Number; V0001781
Metbed o f Analyala te r the Determination o fPerfluorvoctanolc A dd (PFOA) to Soil by LC/MS/MS
Analytical Tenting Facility;
Exygen Research 3058 Reaearch Drive State College. PA 18801
Approved By;
c a L _____
Paul Connolly
'
Technical Leader, LC-MS, Exygen Reaearch
a J / n f U / ________
J & n Flaherty '
/ Vice President, Operation, Exygen Research
ioIla/Q1/
Date
Date
Exygen Research
Total Pages: 7 Page 23 o f65
Page 75 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen P rotocol N um ber: P0001131
E iypi t i n t i
Method N u o t a V0001781
ANALYTICAL METHOD
J
Method o fAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by
LC/MS/MS
Thie method it to be 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 so il
2.0 Safety
2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety
precautions.
3.0 Sample Requirement
3.1 A t least 1$ g o ftest sample for extraction. 3.2 No sampleprocessing is needed for soil samples. 3.3 Samples stored refrigerated should be. allowed to equilibrate lo room
temperature. 3.4 A ll samples mustbe thoroughly mixed before being sampled for extraction. 3.3 Sample collection procedures w ill be specified in the sampling plan for this
project.
4.0 Reagents and Standards
41 W ater-HPLC grade 42 Methanol - HPLC grade 43 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Add - Sigma-Aldrich
5.0 Instrumentand Equipment
S.l A high performance liquid chromatograph capitole o f pumping up to 2 solvents equipped with e variable volume injector capable o f injecting 5-200
2 pL connected to atandem Maas Spectrometer(LC/MS/MS).
5 A devioe to collect raw data for peak integration and quantitation. 5J Analytical balnea capable o freading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifUge tubes. 5.5 15 mL disposable polypropylene centrifUge tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-mL LDPB narrow-mouth bottles. 5.8 2 mL clearHPLC vial k it 5.9 Disposable pipettes. 5.10 Autopipette* (100*1000 pL and 10-100 pL), w ith disposable tip*. 5.11 Waters SepPak Vac 6 cc (lg ) tC t8 SPEcartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath.
p*a*2or7
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Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Bayfwi gj w ireh
Method Number V0QQI78I
I ANALYTICAL MITHOP
Method o fAnalytU far the Determiiutioe o fPerfluorooctmoic Acid (PFOA) in Soil by LC/MS/MS
3.14 Wriet-eotioa eheker. 3.13 Cmtrifbge capable o fq>inaing 30 mL polypropylene lu b a a l 5000 ipm.
6.0 Chromatographie Syatem
6.1 Analytical Column: Fhwphase RP (Keystone Scientific), 2 .1mm x SOmm. 5p (P/N: 82305-032130)
6.2 Temperature: 30"C 6.3 Mobile Phaae(A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phaae(B ): Methanol 6.5 GradientPropam:
T im e fm int
0.0 1.0 8.0 20.0 22.S
3LA 63
65 25
25
65
Flow Rate
2LB 35
fm L /m int
0.3
35 0.3
75 0.3
75 0.3
35 0.3
6.6 IqjectionVohaie: 15 pL (can be increaaed to at much as SOpL). 6.7 Quantitation: PeakArea - external standardcalibration curve. 6.8 RunTime: -2 3 minutei.
The above conditions arc intended a* a 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 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 Phaae
8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 znL o fwater.
Alternate volumes may be prepared.
PspJof?
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol N um ber P0001131
f c ty p a Um weh
Method NumberVOOO1711
ANALYTICAL METHOD
Method o fAnalysis for the Determination ofPcrfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS
9.0 Standard Preparation
9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f ~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 jigfaiL fortifieation solution ofPFOA i 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 l.O p^tiri fortification solution o f PFOA is prepared by bringing iu mL o fthe 10 p^m L solution to a final volume o f 100 with methanol in 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution ofPFOA is prepared by bringing 10 m Loffhe 1.0 pgfaL solution to a final volume o f 100 w ith methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/raL 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 methanolin a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be aimed in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o fpreparation.
9.2 Standard Calibration Solutions
9.2.1 9.2.2
LC/MS/MS calibration standards are prepared in HPLC water The calibration standards are processed through the extraction procedure,
identical to samples. H ie following is a typical example: additional concentrations may be
Final
Fortification V otum s o f
Concentration o f
C alib ratio n
ofFortifioatiaB V olum e Fortified Control
C alib ratio n
Standard ID
Solution iootri
(uL)
Sam olefm L)
Standard foot)*
fexunolct
0 0 40 10 100 40 10 20 0 4 0
0 XCmmddyy*0 25 XCm m ddyy-l SO X C m m ddyy-2
10 40 0 100 100
40 40
100 XCm m ddyyO 250 XCnunddyy-4
100 200 100 400
40 40
500 1000
XCmmddyyO XCmmddw*6
* Theextracted oooeeotretiono fthe calibration standard is equal to 8* its initial
concentration,dueto the concentration o fthe standard during the extraction (SPE).
XC extracted calibration standard.
P ag t4 o f7
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Exygen Study No.: P0001131
f* '
Exygen Protocol Number: P000113!
Exypa ItMNfck
MttkodNamber V000178]
I ANALYTICAL METHOD
I
Method o fAnalysis lo r the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS
9.2.3 A sen standard solution (reagent blank) m int be prepared with each seto fstandards extracted.
9.2.4 Store all extracted calibration standard! in 15-mL polypropylene lubes
92.5 at 2*C to 6*C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed.
10.0 Batch SetUp
10.1 Bach batch o f samples extracted (typically 20 o r less) must include at least ooe reagent control (method blank using 5 mL o f methanol) 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 assuranceplan for Ibis project.
11.0 Sample Extraction
11.I Weigh 3 g o f sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well).
1\2 5 5Add mL o fmetbanol andshakeon a wrist action shaker for 1 minutes. 1U Transferthe tubesto an ultrasonic bath and sonicate for 13 minutes.
11.4 Bring the volume up to 40 mL w ith water in the 50 mL polypropylene centrifiige cube.
11.3 Centrifoge for 10 minutes at >3000 rpm. 11.6 Condition the Cts SPB 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 11.7 Load (decant) the sample on the conditioned Cn SPB cartridge. Discard
11.8 Elute with >5 mL 100% methanol. Collect 5 mL o f eiuste into graduated 15 mL polypropylene centrifoge tubes (final volume - 5 mL).
11.9 Analyze samples using electrospray LC/MS/MS.
12.0 Chromatography
12.1 Inject the same amount o f each standard, sample and fortified sample imo the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples.
12.2 Standards o f PFOA corresponding to at least five or more concentration levels mustbe included in an analytical set.
12.3 An entire set o f extracted calibration standards must be included at the beginning and i t (be end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set of
PqtiofT
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
E x y p a lU i iaich
MethodNumber VQQO1791
ANALYTICAL METHOD
Method o fAnilysie for the Determination o fPerftuofooctanoic Acid (PFOA) in Soil by LC/MS/MS
extracted calibration standards may be iqjected at the beginning o f a set followed by extracted calibration standards interspersed every 5*10 samples (to aeoount for a second set o f extracted standards). In either case, extracted calibration standvds 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 f peak area versus calibration standard concentration using MataLynx 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 km (369 amu)represents the loss o fcarbon dioxide.
13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contents PFOA at levels greater than 50 ng/L, then a new blank sample must be obtainad amithe 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 e control spike foils 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 Teat, may be excluded from the 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
13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (RJ 20.985). I f calibration results fo il outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant seto fsamples should be reanalyzed.
13.6 Retention times between standards and samples must not d rift more than 4 % within ananalytical nm. I f retention time d rift exceeds this lim it within an analytical run then the setmust be reanalyzed.
Pat 7
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No. : P0001131
Exygen Protocol Number: P0001131
ExneaftM M Kli
Method NwafcerVOOQlTil
I ANALYTICAL METHOD
I
Method o fAnalysis for the Determination o fPeriluorooctaooic 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 (io ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program:
PFOA found (ng/L) - (Peak area - intercept) x DF slope
DF - factor by which the final volume was diluted, i f necessary.
14.2 For samples fortified w ith known amounts o f PFOA prior to extraction, use foe following equation to calculate the percent recovery.
Recovery(%) "
[totalanalytefound (ng/L) analyte found in control(ng/L)] analyteadded(n ^L )
14J Use the following equation to convert the amount o f PFOA found in ng/L to ng/g(ppb).
PFOA found (ppb) - fPFQA found fnn/L) x volume extracted (0.04L11 sample weight (5 g)
14.4 Uae foe following equation to calculate foe amount o f PFOA found in ppb basedon dry weight.
PFOA found (ppb) dry weight - PFOA found (ppb) x [100% / total solida(%)]
ptg7or7
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ANALYTICAL METHOD Method Number V01782
Method o f A ialysls fo r the Determination o f PerflaoroocUnoic Add (PFOA) in SedlBMOt by LC/MS/MS
Analytical Testing Facility:
Exygen Research 3038 Rcieaich Drive State College, PA 16801
Approved By:
V -A r_jL
Paul Connolly
|
Technical Leader, LC-MS, Exygen Research
a/fl? / ________
r JohnFlaherty Vice Fieeidcat, Operations,Exygen Research
__IkrM
Did
Mr
Due
Exygen Research
TouIPegn: 7 Page 30 o f63
Page 82 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Method Number VOOOI7B2
[ ANALYTICAL METHOD
Method o fAnalysis for die Determination o fPerfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS
Hue method it to be employed for the isolation and quantitation o fperfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in sediment
2.0 Safety 2.1 Always obseive safe laboratorypractices. 2.2 Consult the appropriate MSD5 before handling any chemical for proper safety
3.0 Sample Requirement 3.1 At least 30 g o ftest sample for extraction s ' No sampleprocessing is needed for sediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate 10 room temperature. 3.4 A ll samples must be thoroughly mixed before being sampled for extraction. 3.3 Semple 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 Acetic Add - Reagent grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.3 Perfluorooctanoic Acid * Signu-Aldrich
3.0 Twtwwqtf nil Equipment
5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume tqjector 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 disposablepolypropylsne centrifUge tubes. 5.5 15 mL disposablepolypropylene centrifuge tubes. 5.6 Disposable micropipets (50>100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.S 2 mL clear HPLC vial kit. 5.9 Disposablepipettes. 5.10 Autopipcttcs (100-1000 pL and 10-100 pL), w ith disposable tips. 5.11 Waters Sep Pak Vsc 6 cc (lg ) tCIS SPE cartridges. 5.12 SPEvacuum manifold.
Pegs2of7
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Exygen Study No.: P0001131
Exygen P rotocol N um ber: P0001131
Exygea Rmwch
Metfcod Number VOQOI7I2
ANALYTICAL METHOD
Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS
5.13 Vortexer. 5.14 Wrist-action ahakar. 5.15 CentriAige capable o f spinning 50 mL polypropylene tubes at 3000 rpm.
6.0 Chromatographic System
6.1 Analytical Column: FtuophaseRP (Keystone Scientific), 2.1mm x SOmm. 5m (P/N: 82505-052130)
6.2 Temperature: 30*C 6.3 Mobile Pham (A ) : 2 mM Ammonium Acetate in Water
Mobile Phase(B ): Methanol Gradient Program:
Tuna faun!
0.0 1.0 8.0 20.0 22.5
A 65 65
25 25
65
Flow Rate fm L/m in)
35 0.3 35 0.3 75 0.3 75 0.3 35 0.3
6.6 Injection Volume: 15 *iL (can be increased to aamuch as 50 pL). 6.7 Quantitation: Peak Ares- external standard calibration curve.
6.8 RunTime: -2 3 minutes.
th e above
are intended as a guide and may be changed in order to
optimise the HPLC system.
7.0 MS/MS System
7.1 Mode: ElectrosprayNegative MRM mode, monitoring 413 369 m/z for PFOA.
The aboveconditions are intended asa 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 it prepared by adding 0.154 g of ammonium acetateto 1000 mL o fwater.
PageJ o f7
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Exygen Study No.: P0001131
Exygcn Protocol Number: P0001131
EaygaaR--wwfc
Mttbod Number V0001712
I 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 IS acetic acid in water is prepared by adding 10 mL o f acetic acid to 1000mL o fwater.
Alternate volumes maybeprepared.
9.0 Standard Preparation
9.1 Stndard Stock/Fortification Solution 9.1.1 Prepare a slock solution o f -100 yg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) aod dilute to 100 mL with methanol in a 125*mL LDPE bottle. 9.1.2 A 10 yg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 100 yg/mL solution to a final volume o f 100 w ith methanol in a 125mL LDPE bottle. 9.1.3 A 1.0 yg/mL fortification solution o f PFOA is prepared by bringing 10 mL o fthe 10 yg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 yg/mL fortification solution o fPFOA is prepared by bringing 10 mL o fthe 1.0 yg/mL solution to a final volume o f 100 w ith methanol in a 12J mL LDPE bottle. 9.1.5 A 0.01 yg/mL fortification eohitioii o f PFOA is prepared by bringing 10 mL o f the 0.1 yg/mL solution to e final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 D ie stock sod fortification solutions are to be stored in refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o fpreparation.
9.2 Standard Calibration Solutions
9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution oftbeO .l yg/mL fortification solution.
9.22 The following is a typical example: additional concentrations may be
Otinvulialton
o fFortification Solution fao/mL)
too
100 100
10
S 2
Votums (mL)
10
5 2 10 10 10
Diluted to (mL)
100 100 100 100 100 100
Final Concentration
(ne/mLl
10.0 5.0 2.0
1.0
0.5 0.2
Pag4of7
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Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
E xypaftsm ich
Method Number VOOO17*2
ANALYTICAL METHOD ............ ~l
Method o fAnelyeii for the DeterminationofPerfluorooctanoic Acid (PFOA) in Sediment by
LC/MS/MS
9.2.3 Store a ll calibration standard* in 125-raL LDPE narrow-mouth bottle* at2*Cto6*C.uplosixm onths.
9.2.4 Alternate volumes and concentrations o f standards may be prepared as
10.0 Batch SetUp
10.1 Each batch o f samples extracted (typically 20 or leas) must include st least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verity procedural recovery for the batch.
10.2 Requirements for field and laboratory duplicates and spikes w ilt be specified in die quality assuranceplan for this project
11.0 Sample Extraction
11.1 Weigh 5 g o f sample into 50 mL polypropylene centriftige tubes (fortify as
\%needed, replace lid and mix well).
11.2 Add 35 mL o f acetic acid, cap, vortex and shake on a wrist action shaker fo r-60 minutes.
113 CentiiAige foe tubes at ~3000 rpm for -20 minutes. 1M Condition the C ii SPB cartridges (1 g, 6 mL) by passing 10 mL methanol
followed by 20 mL o fHPLC water(~ 2 drop/iec). Do not let column run dry 113 Load (decant) the sample on the conditioned C u SPE cartridge. Discard
fliie tft. 11.6 Add 20 mL o f m rthinol to the Kdimcot left in the bottom o f the 50 mL
centriftige lube. Cap, vortex and ahalce on a wriat action thaitei for -30 mimrtaa
11.7 Cantri&ge the tubes at-3000 rpm fo r-2 0 minute. 11.8 Decantthe methanol do the sameSPE cartridge. Collect the duate. 11.9 Wash tiie column w ith 4 mL o fmethanoL Collect the eluate and add it to the
eluate collected in step 11.8. 11.10 Condition a second Cu SPE cartridge (1 g, 6 mL) by passing lOm L methanol
followed by 20 mL o fHPLC water( - 2 drop/sec). Do not let column run dry 11.11 Add the methanol to -200 mL o f water and load on the second conditioned
SPE cartridge. 11.12 Elute w ith -S mL 100K methanoL Collect 5 mL o f duate into graduated
15mL polypropylene centriftige tubes (final volume 5 mL). 11.13 Analyse samples using eiectrospray LC/MS/MS.
Exygen Research
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Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
BxyteaR w eeh
Method Number VOOOI712
ANALYTICAL METHOD
~1
Method o fAnalysU for the Detcmunatta o fPerfluorooctanoic Acid (PFOA) in Sediment by
LC/MS/MS
12.0 Chromatography
12.1 Iqject foe etaie amount o feach standard, eample and fortified lantple into the LC/M8/MS system. A calibration standard must precede and follow all analysed samples.
122 Standards ofPFOA corresponding to at least five or more concentration levels
mustbe included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included st the
beginning and at foe end o f s sample set Standards must be interspersed between every 3-10 samples. Ae an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set of standards). In either case, calibration standards must be the first and last injection in s sample set. 12.4 Use linear standard curves for quantitation. lin e a r standard curves arc generated for foe analyte by linearregression using 1/x weighting o f peak area versus calibration standard concentration using MsssLynx 3.3 (or equivalent)
software system. 12.5 Sample response should not exceed standard response*. Any samples that
exceed standard responses should be Anther diluted and reanalyzed.
13.0 Acceptance Criteria
13.1 Chromatogram must show a peak o f s daughter ion at 369 smu from a parent
o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the
daughter ion (369 emu) represents foe loss o fcarbon dioxide.
armtain*13.2 Method m * "* not contain PFOA at levels greater than the LQQ. If s
h itiif
PFOA at levels greater than 0.2 ng/mL, then a new blank
sample must be obtained and the entire set must be re-extracted.
133 Recoveries o f control spikes and matrix spikee must be between 70-130% o f
their known values. I f a control spike foils outside the acceptable lim its, the
entire set o f samples should be re-extracted. Any matrix spike outside 70
130% should be evaluated by the Analyst to determine i f re-extraction is
warranted.
13.4 Any oattbration standard found to be a statistical outlier by using the Huge
Error Test, may be eluded from the calculation o f the calibration curve
However, foe total number o f extracted calibration standards that could be
yxMiuUH n m t oot exceed 20% o f the total number o f extracted standards
injected.
13.5 The correlation coefficient (R) for calibration curves generated must be
20.992 (R1 20.985). I f calibration results fa ll outside these lim its, then
appropriate steps must be taken to adjust instrument operation, end the
standards or foe relevant set o f samples should be reanalyzed.
Pi|e6ef?
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol N um ber P0001131
B o ia lM w c t
VOOOIMehod Nun*ar
712
I ANALYTICAL METHOD
|
Method o fAnalyse Ibr the Determination ofPerfluoroocianoic Add (PPOA) in Sediment by LC/MS/MS
13.6 Retention times between standards and samples must not d rift more than
4%withinan analytical run. 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 Use the following equation to calculate the amount o f PFOA found (in ng/mL. baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program:
PFOA found (ngftnL) - (Peak area intercept) x OF elope
OF factor by which the final volume was diluted, i f necessary.
14.2 For sample fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate foe percent recovery.
Recovery(% )
f total anaJyfsfound(ng/mL) - analytefound in control (ng/mL)] ^ analyteadded(ng/mL)
14.3 Use tha following equation to convert the amounto f PFOA found in ng/mL 10 ng/g(ppb).
PFOA found (ppb)
(5g)1 v o lu m e /S m D l
sample weight
14.4 Uee foe following equation ( if necessary) to calculate the amount o f PFOA found in ppb basedon dry weight.
PFOA found (ppb) dry weight PFOA found (ppb) x (100% / total soltds{%}]
Page 7 of 7
Page36of65
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Page 88 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ANALYTICAL METHOD
Method Num ber V0001783
Method o fAnalysis fa r the Determlsstiea ofPerffoorooctaaok A dd (PPOA) ie Fhh and Cbms by LC/M&/MS
Anetytical Teetiag Facility:
Exygen Research
3058 Research Drive State College, PA 16801
Approved By.
^ C -JlL _______
Paul Connolly
I
Technical Leader, LC-MS, Exygen Research
MWaif
Date
r aFlaherty Vice President, Operations, Exygen Research
Dale
Exygen Research
Total Pages: 8 Page 37 o f 65
Page 89 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0002131
ExygeaRMMfch
M efcedN uabsr VQQQlTtt
| ANALYTICAL m e t h o d
I
Method o f Analysis fix the Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Clarasby LC/MS/MS
1.0 Scope
This method it to be 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 fish and clams.
2.0 Safety
222.1 Always obeerve safe laboratory practices. Consultthe appropriate MSDS before handling any chemical for proper safely precaution!.
3.0 Sample Requirement
3.1 A t least20 g o ftest sample for extraction. 3.2 Samples abould be processed before extraction. Place the frozen sample in a
food proceaaor and homogenize w ith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide ihK*nytu! Seal and place the aamplee in frozen storage until time o f analysis. 3 J Sample collection procedures w ill be specified in foe sampling plan for this project
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 4.3 Silica gel (60-200meah)* Reagentgrade 4.6 Floriail (60-100 mesh)- Reagent grade 4.7 Superclean LC-NHj - Reagent grade 4.8 I-O ctanol- HPLC grade 4.9 L-Aacorbic acid - Reagent grade 4.10 Dimefoytdichlorositaiie - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. ReagentGrade 4.13 Perfluorooctanoic Add - Sigma-Aldrich
3.0 Instrument and Equipment
3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume iiyector capable o f injecting 5*200
52 pL connected to atandem Mass Spectrometer(LC/MS/MS). A device to collect raw data fix peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g.
Pig* : of*
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ExygtalbM arch
M ttbod Number V0001783
| ...........ANALYTICAL METHOD
|
Method o fAnalyst* ibr the Determination o fPerfloorooctanoic A dd (PFOA) in Fish and Clams by LC/MS/MS
3.4 Rotaty evaporator. 3.3 Tiuumizer.
3.6 123mL pear-shaped flasks. 3.7 50 mL disposable polypropylene centriftige tubes. 3.8 13mL dispoeablepolypropylene centriAige tubes. 3.9 Disposable micropipets (50-1OOuL, 100-200uL). 3.10 125-mL U V E narrow-mouth bottle*. 3.11 2 mL clear HPLC vial kit. 3.12 Disposable pipettes. 3.13 Autopipettes (100*1000 pL aod 10-100 pL), w ith dinotatale tips. 3.14 SPE tubes (20mL) (Supelco cat. no. N057177).
3.13 W rist action taker. 3.16 CentrifUgecapableo ffpinmng SOmL polypropylene tubes at 2000 rpm.
6.0 Chromatographic System
6.1 Analytical Column: Fluophaie RP (Keystone Scientific), 2.1 mm x $0 mm. 5m (P/N: 82305-032130)
6.2 Temperature: 30*C 6.3 Mobile Phase(A) : 2 mM Ammonium Acetate in Water
Mobile Phase (B ): Methanol GradientProyam:
Ttwie im hit
0.0
1.0
8.0 20.0
22.5
63 65 25 25
65
Flow Rate fm L/m inl
35 0.3 35 0.3 75 0.3 75 0.3
35 0.3
6.6 Injection Volume: 1SpL (can be increased to as much as SOpL) 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTime: -2 3 minutes.
The shove conditions are intended as t guide and may be changed in order to optimize the HPLC system.
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygm Research
M tfbod Number VQQ0I7SJ
ANALYTICAL METHOD
Method o fAnalysis for the Determination ofPerfiuorooctanoic Acid (PFOA) in Fish and CUms by LC/MS/MS
7.0 MS/MS System
7.1 Mode: Electroepny Negative MRM mode, monitoring 413 -* 369 nvz for PFOA.
H ie aboveconditions are intended asa guide and may he changed in order to optimize the MSM$ 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 ammonium acetate to 1000mL o fwater.
12 Extraction Solutions
8.2.1 2% ascorbic acid in methanol it prepared by dissolving 2 g o f asooibic
22 acid in 100 mL o fmethanol.
8 30% Dimethyidichloroailane in toluene is prepared by bringing 3 mL o fdimcthyldichloroetlane to a final volume o il 0 mL with toluene.
Alternate volumes maybe prepared.
9.0 Standard Preparation 9.! Standard Stock/Foitification Solution
9.1.1 9.1.2 9.1.3 9.1.4 9.1.3
Prepare a stock solution o f ~100 Hg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) aod dilute to 100 mL with methanol in a 125-mL LOPE bottle. A 1.0 pgftaL fortification solution o f PFOA is prepared by bringing 1 mL o f tiie 100 pg/mL solution to a final volume o f 100 with methanol
in a 123 mL LOPE bottle. A0.1 pgfaiL fortification solution o fPFOA is prepared by bringing 10 mL ofths 1.0 ygtaL solution to a final volume o f 100 with methanol in a 125 mLLDPE bottle. A 0.01 pgfaiL fortification solution o f PFOA it prepared by bringing 10 mL o f the 0.1 pgftnL solution to a final volume o f 100 with methanol in a I2S mL LDPE bottle. 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.
Page 4 o fS
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Page 92 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygea Reeearch
Mecbod Number V0001783
ANALYTICAL METHOD
J
Method o fAnalysis for foe Determination o fPerfluorooctanoic Acid (PFOA) in Fish and
Clams by LC/MS/MS
9.2 Standard Calibration Solutions
9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o ffoe 1.0 pgfaL fortification solution.
9.2.2 The following is a typical example: additional concentrations may be
Coooealration o fFortification Volume
Diluted to
Final Concentration
SolutionfuataL) (mL)
(mL)
(mAnL)
1.0 5.0 100 1.0 23 100
1.0 1.0 100 0.05 10 too
0.05 0.025 0.01
0.005
0.025
10
100
0.1 10 100
0.002S
0.001
0.005
10
100
0.0005
9.23 Store a ll calibration standards in 12$-mL LDPE narrow-mouth bottles
at 2*C to 6*C, up to six months.
9.2.4 Alternate volumes and concentrations o f standards may be prepared as
needed.
10.0 Batch Set Up
10.1 Bach batch o f samples extracted (typically 20 or lass) must include at least ooe attested control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for foe batch.
10.2 Requirements for field and laboratory duplicate and spikes w ill be specified in foe quality assurance plan for this project.
11.0 Sample Extraction
11.1 Weigh 5 g o f frozen sample into SO mL polypropylene centrifuge tubes (fortify aa needed, replace Udand mix well).
11.2 Add 30 mL o facetonitrile and shake on a wrist action shaker fo r-15 minutes 113 Place the tubes in a freezer fo r-1 hour. 11.4 Pack and condition the SPB tubes and silanize foe pear-shaped Basks. 11.5 Pack the 20 mL SPE tuba in sequence w ith 2 g flo risil, 2 g silica gel, 2 g
carbon, and l g LC-NHj. Condition the columns with 20 mL o f methanol, then 20 mL o f acetonitrile. Discard a ll washes. Do not allow the column to
dry. 11.6 Silanizs foe 123 mL pear-shaped flasks by rinsing with foe 30%
duncthyldkhkxosilanc in toluene solution. Rime the flask with toluene once, followed by methanol (fo ra tuna). Dry foe flasks completely before use, either by air-drying or w ith a stream o fnitrogen.
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygea Protocol Number: P0001131
Mrthod Number VOOOPB3
ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Fish and
Clama by LC/MS/MS
11.7 CentrifUge tbs 50 mL polypropylene tubes containing sample at -2000 rpm fbr-lO m m utea.
11.8 Decant the extract on to acooditiooed SPE column fitted intide the mouth of die pear-slupod flask. Collect the eluate in die 125 mL silanized pear-shape flask.
11.9 Add 10 mL o f acetonitrile to the sample in the 50 mL centrifuge tube. Homogenize the frozen fkt phase using a tisaumizer for -30 seconds and rinse the liM i" " iM f w ith -10 mL o f acetonitrile into the tube.
11.10 Shake the sample again for ~10 minutes on a wrist-action shaker. 11.11 Plaoethe tubes in a freezer for -1 hourmore. 11.12 CentrifUge the 50 mL polypropylene tubes containing sample at -2000 rpm
fo r-10 minutes. 11.13 Decant the extract onto the same SPE column. Collect the eluate into the
same pear-chaped flask and combine w ith the eluent from the initial extraction. 11.14 Pass 20 mL o f acetonitrile through the SPE column and combine the eluate in the samepoaMhqted flask. 11.15 Add 3*4 drops o f l-octand to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40*0. 11.16 Make the final volume, by adding 2 mL o f 2% ascorbic acid in methanol to die pear-shaped flask end swirl to mix/dissoive. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze samplesusing electrospray LC/MS/MS.
12.0 Chromatography
12.1 Iqject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples.
12.2 Standards o fPFOA corresponding to at least five or more concentration levels mustbe included in an analytical set.
12.3 An entire set o fcalibration standards must be included at the beginning and ai the end o f a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in u 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 concentration using MsssLynx 3.3 (or equivalent) software system.
F t p 6 o f tf
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Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
BxyfmRMMfch
Ms&od Number V0001713
ANALYTICAL METHOD
Method o fAnalyiU fix the Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS
12.3 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Author diluted and reanalyzed.
13.0 Acceptance Criteria
13.1 Chromatogram must show a peak o f a daughter on at 309 amu from a parent
o f 413 amu. The 413 amu parent correspond to the PFOA anion, while the
daughter ion (349 amu) represents the lots o fcarbon dioxide.
13.2 Method blanks must not contain PFOA at levels greater than die LOQ. I f a
*bt*j**Ablank contain! PFOA at levels greater than 0.3 ppb, then a new blank sample
must be
and the entire actmustbe r*extracted.
13.3 Recoveries o f ooutrol spikes and matrix spikes must be between 70*130% o f
their known values. I f a control spike fhlls outside the acceptable lim its, the
entire set o fsamples should beie>extracied.
13.4 Any calibration standard ibuod to be e 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
mustnot exceed 20% o fthe total numbero f standards injected.
13J The correlation coefficient 0 0 for calibration curves generated must be
20.992 (R2 3&9S5). I f calibration results fU l outside these lim its, then
appropriate steps must be taken to adjust instrument operation, and the
standardsor the relevant sato fsamples should be reanalyzed.
13.6 Retention time between standards and samples must not drift more than
4 % within an analytical run. 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 Use the following equation to caloulate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) gensnted by the Mass Lynx software program:
PFOA found (ng/mL) - (Peak area. intercept) slope
14.2 Use the following equation to convert the amount o fPFOA found in ng/mL to ng/g(ppb).
PFOA found (ppb) - fPFOA found fag/mU final volume (mLi x DF1 sample weight (g)
DF factor by which the final volume was diluted, i f necessary.
Pag* 7 of tt
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Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ExytraRMBardi
MethodNumber V00017SJ
| ANALYTICAL .m e t h o d
|
Method o fAnalysis for the Determination o fPerfluorooctsnoic Acid (PFOA) in Fish and Clams by LC/MS/MS
14.3 For tamp1m fortified with known amounts o f PFOA prior to extraction, use foe following equation to calculate the percent recovery.
Recovery (% )
[totalanalytefound(ng/g) analyte found in control(ng/g)| analyte added (ng/g)
Exygen Research
Page Set'S
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Page 96 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ANALYTICAL METHOD Method Number V0001784
Method o f Analysts fo r the Determination o f Perflnorooetanolc Add (PFOA) In Vegetation by LOMS/MS
Analytical Teetisg Facility:
Exygen Research 3038 Research Drive State College. PA 16801
Approved By:
T U X C i Ly___
Paul Connolly
'
Technical Leader, LC-MS, ExygenResearch
n Z /n /Z l/________
JwnFlaherty
' Vice President, Operations, Exygen Research
iq ) M Date Dat
Exygen Research
Total Pages: 7 Page 45 o f65
Page 97 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ExjrfMRMMich
Method Number VQOOI784
ANALYTICAL METHOD
Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS
1.0 Scope
This method ie to be employed for the isolation and quantitation o f perfluorooctanoic add by High Pcrfornunce Liquid Chromatography coupled to a tandem Mass Spoctromctric Detector (LC/MS/MS) in vegetation.
10 Safety
2.1 Always observe safe laboratorypractices. 12 Consult die appropriate MSDS before handling any chemical for proper safety
precautions.
3.0 Sample Requirement
3.1 A t least 20 g o ftest sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a
food processor and homogenize w ith dry ice. Place the camples in containers and leave open in frozen storage overnight to allow for carbon dioxide yuMnrtm Seal and place the samples in frozen storage until time of analysis. 3.3 Sample collection procedures w ill be specified in the sampling plan for this project
4.0 Reagentsand Standards
4.1 Water - HPLC grade 41 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 4.6 Flooril (60-100 mesh) - Reagent grade 4.7 Superclean LC-NHa - Reagent grade 4.S 1-Octanol-HPLC grade 4.9 L-Ascorbic add - Reagent grade 4.10 Dimethyldichloioailane - Reagent grade 4.11 Toluene-Reagent grade 4.12 Ammonium Acetate-A.CS. ReagentGrade 4.13 Perfluorooctanoic Acid - Sigma-AMrich
5.0 Immanent and Equipment
5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume injector capable o f injecting 5-200 pL connected to a tandem Maas Spectrometer(LC/MS/MS).
51 A device to collect raw data for peak integration and quantitation. 51 Analyticalbalance capable o freading to 0.00001 g.
Pe 2 of 7
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Exygen Study No.: P0001131
Exygen Protocol Number: P000I131
ExypnIUsM ich
Method Number V00017M
ANALYTICAL METHOD
Method o fAnalysis for the Determination ofPerfluorooctaooic Acid (PFOA) in Vegetation by LC/MS/MS
5.4 Rotaryevaporator. 5.5 125 mLpear-chaped flask. 5.6 50 mL disposable polypropylene centriibge tubes. 5.7 15 mL disposable polypropylene centrifuge tubes. 5.8 Disposable micropipets (50*1OOuL, 100-2(XXiL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 mL clem HPLC vial k it 5.11 Duposable pipettes. 5.12 Autopipettes (100-1000 pL and 10*100 pL), w ith disposable tips. 5.13 SPEtubes (20mL)(Supelc cat no. N057177).
5.14 Wrist action shaker. 5.15 Centriflige capable o fspinning 50 mL polypropylene tubes at 2000 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 Temperature: 30*C 6.3 Mobile Phase(A ): 2 raM Ammonium AcotUe in Water 6.4 Mobile Phase(B ): Methanol 6.3 GradientProgram:
T iip t ftTM"* 0.0
1.0 8.0 20.0 22.5
2 iA 65 65
25
25 65
Flow Rate
3 (m L/m inl 35 0.3 35 0.3
75 0.3 75 0.3 35 0.3
6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve.
6.8 RunTime: -2 3 minutes.
The above
are intended asa guide and may be changed in order to
optimize the HPLC system.
7.0 MS/MS System
7.1 Mode: Etoctrospray Negative MRM mode, monitoring 413 -369 tn/z for PFOA.
Page 3 o f7
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exy|a)Um K fc
Method Number V0001784
[ ANALYTICAL .METHOD
]
Method o fA n tlysii for the Determination ofPerfluorooctanoic Acid (PFOA) in Vegetation
by LC/MS/MS
The above conditioni are intended aa a guide and maybe changed in order to optimize the MSMS ayatem.
8.0 Preparation o fSohrtiooe 8.1 Mobile Phase
8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetateto 1000 mL o fwater.
8.2 Extraction Solutions
8.2.1 8.2.2
2% aeeoibic acid in methanol ia prepared by dissolving 2 g o f ascorbic
acid in 100 mL ofmethanoL 30K Dimethyidichlofosilane in toluene ia prepared by bringing 3 mL ofdiinediyldichlorosiiane to a final volume o f 10 mL with toluene.
Alternate vohimee may be prepared.
9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution
9.1.1 Prepare a stock solution o f~ l0 0 p g fa L o f PFOA by weighing lOmg 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 I mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125mL LDPE bottle.
9.1.3 A0.1 pg/mL fortification solution o fPFOA it prepared by bringing 10 mL o fdie t.O pg/mL aolution to a final volume o f 100 with methanol in a 125 mL LDPE bottle.
9.1.4 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL aolution to a final volume o f 100 with methanolin a 125 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 date o fpreparation.
9.2 StandardCalibration Solution
9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o ffoe 1.0 pgfaiL fortification solution.
Page4ui
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Bxygen Protocol N um ber P0001131
ExyiM ftM onb
Method Number V00017W
I a t s a l y u c a l iv u l t h o p
l
Method o fAnalysis for the Determination ofPerfluorooctanoic Add (PFOA) in Vegetation
byLC/MS/MS
9.2.2 The following i< t typical example: additional concentrations may be prepared asneeded.
Coocenfcstiao o f Fortification
Volume
Diluted to
Final Concentration
Solution (utfm L) 1.0 1.0 1.0
OnL) 5.0 2.5 1.0
(mL) 100 100 100
0.05 0.025 0.01
0.05 10 100
0.005
0.025
10
100
0.0025
0.1 10 100
0.001
0.005
10
100
0.0005
9.2.3 Store aU calibration standards in 125-mL LDPE narrow-mouth bonles
d 2*C to 6*C, up to six months.
92.4 Alternate volumes and concentrations o f standards may be prepared as
needed.
10.0 Batch SetUp
10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verity procedural recovery for the batch
102 Requirements for field end 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 asneeded, replace lid and mix well).
11.2 Add 30mL o facetonitrile and shake on a wrist action shaker fo r-15 minutes. 11.3 Centrifuge foe 50 mL polypropylene tubes containing sample at 2000 rpm
for ~10 minutes. 11.4 Pack and condition foe SPE tubes and ailanize foe pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in sequence w ith 2 g flo risil, 2 g silica gel, 2 g
carbon, and 1 g LC-NHj. Condition foe 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 Silanizc the 125 mL pear-shaped flasks by rinsing with the 30% dimetbyldichloroailane in toluene solution. Rinse foe flask with toluene once, followed by methanol (three times). Dry the flaiks completely before use, either by air-drying or with astream o fnitrogen. 11.7 Decant foe extract on to a conditioned SPB column fitted inside the mouth of the pear-shaped flask. Collect the eluato in the 125 mL silanized pear-shape
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygsa Research
Method Number V0Q0I784
a > a l y t ic a l m e t h o d
Method o fA n ily iii for the Determination o fPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS
11.8 Add 20 mL o facetonitrile to the um ple in the 50 mL eentrifiige tube. 11.9 Shake die sample again for -10 minute on a wrist-action shaker. 11.10 Centrifltge the 50 mL polypropylene tubes containing sample at -2000 rpm
fo r-S minutes. 11.11 Decant the extract onto the tame SPE column. Collect the cluate into the
nine peanehapod flask and combine w ith the eluent from the initial extraction. 11.12 Repeat steps 11.8 through 11.11 again. 11.13 Add 3-4 drops o f 1-octand to the extract in the pear-shaped flask and
2%evaporate atreduced pressure using arotary evaporator(at < 40C).
11.14 Make die final volume, by adding 2 m L o f ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve.
11.15 Transferthe extracts to HPLC vials using disposable pipetj. 11.16 Analyxe samples using electrospray LC/MS/MS.
12.0 Chromatography
12.1 Inject die same amount ofsedt 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 fPFOA corresponding to at least five or more concentration levels mustbe included in an analytical set.
12.3 An entire set o f extracted calibration standard! must be included at the hayinnlnfl and ai the end o f a sample set Extracted standards must be interspersed between every 3-10 samplee. As an alternative, an entire set or extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 3-10 samples (to account for a second set o f extracted standards), hi either case, extracted calibration standards mustbe 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 concentration using MsssLynx 3.3 (or equivalent) software system.
12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed.
13.0 Acceptance Criteria
13.1 Chromatoyam 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 daughterion {369 amu) represents the loss o f carbon dioxide.
Pig* 6 of 7
Page SOo f6S
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Method N u o t a V000I7I4
[ ANALYTICAL METHOD Method o fAnalytic to the Determination o fPofluorooctanoic Acid (PFOA) in Vegetation byLC/M S/M S
13.2 Method blank* m int not contain PFOA at levels greater than the LOQ. If a blank contain* PFOA at level* greater than 0.5 ppb, then a new blank sample most be obtained and the entire setmust be re-extracted.
13.3 Recoveries o f control spike* and matrix spikes must be between 70*130% of tbor known values. I f a control spike fails outside the acceptable lim its, the entire seto fsamples should be re-extracted.
13.4 Any calibration standard found to bo a statistical outlier by using the Huge Error Teat, may bo excluded flora 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.
13.5 The correlation coefficient (R) to calibration curves generated must be 0.992 (R* 0.985). I f calibration results fa il outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant seto faampleeshould be reanalyzed.
13.6 Retention tim-- between standards and sanqilea must not d rift more than
14 % within an analytical nut. I f retention time d rift exceed* this lim it within
an analytical nin then die setmust be reanalyzed.
14.0 Calculstionl 14.1 Use die following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters! generated by the Maas Lynx software program:
PFOA found (ng/mL) (Peak area - intercept! slope
14.2 Usethe following equation to convertthe amount o fPFOA found in ng/raL to ngfe (ppb)*
PFOA found fppbl - [PFOA found (naftnLl x final volume fm Ll x DF1 ample weight (g)
DP factor by which the final volume was diluted, i f necessary.
14J For ample fortified w ith known amounts o f PFOA prior to extraction, use tile following equation to calculate thepercent recovery.
Recovery (%) -
[ totalanalytefound (ng/g) - analyte found in control (ng/g)] ^ analyte added (ng/g)
p*er >i?
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ANALYTICAL METHOD Method Number V0001785
Method o f Analysis fo r the Dcttrm Uation o f PerfinorooetM olc Add (PFOA) lo Smell Mammal Uver by LOMS/MS
Analytical Testing Facility:
Exygen Retouch 3058 Research Drive State College, PA 16801
Approvod By:
"wlA c _jlL
ExygenPaul Connolly
Technical Luder, LC-MS,
I R auich
qI L.
' Jl6ohnFFlalahhoerty
/ Vicoe Presseident, Operations, Exygen Research
Date Date
Exygen Research
Total Fegu: 7 Page 52 o f 65
Page 104 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: POOOl 131
E iyfM lU aN K k
Melhod Number VQ0017SJ
I ANALYTICAL METHOD
Method o fAnalytic ft* the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS
1.0 Scope
Thie method is to be employed for the eolation and quantitation o fperfluorooctanok 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 aafe laboratory practices. 2.2 Consultthe appropriate MSDS before handling any chemical for proper safety
precautions.
3.0 Sample Requirement
3.1 A t least 5 gofteat sample for extraction. 3.2 Samples should be processed before extraction. Place foe frozen sample in a
food proceiaor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow fix carbon dioxide sublimation. Seal and place the samples in frozen storage until time o f analysis. Alternately, i f there is an insufficient amoimt o f sample Hess than 3 g), then no processing is oeceasary and the sample can be used as supplied. 3J Sample collection procedures w ill be specified in the sampling plan for this project
4.0 Reagents sad Standvds
4.1 W ater-HPLC grade 4.2 Methanol-HPLC grade 4.3 Acetonitrile-HPLC grade 4.4 Ammonium Acetate-A.C.S. Reagent Grade 4.5 Ferfluorooctnoie Acid - Sigma-Aldrich
5.0 Instrument and Equipment
5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume iqjeclor capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS).
52 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freeding to 0.00001 g. 5.4 50 mL disposable polypropylene centrifoge tubes. 5.5 15 mL disposable polypropylene centrifoge tubes. 5.6 Disposable micropipets (S0>100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 tnL clear HPLC vial IdL
P *j*2oP
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No. : P0001131
Exygen Protocol N um ber P0001131
E xranJU m icb
Msthod Number VOOOI78S
I ANALYTICAL METHOD
Method o fAnalysis for the Dctcnnnation ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS
5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vae 6 cc (lg ) tC18 SPE cartridges.
5.12 SPEvacuum manifold. 5.13 Tissuemizer. 5.14 Wrist-action shaker. 5.15 CentrifUge capable o fspinning 15 mL polypropylene tubes at 3000 rpm.
6.0 Chranatognphic System
6.1 Analytical Cohumi: 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 ): Methanol Gradient Program:
Tfanafwiml
0.0 1.0 8.0 20.0 22.5
&A 65 65
25 25
65
Flow Rate & fm lTm in) 35 0J 35 0.3
75 0.3
75 0.3
35 0.3
6.6 Injection Volume: lSpL(cattbeincreaaedtoasm uchai50pL). 6.7 Quantitation: PeakArea - external standard calibration curve. 6.8 RunTime: 2 3 minutes.
The above condition! are intended asa guide and may be changed in order to optimize the HPLC system.
7.0 MS/MS System
7.1 Mode: EloctrosprsyNegative MRM mode, monitoring 413 --369 m/z for PFOA.
The above conditions are intended as t guide and may be changed in order to optimize the MSMS system.
Page 3 of?
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No. : P0001131
Exygen Protocol Number: P0001131
BaygmlUaNnk
Method Number V000l7*s
I AN/Q.YTICA1- METHOD
Method o fAnalysis fa ' the Determination o fPerfluorooctanoic Acid (PFOA) in Smell Mammal Liver by LC/MS/MS
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 ammonium acetateto 1000 mL o f water.
Alternate volumes may be prepared.
9.0 Standard Preparation
9.1 Standard Stock/Fortification Solution 9.1.1 Pnparoaslockiolutionof-IO O iig/m LofP FO A by weighing lOmg 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 pg/mL fortification solution o f PFOA is prepared by bringing 1 mL o f the 100 uf/m L solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pgAnL fortification solution o f PFOA is prepared by bringing 10 m Lofthe 1.0 ug/mL aolution to a final volume o f 100 with methanol in a 125mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a re&igenuor at approximately 4*C and are stable for a maximum period o f 6 months from foe date o fpreparation.
9.2 StandardCalibration Solutions
9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o fthe 0.1 iitfn L fortificatim i solution.
9.2.2 The following is a typical example: additional concentrations may be
Final
o f F o rtification Volume
Solution In ^reL )
(m L )
D ilu te d to (mL)
C o n cen tratio n
(nc/mL)
100 S.0
100
5.0
100 2.0
100
2.0
100 1 4 100
1.0
5.0 10 100
04
2.0 10 100
0.2
1 4 10 100
0.1
9.24 Store a ll calibration standards in 125-mL LDPE narrow-mouth bottles
at2*C to fi*C, up to six months.
94.4 Alternate volumes and concentrations o f standards may be prepared as
oftPage 4
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygta Research
M ethod Num ber V 0001785
I ANALYTICAL METHOD
Method o fAnalyse for the Determination o fPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS
10.0 Batch SetUp
10.1 Each batch o f simples extracted (typically 20 or less) 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.
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 1 g o f sample into a 50 mL 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 size available for use.
11.2 Add water to the sample for a final volume o f 10 mL. 11J Homogenize sampleusing atissuemizer for ~1 minute. 11.4 Transfer 1 mL o f die sample using a disposable pipette into a IS mL
disposable centrifiige tube. 11.5 Add 5 mL o f acetonitrile and shake for ~20 minutes cma wrist-action shaker. 11.6 Centrifiige foe tubes it ~3000rpm fo r-5 minutes. 11.7 Decant foe supernatant into a 50 mL disposable centrifiige tube and add 35
m Lofwatar. 11.8 Condition foe Cu SPE cartridges (1 g, 6 mL) by passing 10 mL methanol
followed by SmL o f KPLC water(~ 2 drop/sec). Do not let column run dry 11.9 Load foe aampleon conditioned Cu SPE cartridge. Discard eluate. 11.10 Elute w ith 2 mL o f mefoanoL Collect 2 mL o f eluate into a graduated
15mL polypropylene centrifiige tube (final volume 2 mL). 11.11 Analyze samples using eiectroapray LC/MS/MS.
12.0 Chromatography
12.1 Iqject the aame 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 Standard! o fPFOA corresponding to at least five or more concentration levels mustbe included in m analytical set
12.3 An entire set o f calibration standards must be included at the beginning and ai foe 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 iryected at foe beginnmg o f a set followed by calibration standards interspersed every 5-10 simples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in a sample set.
12.4 Use linear standard curves for quantitation. Linear standard curves are generated for foe analyte by linearregression using 1/x weighting o fpeak area
Pag 5 o f 7
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygee Research
M efcod N um ber VO0O17I5
I ANALYTICAL METHOD
Method o fAnalysis for the Determination o fPerfhiorooctanoic Acid (PFOA) in Small MammalLiver by LC/MS/MS
versus catibratioa standard concentration using MastLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceedstandard responses should be Amber diluted and reanalyzed.
13.0 Acceptance Criteria
13.1 Chromatogram muat ahow a peak o f a daughter ion at 369 amu from a parent o f413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o fcarbon dioxide.
13.2 Method blanks muat not contain PFOA at levels pester titan the LOQ. If a blank contains PFOA at levels greater than 10 ng/g, 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 folia 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 warranted.
13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Teat, may be excluded from die calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must notexceed20% o fthe total numbero f standards injected.
13.5 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 appropriate steps must be taken to (just instrument operation, and the standards or the relevantset o fsamples should be reanalyzed.
13.6 Retention times between standards and samples must not d rift more than 1 4 % within an analytical tun. I f retention time d rift exceeds this lim it within an analytical run then the act muat 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 ana) using the standard curve (linear regression parameters) generated by die Mass Lynx software program:
PFOA found (ng/m L)-{Peek area-intercept x DF x aliquot factor slope
DF * factorby which the final volume w u diluted, i f neoessary. Aliquot factor-1 0
Page 6 o f 7
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Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
E xysw fcm eK h
Method Number V0Q017IS
| ........
a n a l y t ic a l m e t h o d
Method o fAnalysis for the Detenmnation ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS
14.2 For uunplea fortified with known amount! o f PFOA prior to extraction, use the following equation to calculate the percent recovery.
Recovery (%) -
[ totalanalyte found (ng/mL) - analytefound in control (ng/mL)] analyte added (ng/mL)
14J Uae the following equation to convert the amount o f PFOA found in ng/mL lo n(/g(ppb).
PFOA found fpph)- fPFOA found (ng/mL) x final volume tm U l sample weight (g)
Exygen Research
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ANALYTICAL METHOD Method Number V00017M
Method o f Analysis fo r the Peterm lnrtoo o f Perfluorooctanolc A dd (PFOA) ie Small Mammal Senna by LC/MS/MS
Analytical Testing Facility:
Exygen Research 3058 Research Drive State College, PA 16801
Approved By:
_C--iAvi___
loda,Pnil Connolly
I
T K h n ictl
LC-MS, Exygen Reeidt
DaU
Johni FFlalahheerty / 'VVicicee President, Operations, Exygen Research
Date
Exygen Research
Total Pages: 7 Page 59 o f65
Page 111 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol N um ber P0001131
Exytee ttM tch
Mechod Number V 000i7t6
I an ajLVt ic a l m e t h o d
Metbod o fAnalysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Semm 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 Spectxometric Detector (LC/MS/MS) in small mammal serum.
2.0 Safety
2.1 Always observe tifo laboratory practices. 2 .2 C o n s u lt f o e a p p r o p r ia te M S D S b e f o r e h a n d lin g a n y c h e m ic a l f o r p r o p e r safety
precautions.
3.0 Sample Requirement
3.1 A t least I mL o ftest sample for extraction. 3.2 No sample processing is needed for serum samples. However, frozen serum
simples must to allowed to completelythaw to room temperature before use. 3.3 Sample collection procedures w ill be specified in the sampling plan for this
project
Reagents and Standads
4.1 W ater-HPLC grade 4.2 Methanol-HPLC grade 4.3 Acetonitrile-HPLC grade 4.4 AfnmomumAoeWe-A.CS. Reagent Grade 4.3 Perfluorooctanoic A dd-S ipne-A ldrich
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 dispoaablc polypropylene ceotrifUge tubes. 5.5 15 mL disposable polypropylene centrifoge tubes. 5.6 Disposable micropipets (50-lOOuL, 100*200uL). 5.7 125-mL LDPE narrow-mouthbottles. 5.8 2 mL clear HPLC vial IdL 5.9 DispoaaMe pipettes 5.10 Autopipettca (100-1000 pL and 10-100 pL), w ith disposable tips. 5.11 Waters SepPak Vac 6 cc (Ig ) tCL8 SPE cartridges. 5.12 SPEvacuum nrenifold. 5.13 Vortaxer.
Page2o f1'
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
E xyfeaieM srcfa
Method Nuotocr V000I7S6
I ............
a n a l y t ic a l m e t h o d
Method o fAnalysis for the Determination o fPerfhiorooctanoic Acid (PFOA) in Small
Mammal Serum by LC^MS/MS
$.14 Wrist-action baker. 5.1$ Centrifuge capable o f^pinning l$ mL polypropylene tubes at 3000 rpm
6.0 Chromatographic System
6.1 Analytical Column: Fluopbsse RP (Keystone Scientific), 2.1 nun x SOmm. Sp (P/N: 82505*052130)
636.2 Temperature: 30*C Mobile Phase(A ): 2 mM Ammonium Acetate in Water Mobile Phase (B ): Methanol Gradient Program:
Time (m ini
0.0 1.0 8.0 20.0 22.5
%A
65 65 25 25 65
Flow Rate %B (m L/m inl
35 0.3 35 0.3
75 0.3 75 0.3 35 0.3
6.6 hyeettoa Volume: 15 pL (can be incseaaed to as much as 50 pL). 6.7 Quantitation: PeakAm t-external standard calibration curve. 6.9 RunTime: -2 3 minutes.
The above conditions am intended as a guide and may be changed in order to optimize the HPLC system.
7.0 MS/MS System
7.1 Mode: ElectrosprayNegative MRM mode, monitoring 413 --369 m/z for PFOA.
The above conditions are intended as a guide and may be changed in order to optimize the MSMS system.
8.0 Preparationo fSolutions 8.1 Mobile Phase
8.1.1 2 mM ammonium acetate in water is prepared by adding 0.IS4 g of mnvMiium acetate to 1000 mL o fwater.
Alternate volumes maybe prepared.
Page 3 o f 7
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ExypofU M veb
M ethod Number V000I7I6
I ...... ANALYTICAL METHOD
Method o fAnelyii for the Determination o fPerfltoiooctnoic 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-100 pg/mL o f PFOA by weighing 10 mg o fanalytical standard (corrected fix' purity) and dilute to 100 mL with methanol in a 12$-mL LDPE bottle. 9.1.2 A 1.0 pgfaiL fortification solutioa o f PFOA is prepared by bringing I mL o f the 100 pgfaL solution to a final volume o f LOOwith methanol in a 125 mL LOPB bottle. 9.1.3 A0.1 ug/mL fortification solutionofPFOAia prepared by bringing 10 mL oftbe 1.0 jigfaiL eotution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in t refrigerator ai approximately 4*C sad are stable for a maximum period o f 6 months from die date o fpreparation.
9.2 Standard Calibration Solutions
9.2.1 LC/MS/MS calibration standard! are prepared in methanol via dilution
922 o ftire 0.1 pgfaiL fortification solutioa The following is a typical example: additional concentrations may be prepared asneeded.
Concentration ofFortification Volume
Diluted to
Final Concentration
Sohtfionfaa/mLl (mL)
(mL)
(ne/mU
100 5.0 100 2.0
100 100
5.0 2.0
100 1.0 100
1.0
5.0 10 100
O.S
2.0 10 100
0.2
1.0 10 100
0.1
9.2.3 Store a ll calibration standards in 125-mL LDPE narrow-mouth bottles
at 2*C to 6*C, up to six months.
9.2.4 Alternate volumes and concentrations o f standards may be prepared as
10.0 Batch SetUp 10.1 Each batch o f saoqries extracted (typically 20 or lees) must include at least one untreated cootrol and two untreated controls fortified at known concentrations (lib control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in foe qualify assurance plan for this project.
Pap 4 o n
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Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exytm RM Nieb
Method Number VOOO17Sd
I ANALYTICAL m e t h o d
forMethod o fAnalysis the Determination o f Perftuorooctanoic Acid (PFOA) in Small
Mammal Saturn by LC/MS/MS
11.0 Sample Extraction
11.1 Measure l mL o f m p le into a 50 mL polypropylene centrifuge tube* (fortify u needed, replace lid and mix well). Note that alternate volumes o f serum may be measureddepending on the sample size available for use.
11.2 Add water to the sample for a final volume o f20 mL. Cap tightly 11.3 Vortex for -1 minute. 11.4 Transfer 1 mL o f the sample using a disposable pipette into a 15 mL
J Add5disposable oentrifiige tube.
11 mL o facetonitrile and shake for **20 minutes on awrist-action shaker. 11.6 Centrifuge the tubes at *-3000 rpm for ~5 minutes. 11.7 Decant the supernatant into a 50 mL disposable centrifoge tube and add 35
mL o fwater. 11.8 Condition foe C SPE cartridges (1 g, 6 mL) by passing 10 mL methanol
followed by 5 m LofHPLC water (-* 2 drop/soc). Do not let column run dry 11.9 Load the sampleon conditioned Cu SPE cartridge. Discard eluatc. 11.10 Elute with -2 mL o f methanol. Collect 2 mL o f eluate into a graduated
15 mL polypropylene centrifoge tube (final volume * 2 mL). 11.11 Analyze samplesusing eloctrospray LC/MS/MS.
12.0 Chromatography
12.1 Iq'oct 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 ofPFOA corresponding to at least five or more concentration levels must be included in an analytical set
12.3 An entire set o fcalibration standards must be included at foe beginning and at the end o f a sample set. Standards must be interspersed between every 5-1o samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5*10 samples (to account for a second set o f standards), in either case, calibration standards must be the first and last injection in a ample set
12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system.
12.5 Sample response should not exceed standard responses. Any samples that exceedstandard responses should be fortber diluted and reanalyzed.
P|eSof?
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
E xyynfcw nreh
Method Number VOOO17W
| ANALYTICAL m e t h o d Method o f Analysis Ibr the Determination o fPerfluorooctanok Acid (PFOA) in Small
M am m al S eram b y L C /M S/M S
|
13.0 Acceptance Criteria
13.1 Chromatogram mast 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 die daughter ton (369 amu) represents the loss o fcarbon dioxide.
213 Method blanks must not contain PFOA at level 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% of their known values. I f a control spike A lls outside the acceptable lim its, the entire set o f tangles should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraciion 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 exceed20% o fthe total numbero fstandards injected. 13.5 The condition coefficient (R) for calibration curves generated must be 20.992 (R1 20.985). I f calibration results foil outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standardsor the relevant set o fsamples should be reanalyzed. 13.6 Retention timea between standards and samples must not d rift more than 4 % within in analytical run. 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 Use the following equation to calculate the amount o fPFOA found (in ng/mL. based on peak area) uaing 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 slope
DF * foctor by which the final volume was diluted, i f necessary. Aliquot foctor 20
14.2 For sample fortified w ith known amounts o f PFOA prior to extraction, use foe following equation to calculate the percent recovery.
Recovery (% )*
[ total analytefound(ng/mL) - analyte found in control (ng/mL)] analyte added (ng/mL)
Page 6 o f 7
Page 64 o f65
Exygen Research
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Interim Report #8-Analysis of Sediment Samples
Exygen Study No. : P0001131
Bxygen Protocol Number: P0001131
Exyfea Research
M ethod Num ber VOQO1786
I A N A L IT IC A ! m e t h o d
Method o fAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS
14.3 Use foe following equation to convert foe amount o f PFOA found in ngtatL to ppb.
xPFOA found foobl - fPFQA found fna/mU final volume ftnLl)
sample volume (mL)
Exygen Research
Pap T op
Page 65 o f65
Page 117 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
3058 Research Drive
Phone: 814-272-1039
S tate College, PA 16801
Fax: 814-231-1580
PROTOCOL AMENDMENT
Am endm ent Num ber. 1
Effective Date: 0 1 /19 /0 5
Exygen Study Number P0001131 Client Study N um ber P a g e lo M
None
D ES C R IPTIO N O F AM ENDED SE C TIO N 1) Analytical Procedure Sum m ary V0001780:Sectk>n 9.1 2 ) Verification o f Analytical Procedure
AMENDEDTQ
1) Add to Section 9.1: Section 9 .1 .6 , A tterrate weights o f standards m ay be used to prepare alternate concentrations o f stock solutions as necessary. A lternate levels of
fortification solutions may also be prepared. 2 ) Low and high spiking levels o f the analytes fo r each m atrix m ay be altered depending on sam ple size available for extraction and/or to cover analyte concentrations expected
in th e 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 available fo r sm all mam mal 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. H igher spiking levels in the bottles w ill cover th e analyte concentrations expected in the w ater sam ples.
IM PA C T O N S TU D Y
T h e LO Q is 100 ng/g for a 0.1 g sam ple o f small m am m al liver and is 1000 n g/m L for a
0.01 mL sam ple o f sm all m am mal serum .
.
Higher levels o f spiking fo r the w ater sam ples w ill ensure that more Q C recovery data
can be used.
UBRARY ID: W 0 0 0 1 2 2 M * /
''
ADMINISTRATIVE FORM
Exygen Research
Page 118 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
BOSS Research Drive
Phone: 814-272-1039
S tate College, PA 16801
Fax: 814-231-1580
Am endm ent Num ber E ffec tiv e D a te: Exygen S tudy N u m b er
PROTOCOL AMENDMENT
2 _______
0 3 /0 7 /0 5
P0001131
C lie n t S tu d y N u m b e r
Pap 1 of 1 None
D ES C R IPTIO N O F AM ENDED S E C TIO N 1) Report, page 11 o f 65 2 ) T est M aterials, page 6 o f 65: PFO S transition monitored 4 99 -> 99.
AMENDED TO 1) Instead o f one Anal report, Interim reports will be issued. 2 ) P FO S transition monitored m ay also be 4 9 9 -> 80.
RATIO NALE 1) Due to th e excessive sizes o f the data sets, interim reports w ill be issued to allow the client to receive data in a tim elier m arten '' w ' 2 ) T he A P I 4 0 0 0 LC /M S /M S system s detect the 4 9 9 -> 80 PFO S transition with greater sensitivity than the 4 9 9 -> 99 transition.
IM PACT O N S TU D Y 1) T he 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: V000122S4- '
Exygen Q AU R eview . ADMINISTRATIVE FORM
Exygen Research
Page 119 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
OS--20-2005 02:34pm FrwHSTON SOLUTIONS
+ T--394 P.002/004 F-437
E^gsnR E S E A R C H
3 0 5 8 R esearch D riv e
Phone: 8 1 4 -2 7 2 *1 0 3 9
S ta te C o lle g e , RA 16801
Fax: 81 4 *2 3 1 -1 5 8 0
General: X Project S p ecific D eviation
DEVIATION FORM
Page 1 of 1
Facility Deviation
D a te o f O ccurrence: 0 3 /1 6 /0 5
Exygen P ro je c t# :
P 760/P 1131
D eviation # :
1/1
C lient Project # :
NA
R eference # :
0 5 -1 2 2
R ag u lateiV -P rlver:
GMP GLP O ther None
Sample Description:
Deviation Type: (Include W fo r methods and SOPs)
______ Protocol
______ M ethod
X SOP
V fc 0001658-3 Notebook reference: NA
Login#: _______ NA
Container#:
NA L o t# :
NA
Summary o f Deviation: Thle deviation pertains to a ll soil and sedim ent sam ples analyzed fo r percent solids before 0 7 /0 7 /0 5 .
a . No blanks o r duplicates w ere run as required by section 9 .3 . b . Som e sam ple w eights exceed the ellow able range (> 10 g ).
Cause:
P re p a ra tio n
A n aly s is
Instrum ent
C lien t R equest X O ther
Im pact; T here has been no negative im pact on th e study. A ll o f the p ercent soiid values th a t w ere determ ined during th e tim e period in question a re considered valid, although th e S O P w as not follow ed. In the new ly revised version o f th e S O P blanks and duplicates are no longer required. A lso, in th e new S O P , the allow able am ount o f sam ple to be used is < 2 0 g. A ll o f th e sam ples in question in this deviation w eighed lass than 2 0 g. T h e technician analyzing d ie sam ples fo r percent solids w as follow ing the new procedure before it w as form ally approved.
C o rrec tiv e A c tio n s: A n ew version o f th e S O P has been Issued and approved (V 0 0 0 0 4 2 7 -3 ).
Slanature:
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Study Dii
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Q ualify Assurance
D ate
Sponsor Managem ent
Exygen Research
Page 120 of 121
Interim Report #8-Analysis of Sediment Samples
Exygen Study No.: P0001131
CHEM EHSR 236 1%
651 733 1958
07-11-Z00E 03:S7pa FrmrlESTOK tOltfTIOM ;r
07/11 '06 14:09 NO.142 02/02
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A pj*n dk I, Analytical Mathod V001782.
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b e i extracted ung Ih a foHpwInq method.
Dtretlhjacdon Mamed: Befere'tuesampiesaraw a tt*|V thaartryUen.
aoatlc add ln methanol la added W each ampia. T h e e a m p t a s a r a t h a n .
hand, vertaaadi and sonicated ferth tf^ minute
W
-1 0 minuted at -3000 rpm. Eaobtam ple.ii analyzed by LC/MS/MS elpetrtapray. ....
------ .
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Mord uaebto data amba obtained by usingan altamaia meiod.
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RECEIVED TIME JUL.ll. 4:27PM
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PRINT TIME
A O M lkW tA T h/O P n * u '
JUL.ll. 4:28PM
Exygen Research
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