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E054210 Interim Re ort #I8 Notthem Alabama Potable Water Systems, Decemger 2005 Interim Report #18: Analysis of Water Samples from Northern Alabama Potable Water Systems, December 2005 Study Title ANALYSIOSF PERFLUOROBUTANESULFON(PAFTBES), PERFLUOROHEXANESULFONATE (PFHS), AND PERFLUOROOCTANESULFONATE (PFOS) IN WATER, SOIL, SEDIMENT, FISH, CLAMS, VEGETATION, SMALL MAMMALIVER AND SMALL MAMMASLERUM USING LC/MS/MS FOR THE 3M DECATURMONITORINGPROGRAM Data Requirement EPA TSCA Good Laboratory Practice Standards 40 CFR 792 Author Michelle D. Malinsky, Ph.D 3M Environmental Laboratory Interim Report Completion Date Date of signing Performing Laboratory 3M Environmental Laboratory Building 2-3E-09 935 Bush Ave. St Paul,MN 55106 ! !E!ACCRtMtED certificate r2052-01 Project Identification E05-0210 Total Number of Pages 122 The testing reportedherein meet the requirementsof ISOfiEC 17025-1999 "General Requirementsfor the Competence of Tesling and CalibrationLaborat&, in accordancewith the A2LA Certificate#205241. Testing that complies with this InternationalStandard also operate in accordancewith IS0 9001flSO 9002 (1994). E05-0210 Interim Report 118 Notthem Alabama Potable Water Systems, December 2005 Page 1 of 122 05-0210 Interim R oti # I 8 Northern Alabama Potable Water Systems, Decem'k,, 2005 This page has been reserved for specific country requirements. E054210 Interfm Report # I 8 NorthernAlabama Potable Water Systems, December 2005 Page 2 of 122 05-0210 Interim Re ori #18 NorthemAlabama Potable Water Systems, W e n & 2005 GLP COMPLIANCSETATEMENT 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 Interim Study: Analysis of Water Samples from Northern Alabama Potable Water Systems, December 2005 Interim Study Identification Number: E05-0210 Interim Report #18 This study was conducted in compliance with Toxic Substances Control Act (TSCA) Good Laboratory Practice (GLP) Standards, 40 CFR 792, with the exceptions listed below: Exceptions to GLP compliance: None Sponsor Representative Jaisimha Kesari P. E., DEE, Study Director Date cemcate me41 The testing repMtedherein meetthe requirementsof ISO/IEC 17025-1999 'General Requirementsb rthe Competence of Testingand Calibration Laboratories'. in accordancewith the A2LA CertificateR052-01. Testingthat compl~swiththis InternationalStandard also operate in accordancewith IS0 9001/ISO 9002 (1994). 05-0210 lnterim Report #18 Notthem Alabama Polable Water Systems, December 2005 Page 3 of 122 05-0210Interim Re ort #I8 Northern Alabama Potable Water Systems, Decem%er 2005 QUALITYASSURANCSETATEMENT 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 InterimStudy: Analysis of Water Samples from NorthernAlabama PotableWater Systems, December 2005 Study IdentificationNumber: E05-0210 Interim Report#8I This study was audited by the 3M EnvironmentalLaboratoryQuality Assurance Unit (QAU), as indicated in the following table. The findings were reported to the study director and laboratory management. Inspection Dates February 15,2006 Phase DatalReport Date Reported to Management 1 Study Director I March 17,2006 March 17,2006 EO5-0210 Interim Report #I8 Notihem Alabama Potable Water System, December2005 Page 4 of 122 05-0210 Interim Re orf # I 8 NotihemAlabama Potable Waier Systems. Decemf e r 2lV5 TABLEOF CONTENTS GLP Compliance Statement ...................................................................................................................... 3 Quality Assurance Statement .................................................................................................................... 4 Table of Contents ....................................................................................................................................... 5 List of Tables............................................................................................................................................... 6 Study Information ....................................................................................................................................... 7 Summary and Introduction......................................................................................................................... 8 Test Samples............................................................................................................................................ 10 Reference Substances............................................................................................................................. 10 Method Summaries .................................................................................................................................. 11 Sample Collection ........................................................................................................................ 11 Preparatory and Analytical Methods ........................................................................................... 11 Extraction...................................................................................................................................... 11 Analysis......................................................................................................................................... 11 Analytrcal Results ..................................................................................................................................... 13 Calibration..................................................................................................................................... 13 Limit of Quantitation (LOQ) .......................................................................................................... 13 Blanks ........................................................................................................................................... 13 Solvent Blank................................................................................................................................ 13 MethodBlank................................................................................................................................ 13 Trip Blank...................................................................................................................................... 14 System Suitab.il.ity......................................................................................................................... 14 ContinuingCalibration.................................................................................................................. 14 Lab Control Spikes (LCSs) .......................................................................................................... 14 Sample Duplicates ....................................................................................................................... 15 FieldMatrix Spikes....................................................................................................................... 15 Data Summary and Discussion............................................................................................................... 15 05-0210 Interim Report #18 Notfhem Alabama Potable Water Systems. December 2005 Page 5 of 722 05-0210 Interim Re ort #18 NorthemAlabama Poteble water systems. Decemier 2005 Statistical Methods and Calculations....................................................................................................... 19 Accuracy and Precision Equations.............................................................................................. 19 Determination of Analytical Uncertainty ...................................................................................... 19 Statement of Conclusion.......................................................................................................................... 20 References ............................................................................................................................................... 20 List of Attachments ................................................................................................................................... 20 Signature Page......................................................................................................................................... 21 LISTOF TABLES Table 1. Sample ResultsSummary ............................................................................................................. 9 Table 2. Sample DescriptionCodes......................................................................................................... 10 Table 3. Study Reference Substances..................................................................................................... 10 Table 4. Sample Collection Information.................................................................................................... 11 Table 5. InstrumentParameters................................................................................................................ 12 Table 6. LiquidChromatographyGradient Program................................................................................ 12 Table 7. MassTransitions.......................................................................................................................... 12 Table 8. MethodBlank Area Counts......................................................................................................... 14 Table 9. Lab ControlSpike Results........................................................................................................... 15 Table 10. Sample and QC Results........................................................................................................... 16 E050210 lnterim Report 118 NorthernAlabama Potable Water Systems. December 2005 EO5-0210 Interim Re ori # I 8 NoIulem Alabama Potable Water Systems, Decemger 2005 STUDY INFORMATION Sponsor 3M Company Sponsor Representative MichaelA. Santoro 3M Company 3M Building 0236-01-8-10 St. Paul, MN 55144 Study Director Jaisimha Kesari, P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Study Location Testing Facilifies Exygen Research 3058 Research Drive State College, PA 16801 3M Environmental Laboratory 3M Building 2-3E-09 935 Bush Avenue St. Paul, MN 55144 Study Dates Study Initiation: 11/5/2004 Interim Experimental Initiation: January 11, 2006 Interim Experimental Completion: January 27,2006 Interim Study Completion: Date of interim report signing Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory according to 40 CFR Part 792. The test substance and analytical reference standard reserve samples are archived at the 3M Environmental Laboratory according to 40 CFR Part 792. E05-0210 Interim Report #l8 NorthernAlabama Potable Water Systems, December 2005 Page 7 of 122 05.0210 Interim Report #lE Northern Alabama Potable Water Systems, December 2005 SUMMARY AND INTRODUCTION The 3M Environmental Laboratory extracted and analyzed water samples collected from six Northern Alabama potable water systems by Weston Solutions personnel on December 13 and 14, 2005. Samples were submitted for analysis as part of 3M Environmental Laboratory project number E05-0210 (3M Decatur Fluorochemical GLP Monitoring Program, Interim Report #18: Analysis of Water Samples from NorthernAlabama Potable Water Systems, December 2005). Water samples were analyzed for perfluorobutanesulfonate (PFBS), perfluorohexane sulfonate (PFHS), and perfluorooctane sulfonate (PFOS) using 3M Environmental Laboratory Method ETS 8-154.1 "Determination of PerfluorinatedAcids, Alcohols, Amides, and Sulfonates in Water by Solid Phase Extractionsand High Performance Liquid ChromatographylMassSpectrometry" in accordance with Exygen Research Protocol POOOl131 (Attachment C). ETS 8-454.1 falls under the 3M Environmental Laboratory's current ISOAEC 17025-1999 scope of accreditation and the results reported herein were compliant with this accreditation. The analytical start date for this interim report was January 11, 2006 and the analytical termination date was January 27, 2006. Sample containers were prepared at the 3M Environmental Laboratory. Sample containers for each sampling location included a field sample, field sample duplicate, low field spike (0.1 ng/mL) and high field spike (1.0 nglmL). Each empty container was marked with a "fill to here" line to produce a final sample volume of 450 mL. Containers designated for field matrix samples were fortified with an appropriate matrix spike solution containing the three target analytes prior to being sent to the field for sample collection. For the samples and field matrix spikes collected and analyzed for this interim report, PFOA [I,213C]was not included as a surrogate. This is a deviation from Protocol POOOl130Amendment #3. A record of deviation has been issued as is included in Attachment C. Table 1 below summarizes the sample results. The average between the sample and the sample duplicate is provided along with the relative percent difference (%RPD), if applicable. The limit of quantitation (LOQ) of PFBS, PFHS, and PFOS for the sample set was 0.0248 nglmL, 0.0252 ng/mL, and 0.0249 nglmL, respectively. All results for quality control samples prepared and analyzed with the samples will be reported and discussed elsewhere in this report. 05-0210 lnterlm Report #I8 NorthernAlabama Potable Water Systems, December 2005 Page E of 122 Table 1. Sample Results Summary 05-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 E05-0210-92258 E05-0210-92259 SWTP PWF W 2 0 051214 SWTP PWFW2DB051214 Average Sample/SampleDuplicate %RPD ~0.0248 4.0248 e0.0248 `"NA co.0252 C0.0252 e0.0252 `"NA ~0.0249 ~0.0249 <0.0249 "NA 054210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Page 9 of 122 E054210Interim Re ort #I8 Northern Alabama Potable Water Systems, Decemger 2OO5 TESTSAMPLES Water samples from six locations were received at the 3M Environmental Laboratory on December 16,2005 from Tim Frinak of Weston Solutions, Inc. Sample containers were received in satisfactory condition at ambient temperatures. A total of twenty-seven containers were submitted for analysis. The samples were logged in by 3M Environmental Laboratory professional services personnel and placed in refrigerated storage on December 16, 2005 until they were removed for extraction on January 11, 2006. Table 2 below provides the codes for the given sample descriptions. I I 1 Table 2. Sample DescriptionCodes. Sample-cription Definition REFERENCSEUBSTANCES Table 3 lists the pertinent information regarding the reference substance used for this study. Table 3. Study Reference Substances. Reference Substance PFBS PFHS 1 PFOS I Chemical Name I Perfluorobutanesulfonate I Pemuorohexanesulfonate I PerRuorooctanesulfonate I (2) All test substances are believed to be soluble in water at the levels to be investigated.No visual precipitates observed. 05-0210 Interim Report 118 Northern Alabama Potable Water Systems, December2005 Page 10 of 722 05-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 METHODSUMMARIES Sample Collection Samples were collected on December 13-14,2005 in rinsed NalgeneTM(low-density polyethylene) bottles prepared at the 3M Environmental Laboratory on December 9, 2005. Containers designated for field matrix spike samples were spiked in the laboratory with a known volume of an appropriate matrix spiking solution containing the target analytes. Four collection bottles were associated with each individual sampling location: sample, sample duplicate, low level field matrix spike (LS), and high level field matrix spike (HS). Additionally, one "Trip Blank" sample set was prepared. A Trip Blank set consisted of three individual bottles: trip blank sample, trip blank low spike, and trip blank high spike. The matrix spike levels for the trip blanks were the same as the samples. Table 4 below details the spike amounts for the four bottles comprising the sample location set. Table 4. Sample Collection Information Nominal Fina Final Concentration(ndmL) I I Sample 450 Sample Dup 450 Low Field Matrix Spike (LS) 450 High Field Matrix Spike (HS) 450 NA NA 0.0992 0.992 NA NA 0.101 1.01 NA NA 0.0997 0.997 (1) The sample description was assigned in the field by Weston Solutions personnel. Preparatory and Analytical Methods Extraction All samples, calibration standards, and associated quality control samples were extracted using the procedure outlined in ETS-8-154.1 "Determinationof PertluorinatedAcids, Alcohols, Amides, and Sulfonates in Water by Solid Phase Extractions and High Performance Liquid Chromatography/MassSpectrometry". Briefly, 40 mL of sample was loaded onto a pre-conditionedWaters C18 solid phase extraction (SPE) cartridge (SepPak, 6 cc) using a vacuum manifold. The loaded cartridges were then eluted with 5 mL of methanol. This extraction procedure concentrates the samples by a factor of eight. (Initial volume = 40 mL, final volume = 5 mL). Samples were initially extracted on January 11,2006 and analyzed on January 14,2006. Because associated laboratory quality control samples did not meet method criteria, samples were reextracted along with a new calibration curve and all associated quality control samples on January 23,2006. The second set of extracts were analyzed on January 26, 2006. All results reported here are from the second extraction set. Analysis All sample and quality control extracts were analyzed for PFBS, PFHS, and PFOS using high performance liquid chromatography/tandem mass spectrometry(HPLC/MS/MS). Pertinent instrument parameters, the liquid chromatography gradient program, and the specific mass transitions analyzed are described in the tables below. 050210 Interim Report #18 Northern Alabama Potable W a W Systems, December 2005 Page 11 of 122 05-0210 Interim Re ori #I8 NorthernAlabama PotableWater Systems, Decemger2005 Table 5. Instrument Parameters Instrument Name Liquid Chromatograph Guard column Analytical column Injection Volume Mass Spectrometer Electrode Ion Source Polarity Software I ETSOllie Agilent 1100 Two Betasil C18 (2.1 mm X 100 mm), 5 pm linked in series before the injection port Befasil C18 (2.1 mm X 100 mm). 5 vm 5 pL Applied BiosystemsAPI 4000 Q Trap Z-spray Turbo Spray Negative Analyst 1.4.1 Table 6. Liquid Chromatography Gradient Program step Number 0 1 2 3 4 5 Total Time (min) 0 1.o 14.5 15.5 16.5 20.0 Flow Rate (Nmin) 300 300 300 300 300 300 PercentA (2 mMammonium acetate) 80.0 80.0 10.0 10.0 80.0 80.0 Percent B (Methanol) 20.0 20.0 90.0 90.0 20.0 20.0 Table 7. Mass Transitions Analyte (~'PFBS (~'PFHS ("PFOS Mass Transition QVQ3 299/80 299199 3991130 399199 399180 49911 30 499/99 499180 Dwell Time (msec) 150 150 150 150 150 150 150 150 (1) The individualtransitionswere summed to producea 'total ion chromatogram"(TIC) for the given analyte. The resultant TIC was used for quantitation. E054210 InterimReport#18 NorthernAlabama PotableWater Systems, December2005 Page 12 of 122 EO5-0210 Interim Re oti #18 Northern Alabama Potable Water Systems, Decemger 2005 ANALYTICARL ESULTS Calibration Calibration standards were prepared by spiking known amounts of stock solutions containing the target analytes into 40 m i of ASTM type I water. Each spiked water standard was then extracted in the same manner as the collected samples. A total of twelve spiked standards ranging from 0.025 ng/mL to 25 ng/mL (nominal) were prepared. A quadratic, l/x weighted, calibrationcurve was used to fit the data. The data was not forced through zero during the fitting process. Calculatingthe standard concentration using the peak area counts and the resultant calibration curve confirmed accuracy of each curve point. Each extracted calibrationstandard used to generate the final calibrationcurve met the method calibration accuracy requirement of 100*25% (100*30% for the LOQ standard). The correlationcoefficient was greater than 0.999 for all analytes. Limit of Quantitation (LOQ) The LOQ for this analysis, as defined in ETS-8-154.1, is the lowest non-zero calibration standard in the curve in which the area counts are at least twice those of the method blank@). This will be presented and discussed in more detail in the section below regarding method blanks. The LOQ for PFBS, PFHS, and PFOS was 0.0248 ng/mL, 0.0252 ng/mL, and 0.0249 ng/mL, respectively. Blanks Three types of blanks were preparedand analyzedwith the samples: method blanks, solvent blanks, and fieldhip blanks. Each blank type is described below. Solvent Blank Several methanol solvent blanks were analyzed to assess system Contamination and/or instrument carryover. One methanol solvent blank analyzed immediately after the highest calibration standard produced PFOS area counts greater than half the LOQ standard. However, the solvent blank analyzed immediately after the high blank demonstrated that the likely instrument carryover observed in the preceding injection had diminished. Analyte peak area counts in all other solvent blank samples were less than half the area counts of the calibration standard used to establish the LOQ for all analytes. Method Blank Eight method blanks were prepared by loading40 mL of ASTM Type I water onto a C18 SPE cartridge and eluting with 5 mL of methanol using the same extraction procedure as the samples. Method blanks were prepared to evaluate the levels of background contamination in the overall extraction process (glassware, SPE cartridges, etc.) Table 8 lists the PFOA area counts for the method blanks and the corresponding LOQ standard. 05-0210 Interim Report 618 Northern Alabama Potable Water Systems, December 2005 Page 13 of 122 EO54210 Interim Re OH W18 Northern Alabama Potable Water Systems, Decemker 2005 Iawe 8. Metnoa manicArea Gounts. ~ PFBS MB-060123-1 Method Blank-1 MB-060123-2 Method Blank-2 MB-060123-3 Method Blank-3 MB-060123-4 Method Blank4 MB-060123-5 Method Blank-5 M E 0 6 0 123-6 Method Blank-6 MB-060123-7 Method Blank-7 MB-060123-8 Method Blank-8 Area counts of the highest method blank multiplied by two Area Counts of the LOQ standard 7923 18778 8061 6404 5688 14035 5693 4299 37556 179926 1 ~ PFHS Area Counts 50210 162287 ~~ PFOS "'51 712 102452 (2) Highestmethodbhnk area count for PFOS is 25856 (Method Blank-2) when Method Blank-5 isexcludedas on outlier. Trip Blank Prior to sample collection, one sample container was filled with 450 mL of ASTM Type I water, sealed, and shipped to the sample collection site along with the empty containers. This sample was analyzed as the fieldhip blank. The trip blank serves as an additional method blank that account for any storage conditions andlor holding time issues that the samples may experience. The resultant field blank concentrations for PFBS, PFHS, and PFOS were <0.0248 ngImL, <0.0252 ngImL, and <0.0249 nglmL, respectively. System Suitability Five replicate injections of the 1.O ppb (nominal concentration) extracted calibration standard were analyzed at the beginning and end of the analytical sequence to demonstrate overall system suitability. The relative standard deviation (RSD) of the peak area counts and the RSD of the peak retention times were less than 5% and 2%, respectively, for all analytes which met method criteria. Continuing Calibration During the course of the analytical sequence, several continuing calibration verification samples (CCVs) were analyzed to confirm that the instrument response and the initial calibration curve were still in control. ETS-8-154.1 states that all CCV recoveries must be within &25%for the data to be acceptable. All analyte CCV recoveries met methodcriteria of &25%. Lab Control Spikes (LCSs) Replicate low (0.05 ng/mL nominal concentration) and high (0.5 nglml nominal concentration) lab control spikes were prepared and extracted on the same day as the samples. LCSswere prepared by spiking known amounts of the analytes into 40 mL of ASTM Type Iwater to produce the desired concentration. The spiked water samples were extracted and analyzed in the same manner as the samples. Individual LCS results, along with the average and percent RSD, for the extraction set are presented in the data table below. All LCS recoveries met method criteria for both accuracy and precision. E054210 Interim Report #l8 NorthernAlabama Potable WaterSystems, December 2005 Page 14 of 122 05-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Lhxemfer 2005 Table 9. Lab Control Spike Results. I LCS-060123-1 LCS-060123-2 LCS-060123-3 LCS-060123-4 LCS-060123-5 LCS-060123-6 Avenge %Recovery f %RSD uJPFBS Conc. Conc. % Recovery 0.0496 0.0496 0.0496 0.496 0.496 0.496 0.0469 0.0473 0.0483 0.485 0.473 0.497 94.6 95.4 97.4 97.8 95.4 100 96.8% f 2.2% "PFHS I I Spiked - Conc. fnNmL) ______ ~ ~~ "PFOS I I ~~ ~ 0.0503 0.0459 91.2 0.0499 0.0453 90.8 0.0503 0.0456 90.8 0.0499 0.0469 94.0 0.0503 0.0465 92.4 0.0499 0.0429 86.0 0.503 0.500 99.4 0.499 0.477 95.6 0.503 0.472 93.8 0.499 0.462 92.6 0.503 0.488 97.0 0.499 0.475 95.2 I 94.1% f 3.6% 92.3%f3.9% ionand percent recoveryvalues (3 signiint figures) and %RSD (2 eportedvalues may m y sliihtly from the raw data. Because a field sample duplicate (separate container) was collected at each sampling location, duplicate/replicate extractions of a given sample were not performed. Overall method precisionwas determined using LCSs. Field Matrix Spikes Low (nominal concentrationof 0.1 ng/mL) and high (nominal concentration 1.O ng/mL) field matrix spikes were collected at each sampling point to verify that the analytical method is applicable to the collected matrix. Field matrix spike recoveries within 100k30% confirm that "unknown"components in the sample matrix do not interferewith the extraction and analysis of the analytes of interest. Field matrix spikes will be presented in the next section with the sample data. DATASUMMARY AND DISCUSSION Table 10 below summarizes the sample results and field matrix spike recoveries for all of the sampling locations plus the trip blank. All field matrix spikes produced recoveries within 100+30% demonstratingthat the analytical methodwas appropriate for the matrices analyzed. 05-0270 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Page 15 of 122 Table I O . Sample and QC Results. I Location I1 DPWS PW FWOI 3M LIMS ID "PFBS 05-021 0 Interim Report f18 Northern Alabama Potable Water Systems, December 2005 "PFHS "PFOS E054210 Interim Report Wl8 Northern Alabama Potable Water Systems, December 2005 Page 16 of 122 Table 10 Continued. E054210 Interim Re ori C l 8 Northern Alabama Potable Water Systems, Decemger 2005 E05-0210-92269 R M P PWFWO2HS 051214 Average Sample Concentration(ng/mL)f%RPd" 0.962 96.9 e0.0248 0.927 92.1 e0.0252 0.811 81.3 e0.0249 E05-0210 Interim ReporiCl8 Northern Alabama Potable Water Systems, December 2005 Page 17 of 122 Table 10 Continued. 05-0210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 (1) The analyticaluncertaintyof the PFBSresutsis 100?5.3% based on methodaccuracy and precision. All resultsand averagesare presented with three significantfigures, %RPDto two significantfigures. Sampleconcentrations.averages, and %RPDvalues mayvary slightly from the raw data. Samples with reported resultsless thanthe LOQ of 0.0248ng/mL may or may nothave PFBSpresent. Resultsless thanthe LOQare notquantified. (2) The analyticaluncertaintyof the PFHSresutsis 10M9.3% based on methodaccuracy and precision. All resultsandaveragesare presentedwith three significantfigures, %RPDto two signifwnt figures. Sampleconcentrations,averages, and %RPDvalues may vary slightlyfrom the raw data. Sampleswith reportedresultslessthanthe LOQof 0.0252nglmLmay or may nothave PFHS present. Resultslessthanthe LOQ are notquantified.. (3) The analyticaluncertaintyof the PFOSresuts is 100~11%basedon methodaccuracy and precision. All resultsand averagesare presentedwith three significantfigures, %RPDto two significantllgures. Sampleconcentrations.averages, and %RPDvalues mayvary slightlyfrom the raw data. Samples with reportedresultslessthanthe LOQof 0.0249 nghLmay or may nothave PFOSpresent. Resultslessthan the LOQ are notquantified. (4) NA: Not applicable. %RPD values not determined when concentrations for the sample and sample duplicate are below the stated LOQ. E054210 lnterfm Report I18 Northern Alabama Potable Water Systems, December 2005 Page 18 of 122 E05-0210 Interim R ort #18 Northern Alabama Potable Water Systems, DecmTeer ZOOS STATISTICAL METHODASND CALCULATIONS Statistical methods used to interpret sample results include averages and standard deviations. The Analyst software program calculated sample concentrations using resultant analyte peak areas and the established quadratic, l / x weighted, calibrationcurve. None of the samples analyzed for this interim report required dilution. Sample calculations and equations used to report method accuracy and precision are described below. Accuracy and Precision Equations LCS/Surrogate Percent Recovery = Calculated Concentration ,,oo% Spike Concentration Sample Spike Recovery = (Spiked Sample Concentration- Average Concentration:Fie Sample & FieldSample Dup !*loo% Spike Concentraton %RPD (RelativePercent Difference) = Absolute differencebetween sample duplicates 100% average sample concentration Determination of Analytical Uncertainty Both the accuracy (percent recovery) and precision (%RSD) of the lab control spikes are used to estimate the analytical uncertainty for a given analyte. The measured precision (%RSD) is then used to determine the range of the accuracy. The following example demonstrates how the analytical uncertainty, as defined here, is calculatedfor PFOS. Example: First, the accuracy range is determined by multiplyingthe average recovery by the precision. 92.35 (average accuracy)*0.0389 (precision) = 3.59 (accuracy range) Next, the accuracy range is added and subtracted from the average accuracy. 92.35 + 3.59 = 95.94; 92.35 -3.59 = 88.76 Thus, LCS accuracy results range from 88.76% to 95.94%. The absolute difference of the low and high ends of this range, when compared loo%, are then calculated. 100%-95.94 = 4.06% 1Q0%-88.76%= 11.23%. The most conservative (largest) absolute difference is then used as the analytical uncertainty for the given analyte. Therefore, the analytical uncertaintyfor PFOS as defined here, is given as 100*11% (2 significant figures) for the data associated with this project. 05-0210 Interim Report 118 Northern Alabama Potable Water Systems, December 2005 Page 19 of 122 EO5-0210 Interim Report #18 NotthemAlabama Potable Water Systems, December 2005 STATEMENT OF CONCLUSION Sample results for this project were presented in Table 1. Laboratory control spikes were used to determine the method accuracy and precision for the target analytes for this data set. The accuracy and precision were then used to estimate the analytical uncertainty for each of the analytes (PFBS: 100+5.3%; PFHS: 100f9.3%, and PFOS: 100+11%). Recoveries of field matrix spiked samples demonstrated that the overall analytical method was appropriate for the matrix collected. REFERENCES Rorabacher, David B. "Statistical Treatment for Rejection of Deviant Values: Critical Values of Dixon's 'Q' Parameter and Related Subrange Ratios at the 95% Confidence Level." Anal. Chem. 1991, 63,139-146. LISTOF ATTACHMENTS Attachment A: Selected Chromatogram and Calibration Curves Attachment B: Extraction and Analytical Methods Attachment C: Protocol and ProtocolAmendments E059210 Interim Report # I 8 Notthem Alabama Potable Water Systems, December 2005 Page 20 of 122 05-0210 Interim Re ott #18 Northern Alabama Potable Water Systems, Decemger 2005 SIGNATURPEAGE We certifythat this reportis a true and completerepresentation of the data for this study: JaisimhaKesari, P.E.,DEE, Study Director 3,',//06 Date William K. Reagen, Ph.D., MichaelA. Santoro, 3M EnvironmentalLaboratoryManager Date Sponsor Representative / Date E054210 Interim Report 118 Northern Alabama Potable Water Systems, December 2005 Page 21 of 122 EO54210 Interim R ort #18 Northern Alabama Potable Water Systems, Decemyer 2005 ATTACHMENT A: SAMPLE CHROMATOGRAMSAND CALIBRATION CURVES E054210 lnierim Report #le Northern Alabama Potable Water System, December 2005 Page 22 of 122 ,,,,. """I,"."? - r-....*..--..-...--...r.....-..T.........*..-....-..z.......~.......-.....-....s.."_"..*,-!% " --= .I r". I: . .. . ~ ..A. .l . . . . .1.7...~9..... .. . .. ,.s. I n u .... I: . . . . . . ... l.....,,l,.? .l......l..".. .>......,...,.._.... u E050210 Interim Re ott #18 Northern Alabama Potable Water Systems, Decemger 2005 R..",t. *..e: 0060126. 6U5-021u.rdb - 0060126. -I 05-0210Interim Repod #18 NorthernAlabama Potable Water Systems, December 2005 Page 23 of 122 . . I, .... .,-.... ... .. . . .,*',./*<., I. . <. I .., 6 I ,~~ ,..*. 3 . 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'I... .., "1"L. 1 05-0210 Interim Reporl #I8 Northern Alabama Potable Water Systems, December 2005 05-0210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Page 29 of 122 05-0210 Interim Re ort U18 Northern Alabama Potable Water Systems, Decemger 2005 " I' i "5 EOS-0210Interim Report #la Northern Alabama Potable Water Systems, December 2005 Page 30 of 122 E054210 Interim Re ort #I8 Northern Alabama Potable Water Systems, Decemger 2005 E054210 lnterfm Report #18 Northern Alabama Potable Water Systems, December 2005 Page 31 of 122 05-0210 Interim Report 118 Northern Alabama Potable Water Systems, December 2005 05-0210 lntetfm Report #I8 NorthernAlabama Potable Water Systems, December 2005 Page 32 of 122 05-0210 Interim Re ort #18 Northern Alabama Potable Water System, Decemger 2005 05-0210 Interim Repori 618 Northern Alabama Potable Wafer Systems, December 2005 Page 33 of 122 05-0210 Interim Re ort 618 Non%ernAlabama Poable Water Systems, Decemker 2005 ATTACHMENBT: EXTRACTIOANND ANALYTICAML ETHODS E054210 lnierim Report #18 Nottftern Alabama Potable WaterSystems, December 2005 Page 34 of 122 05-0210 Interim Re ort 118 NortnemAlabama Potable Water Systems, Decemter 2005 3M EnvironmenMLabomby Method Determination of Perfluorinated Acids, Alcohols, Amides, and Sulfonates In Water By Solid Phase Extraction and High Performance Liquid Chromatography/Mass Spectrometry Method Number: ETS-8-154.1 Adoption Date: 28 Apr 2000 Revision Date: 5 Mafi 2003 Effective Date: 5 May, 2003 Approved By: William K. Reagen Manager OJ/os/a3 Date ETs-8-154.1 PaDe 1 of 17 Determinationof Perfluorinated Compounds in Water Using SPE and LCIMS. 05-0210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Page 35 of 122 EO5-0210 Interim R orf #18 Northern Alabama Potable Water Systems, Decem%r2005 I Scope and Application This methodwas validated for the collection, extraction, and analytical procedure for the determination of Petfluorooctane sulfonate (PFOS), Perfluorooctanesulfonylamide (FOSA), and Peifluorooctanoate(PFOA) in groundwater, surface water, and drinking water samples. This methodmay also be applied to the determination of other petfluorinated acids, alcohols, amides, and sulfonates in similar matrices, as long as the defined QC elements are satisfied and with the understandingthat the methodis not validated for compounds outsiie the scope of the original protocol This methcd is based in part on the report "Method of Analysis for the Determination of Pertluorooctane sulfonate (PFOS), Petfluorooctane sulfonylamide (PFOSA), and Peffluorooctanoate (POAA) inWater" (seeSection 17), as developed and validated by Exygen Research (formerly Centre Analytical Laboratories, Inc.). 2 Method Summarv Water samples are collectedfrom a site of interestand shipped cold to an analybcalfacility. Peffluorinatedacids, alcohols, amides, and sulfonates are extractedfrom 40mLwater samples using C18 solid phase extraction (SPE) cartridges. The compounds are eluted from the C18 cartridge, using methanol. Separation, identification, and measurement are accomplished by highperformance liquidchromatography/tandem massspectrometry (HPLC/MS/MS) analysis. Highperformance liquid chromatographyhnass spectrometry (HPLC/MS) may be used if the defined QC elements are satisfied. The concentration of each identified componentis measuredby comparing the MS response of the quantitation ion produced by that compoundto the MS response of thequantitation ion producedby the same compound in an extracted calibration standard (externalstandard). 3 Definitions 3.1 Analytical Sample A portion of an extracted Laboratory Sample prepared for analysis. 3.2 Calibration Standard A solution preparedfrom the Working Standard (WS) and extractedaccording to this method. The calibrationstandard solutions are used to calibrate the instrument responsewith respectto analyte concentration. 3.3 Duplicate Sample (DS) A OS is a separate afiquotof a sample, taken in theanalytical laboratory that isextractedand analyzed separately with identicalprocedures.Analysis of DSs comparedto that of the first aliquot give a measure of the precision associated with laboratory procedures, but not with sample collection, preservation, or storage procedures. 3.4 Field Blank Control Sample (FB) ASTM Type I water placed in a sample container in the laboratory and treated as a sample in all respects, includingexposure to samplingsite conditions,storage, presentatin and all analytical procedures. The purpose of the FB is to determine if test substances or other interferences are present in the feld environment ETS-8-154.1 Page 2 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/Ms. E050210 Interim Report # I 8 NorthernAlabama Potable Wafer Systems, December 2005 Page36of122 05-0210 Interim Re ort # I 8 Nortrwmr Alabama Poebh? Water Systems, Decem!erZooS 3.5 Field Duplicate (FD) A sample collected in duplicate at the same time as the sample and placed under identical circumstances and treated exactly the same throughout field and laboratory procedures. Analysis of FD comparedto that of the first sample gives a measure of the precisionassociated with sample collection, preservationand storage, as well as with laboratory procedures. 3.6 Field Matrix Spike (FMS) A sample collected induplicateto which knownquantities of thetarget analytes are added in the feld at the time of sample collection. Alternatively, the knownquantity of target analytes may be addedto the sample bottle in the laboratory before the bottles are sent to the field. A known, specifcvolume of sample must be addedto samplecontainer without rinsing. This may be accomplished by making a "fill to this level" line on the outside of the sample container. The FMS should be spiked at approximately 50 150% of the expected analyte concentration in the sample. If the expected range of analyte concentrations is unknown, a low and a high spike may be preparedto increasethe likelihoodthat a spike at an appropriate range is made. The FMS is analyzed to ascertain if any matrix effects, interferences, or stability issues may complicate the interpretation of the sample analysis. 3.7 Field Spike Control Sample (FSCS) An aliquot of ASTM Type I water to which known quantities of the target analytes are added in the k l d at the time of sample collection(at an appropriateconcentrationto be determined by the prqect lead) or in thelaboratoryprior to the shipment of the collection bottles. The FSCSis extracted and analyzed exactly like a sample to determine whether a loss of analyte could be attributed to sample storage andlor shipment. A low and high FSCS may be appropriate when expected sample concentrations are not known. 3.8 Laboratory Control Sample (LCS) An aliquot of ASTM Type Iwater to which knownquantities of the target analytes are added in the laboratory. Two levels are included, one at the LLOQ (approx. 25 pg/mL), the other at a concentration of approx. 100 250 pg/mL or another concentration to be determined by the project lead. The LCS is extracted and analyzed exactly like a laboratory sample to determine whether the methodology is in control, and whether the laboratory is capable of making accurate measurementsat the requiredmethoddetection limitand higher. 3.9 Laboratory Sample A pottionof a sample receivedfrom the field for testing. 3.10 Limit of Detection (LOD) The LOD is the lowest concentrationof an analyte that can be measuredand reportedwith 99% confdence that theanalyte concentration is greater than zero. If required, the LOD may be determined in several ways, including signal-tenoise ratio and statisticalcalculations. 3.1 1 Limit of Quantitation (LOQ) The LOQ for a dataset is the lowest concentration (LLOQ) or highestconcentration (ULOa) that can be reliably achieved within the specitied limits of precision and accuracy during routine operating conditions. Note: For many analytes, the LLOQ analyte concentration is selected as the lowest non-Zero standard in the calibration curve to simplify data reporting. Sample LLOQs are matrixdependent. 3.12 Matrix Spike (MS) ETS-8-154.1 Page 3 of 17 Determinationof Perfluorinated Compounds in Water Using SPE and LCIMS. 05-0210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Page 37 of 122 EO5-0210 Interim Re ort # I 8 Norfhem Alabama Potable Water Systems, Decemger 2005 A matrix spike is an aliquot of a sample, to which knownquantities of target analytes are added in the laboratory. The MS is extractedand analyzedexactly like a laboratory sampleto determine whether the sample matrix contributes bias to the analytical results. The background O O R O ~ R ~ ~ ~o~fOthReSanalytesin the sample matrix must be determinedin a separatealiquot and the measuredvalues inthe MS corrected for backgroundconcentrations. 3.13 Method Blank An aliquot of ASTM Type Iwater that is treated exactly likea laboratory sample including exposure to all glassware, equipment, solvents, and reagents that are used with other laboratory samples.The methodblank is used to determine if test substances or other interferences are present inthe laboratory environment, the reagents, or the apparatus. 3.14 Method Detection Limit (MDL) Determination A MDL is the statisticallycalculatedminimumamount of an analyte that can be measuredwith 99% confdence that the reportedvalue is greater than zero. One of several processes that may be used to establish a LOD value is found in 40 CFR Part 136 Appendbc B. 3.15 Sample A sample is a man portion collect& rfom a larger quantity d materialintendedio representthe original source material. 3.16 Spiking Stock Standard (SSS) A solution prepared from stock standards used to prepare the working standard. 3.17 Stock Standard (SS) A concentrated solution of a single analyte prepared in the laboratory with an assayed reference COmpOUnd. 3.18 Working Standard (WS) A solution of severalanalytes preparedin the laboratoryfrom SSs and dilutedas neededto prepare calibration standards and other required analyte solutions. 4 Warnings and Cautions 4.1 Health and Safety The acute and chronic toxicity of the standards for this method have not been precisely determined; however, each should be treated as a potential health hazard. Unknownsamples may contain high concentrationsof volatile toxic compounds. Sample containers should be opened in a hood and handled with gloves to prevent exposure. The laboratoryis responsible for maintaining a safe work environment and a current awarenessof local regulationsregarding the handling of thechemicals used in this method. A referencefile of material safety data sheets (MSDS) should beavailableto all personnelinvolved in these analyses. 4.2 Cautions None 5 Interferences ETS-8-154.1 Page 4 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LCIMS. E054210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Page 38 of 122 05-0210 Interim Re ort # I 8 Non8hernAlabama Po&ble Water Systems,Decemger 2005 Duringextractionand analysis, major potential contaminant sources are reagents and solid phase extraction devices. All materials used in the analyses shall be demonstrated to be free from interferences under conditions of analysis by running method blanks. Parts and supplies that contain TeflowB should be avoided due to the possibility of interference andlor contamination. These may include, but are not limitedto: wash bottles, Teflon@linedcaps, autovial caps, HPLC parts, etc. The use of disposable micropipettes or pipettes to aliquot standard solutions is recommended to make calibration standards and matrix spikes. 6 Instrumentation, Supplies, and Materials Note: Brand names, suppliers, and part numbers are for illustrative purposes only. Equivalent performance may be achieved using apparatus and materials other than those specified here, but demonstration of equivalent performance that meets the requirements of this method is the responsibility of the laboratory performing the analysis. 6.1 Instrumentation Balance, analytical (display at least O.OOOlg), Meffler HPLClMSMS or HPLCMSsystem, as described in Section IO. 6.2 Supplies and Materials. Sample collection botHes-LDPE (e.g., Nalgene") narrow-mouth bottles with screw cap. Note: Do not use Teflon bottles or Teflon lined caps. Coolers for sample shipment Icefor sampleshipment. Vacuum pump, BOchi. visiprep vacuum manifold, Supelco. Sep Pak Vac 6cc (lg) tC18 cartridges (part # WAT 036795),Waters. 50mLdisposable polypropylene centrifuge tubes, W R . 15mLdisposable polypropylene centrifuge tubes,W R . Disposable micropipefles(50 I-L, 100 ZOOpL), Drummond. Class A pipettes and volumetric flasks, various. Hypercarb drop-in guard column( h m ) (part # 844017 400), Keystone. Stand-alone drop-in guard cartridge holder, Keystone. 125mL LDPE narrow-mouth bottles, Nalgene. 2mL clear HPLCvial kit (cat # 5181 3400),Agilent/Hewlett Packard. Standard labequipment(graduatedcylinders, disposable tubes, etc.),various. 7 Reagents and Standards N o k Suppliers and catalog numbers are for illustrative purposes only. Equivalentperformance may be achieved using chemicals obtained from other suppliers. Do not use a lesser grade of chemical than those listed. ETS-8-154.1 Page 5 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LCIMS. 05.0210 Interim Report #I8 Northern Alabama Potable Water Systems, December 2005 Page 39 of 122 EO5-0210Interim Re ort #I8 Northern Alabama Potable Water Systems, Decemker 2005 7.1 Chemicals Methanol (MeOH), HPLCgrade, JT Baker, Catalog No.JT9093 2. AmmoniumAcetate, Reagentgrade, Sigma-Aldrich, Catalog No. A-7330 ASTM Type I Water, prepared in-house. Sodium Thiosulfate, Reagent grade, JT Baker. 7.2 Standards Potassium perfluorooctane sulfonate Peffluorooctane sulfonylamide Ammonium peffluorooctanoate Others as required. 7.3 Reagent Preparation 25OmghnL sodium thiosulfate solution - Dissolve 259 of sodium thiosulfate in 1OOmL reagent water. 40% methanolwash solution-Measure4OOmL methanoland adjust volume to 1.OLwith reagent water. 100mMammoniumacetate solution (AnalysispWegh 7.719 of ammonium acetate and dissolve in 1.OL of reagent water. Dilutethe 1OOmMsolution by a factor of 50 to makethe 2mM ammonium acetate solution usedfor mobilephase A. Note: Alternativevolumes may be prepared as long as the ratiosof the solvent to solute ratios are maintained. 7.4 Spiking Stock Standard (SSS) Preparation The following standardpreparation procedure serves as an exampleand may be changedto suit the needs of a particular study. For example, pL volumes may be spiked into volumetric flasks when diluting stock solutions to appropriate levels. IOOpglmLeach PFOS, PFOSA,and POAA SSSeWeigh out 1Omgof analytical standard (correctedfor percentsalt and purity-i.e., 10 mg CBF1,S03Kpurity 90% = 8.35mg C8F17S&-) and diluteto 1OOmLwith methanol in a IOOmLvolumetric flask. Transfer to a 125mLLDPE bottle or other suitable container. Prepare a separate solution for each analyte. Solutions may be stored in a refrigerator at 4'r+2"C for a maximum p e r ' d of 6monthsfrom the date of preparation. l d m L mixedSSS-Add 1.OmL each of the loOpg/mLSSSs (frcm7.4.1) to a lOOmL volumetric flask and bring up to volume with methanol. O.lpg/mL mixed S S S d d d 10.0mL ofthe l.Oclg/mLmked solution (from7.4.2) to a 1OOmL volumetric flask and bring up to volume with methanol. 0.0llrglmL mixed SS!LAdd 1O.OmL ofthe 0.lpghnL-mixedsolution (from 7.4.3) to a 100mL volumetric tlask and bring up to volume with methanol. Storage Condtlons-Store all SSSs in a refrigerator at 4"-C for a maximumperiodof 6 monthsfrom thedate of preparation. 7.5 Calibration Standards The following standard preparation procedure serves as an exampleand may be changedto suit the needs of a particular study, provided the concentrations are calculated correctly. ETS-8-154.1 Page 6 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LCfMS. 05-0210 Interim Report 618 Northern Alabame Potable Water Systems, December 2005 Page 40 of 122 05-0210 Interim Re ort #18 NonMmAlabama Potable Water Systems, Decemier 2005 1 W m Leach PFOS, PFOSA, and POAAstock standardsolutions-Weigh out 1Omgof analyticalstandard (correctedfor percentsalt and purity) and diluteto 1OOmLwith methanolin a 100mL volumetric flask. Transfer to a 125mL LDPE bottle or other suitable container. Prepare a separatesolutionfor each analyte. Store solutionsin a refrigeratorat 4 " e T for a maximum period of 6 months from the date of preparation. 1pg/mL Working S t a n d a M d d l.OmL each of the IOOpg/mL SS solutions (from7.5.1) to a 100mL volumetric flask and bring up to volume with methanol. O.Ipg/mL Working Standard- A d d 1O.OmLof the 1.Opg/mL mixed solution(&om7.5.2) to a IOOmL volumetric flask and bring up to volume with methanol. 0.01pglmL Working Standard- A d d 1O.OmLof the 0.1p g h L mked solution (from 7.5.3) to a 100mL volumetric flask and bring up to volume with methanol. Storage C o n d i t i o M t o r e allWSs in a refrigeratorat 4WC for a maximum period of 6 months from the date of preparation. Calibration StandaKCPreparecalibrationsolutions inASTM Type Iusingthe following table as a guideline: 0.0 0 40 0.010 100 40 0.010 200 40 0.010 400 40 0.10 100 40 0.10 200 40 0.10 300 40 0.10 400 40 1.o 100 40 I.o 400 40 1 .o 1000 40 0 25 50 100 250 500 750 I000 2500 10000 25000 The stancJards are processedthroughthe extractionprocedure (Section 1I)id,entical to the laboratorysamples. The concentrationof the calibrationstandard inthe final extractis equal to 8X the initial concentration.due to the concentrationof the standard duringthe extraction process. Storage Conditions-Store all extractedcalibrationstandards in 15mL polypropylenetubes at 4"&'C, for a maximum period of two weeks from the date of preparation 8 Sample Collection and Handling Note: Sampling equipment, includingautomaticsamplers, must be free of Teflontubing, gaskets, and other parts that may leach interfering analytes into the water sample. Automatic samplers that compositesamples over time should use refrigeratedpolypropylenesample containers if possible. Sample bottles should not be rinsed before sample collection. Labeling: Eachsample bottlemust display informationregardingthe collectionof that sample, the individualcollectingthe sample, and any matrix spike that has been added to the sample. ETS-8-154.1 Page 7 of 17 Determination of PetfluorinatedCompounds in Water Using SPE and LC/MS. E050210 Interim Report C18 NotihemAlabama Pofable Water Systems, December 2005 Page 41 of 122 05-0210 Interim Re ort 618 Northern Alabama Potable Water Systems, Decemier 2005 This includes the volume and concentration of any spiking solution added and the volume and identificationof any preservatives added inthe field. Spiking: The spiking schemewill be clearly outlined in the samplingplan, including whether the sampleswill be spiked in the fEld or in the laboratory prior to the shipment of the bottles to the site. If spiking is to be performed in the field, materials and specific instructions will be included in the sampling kit. Be sure to clearly labeleach bottlewith spiking information if applicable. Tap Water: Open the tap and allow the system to flush until the water temperature (15klo"C) has stabilized(usually about two minutes). Adjust the fEow to about 500mUmin and collect samples from the flowing stream. Ground Water: Purge the well of standing water usinga pump or a bailer. Collectthe sample directly from the pump or from the bailer. SurfaceWater: When sampling from an open body of water, fill the sample container with water from a representative area. Sample Dechlorination:All samplesshould be iced or refrigerated at 4"%2'C and kept in the dark from the time of collection untilextraction. Residual chlorineshould be eliminatedby adding 200pL of a 250mg/mLsodiumthiosulfate solution to each tapwater sample and associated FB and FSCS (which may be placed in each bottle before leaving for the sampling site or done in the field.). HoldingT i m (HT): Resultsof the timektorage study of all target analytes showedthat the three cumpoundsare stablefor 14days in water samples when the samplesare dechlorinated and storedas described in the previous section(seealso references in section 17).Therefore, laboratory samples must be extracted within 14 days and the extracts analyzed within 30 days of sample collection. If the HT exceeds 14 days, great care is usedwhen evaluating field spikes to avoid misrepresentation of the sample concentration. 8.1 Field Blanks Process a Field Blank ControlSample(FE) along with each sample set (samples collectedfrom the same general sample site at approximately the same time). At the laboratory, priir to sample collection, fill a sample containerwith ASTM Type Iwater, seal, and ship the FBto the sampling site along with the empty sample containers. Retum the FB to the laboratory with the filled sample bottles. When sodiumthiosulfate is added to samples, use the same procedureto presenrethe FB. 8.2 Field Duplicates Collecta Field Duplicate (FD) for every ten (IO) samples collected or per each samplingset, if less than 10 samples are collected. Separate FDs must be collected for each type of water sample (ground, tap, etc.) collected. Collectthe FD immediatelya k r the sample. Preserve, store and ship FD using the same procedures as used for the samples. 8.3 Field Spike Control Sample (FSCS) A Field Spike Control Sample (FSCS) must be prepared for each sample shipment If multiple coolers are used to ship a set of samples, each cooler must contain a FSCS. At the laboratory, fill a samplecontainer with 100mLof ASTM Type Iwater. Seal and ship to the sampling site along with the emptysample containers and FEs. Samplesmay either be spikedin the feld or in thelaboratory prior to shipment The methodemployedshould be consistent throughout the study. If the samples are to be spiked in the feu, be sure to send appropriate supplies and instructionsfor the field personnelto follow. ETS-8-154.1 Page 8 of 17 Determinationof PertluorinatedCompounds in Water Using SPE and LCIMS. EO5-0210 Interim Report #18 NorthernAlabama Potable Water Systems, December 2005 E054210 Interim Re oti U18 Northern Alabama Potable Water Systems, Decemger 2005 Seal and gently invert the FSCS to mix. Store and ship the FSCS using the same procedures as used for the samples Provide information on sample collection, preservation, shipment and storage. List applicable holding times. Include sample stabildy and extractstorage requirements. Reference the method used for sample preparation, if applicable. 8.4 Field Matrix Spike (FMS) A Field Matrix Spike (FMS) must be prepared for each sampling location. One unspiked sample from the same location must accompany the FMSto determine endogenouslevels in the sample. The samples should be clearly identiable as being from the same location. Samplesmay either be spiked inthe field or in the laboratoryprior to shipment. The method employedshould be consistent throughout the study. Ifthe samplesare to be spiked in the feld, be sure to send appropriate suppliesand instructions for the field personnelto follow. 9 Quality Control and Data Quality Objectives Analytical results of the FB, FMS, FD, and FSCS should be evaluated at the conclusion of the study to help interpret the quality of sample data. Analytical results for these controollduplicate samplesmust be reportedwith the sample data. 9.1 Solvent Blanks Solvent blanks are analyzed with each sample set to determine contamination or carryover. Aliquots of methanol represent the solvent used for the standard curve and the sample extraction. Solvent blanks should have area counts that are less than 50% of the area count of the lowest calibration standard. Solvent blanks should be analyzed prior to and following each calibration curve, each set of system suitability samples, and after no more than 10 unknown sample extracts. If instrument carryover is a problem consecutive solvent blanks may be necessary. Inthis case the area counts of the solvent blanksshould returnto ~50%of the lowest calibration standard prior to the injection of further standards or samples. 9.2 Method Blanks A method blank consists of an aliquot of ASTM Type Iwater, equal in volume to the samples, and extractedin thesame manneras the samples. At least two methodblanksshouldbe prepared and analyzed each day that extractions are performed for a particular study or project. When analyzedt h e area counts of t h e s e samples must be less than 50% ofthe area count of the lowest calibration standard. 9.3 Sample Replicates All samples, including field spikes, trip blanks, etc., shouldbeextractedat leastin duplicate, and in triplicate if diffcultieswere encountered in the sampling and/or holding condiiionsof the samples. The relative percent difference(RPD) of duplicate samples or relativestandarddeviation (RSD) should be less than 15%for the precision of samplepreparationand analysis to be considered in control. 9.4 Matrix Spike Matrix spikes are preparedfor each sampletypeand analyzedto determine the matrix effecton the recoveryefficiency. Matrixspike recoveriesshould fall within f25% of expected values. If the matrix spikes fail, evaluate the lab control spikes. If the LCS are within acceptance criteria there may be matrix issues in the samples. Discuss these in the final report. ETS8-154.1 Page 9 of 17 Determinationof Perfluorinated Compounds in Water Using SPE and LCIMS. EO5-0210 lnlerm Report #l8 Northern Alabama Potable WaterSystems, December 2005 Page 43 of 122 05-0210 Interim Re ort #le NorthernAlabama Potable WaterSystems, Decemier 2005 Matrix spikeduplicates are prepared periodically to measurethe precisionassociated with the analysis. Analyze a matrix spike and matrix spike duplicate (if prepared) in the same run as the original sample. Matrix spike and matrix spike duplicate concentrations should fall in the mid-range of the initial calibration curve or shouldbe prepared at 1.55times the endogenousconcentration of the analyte. Spike concentrations should fall inthe low-range of theinitialcalibrationcurve if extremely low-levels are expected. Generally two or more levels are prepared, one in the low range of the curve and one in the mid-range. This avoids the needto pre-screen unknown samples prior to preparation. 9.5 Laboratory Control Spike Lab controlspikes are prepared for each study to ensure recoveryof the target analytes. These should be prepared at a minimum of 2 levels and in duplicate or triplicate. Recovery of these samplesshould be within j:25% of expected values, and the RPD (orRSD)be 5 15%. If recoveries fall outside these limits the samples should be addressed in the final report. 10 Calibration and Standardization 10.1 Instrument Setup Note: Inthis example, a MicroMass Ultimam Liquid ChromatographyTandem Mass Spectrometer (LC/Ms/MS) is used. Other brands of LC/MS/MSs as well as single quadrupole mass spectrometers (LC/MS)may be used as long as the methodcriteriaare met Brand names, suppliers, part numbers, and models are for illustrative purposes only. Equivalent performance may be achieved usingapparatus and materialsother than those specifiedhere, but demonstration of equivalent performance that meets the requirements of this method is the responsibilty of the laboratory. The operator must optimize and document the equipment and settings used. Establishthe LC/MS/MSsystem and operating conditions equivalent to the following: Mass Spec: Micromass Ultima (Micromass) Interface: Electrospray (Micromass) Mode: Electrospray Negative, Multiple Response Monitoring (MRM) Harvardinfusionpump (Harvard Instruments),for tuning Computer: COMPAQ ProfessionalWorkstation AP200 Software: W i n d m NT, MassLynx3.3 HPLC: Hewlett Packard (HP) Series 1100 HP Quaternary Pump HP Vacuum Degasser HP Autosampler HP Column Oven Note: A 4 x 1Omm Hypercarbdropin guard cartridge (Keystone, part# 844017 400) may be attached on-line after the purge valve and before the sample injector port to trap any residue contaminantsthat may be in themobilephaseand/or HPLC system. HPLC Column: Genesis C8 (Jones Chromatography), 2.1mm x 50mm, 4pn ETS-8-154.1 Page 10 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LCIMS. 05-0210 Interim Report d18 Northern Alabama Potable Water Systems, December 2005 Page 44 of 122 E050210 Interim R 017#18 Notthem Alabama Potable Water Systems, DecemTer 2005 Column Temperature: 35C Injection Volume: 15pL Mobile Phase (A): 2mM Ammonium Acetate inASTM Type Iwater (See7.3.1) Mobile Phase (B): Methanol 60 40 0.3 0.4 60 1 .o IO 40 0.3 90 0.3 7.0 10 90 0.3 7.5 0 100 0.3 9.0 0 100 0.4 9.5 60 40 0.4 13.5 60 40 0.4 14.0 60 40 0.3 Note: Other HPLC gradients may be used as long as the me id criteria are met. Itmay be necessary to adjust the HPLC gradient in order to optimize instrument performance. Columnswith different dimensions (e.g. 2.1mm x 30mm) and columnsfrom different manufacturers (Keystone BetasilC18 etc.) may be used. 1 PFOA 41 3 169 5.0 PFOS 499 99 5.2 FOSA 498 78 5.8 Other product ions may be chosen at the discretion of the analyst, although mh 99 is suggested for PFOS. Use of the suggested primary ion is recommended. Retention times may vary slightly, on a day-to-day basis, depending on the batch of mobile phase etc. Drift in retention times is acceptable within an analytical run, as long as the drift continues through the entire analysis and the standards are interspersed throughout the analytrcal run. 10.2 Tune File Parameters The following values are provided as an example. Actual values may vary from instrument to instrument.Also, thesevalues may be changedfrom time to time in order to optimize for greatest sensitivity. PFOA PFOS FQSA 0.2 0.4 0.2 0.4 0.2 0.4 10 25 30 60 20 50 20 30 50 80 30 60 ETS-8-154.1 Page 11 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LCMIIS. 05-0210Interim Report #18 Notthem Alabama Potable Water Systems, December 2005 Page 45 of 122 EOIO210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 ~~ Capillary Hexapole 1 Aperture 1 Hexapole 2 Source Block Temp. Desolvation Temp. LM Res 1 HM Res 1 IEnergy 1 Entrance Exit LM Res 2 HM Res 2 IEnergy 2 Multiplier Cone Gas Desolvation I Gas Cell 2.6 3.5kV 0.5V 0.2v 0.8V 100-1 50C 250400C 12.5 15.0V 12.5 15.0V 0.N -2v IV 11.ov 11.ov 1 .ov 650V 15OVhr 700Uhr 3.0-3mbar 1 ETS-8-154.1 Page 12 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LC/MS. EO5-0210 Interim Report #18 Northem Alabama Potable Water Systems, December 2005 Page 46 of 122 05-0210Interim Re ort #18 NorthernAlabama Potable Water Systems, Decemhr 2005 10.3 Calibration Curve s Analyzethe standard curves priorto each set of samples. The validated methodspecifiesthat the standard curve should be plottedusing a linearffi, wei hted Ihc or unweighted. However, the standard curve may also be plottedby quadratic fit (y = a + bx + c), weighted l / x or unweighted, using suitable software. The calibration curves may include but should not be forced through zero. The mathematical method used to calculate the calibration curve should be applied consistently throughout a study. Any change should be thoroughly documentedin the raw data. If the calibrationcurve does not meet acceptance criteria perform routine maintenance or prepare a new standardcurve ( inecessary)and reanalyze. For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range. For example, when attempting to quantitate approximately50 w/mL of analyte, generate a calibration curve consisting of the standards from 25 pg/mL to IO00pg/mL rather than the full range of the curve (25 pg/mL to 25OOO p@mL). This will reduce inaccuracy attributedto linear regressionweighting of high concentration standards. Highand/or low points may be excluded from the calibrationcurves to provide a better ffi over the linear range appropriate to the data or because they did not meet the predetermined acceptance criteria. Low-level curve points should also be excluded if their area counts are not at least twice that of the method andlor solvent blanks. Any curve point may be rejected due to a bad injection or failing to meet accuracy requirements of & 25% (and & 30% for the LLOQ). Justification for exclusionof calibration curve points will be noted in the raw data. A minimumof 6points wiff be used to construct the calibration curve. 10.4 Continuing Calibration Verification (CCV) Continuing calibration verifiitions (CCV)are analyzed to verify the accuracy of thecalibration CUM. Analyze a mid-range calibration standard, one of the same standards used to construct the calibrationcurve, at a minimumafter every tenth sample, not including solvent blanks,with a minimumof one per sample set. Calibrationverification injections must bewithin f25% to be consideredacceptable. The calibration curve and the last passing CCV will then bracket acceptable samples. MultipleCCV levels may be used. 10.5 System Suitability A minimum of three system suitability samples will be injected at the beginning and end of each analyticalrun. Typically these samplesare run priorto the calibration curve. The system suitabilityinjections must have area counts with an RSD of 55% and a retention time RSDof S2% when evaluated independently. I 1 Procedures 1I.I Extraction Scheme Allow samplesto equilibrate to room temperature. Thoroughly mix samples by gently invertingthe sample bottle. Measure4OmL of sample into 5OmL polypropylene centrifuge tubes (Spike the Matrixspikes as required*, replace lid and mix well). Note: Samplesmay needto be prescreenedto determine an appropriate matrix spike level (typically50 150%of sample concentration). Alternatively the samplescould be spikedat more than one level, allowingfor the inappropriate spike levelto be eliminated. ETS-6-154.1 Page 13 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LCMS. E054210 Interim Report 118 NotihernAlabama Potable Wafer Systems, December 2005 Page 47 of 122 05-0210 Interim Re 011 #18 AbfYhemAlabamaPotpbe W a f Systems,Decemger2005 Condition the C18 SPE cartridges (lg, 6mL) by passing approximately 1OmLmethanolfollowed by approximately 5OmL ASTM Type Iwater (flow rate approximately 2 droplsec). Do not let column run dry. Note: For the following steps, maintaina -1 droplsecflow rate. Do not allow the columnto run dry at any time. Loadthe analytical sampleonto the C18 SPE cartridge. Discardeluate. Ten mL of the 40% methanol in water wash mixtureis passedthrough the C18 SPE cartridge to rinseaway potentialinterferencesand then discarded. This step must be omitted if perfluorinated compoundswith chain lengths less than C8 are targeted since these will be lostduringthis wash step. Elute with exactly 5mL of 100% methanol. Collect eluate into graduated 15mL polypropylene centrifuge tubes. This is the target elution fraction (Final volume approximately 4.5 mL as not all of the solvent will leave the SPE column. This will not affectthe calculations in any way since the curve is also extracted). Analyze a portionof the target elutionfractioneluentusingnegative electrospray HPLCAG"VIS or HPLCMS. + Note: Samplesare concentratedby a factor of eight duringthe extraction; InitialVol = 4OmL Final Vol. = 5mL. Samples are stable at room temperature for at least 24 hours. Analytical samples may be stored in a refrigerator at 4"SC until analysis. Standardizationof C18 SPEcolumns-lf poor recoveries are observed, it may be necessary to standardize the C18 SPE columns in the following manner before analyzing samples. Use a standard with an analyte concentration between IO00 and 4000 pg/mL. Repeat the extractionschemefrom the beginning up through the eluting with -5mL 100% methanol. After the eluting with -5mL 100% methanol step, collect an additional postelutionfractionby eluting with an additional 5mL of 100% methanol. Analyze both fractions by H P L W S M S or HPLCMS. If the target fraction contains a minimumof 85% of the respective analytes, it may be considered acceptable. If the wash contains significant standard (>15%), either the wash volume or percentage of MeOH should be decreased. If the post-elution fraction contains significant standard (>15%), the target elution volume should be increased. 11.? Sample Analysis Set up analysis sample queue. Inject the same volume (between 5 25pL) of each standard. analytical sample and blank into the instrument. All sampleswith a concentration > ULOQ must bedilutedand reanalyzed. If dilutionof the final extract fails to produce acceptable results (e.g. poor MS recoveries) dilute the original sample and eextrad 12 Data Analysis and Calculations Calculate the analytical sample (extract) concentration from the standard curve using the following equation: ETS-8-154.1 Page 14 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LCNS. 05-0210 Interim Report #I&? Northern Alabama Potable Water Systems, December 2005 Page 48of 122 E054210 Interim Re OH I18 Northern Alabama Potable Water Systems, Decemger 2005 ExtractConcentration, pg/mL = JPeak area - intermt) (slope) Calculate the percent recovery of the FSCS using the following equation: FSCS % rec. =/FSCS conc.. wlmL) x 100 (Conc. added, pg/mL) Calculate the percent recovery of the MSs using the following equation: M S % rec. = (MSconc., w/mL -Sample conc., pglmL) x 100 (Conc. added, pg/mL) 13 Method Performance Note: Any method performance parameters that are not achieved must be considered in the evaluation of the data. Nonconformanceto any specified parameters must be described and discussed in any reporting of the data. If criteria listed in this method performance section are not met, maintenance may be performed on the system and samples reanalyzed, or other actions taken as determined by the analyst Documentall actions in the raw data. Ifdata are to be reportedwhen performance criteria have not been met, thedata mustbe footnoted on tables and discussedin the text of the repott 13.1 System Suitability A minimumof three system suitability sampleswill be injected at the beginning and end of each analyticalrun. Typically these samples are run prior to the calibration curve. The system suitability injections must have area counts with an RSDof 55% and a retention time RSDof 12% when evaluated independently. 13.2 Quantitation Calibration Curve: The coefficient of determination (8)value for the calibrationcurve must be greater than or equal to 0.990. Each point in thecurve must be within 325% ofthe theoretical concentrationwith the exception of the LLOQ, which may bewithin SO%. Demonstrationof Specificity: S p e u f i i is demonstratedby chromatographic retentiontime (within 3% of standard) and the mass spectral response of unique ions. 13.3 Sensitivity SotventBlanksand Method Blanks: Solvent and methodblank area counts mustbe c 50% that of the lowest standard used in the calibration curve. Limits of Quantitation (LOQ): The lower LOQ (LLOQ) is the lowest non-zero active standard in the calibration curve; the peak area of the LLOQ must be at least W that of the extraction blank. By definition, the measuredvalue of the LLOQ must bewithin 30%of thetheoretical value. ETS-8-154.1 Page 15 of 17 Determinationof Perfluorinated Compounds in Water Using SPE and LCIMS. EO5-0210Interim Report 118 NorthernAlabama Potable WaterSystems, December2005 Page 49 of 122 05-0210 Interim Re ort 118 Northern Alabama Potable Water Systems, DecetnLr 2005 13.4 Accuracy CCV Perfotmance: Calibration verification injections must be within E25%to be considered acceptable. The calibrationcurve and the last passing CCV will then bracketacceptable samples. Multiple CCV levels may be used. Matrix Spikes: Matrix spike percent recoveries must be within f 25% of the spiked concentration. Ifmatrix effectsare suspected, evaluate the LCS results to determine if a matrix effects are present and if the methodis in controlbasedon compliant LCS results. Discuss all results in the anatytical report 13.5 Precision Reproducibility: Reproducibility of themethodis defined by the resultsof duplicate or triplicate analysis of samples. A RPD or RSD of 5 15% will be considered acceptable. System Suitability: The system suitability injections must have area counts with an RSDof s5Y0 and a retention time RSDof 9%when evaluated independently. 14 Pollution Prevention and Waste Management Sample extract waste and flammable solvent is discarded in high BTU containers, and glass pipette waste is discarded in broken glass containers located in the laboratory. 15 Records Eachdata packagegenerated for a study must havethe following informationincluded: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. Printthe tune page, sample list, and acquisition methodto include in the appropriate study folder. Copy these pages and tape into the instrument run log. Plot the calibration curves as described in this method, then print these graphs and store in the study folder. Printdata integration summary, integration method, and chromatogramsand store in the study folder. Summarizedata using suitable software and store inthe study folder. 16 Attachments None. 17 References "Method of Analysis for the Determinationof PerRuorooctanesulfonate (PFOS), Perfluorooctane sulfonylamide (PFOSA), and Perfluoroodanoate(POAA) in Water", E. Wdremesinhe and J. Flaherty, Study Number 023 002, Centre Analytical Laboratories, Inc., State College, Pennsylvania, January 2000. Validation reportfor the "Method of Analysis for the Determinationof Perfluoroodanesulfonate (PFOS), Perfluorooctanesulfonylamide (PFOSA), and PerRuorooctanoate(POAA) in Water", E. W~ckremesinheand J. Flaherty, Study Number 023 002, CentreAnalyticalLaboratories,Inc., State College, Pennsylvania. ETS-8-154.1 Page 16 of 17 Determinationof PerfluorinatedCompounds in Water Using SPE and LCMS. E05-0210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Page 50 of 122 05-0210 Interim Re ott U18 Notthem Alabama Potable Water Systems, Decemger 2005 18 Affected Documents None. 19 Revisions Revision Number 1 Revision Number Updated to the new format. Changed Title. Section I : States the validation of 3 analytes, removes reference to PA document that's no longer applicable. Section 2: Provided for the extraction of more than the 3 validatedanalytes, allows the use of a LC/MS system, not only the LUMSMS previously mentioned. Section 3: Revised definitions for field matrix spike, field control spike, LLOQ, method blank, and MOL. Section 5: Reworded the interferences, added recommendationto use disposable pipettes. Section 6: Recategorized and pared down. Section 7: Changed storage time to 6 months. Added more calibration points to the table. Section 8:Added statement addressing labeling requirements and spiking procedures. Expanded section 8.8. Section 9: New Section Section IO: Changed some of the parameters in the tables. Allowed for use of different instrumentation. Added information from section 12 of previous version, extensively revised. Section 11 (section 9 in previous version): Clarificationof wash step, stated exact volume of eluate is 5 mL, revised standardizationprocess, removed requirement to use LWMWMS. Section 12 (section 13 in previous version: no changes Section 13 (section 14 in previous version):Extensively rewritten. Section 14 (section 15 in previous version): no changes Section 15 (section 16 in previous version): Minor changes to recording requirements. Section 16 (section 17 in previous version): Removed attachment. Section 17 (section 18in previous Version): Removed reference to PA document that no longer applied to this SOP. Section 18: New section. - Revision Date 7 ETS-8-154.1 Page 17 of 17 Determinationof Perfluorinated Compoundsin Water Using SPE and LC/MS. E054210 InterimReport W18 NorthernAlabama Potable WaterSjfstems, December 2005 Page 51 of 122 EO5-0210 Intetfm Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 ATTACHMENTc: PROTOCOLAND PROTOCOLAMENDMENTS 05-0210 Interim Report #18 NorfhemAlabama Potable Water Systems, December 2005 Page 52 of 122 05-021 0 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 13 1 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 .c Exygen Protocol Number: PO001131 Performing Laboratorv: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 SDonsor Rmresentative: Michael A. Santoro Director of Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 E054210Interim Report I18 NorthernAlabama Potable Water Systems, December 2005 -___- pigc I I!\ 6.i Page 53 of 122 - E05-0210Interim Re ort # l 8 Northern Alabama Potable Water Systems, Decemfer 2005 Exygen Protocol Number: PO00I 13 I DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit EOS-0210 Interim Report # l a Northern Alabama Potable Water Systems, December 2005 Page 2 of65 Page 54 of 122 E05-0210 lnterim Re ort # l U Northern Alabama Potable Water Systems, Decem& 2005 Exygen Protocol Number: PO001 131 PROTOCOL APPROVAL Study Title: Analysis of Perfluorobutanesulfonate(PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate(PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: PO001 131 APPROVALS Weston Solutions *- /$/I& toro, Sponsor Representative John M. Flaherty, hincipal Investigator / Exygen Research Exygen R e s e u Facility Management /?hh Date 24 - &T- Date EOS-0210lnterim Report #18 NorthernAlabama Potable Water Systems, December 2005 Puge 3 7 % Page 55 of 122 05-0210Interim Re ort #18 Northern Alabama Potable Wafer Systems, Decemger 2005 Exygen Protocol Number: PO00 1 15 1 TABLE OF CONTENTS TITLE PAGE .................................................................................................................................. DISTRIBIJTION ................................................................................................................................................ 2 PROTOCOL APPROVAL................................................................................................................................. 3 TABLE OF CONTENTS............................................................................................................ MTRODUCTION........................................................ ........................................................ ...... TEST MATERIALS ......................................................................................................................................... 5 OBJECTIVE.............................................._........................ ......................................................... TESTING FACILITY ................................,..._........._.................................................... STUDY DIRECTOR......_................................._............................................... ....................... SPONSOR REPRESENTATIVE...................... ...._....._......._......_......_.._..................................... PRINCIPAL INVESTIGATOR............._........................................ ................................. PROPOSED EXPERIMENTAL START AND TERMINATION DATES ..................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM ...................... SAMPLE PROCUREMENT, RECEIPT AND RETENTION .._................_.....,.......... c SAMPLE IDENTIFICATION............................,......................................................................... ANALYTICAL PROCEDURE SUMMARY............................... ....................................................................9 VERIFICATION OF ANALYTICALPROCEDURE............................._.............................._........................9 METHOD FOR CONTROL OF BIAS ............................................................................................................. 1 1 STATISTICALMETHODS ............................................................................................................................. I I GLP STATEMENT .......................................................................................................................................... 1 1 REPORT ........................................................................................................................................................... I I SAFETY AND HEALTH ................................................................................................................................. I2 AMENDMENTS TO PROTOCOL ............ ..............................._..... ........................................ .......... ......... 13 DATA RECORD KEEPING ............................................................................................................................ 13 QUALITY ASSURANCE....................................... ............................................................... ...........................I4 RETENTION OF DATA AND ARCHIVING ................................................................................................. 14 APPENDIX I, ANALYTICAL METHODS..................................................................................................... I5 E054210Interim Report #18 NorthernAlabama Potable Water Systems, December 2005 P q c 4 of 65 Page 56 of 122 - 05-0210Interim Report #l8 NorUlemAlabama Potable Water Systems, December 2005 Exygen Protocol Number: PO001 13I INTRODUCTION The purpose of 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 of 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 c Molecular Weight: 338 supplied as the potassium salt (C4F603-K') Lot Number: 101 Punty: 96.7% Transitions Monitored: 299 + 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CbF&OiK') Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 3 80 Structure: 05-0210 Interim Report t18 Nortnem Alabama Potable Wafer Systems, December 2005 Pflge 5 qf 65 Page 57 of 122 E05-0210Interim Re ort # I 8 NotthemAlabama Potable Water Systems, DecemLr 2005 Exygen Protocol Number: PO00 I 11I c PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CsF17SOjK+) Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 -+ 99 Structure: OBJECTIVE The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions of the followingExygen analytical methods: VOOOl780: V0001781: V0001782: V0001783: V0001784: vooo 785: vooo 786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" "Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetationby LCMSMS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Page 6of65 05-0210 Interim Report #I8 Northern Alabama Potable Water Systems, December 2005 Page 58 of 122 E054210 Interim Re rt #18 Northern Alabama Potable Wafer Systems, Decem& 2005 Exygen Protocol Number: PO00I 13 1 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400Weston Way West Chester, PA 19380 Phone: (610)701-3761 Fax: (610)701-7401 j .kesari@westonsolutions.com SPONSOR REPRESENTATrVE Michael A. Santoro 3M Company Director ofRegulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651)733-6374 PRINCIPAL INVESTIGATOR John M.Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 john. flaherty@exygen.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion of this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and termination dates will be included in the final report. h 05-0210 Interim Report #18 NorthernAlabama Potable Water Systems, December 2005 Puge 7 o/ 65 Page 59 of 122 05-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 13 1 n IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: 0 Water (groundwaterand surface water) Soil 0 Sediment Fish 0 Clams 0 Vegetation 0 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 of 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 of sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types of samples collected will vary depending availability in the field. The total number of samples received and analyzed for each matrix will be documented in the final report associated with this study. Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at 2OC-8"C. 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 I -10C. Small mammal whole blood sampleswill be centrifuged in the field at the time of 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 of the sampIes will be documented in the final cc report and raw data associated with the study. 05-0210 Interim Report #l8 NorthernAlabama Potable Water Systems, December 2005 Pngr 8 of65 Page 60 of 122 05-0210 Interim Re ort # I 8 Northern Alabama Potable Water Systems, Decem& 2005 Exygen Protocol Number: PO00I 131 SAMPLE IDENTIFICATION Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Sample storage conditions and locations will be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: VOOOl780: V0001781: VOOOl782: V0001783: V0001784: V0001785: VOOOl786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/Ms" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS' "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS' The above methods use analytical conditions capable of separating the isomers of PFBS,PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANfiYTICAL PROCEDURE A laboratory control sample will be used for the preparation of 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 ScUSpec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample I - 05-021. 0 Interim Report #18 Northern Alabama Potable WaterSystems, December 2005 Page 61 of 122 E050210 Interim Re ort 118 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number:PO001 I3 I C llecti n at the testing facility and have been shown to be free of PFBS, PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike of each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (13C PFOA). PFOA and I3C PFOA are included in the solutions used to spike the samples. The results for PFOA and I3C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containershags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at h both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. For small mammal liver, Exygen will supply a 50 mL polypropylene centrifitge 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. Water I 500 n d L 5000 n d L c E05-0210hrfsrm Report #18 NorthernAlabama Potable Water Systems, December 2005 Page IO oj 65 Page 62 of 122 --- 05-0210Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001131 s-r Recoveries are anticipated to be between 70% and 130% of the fortified levels; however, the exact precision and accuracy will be determined by the analysis of the quality control samples described above. A statement of accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control of bias will be addressed by taking representative sub-samples from a homogeneous mixture of each matrix from untreated control samples, and by analyzing at least two levels of fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation of average recoveries, as applicable. GLP STATEMENT All aspects of 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 of the study. The report will include, but will not be limited to, the following: 0 The name and address of the Study Director, Sponsor Representative, and of the testing facility. A statement of GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). E054210Interim Report 111 NorthernAl8b8m8 Potable Water Systems, December 2005 Pngr I t ($65 Page 63 of 122 05-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 13 1 The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates of study inspections and dates findings were reported to the Study Director and Management. A description of the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy of the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page. Descriptionof 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 chromatogramsof a standard and a control sample, and a chromatogram at a fortification level. The location of the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity of 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 of an amendment signed by the Study Director. The amendment shall clearly identify that part of 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. 0 All applicable requirements for reporting of study results as per 40 CFR 792.18 5 . SAFETYAND HEALTH Laboratorypersonnel will practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure of personnel and the environment to the test or reference substance(s). 05-0210 lnterh Report #18 Northern Alabama Potable WaterSystems, December 2005 Page 12 of 65 Pf?ge84 of 122 EO54210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO00 1 I 3 I 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 of study plan change. However, when a change is required without sufficient time for the issue of a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date of the written amendment will be the date of the documented change. Copies of 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): e Sample tracking sheet@) e Sample receipt records, storage history, and chains of custody e History and preparation of standards (stock, fortification, calibration) Description of any modifications to the method e Instrument run sheets, bench-sheets or logs e Analytical data tables e All chromatographic and instrumental conditions e Sample extraction and analysis dates e A complete listing of study personnel, signatures and initials e Chronologicalpresentation of all study correspondence e Any other documentation necessary for the reconstruction of the study Chromatograms- All chromatograms will contain the following: e Sample identification, injection date, arrow or other indication of the area of interest, and injection number corresponding to the run. e Additionally, fortifications will include the amount of analyte added and the sample number of the sample that was fortified. C. e Analytical standard chromatograms will additionally include the concentration (e.g., pdmL). E05-0210Interim Report #I8 NorthernAlabama Potable Water Systems, December 2005 Page 65 of 122 E054210 Interim Re ort#18 Northern Alabama Potable Water Systems. DecemLr 2005 Exygen Protocol Number: PO00I 13 1 .a As part of 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 of Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings of 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 of time specified in 40 CFR 792.195. An exact copy of the materials submitted to the study director will also be kept at Exygen Research. Exygen will obtain permission h m the study director before discarding or returning samples. -__05-0_210 Interim Repott #18 NorthernAlabama Potable Water Systems, December 2005 Puge 14 ($63 Page 66 of 122 -. h _- EO5-0210 lnterim Re ort#18 Northem Alabama Potable Water Systems, Decernler 2005 Exygen Protocol Number: PO00I I3 1 APPENDIX I ANALYTICAL METHODS VOOOl780: V0001781: V0001782: V0001783: V0001784: V0001785: V0001786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" E05iOzlO Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Page 15 01'6.5 Page 67 of 122 -- -- E05-0210Interim Re ort t l 8 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO00I 13 1 ANALYTICAL METHOD Method Number: VOOO1780 Method of Anrlyrlr for Ute Det~~1in8tiOoDf Perfluorooctanoic Acid (PFOA)in Water by LC/Ms/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: CA Paul Connoll~ Technical Leader. LC-MS. Exygm Research 'Vi& Preside&, Operations. ExygmResearch .4 -- TotalPagcr: 7 05-0210Interim Report #I8 Northern Alabama Potable Water Systems, December 2005 Page 16 of65 Page 68 of 122 E05-0210Interim Re ori #I8 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 131 Exygcn Research M c W Numbcr V0001780 1 I ANALYTICAL METHOD Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Water by LC/MSiMS 1.0 scope This method is IO be anployed for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass SpectrometricDetector(LCIMSIMS)in water. 2.0 safery 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 40 mL of test sample for extraction. 3.2 No sample processing is ncedcd for water samples. 3.3 Samples stored refrgeratcd should be allowed IO equilibrate ro room temperature. 3.4 All samples must be thoroughly mixed before being sampled for exuaclion. 3.5 Any samples containing particles should be centrifuged at -3000 rpm for -5 minutes and the supematant used for the extraction. 3.6 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards - 4.1 Water - HPLC grade 4.2 Methvlol HPLC &e - 4.3 Ammonium Acetate A.C.S. Reagent Grade - 4.4 PerfluorooctanoicAcid Sigma-Aldrich 5.0 Instrument and Equipmeat 5.I A high performance liquid chromatograph capable of pumping up to I solvents equipped with a variable volume injeclor capable of injecting 5-200 pL connected IO a tandem Mass Spechumeta (LCMs/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable of readingto O.oooO1 g. 5.4 50 mL dispoarble polypropylenecentrifuge tubes. 5.5 15mLdisposablepolypropylenecentrifuge tubes. 5.6 Di.posable micropipets(50- 1M)UL. lOO-2OOuL). 5.7 125-mL LDPEnarrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettcs(100-1OOO pL and 10-100 )rL).with disposable tips. 5.1 1 Waters Scp Psk V u 6 IX (le) tC18 SPE cartridges. E05-0210 lntedm Report #I8 Northern Alabama Potable Water Systems, December 2005 Page I7 of 65 Page 88 of 122 05-0210 Interim Re ort #18 NorthemAlabama Potable Water Systems, DecemIer 2005 Exygen Protocol Number: PO001 131 Eaypen R-h MclhodNumbcr V0001780 I I AhALYTlCAL ,METHOD Method of AnalvSis for the Determinationof Pnfluorooctanoic Acid (.PFOAI,in Water bv LCIMS/MS 5.12 SPE vacuum manifold. 5.13 Ccntri'itge wpable of spinning 50mL polypropylene cubes at 3000 rpm. 6.0 chromatographic System 6.1 Analytical Column:FluophaseRP (Keystone Scientific), 2.1 mm x 50 mm. 511 (PN 82505-052130) 6.2 Temperature: 30-C 6.3 Mobile Phase (A) : 2 mM AmmoniumAcetate in Water 6.4 Mobile Phase(B) : Methanol 6.5 Gradient Program: Time fminl 0.0 65 1 .o 65 8.0 25 20.0 25 22.5 65 a 35 35 75 75 35 Flow Rate bUminJ 0.3 0.3 0.3 0.3 0.3 - 6.6 Injection Volume: I5 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Pcak Area - cxtcmal standard calibration e w e . 6.8 Run Time: 23 minutes. The above conditions uc intended as a guide and may be changed in order to optimize the HPLC system. 7.0 M U M S S y r l m 7.1 Mode: Elatrospray NegativeMRM mode, monitoring 413 -+ 369 miz. The above conditions arc intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation of Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154g of ammonium acetate to loo0m L ofwater. Alternate voluma may be prepared. 05-0210 Interim Report #l8 NorfhemAlabama Potable Water Systems, December 2005 Page 18 of 65 Page 70 of 122 EO5-02lOlnterim Re rt#l8 Notthem Alabama Potable WaterSystems, D e c e m G 2005 Exygen Protocol Number: PO001131 Exysen Rcreuch McthodNumkr VooO1780 1 I ANALYTIC.U METHOD i Method of Analysis for the Dctcnnination of P~uoroootanoicAcid QFOA) in Water by Lmms 9.0 SlDndardPrepoRtion 9.1 Standard StockIFortifiution Solution 9.1.1 Prepare a stock solution of -100 p g h L of PFOA by weighing 10 nir. of analytical standard (corrected for punty) and dilute to 100 m L with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 gg/mL fortification solution of PFOA is pr&d by bringing I IJ mL of the 100 p g h L solution to a final volume of 100 with merhonol in a 125 mL LDPE bottle. 9.1.3 A 1.O p#mL fortification solution of PFOA is prepared by bnnging I l l mL ofthe IO p#mL solution to a final volume of 100with rncthdnol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pglmL fortification solution of PFOA is prepared by bringing IO mL of the 1.0 p#mL solution to a final volume of 100with merhanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 gB/mL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortificationsolutions are lo be stored in a rekigerator at approximately 4OC and am stable for a maximum period of 6 months from the date of preparation. 9.2 Standard Calibntion Solutions 9.2.1 LC/MS/MS calibration standards am p r e p d in HPLC water. The calibratioa standards arc processed through the extraction procedure, identicalto samples. 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. ~ Final ConccneWcm Fortification Volume of Concenhationof Calibration of Forhfication Volume Fort~ficdControl Calibration Standard ID Solution(ppb) (cL) 0 0 Sample (mL) 40 Standard (ppt)' 0 (example) XCmmddyy-0 IO 100 40 25 XCmmddyy- I IO 200 40 50 XCmmddyy-2 IO 400 40 100 XCmmddyy-3 100 100 40 250 XCmmddyy-J 100 200 40 500 XCmmddyy-5 * 100 400 40 1000 XCmmddyy-6 The extracted concentrationofthe calibration standard is q u a l to 8x its initial concentration,due lo the concenkation ofthe standard during the extraction (SPE) XC = extracted calibrationstandard. _- E054210 Interim Report #18 Notthem Alabama Potable Water Systems, December 2005 Page 19 of65 Page 71 of 122 E054210 Interim Re ori # I 8 Northem Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 131 Exygcn Rcacueh Method Number VOOO I780 1 I ANALYTICAL METHOD Mahod of Analysis for the Determinationof Perfluomoclanoic Acid (PFOA)in Water by LC/MS/MS 9.2.3 A zcro stzndard solution (reagent blsnk) must be with each ret of standuds exescted. 9.2.4 Store all cxfractcd calibration standards in 15-mL polypropylene tubes at 2C to 6C. up to two weeks. 9.2.5 Altermte volumes and concentrations of standards may be prepared as needed. 10.0 Batch Sa Up 10.1 Each batch of samples extracted (typically 20 or less) must include at leilsi one ragent control (mahod blank using HPLC water) and two reagent controls fortified at known concentrations (lab control spike) IU veril'y procedural recovery for the batch. 10.2 Rcquircments for field and laboratory duplicates and spikes will be speolitd in the quality assurance plan for this project. 11.O Sample Extraction 11.1 Measure 40 mL of sample or a portion of sample diluted to 40 mL with water into SO mL polypropylcne cenhifige tubes (fortify as necdcd, replace lid and mix well). 11.2 Condition the Cti SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followedby 5 mL of HPLC water (-- 2 dropkc). Do not let column rundry - 11.3 Load sample on conditionedCISSPEcartridge. Discard eluate. 11.4 Elute with -5 mL 100% methanol. Collect S mL of eluate into graduated IS mL polypropylene centrifiige tubes (final volume 5 mL). I 1.5 Analyze samples using electrosprayLCh4sIMS. 12.0 Chromatography 12.1 Inject the same amount of 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 of PFOA correspondingto at least five or more concentration levels must be included in M analytical set. 12.3 An entire set of cxtncted calibration rtandvdr mwt be included at the beginning and al lhe end of a sample set. Extracted standards must bc interspersed betwtcn way 5-10 samples. As an alternative. an entire set of C X t n o t d cdibntion S l a n d a d s may be injected at the beginning o f a SCI followed by exuected calibration standards interspersed every 5-10 samples (to account for a scwnd u t of exhacted standards). In either case, extracted calibration standards must be the firsl and last injection in a sample set 12.4 Use linear standard curvu for quantilation. Linear standard curves are generated for the d y t c by linear regrcarion using I/x weighting o f peak area 05-0210 Interim Report #I8 NordhemAlabama Potable WaterSystems, December 2005 Page 20 4 6 5 Page 72 of 122 E05-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 13 1 EX.Y_ ECDRerearch Metbod Number V0001780 I I AN4LYTICAL METHOD Methodof Analysis for the Determination ofPerfluorooctanoicAcid (PFOA)in Water by versus calibration standard concentration using MassLynx 3.3 (or equivaleni) sofhvarc system. 12.5 Sample response should not a c e d standard responses. Any samples rhal exceed standardresponses should be furtherdiluted and reanalyzed. 13.0 AccepIance Criteria 13.1 Chromatogrammust show a peak of a daughter ion at 369 a m u from a parent of 413 m u . The 413 amu parent corresponds to the PFOA anlon. while 1 1 1 ~ daughter ion (369 amu) represents the loss of carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. I C d blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set must be recxtracted. 13.3 Recoveries of control spikes and matrix spikes must be between 70-130/, ol' their known valuca. If a control spike falls outside the acceptable limits. tlic entire set of sampler should be re-extracted. Any manix spike outside 7013Wh should be waluated by the analyst to determine if re-extraction is WlUTantCd. 13.4 Any calibtation standard found to be a statistical outlier by using the Huge E m Tat, may be excluded from the calculation of the calibration curve. Howmr, the total number of extracted calibration standards that could be a r c l u d ~murt not e x d 20% of the total number of extracted standards injected. 13.5 The correlation coefficient (R)for calibration curvm generated must be 20.992 (R' 20.985). If calibration results fall outside these limits. then appropriate steps must be taken to adjust instrument operation. and the standards or the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples must nor drift more than k 4 % within an analyticalmn. If retention time drifl exceeds this limit within an analytical run then the sct mu81 be reanalyzed. 14.0 Cakulationc 14.1 Use the following c q d o n to cllculate the amount of PFOA found (in nuL. based on peak area) using the standard curve ( l i n w regression paramelerr) genaatedby the Mass Lynx software program: PFOA found (ng5.L) -@e& - area intcrceDtl x DF slope DF = factorby which the f i ~voIlume was diluted. if necessary 05-0210 lnterfm Report #18 Northern Alabama Potable Water Systems, December 2005 Page 21 of65 Page 73 of 122 05-02lOlnterim Re r t # l U Northern Alabama Potable Water Systems, O e c e m E 2005 Exygen Protocol Number: PO001 13 1 E x y pRuevch Method Number V0001780 1 i ANALYTICALMETHOD Method of Analysis for the Determinationof PerfluorooctanoicAcid (PFOA) in Water by LC/MS/MS 14.2 For samples fortified with known amounts of PFOA prior to extraction. use the followingequationto calculatethe percent recovery. Raovay (Ye) * I total analytefound(nfi) - annlytefoundin control(nglL)l ,oo analyte addcd (ng/L) .- 054210 Interim Report #la Northern Alabama Potable Water Systems, December 2005 Page 22 of65 Page 74 of 122 EO5-0210 Interim Report #18 N o m e mAlabama Potable Water Systems, December 2005 Exygen Protocol Number: PO001 13 1 ANALYTICAL METHOD MethodNumk V0001781 Method of Andyak for Le Dcterdmtion of Ptrfluorooctmoic Acid (PFOA)L Soil by LClMSlMS Analytical TestingFacility: Exygen Research 3058 Research Drive state College. FA 16801 Appmvcd By: Paul Connolly Technical Leader, LC-MS, Exygen Research ' /6hn Flahuty /Vice Presidmt, Operations.Exygen Research lOl7-blall Date PA74 Date TotllPagn;: 7 E054210 Interim Report # l a NorthernAlabama Potable Water Systems, December 2005 Page 23 oj65 Page 75 of 122 05-0210 Interim Re ort #18 NorthernAlabama Potable Water Systems, Decemler 2005 Exygen Protocol Number: PO001 13 1 r.4 Exvmeo Rctcuch Method Number VOOO1781 .- ANALYTICAL METHOD I I Method of Analvsis for the Detenninationof Perfluorooctanoic Acid (PFOA) in Soil by 1.o Scope This method is to be employed for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chmatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in soil. 2.0 safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriateMSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At lcasr IS g of test sample for extraction. 3.2 No sampleprocessing is needed for soil spmpla. 3.3 Samplw stored refrigerated should be allowed to equilibrate to room tempcram. 3.4 All samplw must be thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standarda - 4. I WUtu - HPLC grade 4.2 Methanol - HPLC gde 4.3 Ammonium Acetate -AC.S. Reagent Grade 4.4 PcrfluorooetanoicAcid Sip-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable of pumping up to 1 solvents quipped with a variable volume injector capable of injecting 5-3KJ pL connected to a tandem Mass Spectrometer(LCIMSIMS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analyticalbalance capable of reading to O.oooO1 g. 5.4 50 mL disposablepolypropylenecentrifuge tubes. 5.5 I 5 mL disposablepolypropylene centrifuge tubes. 5.6 Disposablemicropipets(50-1OOuL. 100-2OOuL). 5.7 125-mL LDPE nmw-mouth bottles. 5.8 2 mL cleu HPLC vial kit. 5.9 Disposablepipenu. 5.10 Autopipettes(100-1OOO pL and 10-100 pL), with disposable tips. 5.11 Watm Sep Pak Vac 6 cc (le) IC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 UltrasoNc bath. 05-0210 lntetjm Report #18 Northern Alabama Potable Water Systems, December 2005 Page 24 4 6 s Page 76 of 122 05-0210 Interim Re ort #18 Northern Alabama Poiable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 13 I Ba.yp-en Rcrureb Method Number VooUl7UI 1 1 ANALYTICAL METHOD Method of Analysis for the Determinationof PduorooctPnoic Acid (PFOA) in Soil by 5.14 Wrist-action shaker. 5.15 Centrifugecapable of spinning 50 mL polypropylene tubes at 5000 rpm 6.0 ChromatographicSystem 6.1 Analyticid Column: FluophascRP (Keystone Scientific). 2.1 m m x 50 mm. 511 (PM:82505-052130) 6.2 Temperature: 30'C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phasc (B) : Methanol 6.5 Gradient Program: Time (min) 0.0 65 1 .o 65 8.0 25 20.0 25 22.5 65 Flow Rate &Q [rnUmin) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 - - 6.6 InjectionVolume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area e x t d standard calibration curve. 6.8 Run Time: 23 minutes. The above wnditiona am intended 88 a guide and may be changed in order to optimize the HPLC system. 7.0 MSMS System 7.1 Mode: ElcctrosprayNtgaIive MRM mode, monitoring 413 +369 miz Ibr PFOA. The above conditions ~nintended as a guide and may be changed in order IO optimize the MSMS system. 8.0 Preparation of Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acolotc in water is prepared by adding 0.154 g or ammonium acetate to 1000 mL of water. Alternate volume$may be prepared. 05-0210 Interim Report 118 Northern Alabama Potable Water Systems, December 2005 Pflge25 of 65 Page 77 of 122 05-0210 Interim Report # l a Northern Alabama Potable Water Systems, December 2005 Exygen Protocol Number: PO001131 Exygcn Rauueh Method Number VOOO17BI I I ANALYTICAL METHOD Method ofAnalyrir for the DeterminationofPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.0 Standard Preparation 9.1 Standard StocWFortificationSolution 9.1.1 Prepue a stock solution of -100 pg/mL of PFOA by weighing IO inp of analytical ctandrrd (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pdmL fortificationsolution of PFOA is prepared by bringing 111 mL of the 100 pghnL solution IO a final volume o f 100 with meikinol in a 125 mL LDPE bottle. 9.1.3 A 1.O p g h L fortificationsolution of PFOA is prepared by bringing It! mL of the IO p g h L solutionto a final volume of 100 with m i h a n d iii a 125 mL LDPE bottle. 9.1.4 A 0.1 p g h L fortificationsolution of PFOA is prepared by bringing 1 0 mL of fhe 1.0 p g h L solution to a final volume of 100 with methanol in a 125mL LDPE bottle. 9. I .5 A 0.01 p@mLfortification solution of PFOA is prepared by bringing 10 mL of the 0.1 &mL solution to a final volume o f 1 0 0 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortificationsolutions are to be stored in a rehiperator at approximately 4'C and arc stable for a maximum period of 6 months from the date of preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in HPLC water Thc calibration standards are processed through the extraction procedure. identical IO samples. 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Volume of Concentrationof ofFortification Volume Fortified Conuol Cilibration Calibration Standard ID Solution (ppb) ( pL) Sample(mL) Standard@p)* (example)- 0 0 40 0 XCmmddyy-0 10 100 40 25 XCmmddyy- I IO 200 40 50 XCmmddyy-2 IO 400 40 I00 100 40 100 XCmmddyy-3 250 XCmmddyy-4 IO0 200 40 IW 400 40 SO0 XCmmddyy-5 IO00 XCmmddyy-6 The extracted concentrationof the calibrationstandard is q u a l to 8x its initial concentration,due to the concentrationof the standard during the extraction (SPE). XC = extracted calibration standard. Page 4 of 7 E054210 lnterfm Report # l a Northern Alabama Potable Water Systems, December 2005 Page 26 4 ' 6 5 Page 78 of 122 - .- E054210 Interim Re ort # I 8 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number:PO001 131 9.2.3 A zero standard solution (reagent blank) must be oreoared with each set of standards extracted. 9.2.4 Store all extracted calibration standards in 15-mL polypropylene tubes at 2C to 6 T . up to two weeks. 9.2.5 Altmate volumes and concentrations of standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of sampled extracted (typically 20 or Icss) must include at leas! one reagent control (method blank using 5 mL of methanol) and two reagent controls fortified at known concentrations (lab control spike) to verily procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be spccifisd in the quality assuranceplan for this project. 11.0 SamplI Extraction 11.1 Weigh 5 g of sample into 50 mL polypropylene centrifuge tubes (fortify as needed. replace lid and mix well). 11.2 Add 5 mL of methanol and shakeon a Wrist action shaker for -I 5 minutes. 11.3 Transfer the lubes to an ultrasonic bath and sonicate for -1 5 minutes. 11.4 Bring the volume up to 40 mL with water in the 50 mL polypropylene centrifuge tube. 11.5 Centrifuge for -10 minuted at -3000 Qm. 11.6 Condition the CII SPE c d d g e s (1 g. 6 mL) by passing 10 mL methanol followed by 5 mL of HPLC water (- 2 droplsec). Do not let column run dry 11.7 Load (decant) the sample on the conditioned CII SPE cartridge. Discard eluate. 11.8 Elute with -5 mL 100% methanol. Collect 5 mL of eluate into graduated 15 mL polypropylenecentrifuge tubes (final volume = 5 mL). 11.9 Analyze samples using electrospray KMSMS. 12.0 Chromatography 12.1 Inject the same amount of each standard, sample and fortified sample into the LUMSh4S systrm. A calibration standard must precede and follow all Mal@ ..mplor. 12.2 Standardr of PFOA wrrcspondingto at least five or more concentration levels must be included in an analytical set. 12.3 An entire set of extracted calibration standards must be included ai the beginning and at the end of a sample set. Extracted standards must be interspersedbetween wery 5-10 samples. As an alternative. an entire set of Page J of 7 05-0210 Interim Report #la NotihemAlabama Potable WaterSystems, December 2005 Page 27 f7#'65 Page 79 of 122 EO5-0210 Interim Re ort#18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 13 1 Exygen Research M c l b d Number VooO1781 L 1 ANALYTICAL METHOD Method of Analysis for the D e w m h t i o n of Penluorooctanoic Acid (PFOA) in Soil by LC/MS/MS extracted calibration standards may be injected at the beginning of a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a wcond ret of extracted standards). In either case, extracted calibration stmdprdc must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are genenrtedfor the aanlyte by linearregression using I/* weighting of peak area vcrsup calibration standard wncenlration using MassLynx 3.3 (or cquivalcni) Jofhvare system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard nsponses should be furtherdiluted and reanalyzed. 13.0 AcceptanceCriteria 13.1 Chromatogrammust show a peak of a daughter ion at 369 m u from a parent of 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 MU) represents the loss of carbon dioxide. 13.2 Mehod blanks must not contain PFOA a1 levels greater than the LOQ. 11a blank contain8PFOA at levels greater than 50 ng/L. then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries of control spikes and matrix spikes must be between 70-130% of their known values. If a control spike falls outside the acceptable limits. the entire set of samples should be re-extmcted. Any matrix spike outside 7013G% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration a t a d a d found to be a statistical outlier by using the Huge Error Test, may be excluded from the cdculation of the calibration curvc. However, the total number of cxtractcd calibration standards that could be excluded must not exceed 20?h of the total number of extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must hc 20.992 (R' 20.985). If calibration results fall outside these limits. tlirii appropriate steps must be taken to adjust instrument operation, and tlir standardsor the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than f 4 %within an analytical run. If retention time drifl excceds this limit within Manalyticalrun thcn the set must be reanalyzed. E059210 Interfm Report #18 Nodhem Alabama Potable Water Systems, December 2005 Page 28 of 65 Page 80 of 122 05-0210Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 13 1 Eaygcn Rucvch MecbodNumberV00017111 1 ASALYTICAL METHOD 1I Method of Analysis for the Determinationof Pcrfluorooctanoic Acid (PFOA) in Soil by LCIMSIMS 14.0 Calculations 14.1 Use the following equalion to calculate the amount of PFOA found (in nglL. basad on peak area) \wing the standard curve (linear regression parameters) generated by the Maas Lynx sofhvarc program: PFOA found (na=)fPeak area - intemeDt) x DF - slope DF factor by which the final volume was diluted, if necessary. 14.2 For samples fortified with known amounts of PFOA pnor to exrraction, use the following equation to calculate the percent recovery. Rccovery (%) = 1 (e- ) total d y t e found analytefound in control (ng&)l Y 100 analyteadded (n@) 14.3 Use the following equation to convefi the amount of PFOA found in ng/L IO n%s @Pb). PFOA found (ppb) - ] - f sample weight (5 g) e 0.04 14.4 Use the following equation to calculate the m o u n t of PFOA found in pph based on dry weight. PFOA found (ppb) dry weight - PFOA found (ppb) x [ 100% / total solids(%)] Page 7 or7 E054210Interim Report #18 Northern Alabama Potable Wafer Systems, December 2005 Fuge -79of.5 Page 81 of 122 EOW210lnferim Re rt#18 Northern Alabama Potable Water Systems. Decemer 2005 Exygen Protocol Number: PO001 131 ANALYTICAL METHOD Method Number: V0001782 Method olAnalyd8 for tbe DetermlnMionof Pcrfloarooetrnoic Acid (PFOA)in Sediment by LC/MsMS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: ?JGIL. Paul Connolly Technical Leader, LC-MS. EXY- Research Date / Vice Prcsi&t, Operptionr, Exygcn Research TotnlPagn: 7 05-0210 Interim Report #18 NotthemAlabama Potable Wafer Systems, December 2005 Page 30 of65 Page 82 of 122 05-0210Interim Report #I8 Northern Alabama Potable Water Systems. December 2005 Exygen Protocol Number: PO001 13 I Exygcn Resanrcb McthodNumbcr VW01782 1.0 scopc This method is to be CmpkIycdfor the idation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass SpectrometricDetector (LCIMSIMS)in &mt. 2.0 safety 2.I Always observe safe laboratorypractkes. 2.2 Consult the appropriate MSDS before handling any chemical for pmper safety precautions. 3.0 Sample Requirement 3.1 At lust 30 g of test sample for extraction. 3.2 No sample processing is needed for sediment samples. 3.3 Samples stored refiignated should be allowed to equilibrate IO room temperature. 3.4 AI1 samples must be thoroughlymixed before being sampled for extracrion. 3.5 Sample collection procedures will be specified in the sampling plan for this project. 4.0 RcagcntsandStandardr 4.1 Water - HPLC grade 4.2 Methanol - HPU: - 4.3 Acetic Acid -Reagent grade - 4.4 Ammonium A.C.S. R u g a t G d e 4.5 PerfluotwctanoicAcid Sigma-Aldrich 5.0 Iwrmmmt and Equipment 5.1 A high perfomwce liquid chromatograph capable of pumping up to 2 solvenu equipped with I ) variable volume injector capable of injecting 5-200 pL connected to a tandem Mass Spectrometcr(LCIMSIMS). 5.2 A device to collect raw data for p a k integration and quantitation. 5.3 Analytical balance capable ofreading to O.oooO1 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposablepolypropylenecentrifuge tubes. 5.6 Disposable micropipets(50-1OOuL100-20OuL). 5.7 125-mLLDPE nmw-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposablepipcltes. 5.10 Autopipettes(100-1000 p L d 10-100 pL). with dirposable tips. 5.1 1 WatersScp Pak Vac 6cc (le) tC18SPEcartridges. 5.12 SPE vacuum manifold. E054210 Interim Report #lU NorthernAlabama Potable WaterSystems, December 2005 Page 31 of 65 Page 83 of 122 05-021 0 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001131 Exygm Rclcrrsh Method Number VOOOl702 1 ANALYTICAL METHOD J Method of Annlysis for the Determinationof Pertluomctanoic Acid (PFOA) in Sedimenib) LC/Ms/MS 5.13 Vortcxer. 5.14 Wnrt-oCti~n&kW. 5.15 Centrifulre capable of spinning 50 mL polypropylene tubes at 3000 rpm. 6.0 ChromatographicSystem 6.1 AnalyticalColumn: FluophascRP (Keystone Scientific). 2.I m m x 50 mm. 511 (PRJ: 82505-052130) 6.2 Tanpaatwe: 300C 6.3 Mobile Phasc (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 GrPdimtPmgrm: u Time (mi& 0.0 65 1.o 65 8.0 25 20.0 25 22.5 65 Flow Rate (mUminj 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be innured to IU much as 50 pL) - 6.7 Quantitation: PeakA n a -external standard calibration curve. A 6.8 Run Time: 23 minutes. The above conditions an intended BP a &de and m a y be changed in order to optimize the HPLC sptem. 7.0 MSlMS System 7.1 Mode: Elechospr~yNegativeMRM mode, monitoring 413 --t 369 dz for PFOA. The above Conditiom arc intendedM a &de and may be changed in order to optimize the MSMS system. 8.0 F?epmtion ofSolutionr 8.1 MobilePhuc 8.1.1 2 mM UNnonium SCctDtc in water is prepared by adding 0.I54 g 01' ammoniumacetateto I OOO mL of water. E050210 Interim Report #78 NorthernAlabama Potable Water Systems, December 2005 Page 32 of 65 . - -. Page 840f ?22---- E054210 Interim Re orl#18 Northern Alabama Potable Water Systems, Decemler 2005 Exygea Protocol Number: PO001 13 1 Erypm Rawarch Method Number V0001782 1 I ANALYTICAL METHOD Method of Analysis for the Determinationof Pduorooctanotc Acid (PFOA) in Sedimenl b\ 8.2 Extraction solutio^ 8.2.1 1% acetic acid in water is prepared by adding 10 mL of acetic acid IO IO00 mL of watn. Alternate volumecl may be prepared. 9.0 StandardPrcparation 9.I StandardStocWFortificationSolution 9.1.1 Prrp.re a stock solution of -100 p&lmLof PFOA by weighing 10 mg of analytical standard (corrected for purity) and dilute to 100 mL wilh methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution of PFOA is prepared by bringing l(1 mL of the 100 p g h L solution to a final volume of 100 with m t h s n d in a 125 mL LDPE bottle. 9.1.3 A 1.0p&lmLfortification solution of PFOA is prepared by bringing IO mL of the 10 p#mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 p g h L fortificationsolution of PFOA is prepared by bringin6 I O mt ofthc 1.0p g h L solutionto a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01pg/mL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 p&lmL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions arc to be stored in a refrigerator at appmximately 4 T and arc stable for a maximum period of 6 months from the date of preparation. 9.2 Standud Calibration Solutionr 9.2.1 Lc/MS/MS calibration stpndards are prepared in methanol via dilution of the 0.1 p@L fortification solution. 9.2.2 The following is a typical example: additional concentrations may he prepared as needed. ConcmWation Final ofFmifmtion Volume Diluted to Concentration Solution (ng/mL) (mL) (mL) (ng/mL) 100 10 100 10.0 too 5 too 5.0 100 2 100 2.0 10 10 100 I .o 5 10 100 0.5 2 10 100 0.2 Paee 4 ut' 7 05-0210 Interim Report 118 Northern Alabama Potable Water Systems, December 2005 Page 33 of65 Page85of122 05-0210 Interim Re ti#18 Northern Alabama Potable Water Systems, Decemcr 2005 Exygen Protocol Number: PO001131 Exygm Rcaarcb Memod Number VOW1782 1 I ANALYTICAL METHOD Method of Anal)ktis for the Determiaationof Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS . 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bonles at 2'C to 6C. up to six months. 9.2.4 Alternate volumes and conccntrntiOns of s*mdards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of ~ m p l e dextmcted (typically 20 or less) must include at least om untreated control and two untreated controls fortified at known concentrations(lab control spike) to verify procedural recovery for rhe batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality ~ u u n n c pelan for tbis project. 11.0 Sample Extraction 11.1 Weigh 5 g of sample into SO mL polypropylene centrifuge rubes (fortify as needed, replace lid and mix well). 11.2 Add 35 mL of 1% a c d c acid, cap. vortex and shake on a wrist action shaker for -60 minutes. 11.3 Centrifugethe tubed at -3000 rpm for -20 minutes. 11.4 Condition the Cta SPE cartridges (I g, 6 mL) by passing IO mL methanol followedby 20 mL of HPLC water 1- 2 droplscc). Do not le! column run dry . 11.5 Load (decant) the sample on the conditioned CI: SPE cartridge. Discard .- eluate. 11.6 Add 20 mL of methanol to the sediment I& in the bottom of the 50 mL centrifuge tube. Cap, vmex and shake on a wrist action shaker for -30 minutes. 11.7 Centrifugethe tubesat -3000 rpm for -20 minutes. 11.8 Dccant the metbanol onto the same SPE cartridge. Collect the eluate. 11.9 Wash the ~olvmnwith 4 mL ofmethanol. Collect the eluate and add it to the eluate collected in step 11.8. 11.10 Condition a second Cia SPE cartridge (I g, 6 mL) by passing 10mL methanol followedby 20 mLof HPLC water 1- 2 drop/scc). Do not ler column run dry 11.1 1 Add the methanol to -200 mL o f watm and load on the second conditionnl SPE cartridge. 11.12 Elute with -5 mL 100% methawl. Collect 5 mL of eluate into graduared 1s mL polypmpyleneccnbifuge tubes(final volume = S mL). 1I. 13 Analyze urnpias using clatmsprsy LC/MS/MS. E054210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Page 34 of 65 Page 86 of 122 05-0210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Exygen Protocol Number: PO001 131 Exygen Rcreueb M c W Numki VooOl702 1 ANALYTICAL METHOD 1 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 12.0 Chromatopphy 12.1 lnjcct the same amount of each standard, sample and fortified sample into thc LC/MS/MS syatctn. A calibration standard must precede and follow all analyzed samples. 12.2 S e of PFOA corresponding to et least five or more concentration Icvel~ must be included in an analytical set. 12.3 An entire set of extracted calibration standards must be included at the beginning and at the end of a sample set. Standards must be interspersed between every 5-10 samples. As an alternative. an entire s e ~of calibrdliun standards may be injected at the beginning of a set followed by calibration standards interspersed evny 5-10 samples (to account for a second sel o f standards). In either case. calibration standards must be the first and last injection in a sample set. 12.4 Use lincar standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using l/x weighting of peak area versus calibration standard concentration using MasrLynx 3.3 (or quivalenl\ SORWarC system. 12.5 Sample response should not excccd standard responses. Any samples that exceai standardresponses should be htther diluted and reanalyzed. 13.0 Acceptrnce criteria 13.1 ChmatoIpMl must show a pcrL of a daughter ion at 369 mu from a parent of 413 mu. The 413 m u parent corresponds to the PFOA anion. while thc daughter ion (369 mu) represents the loss of carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. IC a blank con(ains PFOA at levels greater than 0.2 WmL. then a new blank sample must be obtnined and the entire set must be m-extracted. 13.3 Rccovcries of control spikes and matrix spikes must be between 70-130?/0 of their known vduec. if a eontrol spike falls outside the acceptable limits, the entire set of w p l c s should be m-attracted. Any matrix spike outside 70130% should be evaluated by the analyst to determine if re-extraction is Wlumntcd. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test,m y be excluded &om the calculation of the calibration cuwc However. the total number of exeaCted calibration standards that could be excluded must not excccd 20% of the total number of extracted standards injected. 13.5 The wml.tion coefficient (R) for dibmtion c w a generaIcd must he 20.992 (Ra 20.985). If calibration results fall outside these limits. then appropriate acps must be taken to adjust insnument operation. and the standards or the relevant set of samples should be reanalyzed. Page 6 of 7 E054210 Interim Report #la Northern Alabsms Potable Wster Systems, December 2005 Page 35 0/65 Page 87 of 122 05-0210Interim Re ort#18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001I 3 I Exygcn Remuch Method Number VOW1782 1 1 ANALYTlCAL METHOD Method of .Analysis for the Determination of Perfluonwxtanoic Acid (PFOA) in Sediment b) LC/MS/MS 13.6 Retention tim between standards and samples must not dnfi more than f 4 %within an analyticalrun. If retention time drift exceeds this limit within an analytical run thcn the set must be reanalyzed. 14.0 Calculations 14.1 Use the followingequationto calculate the amount of PFOA found (in n#mL. b a d on peak area) using the standard curve (linear regression parameters) generatedby the Mass Lynx softwpre program: PFOA found (n&L) =[Peak area - intercmtl x DF slope DF = factor by which the final volume was diluted. if necessary. 14.2 For sample fortified with known mounts of PFOA prior to extraction. use the following equation to calculate the percent recovery. Recovay (%) = 1totalanalytcfound(nghL) - analytefound in control ( n g h L ) l aaalyte added (ng/mL) 14.3 Use the following equation to convcrl the mount of PFOA found in n g h L to n%e @Pbh PFOA found (ppb) = PFOA found (ndmL) x final volume ( 5 mLU sample weight (5 g) 14.4 Use the following quation (if necessary) to calculate the amount of PFOA found in ppb bared on dry weight. PFOA found @pb)dry weight = PFOA found (ppb) x [lW/.I total solids('/dl Page 7 of i E05-0210 Interim Report #18 NorthernAlabama Potable Wafer Systems,December 2005 Page 36 of65 Page 88 of 122 EO5-0270 Interim Re ort t78 Northern Alabama Potable Water Systems, Decemier 2005 Exygen Protocol Number: WOOI I3 1 ANALYTICAL METHOD MethodNumbcr: VWO1783 Method of Analysis for tbe Determination of PerfluorooetPnoicAcid (PFOA) in Fish and Clam by LCIMSNS Analytical Testing Facility: Exygcn Research 3058 Rescarch Drive StateCollege,PA 16801 Approved By: Paul Connollv CAL Technical Lckcr,E-MS. ExygenResearch L Date Total Pages: 8 E054210Interim Report #18 Northern Alabama Potable Water Systems, December 2005 P q p 37of65 Page 89 of 122- - 05-0210 Interim Re ort #I8 Northern Alabama Potable Water Systems, Decemfer 2005 Exygen Protocol Number: PO001131 Exygm Rcrlrch I Mahad of -lysis McWNumber V0001783 I ~ A L Y C I C A LMETHOD for tbc Dctemtinationof P d w m t a n o i c Acid (PFOA) in Fish and Clams by LCIMSIMS 1.0 scope This method is to be employed for the isolation and qurntitation of pcrnuorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mars Spacbomctric Dctcctor (LC/Ms/MS) in fish and clams. 2.0 safety 2.I Always obscrve safe laboratory practices. 2.2 Consult the appropriateMSDS before handling any chemical for proper saki) precautions. 3.0 Sample Requiment 3.1 At lcast 20 g of test sample for extraction. 3.2 Samples should be processed before exaction. Place the frozen sample in a food pmccssor and homogcnizc with dry ice. Place the samples in containers and leave open in rozm storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time ol' analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 RwgcntsandStsnduds - 4.1 W+t--HPLCgrodc 4.2 ACCtonihilC- HPLC @e - 4.3 Carbon (120-400mesh) Reagent grade 4.4 Methmol HPLC @e - 4.5 Silica gel (60-200 mesh) -Reagent grade - 4.6 Plonsil(60-100 mesh) Reagent grade - 4.7 S ~ p ~ ~ lLeCa-NnHz RePgmt grade 4.8 1-oetanol- HPLC grade - 4.9 L-Ascorbic acid Reagent grade 4.10 Dimethyldichlorosilane RCagCnl grade - 4.11 TOIUOIIC R-gat grade 4.12 4.13 - Ammonium Acetate- A.C.S. Reagent Grade Pafluorooctanoic Acid Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chrnalagraph capnble of purnphg up ro 3 solvcnts equipped with a vuiable volume injector capable of injecting S-2OU pL connectedto a tandem Maas Spectrometer (LCIMSiMS). 5.2 A device to collect raw data for ptrk integration and quantitation. 5.3 Analyticalbalancecapable of d i n g to O.oooO1 g. Page 2 af S c 05-0210 Interim Report # l U Northern Alabama Potable Water Systems, December 2005 05-0210 lnferim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 ExygenProtocol Number: PO001 I 3 I ,-- E-x.,vs-cn-Rn-ar~ cb M...e.tho~ d Nu.mher VM..W..)I_ 'IR_I I I ANMYTICAL METHOD Method of Analysis for the Determination of Pnfluorooaanoic Acid (PFOA)in Fish and Clamsby LCMS/MS .-. 5.4 Rotaryevapzator. 5.5 Tissumiza. 5.6 125mL pcar-shsped h k s . 5.1 50 mL disposablepolypropylenecentrifugetubes. 5.8 I5 mL disposablepolypropylenecentrifuge tubes. 5.9 Disposable micropipets (50-1OOuL, 100-2OOuL). 5.10 125-mL LDPE nsrrow-mouthbottles. 5.1 1 2 mL clear HPLC vial kit. 5.12 Disposable pipettes. 5.13 Autopipates (100-1OOO pL and 10-100pL). with disposable tips. 5.14 SPE tubes (ZOmL) (Supclw cat. no. NOS7177). 5.15 wrist action shaker. 5.16 Centrifugecapableof spinning 50 rnL polypropylenetubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analyricsl Column: FluophascRP (KeystoneScientific). 2.1 mm x SO mm. 5p (PM: 82505-052130) 6.2 Temperature: 3 0 T 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: zIE6Jd %LA 0.0 65 1.0 65 8.0 25 20.0 25 22.5 65 Flow Rate !LLBfDuaiQl 35 0.3 35 0.3 15 0.3 75 0.3 35 0.3 - - 6.6 tnjection Volume: 15 pL (canbe increased to as much as 50 pL). 6.7 Quantitation: Peak A n a external standard calibration curve. 6.8 Run Time: 23 minutes. The above conditions arc intended as a guide and may be changed in order to optimize the HPLC system. Page 3 of 8 .- EOS-0210 Interim Report 118 Northern Alabama Potable Water Systems, December 2005 Page 39 01.65 Page 91 o f 7 EO5-02fO Interim Report #18 Northern Alabama Potable Water Systems. December 2005 Exygen Protocol Number: PO00I 13 1 Exygen Rsrerrch 1 Methodof Anal- MabodNumbcr VOW1783 I ANALYTICAL METHOD for the DeterminationofPcrfluorooctanoic Acid (PFOA) in Fish and Clamsby LCMs/MS _- 7.0 MS/MS System 7.1 Mode: ElectrosprayNegative MRM mode, monitoring 413 -+ 369 wvy I'or PFOA. Thc above conditions rre intended as a guide and may be changed in order IO optimize the MSMS system. 8.0 Preparationof Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetateto lo00 mL of water. 8.2 Extraction Solutioo?l 8.2.1 2% ascorbic .cid in mctbanol is prepared by dissolving 2 g of ascorbic acid in Io0mL of methanol. 8.2.2 30% Dimcthyldichlorosilannein toluene is prepared by bringing imi. of dimethyldichlorosilaeto a final volume of 10 mL with toluene. Atanate volumes may be prcparrd. 9.0 Standard Preparation 9.1 Standard StocWortificationSolution 9.1.1 9.12 9.1.3 9.1.4 9.1.5 Prepare a stock solution of -100 & n L of PFOA by weighing 10 my of analytical standard (corrected for purity) and dilute IO 100 mL with metha1101 in a 125-mL LDPE bottle. A 1.0 Mg/mL fortification solution of PFOA is prepared by bringing I mL of the 100 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. A 0.1 pg/mL fortificationsolution of PFOA is prepared by bringing l(1 mLofthe 1.0 p g h L solution to a final volume of 100 with methdnol III a 125mL LDPE bottle. A 0.01 pUmL fortificationsolution of PFOA is prepared by bringing 10 mL of the 0.1 &mL solution Io a final volume of 100 usith mcthnolin a 125 mL LDPE bottle. The rtock and fortificationsolutionsuc b be stored in a refrigerator ar approximately4OC and arc stable for Imaximum period of 6 months from the date of preparation. e 05-0210 Interim Report #18 NorthernAlabama Potable Water System.?,December 2005 Page 40 0/.6> Page 92 of 122 05-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 13 1 Exygrn ReIcucb Mecbod Number VMw1781 1 1 ANALlTICAL kiETIIOD Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 9.2 Standard Calibration Solutions 9.2.1 LClMSlMS calibration standards arc prepared in methanol via dilution of the 1.O pghnL fortificationsolution. 9.2.2 The following is a typical example: additional concentrations may be prepared u)needed. ~ Concmartion Final of Fortifisahon Solution (rdmL) 1 .o 1 .o 1 .o Volume (mL) 5.0 2.5 I .o Diluted to (Id) 100 100 100 Concentration (pe/mL, 0.05 0.025 0.01 0.05 IO 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 all calibration standuds in 125-mL LDPE narrow-mouth bottles at 2OC to 6OC. up to six months. 9.2.4 Altmate volumes and concentrations of standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of samples extracted (typically 20 or ISS) must include at least one untreated control and hvo untreated controls fortified at known concentrations (lab control spike) to veri& procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. I 1.O Sample Extraction 11.1 Weigh 5 g of frozen sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 30 mL of acetonitfileand shake on a wrist action shaker for -1 5 minutes. 11.3 Place the tubes in a frcccer for -1 hour. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in sequence with 2 g florid, 2 g silica gel. 2 g carbon. and 1 g LC-NHp Condition the columns with 20 mL of methanol. *.then 20 mL of acetonitrile. Discard all washa. Do not allow the column to 11.6 Silanizc the 125 mL pcar-lhaped flasks by rinsing with the 30% dmcthyldichlomilane in tolunc solution. Rinse the fl-k with toluene once. followed by mcthanol (lhrcc tima). Dry the flasks completely before use. cithm by airdryingor with a atreamof nitrogen. 05-0210 Interim Report 118 NorthernAlabama Potable Water Systems, December 2005 P o p 41 of 65 . - Page 93uf la-- 05-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygcn Protocol Number: PO00 1 I3 I Exygn Rercueb McthodNumber VoM)1782 1 I ANAL.'tl'lCAL METHOD Method of Analysis for the Determinationof Peffluorooctanoic Acid IPFOA) in Fish and Clams by LCIMSIMS 1 1.7 11.8 11.9 11.IO 11.1 1 I I. I2 11.13 11.14 11.15 11.16 11.17 11.18 CcntrirUge the 50 mL polypropylene tubes containing sample at -2000 rpm for -1 0 minutes. Decant the extract on to a conditioned SPE column fitted inside the mouth of the pear-shaped flask. Collect the eluate in the 125 mL silanized pear-shape flask. Add IO mL of acetonitrile to the sample in the 50 mL centrifuge tube. Homogenize the frozen fat phase using a tissumizcr for -30 seconds and rinse the tissumizer with -10 mL of acetonitrile into the tube. - Shake the sample again for -10 minutes on a wrist-action shaker. Place the tuba in a freezerfor 1 hour mom. Centrifuge the 50 mL polypmpylene tubes containing sample at -2000 rpn~ for -10 minutes. Docant the extract onto the same SPE column. Collect the eluate into the same pear-shaped flask and combine with the eluent from the initial CXtractiOh Pass 20 mL of acetonitrilethrough the SPE column and combine the eluate In the SPIIIC p e ~ ~ ~ h a fpisekd. Add 3-4 drop of I-octanol to the extract in the pear-shaped flask and evaporateat reduced pressure wing a rotaq evaporator (at < 40'C). Make the final volume, by adding 2 mL of 2% ascorbic acid in methunol to the peu-Jhapcd flask and swirl to mix/dissolve. Transfer the extractr to HPLC vials using disposable pipets. Analyze sampler using elcctrosprayL C M W S . 12.0 Chromatography 12.1 Injcct the same mount of each standard, sample and fonified sample into the LCIMSMS system. A calibration standard must precede and follow all analyzed sampler. 12.2 Standards of PFOA comspondingto at Icast five or mom concentration levels must be included in an analytical set. 12.3 An entire set of calibration standards must be included at the beginning and 81 the end of a sample act. Standards must be interspersed between every S- IO samples. As an alternative. an entire set of calibration standards may be injected at the beginning of a set followed by calibration standards i n t c q x r s a l every 5-10 sampler (to account for a second set of standards). In either came, calibration standards must be the first and last injection in il Umpk .cI. 12.4 Use linear standard c w e r for quantitation. Linear standard curves arc generated for the analytc by linear regmuion using I/x weighting of peak area versus calibration standard conccntmtion using MamLynx 3.3 (or equivalent) software system. .. . Payc 6 d % .- EO5-02fO Interim Report #llJ Northern Alabama Potable Water Systems, December 2005 Page 42 01'65 Page 94 of 122 EOCOZIO Interim Re orf # I 8 Northern Alabama Potable Water Systems, Decemger ZOO5 Exygen Protocol Number: PO00I I3 1 1 I ANALYTICAL METHOD Method of A d d s for the Determinationof Perfluorooctanoic Acid (PFOA) in Fish and Clams bv LCIMSIMS 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 musl show a peak of a daughter ion at 369 amu from P parem of 413 m u . The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 mu) represents the loss of carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.5 ppb. then a new blank sample must be obtained and the entire scl must be re-exrracted. 13.3 Rccovnies of conirol spikes and matrix spikes must be between 70- I30?4 of their known values. If a control spike falls outside the acceptable limits, the entire set of samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test. may be excluded from the calculation of the calibration curve However, the total number of calibration standards that could be excluded must not excced 2Wh of the total number ofstandards injected. 13.5 The correlation coefficient (R) for calibration curves generated must he 20.992 (R' a.985). If calibration results fall outside these limits. then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set of samples should be reanalyzed. 13.6 Retention timer behucen standards and samples must not drift more than f 4 % within an analyticalrun. Ifretention time drift exceeds this limit wilhiii an analytical run then the ret must be reanalyzed. 14.0 Calculations 14.1 Use the followingquation to calculate the amount of PFOA found (in ng/niL, bascd on peak area) using the standard curve (linear regression paramelem) generated by h e Mass Lynx software program: PFOA found (ng/mL) ) -= { slope 14.2 Use the followingequation to m v c t t the amount of PFOA found in nglmL to noln @pb). - PFOA found @pb) JPFOAfound(nP/rnLl x final volume h L I x DFI sample weight (8) -DF factorby which thc !idvolume was diluted, I f neeesoary E05-0210 Interim Report #I8 NorthernAlabama Potable Water Systems, December 2005 Page 43 of 65 -Page 95 a??n - EO5-0210Interim Report# I 8 NorthernAlabama Potable WaterSystems, December2005 Exygen Protocol Number:PO001 131 Fxvucn R-h Method Numbcr VWOI 7R1 Clams by LCIMSIMS 14.3 For sampler fortified wilh known amounts of PFOA prior to extraction. use the followingequationto calculate the percent recovery. Recovery (Yo)= 1 - total uulytefound (nglg) annlyte foundin control (ng/g)l oo analyte added (ng/g) Page 8 uiX - EO5-0210Interim Report #I8 NorthernAIahbsme Potable WaterSystems, December 2005 Page 44 of 65 ~ -Page884ttm--- EO5-0210Interim Re ort #18 Northern Alabama Potable WaterSystems, Decemger 2005 Exygen Protocol Number:PO001131 ANALYTICAL METHOD Method Number: VOW1784 Method of Analysis for the Determinationof Periluorooctaaoic Acid (PFOA) in Vegetation by L C M S N S Analytical Testing Fscility: Exygcn Research 3058 Research h i v e State College. PA 16801 Appmvcd By: Paul Connolly I Technical Leader, LC-MS, Exygcn Research lakbI 4 Date dbhnFlahertv ' Date - TotalP.ger: 7 .- E05-0210 Interim Report #18 Northern Alabama Potable Water Systems. December 2005 Page 45 of 65 Page 97 of 122 - 05-0210 Interim Report #I8 Northern Alabama Potable Water Systems, December 2005 Exygen Protocol Number: PO001 13 1 Exvncn Research ~ Mcihod Number VOW I 'X4 I SSALPI'ICAL METHOD 1 Method of Analysis for the Determinationof P e r f i u o ~ l a n o i cAcid (PFOA) in Vegetation by LCMSIMS I .o scope This method is to be employed for the isolation and quantitation of pcrfluorooctanoic acid by High Performance Liquid chromatography coupled to a tandem Mass SpectrometricDetector (LCIMSIMS) in vegetation. 2.0 safe 2.1 Always obscrvc safe laboratorypractices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 SampleRequirement 3.1 At least 20 g of test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in il food pmccssor and homogenizewith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and plicc the samples in f r o m storage until time of analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Stmdardc - 4.1 Wata - HPLC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh)- Reagent grade 4.4 M a 0 1 HPLC @e 4.5 Silicagel (60-200 mesh) - Reagent pade 4.6 Florisil (60.100 mesh) - Reagent grade 4.7 Superclean LC-NHz-Reagent @e 4.8 1-0ctanol- HPLC grade - 4.9 L-Ascorbic acid Reagent @e - 4.10 Dimcthyldichlorosilane Reagent grade 4.11 Toluene - Reagent grade - 4.12 A m r n ~ N ~Amcetate A.C.S. ReagCnt h d e - 4.13 Pcrtlwmoctanoic Acid Sigma-Aldrich 5.0 Insbumen( and Equipment 5.1 A hi@ perfonnanw liquid chmrrtostrph capable of pumping up to 2 solvents quippod with a variable volume injectorcapabk of injecting S-ZH) pL C O M C C ~ C ~to s tpdmtMma Spcoirumctcr(LCMSMS). 5.2 A device tocollect raw data for peak integration and quantitation. 5.3 Analytical balance capable of readingto O.oooO1 g. Page 2 oi 7 .-.- E054210 Interim Report #I8 Northern Alabama Potable Water Systems, December 2005 Page 46 of 65 Page 98 of 722 05-0210 Interim Re ort #I8 No?themAlabama Potable Water Systems,Decemger2005 Exygen Protocol Number: PO00I I3 I r 1 ExygmRcrarob Method Number VOLMI 784 AiiALYTICAL METHOD Method of Analysis for the Determinationof PcrIluorooctanoic Acid (PFOA)in Veseiation by LC/MS/MS 5.4 Rotary evaporator. 5.5 125 mLpe~-shppcdflsskr. 5.6 50 mL disposable polypnpylmc centrifugeti~bes. 5.1 15 mL disposable polypropylenccentrifugetubes. 5.8 Disposablemicropipets(50-100uL.100-200uL). 5.9 125-mLLDPE narrowmouth bottlts. 5.10 2 mL clear HPLC vial kit. 5.11 Disposable pipettes. 5.12 Autopipnu (100-1OOO JILand 10-100pL). with disposable tips. 5.13 SPE tubes (2hnL) (Supelmcat. no.N057177). 5.14 Wrist action shaker. 5.15 Centrifugecapable ofspinning 50 mL polypropylene tubes at 2000 rpm. 6.0 Chmatographic System 6.1 Analytical Column: Fluophare RP (KeystoneScientific), 2.1 mm x 50 mm, 5t1 (PRJ: 82505-052130) 6.2 Temperahue: 3OoC 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B): Methanol 6.5 Gradient Program: Time (mi& 0.0 I .o 8.0 20.0 22.5 %A 65 65 25 25 65 a 3s 35 75 75 35 Flow Rare (mUmin) 0.3 0.3 0.3 0.3 0.3 6.6 Injection Vohune: I5 pL (can be increased to IU much IU 50 pL). - 6.7 Quantitation: Peak Area externalstandard calibration curve. - 6.8 Run Time: 23 minuter. The above conditions are intended PI a guide and may be changed in order to optimize the HPLC system. 7.0 MSlMS Syatem 7.1 Mode:Electrolproy Negative MRM mode. monitoring 413 + 369 m/z for PFOA. EO5-0210 Interim Report # I 8 NorthernAlabama Potable WaterSystems, December 2005 Page 47 uj'6S - -Psse--m---- 05-0210 Interim Report #18 Nolthem Alabama Potable Water Systems, December 2005 ExygenProtocolNumber: PO001131 E x y p R-b Mehod Nvmbcr VDou178J I I AWALYT1C;rL METHOD Method of Analysis for the Determinationof Perfluomoctanoic Acid (PFOA) in Veyetation by LC/MS/MS The above conditions arc intended as a guide and may be changed in order IO optimize the MSMS system. 8.0 Preparationof Solutions 8.1 MobilePhaae 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to IO00 mL of water. 8.2 Extraction Solutions 8.2.1 2% ascorbic acid in methanol is prepared by dissolving 2 g of ascorbic acid in 100mL of methanol. 8.2.2 30?4 Dirnethyldichlomilane in toluene is prepared by bringing 3 mL ofdmcthyldichlomiilane to a final volume of 10 mL with toluene. Altcmate voluma may be prepand. 9.0 Standard Preparation 9.I StandardStocWFortification Solution .- 9.1.1 Preparea rtocl: rolution of400 & n L of PFOA by weighing 10 ing of analytical standard ( c o m t c d for punty) and dilute to 100 rnL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/rnL fonification solution of PFOA is prepared by bringing I mL of the 100 pg/mL solution to a final volume of 100 with niethanol in a 125 mL LDPE hottle. 9.1.3 A 0.1 pg/mL fortification solution of PFOA is prepared by bringing IO mL of the 1.0 p@mLsolution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.01 pglmL fortification solution of PFOA is prepared by bringing 10 mL of the 0.1 p@mL solution to a final volume of 100 with muunOl in a 125 mL LDPE bottle. 9.1.5 The stock lrnd fortification solutionsare to be stored in a refrigeratorai appmximntcly4OC and arc stable for a maximum period of 6 months &om the date of preparation. 9.2 Standard Cdibration Solutions 9.2.1 LC/MS/MS calibration rtmdudr arc prep@ in methanol via dilution of the 1.O pg/mL fortificationsolution. EOS-0210IntetfmReportiVl8 Northern Alabama Potable WaterSystems, December 2005 05-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 131 Exygen Rcrcuch Merhod NwnberVWOt784 I i AhALYl ICAL ILlh'THOD Method of Analysis for the Determinationof Pcrfluomoctanoic Acid (PFOA) in Vegetation by LCMSIMS 9.2.2 The following is a typical example: additional concentrations may he prepared aa needed. Conembation Final ofF M i f d i o n Solution (pdml,) 1 .o I .o 1.0 Volumc (mL) 5.0 2.5 I .o Diluted to (mL) 100 100 100 Concmhation (pdmL) 0.05 0.025 0.01 0.05 10 100 0.00s 0.025 IO 100 0.0025 0.1 10 100 0.001 0.005 10 100 o.Ooo5 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2OC to6C.up to six months. 9.2.4 Altcmate volumes and concentrations of standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of sunplcs cxb-actd (typically20 or less) must include at least one untreated control and huo untreated controls fortified at known concentrations(tab control spike) to verify procedural recovery for the b a ~ h 10.2 Requirements for field and laboratory duplicates and spikes will be specilied in the quality assurance plan for this project. I I .O Sample Extraction 11.1 Weigh 5 g of frozen sample into 50 mL polypropylene centrifuge tubes (fodfy as needed. replace lid and mix well). 11.2 Add 30 mL of acetonitrile and shake on a wrist action shaker for -1 5 minutes. 11.3 Centrifuge the 50 mL polypropylene tuba containing sample at -2000 rpni for -10 minutes. 11.4 Pack and conditionthe SPEtubes and silanize the pear-shaped flasks. 11.5 Pack the 20 mL. SPE tuba in sequence with 2 8 florisil. 2 g silica gel. 2 g carbon. and 1 g LC-NHz. Condition the columns with 20 mL of methanol. then 20 mL of acetonitrile. Discard all washes. Do not allow the column IO dry. 11.6 Sillnize the 125 mL. pcar-shapcd flasks by nnring with the 30% dimethyldichlodlme in t o h e solution. Rinse the flask with toluene once. followed by mc(h.aol (thrce times). Dry the flasks completely before use. either by &-drying or with a stream of nitrogen. 11.7 Dccant the extract on to a conditioned SPE column fitted inside the mouth 01' the par-rhapod flask. Collect the eluate in the 125 mL rilanizcd pear-shape flask. E054210 Interim Report #I8 Northern Alabama Potable Water Systems, December 2005 Page 49 0165 Page 101 of 122 - - - 05-0210 lnterim Re ort #18 Northern Alabama Potable WaterSystems, Decemger 2005 Exygen Protocol Number: PO001 I3 1 Exypen Remuch M c b d Number VOiXJI7E.4 I Methodof Analysis for the Determinationof Pertluorooftanoic Acid (PFOA) in Vepiatioti by LCIMS/MS 11.8 11.9 11.10 11.1 1 11.12 11.13 11.14 11.15 11.16 Add 20 mL of acaonitrileto the sample in the 50 mL centrifuge tube. Shake the sample again for -10 minutes on a Wrist-action shaker. Centrifuge the 50 mL polypmpylcne tuba containing sample at -2000 rpm for -5 minutes. Decant the extract onto the same SPE column. Collcct the eluate into the same pear-shaped flask and combine with the eluent horn the initial extraction. Repeat steps 11.8 through 1I. I I again. Add 3 4 drops of I-octanol to the ubact in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40'C). Make the tinal volume, by adding 2 mL of 2% ascorbic acid in melhanol IO the pear-shapedflaskand swirl to mix/dissolve. Transfer the extracts to HPLC vials using disposable pipets. Analyze~ampleussingelcetrorpray LCIMSMS. 12.0 Chromatography 12.1 lnjcct the sune amount of each standard, sample and fortified sample into thc LCIMSIMS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards of PFOA correspondingto at least five or more concentration levels must be included in an analytical set. 12.3 An entire set of extracted calibration standards must be included at the beginning and at the end of a sample set. Extracted standards must hc intmpcrscd between every 5-10 aamplss. As an alternative. an entire set of extracted calibration standards may be injected at the beginning of a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a scwnd set of extracted standards). In either case, extracted calibration nnndsrdrmust be the first and last injection in a sample set. 12.4 Use linear standard curved for quantitation. Linear standard curves are generatedfor the analytc by linear regression using I/xweighting of peak area versus calibration standard concentration using MsssLynx 3.3 (or equivalent) sonwan system. 12.5 Sample reoponse shouId not exceed standard responses. Any samples that exceed standardresponsesshouldbe furtherdiluted and reanalyzed. 13.0 ArrapclaccCrifari. 13.1 Chromatogram mud show Ipeak of a daughter ion at 369 a m u from a parent of 413 mu. Thc 413 unu pucnt Comrponds to the PFOA anion. while the daughter ion (369m u ) rcpmcnts the IOUof carbon dioxide. 05-0210 Intedm Report #la Northern Alabama Potable Water Systems, December 2005 Page 50 of 65 page 102or fm-- 05-0210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Exygen Protocol Number:PO00I I3 1 Exy~coR-h Motbod Number VOW1784 ."- AYALYTICIU METHOD 1 I Method of Analysis for the Determinationof Pertluorooctanoic Acid (PFOA) in Vegetation by LCIMSIMS 13.2 Method blanks must not contain PFOA at levels m a t e r than the LOQ. If Q blank contains PFOA at levels greater than 0.5 ppb. then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries of contml spikes and matrix spikes must be between 70- 130% 01' their known valuw. If a control spike falls outside the acceptable limits. the entire set of samplu should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation of the calibration curve. Howcvn. the total number of calibration standards that could be excludrd must not excecd 20% of the total number of standards injected. 13.5 The correlation cocficient (R) for calibration CUNCS generated must be 20.992 (R' 20.985). If calibration results fall outside thew limirs. then appropriate steps must be lakcn to adjust insbument operation, and the standar& or the relevant set of samplu should be reanalyzed. 13.6 Retention times bawem standard3 and samples must not driA more than f 4 %within an analyiicalrun. Ifretention time drifi exceeds this limit within an analyticalrun then the set must be reanalyzed. 14.0 Calculations 14.1 Use the followingequation to calculate the amount of PFOA found (in ng/mL. bascd on peak area) using the standard curve (linear regression parameters) generated by the Mess Lynx soAwan program: PFOA found inghnl) = slope 14.2 Use tho followingequation to convat the amount of PFOA found in ng/mL to ngle @pb). PFOA found (ppb) =lpF04 found1-nf volume ImL) x DF] DF - factor by which the final sample weight (9) volume was diluted, if necessary. 14.3 For rmplcs foaified with known mounts of PFOA prior to extraction. use the following equation to calculate the pcrcent recovery. Rccovcry (On) = - total d y t efound(nlys) analfiefound in control (nugll oo malyreadded (ng/g) P a p 7 UI 7 c E05-0210 Interim Report #18 Notthem Alabama Polable Water Systems, December 2005 Page 51 of 65 Page 103 of 2l- EO5-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemhr 2005 Exygen Protocol Number: PO00I I3 I e ANALYTICAL METHOD Method Number: V000178S Method of Analyrls for the Determinationof Perfluorooetanole Acid (PFOA) in Small M8mmalLiver by LCIMSIMS Analytical Twting Facility: Exygen Rwcareh 30S8 Research Drive State College. PA 16801 Approved By: TechnicalLeader, LC-MS,Exygm Research Vice President,OpaPtions. Exygm Research /flAi Date TotalPagw: 7 E054210 Interim Report # l a Norfhern Alabama Potable Water Systems, December 2005 Page 52 of 65 - .- --Page 1tU oft22 - 05-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 131 1 ' ExygcnRcscuch Mcrhod Number VUWl785 &LYTIC& METHOD Mcthod of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LCIMSIMS 1 .o This method is to be employed for the isolation and gumtitalion of perfluorowtanoic acid by High Pcrfomrncc Liquid Chromatography coupled to a tandem Mass SpectrometricDetector (LCIMSIMS)in small mammal liver. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appmpnate MSDS bcbrehandling m y chemical for pmpcr safely precautions. 3.0 Sample Rcquircmcnt 3.1 At Itpat 5 g of test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave opcn in frozen storage overnight to allow for carbon dtoxlde sublimation. Sal and place the samples in frozen storage until time of analysis. Alternately, if t h a t is an insuffcient amount of sample (-less llidn 5 9).then no processing is necessary and the sample can be used as supplied 3.3 Sample collection procedures will be specified in the sampling plan for thls project. 4.0 Reagents and Standards - 4.1 Wata HPLC &C 4.2 McthaI~ol-HPLC grsd~ 4.3 Acctonidle - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade - 4.5 PerfluorooctnnoicAcid Sigma-Aldrich 5.0 Instnunent nnd Equipment 5.1 A high pdonnancc liquid chromatograph capable of pumping up to 2 rolventli equipped with a variable volume injector capable of injecting 5-200 pL C O M C C ~ Ct~o a tandem Mass Spectrometer(LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Anplyticalbalance capable of d i n g to 0.M)OI g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposablepolypmpylcnc centrifuge tubes. 5.6 Disporrblc micropipets (SO-lOOuL, 100-2OOuL). 5.7 125-mLLDPE narrow-mouthbottles. 5.8 2 mL ckar HPLC vial kjt. EO5-0210 Interim Report#?8 Northern Alabama Potable Water Systems, December 2005 Page 53 of 65 Page 105bf1;Er 05-0210 Interim Report #18 Northern Alabama Potable Water Systems, December 2005 Exygen Protocol Number: PO00113 I Exygonl W c m b Method Number V0001785 1 D - P AKALYTICAL I\.IETHOD Method of Analysis for the D e t M n i d o n of Pdwrooctanoic Acid (PFOA) in Small Mammal Liver by LClMSlMS 5.9 Disposable pipettes. 5.10 Autopipettes (100-1OOOpL and 10-100pL). with disposable tips. 5.1 1 Waters Sep PaL Vac 6 cc (lg) tC18 SPE c u u i d g a . 5.12 SPE vacuum manifold. 5.13 Tissuanizer. 5.14 Wrist-action shaker. 5. I5 Centrifuge capable of spinning I5 mL. polypropylene tubes at 3000 rpm. 6.0 ChromatographicSystem 6.1 Analytical Column: Fluophrcc RP (Keystone Scientific). 2.1 mm x 50 mnl. 511 (P/N: 82505-052130) 6.2 Tempcnture: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: ximflw LA 0.0 65 1.o 65 8.0 2s 20.0 25 22.5 65 Flow Rate Q (rnmYrnin) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 - 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: 23 minutes. The above conditions m intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS system 7.1 Mode: Elcctrospy NegativeMRM mode. monitoring413 -+ 369 m/z for PFOA The above conditions an intended as a guide and may be changed in order to optimize the MSMS system. 05-0210 Interim Report #I8 NorthernAlabama Potable Water Systems, December 2005 Pnge 54 of45 Page 106 of 122 .._- EO5-0210 Interim Re ort #18 Northern Alabama Potable Water Systems, Decem& 2005 Exygen Protocol Number: PO001 131 Exygm Rcsnrsh McthodNumbcr VooO1785 1 - - I 1 A N ~ Y T I C A LMETHOD Method of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 8.0 Proparationof Solutions 8.1 Mobile P h w 8.1.1 2 mM ammoNum acetate in water is prepared by adding 0.154 y 01' ammonium acetate to IO00 mL of water. Alternate volumes may be prsparad. 9.0 Standard Preparation 9.I Standard SIocWFortification Solution 9. I.I Pnpan a stock solution of -100 p v m l of PFOA by weighing IO m y of analytical standard (comcted for purity) and dilute to 100 mL with methanol in a 125-mLLDPE bottle. 9.1.2 A 1.0 pe/mL fortification solution of PFOA is prepared by bringing I mL of the 100 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pdmL fortificationsolution of PFOA is prepared by bringing I I ) mL of the 1.0 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock aud fortificationsolutions arc to be stored in a refrigeratora1 approximately 4'C and am stable for a maximum period of 6 months from the date of preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/Ms/MS calibration standards are prepared in methanol via dilution of the 0.1 p e / d fortification solution. 9.2.2 The following is a typical example: additional eoncentrations may hr prepared as nccdcd. Concentration Final of Fortification Volume Diluted lo Concentration Solution (ng/mL) (mL) (mL) (ngImL) 100 5.0 I00 5.0 100 2.0 100 2.0 100 I .o I00 I .o 5.0 10 100 0.5 2.0 IO 100 0.2 J.0 10 100 0.1 9.2.3 S t o a~ll calibration standards in 125-mL LDPE narrow-mouth bottles at 2'C to 6-C. up to six months. 9.2.4 Altcmate volumed and concentrations of standards may be prepared as nccdcd. 05-Q2lVI-8 Northern Alabama Potable Water Systems, December 2005 Puge 55 qf 65 Page 107 of 122 ---I EO5-OZlOlnteritn R Northern Alabama Potable Water Systems, Decem%$Zti$ Exygen Protocol Number: PO001131 Exygcn Reseuch M c W Number VOOO 1715 L 1 AhALYTICAL METHOD Method of Analysis for the Determinationof Pafluorooctanoic Acid (PFOA)in Small Mammal Liverbv LCMSMS 10.0 Batch Set Up 10.1 Each batch of wnplm extracted (typically 20 or less) must include at leas1 one untreated control and two unlrurted controls fortified at known concentrations(lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assuranceplan for this project. 11.O Sample Extraction 11.1 11.2 11.3 11.4 113 1 I .6 11.7 11.8 11.9 11.10 I 1.I1 Weigh I g of sample into a 50 mL polypropylene centrifuge tubes (fonify us needed. replace lid and mix well). Note that alternate weights o f liver may he measured dependingon the sample size available for use. Add water to the m p k for a fmd volume of 10 mL. Homogenize 8amplc using a tissucmizff for -1 minute. Transfer 1 mL of the sample using a disposable pipene into a I5 mL disposrble centrifuge tube. Add 5 mL of acetoni~leand sh8ke for -20 minutes on a wrist-action shaker. Centrifugethe tubes at -3000 rpm for -5 minutes. Decant the supernatant into a 50 mL disposable centrifuge tube and add 35 mL of water. Condition the CI:SPE cartridges (1 g, 6 A)by passing 10 mL methanol followedby 5 mL ofHPLC water I- 2 dropkc). Do not let column run dry Load the sample on conditioned Cl, SPE cubidge. Discard eluate. Elute with -2 mL of methanol. Collect 2 mL of eluate into a graduated 15 mL polypropylenecentrifugetube (final volume = 2 mL). Analyze samples using electrospray LCIMSIMS. 12.0 Chromatography 12.1 hjcct the siuue amountof each stand.rd sample and fortified sample into the Lc/Ms/MS system. A calibration Itandard must precede and follow all analyzed samples. 12.2 Standards of PFOA corresponding to at least five or more concentration levels must be included in an pnalficd set. 1 2 3 An entire 6et ofcalibration standardsmust be included at the beginning and dI the end of a sample set. Standardsmust be interspersed between every 5-11) samples. As an alternative, M entire set of calibration standards may hc injected pt the bcgnning of a set followed by calibration standards intmpcnod every 5-10 rampla (to account for a second set of standards). In either case. calibration standatdr must be the first and last Injection in a sample set. 12.4 Use linear standud cuwu for quantitation. Linear standard curves arc generated for the d y t e by lineu regression using Ihc weighting of peak area E054210 InterimReport #I8 Norulem Alabama Potable WaterSystems, December 2005 EO5-0210 Interim Report #18 Northern Alabama Potable Wafer Systems, December 2005 Exygen Protocol Number: PO001I3 I Exylca Rucarcb Metbod Numbcr V0001785 L I ANALYIICAL METHOD Method of Analysis for the Determination of PcrfluorooctanOic Acid (PFOA) in Small Mammal Livcr by LCIMSIMS v m u s calibration standad concentration using MassLynx 3.3 (or equivalent) sofhvarc system. 12.5 Snmple response should not excccd standard responses. Any sampler I h d exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 ChmmaIogram must show a peak ofa daughter ion at 369 a m u from B parent of 413 mu. The 413 amu p m t cornsponds to the PFOA anion. while the daughter ion (369 m u ) representsthe loss of carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 10 ndg, thcn a new blank sample must be obtained and the entire set must be re-extracted. 13.3 RccovCriw of control spikes and mahx spikes must be between 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set of s ~ l p l c should be racxtracted. Any matrix spike outside 70130.9 should be evaluated by the analyst to determine if re-exlracfion is warranted. 13.4 Any calibration standani found to be a Statistical outlier by using the Huge Error Test, may be excluded &om the calculation of the calibration curve However, the total number of calibration standards that could be excluded must not exceed 20% of the total number of standards injected. 13.5 The cornlation coenicient (R) for calibration curves generated must be 20.992 (R2 20.985). If calibration results fall outside these limits. then appropriate steps must be taken to adjust instrument operation. and the standads or the relevant set of samplesshould be reanalyzed. 13.6 Retention times between standards and samples must not dnR more than f 4 %within an analytical run. Ifretention time driR exceeds this limit within an analytical run thcn the set must be reanalyzed. 14.0 Calculations 14.1 Use the followingequation to calculate the amount of PFOA found (in ng/mL. b a e d on perk area) using the standard curve (linear regression parameters) generated by the Mass Lynx sofiwarcprogram: PFOA found (nglmL) = slope x DF x aliquot factor DF = f s t o rby which the final volume was diluted, ifnccuury. Aliquot factor * 10 059210 Interim ReHrt #78 Northern Alabama Potable WaterSystems, December 2005 Pnge 57of 65 - --7e7uTgofm E054210 Interim Re ort #I8 NorthernAlabama Potable WaterSystems,DecemLr 2005 Exygen Protocol Number: PO00 1 13 I '- Exygcn Raemb Method Numbcr VOOUl~85 I I ANALY i IC& METHOD Mdhod of Ana-lw-,is for the Determinationof PerfluorooctanoicAcid (PFOA) in Small Mammal Liver by LcmiIslMS 14.2 For samples fdfied with known amounts of PFOA prjor IO extraction. use the followingquation to calculatethe percmt recovery. 1total analytefound(nghnL) - andytefoundin control(nglmL)l analyte addcd (ng/mL) 14.3 Use the following c q d o n to convert the amount of PFOA found in n g h L 11) ntde @PU. PFOA fouund (pp-b) = p nvu m sample weight (g) .- E05-0210 lnterinrRepart#l8 NorthernAlabama Potable WaterSysfems, December2005 Page 58 of 65 Page 110 of 122 - .- 05-0210 Interim Re ort # I 8 Norfhem Alabama Potable Water Systems, Decem/er 2005 Exygen Protocol Number: PO00I 131 ANALYTICAL METHOD Method Number VOOOl786 Method of Analysis for the ~ t t r m i n r d o aof Perlluormtaaoic Acid (PFOA) in Smull Mammri Serum by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College. PA 16801 Approvcd By: Paul Connolly I Technical Leader, LC-MS. Exygcn Research /Vice Prcsidenl, Operations, Exygcn Research ToulPages: 7 05-0210 Infenlm Report #18 Norfhem Alabama Potable Water Systems, December 2005 05-0210Interim Re rt #I8 Northern Alabama Potable Water Sysfems, DecemEr 2005 Exygen Protocol Number: PO001 13 1 c ExygmResarcb Method NumberVOLUJ1786 1 1 ANaiTJCAL MJZTHOD Mahod of Analysis for the Determinationof Perfluormtnnoic Acid (PFOAI in Small Mammal SCNm by LCIMSIMS 1.0 scope This method is to be employed for the isolation and quantitation of peffluorooctano~c acid by High Performance Liquid Chromatography coupled to a tandem Mass SpectromemC Detector (LCIMSIMS) in small mammal semm. 2.0 Safety 2.I Always observe safe laboratorypractices. 2.2 Consult the appropriateMSDS bcforc handling any chemical for proper saliry precautions. 3.0 Sample Requirement 3.1 At lurt 1 mL of lest sample forextraction. 3.2 No sample processing is needed for aenun samples. However, frozen serum samplesmust to allowed to completely thaw to roomtemperature before use. 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Stnndvdr 4.1 Water-HPLCgredc 4.2 M a h ~ n o l -HPLC g r s d ~ 4.3 Acetonitrile- HPLC grade 4.4 Ammonium Acetate- A.C.S. Reagent Grade 4.5 PerfluorooctanoicAcid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable of pumping up IO Z solvents equipped with a variable volume injector capable of injecting 5 - 2 0 0 pL connected to a tandem Mass Spectrometer (LCMSIMS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analyticalbalance capable of reading to O.OOOO1 g. 5.4 50 mL disposablepolypropylenecentrifuge tubes. 5.5 15 mL disposablepolypropylenecentrifuge Nbcs. 5.6 Disposablemicropipets(SO-IOOuL.100-2ohcL). 5.7 125-mLLDPE nanow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Di.poublcpipcna. 5.10 Autopipettea(100-1OOO pLMd 10-100 pL), with dirposable tips. 5.11 WaterrSepPPLVPC6CE(le)lClSSPEccutridges. 5.12 SPE vacuum manifold. 5.13 Vortcxer. - - E O S ~ 2 t O ' l n ~ ~ l i ?NorthernAlabama Potable Wafer Systems, December 2005 EO50210 Interim Re rt M 8 Northern Alabama Potable Water Systems, Decemer 2005 Exygen Protocol Number: PO001 131 Exypcn Rnureh Method Number VooU17U6 I I A L L Y ' l l C A L METHOD Method ofAnalysi8 for the Detmination of Perfluorooclanoic Acid (PFOA)in Small Mammal Serum by LClMSlMS 5.I4 Wrist-actionshaker. 5.15 Centrifuge capableof spinning 15 mL polypropylene t u b a at 3000Ipm. 6.0 ChromatographicSystem 6.1 Analytical Column: FluophaccRP (Keystone Scientific). 2.1 m m x 50 mm.5p (PM: 82505.052130) 6.2 Temperature: 30-C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Pholre (B) : Metheno1 6.5 Gradient Program: Time rq&l) .%ti 0.0 65 1 .o 65 8.0 25 20.0 25 22.5 65 Flow Rate ImUmin) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 - 6.6 Injection Volume: I5 pL.(can be increased to as much as SO pL). 6.7 Quautitation: Peak Am - extemal standard calibration curve. 6.8 Run Time: 23 minutes. The above conditions am intended as a guide and may be changed in order Io optimize the HPU: system. 7.0 MSMS System 7.1 Mode: Electrorpray Negative MRM mode, monitoring 4 I3 + 369 m'r for PFOA. The above conditions am intended as a guide and may be changed in order IO optimize the MSMS system. 8.0 Preparationof Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammoniumacetate to IO00 mL of water. Alternate voluma my be pnpucd. E05-0210Intetfm-RqWr#ir Northern Alabama Potable Water Systems, December 2005 Page 61 of65 Page ii3of122 05-0210 Interim Re orl#18 Northern Alabama Potable Water Systems, Decemger 2005 Exygen Protocol Number: PO001 13 I Exygen RcKuch M C W N&I V000178b I ANALYTICAL !blhTHOD 1 Method of Analysis for the Determination ofPduorooctanoic Acid (PFOA) in Small Mammal Serum by LCIMSiMS 9.0 StandardPnparstion 9.1 StandardStocWFottificationSolution 9.1.1 Prepare a stock solution of -100 p g h L of PFOA by weighing 1 0 m g of rnalyiical standard (corrected for purity) and dilute ID 100 rnL u ill1 methanol in a 125-mLLDPE bottle. 9.1.2 A 1.0 pg/rnL fottification solution of PFOA is prepared by bringing I mL of the 100 (rg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution of PFOA is prepared by bringing I O mL. of the 1.0 &mL solutionto a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortificationsolutions arc to be stored in a refrigerator at approximately 4C and am stable for a maximum period of 6 months from the date of preparation. 9.2 Standard Calibration Solutions 9.2.1 L C / M S M S calibration standards u e prep& in methanol via dilution of the 0.1 pghnL fortificationsolution. 9.2.2 The following is a typical example: additional concentrations may be prepared asneeded. Concmtmtion Final of Fortification Volume Diluted to Concentration Solution(ng/mL) [mL) (Id) (ng/mL) 100 5.0 100 5.0 100 2.0 100 2.0 100 1 .o 100 I .o 5 1) IO 100 0.5 2.0 10 100 0.2 1 .o 10 100 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bollles at 2C to 6C.up to six months. 9.2.4 Altanate volumes and concentrations of standards may be prepared lis needed. 10.0 Batch Set Up 10.1 E A bat& of .p"pla exmcrcd (tyPic.Uy 20 or l a ) must include at lens^ one unmated contrOt and two untreated controls fortified at known coneentntiotu (lab controlspike)to verify procedural recovery for the batch 10.2 Requirements for field and Iabor8tOry duplicates and spikes will be spccl(ied in the quality wmcc p l fo~r this project. 05-0270 In*-m ReMti #la Northern Alabama Potable Water Systems, becember 2005 05-0210 lntetim Report #I8 Northern Alabama Potable Water Systems, December 2005 Exygen Protocol Number: PO001131 I 1 ExypnResmcb MClbodNumberV000171b AhALYTICAL METHOD Mahod of Analysis for the Determinationof Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MSflUIS 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 11.10 11.11 Measure I mL of sample into a 50 mL polypropylene centrifuge tubes (fortify as needed, nplace lid and mix well). Note that altcmate volumes of serum may be measured depending on the sample size available for use. Add water to the sample for a final volume of 20 mL. Cap tightly Vortex for -1 minute. Trmsfcr 1 mL of the sample using a disposable pipate into a 15 mL disposable centrifuge tube. Add 5 mL of acetonitrileand shake for -20 minuter on a wrist-action shaker Centrifuge the tubes at -3000 rpm for -5 minutes. Decant the supernatant into a 50 mL disposable centrifuge tube and add 35 mL of water. Condition the C,, SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followedby 5 mL of HPLC water (- 2 dropkc). Do not let column run dry Load the sample on conditioned Clt SPE cartridge. Discard eluate. Elute with -2 mL of methanol. Collect 2 mL of eluate into a graduated 15 mL polypmpylene centrifuge tube (final volume = 2 mL). Analyze samples using elcctrosprayLCMSIMS. 12.0 Chromatography 12.1 Inject the y ~ m camount of 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 of PFOA corresponding to at least five or more concentration Icvcls must be included in an analytical set. 12.3 An entire set of calibration standards must be included at the beginning and PI the end of a sample set. Standards must be interspersed between every 5 - 1 0 samples. As an alternative, an entire set of calibration standards may he injccted at he beginning of 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 3 sample set. 12.4 Use linear standard curves for quantitation. Linear standard CUNS are generated for the uulfle by linear regression using Ilx weighting of peak area vmus calibration standard concentration using MMsLynx 3.3 (or equivalent) sonware system. 1 2 3 Sample mp0ll.c ahodd mi exceed sLs%Urd raponsea. Any samples Ihai a c d standardmponsca should be further diluted and reanalyzed. --- Page 5 01.- - - - V m t B m Riieportma NorthernAlabama Potable Water Systems, December 2005 Pogc 63 oj 65 -Pii@i7r5imn 05-0210 Interim Re rt #18 Northern Alabama Potable Water Systems, Decemgr 2005 ExygenProtocol Number: PO001131 Ex.w-enReKucb McthodNumber VW17116 1 i ANALYTICAL METHOD Method ofARnlvsis for the Damnination of Perfluorooetsnoic Acid (PFOA) tn Small Mammal Serum by LCIMSMS ~~ ~ 13.0 AcceptanceCriteria 13.1 Chromatogrammust show a peak of a daughter ion at 369 a m u from a parent of 413 mu. m e 413 amu parent eomsponds to the PFOA anion, while tlic daughter ion (369mu) reprcsents the loss of carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 10 ng/mL. then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recovcri~of control spikes and matrix spikes must be between 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set of samples should be re-cxhnctcd. Any matrix spike outside 70130% should be cvphutcd by the analyst to dcterminc if re-extraction is wmtcd. 13.4 Any calibdon standard found to be a statistical outlier by using the Huge Error TUr, may be ucludcd from the calculation of the calibration curve. However. the total numba of calibntion standards that could be excluded must not exceed 20%" of the total number of standards injected. 13.5 The correlation coeficient (R) for calibration curves generated must he 20.992 (R' 20.985). Ifcalibration results fall outside these limits. then appropriate steps must be taken to adjust instrument operation. and thc standards or the rclevant set of samples should be reanalyzed. 13.6 Retention times behvcen standards and samples must not drift more lhan f 4 %within an analytical run. If retention time drifi exceeds this limit within an analyticalrun then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equationto calculate the amount of PFOA found (in ngimL. bared on peak area) using the standard curve (linear regression parmerersr generated by the Mass Lynx softwareprogram: PFOA found (nghL) = p ! slope Lx DF x aliquot factor DF = factorby which the finalvolume was diluted, if necessary. Aliquot factor = 20 14.2 For urnplea fottificd with known amounts of PFOA prior to extraction. use - Ihe following Recovery (%) equation to cdculate the p m m t recovery. A E054210 Interim Report #18 NorihemAlabama Potable Water Systems, December 2005 Page 64of65 Page 116 of 122- 05-0210 Intefim Re rt #18 Northern Alabama Potable Water Systems, Decemgr 2005 Exygen Protocol Number: PO00113 1 --- Aii&ILYTICAL !METHOD 1 I Method ofAnalysis for the DeIermination ofPduoroocIanoic Acid (PFOA)in SrndlI Mammal Serum by LCIMSIMS 14.3 Use the followingequation to convert the amount of PFOA found in n g h L IO PPb. PFOA found @pb) =JPFOAfound h d m U x final volume (mLu ample volume (mL) E05-0210Interlm Report#l8 NorthernAlabama Potable WaterSystems, December 2005 Page 65 oj 6j Page 117of 122-- E050210 Interim Re OH #78 Northern Alabama Potable Water Systems, Decemfer 2005 Protocol Exygen PO001131; 3M Study Number E050210 Amendment 3 Study Title ANALYSISOF PERFLUOROBUTANESULFO(NPFABTSE), PERFLUOROHEXANESULFONATE(PFHS), AND PERFLUOROOCTANESULFONATE(PFOS) IN WATER, SOIL,SEDIMENT. FISH, CLAMS, VEGETATION, SMALL MAMMALLIVERAND SMALL MAMMALSERUM USING LC/MS/MS FOR THE 3M DECATURMONITORING PROGRAM PROTOCOLAMENDMENT NO. 3 Amendment Date: May 16,2005 Performing Laboratory 3M Environmental, Health, and Safety Operations 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification E05-0210 E05-0210 GLP Protocol Amendment #3 Page 1 of 4 E059210 lnterim Report 118 NorthernAlabama Potable Wafer Systems, December Zoos Page 718 of 122 E050210 Interim Re ort # I 8 NorthernAlabama Potable Water Systems, Decemf e r 2005 Protocol Exygen PO001131; 3M Study Number E050210 Amendment 3 This amendment modifies the following portion(s) of the protocol: PROTOCOL READS: TESTINGF A C / L t W Exygen Research 3058 Research Drive State College, PA 15801 Phone: (814) 272-1039 AMENDTO READ: TESllNG F A C I ~ E S Exygen Research 3058 Research Drive State College, PA 15801 Phone: (814) 272-1039 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 I REASON: Addition of 3M Environmental Laboratory as a testing facility allows selected water samples to be sent to the 3M facility for analysis of PFBS, PFHS, and PFOS using the most current version of ETS 8-154 "Determinationof PerfluorinatedAcids, Alcohols, Amides, and Sulfonates in Water by Solid Phase Extractionsand High Performance Liquid ChromatographyhlassSpectrometry". The following modifications to ETS 8-154.1 will be incorporated into the study: (1) The sample volume extracted and the final SPE elution volume may be adjusted, at the analyst's discretion, to better achieve the detection limits specified in study objectives. As written, ETS 8154.1 calls for a 40 mL extraction volume with a 5 mL elution volume which results in an eightfold sample concentration. The actual volumes used and the resulting overallconcentration factor will be given in the final report. Associated method quality control samples will demonstratethat the volume modificationsdo not impact method accuracy and precision. (2) A solvent (unextracted) calibration curve may be analyzedwith the extracted calibration curve to ascertain extraction efficiency. Samples will be analyzed using the extracted curve unless otherwise stated in the final report. Data from the solvent calibration curve will not be induded or discussed in the final report unless it is used to strengthen experimental observationdexplanations. (3) ETS 8-154.1 makes no mention of sample surrogate spikes; however, all samples will be spiked - with at least one of the followingradiolabeledsurrogates, PFOA [1,2 13C](perfluorooctanoicacid), PFOS ["02] and/or PFNA [ I,2 3C](perfluorononanoicacid C9).The concentrationof the surrogate spike may vary depending on the collection event, but typical samples are spiked with a nominal concentrationof 0.05 ng/mL. Surrogate spike recoverieswill be documented and discussed in the final report. Surrogate recoveriesbetween 100e5%will be deemed as meeting method criteria for accuracy. (4) Several method blanks may be prepared and analyzed to better determine a "representative" area count value for the method blank. The method blank area count is instrumental in determiningthe method limit of quantitation and understandingthe average and range of area counts possible is critical. If a method blank value is used as a "zero point" in the final calibration curve, then a brief explanation in the final report or in the raw data as a Note to File must be E05-0210 GLP Protocol Amendment #3 Page 2 of4 E050210 lntedm Report # I 8 Northern Alabama Potable Water Systems, December 2005 Page 119 of 122 05-0210 Interim R ort # I 8 Northern Alabama Potable Water Systems, Decern"g,, 2005 Protocol Exygen PO001131; 3M Study Number E050210 Amendment 3 included to describe why that specific method blank was selected as the zero point (closest to the average value, most conservative (largest) value, etc.) (5) The overall analyticaluncertaintyfor each target (non-surrogate) analyte will be estimated using both the method accuracy and precision. The method accuracy and precision will be calculated using data from laboratory contorl spike (LCS) samples prepared and analyzed with the sample set(s). Replicate LCSs must be prepared each day samples and/or other quality control samples are prepared. A sample calculationof how the analyticaluncertainty was determinedwill be provided in the final report and/or raw data. A new revision of ETS 8-154 may incorporate all or several of the modificationslisted above. If a new version of ETS 8-154 is issued during the course of this study, sample results must meet the new method requirementsregardlessif they are listed above. 3M EnvironmentalLaboratory managementwill approve all documented deviations to the 3M EnvironmentalLaboratoryquality system (SOPS,methods, etc.) that occur during the course of this investigation. E05-0210 GLP Protocol Amendment #3 E054210 Interim Report # I 8 Northern Alabama Potable Water Systems, December 2005 Page 3 of 4 Page I20 of 122 E050210 Interim Re ort # l 8 Northern Alabama Potable Water Systems, Decemger 2005 Pd& Exvgen pooOll31; 3M Study Number E050210 Amendment 3 . Amendment Approval MichaelA. SantA, 3M, c WilliamK Reajen, ph.D. Sponsor Representative Date UJ*MJ-- 3M Environmental Laboratory Manager Date W Jaisimha Kesari, Wesion Solutions, Study Director ' Date EO54210 GLP Protocol Amendment #3 Page 4 of4 E050210 Interim Report #18 Nodtern Alabama Potable Water Systems, December 2005 Page 121 of 122 05-0210 Interim Re ori #18 NotthemAlabama Potable WaterSystems, Decemger 2005 3M Confidential RECORDOF NONCONFORMAN/CDEEVIATION Study I Project No. E050210 Interim Report #18 Deviation type 0 SOP {Check one) El Protocol Protocol Requirements: 1. Identification Date@)of Occurrence: Document Number: 12/9/2005-January26,2006 Exygen Protol #PO001131 Amendment #3 (3M) 0 Equipment Procedure 0 Method 0 GPO 0 Other: 11. Description (3) ETS 8-154. I makes no mention of sample surrogate spikes; however, all samples will be spiked with a radiolabeled surrogate, PFOA [1,2 The concentrationof the surrogate spike may vary depending on the collection event, but typical samples are spiked with a nominal concentration of 0.05 ng/mL. Surrogate spike recoveries will be documented and discussed in the final report. Actual procedurelprocess: PFOA[1,2 13C]was not included as a surrogate spike in any of the samplelsampleduplicates, or field matrix spikes collected for this project. Surrogate recoveries from the other water projects and previous interim reports for E050210 indicate that there are solubilitylstabilityissueswith this surrogate in water. The 3M EnvironmentalLab has started an official investigation(which is not part of E05-0210) to determinethe root cause for the low recoveries. Untilthis situation is fully understoodand a method modification has been incorporatedto eliminate or circumventthe issue, PFOA[1,2 13C]will not be added as surrogate. Ill. Actions Taken (such as amendment issued, SOP revision, etc.) CorrectiveAction (0 Yes 13lNO) Reference: Acceptability of the nonconforming work: Surrogateswere originally used as an additionalquality control element in conjunctionwith lab control spikes and field matrix spikes. The surrogate recoverywas never intended to be the sole confirmation of that the analyticalmethodwas appropriatefor the given matrix. Non-inclusionof the surrogate spike has no impact on the quality and integrity of the data presented here. Actions: 0 Halting of Work 0 Client Notification 0 Work Recall 0 Withholding of Report El Other: Non-inclusionof the surrogatewill be addressed in the final report. Recorded by: Date: Authorization: Date: Sponsor Approval (GLP Protocols): Date: Technical Manager Approval (n halting of work w w ~ Ik-WMemindicated): Date: IV. Authorization to Resume Work Wherehaning of work occurred, resumption of work must first be approved by TechnicalManagement Technical Manager Approval: Date: Deviation No. (assigned by StudyDirsctoror Pmject Lead at the end of studyorpmject) Protocol Deviation Page 1 of 1 054210 Interim Report 118 NorthernAlabama Potable WaterSystems, December 2005 Page 122 of 122