Document RabeMamRmK1eKz1b1MNDOodY8

Jean B. Sweeney Staff Vice President - 3M-JEowronmental, Health and Safety Operations I 900 Bush Avenue, g ild in g 4?-2^-26 PO Box 33331 St. Paul, MN 55133-3331 651 778 5488 CERTIFIED MAIL April 9, 2009 NO CBI Document Processing Center EPA East - Room 6428 Attn: Section 8(e) Office of Pollution Prevention and Toxics, U.S. EPA 1200 Pennsylvania Avenue NW Washington, DC 20460-0001 Re: TSCA 8(e) Substantial Risk Notice: Supplemental to Docket No. 8EHQ-0598-373; Sulfonate-based and Carboxylate-based Fluorochemicals To whom it may concern: 3M is submitting this notice to supplement its previous submissions on sulfonate and carboxylate-based fluorochemicals. 3M recently received data from a fluorochemical (FC) analytical method validation study in which fish tissues, purchased from Osage Catfisheries, Inc. (Osage Beach, MO), were used as control samples. As detailed in the following table, endogenous levels of various FCs were detected in these tissues. All levels were in the ng/g range and are provided in the enclosed report. w hole-body largem outh bass (M icropterus salm oides) perfluorooctane sulfonate (PFOS) perfluorobutanoic acid (PFBA) perfluorodecanoic acid (PFDA ) perfluoroundecanoic acid (PFU nA ) perfluorododecanoic acid (PFDoA) w hole-body channel catfish (Ictalurus punctatus) PFOS PFBA perfluoropentanoic acid (PFPeA) perfluorononanoic acid (PFNA) PFDA PFUnA PFDoA w hole-body bluegill sunfish (Lepom is m acrochirus) and fillets o f rainbow trout (O ncorhynchus m ykiss) PFOS PFBA PFUnA r- 1 While 3M does not believe that any of these data taken alone or cumulatively meet the "substantial risk" reporting threshold, we nevertheless recognize the ongoing work by U.S. EPA to assess fluorochemical exposure pathways. Therefore, we are placing these results in the 8(e) docket as a supplement to previous submissions. If you have any questions or would like any additional information, please contact Deanna Luebker at (651) 737-1374 or diluebker@mmm.com. Sincerely, Jean B. Sweeney Staff Vice President, 3M Environmental, Health and Safety Operations Enclosure CONTAINS NOCBI P-2 3M ENVIRONMENTAL LABORA TORY REPORT NO. E08-0261 Final Analytical Report Method Validation of E TS -8-45 "Determination of Fluorochemicals via Protein Precipitation of Fish Tissues (Fillet or W hole Body) and Analysis by High Performance Liquid Chromatography with Tandem Mass Spectrometry" Laboratory Request Number: E08-0261 Testing Laboratory 3M EHS Operations 3M Environmental Laboratory 3M Center Building 260-5N-17 Maplewood, MN 55144 Requester William Reagen 3M EHS Operations 3M Environmental Laboratory 3M Center Building 260-5N-17 Maplewood, MN 55144 3M ENVIRONMENTAL LABORATORY PAGE 1 OF 25 P-3 3M ENVIRONMENTAL LABORATORY REPORT NO. E08-0261 3M Environmental Laboratory 3M Environmental Laboratory Manager: W illiam K. Reagen, Ph D. 3M Project Coordinator. Cliffton B. Jacoby, Ph.D. 3M Principal Analytical Investigator and Report Author: Michelle D. Malinsky, Ph D. Analytical Report E08-0261 Method Validation of ETS 8-45 "Determination o f Fluorochemicals via Protein Precipitation of Fish Tissues (Fillet or W hole Body) and Analysis by High Performance Liquid Chromatography with Tandem Mass Spectrometry* 1' ri&Qdtoh Report Date: March 12,2009 . ?s ---------- 'V -- This report summarizes the method validation of 3M Environmental Laboratory analytical method ETS 8-45 "Determination of Fluorochemicals via Protein Precipitation of Fish Tissues (Fillet or Whole Body) and Analysis by High Performance Liquid Chromatography with Tandem Mass Spectrometry1. The method validation protocol was detailed in the general project outline (Attachment 1) and was developed using the 2001 FDA Bioanalytical Method Validation Guidance for Industry as an analytical reference which requires the use of matrix-matched calibration^ 1) The extraction procedure employs acetonitrile protein precipitation and incorporates a cryogenic incubation step. Extracts were analyzed using liquid chromatography tandem mass spectrometry (LC/MS/MS) for perfiuoroalkane carboxylic acids (PFCAs) ranging from C4 to C12, perfiuoroalkane sulfonates (C4, C6, and C8), and perfluorooctanesulfonamide. A comprehensive validation is presented for whole-body homogenates of largemouth bass (Micropterus salmoides) and method cross-validation results are shown for wholebody channel catfish {Ictaiuruspunctatus), whole-body bluegill sun-fish {Lepomis macrochirus), and rainbow trout fillets (Oncorhynchus mykiss). All data presented here were generated using fish from a supplier for scientific studies. No environmental samples were used. For the full method validation, linearity, precision, and accuracy were determined in three separate extraction batches prepared over the course of two separate days which provided inter-day and intra day statistics. Each preparatory batch consisted of the following samples: thirteen point matrixmatched calibration curve ranging from 0.025 ng/g to 25 ng/g spiked tissue concentrations, four matrix blanks (two with internal standards (iSs) and surrogates, two without ISs and surrogates), four method (aqueous) blanks (two with ISs and surrogates, two w ithout ISs and surrogates), two acetonitrile solvent blanks with ISs and surrogates, triplicate lab control matrix spikes at three levels, and triplicate lab control matrix spikes o f perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) from 3M electrochemical fluorination (ECF) production lots. On the first extraction day, additional PFOS "dilution" QC samples were also prepared where the PFOS concentration (ppm range) was two to three orders of magnitude higher than the rest of the target analytes (ppb range) Additionally, an alternate approach to PFOS quantitation was explored using a species specific matrix-matched calibration curve prepared using [1,2,3,4- 13C4]PFOS. The cross validation procedures for the three other fish species represented an abbreviated approach to the full validation performed for the wholebody largemouth bass. Finally, this study explored the use of a solvent (unextracted) calibration curve for quantitation of the fish extracts. 3M ENVIRONMENTAL LABORATORY PAGE 2 OF 25 Tt p.4 3M ENVIRONMENTAL LABORATORY REPORTNO EOB-0261 T K3SA. : :> .k s. s * f * :; j a * (i "' ' i'.iP T ; il 2.1 Target Analytes. Table 1 below provides pertinent information regarding the target analytes, internal standards, and surrogate compounds investigated in this method validation. Table 2 lists the 3M Environmental Laboratory identification numbers fo r the control tissues used. Table 1. Target Analyte Summary. Compound Name Perfiuoiobutanoic acid Perfluoropentanoic add Perfluorohexanoic add Perfluorcheptanotc add Synonym o r Acronym Analytical Purpose PFBA (C4 Acid) PFPeA (C5 Add) PFHxA (C6 Add) PFHpA (C7 Add) Target Target Target Target Perfluonooctanoic acid PFOA (CSAcid) Target Perfluoranonanoic add Perfluorodecanoic add Perfluoroundecanoic add PerDuorcdodecandc add Perfluorobutane sulfonate Perfluorohexane sulfonate PFNA (CSAdd) PFDA(CIOAcid) PFUnA(C11 Acid) PFDcA(C12 Acid) PFBS (C4 Sulfonate) PFHS (C6 Sulfonate) Target Target Target Target Target Target PerfluoTOOctane sulfonate PFOS (C8 Sulfonate) Target Perfluorooctanesulfonamide FOSA (C8 Sulfonamide) Target Formula C jF tC O O H c <f 3c o o h CjFnCOOH CsFoCOOH CTF 1sC O O H ICtF isC O O IN H I C ,FitCOOH CFisCOOH CioFuCOOH CuFaCOOH [ C f & X ] [K*] IC e F ijS O jIN a *) (CoFnSOjJK*) (CrfoSOsUlC) C jF n S O jN H j Reference Standard Source Aldrich AliaAesar Oakwood Products Aldrich L-PFOA (Inear) WeBngton Labs EOF PFOA (branched) 3M Production Lot 332 Oakwood Products Oakwood Products Oakwood Products Oakwood Products 3M Production Lot 2 L-PFHS (linear)Wellington Labs L-PFOS (linear)Wellington Labs EOF PFOS (branched) 3M Production Lot 217 3M (L-157D9) 3M ENVIRONMENTAL LABORATORY PAGE 3 OF 25 P-5 3M ENVIRONMENTAL LABORATORY REPORT NO. E08-026 Table 1 Continued. Compound Name Synonym or Acronym Analytical Purpose " G,-Perfluorobutandc add [1,2,3,4-13C]PFBA ^CL-Perflgorooctenoic add [1,2,3,4-13C4]PFQA ' 'CrPerfuorodecanoic add "'OzYVnmonium Perfluorobutane sulfonate "OrAmmonium Perfluorqhexane sulfonate "OrAmmonium Perfluorooctane sulfonate l3C4-Sodium Perfluorooctane sulfonate [1 ,2 -I3C J P F D A ["OJPFBS fC yPFHS [" OJPFOS f1,2,3,4-l3C4]PFOS Surrogate Internal Standard Surrogate Surrogate Surrogate . Internal Standard Calibration Formula "CF^CF^CO O H C F 3fC F 2)3(13CF2) j ' 3C O O H CF3<CFjM '3CF2),3COOH [C4FsS 'aO?0' ][NH /l [C,F,3S ,e0 2O ][N R f) (C8F ,,S 1,0 201 [N H /1 [CFjCCFjM ^ C F ^ C F ^ O jI [N a l Reference Standard Source Wellington t.abs Wellington Lab3 Wellington Labs RTI international Wellington Labs RTI International Wellington Labs Table 2. Control Tissue Summary Spedes Whole-body largemouth bass Whole-body catfish Whale-body bluegill Rainbow trout filet 3M Environmental Lab ID Number TN08-0194-1/1 TN06-0199-1/1 TNOS-0203-1/1 TN08-0196-1/1 3.1 Tissue Preparation W hole-body largemouth bass {Micmpterus salmoidas), whole-body channel catfish (ictalurus punctatus}, whole-body bluegill sunfish (Lepomis macrochirus), and rainbow trout fillets (Oncorhynchus mykiss) were purchased from Osage Catfisheries, Inc. (Osage Beach, MO). W hole-body and fillet tissues were homogenized frozen in a two-step process. The initial homogenization step occurred at MPI Research (State College, PA). Frozen fish tissue was homogenized with dry ice until a coursemeat consistency was reached. After the initial homogenization, the ground fish was transferred to a polyethylene bag which was placed in the freezer unsealed overnight to allow the residual carbon dioxide to subtime. After sublimation, the bag was sealed and homogenates were kept frozen and shipped to the 3M Environmental Laboratory for sample preparation and analysis. Approaching the tim e of extracten, the frozen course-ground fish tissue was added to a pre-chilled stainless steel bowl o f a Robot Coupe RSI 2Y-1 vertical batch processor (Joliet, IL) and re-homogenized with dry ice until a powder-like consistency was reached. Again, the tissue homogenates were transferred to a pre-chilled polyethylene bag and the residual dry ice was allowed to sublime overnight. 3.2 Sam ple Extraction Polyethylene centrifuge tubes (50 mL) were chilled in a cooler with dry ice for approximately thirty minutes prior to weighing homogenate aliquots to prevent the tissue powder from thawing and congealing during the weighing process. A 0.5 g sub-sample of the fish tissue homogenate was accurately weighed in a pre-chilled 50 mL polyethylene centrifuge tube. Tissue aliquots were then spiked with internal standard (IS) to produce an approximate concentration of 1 ng/g. Samples designated as matrix-matched calibration standards were additionally spiked with the target and surrogate analytes at appropriate levels to produce the desired tissue concentrations. Samples 3M ENVIRONMENTAL LABORATORY PAGE 4 OF 25 I P-6 3M ENVIRONMENTAL LABORATORY REPORT NO. E0B-0261 intended for laboratory QC {m atrix blanks and laboratory control matrix spikes) were spiked with surrogates at 1 ng/g and the other target analytes at appropriate levels. A fter spiking, the tissue homogenates w ere allowed to sit for 30 minutes before 5 mL of acetonitrile was added. The samples w ere then thoroughly homogenized with the acetonitrile using an Omni Prep multi-place homogenizer with disposable plastic probes (15,000 rpm for 2 minutes). The acetonitrile/tissue extracts were then placed in a freezer at -20C for at least one hour. Upon rem oval from the freezer, sample extracts were centrifuged at -5C fo r 20 m inutes at 3000 rpm. If any delays occurred, samples were returned to the 20C freezer and then re-centrifuged prior to resumption o f preparation and analysis. After centrifugation, 1 mL o f clarified supernatant was transferred to a 2 mL autovial spiked with 10 pL of 10% form e acid. Autovials were then capped and vortex mixed. 3.3 Analysis Analysis fo r the suite o f PFCs listed above was performed using high performance Squid chromatography-tandem mass spectrometry (HPLC/MS/MS). For this investigation, samples were analyzed using an Agilent 1100 series (Palo Alto, CA) HPLC system interfaced to either a PE SCIEX API 4000 triple quadrupole o r SCIEX API 4000 Q-Trap mass spectrometer (Foster City, CA). Both instruments were equipped with a SCIEX Turbo V ion-spray interface operating in the negative ion MS/MS mode using m ultiple reaction monitoring (MRM). AnalystTM 1.4.2 software was used for all data collection and reduction. Table 3 lists the MRM transitions monitored fo r each analyte. A Thermo Scientific PRISM RP guard column (2.1mm * 50 mm; 5p particle size) was placed in-line after the purge valve and before the sample injection port to trap any PFC contaminants coming from the HPLC instrum ent and/or the m obile phases. This sufficiently separated the elution of the 'system ' PFC peaks from those present in the sample extracts. Table 3. MRM Transition Summary. Compound PFBA (C4 Acid} PFPeA (C5 Add) PFHxA (C6 Acid) PFHpA (C7 Acid) PFOA <C8 Acid) PFNA {C9 Add) PFDA (C10 Acid) PFUnA (C11 Acid) PFDaA (C12 Acid) PFBS (C4 Sultanate) Analyte Description Target Target Target Target T arget Tanjet Target Target Target Target mMRM TransWon(s) 213>169 263>219 313>269 3 1 3 > 1 19 363>319 363M69 413>369 413>219 413>169 463>419 463>219 4 6 3 > 169 513>469 513>269 513>219 563>519 563>269 563>219 613>569 613>319 613>169 299>80 299>99 Dwelt Time (ms) 100 100 100 100 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 3M ENVIRONMENTAL LABORATORY PAGE 5 OF 25 p.7 3M ENVIRONMENTAL LABORATORY REPORT NO. E08-0261 Table 3 Continued. Compound Analyte Description mMRM Transitionis) Dwelt Time PFHS (C6 Sulfonate) Target 399>80 399>99 50 50 499>a0 50 PFOS (C8 Sulfonate) Target 499>99 499>13Q 50 50 FOSA (C8 Sulfonamide) Target 498>78 50 (1,2.3.4 -'3C<]PFBA Surrogate (Smalt PFCAs; C4-C6) 217>172 100 [1,2- " CJPFDA Surrogate (Large PFCAs: C7-C12) 515>470 50 f'O jlP F B S Surrogate (Sulfonates, FOSA) 303>84 50 [ 1 ,2 ,3 , 4 - ,5C*]PFOA Internal Standard (A# PFCAs) 417>372 50 fcyP FO S Internal Standard (Sulfonates, FOSA) 5Q3>84 50 (1) Individual transitions were summed to produce a "total ion chromatogram"(TIC). The TICs were used for quantitation. Analysts o f the small acids (C4 through C6) was performed using a Thermo Scientific PRISM RP column (2.1mm x 50 mm; 5 p particle size) held at 30aC with the mobile phase system consisting of 5 mM ammonium acetate with 0.01% acetic acid irt water (A) and methanol (B). The gradient used to elute the analytes of interest is presented in Table 4, The f ow rate was held at 300 pL min"1throughout the run and a 20 pL injection volume was used. The outlet o f the analytical column was directed to a column switching valve where the first three minutes of the run were diverted to waste. After three minutes, the valve was switched to direct the effluent to the mass spectrometer for analysis. The large acids {C7 through C12), the sulfonates, and FOSA Wei's analyzed using a Thermo Scientific BetasilTM Cis analytical column (2.1mm x 100 mm; 5p particle size) held at 30C with the mobile phase system consisting of 2 mM ammonium acetate in water (A) and acetonitrile (B). The gradient used is also provided In Table 4. The flow rate was held at 400 pL m in'1throughout the run and a 25 pL injection volume was used. Samples were injected onto a W aters (Milford, MA) Oasis HLB on-line extraction column (20 mm x 3.0 mm, 25 p particle size) with the outlet directed to a column switching valve where the first five minutes were diverted to waste. After five minutes, the valve was switched and the effluent was directed to the analytical column for separation of the target analytes and analysis by the mass spectrometer. Table 4. LC Gradient Parameters. Small A dd s: C4-C6 Large A d d s ' C7-C12- Sulfonates, FOSA Step 0 1 2 3 4 5 6 Total Time (min) 0.0 3.0 3.5 9.0 15 0 15 1 190 %A 90 90 30 5.0 5.0 90 90 Total Time %B Step (min) %A 10 0 0.0 10 1 3.0 70 2 3.5 95 3 15.0 95 4 17 0 10 5 17.1 10 6 20.0 97 97 75 10 10 97 97 A 5 mM ammonium acetate with 0.01% acetic acid (aq) A 2 mM ammonium acetate (aq) B Methanol B Acetonitrile %B 3.0 3.0 25 90 90 30 3.0 3M ENVIRONMENTAL LABORATORY PAGE 6 OF 25 !t p.8 3MENVIRONMENTAL LABORATORY REPORT NO. E08-026I To manage the large num ber of transitions required for the large acids, sulfonates, and FOSA, two separate injections/anaiyses were acquired. Inclusion of all transitions in one chromatographic run made it challenging to get a sufficient number o f scans across the chromatographic peak while m aintaining a long enough dwell tim e to achieve the sensitivity needed for accurate and reproducible quantitation. The first injection acquired the MRM transitions for the C8-C12 acids (17 transitions) and the second injection acquired the MRM transitions needed for the anlaysis of C7 acid, the sulfonates, and FOSA (13 transitions). Chromatographic conditions were identical for both injections. For analytes where m ore than one transition w as acquired, a total ion chromatogram (TIC) which summed the respective transitions for that analyte was used analyze the data. A table summarizing the extraction and analysis dates is provided in the supplemental information. jD m ta 1 1 2 1 1 * is * *. 2 -f * -Ere^SSS: &f,-S g i5 *i. *if f* * b*.*v ** W-fcj 4.1 Calibration Species-specific matrix-matched calibration standards were prepared by spiking known amounts of target analytes, surrogates, and internal standards into individual 0.5 g aliquots o f fish tissue homogenate. Each spiked fish aliquot was then extracted using the procedures outlined in Section 3.2, A total of thirteen spiked standards ranging from 0.025 ng/g to 25 ng/g (nominal) were prepared. A quadratic, 1lx weighted, calibration curve was used to fit the data for each analyte. Internal standard quantitation was used. The data yyas not forced through zero during the fitting process. The accuracy o f each curve point was verified by back-calculating the concentration using the area count ratio o f the target analyte tc the internal standard. Each extracted calibration standard used to generate the final calibration curve m et the method calibration accuracy requirement o f 10025%, except the LOQ standard (100+30%). The coefficients of determination ( r ) were greater than 0.990 fo ra l analytes. As required by ETS 8-45, a minimum of six calibration points were used to generate the final calibration curve. 4.2 Method of Standard Addition Some target analytes, m ost notably PFBA and PFOS, had significant levels present in the control matrix. This resulted in the exclusion of several low-level points from the final calibration curve. When this occurred, the method o f standard addition was used to determine the endogenous amount in the matrix, which was then used to correct the calibration and QC spiked concentrations. The two matrix blanks that were spiked w ith IS were included In the final calibration curve with concentration values assigned as the determined endogenous level. The calibration curve, with the adjusted concentrations, was then used to quantitate the QC samples. 4.3 Limit of Quantitation (LOQ) The LOQ as defined in ETS 8-45 is the lowest non-zero calibration standard in the curve in which the area counts o f the target analyte are a t least twice those of the matrix blank(s). The limit of quantitation for each analyte varied from extraction date and instrument batch. If the percent relative standard deviation (RSD) of the m atrix blanks area counts was less than 30%, then the average area counts was used to determine the LOQ, If the RSD was greater than 30%, then the m atrix blank with the largest area counts was used for the LOQ determination. The resulting LOQs are provided in the Data Summary and Discussion section. 4.4 System Suitability Five replicate injections of the solvent calibration standard were analyzed at the beginning of the analytical sequence to demonstrate overall system suitability. Method criteria states that system suitability injections shall produce a RSD of less than 7% for the ratio of target analyte area counts to Internal standard area counts and an RSD of less than 2% for the retention time. In general, method 3M ENVIRONMENTAL LABORATORY PAGE 7 OF 25 P-9 3M ENVIRONMENTAL LABORATORY REPORT NO E08-0261 acceptance criteria were m et for both area counts and retention times. Method deviations have been issued for the instanoes of non-compliant system suitabilities and are documented in the Supplemental Information. 4.5 Continuing Calibration During the course o f the analytical sequence, several continuing calibration verification samples (CCVs) were analyzed to confirm that the instrument response from initial calibration curve was still in control. In general, CCV injections produced recoveries within 100%25%, which m et method criteria. Non-compliant CCV recoveries were documented in method deviations in the raw data package and are provided in the Supplemental Information. 4.6 Blanks Five types o f blanks were prepared and analyzed with the samples: matrix blanks {two with IS and surrogate, two w ithout IS and surrogate), aqueous method blanks (two with IS and surrogate, two without IS and surrogate), acidified acetonitrile solvent blanks, acetonitrile blanks with internal standards and surrogates, and straight acetonitrile blanks (no acid, IS, or surrogate). Each blank result was reviewed and used to evaluate method performance to determine the LOQ for each analyte. Surrogate recoveries o f spiked blanks are provided In the Supplemental Information. 4.7 Lab Control Spikes (LCSs) Triplicate lab control spikes at three different levels were prepared each extraction day. For PFHS, PFOS, and PFOA, the standard reference material used for the LCS spikes was the linear isomer. Separate ECF spikes of branched PFOS and PFOA were prepared to evaluate any potential bias from quantitation against a linear standard. For Day 1 only, triplicate PFOS dilution QC samples were prepared where PFOS was spiked at ppm levels whereas the rest of the analytes were at low ppb levels. Table 5 provides the approximate spike levels for the prepared LCS samples. The lab control spikes were prepared to evaluate method accuracy and precision. LCS recoveries wiii be presented and discussed in the following section. Table 5. Validation QC Spike Levels. QC Sample Description (1)PFBA A pproxim ate Spike Concentration (ng/g) All Other Target (2)p f o s PFOA Analytes Surrogates LCS Low 4 4 0.3 0.3 1 LCS Mid 12 12 1.5 1.5 1 LCS High ECF LCS 20 20 NA c 88 K NA 1 1i wPFOS Dilution QC 12 4000 1,5 1.5 1 (1) Initial screening of the largemouth bass control matrix indicated that PFBA and PFOS were at levels higherthan the rest of the target analytes. Spike levels were adjusted accordingly based on the endogenous level so that the low level spike was approximately twice that of the endogenous leve!. The spike concentration listed reflects the amount of analyte spited into the tissue and does not aocount for the endogenous level. (2) PFOS dilution QC samples were only prepared on Day 1 3M ENVIRONMENTAL LABORATORY PAGE 8 OF 25 3M ENVIRONMENTAL LABORATORY REPORT NO. E08-0261 D m tw m n rg-* * * * * * * * rT ?-3 y ja B B B Z .j' 5.1 Whole body Largemouth Bass Validation Results 5.1.1 Accuracy and Precision Table 6 below displays the accuracy (average percent recovery) and precision (%RSD) results for the three separate levels o f LCSs (N=3) prepared in the whole-body largemouth bass control matrix for each preparation batch o f the method validation. Internal standard, species-specific matrix-matched calibration was used for quantitation. Table 7 provides the average batch accuracy and precision when all three sp ire levels are considered collectively (N=9) as well as the LOQ, inter-day, and intra-day statistics. Additionally, the validation results are summarized graphicaly in Figure 1. For simplicity, the results of the ECF QC spikes are provided separately (N=3) for a given batch. In general, alm ost all o f the analytes demonstrated excellent accuracy and precision with average percent recoveries within arbitrary method acceptance criteria o f 100*30% and %RSDs less than 20% when individual batch, inter-day, intra-day, and ali data collectively were considered. W ith the exception of the sm all PFCAs(PFBA, PFPeA, and PFHxA), all analytes demonstrated average accuracies within 100*15% with %RSDs less than 10%. Endogenous levels were detected m the control matrix fo r the following analytes: PFBA, PFHpA (Day 2 Analyst B only), PFDA, PFUnA, PFDoA, and PFOS. For these analytes, the method of standard addition was performed to determine the endogenous concentration. The resulting concentration was then used to correct the spiked am ount for the calibration standards and QC samples 3M ENVIRONMENTAL LABORATORY PAGE 9 OF 25 3M ENVIRONMENTAL LABORATORY REPORT NO. E03-0261 Table 6. Whole-body Largemouth Bass Accuracy and Precision Results Summary By Spike Level. Analyte Accuracy (Average % Recovery) Precision (%RSD) Low Day 1 Analyst A M id High Low Day 2 Analyst A Mid High Low Day 2 Analyst B M id High roPFBA PFPeA PFHxA PFHpA PFOA PFNA n PFDA `''PFUnA ;,'PFCoA PFBS PFHS (1)PFOS FOSA 124 4 .9 151 5 .4 115 31 126 6 .4 86.3 13 111 1 ,7 111 5 .8 100 8.1 100 10 113 0 0 99.6 3.8 9 5 5 *3 6 101 4 .6 125 4 .8 137 3 .6 118 5 .5 107 2 .2 95.5 3 .6 114 2 .8 100 5 4 102 2.1 1105.1 106 7 .8 104 3 .3 102 6 .8 109 4 .5 121 0.83 128 3.7 121 3.3 103 4 .4 101 7 .7 113 10 94.8 6.3 1101,0 1 M it 10 102 11 100 9.6 104 5.6 109 11 110 3 .3 134 6 .8 129 2 .7 90.8 1 .7 82.1 8 .2 114 4.6 104 8.0 100 9 .4 105 4 .4 122 3 2 103 5.6 98 2 2 .9 120 4 .7 117 2 .7 124 3.7 128 2 .8 104 0 .44 95.6 2.4 111 3 .4 94.5 2.5 103 5 .9 105 0 .5 5 110 3 .0 104 1.5 93.7 2,4 r8 0 .4 3 1161 3 120 2 .2 119 3 .5 1 0 9 1.1 92.6 1 .6 10712.0 91.7 1.9 97.9 1.9 100 12 98 5 2 1 95.9 2.5 96.7 5 5 1123.9 103 5.1 85.0 9 .6 11418 112 5.1 b <0.920 112 3.7 102 2.0 106 4 .8 105 1 .7 106 11 104 1.G 95.9 1.3 98.6 4.4 103 3.9 103 5.3 106 8 .2 10614.4 102 2 .2 111 2 .0 96.7 0.45 110 + 1.6 108 2 .4 97 5 *1 .1 100 2 .6 96.0 3.2 1 0 3 + 4 .0 107 0 93 102 2.2 96 8 4 4 104 2 .9 89.4 1.5 108 5 .2 93 9 1 6 1033.5 101 2 .2 92.4 1.6 94.5 0.92 964 4 1 97.4 2 6 [1,2,3,4',3C,]-PFBA 114 2 .5 113.0.54 108 1.1 106 4 .7 110 1.4 108 0 .93 95.3 4 .2 94.2 3 .3 91 3 1 7 [1.-^Cy-PFDA 106 6 .8 110 3 .2 109 2 5 105 7.5 97.6 1.9 102 3 .5 101 2 .3 10011.8 104 2.7 ("OjJ-PFBS 96 2 6.1 99.8 3.7 102 4 .2 1 0 0 1.3 102 2.1 98.3 2.1 101 5 .2 96.1 6 .6 10 8.0 (1 ) tndogenous levels detected in the control matrix. endogenous values. Method of standard addition was used to determine endogenous concentration. Ali QC spike concentrations were c (2) Matrix blanks exhibited contamination and several low-level calibration standards were deactivated. Consequently, the low-level LCSs were below the resultant LOQ. p. 11 3M ENVIRONMENTAL LABORATORY PAGE 10 OF 25 (L) If the notice includes a health and safety study concerning the new chemical substance, the submitter must also answer the question; in Sec. 720.90(b)(2). See below. 720.90 (b) (2) (i) Would disclosure of the chemical identity disclose processes used in the manufacture or processing of a chemical substance or mixture? Describe how this would occur. Yes. Disclosure of the chemical identity would enable a chemist to identify feedstock chemicals and the manufacturing process. Because of the type of chemical, its identity would also enable a chem ist to identify processing and use information. (ii) Would disclosure of the chemical identity disclose the portion of a mixture comprised by any of the substances in the mixture? Describe how this would occur. The notified substance is not a mixture. (iii) Do you assert that disclosure of the chemical identity is not necessary to interpret any of the health and safety studies you have submitted? If so, explain how a less specific identity would be sufficient to interpret the studies. The health and safety studies stand by themselves. The generic name and the toxicology studies should enable any toxicologist to comment on the safety of the chemical. CO | Q. 3M ENVIRONMENTAL LABORA TORT REPORT NO E08-026I A ccu ra cy (% R eco very) P re cisio n (%RSD) 160 140 120 100 80 60 40 20 0 i '* CM cO CM F igure 1. M ethod ETS 8-45.0 W hole-body la rg e m o u th B ass V a lid a tio n R e su lts S um m ary. Average percent recovery and %RSD reflect all LCS spike levels collectively. Day 1 Analyst A Day 2 Analyst A S Day 2 Analyst B Inter-Day Intra-Day ESAll Batches 3M ENVIRONMENTAL LABORATORY PAGE 12 OF 25 p. 14 3M ENVIRONMENTAL tABORATORV REPORT NO. E08-0261 Table 7. Whole-body Largemouth Bass Results S um m ary: LOQ, Batch, Inter-Day, and Intra-Day Statistics. Analyte Day 1 Analyst A 01A c c u ra c y Predsion (N=S) L O Q (n c /g ) D ay 2 Analyst A wA c c u ra c y iPrec/sron (N=9> LOQ (ng/0 Day 2 Analysts mA c c u ra c y tP re d s io n LOQfngfi.at (,)Accuracy Predsion Inter-Day (N=18) mA c c u ra c y 1Precision Intra-Day (N=18) PFBA PFPeA PFHxA PFHpA PFOA PFNA PFDA PFUnA PFDoA PFBS PFHS PFOS FOSA [1,2,3,4'1}C]-PFBA 12313.8 13918.4 118115 112110 9 4 .4 1 1 0 11315.4 10218.5 10416.1 10518.5 10717.8 10115.2 10C 6 . 10617.4 11213.0 E=2.01 0.248 0.248 0.096.9 0.0504 : 0.0512 " E=0184 : **=0.285 E=00747 0.0994 0.0509 E=189 0.0511 0.0245 11413.7 12616.4 12614.3 101 8 .2 901 7 9 110142 96.7 7 3 10 0 16 .0 10313.8 11019.4 101 4 .9 9 7 .9 1 3 .4 11714.4 10812.9 *E=3.43 0.259 0.259 0.102 00493 0.0501 E=0.224 *E=0.392 *E=0.0693 0.105 0.0468 E=2.77 0.105 0.0257 10413.7 96.6110 106113 10714.9 9 5 .5 1 7 .2 11013.7 97.413.7 10614.2 10513.6 9 8 .7 1 8 .6 10014.5 96.1 2 .7 10014.0 93.613.4 *E2.37 0.246 0.347 *=0.0332 O:0.920 0.0490 *E=0.155 *E=0.393 *=00537 0.246 0.0457 E=1.96 0.049 0.0221 11915.4 13218.9 121111 107110 92.319.2 11214.8 9 9 .3 1 8 .2 10 216 .2 10416.5 10918.5 101152 9 9 .0 1 4 .9 11117.6 11013.4 10915.8 111 1 6 115112 10417.1 92.217.9 . 110*3.9 97.015.6 10315.8 10413.7 104110 10014.6 97 0 1 3.1 10819.1 101 7.9 [l^ .-'^ P F D A 10814.1 0.0504 101 5 .3 0.0257 10212.5 0.0221 10515.7 101 4 .0 T O jI-PFBS 9 9 .9 1 4 .9 0.0471 10012.2 0.0241 9 9 .7 1 5 .6 0.0207 99.613.6 9 9 .5 1 4 .5 (1) Accuracy = average percent recovery all three spike levels combined. Precision = percent relative standard deviation all three levels combined. (2) E = endogenous. Analyte present in the control matrix. Endogenous concentration determined by method of standard addition. Endogenous value = LOQ. (3) Matrix blanks exhibited contamination and several lew level calibration standards were deactivated resulting in an elevated LOQ tor this analysis. ^Accuracy Precision All B atches (N=27) 11417.8 120117 1161 13 10718,9 93 1 8 7 111 4 ,5 9 8 .7 1 7.0 10415 8 10415 6 10519.5 101 5 .0 98.1 1 4.5 10718.5 10418.1 104151 99.414.5 3M ENVIRONMENTAL LABORATORY PAGE 11 OF 26 3M ENVIRONMENTAL LABORATORY REPORT NO EQ8-0261 5.1.2 ECF PFOA and PFOS QC Spikes Table 8 provides the accuracy (percent recovery) and precision (%RSD) results of the 3M ECF QC spikes of PFOS and PFOA quantitated against the linear reference standard. For PFOS, the mean recovery of all three batches collectively was 95.9% with a RSD of 3.4% demonstrating that no measurable bias could be attrfouted to the use o f the linear reference material. Alternatively, the average PFOA recovery o f the ECF spike was consistently approximately 87% for a ll three batches indicating that a potential bias may exist when branched PFOA isomers are quantitated using a linear reference material. Potential explanations of the observed decrease in recovery may be differences in the response factor from the linear to the branched isomers or co-extracted matrix components may suppress the signal of the branched isomers relative to the linear Even though the PFOA demonstrated some decrease in recovery, the results were s tii within 10015%. Table 8. Results Summary: ECF (branched) PFOA and PFOS QC Spikes Quantitated Against a Linear Reference Standard. Accuracy (Average Percent Recovery) t Precision (%RSO) Analyte PFOS PFOA Day 1 Analyst A 97.6 2.0 87.4 4.0 Day 2 Analyst A 95.7 2 6 8 7 .0 * 1.1 Day 2 Analyst B 94 5 * 53 8 6 .6 *4 .1 tnter-Day 9 6 .6 *2 .3 8 7 .2 *2 .7 Intra-Day 95.1 3 .8 8 6 .8 *2 .7 A ll Batches 95.9 3.4 8 7 .0 *3 .0 5.1.3 PFOS Dilution QC Spikes On Day 1, additional QC spikes identified as 'PFOS dilution QC" were also prepared. These QC samples were prepared with PFOS at approximate levels of 4000 ng/g with all other analytes at 1.5 ng/g, with the exception o f PFBA at 12 ng/g. The undiluted extract was analyzed for all analytes except PFOS. A 1:1000 acetonitrile dilution of the final extract was prepared for the PFOS analysis. The PFOS analysis o f the diluted extract was performed using external standard caSbration as the internal standard spiked into the tissue prior to extraction was diluted below the lim it o f detection. Spiking these QC samples w ith IS a t a level that could be diluted into a usable range was not feasible as the isotopically labeled standards come from the vendor already in solution at a concentration o f 50 jig/m L. Table 9 lists the results o f the PFOS dilution QC. With the exception o f PFBA and PFPeA, the average recoveries were w ithin 100+20% with RSDs less than 15%. The internal standard for PFBS, PFHS, FOSA and [180 2]PFBS surrogate was switched from [18Os]PFOS to (1,2,3,4-13C4)PFOA for these samples only. W hen [1*0 2]PFOS was used as the internal standard, recoveries ranged from approximately 65-75% because the internal standard area counts increased by about 32% in these samples. The IS signal increase was attributed to the co-elution o f the [^O JP FO S spiked at 1 ng/g with the unlabeled PFOS spiked at 4000 ng/g. The combination o f MS and a single 10 in the unlabeled PFOS would also produce the same 503 m/z parent ion as the [180 2]PFOS. Based on the natural abundance o f (4.4% ) and ,80 (0.2%), the probability o f both MS and a single 180 present in the unlabeled PFOS spike is 0.0088%.(2) W ith the PFOS spiked at 4000 ng/g, this contribution would be equivalent to approximately 0.35 ng/g or 35% o f the spiked IS and explains the observed increase in IS area counts, When the IS was switched to [1,2,3,4-13C4]PFOA, which does not co-elute with PFOS, the recoveries improved dram atically demonstrating that the extraction procedures were not responsible fo r the low recoveries when the [,8Q2]PFOS IS was used. The average recovery o f the post-extraction dilutions for PFOS was lower than the other analytes at 84.4%. Possible explanations include dilution o f m atrix components along with the PFOS that alter the signal enhancement against the matrix-matched curve or simple variability in dilution technique. 3M ENVIRONMENTAL LABORATORY PAGE 13 OF 25 3M ENVIRONMENTAL LABORATORY REPORT NO. EG8-026I Table 9. PFOS Dilution QC Results. Analyte PFBA PFPeA PFHxA PFHpA PFOA PFNA PFDA PFUnA PFDoA ^PFBS PFHS lJ>PFOS FOSA 13Cx-PFBA ' ' Ct-P FDA <!),` o 2-p f b s '^Accuracy Precision 129 7 .4 144 9 .6 1 1 5 12 93.8 S.6 93.2 7 .9 106 13 105 5 .3 96.4 6 .7 106 1 0 99.1 1 .9 91.7 7 .6 84.4 12 96.4 5.0 120 6.2 117 12 98.0 6.1 (1) Accuracy = average percent recovery. Precision = %RSD. (N=3) (2) [1,2,3.4-13CJPFOA was used as the internal standard. (3) PFOS concentration determined by external standard calibration of 1:100C dilution of the final extract 5.1.4 Alternate PFOS Quantitation In a separate preparation batch, a whole-body largernouth bass matrix-matched calibration curve was prepared using [1,2,3,4-,3C4]PFOS as the calibrant, [^O-JPFBS as the internal standard, and [10O2]PFHS as the surrogate. Thirteen calibration standards ranging from 0.025 ng/g to 25 ng/g spiked tissue concentrations were generated along with four matrix blanks (two with [S and surrogate and two w ithout IS and surrogate). Triplicate lab control matrix spikes were also prepared by spiking unlabeled linear PFOS at approximate levels of 4 ng/g (low), 12 ng/g (mid), and 20 ng/g (high) along with triplicate ECF PFOS spikes at 5 ng/g. Consistent with previous validation QC samples, the surrogate was spiked at an approximate level of 1 ng/g for all samples. The three 1,2,3,4- 'Q P F O S transitions (503>131, 503>99,5Q3>80) as well as the corresponding unlabeied PFOS transitions (499>130, 499>S9, and 4992-80) were monitored. The calibration curve was constructed using the total ion chromatogram (summation) o f the three [1,2,3,4-l3C4]PFOS transitions. The three unlabeled PFOS transitions were also summed and the total area counts were plugged into the resulting regression equation for [1,2,3,4-13C4lPFOS. The average endogenous PFOS concentration in the control matrix was calculated for the thirteen matrix-matched calibration standards and the two matrix blanks spiked with IS. The mean endogenous PFOS concentration in the whoie-body largernouth bass control matrix was 2.76 ng/g 10%(RSD) (N=15). This concentration was then used to correct the spiked values of the QC spikes. Table 10 provides the accuracy and precision data of the QC spikes. All QC results met method acceptance criteria for accuracy (100*30% ) and precision (RSD<20%). 3M ENVIRONMENTAL LABORATORY PAGE 14 OF 25 3M ENVIRONMENTAL LABORATORY REPORT NO. E08-0261 Table 10. QC Results for Alternate PFOS Quantitation using [1,2,3,4-13C,,]PFOS as a Calibrant. Low Accuracy (Average % Recovery) Precision (%RSD Mid High ECF Batch Average (N=3I m i -------- -------- m -------- ---------- a t m ---------- PFOS (Linear) 107 5.6 114 3.8 111 5.1 '^ O * PFHS surrogate 111 1.8 117 2 5 112 4.4 7T) '"O jP FH S surrogate was spiked at approximately 1 ng/g for a# QC levels. 105 2.4 112 4.8 109 5.0 113 3.8 5.2 Multi-Species Cross Validation The method presented here was subsequently cross-validated for three additional fish species: wholebody channel catfish (Ictalurus punctatus), whole-body bluegill sunfish (Lepomis macrochirus), and rainbow trout fillets (Oncorhynchus mykiss) using spedes-specific matrix-matched calibration curves. The cross-validation procedures represented an abbreviated approach to the full validation performed for the whole-body largemouth bass. The cross-validation for the three additional species included species-specific matrix-matched calibration with triplicate lab control m atrix spikes at three levels to assess accuracy and precision. Additional ECF spikes o f PFOS and PFOA were prepared and analyzed against predominantly linear reference materials. PFOS dilution QC and inter- and intra-day evaluations were not included. The results from the m ulti-species cross-validation are summarized below {whole-body catfish: Table 11, whole-body bluegill: Table 12. and rainbow trout fillet: Table 13). The average percent recovery and %RSD for all spike levels combined is presented graphically in Figure 2. For all three species, the average percent recovery (all levels combined) was within 100+30% with a %RSD less than 20% for all analytes, with the exception of PFBA for rainbow trout fille t Rainbow trout fillet PFBA results were not reported because several o f the calibration curve points did not meet method acceptance criteria once the concentrations were adjusted for endogenous levels. Rainbow trout cross validation samples were re-extracted and analyzed for the small PFCAs to see if a better calibration curve could be achieved for PFBA. For the re-prepared trout samples, the area counts of the [1,2,3,4-13C4]PFOA internal standard in the LCS samples dropped by approximately 30% when compared to the IS area counts o f the curve resulting in high recoveries. No results.were reported from the reanalysis; however, external standard quantitation of the samples produced acceptable recoveries in general. The variability o f the [1,2,3,4"C 4]PFOA IS during the sm all acid analysis was observed throughout the cross validation analyses and was more pronounced as the PRISM analytical column aged. This variability was not observed during the analysis of the large PFCAs using the Betasil C l 8 column, therefore, the signal degradation and variability over the course of an instrument batch is largely attributed to the IS's lack of robustness on the analytical column and not to extraction issues. Analysis o f the sm aller PFCAs may benefit by selecting a different isotopically labeled internal standard with a sm aller chain length, more representative of the target analytes. As observed with whole-body largemouth bass, the ECF spikes o f PFOS did not exhibit any measurable bias when quantitated against a linear reference standard for the three additional species (average recoveries were greater than 95%). The ECF PFOA LCSs exhibited lower recoveries for the whole body catfish and bluegill species, 90.9% + 0.82% and 83.0% 3.0%, respectively; however, the rainbow trout fillets produced an average recovery of 96.4% 3.0%. This may suggest that a co extracted matrix component present in whole-body tissues, but not in fillets, is affecting the PFOA isom er response. Because quantitation of the ECF materials against a linear reference standard produced variable recoveries in the different species and tissue types, it is recommended that this QC component be evaluated a t a minimum each tim e this method is applied to a new species and ideally with every preparation batch. 3M ENVIRONMENTAL LABORATORY PAGE 15 OF 25 3M ENVIRONMENTAL M O RATO RY REPORT NO. E08-0261 Table 11. Method Cross-Validation Results for Whole-Body Catfish. Accuracy (Average %Recoveryj t Precisian PARSD) Analyte Low Mid High A ll Levels Combined PFBA PFPeA PFHxA PFHpA PFOA PFNA PFDA PFUnA PFDoA PFBS PFHS PFOS FOSA EOF PFOA ECF PFOS [1,2,3I4',*CJ-PFBA [1,2,-I3CJ-PFDA 121 1 .0 102 3 .3 1133.7 1092.1 w 126 IS 101 1,7 107 3.1 (,>1 2 4 1 4 116 4 .4 94.3 1 0 94.4 7.7 97 6 3 8 118 2 .9 NA NA 116 1.1 109 2.6 120 2.1 108 3 .0 117 2 .8 114 1.6 111 8 .4 114 4.2 109 3.4 120 6.3 1126.9 99.9 1 .4 1063-3 1032.9 117 3 .7 NA NA 102 2.9 107 8 .8 123 4 .6 115 3 .5 111 5 .0 110 3 .2 1C2 1.2 113 3 .2 96.0 1.4 106 3 8 104 4.2 951 2.2 85.1 3 .2 98.7 3 1 105 3.1 NA NA 114 2 7 111 5 .7 121 2 .9 109 5 .9 113 4.2 111 2 .9 113 1 3 109 6 .3 104 6 .3 117 11 111 6 .5 96.5 5 .7 98.6 7 .3 99 7 3.7 113 6 .4 90.9 0.82 99.1 2.1 111 6 .2 109 5.6 [,aOj}-PFBS 101 6 .2 (1) One LCS exceeded 130% recovery. 98,7 3 .4 101 1 .7 100 3 .8 E= endogenous. The endogenous concentration listed is the IO Q for the control matrix studied. NA = Not applicable. LOQ (ngfgi E=2'29 E=0.376 0 103 0.0486 0.265 E=0.332 E=0.0784 E=0.142 E=0.0376 0103 0.0466 E = 0 .166 0.268 NA NA 0.0244 0.0245 0.0229 3M ENVIRONMENTAL LABORATORY PAGE 16 OF 25 3M ENVIRONMENTAL LABORATORY REPORT NO. E08-0261 Table 12. Method Cross-Validation Results for Whole-Body Bluegiil. Accuracy (Average %Recovervi Precision (%RSD) Analyte Lew AM High A ll Levels Combined LOQfngfg) PFBA PFPeA PFHxA PFHpA PFOA PFNA PFDA PFUnA PF'DoA PFBS PFHS PFOS FOSA EOF PFOA ECFPFOS 121 1 1 P)NA P>NA 104 5 .3 93.5 6 .9 112 9 .4 93.5 8 .0 99.1 2 .6 103 12 112 3.8 93.8 9.9 99.7 0 .9 3 122 8 .7 NA NA 111 3 .7 109 7 .0 133 3 .2 100 2 .2 99.8 7 .4 111 3.0 97 2 8.9 107 13 116 3.8 105 7.0 101 2.1 98.3 0.85 114 3.9 NA NA 1057.5 1004.5 109 3 .9 95 8 2.5 '84.0 0.46 ^O.B 0.60 87.1 3.9 99.0 2 .3 94.5 1 .6 96.2 3 5 1068.8 NA NA 11294 104 7 1 121 11 100 4.6 93.5 9.4 1 0 8 8.5 91.7 10 99.1 1 1 107 10 105 6 .8 96.5 6 .2 98.0 2.4 114 8 9 83.0 3 .0 98.1 0 .46 E=1.45 0.359 0.505 0.251 0.255 0.259 0.248 E=0.382 0.251 0.268 0.0977 E=1.99 0105 NA NA [1,2,3,4" C^PFBA 110 11 99.2 5.4 93 3 7.8 101 1 0 0.0247 [1,2,-,:,C2]-PFDA 92.2 4 .0 100 5.8 93 6 5.2 95.7 5.9 0.051 ("Chl-PFBS 98.8 3 .0 98.0 3.4 99.0 3 .2 98.6 2.8 0.0231 {1) Spike concentration less than the resultant LOQ, recoveries not reported. (2) Bad instrument injection for one of the replicates, data not generated. Precision evaluated as percent relative difference (N=2). NA= Not applicable. 3M ENVIRONMENTAL LABORATORY PAGE 17 OF 25 3M ENVIRONMENTAL LABORATORY REPORT NO. EOS-0261 Table 13. Method Cross-Validation Results for Rainbow Trout Fillets. Accuracy (Average %Recovery) t Precision (%RSD) Analvte Low M id High A ll Levels Combined LOQ (no/m PFBA PFPeA PFHxA PFHpA PFOA PFNA PFDA PFUnA PFDcA PFBS PFHS PFOS FOSA EOF PFOA EOF PFOS NR (,|131 1 9 124 7.8 109 0.27 105 4 .0 110 2.2 102 1 .6 108 7.5 108 6.9 102 6 .7 99.0 3.3 103 1.7 124 1.5 NA NA NR <1,127 6 7 122 1 .8 110 4 .5 108 5 .6 117 2.1 1 0 2 *2 .8 112 3 6 112 1 .8 106 4 .9 102 3 .5 102 3.b 121 5 .7 NA NA NR ">131 1 4 1 1 5 12 104 5 .9 103 6 .5 114 4 .9 100 3 1 m *1.1 1 0 9 *4 .5 93.2 15 93.8 8 .4 104 3 .8 124 9 .5 NA NA HR 129 1 3 120 7 .8 108 4 .4 106 5 .2 114 3 .9 101 2 .5 110 4 .5 110 4 .5 101 1 0 98.1 5 .9 103 2 .8 123 5.8 96.4 3 0 96.5 2 .5 NR 0.252 0.252 0.0243 0.0501 E=0.110 0.0487 0.0495 0.0493 0.0249 0.0234 E=0.287 0.104 NA NA [1,2,3,4`13C<]-PFBA 113*12 114 2.2 107 2 1 111 7 .0 0.0246 [1 ,2-,3Ci]-PFDA 105 3.2 107 2 .6 113 4 1 108 4 .5 0.0247 ["OJ-PFBS 103 2.7 103 3 .2 96.8 13 101 7.1 0.0231 (1) One or more of the LCS replicates produced a recovery greater than 130% NR = Not reported. The calibration curve once adjusted for the endogenous concentration did not meet method acceptance criteria for several points. IMA = Not applicable. 3M ENVIRONMENTAL LABORATORY PAGE 18 OF 25 3M ENVIRONMENTAL LABORATORY REPORT NO. 0 3-026 i 5.3 Solvent Curve Analysis. For each of the four species studied here, triplicate m atrix blanks along with triplicate laboratory matrix spikes at three levels were prepared in a separate preparation batch and analyzed against an acetonitrile solvent (un-extracted) calibration curve that was acid adjusted in a sim ilar fashion to the sample extracts. The results from the solvent (un-extracted) calibration are presented in Table 14 and graphically in Figure 3 for all the target analytes and surrogates except fo r the small PFCAs (PFBA, PFPeA, PFHxA, and [1,2,3,4 -13C4]PFBA Results for the sm all acids were not reported as the [1,2,3,4 -13C4JPFOA IS signal response 'was significantly suppressed when compared to IS signal in the soivent curve. Furthermore, the small acid target analyte signals were also suppressed when compared to the solvent response, but at different percentages than the IS. The combined target analyte and IS signal suppression observed in the extracted samples for the small acids resulted in recoveries that greatly exceeded method acceptance criteria of 10030% for PFBA, PFPeA, and [1,2,3,4 -13C4jPFBA. Acceptable results were observed for PFHxA as it was the largest acid analyzed and structurally m ost resembles the C8 internal standard. For simplicity, none of the PFHxA results are reported. Samples were prepared and analyzed twice for the sm all adds using newer PRISM columns to see if improved results could be achieved. Varying degrees of IS signal suppression relative to the solvent curve were observed during both analyses. The [1,2,3,4 -13C4]PFOA IS signal response did not demonstrate near the level of suppression in the large acid and sulfonate analyses for most species which indicates that the low S response was not an extraction efficiency issue. These results again suggest that the PRISM analytical column may not be suitable for analysis o f the larger PFCAs and selection o f a different internal standard for the smaller adds may improve the analysis. Although the ISs for the large PFCAs and sulfonates exhibited some suppression in some species, the target analyte signal w as suppressed by approximately the same amount. This produced a '`self-correcting' result and most recoveries For the target analytes were within 10030% (Table 14). However, it should be noted that the average overall recovery o f FOSA in the whole-body biuegill samples was less than 40% when quantitated against the solvent curve. This result emphasizes the criticality o f inclusion of appropriate QC samples to verify the method applicability and quantitation approach for each species for each analyte. 3M ENVIRONMENTAL LABORATORY PAGE 20 OF 25 p. 22 PFUnA F igure 3. S o lve n t C urve A n a ly s is R e su lts Sum m ary. ENViPOMMfcN ' Al. LABOR* 1OHY 3M ENVIRONMENTAL LABORATORY REPORT NO 08-0261 T WB Bass WB Catfish WB Biuegili Rainbow Trout Fillet < < (ft ffi( f t Q o u. u. u . 0 . CL F" ** CM CM O VO o< 0 T- 21 w W>* PAGE 22 OF 25 PFOS p. 23 3M ENVIRONMENTAL LABORATORY REPORT NO. E08-0261 Table 14. Solvent Curve Analysis Results Summary. Whole-Body Largemouth Bass mAccuracvPrecision N Whole-Body Cattish mAccutacytPr*cision N mWhole-Body Bluegill mAccuracy*Pradsion N Ra/nbotv TroutfWet a AccuraoyPrecision PFBA PFPeA PFHxA PFHpA PFQA PFNA PFOA RFUnA PFDoA PFBS PFHS PFOS FOSA NR NR NR 120115 115110 12716.4 104120 92 5 1 1 4 9 1 .1 1 5 6 11414.6 99 4 * 9 0 1 0 9 *7 .5 9 0 .3 *5 .5 NR NR NR NR NR NR 9 101 5 .3 9 r6 11819.2 >8 f8 125*14 9 1 3 5 *5 .9 1 0 9 *5 .7 1 1 9 *6 .4 "8 8 1 .3 *6 .4 "V (Jig 103*12 9 Kl? 9 5 .0 1 6 5 M)g 9 8 .5 *1 1 127*13 94.1 1 7 .2 9 1 1 5 *7 .6 9 1 2 9 *6 2 7 101 8.1 <*>8 1 1 3 *6 .4 9 10316.2 9 1 0 4 *5 .8 9 7 3 .8 *1 .6 % 36.1 * 12 9 t.i8 9 9 9 9 9 9 9 9 NR NR NR 1 0 3 *3 .9 118113 141 1 1 5 3 6 .7 *8 .6 101 1 2 9 8 4 1 7.5 111 4 .3 100113 10816.4 76.912.2 [1,2,3,4'I3C*}-PFBA NR NR NR [1,2,-,:>Cz]-PFDA 94,914.6 P>11 8 0 .7 *1 2 12 1 1 0 *6 .2 12 ("Ojl-PFBS 1 1 4 *4 .2 12 112*3.8 12 1 3 5 *6 .7 12 (1) Whole-body bluegill were (prepared and analyzed ma separate batch as tour or the loss were not spea wnn to. (2) Accuracy - average percent recovery (all levels combined). Precision * percent relative standard deviation (alt levels combined). (3) Instrument injection error observed for one low-level LCS. (4) One or more of the low-lsvel LCSs produced a final concentration below the limit of quantitation and were excluded from statistical calculations. (5) Sample preparation error. One of the matrix blanks was not spiked with internal standardisqrrogate prior to extraction N R -N ot reportable. NR 91.1 7.7 1 0 8 *5 .8 N S 9 9 (4>7 8 .>7 9 9 9 % 11 3M ENVIRONMENTAL LABORATORY PAGE 21 OF 25 p. 24 PFUnA F igure 3. S o lve n t C urve A n a ly s is R e su lts Sum m ary. 5M ENVIPONMfcN AL LABGRAIGRY 3M ENVIRONMENTAL LABORATORY REPORT NG E08-026! FOSA m W8 Bass WB Catfish BW B Biuegii! Rainbow Trout Fillet <m ou. EC u. a. CL M fjLm (st oto Q00 ci V* PAGE 22 OF 5 3M ENVIRONMENTAL LABORATORY REPORT NO. E08-0261 i i I ! i i * ( * . , . " ; ; I I 1 M f The method presented here produces excellent accuracy (average percent recovery) and precision (%RSD) when species-specific, matrix-matched calibration is used for the C7 through C12 perfluorocarboxylic acids, PFBS, PFHS, PFOS, and FOSA. In general, the method used here produces acceptable results (accuracy 1QQ30%, precision <20% RSD) for the sm all PFCAs (C4-C6); however, it is hypothesized that improved results can be achieved if a different internal standard is selected tor the sm all PFCA analysis in future studies. Analysis against a solvent curve produced more varied results than the species-specific m atrixmatched calibration. The solvent curve analysis may be an acceptable approach for screening level analyses or when a suitable control m atrix is not available. If the solvent curve calibration is used for generating quantitative values, enough QC elements (lab control and lab m atrix spikes) should be included to verify that m atrix effects are not artificially biasing the sample results. When quanlitated against a linear reference standard, QC spikes o f 3M ECF PFOS and PFOA produced recoveries within method acceptance criteria o f 10030%, although foe recoveries for the ECF PFOA exhibited a small, but measurable, bias in the whole body largemouth bass, catfish, and bluegill tissues (average recoveries ranging from 83.0-90.9%). No bias was observed for the ECF PFOS (all species) and PFOA in rainbow trout fillets with average recoveries greater than 95%. i m r ?inr m 94 * * *r,.i , 11.1 J * e-!*TT ' * . .* r i s .iU 'l All remaining sample and associated project data (hardcopy and electronic) w ill be archived according to 3M Environmental Laboratory standard operating procedures. 3M ENVIRONMENTAL LABORATORY PAGE 23 OF 25 kflOs 3M ENVIRONMENTAL LABORATORY REPORT NO. 08-0267 -Jriirrur-,-- rt w S-.# -kw ---- ________ - . . . .' ti'3 K W ;~ l ir IS B * * g wi. .-i .31'S-i rt-'* ' l I f i l i fc./b^il-*\:S..5u&*' ^ 3 //5 /^ (^ Principal Analytical Investigator Date /J William K. Reagen, Ph.D., Environmental Laboratory Management _____________________ __________________________ Cliffton $/3adbby, P h.D .,^/3M Technical Reviewer, Project Coordinator Date Date The 3M Environmental Laboratory's Quality Assurance Unit has audited the data and report for this project. QAI Representative Date 3M ENVIRONMENTAL LABORATORY PACE 2A Of- 21 3M ENVIRONMENTAL LABORATORY REPORT NO. E08-0261 K %' 9.1 3M Environmental Laboratory Michelle D. Malinsky, Ph.D Research Specialist 9.2 Pace Lab Ops Jonathan Steege c- r-*r'^T^CTiM no*R fL., s aanss?: r-'n s{ > at m-w s- a ' > X VV 5J... a v : . . ^ y S T !::K 7 " ;:'l [1] Guidance for Industry: Bioanalytical Method Validation, U S. Department o f Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), May 2001. [2] F. W. McLafferty, Interpretation of Mass Spectra, Third Edition 1980, University Science Books. iE ! : "f!r'Tii '`Tr'*--TV'ff.'lt >i t is \ ..3 .:- _ I 1.1 General Project Outline t Sv*.- 51 : ji f . $ H * *!' t' >S1'-*?i Ia ,f 3M ENVIRONMENTAL LABORATORY PAGE 25 OF 25 Environmental Health & Safety Operations, Environmental Laboratory Amended General Project Outline To: Bill Reagen, 3M EHS&Opns; Environmental Lab From : cc: Michelle Malinsky, 3M EHS&Opns; Environmental Lao C liff Jacoby, 3M EHS&Opns; Environmental Lab Date: Subject: January 14, 2009 Method Validation - Determination of Ftuorochemicals via Protein Precipitation of Fish Tissues (Fillet and W hole Body) and analysis by High Performance Liquid Chromatography with Tandem Mass Spectrometry 1 General Project Information Project Requester Project Coordinator Principal Analytical Investigator Contract Facility/Laboratory Lab Request Number Six Digit Department Number -- ..... ' | Project Schedule/Test Dates i. ..... W illiam Reagen 3M EHS Opns - Environm ental Laboratory 2 6 0 -5 N -1 7 St. Paul, MN 55144-1000 651-733-9739 wkreagen@ mm m .com C liff B. Jacoby 3M EHS&Opns, Environm ental Laboratory 2 6 0 -5 N -1 7 St. Paul, MN 55144-1000 651-733-2533 cbiacobv2@ mmm.corn M ichelle D, M alinsky 3M EHS&Opns. Environm ental Laboratory 260-5N 17 St. Paul, MN 55144-1000 651-733-9859 m maiinsky@ rnmm.com 3M Environm ental Health and Safety Operations, Environm ental Laboratories E08-0261 832202 Starting August, 2008 .. All verbal and written correspondence will be directed to Cliff Jacoby. 3M Environmental' Laboratory GPO 00-0261 Page `i o i 10 2 Background Information and Project Objective's) The purpose of this project is to validate method ETS-08-045. The validation of this method will generally follow the FDA Guidance for Industry; Bioanalytical Method Validation, May 2001. Method ETS-08-045 is used to extract perfluorochemicals (PFCs) from fish fillet and whole body sample homogenates, followed by quantitation by LC/MS/MS methodology. In general, this method involves the homogenization of fish fillet or whole body fish samples with acetonitrile at a 1:10 fish mass to solvent volum e ratio (i.e. 0.5 g of tissue to 5 mL acetonitrile). Note: all fish fillets and whole body samples w ill be pre-homogenlzed before weighing out aliquots for extraction. The centrifuge tubes containing the protein precipitated fish tissue and solvent are then placed in a freezer for at least one hour. After removal from the freezer, samples are centrifuged at -5C to pelletize the fish solids. Then, a measured volume o f the acetonitrile supernatant is removed to an autovial and acidified with a known volume of 10% form ic add (necessary fo r the analysis o f sm all acids). PFCs in the acetonitrile protein predpitate extract are analyzed using LC/MS/MS where the m ass transitions appropriate for the analytes o f Interest are monitored. Stable isotopes of various analytes will be incorporated into the method for use as internal standards and surrogates to allow for matrix internal standard quantitation and monitoring o f method performance via surrogate recoveries. The method validation proposed here will explore PFC quantitation in fish tissues via the following approaches; (1) internal standard calibration against a matrix-matched calibration curve. (If quantifiable levels of target analytes are present in the control matrix, method of standard addition w il be used to determine the endogenous concentration.) ' (2) internal standard quantitation o f endogenous PFC using a matrix-matched calibration curve of the target analyte's isotopically labeled counterpart. (This approach will be performed for quantitation of PFOS only using a matrix-matched calibration curve o f [1,2,3,413C]PFOS.) (3) internal standard calibration against a solvent (unextracted) calibration curve. The target quantitative range of this method validation w il be 0.2 ng/g (0.2 ppb) to 10 ng/g (10 ppb), with . selected QC samples spiked at levels up to 4000 ng/g of fish tissue for PFOS. Flowever, if the endogenous concentrations of the test matrices are sufficiently low, quantitation down to 0.025 ng/g w il be attempted. The validation of this method w il proceed utilizing whole body homogenates from largemouth bass samples purchased from Osage Catfisheries (Osage Beach, MO), a supplier o f fish for scientific testing purposes. These samples have been sent to MPI (State College, PA) for initial processing, then returned to the 3M Environmental Lab for further processing, storage and utilization. Following the foil validation of this method fo r whole body largemouth bass homogenates, tissues from other species will be cross validated using abbreviated procedures. The planned species cross validation includes fillet and/or whole body homogenates o f channel catfish, bluegill, and rainbow trout. The application of this method fo r additional sample types, ie. shellfish, may be appropriate, providing sufficient quality control components are included in the analysis of those sample types. This project will not include any type o f shellfish as part of the validation. This validation project does not address the process of the initial whole body or fillet homogenization, only the preparation and analysis of individual aliquots of the homogenized whole body or fillet. 3M Environmental Laboratory GPO E08-0261 Page 2 of 10 3 Project Schedule The project is scheduled to start in August 2008. The validation of this method utilizing largemouth bass is projected to take t month. Cross validation of this method to other species w ill depend on the number of species analyzed. 4 Test Parameters The goal of this project is to validate analytical method ETS-8-45 for whole body largemouth bass homogenates. This method can be used to quantitate the levels of perfluorochemical analytes in both fish fillet and whole body fish homogenates. This method utilizes solvent extraction of the PFCs via protein precipitation, followed by LC/MS/MS analysis. The analytes to include in this method validation are the C4to C12 perfluorocarboxylic acids, PFBS, PFHS, PFOS and FOSA. A list of the formulae and m olecular weights of these analytes can be found in Table 1. The stable isotope labeled compounds [1,2,3,4~13C4]PFBA, [1,2;3.4-13C4]PFOA, [1,2 -l3C2]PFDA, [180 2]PF0S, [1B0 2]PFHS, and [180 2]PFBS w ill be spiked for potential use as internal standards (IS) and surrogates. The sample spike level for each IS and surrogate w ill be approximately 1.0 ng/g (1.0 ppb). The reference m aterials to be used in this project are all commercially available. This method validation will incorporate the commercially available linear forms o f PFOA, PFHS, and PFOS. However, select quality control samples spiked with 3M electrochemical fluorination (EOF) production lots of PFOA and PFOS will be analyzed with the intent to show that these branched materials may be accurately quantitated against the linear standard. The target quantitation range o f th e validation for all analytes is 0.2 ng/g (200 ppt) to 10 ng/g (10 ppb) for each analyte, or as defined/limited by the endogenous levels of any of these analytes in the blank fish tissues, if the endogenous levels o f any analytes are below the target level of 0.2 ng/g, the actual LOQ will be the iowest standard point o f the curve. In this case, the next target LOQ will be 0.025 ng/g (25 ppt). The ability o f this method to quantitate high levels of PFOS in the presence of lower levels of other PFC analytes will be evaluated via dilution QC. The dilution QC samples will be spiked with PFOS at levels up to 4000 ng/g (4 ppm) in the presence of the other analytes spiked at lower levels. Because PFOS may be present in the endogenous fish samples at levels above 0.2 ng/g, a separate analysis will be performed where a matrix calibration curve of [1,2,3,4-13C4jPFOS will be prepared using [180 2]PFBS as an internal standard and [1B0 2]PFHS as the surrogate. The purpose of this analysis will be to investigate if a lower quantitation lim it o f PFOS can be achieved using a calibration curve prepared from a stable isotopically substituted analog of PFOS. Area counts of the unlabeled PFOS transitions will be monitored and entered into the calibration equation o f [1l2,3,4-13C4]PFOS to quantify the endogenous and spiked levels. 3M Environmental Laboratory GPO '05-026/ Page 3 of 10 Table 1. Target Analytes. Name Perfluorobutanoic acid Perfluoropentanoic acid P erfluorohexanoic acid Perfluoroheptanoic acid Perfluorooctanoic acid Perfluorononanoic acid Perfluorodecanoic acid Perfluoroundecanoic acid Perfluorododecanoic a dd P erfluorobutane sulfonic acid P erfluorohexane sulfonic acid Perfluorooctane sulfonic acid Perfluorooctanesulfonam ide 13C4"P erfluorobutanoic acid l3C4-Perfluorooctanoic acid 13C2-P erfluorodecanoic acid 180 2-Am m onium Perfluorobutane sulfonate 180 2-Am m onium Perfluorohexane sulfonate 180 2-Am monium Perfluorooctane sulfonate 13C4-Sodium Perfluorooctane sulfonate Synonym or Acronym Formula PFBA (C4 Acid) PFPeA (C5 Acid) PFHxA (C6 Acid) PFHpA {C7 Acid) PFOA (C8 Acid) PFNA (C9 Acid) P FD A {C 10 Acid) PFUnA (C 11 Acid) PFDoA (C l2 A dd) PFBS (C4 Sulfonate) PFHS (C6 Sulfonate) PFOS (C8 Sulfonate) FOSA (C8 Sulfonam ide) [1 ,2 ,3 ,4 -13C ,,]P F B A [1 ,2 ,3 ,4 -13C 4]P F O A [1,2-13C 2]PFDA [' "OJPFBS C 3F 7C O O H c 4f 9c o o h CsFuCOOH c sf 13c o o h c 7f 15c o o h c 9f 17c o o h c 9f 13c o o h c 10f 21c o o h Ci i F23COOH c 4f 9s o 3h c 8f 13s o 3h c 8f 17s o 3h c 8f 17s o 2n h 2 13C F 3(13C F 2)213COOH C F 3(C F z)3(13C F 2)313CO O H C F3(C F 2)7( ,3C F 2) ,3C O O H C4F9S ,b0 20 'N H / [,80 2]PFHS [C6F i3S180 20 rN H 4* [180 2JPF0S [C8F i 7S180 20 ]" N H /" [1 ,2 ,3 ,4 -13C 4]P FO S CF3(CF2)3(13CF2)313CF2S 0 3"Na* Molecular Weight (Anionic Form) 213 [M -H ]' 263 [M -H ]' 313 (M -H f 363 [M -H ]' 413 [M -H ]' 463 [M -H ]' 513 [M-HJ563 [M-H]" 613 [M -H ]' 299 [M -H ]' 399 [M -H ]' 499 [M -H ]' 498 [M -H ]' 217 [M-H]" 417 [M -H ]' 515 [M-H]" 303 [M -H ]' 403 [M -H ]' 503 [M-H]" 503 [M -H ]' Table 2 lists the mass transitions (MRMs) typically monitored for these analytes. M ultiple MRM transitions can be summed for an individual analyte to improve the sensitivity for that analyte. However, care should be taken to use a reasonable number o f transitions within any one tim e period, as the inclusion of a large number of transitions can affect the precision and accuracy of all results obtained during that tim e period For this reason, it is recommended that the analyte list be split between two or more injections to optimize the overall performance of the method when analysis o f all the listed analytes is required. Division of the analyte list also allows the analyst to vary chromatographic conditions to separate m atrix interferences from the target analyte 3 M Environmental Laboratory GPO EOS-026 f Page 4 of 10 when present, which often cannot be done for simultaneous analysis of all compounds in a single run. For this validation, three separate injections of each sample, QC, blank, etc. are planned. Table 3 provides a summary of the analytes to be monitored in each injection and a brief description of the chromatographic conditions Table 2 Mass Transitions of Analytes. Compound Q1 Q3 PFBA (C4 A cid) 213 169 PFPeA (C5 Acid) 263 219 PFHxA (C6 A d d) 313 269, 119 PFHpA (C7 Acid) 363 319, 139 PFOA (C8 Acid) 413 369, 219, 169 PFNA (C9 Acid) 463 419, 219, 169 PFD A(C 10 Acid) 513 469, 269, 219 PFUnA {C 11 Acid) 563 519, 269,219 PFD oA(C 12 Acid) 613 569, 319, 169 PFBS (C4 Sulfonate) 299 99, 80 PFHS (C6 Sulfonate) 399 99*', 80* PFOS (C8 Sulfonate) 499 130, 99, 80** FOSA (C8 Sulfonam ide) 498 78 13c *-p f b a 217 172 13c 4- p f o a 13Cr PFDA 18o 2- p f b s ^ 417 515 303 372 470 84 18o 2- p f h s ,8o 2- p f o s 403 5Q3 84 84 13c 4-p f o s 503 131, 99, 80 * The MRMs of 399 to 80 and 99 have been documented in literature to result in interferences in some biological tissues, arising from the presence of 5-pregnan-3,2C-diol-3-suIfate isomers[2], **The MRM of 499 to 80 for PFOS has been documented in literature to result in interferences in some biological tissues, arising from the presence of taurodeoxycholate isomers (bile sa!t)[2]. 3M Environmental Laboratory GPO E08-0261 Page 5 of to Table 3. Analysis Summary. Method Transitions Monitored Chromatographic Conditions Small Acids (6 transitions) 213>169 (PFBA) 217>172 ([1,2,3,4-13C4]PFBA surrogate) Analytical C olum n: Prism RP (2 x 5 0 mm, 5pm particle size) Mobile Phases 263>219 (PFPeA) 313>269, 313>119 (PFHxA) 417>372 ([1,2,3,4-13C4]PFOA internal standard) A: 5 mM ammonium acetate in 0.01% acetic acid B: Methanol PFHpA, Sulfonates, and FOSA (13 transitions) *363>319, 363>169 (PFHpA) 299>99,299>80 (PFBS) 399>99, 399>80 (PFHS) 499>99, 499>80, 499>130 (PFOS) 498>78 (FOSA) 304>84 ([180 2]PFBS surrogate) 504>84 ([^O JPFO S internal standard for sulfonates, FOSA) Extraction Pre-Column: W aters HLB Online Column (3x20mm, 25pm particle size) Analytical C olum n: BetasilC 18 (2.1x100 mm, 5pm particle size Mobile Phases A: 2mM ammonium acetate B: Acetonitrile 417>372 ([1,2,3,4-13C4]PFOA internal standard for PFHpA) Large Acids (17 transitions) 413>369, 413>219, 413>169 (PFOA) 463>419, 463>219, 463>169 (PFNA) 513>469, 513>269, 513>219(PFDA) 563>519, 563>269, 563>219 (PFUnA) 613>569, 613>319, 613>169 (PFDoA) 417>372 ([1,2,3,4-13C4)PFOA internal standard) Extraction Pre-Column: Waters HLB Online Column (3x20mm, 25pm particle size) Analytical Column: BetasilC18 (2.1x100 mm, 5pm particle size Mobile Phases A: 2mM ammonium acetate B: Acetonitrile 515>470 ([1,2-13C2]PFDA surrogate) '([1 ,2-13C2]PFDA surrogate recovery for PFHpA w ill be recorded from the large acid analysis. The ({1,2X J P F D A transition will not be monitored in the sulfonate analysis. 5 Method Validation The individual components of this method validation are listed below. It is anticipated that all of these aspects will be evaluated, addressed and discussed in the method validation report. 5.1 Species and Sample Types The method validation w ill include analysis of whole body homogenates of largemouth bass (Micropterus salmoides). Method cross validation may include whole body and/or fillet homogenates from the following species: channel catfish (Ictalurus punctatus), bluegill (Lepomis macrochirvs), and rainbow trout (Oncorhynchus mykiss). 3 M Environmental Laboratory GPO E08-0261 Page 6 of 10 5.2 Analytes The analytes to be evaluated in this validation are PFBA, PFPeA, PFHxA, PFHpA, linear and branched PFOA, PFNA, PFDA, PFUnA, PFDoA, PFBS, linear PFHS, linear and branched PFOS and FOSA (C4 to C12 PFCAs,' C4, CBand Cg sulfonates, and FOSA). 5.3 Internal Standards and Surrogates The isotopically substituted compounds [1,2,3,4-13C4]PFBA, [1 ;2,3,4-riC4]PFOA, [1,2 -V3C2]PFDA, [13Oj]PFOS, [ ,80 2]PFHS, and {^O ^PFBS w ill be used as internal standards and surrogates. Internal standards, surrogates, and calibration spikes {where appropriate) will be spiked into individual aliquots of homogenized fish tissue prior to extraction. The surrogate concentration in extracted calibration standards wilt be at the same level as the other target analytes, while the internal standard concentration will be 1 ng/g for each individual standard ti e. a m ulti-level surrogate calibration curve will be generated for accurate quantification o f surrogate recovery). QC samples, appropriate blanks, and "samples" will have surrogate spiked at 1 ng/g. [1,2,3i4-13C4lPFOA will be used as the internal standard for the quantitation of all the target carboxylic acids. [1 ,2,3;4-1sC4]PFBA will be used as a surrogate to estimate the recoveries of the C4-C6 carboxylic acids [1,2-13C2]PFDA w ill be used as a surrogate to estimate the recoveries o f the C7-12 carboxylic acids. [18C>2]PFOS w ill be used as the internal standard for the quantitation of the target sulfonates and FOSA. [180 2jPFBS w ill be used as a surrogate to estimate the recoveries o f the target sulfonates and FOSA. For the separate PFOS analysis, {1,2,3,4-13C4]PFOS w ill be used to construct the calibration curve with [,b0 2]PFBS serving as the internal standard and [1sU2jPFHS as the surrogate. 5.4 Endogenous Levels of Target Analytes in Control Tissues The endogenous levels o f the target analytes wilt be determined in the whole body largemouth bass in triplicate (x3) by quantitation against a solvent curve and by the method of standard addition. These data can be collected as part of the evaluation of Linearity, Precision and Accuracy (Section 5.5). The endogenous levels of each analyte in the tissue will be evaluated. These values, if significant, may need to be accounted for in the rest o f this validation project, and in future quantitative studies. The endogenous values w ill be considered specific to the validation tissue used for the study (ie. date of sample receipt, lot#). 5.5 Linearity, Precision and Accuracy The intended quantitative range for this method is 0.25 ng/g (0.25 ppb) to 10 ng/g (10 ppb) comprised of at least six standard points in the final calibration curve If the endogenous level of the target analyte is significant, then the endogenous level of that analyte in that sample w ill be the LOQ. If the endogenous level of the analyte is sufficiently low, then the lowest calibration standard satisfying the method criteria for LOQ will be the LOQ {area counts > two times the area counts of the blank samples and accuracy of 1CC>30%). Quantitation down to 0.025 ng/g w ill be attempted when endogenous levels are sufficiently low. Using whole body largemouth bass homogenates, three curves w ill be prepared over the range of 0.025 ng/g (25 ppt) to 25 ng/g (25 ppb). The final LOQ for each analyte will largely depend on the endogenous amount detected in the control tissue, and for some analytes w ill be significantly greater than 0.025 ng/g. Each curve point w ill include internal standards and surrogates. (IS concentration w ill be constant at 1 ng/g, surrogate concentrations will be at the same levels as the other target analytes.) Each run will also include duplicates of the following control samples: control matrix blanks with the ISs and surrogates control matrix blanks without the iSs and surrogates, aqueous blanks with the ISs and surrogates, aqueous blanks without the ISs and surrogates 3M Environmental Laboratory GPO E08-0261 Page 7 of 10  acid adjusted acetonitrile extraction solvent blanks w ith'trie lS and surrogates acid adjusted acetonitrile extraction solvent blanks without the ISs and surrogates. The full validation o f whole-body bass w ill include the following preparations to assess linearity, precision, and accuracy; Duplicate m atrix-m atched curves prepared on a single day to evaluate the intra-day precision and accuracy. {Two separate analysts may prepare the curves; however, each analyst must have his or her own set of accompanying QCs as outlined in Section 5.6.) A single matrix-matched curve prepared on a different day to evaluate the inter-day precision and - ; - accuracy. For each target analyte and surrogate, the data w ill be reduced according to ETS 8-45 using the following approaches: (1) internal standard calibration against a matrix-matched calibration curve using the method o f standard addition when appropriate. (2) internal standard quantitation of PFOS using a matrix-matched calibration curve o f I1,2,3,4-13C<]PFOS for a separate single preparation batch 5.6 Quality Control Samples Each day an extracted curve is prepared, four levels o f Q samples should be prepared for the whole body bass. An additional fifth level of QC will be included on the extraction day in which the single extracted curve is prepared for inter-day comparisons. For PFO A PFHS, and PFOS, the standard curves w il use the linear reference materials. Aspects of the QC samples w ill utilize both the linear and 3M ECF production lot branched reference m aterials Each QC sample w ill be analyzed against the relevant species-specific matrix matched curve. As discussed previously, samples w ill include appropriate internal standards and surrogates spiked at 1 ng/g. Internal standard quantitation w il be performed. The QC spike levels should be at different concentrations than the curve points. The first four QC levels need to be prepared each tim e an extracted curve is generated. Dilution QC need to only be prepared once during the validation. (1) The low-level spiked QC should be approximately 200% o f the LLOQ or approximately 0.3 to 0.4 ng/g if the LLOQ is ^ 0.5 ng/g. If the LLOQ is >0.5 ng/g, then the low-level spikes w ill be adjusted accordingly for the affected analytes. (PFOA, PFHS, and PFOS in these QC samples will be the linear reference materials). (2) The m id-level spiked QC should be in the middle part of the curve range. (PFOA, PFHS, and PFOS in these QC samples w ill be the linear reference materials.) . (3) The high-level spiked QC should be greater than.the mid-levei spike but still w ithin 80% of the ULOQ. (PFOA, PFHS, and PFOS jri thse QC samples w ill be the linear reference materials.) (4) Separate triplicate mid-level QCs at approximately 5 ng/g will be prepared that contain the 3M ECF PFOA and PFOS only (no other target analytes). These samples will only be quantitated for PFOA and PFOS (i.e. endogenous levels and impurities o f other analytes will not be evaluated). (5) The dilution QC should be spiked with PFOS at a level of approximately 4000 ng/g (4 ppm). The spike levels o f the other analytes should be approximately 1ng/g These QCs w ill be analyzed without dilution for all analytes excluding PFOS and then post-extraction dilutions w ill be performed to bring the PFOS levels w ithin cafibration range. These high-level QCs are intended to show that low levels of PFCs can be accurately quantitated in the presence of high levels of PFOS. Note: the quantitation of the high levels of PFOS will be done via external standard calibration only. Spiking the internal standard at a comparable ppm level is not practical given that the laboratory's labeled PFOS reference material is only available as a 50 ppm solution. The dilution QC only needs to be prepared once during the validation. Care should be taken to run the dilution QC near the end of the run after any low-level samples. Additionally, several blanks should be analyzed after the dilution QC to verify that PFOS instrum ent carryover from the high level sample is not present. 3M Environmental Laboratory CPO E06-0261 Page 8 of 10 For the alternate PFOS analysis using the [1,2,3,4- ':jC,!]PFOS calibration curve, a minimum of triplicate LCSs of non-labeled PFOS at a level 200% the endogenous concentration will be included. 5.7 Solvent Curve Analysis In a separate preparation batch, triplicate matrix blanks (with IS and surrogate) and triplicate lab control spikes at the low, mid, and high levels will be prepared and analyzed against an acid-adjusted solvent curve in acetonitrile. The preparation and analysis batch w ill include the whole body largemouth bass and all the species/tissue types selected for the cross-validation discussed below. Internal standard quantitation will be perform ed. 6 C ross Validation of Other S p ecies Cross validation procedures of this method to other tissue matrices will depend on the results obtained as part of this formal validation of whole body largemouth bass. The nine additional m atrices proposed for cross validation include the following: largemouth bass fillet, whole body catfish, catfish fillet, whole body bluegill, bluegill fillet, whole body carp, carp fillet, whole body rainbow trout, and rainbow trout fillet. The priority and inclusion/exclusion of some matrices may be reassessed after the full validation o f the whole body bass. The cross validation procedures will include all preparation, analysis, and quantitation aspects o f the full validation described in Sections 5 with the following exoeptions: (1} Only a single species-specific, matrix-matched calibration curve w ill be prepared on a single day (no intra-day or inter-day comparisons will be performed}. (2) Dilution QC of PFOS will not be included. (3) Alternate PFOS quantitation via calibration with i1,2,3,4- JC^]PFOS wili not be included. 7 Method Validation Acceptance Criteria The applicability and acceptability of the method towards each species and tissue type wili be determined by evaluation of the results from this project. The aspects to be evaluated and their proposed acceptance criteria are listed below. (1) Precision - The precision of each analyte in each control tissue w ill be a %RSD of s 20%. (2) Accuracy - The accuracy of each analyte in each control tissue will be 10Q25% (10030% at the LLOQ). (3) Correlation Coefficient - The correlation coefficient (r) of each analyte in each control tissue will be 0.995. (4) QC Samples - The precision and accuracy of the QC samples will be 10030% for each analyte and the %RSD of +20% for each analyte, respectively. Based on the results of the method validation results, the acceptance criteria of this method may be modified based on the analytical needs of a project. Because the intent of the method validation is to determine what acceptance criteria is plausible for this method, method deviations will not be issued for QC samples not meeting the arbitrary requirements listed above. 8 Attachments None 3A- Environmental Laboratory GPO 05-0267 Page 9 of to 9 References 1. FDA Guidance for Industry, Bioanalytical Method Validation, May 2001. 2. Benskin, J.P.. Bataineh, M, Martin, J.W.. Anal. Chem 2007, 79.6455-6464. 10 Revisions The original GPO stated that m ethod ETS 8-49 would be validated. W hile writing the validation report, management decided that the m ethod validated was sufficiently different from ETS 8-49 and requested that a new method number be assigned (ETS 8-45). The original GPO included four different aspects of quantitation (internal standard quantitation with a m atrixmatched calibration curve, external standard quantitation against a matrix-matched curve, internal standard quantitation against a solvent curve and external standard quantitation against a solvent curve). As the project progressed, it became apparent that the amount o f instrument analysis time and data reduction needed to fulfill the original proposal was not going to be feasible in the tim e allotted. After discussions w ith management, it was decided to elim inate ail external standard and solvent curve quantitation requirements. After analysis was completed for the three selected species for cross-validation, management reconsidered the need for internal standard solvent curve analysis. The samples outlined in Section 5.7 were then prepared. PFOS dilution QC was intended to be 10,000 ng/g {10 ppm). Due to a calculation error, the PFOS dilution QC were prepared at 4,000 ng/g. It was decided that this concentration was sufficiently high. The GPO was updated to reflect the concentration prepared. The original GPO detailed proposed analyses for NIST SRMs of Lake Michigan and Lake Superior trout fillets. Analyses of these samples was moved to a separate project with its own GPO (E07-0295). 3M Environmental Laboratory GPO E08-0261 Page 10 of 10 3MENVIRONMENTAL LABORATORY REPORT NO. E08-0261 SUPPLEMENTAL INFORMATION Supplemental Information Method Validation of ETS -8-45 "Determination of Fkiorochemicals via Protein Precipitation of Fish Tissues (Fillet or Whole Body) and Analysis by High Performance Liquid Chromatography with Tandem Mass Spectrometry" Laboratory Request Number: E08-0261 Testing Laboratory 3M EHS Operations 3M Environmental Laboratory 3M Center Building 260-5N-17 Maplewood, MN 55144 Requester William Reagen 3M EHS Operations 3M Environmental Laboratory 3M Center Building 260-5N-17 Maplewood, MN 55144 3M ENV'RONMENTAL LABORATORY PAGE 1OF 6 3MENVIRONMENTAL LABORATORY REPORT NO. 0 8-02 6 J SUPPLEMENTAL INFORMATION f; Extraction andAhifyss Dfs' : - Mg Vf .L B S ) Table 1. Extraction and Analysis Dates and Instrument Information. W B Largemouth Bass Day 1 W B Largemouth Bass Day 2 W B Largemouth Bass Day 2 W B Largemouth Bass - Alternate PFOS Quantitaion WB Catfish WB Bluegill Rainbow Trout Fillet Prep A nalyst A A B A A A A 8/20-8/21/2008 8/27-8/28/2008 8/27-8/28/2008 A nalyte G roup Instrum ent C7 Acid/Sulfonates/FOSA Large Acids Small Acids C7 Add/Suifonates/FOSA ---L--a-r-zg-e--A---d--d--s-------------------------------------------------Small Adds C7 Acld/Sulfonates/FOSA Large Adds Small Adds Stan Ollie Ollie Ollie Ollie Ollie Ollie Ollie Ollie Safe/) o080821a s080824a o080910a 0080828a 0080830a o080905a 0080829a o080831a o080906a 9/3-3/4/2008 PFOS only Stan s080919a 9/9-9/10/2008 9/26/2008 & 9/29/2008 9/16-9/17/2008 C7 Add/Sulfonates/FOSA Large Acids Small Acids C7 Acid/Sulfonates/FOSA Large Acids Small Adds (re-analysis) C7 Acid/Sulfonates/FOSA Large Adds Small Adds (re-analyss) 11/17-11/18/2008 (small add analysts only) Small Adds Ollie Ollie Ollie Ollie Stan Ollie . Ollie Ollie Ollie Ollie 0080912a 0080913a o080911a 0080930a 8080929a o081031a 0080918a o080919a o081110a o081118a p. 39 3M ENVIRONMENTAL LABORATORY PAGE 2 OF 6 Table 1 C o n tin u e d . P rep Batch Description P rep A nalyst P rep/Extraction D ata Solvent Curve Analysis 10/1-10/2/2008 {all species) A 10/26-10127/2008 W B Blueglll only 1 1/20/2005-11/21/2208 (small acids only) 3M ENVIRONMENTAL LABORATORY REPORT NO. E08-02S1 SUPPLEMENTAL INFORMATION Analyte Group C7 Add/Sulfbnates/FOSA Large Acids Small Acids Small Acids (re-analysis) C7 Add/Sulfonates/FOSA Large Acids Small Acids Small Acids (re-prep) Instrum ent Oil Stan Olile Ollie Olile Ollie Olile Olile B atc h o081006a s081003b 0081016a o081111a o081105a 0 0 8 1 105b o081030a o081121a 3M ENVIRONMENTAL LABORATORY PAGE 3 OF 6 U O 3M ENVIRONMENTAL LABORATORY REPORT NO. E08-O261 SUPPLEMENTAL INFORMATION i2 ' ; iSfe . ; -, V Table 2 System Suitability Non-Compliances. Prep Batch Description WB Laigemoutli Bass Day 1 Rainbow Trout Fillet Rainbow Trout Fillet Solvent Curve Analysis Analyte Group Large Acids C7 Add/Sutfonates/FOSA Small Acids C7 Add/Sutfonates/FOSA Large Acids C7 Add/Sulfonates/FOSA (WB Bluegiil reprep) Large Acids (WB Bluegilf reprep) Instrum ent Stan Ollie Olire Ollie Stan Ollie Oltre Batch s080824a o080918a o081110a o081006a s081003b o081105a o081105b Non-compliance Description Area Counts %RSD PFN A 8.8% FO SA 14% PFBA: 12%, PFPeA: 9.0%, [1,2,3,4-" C,]PFBA: 12% FO SA 8.6% PFNA 7.6%, PFUnA 7.9% FO SA 10% PFDA: 8.1% Table 3. Continuing Calibration Verification (CCV) Non-Compliances. Prep Batch Description WB Larqemouth Bass Day 1 A n alyteG ro up Small Acids Instrument . Ollie Batch o080910a Non-compliance Description CCV Recovery PFPeA 127%, 13C PFBA 127% I V 'd 3M ENVIRONMENTAL LABORATORY PAGE 4 OF 6 p. 42 3M ENVIRONMENTAL LABORATORY REPORT NO, 08-0267 SUPPLEMENTAL INFORMATION 3 Surrogate-Recoveries inSpikd WanMli y ; Table 4. Surrogate Recoveries: Small Acids. [1.2,3,4-" CJPFBA Surrogate Recoveries in Spiked Blank Samples (Small Acids) Matrix Blank Aqueous Blank Prep Batch Description Rep 1 Rep 2 Rep 1 Rep 2 W B Largemouth Bass Day 1 WB Largemouth Bass Day 2A WB Largemouth Bass Day 2B WB Catfish WB Bluegill Rainbow Trout Fillet 112 112 89.6 89.9 101 102 113 104 104 101 98.6 95.4 108 105 85.7 86.6 131 107 132 110 124 114 140 156 Table 5. Surrogate Recoveries: Large Acids. fl,2 -1,CJPFDA Surrogate Recoveries in Spiked Blank Samples (Large Adds} Matrix Blank Aqueous Blank Prep Batch Description Rep 1 Rep 2 Rep 1 Rep 2 WB Largemouth Bass Day 1 WB Largemouth Bass Day 2A WB Largemouth Bass Day 2B WB Catfish WB Bluegill Rainbow Trout Fillet 121 109 90.3 106 110 104 103 100 103 105 104 100 92.6 82.6 109 106 104 103 131 101 99.4 102 114 109 Table 6. Surrogate Recoveries: Sulfonates, FOSA. f ' OJPFBS Surrogate Recoveries in Spiked Blank Samples (Sulfonates, FOSA) Matrix Blank Aqueous Blank Rep 1 Rep 2 Rep 1 Rep 2 W B Largemouth Bass Day 1 97.5 99.0 WB Largemouth Bass Day 2A 38.3 106 WB Largemouth Bass Day 2B 104 102 WB Catfish 102 99.2 WB Bluegill 97.5 101 Rainbow Trout Fillet 92.4 114 96.6 104 97.4 103 95.9 107 106 95.5 103 S4.9 98.9 98.3 Acetonitrile Blank R ept Rep 2 78.6 110 92.2 83.2 95.5 136 80.9 101 86,4 84.0 988 120 Acetonitrile Blank Rep 1 Rep 2 132 121 106 95.0 106 99.2 103 96.8 120 115 114 108 Acetonitrile Blank Rep 1 Rep 2 94.7 103 91.9 101 88.5 104 90.4 96.1 90.6 93.5 90.7 98.8 3V ENVIRONMENTAL la b o r ato r y PAGE 5 OF 6 p. 43 3M ENVIRONMENTAL LABORATORY REPORT NO E08-0261 SUPPLEMENTAL INFORMATION Table 7. Surrogate Recoveries: Solvent Curve Analysis. S olvent C urve A nalysis Large Acids Large Acids W B Bluegitl Reprep C7 Acid, Sulfonates, FOSA C7 Acid, Sulfonates, FO SA W B BluegiH Reprep Rep 1 896 99.3 106 110 Aqueous B lank Rep 2 Rep 3 101 106 98.3 109 100 NA 97.1 NA Rep 4 108 NA 105 NA A cetonitrile B lank Rep 1 Rep 2 109 101 98.4 110 98.2 100 92.5 101 3M ENVIRONMENTAL LABORATORY PAGE 6 OF 6