Document vxNp8JgdjmBwedmOvjgb0q0E

LaboratmyReport-Revisionl Analytical Report of Data for PFOS Dietary LC50 Study with Mallards (Sera and Liver) Laboratory Report No. FACT-TOX-130 (W1902) Testing Laboratory 3M Environmental Technology& Safety Services 3M Environmental Laboratory Fluorine Analy-hcalChemistry Team (FACT) 2-3E-09 935 Bush Avenue, St. Paul,MN 55106 Kris Hansen, Ph.D. Bldg. 2-3E-09 P.O.Box 3331 St. Paul, MN 55133-3331 Phone: (651) 778-6018 FAX: (651) 778-6176 Revision Date: March 19,2001 ,. page 1 of6 3M ENVIRONMENTAL LABORATORY REPORTNO. FACT-TOX-I30 1 Introduction This study was designed to quantitatively assepsesrfluorooctanedfonate(PFOS, C8F17S03-, CAS ## 279539-3) levels in thesera and liver of mallard ducks exposed to PFOS through diet. Details of dosing conditions, clinical observations and other toxicity data were generated by the in-life testing facility (Wildlife International) and are not includedthiins report. Subsetsof the nine groups of taenstimalswere sacrificedon day 8 and day22, postdose. Sera and liver collected fiom the sacrifiacneidmalswere sent to the 3M Environmental Lab and analyzed quantitatively for PFOS. Tissfuieosm someanimals that died befortehe scheduled sacrifice date were collected and analyzed also; results are incthliusdreedpoinrt. With few exceptions, PFOS leveinlsthe sera and livers of the mallards increawseitdh dose group. Additionally, considering the average PFOS levweiltshineach dose group, both liver saenrda levels of PFOS declined dramatically between da8yand day22. In most animals,for a specific timpeoint,the ratioof PFOS concentration insera ( p g t d ) to PFOS concentration in liv(eprgtg) was greater than1. No data was collected for analytes otthhearnPFOS. Although rigorousqual~tycontrol measures and3M EnvironmentalLaboratory Standard Operating Procedures were followed, the acquisitionthoisf data was not necessarily collected accordtiongapplicable Good Laboratory Practices. Data presented heisrethe highestquality data availableat t i i s time. In-phase testing was conducted accordintogapplicable Good Laboratory Practices.The conduct of the in-life phase is the sole responsibility of Wildlife International Ltd. See attachmCenfot r the in-life protocol. The Environmental Lab receive6d0liver, 58 bile, 46blood, and46 serum specimens, frozeno,n 05/19/99. Upon specimen receipta, 3M chain of custody wasinitiated The Environmental Lab receive5d0 liver, 50 bile, 50 blood, and50 serumsamples, fiozen, on 06/29/99. Upon specimen receipta, 3M chain of custody wasinitiated Acopy of eachfonn can be found inan Attachment to this report. There is no holding time criteria associated with these samples LfoCr ESMSMS analysis. Analytical methods ETS-84.1,Extraction of PotassiumPduorooctanedonate or Other FluorochemicaClompounds fiom Serum for Analysis Using HPLC-Electrospraybfass SpectromwetirtyhthefolZow'ngexceptions: Due to the small volume of mallard sera available, standard curves were preipnamreedthanol insteadof fiom mallard sera. Available sample volumes werseo small, there wasnot enoughsera for matrix spike studies. As an indicator ofgross error, rabbitserumwas usedas a surrogate matrix. Sixteen matsrpixikes were preparedin rabbit sera. PFOS lot171was used as reference material. ETS-8-6.0,Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compofuinodms Liver for Analysis Using HPLC-ElectrosprayMass Spectromwetirthy thefollowingexception: -2of6 3M ENVIRONMENTAL LABORATORY REPORTNO. FACT-TOX-I30 Due to the small amount of mallard liver available, standard curves were prepared in methanol instead of fiom mallard liver. Twelve matrix spikes were prepared fiom the mallard Alisvedre.scriied in the method, all results are reported in teorfmpsg of analyte pergram of liver (wet weight). PFOS lo1t71was used as reference material. ETS-8-5.1, Analysis of PotassiumPerfluorooctanesulfonateor Other Fluorochemicals iSnerum Extracts Using HPLC-ElectrosprayblasSspectrometry ETS-8-7.0, Analysis of PotassiuPmerfluorooctanesulfonateor Other Fluorochemical in Liver Extracts Using HPLC-ElectrosprayblassSpectrometry Sample preparation-HPLC/ESMSMS: ion-pairing extraction Analyte is extracted h m a sample matrix with ion pairing reagent [tetmbaumtyml oniumhydrogen sulfate (TBA)] in a pH-controlled environment The cationic reagent selectively targets anionfliucorochemicals. Once the auion-TBApair is formed, the analyteis transferred intoa non-polar organic solven(tmethyl-td- ,". butyl ether), dried, and reconstituted in methanol for MS analysis. HPLCESMSMS In HPLC, an aliquootf extractis injected and passedthrougha reverse phase liquid chromatographic column. Based on thseanity of the analyte for the stationary phase in the column rethlaetilviqeutiod mobile phase, the analyitse retained for acharacteristic amountof time. For example, ina standard solution PFOS may elute at 8.0 minutes. Retention times between a standard PFOS solution and the analyte extractedfrom tissue in thisanalysis were matched on the HFLC systtoemwithin 1%. Following HPLC separation, ESMSMS provides a rapid and accurate means for anwaildyezirnagngae of organic compounds, including fluorochemicals. Electrospraayn,ionization technique usedprimarily for detection of molecular ionis,generally operatedat relatively mild temperatures. Moleculeasre ionized, and aprimary ion, characteristic of the analiystese, lected Ions are accelerated for collision with argon gas resulting in hgmentation of the selected species; subsequent collisions csrmeaatleler secondary ionic fragments uniqueto the primary ion, which are detected. For example, for PFOS (CSF1,SO3-) analysis,49io9nis selected as the characteristic primary ionT. his ion is hgmented intoother ions suchas 80 amu (corresponding toSO;), and 230 amu (C&SO,-). Each of these secondaryhgments is detected and can be used to differentiate fProFmOSother compounds that might have the same characteris4t9ic9 amu primary ion, but different chemical compositioansd secondary ionhgmentation patterns. 5.1 Calibration For quantitative determinations, a mid-level, caliiration curve verification (sCtaCnVda)rdin methanol was analyzed every t5o 10samples to monitor instrumentdarlift Caliiration curve verificatiostnandards are acceptable ifinstrumental responseis within+/- 30% of the expected valueI.n thisstudy, allCCV standards were acceptable. Quantitation of the target analiystbeassed on linear regression analysis (weighlt/exd) of two curves, prepared in methanol, bracketing each group of samples. Quantitation of eachis banasaelydteon the response of one morore specific daughter ions using the multiple response-monmitoordinegof the instrument. Page3of6 3M ENVIRONMENTAL LABORATORY REPORTNO. FACT-TOX-130 3M lot #217 (purity 86.9%) of PFOS was used for caliirastitoandards. As purity information was not available at the time of data collection, reported results have not been corrected. 5.2 Blanks Two to four extraction blanks, utilizing waastseurrrogate malrix,were extracted with each batch of samples. Blanks were acceptabilefno target analyte was detected above the limit of detection for a specific analyte.In this study, all blankws ere acceptable. 5.3 Surrogates 1,1,2,2-Tetra-hydroperfluor~tane~onwataseused as a surrogate in thisanalysis. Surrogate response was monitoredto confirm gross instrumental failure,if necessary. Surrogate response was acceptable for all analyses. 5.4 Matrix Spikes Over the coursoef the analysis,twenty-eight matrix spikes were prepared in the mallard mlivaetrrix With two exceptions,all matrix spikesshowed acceptable recoveries of greattehran50%. Ofthe four matrix spikes extractedon 9/27/99, twowere recovered outside the acceptable raonfg+e/-50%. One sample had a PFOS recoveroyf 49% and a second sample showed PFOS recovoefr3y25%. These outlying recoveries were confirmed, bunto obvious errorwas readily identifiedAn error in spiking thmaetrix is suspected. Because the remaining 2m6atrix spikes were within the acceptacbrlieteria, no further action wastakento explain thesetwo anomalies. Finally, due to a miscalculation earlyin the data reduction process, eight additionalmatrix spikes were extracted to confirm what was thought to be low rUecpoovnedryis.covery of the calculation error, the origmiantarlix spikes were confirmed to be acceptable and the additional reextractions were not used. Because there was so little mallard sera available, rabbit sera aws assuursreodgatematrix in this study. Sixteenmatriv spikes were prepareidn rabbit sera.All matrix spikes showed acceptable recovery 7o0f130%. 5.5 Laboratory Control Samples Laboratory Control Samples are not a compoofntehnist study. 5.6 Sample Related Comments Samples were not analyzed for any residual or potential metabolite other thanPFOS. Other matrices submitted with thisstudy will be analyzedasneededupon written request fiomthe sponsor.Inthe eventof further analysis, an additionarleportwill be generated. With few exceptions, PFOS leveltsheinsera and liversof the mallards increased with dose group. Additionally, considering the average PFOS levels within each dose group, both sleivraerlevaenlds of PFOS declined dramatically between d8ayand day 22. Sera levels of PFOS measuraendiminals sacrificed on day 22 are between 20-35% otfhe sera levels of PFOS measureodn day 8. Levels of PFOS in the liovfers animalssacrificedon day 22 were, on average, 30% lowetrhanthose levels determined in the livoefrs animalssacrificedon day 8. In most anm i asl,for a specific time point, theorfaPtiFoOS concentrationmsera (pg/mL,) to PFOS concentration in live(rpglg) was greaterthan 1. .. Page40f6 3M ENVIRONMENTALLABORATORY REPORTNO. FACT-TOX-130 No circumstances existed during the present study that would have adversely affectesduatlhltey or integrity of the data. The following data quality objectives were followed during the study: Lineaxity - Thecoefficientofdetermination (r"2) ofthestandard curve was equal to or greater than 0.985 using l/x weighting. Limits of quantitation- PFOS in sera is 0.005-0.010 pg/mL; PFOS in liveris approximately0.030 pg / g 0 Acceptable precision of - duplicates c 20%, determined byMSMSD analysis Acceptable recoveries of spikes-70% to 130%for sera; 50% to 150% for liver 0 Use of confirmatory methods- Given the selectivity of the analytical tool used (HPLC-ESMSMS) and lack of a viable alternative for ananlyosicso,nfirmatory methods weruesed. 0 Demonstrationofspecificity - Specificitywasdemonstratedbychromatographicretentiontime (matched to standards to within3%) and the response of at least one characteristic production arising from collisions ofan analyte-specific pareniton. 0 The characterization of thereferencematerial (PFOS, lot #171), withrespect to purity,stability,and homogeneity, was not complete on thedate this report wasissued.Oncethecharacterization is complete, a copy of rtehpeort will on file in the 3M EnvironmenLtaalb archives. Given the parameters listed above, assuming spike recoveries form a suitable indication of endogenous analyterecovery,andassumingthatrabbit sera is asuitablesurrogate for mallard sera, sera data are quantitative to+/- 30%; liver datais quantitative to+/- 50%. The validity of these assumptionhsas not been verified by other techniques. If more accuracyis required, additional matrix shouldb e ' supplied so samples can be evaluated versus a standard curve extracted fiom the sameasmthaterixsamples. 0 Attachment A: Table of results of sera analysis 0 Attachment B: Table of results of liver analysis Attachment C: In-lifeprotocol Attachment D: Samplereceipt 0 Attachment E: Extraction and analytical data PageSof6 3M ENVIRONMENTAL LABORATORY REFORTNO. FACT-TOX-I30 Mark Ellefson, Technologist Date - QAU revikw by R o M Voyksna\ 3MENVIRONMENTAL LABORATORY REPORTNO. FACT-TOX-130 Kris Hansen, PhD., principalAnalytical Investigator b6/OB/bd Date QAU review by Robert Voyksner 3MENVIRONMENTAL LABORATORY REPORTNO. FACT-TOX-130 LaboratoryReport Analytical Report of Data for PFOS Dietary LC50 Study with Mallards (Sera and Liver) r Laboratory Report No. FACT-TOX-130 (W1902) Attachment A Sera Table of Results Day 8 Day 22 Testing Laboratory 3M EnvironmentalTechnology & Safety Services 3M Environmental Laboratory Fluorine AnalyticaCl hemistry Team (FACT) 2-3E-09 935 Bush Avenue, St. Paul, MN 55106 -.... FACT-TOX-130 study: PFOS Dietary LC50 Study with Mallards Product Numkr(Tat Substance): PFOS Mamx: Mallard S m MCthOdiRniriC4l: ETS-8-4. I and ETS-8-5. I Analytical EquipmentSystem Number Soup 020199. Amclia 062498 Insbumrnt S o f ~ ~ u J v m i o n : Marrlynx 3.2 Filename: S a Anachmmu R-Squared Value: See Anachmmu Slope: SeeA m h m m u Y-lntacepl: D a ~ aof Exbac~ion/Analyn: Data of AnalysisIAnalm SaA n a c h u 06/29/99 Mcn 07/01/99.07/06/99.07/08/99 DRBCUEE Date of Data RedudodAnayln: 07/02/99.0707/99.07/09/99 DRBMEE Sample Data IALLARD SERUM Day 8 Croup DOX Method BIk Matrix Blk Sampk I I H20 Blk-I H20 Blk-2 E O BIk-3 HZ0 BIk-4 Rabbit Serum Blk-I IPFOSl Avenge Reported (ug/mL) or KRec ./.Re [PFOSIng/mL or <Loo <LO6 <LOQ <LOQ I aOQ 0.00742 ED(%) Std. Dev.(ug/mL NA Rabbit hBIk-2 <LOQ Rabbit Serwn Blk-3 0.00730 16.4 - Rabbit hBlk-4 0.00967 0.00813 I Outlier 0.00133 - QC 50 ppb-MS RBs06299-Ms-I 107% - 1.0 ppn MSD RBs06299-MSC-I 74% f RBso6299-Ms-2 103% m+ RBS06299-MsD2 7% 101% 5% RBs06299-MS-3 %% ~~s06299-~sn3 126% Group1 RBs0629pMs-4 I RBS06299-MSD-4 5938 I 99% 119% 1.0 p IOOX I - 27% 0.0 m%lig 5939 5940 5941 5942 9195 91% 9197 9198 9199 92W 8401 &002 8403 8404 0.0230 < L o a - 5 Oluliat NA Group2 9190 8.81 9.10 mgkg food 9191 7.68 9192 8.79 9193 4.04 26.5 9194 7.60 7.38 1.96' 5958 9.31 18.3m& food 5959 25.6 5960 5961 S%2 11.45 8.89 13.7 49.8 13.8 6.86 hd 5x8 21.3 36.6 mgntg food 5%9 29.2 5970 17.7 5971 44.3 31.9 5972 34.2 - 30.5 9.75 -.g 5988 37.8 73.2 m& food 5989 41.6 5990 51.3 5991 57.8 17.0 5992 52.0 48. I 8.19 *Ues 5998 63.6 146 HI& food 5999 50.6 6ooo 35.7 9173 81.2 34.8 9174 38.6 53.9 18.8 Group7 9180 67.2 293 m& food 9181 58.9 9.57. . 9183 71.2 65.8 6.29 Croup8 5978 68.4 586 @g food 5979 94.5 e43.4 598 I I59 107 46.5 Group 9 1I71 mgkgfood Limit of Quantitadm(LOPFQO)S -NS 0.005 ue/mL NA Dale EntauVBy: DatcVaifia!lBy: 7/06199.7/07/99.07/1499 LAC 08/09/99 GML NA NA -PFOS P d u o m o c m a u l f ~ Emactioa volume ratioequals initialv d d n f ia lvolume. -NA uci applicable -NS DO sampleanalyzed ITS-8-5. I Excel Version 5.0195 3/8/01 FACT-TOX-130 PFOS Dictuy LC50 S M y with Mllllrdr PFOS Mallard S m ETS-84. I and ETS-8-51 soup 020199 Maslynx 3.2 see A I U d u n m ~ See ALllchmmQ See Auachn~nM See Amchmolu 07/07/99 RWW 07/12/99.07/15/99 D W G M L 07/13/99.07/16/99 DRB/KIH croup2 I 9.10 m& fmd Croup3 18.3 mgkg fmd *croup4 36.6 food 5930 593 I 5932 5933 5934 5935 5936 5937 9185 9186 9187 9188 9189 5953 59% 5955 5956 5957 5963 5964 5965 5966 6.82 5967 5983 hD6 1 I 4 6 In& food 19116 19117 5993 I I .os I .09 268 1.04 1.58 4.40 3.81 4.70 4.92 3.75 4.30 5.83 7.17 7.01 9.69 I 14.S 7.23 1.43 I I I NA <LOQ NA I I 47.3 1.49 0.704 12.0 , . 4.32 a520 29. I 1.99 I I 66. I 11.3 7.49 I I 293 In& rood 41.2 9176 9178 9179 5943 I 5947 *Croups 5943 I171 mglkgfmd Limit of Qvllltiptim (LOQ) PFOS = 0.010 16.5 21.5 I 29.7 I 51.6 25.5 13.4 5.51 I I 1 I 34.0 15.4 . NA NA -PFOS P a f l u o n u m w d f a u n 3M ENVIRONMENTAL LABORATORY REPORTNO. FACT-TOX-I30 LaboratorryReport Analytical Report of Data for PFOS Dietary LC50 Study with Mallards (Sera and Liver) Laboratory Report No. FACT-TOX-130 (W1902) Attachment B Liver Tableof Results Day 4-7 Day 8 Day 22 Testing Laboratory 3M Environmental Technology& Safety Services 3M Environmental Laboratory Fluorine Analytxal ChemistryTeam (FACT) 2-3E-09 935 Bush Avenue, St. Paul, MN 55106 Study: Product Number(Test Substance): Matrix: FACT-TOX-130 PFOS Dietary LC50 Study with Mallards PFOS Mallatd Liver MALLARD LIVER DAY 4-7 Std. Dev.(ug/g) ug/g or YORecM. SlMSDRPD 55% 23% Limit of Quantitation (L.OQ) PFOS a w x . O.O30ug/g NA = not applicable "The M W S D and group 1 sampleswere re-extncted on 10/0&99 to confirmmatrix effects. LAC 10/08/99 145 I 26.6 I FACT-M-2.1 Excel Vmion 5/95 Study: Product NumbeQest Substance): Mahix: FACT-TOX-130 PFOS Dietary LC50 Study with Mallards PFOS Mallard Liver MALLARD LIVER DAY 8 Group I Sample # I PFOS Coaceatratron RSD (%) - Method Blk H20 Bk-I of PM)S ap/g 0.00 Std. Dev.(ng/g) " S D RPD HZ0 Bk-2 0.00 H20 Blk-3 0.00 - HZ0 Bk-4 0.00 <LOQ NA MLL06299-5938-0-MS1 88.0 MLL06299-5938-0-MSE-1 89.4 1.O ppm MLLG6299-5940-0-MS-2 802 100 ppb MLL06299-5940-0-MSD-2 799 73% 5% MLL06299-91964-MS-3 84.4 MLLO6299-91960-MSD-3 73.3 - 1.0 ppm Group1 MLL06299-84014MS-4 915 MLLO6299-84014MSD-4 894 5938 0.00 1.0 ppm 74% 2% 0.0mgkg food 5939 0.00 5940 0.00 5941 0.00 5942 0.00 9195 0.00 9196 92.8 9197 0.00 9198 0.00 9199 132 92 00 0.00 8401 0.00 8402 0.00 - Group2 9.10 rngkg food 8403 0.00 8404 0.00 9190 6828 9191 3714 - <LOQ 2 O u t l i e r s NA NA 9192 4690 - Group3 mgkg food 2902 9193 2.90 32.1 9194 5217 5.22 4.67 1.50 5958 4760 4.76 11542 5959 18.3 11.5 6158 5960 6.16 - Group4 36.6 mgkg food 5969 14901 4202 5961 4.20 43.7 6562 5962 6.56 6.65 2.90 14690 5968 14.7 14.9 5970 9864 9.86 5971 I 5972 . - 1' , Group5 15347 1 5988 ~. 16890 598793.2 mgkg food I . 5992 - Group6 20377 20.4 16505 16.5 15.3 16.9 29129 29.1 51544 51.5 34849 38069 5998 38.1 i 15.3 29.7 34.8 24.8 3.78 49.8 14.78 mgkg food 5999 146 35398 35.4 6ooo 32775 32.8 - 9173 55034 , ' 55.0 28.1 32084 32.1 38.8 10.9 59768 56336 293 mgkg food 50650 . `9183 8.29 248075 84. 91 . 55.6 4.61 32469 586 mgkg food 75542 5979 39.6 5981 59.5 10474 70.5 23.5 Croup94 171 mgkg food NS NA NA 1 1'FOS PClikOrooC~CSdfOMtC Limit of Quantitation(LOQ)PFOS approx. O.O3Oug/g NA = not applicable NS = no sample analyzed One outlier,not includedin any calcolations FACT-M-2.1 Excel Version 5/95 3/1vo1 1044 AM study: Product NumbafTat Substance): Mabix: FACT-TOX-130 PFOS Dietary LC50Study with Mallards PFOS Mallard Liver IMALLARD LIVER DAY 22 Group Dose Sample i# PFOS Conc Concentration of PFOS Calc. Mean PFOS RSD (Ye) Itd. Dev.(ug/g WMSD RPC NA QC-l00ppb H20 Bk-3 1.07 HZ0 Bk-4 1.51 6495-0-MS-1 107 64950MSD-1 101 <LOQ CLOQ 90% 85% CLOQ NA 64964-MS-2 111 94% 64964-MSD-2 126 107% 94% ??A 1.0 ppm 64970MS-3 1033 8m 6497OMSD-3 1111 93% 64980MS-4 1093 91% 64980MsD-4 1053 88% 90.7 3% Croup1 0.0mp/Lp food 22.2 6495 <LOQ 6496 19.3 CLOQ <m 9497 6499 12.8 13.2 6498 8.53 CLOQ CLOQ 6500 13.4 <LOQ 5.63 5929 <LOQ 4.51 5930 <LOQ 593 1 I .72 <LOQ 5932 3.10 CLOQ 5933 8.99 <LOQ 5934 8.49 <LOQ 5935 9.36 <LOQ 5936 4.83 <LOO NA 5937 5.51 <LOQ CLOQ NA Groopt mgkg food 91 85 911 9186 9.10 876 0.91 1 0.876 91 87 1569 1.57 735 9188 0.735 31.4 1.04 1038 9189 1.03 0.322 Grow 18.3 mgkg food 1802 5953 ' 1.80 5954 2186 2.19 1930 5955 I .93 5956 2667 2.67 20.3 5957 1594 1.59 2.04 0.4 13 crow 36.6 mgkg food 1626 5963 1.63 5964 3035 3.03 5965 3154 3.15 I I I I I 5966 3639 3.64 40.0 .. 5961 5366 5.37 3.36 1.35 .- 1.. 10.3 10254 5983 I I I I I 14.0 G1r3o97w6 . 146mgntg food 13.9 5987 8195 8.20 9.21 1.28 5993 7737 5994 7.74 5995 8374 8.37 5996 14015' 14.0 38.1 5997 19574 19.6 12.7 4.85 Croup7 293 mgkg food 9175 9176 5633 7.49 7494 5.63 9177 7.69 7687 9178 12522 12.5 34.8 91 79 12663 12.7 9.20 3.20 Group8 5943 18754 18.8 *' 586 mgncg food 9.76 9163 5944 I I I I I 5945 24677 24.7 38.8 I I roup9-1171 mgk1g6.f3ood 162I61 5947 5977 27.4 27443 20.2 7.82 NA NA - .. PFOS Per!lwnmctandouatc Limit of Quantitation(LOQ) PFOS: appmx. 0.03Oug/g N A =not applicable FACT-M-2.1 Excel Version 5/95 3M1 1044 AM