Document jmze3oRJ3zY4jkv1mYG8q37V9

Laboratory R eport-R evision 1 Analytical Report o f Data for PFOS Dietary LC50 Study in Northern Bobwhite (Sera and Liver) Laboratory Report No. FACT-TOX-129 (U2723) Testing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory Fluorine Analytical Chemistry Team (FACT) 2-3E-09 935 Bush Avenue, St. Paul, MN 55106 Laboratory Contact Kris Hansen, Ph.D. Bldg. 2-3E-09 P.O. Box 3331 S t Paul, MN 55133-3331 Phone: (651)778-6018 FAX: (651) 778-6176 Revision Date: March 19, 2001 Page 1o f / 7 03litio I C00663 1 Introduction This study was designed to quantitatively assess perfluorooctane sulfonate (PFOS, Q F 17SO 3', CAS # 2795- 39-3) levels in the sera and liver o f die northern bobwhite (Colinus virginianus) exposed to PFOS through diet. D etails o f dosing conditions, clinical observations and other toxicity data were generated by the in-life testing facility (W ildlife International) and are not included in this report. Subsets o f the eight groups o f test anim als w ere sacrificed on day 8 and day 22, post-dose. Sera and liver collected from the sacrificed anim als w ere sent to the 3M Environmental Laboratory and analyzed quantitatively for PFOS. Tissues from animals that died before the scheduled sacrifice date w ere collected and analyzed also; results are included in this report Elucidation o f data trends was com plicated by the sporadic nature o f the PFOS levels in sera. W ithin a particular dose group there was dram atic variation between individual animals. The relative standard deviation o f PFOS sera levels w ithin a dose group ranged from greater than 20% to over 200% . By com parison, the results o f PFOS levels in the livers o f the bobwhites w ere far more consistent w ithin a particular dose level (group RSD betw een 10-55%) In general, both sera and liver levels o f PFOS increased w ith dose level, although due to anim al to animal variability, this trend is not pronounced in the sera. W ith the exception o f group 4 anim als, average levels o f PFOS in the sera o f the bobwhites generally increased or stayed the sam e betw een day 8 and day 22. In all dose groups, the group average for PFOS concentration in liver decreased dram atically betw een day 8 and day 22. Livers w ere collected from bobw hites that died between day 1 and day 8, prior to the scheduled sacrifice. In every case, PFOS levels in the livers o f these birds were over 110 pg/g w et w eight A lthough rigorous quality control m easures and 3M Environmental Laboratory Standard O perating Procedures w ere followed, the acquisition o f this data was not necessarily collected according to applicable G ood Laboratory Practices. D ata presented here is the highest quality data available at this tim e. No data w as collected for any analytes other than PFOS. It is recom m ended that bobw hite quail be avoided in future studies because the sm all am ount o f tissue available causes analytical challenges that m ake verification difficult. 2 Sample Receipt T he Environm ental Laboratory received 65 liver, 34 blood, and 34 serum specim ens, frozen, on 05/19/99. U pon specim en receipt a 3M chain o f custody w as initiated. The Environm ental Laboratory received 35 liver, 35 blood, 35 serum, and 6 bile specim ens, frozen, on 06/29/99. U pon specim en receipt a 3M chain o f custody was initiated. The Environm ental Laboratory received 30 pooled bobwhite quail serum tubes, frozen, on 06/30/99. U pon specim en receipt a 3M chain o f custody w as initiated. A copy o f each form can be found in an A ttachm ent to this report. 3 Holding Times There is no holding tim e criteria associated w ith these samples for LC/ESMSMS analysis. Page 2 of^ 7 lAt, 0 000664 4 M ethods - Analytical and Preparatory A nalytical m ethods E TS-8-4.1, Extraction o f Potassium Perfluorooctanesulfonate or O ther Fluorochem ical Compounds from Serum for Analysis Using HPLC-Electrospray/M ass Spectrometry with thefollowing exceptions: D ue to die sm all volume o f bobwhite sera available, standard curves were prepared in m ethanol instead o f from bobw hite sera. Four matrix spikes w ere prepared from the pooled bobwhite quail serum received on 06/30/99. PFOS lot 171 was used as reference material. FA CT-M -1.1, Extraction o f Potassium Perfluorooctanesulfonate or O ther Fluorochem ical Compounds from Liver for Analysis Using HPLC-Electrospray/M ass Spectrometry with thefollowing exception: The extraction w as performed following ETS-8-6.0; this method had been validated but not approved at the tim e o f extraction. PFOS lot 171 was used as reference material. ETS-8-6.0, Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochem ical Compounds from Liver for A nalysis Using HPLC-Electrospray/M ass Spectrometry with thefollowing exception: D ue to die sm all amount o f bobwhite liver available, standard curves were prepared in m ethanol instead o f from bobw hite liver. Li some cases, liver sam ples were very lim ited and tw o or m ore sam ples had to be com bined for extraction. These com binations are indicated in die results. Twenty-two m atrix spikes w ere prepared from die bobwhite liver. PFOS lot 171 was used as reference material. ETS-8-5.1, A nalysis o f Potassium Perfluorooctanesulfonate or O ther Fluorochem icals in Serum Extracts U sing HPLC-Electrospray/M ass Spectrom etry. FACT-M -2.1, Analysis o f Potassium Perfluorooctanesulfonate or O ther Fluorochem icals on Liver Extracts U sing HPLC-Electrospray/M ass Spectrom etry with thefollowing exception: T he analysis was perform ed following ETS-8-7.0, which had been validated but not approved a t the tim e o f extraction. As described in the method, all results are reported in terms o f pg o f analyte per gram o f liver (w et weight). ETS-8-7.0, A nalysis o f Potassium Perfluorooctanesulfonate or Other Fluorochem icals on Liver Extracts U sing HPLC-Electrospray/M ass Spectrometry. Sam ple p rep a ra tio n - H PLC/ESM SM S: ion-pairing extraction A nalyte is extracted from a sample m atrix w ith ion pairing reagent [tetrabutyl am monium hydrogen sulfate (TBA)] in a pH-controlled environm ent The cationic reagent selectively targets anionic fluorochem icals. O nce the anion-TBA pair is formed, the analyte is transferred into a non-polar organic solvent (methyl-tertbutyl ether), dried, and reconstituted in m ethanol for MS analysis. H PLC /ESM SM S In HPLC, an aliquot o f extract is injected and passed through a reverse phase liquid chrom atographic colum n. Based on die affinity o f the analyte for die stationary phase in the colum n relative to the liquid m obile phase, die analyte is retained for a characteristic amount o f tim e. For example, in a standard solution PFOS m ay elute at 8.0 m inutes. Retention times between a standard PFOS solution and die analyte extracted from tissue in this analysis were matched on the HPLC system to w ithin 1%. Following HPLC separation, ESMSMS provides a rapid and accurate m eans for analyzing a wide range o f organic compounds, including fluorochem icals. Electrospray, an ionization technique used prim arily for detection o f m olecular ions, is generally operated at relatively m ild temperatures. M olecules are ionized, and a prim ary ion, characteristic o f the analyte, is selected. This ion is bombarded with high-energy gas; subsequent collisions create smaller secondary ionic fragments unique to die prim ary ion, which are detected. For example, for PFOS (Q F 17SO3') analysis, ion 499 is selected as die characteristic prim ary ion. This ion is fragmented into other ions such as 80 am u (corresponding to S 0 3), and 230 amu (CjFaSOj-). Each o f these secondary fragments is detected and can be used to differentiate PFOS from other com pounds that m ight have the same characteristic 499 amu prim ary ion, but different chem ical com positions and secondary ion fragmentation patterns. Page 3 ofif ? Uc o link. C00S65 5 Analysis j'i,-.. SfV*! JJr- 5.1 Calibration A m id-level calibration check standard is analyzed every 5-10 samples to m onitor instrumental d rift Calibration check standards were com pliant if instrumental response was w ithin +/- 30% o f the expected value. Q uantitation o f the target analytes was based on linear regression analysis (weighted 1/x) o f two unextracted methanol curves bracketing each group o f sanqrles. Quantitation o f each analyte was based on the response o f one or more specific daughter ions using die multiple response-monitoring mode o f die instrum ent 3M lot #217 (purity 86.9%) o f PFOS was used for calibration standards. As purity inform ation was not available at the tim e o f data collection, reported results have not been corrected. 5.2 Extraction and Method Blanks A t least tw o extraction blanks, utilizing w ater as surrogate matrix, were extracted w ith each batch o f sam ples. Additionally, tw o m ethod blanks, utilizing control sample matrix (if available), were extracted along w ith each batch o f samples and curves. Blanks w ere com pliant if no target analyte was detected above the lim it o f detection for a specific analyte. In this study, all blanks were com pliant 5.3 Surrogates Tetra-hydroperfluorooctanesulfonate was used as a surrogate in this analysis. Surrogate response was m onitored to confirm gross instrum ental failure, if necessary. Surrogate response was acceptable for all analyses. 5.4 Matrix Spikes O ver the course o f the analysis, tw enty-six m atrix spikes were prepared in the bobwhite liver matrix. A ll o f the m atrix spikes were recovered at greater than 50% o f the expected value w ith two exceptions. The two 50 ng/g m atrix spikes that were prepared on September 24,1999 with the day 3-7 liver samples showed analyte recovery o f approximately 350% o f the expected value. These samples and matrix spikes w ere re extracted on October 08,1999. The re-extracted m atrix spike recoveries were greater than 50% (104% - 110%), therefore day 3-7 data are reported from extraction date October 08,1999 - refer to extraction sheet for anim al numbers. Because the bobw hite sera were so lim ited, only four m atrix spike samples were prepared. A ll m atrix spikes showed analyte recovery o f greater than 90% o f the expected value. 5.5 Laboratory Control Samples Laboratory Control Samples were not a com ponent o f this study. 5.6 Sample Related Comments Samples were not analyzed for any residual or potential metabolite other than PFOS. Other matrices subm itted w ith this study w ill be analyzed as needed upon written request from the requestor. In the event o f further analysis, an additional report w ill be generated. Page 4 of^ f ^ o W a j/o i G00666 6 Data Summary : i Elucidation o f data trends was complicated by the sporadic nature o f die PFOS levels in sera. W ithin a particular dose group there was dramatic variation between individual animals. The relative standard deviation o f PFOS sera levels within a dose group ranged from greater than 20% to over 200% . By com parison, the results o f PFOS levels in the livers o f the bobwhites were far more consistent w ithin a particular dose level (group RSD between 10-55%) In general, both sera and liver levels increased with dose level, although due to animal to animal variability, this trend is not pronounced in die seta. W ith the exception o f group 4 anim als, average levels o f PFOS in die seta o f the bobwhites generally increased or stayed the same between day 8 and day 22. In all dose groups, the group average for PFOS levels in liver decreased dramatically between day 8 and day 22. Livers were collected from bobwhites that died between day 1 and day 7, prior to the scheduled sacrifice. In every case, PFOS levels in the livers o f these birds were over 110 pg/g. Data Q udity Obja N o circum stances existed during the present study that would have adversely affected die quality or integrity o f the data. The following data quality objectives were followed during die study: Linearity - The coefficient o f determination (^ 2 ) o f the standard curve was equal to or greater than 0.985 using 1/x weighting Lim its o f quantitation - PFOS in sera is 0.010 pg/raL; PFOS in liver is approxim ately 0.070 pg/g Acceptable duplicate precision - < 30%, assessed by analysis o f die MS/MSDs. A cceptable spike recoveries - sera: 70% to 130%; liv er 50% to 150% U se o f confirm atory methods - Given the selectivity o f the analytical tool used (HPLC-ESM SM S) and lack o f a viable alternative for analysis, no confirmatory methods were used. D em onstration o f specificity - Specificity was dem onstrated by chromatographic retention tim e (matched to standards to within 3%) and the response o f at least one characteristic product ion arising from collisions o f an analyte-specific parent ion % The characterization o f die reference material (PFOS, lot #171), with respect to purity, stability, and hom ogeneity, w as not complete on the date this report w as issued. Once die characterization is com plete, a copy o f the report w ill on file in die 3M Environmental Lab archives. G iven the param eters listed above and assuming spike recoveries form a suitable indication o f endogenous analyte recovery, sera data are quantitative to +/-30 % and liver data are quantitative to +/-50 %. The validity o f these assumptions has not been verified by other techniques. If a greater degree o f accuracy is required, additional matrix should be provided so samples can be evaluated versus a standard curve extracted from the sam e m atrix as the samples. It is recommended that bobwhite quail be avoided in future studies because the small am ount o f tissue available causes analytical challenges. Page 5 of U C 07/23io( C00667  :~??r 8 A ttachm ents- A ttachm ent A : T able o f results o f liver analysis A ttachm ent B: T able o f results o f sera analysis A ttachm ent C: Sam ple R eceipt A ttachm ent D: In-life protocol from W ildlife I n t, LTD A ttachm ent E: E xtraction and analytical data <*s?7< C00S68 9 Signatures . A__C W __ Lisa Clem en, A dvanced Chemist U ^ - -- ~~~ K ris P h D ., Princqipwal Analytical Investigator Q uality A ssurance U nit Representative *'*>'*; rfc. tn ln L D ate O Z jlZ IO t D ate D ate Page'/oijf 01 C00669 9 Signatures A - CArKK. L isa Clemen, A dvanced Chemist J - tL * -------K ris H ansen, Ph.D ., Principal Analytical Investigator D u lfih liya D ate 0 C / o t /o -o D ate Q uality A ssurance U nit Representative G " *if" O o D ate -V - ^ ,at r0\ G00G70 Laboratory Report Analytical Report of Data for PFOS Dietary LC50 Study in Northern Bobwhite (Sera and Liver) Laboratory Report No. FACT-TOX-129 (U2723) Attachment A Table o f Results - Liver Analysis Day 3-7 Day 8 Day 22 000671 FACT-TOX-129 Study: Product NumberiTesl Substance): Matrix: Method/Revision: Analytical Equipment System Number Instrument Software/Veision: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Dale o f Data Reduction/Analyst Simple D ili 454-103 Bobwhite Quail LC50 PFOS Bobwhite Quail Liver ETS-8-6.0 A ETS-8-7.0 Amelia 062498 Massiynx 3.3 10/01*9 SEE/MCH 10/21/99,01/18/00 IAS/MMH 10/22/99,01/19/00 IAS/MMH Filename: R-Squared Value: Slope: Y-Intercept: See Attachments See Attachments See Attachments See Attachments BOBWHITE LIVER DAY 3-7 Croup Dose Sam ple! Method BUc QC - 50 ppb BWL100S9-H2O Btk-U BWL10089-H20 Btk-12 R342--0-Day8-50ppb-MS-11 R342-0-Dey8-50ppb-MSD-l 1 Q C - lOOOppb B439-0-DayS-lppro-MS-12 B439-0-Dy8-lppm-MSD-l2 G roupl 0.0 mg/kg lood R342-0-Day8 B439-0-Day8 G roups 146 mgAg food G355-Day7 Group6 R352, B450-Day5 293 mg/kg food Y417, R353-Day6 G357, G358-Day7 B449, Y416-Day7 Group7 AL354-Day3 586 mg/kg food R354-Day4 R355, Y401, B452-Day5 Y4I8, B451, G360-Day6 G359, AL355-Day7 G roup! 117! mg/lcg food AL356, R357, Y419-Dey3 AL357, B453, G361-Day4 B454, G362-Dey4 R356, Y420-Day4 PFOS ~ Perfluorooctanesulfonate Limit of Quantitation (LOQ): PFOS is approximately 0.070 ug/g NA - not applicable 01 Day 8 data is included to support QC NE - not extracted Date Entered/By: 01/18100,01/26/00 LAC Date Verified/ By: 04/12/00 KJH PFOS Calc. Couc. tft 0.00 0.00 6S.7 64.6 980 1034 229 383 110527 248693 201523 153461 123521 NE . NE 125957 235179 111299 133852 131788 122934 124333 Concentration of pros -ug/1 o r % Ree. <LOQ <l o q 110% 104% 82% 86% 0.229 0.383 111 249 202 153 124 NE NE 126 235 in 134 132 123 124 Mean pros ng/g o r %Rec <LOQ 107% 84% 0.306 111 182 157 128 R SD (% ) Std. Dev.(ag/g) MS/MSD RPD NA 6% 5% 3.7 0.109 NA 30.2 55.0 43.0 67.7 4.21 5.40 FACT-M-2.1 Excel Version 5/95 TOX129 liver-3.xls 3/8/01 9:40 AM % j*j / i i FACT-TOX-129 Study: Product Numb<Test Substance): Matrix: Mcthod/Revision: Analytical Equipment System Number Instrument Software/Venion: Date of Extraction/Analyst: Date of Analysis/Analyst Date of Data Reductkm/Analyst: Sample Data 454-103 Bobwhite Quail LC50 PFOS Bobwhite Quail Liver FACT-M-1,1 and FACT-M-2.1 Amelia 062498 Masslynx 3.2 06/30/99 SAH 07/02/99,07/22/99, 09/01/99 MEE/PTF 07/20/99,07/23/99, 09/02*9 DRB/MEE/PTF Filename: R-Squared Value: Slope: Y-Inlencepl: See Attachments Sec Attachments See Attachments See Attachments BOBWHITE LIVER DAY 8 Group Dose Sim ple* Method Blk BWL-H20 BIk-6/30-1 BWL-H20 BIk-6/30-2 BWL-H20 BIk-6/30-3 BWL-H20 BDc-6/30-4 QC- 1000ppb G347-MS-I G347-MSD-1 Y405-MS-2 Q C-50ppb Y405-MSD-2 G346-0-MSI1-I G348-0-MS11-2 G347-0-MS11-3 Y40-0-MS11-4 QC-lOOOppb G rovpl 0.0 mg/kg food G346-0-MS1 l-l G348-0-MS11-2 G347-0-MS11-3 Y405-0-MS11-4 G347 B438/AL34I Y405 AL343 B440 Y406 Y407 R34I R342 B439 G346/R346 Day6 G34S/AL342 G roup! 18.3 mg/kg food B442/AL345 R340 G350/Y4O9 Groups 36.6 mg/kg food Group# 73.2 mg/kg food Y4I1 B444/R347 AU47/G352 R348/B446 G354/AL349 Y413 Groop5 146 mg/kg food G roup 6 293 mg/kg food AL351/Y4I5 G356/B448 AL353-Day8 PFOS - Petfluoraoctanesulfonate Limit olQuantitation (LOQ): PFOS is approximately 0.070 ug/g NA - not applicable ME - not extracted Date Enteted/By: Date Verified/ By: 07/21/99.07/23/99,09/03/99 LAC 08/09/99 OML. 04/12*0 KJH PFOS Calc. Cone. "S'* 0.00 0.00 0.00 0.00 977 972 920 872 47.2 72.1 42.8 51.7 841 955 680 804 0.00 34.7 0.00 86.2 39.1 0.00 60.6 9.72 16.5 0.00 147 0.00 22780 15691 17080 28909 20463 28049 43414 48741 39880 65404 75293 NE Concentradou of PFOS u t f e o r K Ree. <LOQ <LOQ LOQ <LOQ 82% 81% 76% 72% 79% 120% 71% 86% 70% 79% 57% 67% <LOQ <LOQ <LOQ 0.0862 <LOQ <LOQ <LOQ <LOQ <LOQ <l o q 0.147 <LOQ 22.8 15.7 17.1 28.9 20.3 28.0 43.4 48.7 39.9 65.4 75.3 NE Mean PFOS ug/t or %Rec <LOO 81% 74% 89% 68% <LOQ 18.5 25.8 44.0 70.3 NA RSD (% ) Std. Dev.(ug/g) MS/MSD RPD NA 0% 5% 21% 9% NA NA 20.3 3.76 18.0 4.65 10.1 4.46 9.94 6.99 NA FACT-M-2.I Excel Version 5/95 TOXI29 liver-3.xls 3/8/01 9:01 A M 0-00673 FACT-TOX-129 C00G74 OOOG7 C00676 C00677 C00678