Analytical improvements shown over four interlaboratory studies of perfluoroalkyl substances in environmental and food samples

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1 Trends Trends in Anlyticl Chemistry, Vol. 43, 2013 Anlyticl improvements shown over four interlbortory studies of perfluorolkyl substnces in environmentl nd food smples Jn M. Weiss, Ike vn der Veen, Stefn P.J. vn Leeuwen, Wim Cofino, Steven Crum, Jcob de Boer An incresing number of reports confirm the world-wide presence of the perfluorolkyl substnces (PFASs). As consequence, the demnd for qulittive nd quntittive environmentl occurrence dt requires ccurte risk ssessments. To improve the nlyticl qulity of the determintion of PFASs in food nd environmentl smples, 4 th interntionl interlbortory study (ILS) ws conducted in A totl of 31 prtners prticipted, nd, depending on the smple mtrix, up to 29 dt sets were submitted. The ILS focused on food smples, s it ws orgnized by the PERFOOD consortium in collbortion with QUASI- MEME. The results showed tht the cumultive experience of the prticipnts hs improved their nlyticl qulity over four interntionl ILSs. Severl sources of errors were identified nd methods to void them re suggested. ª 2012 Elsevier Ltd. All rights reserved. Keywords: Anlyticl qulity; Environmentl smple; Food smple; Interlbortory study (ILS); Perfluorolkyl substnce (PFAS); PERFOOD consortium; QUASIMEME; Wter smple Jn M. Weiss*, Ike vn der Veen, Jcob de Boer Institute for Environmentl Studies, VU University, De Boeleln 1085, 1081HV Amsterdm, The Netherlnds Stefn P.J. vn Leeuwen RIKILT Institute of Food Sfety, Wgeningen UR, The Netherlnds Wim Cofino, Steven Crum Wgeningen University nd Reserch Centre, Alterr, QUASIMEME Lbortory Performnce Studies, The Netherlnds * Corresponding uthor. Fx: ; E-mil: jn.weiss@vu.nl 1. Introduction The nlysis of perfluorolkyl substnces (PFASs) in environmentl smples hs proved chllenging, nd the qulity of dt obtined hs been mjor issue of concern [1]. Issues include the lck of high-qulity stndrds (e.g., mss lbeled, liner or brnched), bckground contmintion nd chromtogrphic interferences, nd poor recovery. At the sme time, the nlyticl demnds regrding limits of detection (LODs) nd ccurte quntifiction hve incresed since the PFASs hve been shown to be omnipresent in our surrounding environment [2], nd the toxicologicl informtion points towrds more complex picture of compound group tht exhibits developmentl neurotoxicity [3], endocrine disruption [4,5] nd reproduction disturbnces [6]. These obstcles hve been extensively investigted in order to stisfy the incresing demnds for quntittive estimtes of environmentl occurrence for risk ssessments, mong others, vi series of interntionl interlbortory studies (ILSs) nd workshops ddressing the nlyticl spects [7 10]. Severl lbortories hve developed methods for nlysis of PFASs in food nd environmentl mtrices to study the distribution of these chemicls in the environment nd to ssess the humn nd environmentl exposure. Food intke is the mjor source of PFAS exposure to humns due to the mount consumed [11,12]. The Europen Food Sfety Authority (EFSA) set tolerble dily intke (TDI) of 150 ng PFOS/kg nd 1500 ng PFOA /kg in 2008 [13]. In 2012, EFSA performed dietry intke estimtion bsed on 54,195 nlyticl results obtined for 7560 food smples reported by Europen member sttes [14]. EFSA concluded tht the low proportion of quntified results (<LOQ) prevented clcultion of more relistic dietry exposure. As the concentrtions in individul food items re often reported s below the limit /$ - see front mtter ª 2012 Elsevier Ltd. All rights reserved. doi:

2 Trends in Anlyticl Chemistry, Vol. 43, 2013 Trends of quntifiction (LOQ), this cn cuse underestimtion of the dietry exposure scenrio. It ws only in the lte 1990s, fter liquid chromtogrphy coupled to mss spectrometry (LC-MS) becme commonly vilble, tht it ws possible to determine PFAS levels in the lowng/ml rnge, llowing for the first time the ccurte evlution of bckground levels of PFASs in biologicl nd environmentl mtrices. The mjor PFASs re regulrly monitored in food, nd low LOQs re needed to determine their concentrtions [15]. Typicl PFAS levels in fish re t the ng/g wet weight (ww) level, the mjor determinnd being perfluorooctyl sulfonte (PFOS) [16], wheres other food items re in the pg/g ww rnge [17]. Mny lbortories hve LOQs just round this level, so tht improvements re often needed to be ble to report meningful observtions. Such low levels re considered to be sfe in comprison to existing toxicologicl evlutions, but still little is known bout the combintion effects between the cocktil of compounds present (e.g., in food nd wht is emigrting from food pckging). Hence, it is importnt to nlyze the full spectr of contminnts. It is therefore importnt to improve the LODs nd nlyticl qulity of reported dt. Inviting lbortories to perform interlbortory comprisons is one importnt tool to identify problems nd to stimulte improvements within the nlyticl field. A1 st ILS, conducted in 2004/2005 showed unstisfctory results for the determintion of PFAS concentrtions in humn nd environmentl mtrices [7]. The humn mtrices gined less unstisfctory z-scores thn the environmentl mtrices. The environmentl smples (i.e. wter, fish-liver extrct nd fish tissue) were nlyzed by 27 lbortories nd stisfctory z-scores ( z < 2) were obtined by, respectively, 31%, 55% nd 17% of the prticipnts for PFOS nd 22%, 40% nd 25% for PFOA. The ssigned vlues for PFOS were 37 ng/g ww in fish tissue nd 20 ng/l in wter. Assigned vlues for perfluorooctyl sulfonic cid (PFOA) were 10 ng/g ww in fish tissue nd 19 ng/l in wter. The reltive stndrd devitions (RSD) between lbortories were %. The result indicted the need for the lbortories to ssess their nlyticl procedures criticlly in order to reduce possible sources of error. Menwhile, lrge number of high-qulity stndrds hd become commercilly vilble, s hd wide rnge of mss-lbeled stndrds. A follow-up study on wter nd fish demonstrted in 2008 tht significnt improvements were obtined if the prticipnts employed high-qulity ntive stndrds nd multiple mss-lbeled internl stndrds (ISs) provided by the coordintor [9]. In ddition, since limited precision cn be cused by low concentrtions, the fish nd wter smples spiked with set of PFASs were distributed to focus the interlbortory evlution on other nlyticl spects (e.g., mtrix effects, quntifiction principles nd the determintion of the ccurcy of the lbortories). The stisfctory results, expressed s between-lbortory RSD to demonstrte precision, showed tht the usge of mss-lbeled stndrds is cpble of correcting for different in-house nlyticl methods. Z-scores were not clculted in tht study. Environmentl concentrtions re often close to the LOQs of lbortories. The next chllenge would therefore be to mintin the sme level of performnce t relistic (low) concentrtions. The 3 rd ILS on PFASs ws thus orgnized in 2009 to ssess if this could be chieved [8]. Agin, the nlyticl performnce of PFASs in humn smples ppered to be better thn tht in the environmentl smples studied (fish, wter nd sludge). Despite recommendtions, mny lbortories used only limited number of mss-lbeled stndrds. For PFOS, specificlly, significnt mounts of brnched isomers present in the wter, fish nd blood smples ppered to be significnt source of vrition, due to clibrtion procedures being bsed on only the liner isomer. Also, some results reported might hve been bsed on the slt rther thn on the nion. For the first time, sewge sludge ws included in the study. The vrince for the results in this mtrix ws substntil, showing tht more effort ws needed to improve methods for sludge [8]. Compring the results between the 2008 ILS nd 2009 ILS is difficult since the 2008 study ws conducted in very controlled sitution in which the results were chieved. In ddition to mss-lbeled ISs being distributed, the dt were lso criticlly ssessed in meeting nd outliers removed if technicl resons were found. Hence, the 2008 study represents sitution of best possible prctice. In generl, the between-lbortory RSDs reported in 2009 were higher thn those in The 4 th ILS focused on humn smples nd reported in 2009 [10]. Two serum smples nd one stndrd solution were nlyzed by 17 prticipnts. The conclusion from the 4 th ILS sttes tht experienced lbortories world-wide re tody cpble of determining the most prevlent PFASs in humn blood with ccurcy nd precision suited to serve the monitoring nd exposure ssessment of these compounds. As hd lredy been indicted in the erlier ILS studies, it seems tht humn smples re less chllenging to nlyze, thn environmentl nd food smples, which re not yet fully under control, possibly becuse mny lbortories hve longer experience in nlyzing blood thn food. The present ILS on PFASs, which is the 5 th ILS on PFAS nlysis but the 4 th in food nd environmentl smples, ws orgnized within the frmework of the Europen Union (EU)Õs PERFOOD project nd in collbortion with QUASIMEME (EUÕs Qulity Assurnce of Informtion for Mrine Environmentl Monitoring in Europe project). The objectives were (1) to ssess the intercomprbility of PFAS dt produced by nlyticl lbortories, focusing on those nlyzing food nd drinking wter, since these 205

3 Trends Trends in Anlyticl Chemistry, Vol. 43, 2013 mtrices re especilly chllenging due to low levels nd demnds from ssessors for regultory purposes; nd, (2) to follow-up on the erlier ILSs to mintin the efforts to improve the nlyticl qulity of the PFASs. The ILS mtrices were crefully selected to cover the min contributors to the dietry exposure pthwy [12] [i.e. wter, which is the min contributor to the liquid intke, fish (two smples) nd met (pig liver, not due to high consumption but representing the mtrix) s being the mjor dietry contributor], except for perfluorooctnoic nd perfluorohexnoic cid. The two crboxyliccid exposure pthwys re primrily from drinking wter nd other high-consumption food ctegories (e.g., cerels, diry products, vegetbles nd fruits), so vegetble mixture ws included in the study to evlute the nlyticl performnce of this mtrix. In totl, 31 lbortories registered their interest in prticipting in the present ILS, of which 29 submitted dt. Since prticipting lbortories were ble to choose between pckges of smples, the number of prticipting lbortories vried between mtrices. The ILS study focused on the following PFASs: perfluorobutnoic cid (PFBA), perfluoropentnoic cid (PFPeA), perfluorohexnoic cid (PFHxA), perfluoroheptnoic cid (PFHpA), perfluorooctnoic cid (PFOA), perfluorononnoic cid (PFNA), perfluorodecnoic cid (PFDA), perfluoroundecnoic cid (PFUnDA), perfluorododecnoic cid (PFDoDA), perfluorotridecnoic cid (PFTrDA), perfluorotetrdecnoic cid (PFTeDA), liner perfluorobutne sulfonte (L-PFBS), liner perfluorohexne sulfonte (L-PFHxS), liner perfluoroheptne sulfonte (L-PFHpS), liner perfluorooctne sulfonte (L-PFOS), brnched PFOS (br-pfos), totl PFOS (tot-pfos), liner perfluorodecne sulfonte (L-PFDS), perfluorooctnesulfonmide (FOSA), nd 6:2 fluorotelomer sulfonte (6:2 FTSA). 2. Methods nd mterils 2.1. Mteril preprtion The smples were selected s representtive of PFAS sources vi food. One fish smple identicl to one used in the most recent ILS [8] ws included to enble comprison of performnce between the studies. The invittion ws sent out in June 2011 nd the smples were distributed in August Pig-liver test mteril. A pig-liver smple ws collected from locl Dutch mrket. The bulk mteril ws ground nd thoroughly homogenized fter ddition of PFASs to rech detectble concentrtion ( ng/g ww, 15 PFASs), nd 0.02% butylhydroxytoluene (BHT) s n ntioxidnt. Jrs were filled with 45 g liver homogente. The jrs were sterilized by gmm irrdition, which llowed storge t room temperture nd convenient trnsporttion. The stbility of the PFASs during gmm rdition ws estblished before the smples were sterilized Drinking wter test mteril. A bulk drinking wter smple (20 L) ws tken out of tp of the VU University in Amsterdm in July After dding 10 ml of methnol contining PFASs to rech detectble concentrtion of PFASs ( ng/l, 8 PFASs), it ws homogenized by stirring for 2 h with stinless-steel mixing device (RW20, IKA-Lbortechnik, Stufen, Germny). Individul high-density polyethylene (HDPE) bottles) were filled with pproximtely 500 ml wter nd distributed to the prticipnts Fish test mteril. Fish A ws crp (Cyprinus crpio), collected from the North Se by locl fishermn from The Netherlnds. Fish B ws pike perch (Stizostedion lucioperc), smpled in the Lke IJssel, The Netherlnds, nd ws obtined from the QUASIMEME Lbortory Performnce Studies (LPS). The ltter smple is identicl to the fish smple used in the ILS in 2009 [8]. The muscle tissue ws collected by filleting the fish. The bulk mteril ws ground nd thoroughly homogenized fter ddition of 0.02% butylhydroxytoluene (BHT) s n ntioxidnt. In ddition, Fish A ws spiked with 1.16 lg/g turodeoxycholic cid (TDCA) s n interfering compound. TDCA is bile cid, often present in biologicl smples, which cn co-elute with PFOS. The moleculr mss of TDCA (498.3 g/mol) is similr to tht of PFOS (499.0 g/mol), nd it is impossible to distinguish the two compounds using nominl-resolution MS. TDCA contins, just s PFOS, sulfonte group, which delivers the sme frgment (SO 3 ; m=z80), which is often used s quntifier frgment for PFOS. Therefore, co-elution, especilly in the presence of high concentrtions of TDCA, cn led to overestimtion t the m/z trnsition [18]. Individul jrs were filled with 45 g muscle homogente. The jrs were sterilized t 3 br, 120 C, which llowed storge t room temperture nd convenient trnsporttion. Detils on the preprtion process of similr mterils cn be found elsewhere [19] Vegetble test mteril. The selected vegetble in this study ws lettuce/pe mixture. The lettuce ws supplied by the University of Amsterdm (UvA) from greenhouse experiment, where lettuce ws grown in spiked nutrient solutions [20]. The nominl PFAS concentrtions in the nutrient solution were 10 ng/l 10 lg/l (14 PFASs). The lettuce ws mixed with green pes (purchsed from supermrket in Amsterdm) to bind the moisture. After dding 0.02% BHT, the mteril ws ground nd thoroughly homogenized. Individul 206

4 Trends in Anlyticl Chemistry, Vol. 43, 2013 Trends jrs were filled with 40 g homogente. These jrs were lso sterilized t 3 br nd 120 C Stndrd solution. A stndrd solution contined 17 PFASs in methnol. The concentrtions of individul PFASs were in the rnge 1 10 ng/ml Methods used by prticipnts The prticipting lbortories were free to use their own in-house methods, nd different extrction, clen-up nd nlysis methods, fine-tuned to chieve the best results were reported. The methods re described in detil elsewhere [21]. In summry, solid-phse extrction (SPE) ws the most commonly employed extrction method for the drinking-wter smple. This combined extrction nd clen-up method ws used by 86% of the prticipnts for the wter smples. The two mjor types of SPE columns used were the WAX column (32%) nd the HLB column (23%). The extrction of the pig liver, the fish-muscle tissue nd the vegetbles ws performed by most of the prticipnts by liquid-liquid extrction with cetonitrile or methnol. Only limited number of lbortories used ion pir or different kind of extrction. After the extrction, the mjority of the prticipnts used SPE (ctive crbon or other) to further clen up the extrct nd thus reduce interferences during the finl nlysis. A frction of the lbortories injected the extrct directly for chemicl nlysis (12.5% of the pig-liver smples nd 8% of fish smples). The finl detection method of the trget compounds used by ll prticipnts ws LC-MS. The mjority of the lbortories used LC triple-qud MS (LC-MS/MS) (53%), nd few lbortories used Q-trp (13%), Orbitrp (7%) or different LC-MS method. All prticipnts used mss-lbeled ISs but not for ll trget compounds. Some lbortories used only one lbeled IS, while others used whole rnge. About hlf of the prticipnts used dditionl lbeled recovery stndrds. 35% of the prticipnts did not correct for blnk levels, with or without explntion, s reported in the full report [21] Dt ssessment The dt ssessment ws crried out ccording to the principles employed in the dt ssessment of the QUASIMEME lbortory-performnce studies nd is described in detil in the supporting informtion (SI-Dt Assessment). All dt received from the prticipnts were entered into dtbse nd ssessed using stndrd procedure enbling direct comprison between prticipnts. The pproch to the ssessment ws bsed on stndrd ISO [22]. However, the ssigned vlue nd the lbortory ssessment using z-scores ws clculted using the model developed by Cofino et l. [23]. The stndrd (ISO 13528) includes sttistics for proficiency-testing schemes, nd uses robust sttistics s bsis for the ssessment. However, it is generlly cknowledged tht robust sttistics cnnot cope with more thn 10% extreme vlues, prticulrly with skewed distribution. The Cofino model is ble to hndle these types of distribution routinely nd provides the best estimte of the consensus vlue, which my be used s the ssigned vlue. The between-lbortory coefficient of vrition (CV) is clculted bsed on the Cofino pproch. The Cofino model is used routinely for QUASIMEME ssessments. In this study, the Norml Distribution Approximtion (NDA) ws employed [23]. This pproch included ll dt in the evlution nd no subjective trunction or trimming ws mde. This model ws further developed to include Left Censored Vlues (LCVs, which is the correct nomenclture for less thn vlues). The development of these models hs been fully documented nd published [23 25] nd ccepted by the Dutch Accredittion Body. An overview of the ssessment with explntion nd exmples is given in the Assessment Rules for the Evlution of the QUASIMEME LP Studies Dt [26]. The z-score ws clculted s described in supporting informtion (SI-Dt ssessment) for ech prticipntõs dt for ech mtrix or determinnd combintion, which is given n ssigned vlue [27]. Following usul prctices (e.g., ISO 17043), the z-scores cn be interpreted s follows for lbortories tht tke prt in ILSs: Z < 2 Stisfctory performnce; 2 < Z < 3 Questionble performnce; Z > 3 Unstisfctory performnce; nd, Z > 6 Frequently points to gross errors (e.g., mistkes with units during reporting, clcultion or dilution errors). It is not possible to clculte z-score for LCVs, s LCVs represent cut-off vlue rther thn continuous dt. The percentge of z-scores reported here represents the ssigned vlues clculted nd does not include the indictive vlues or the percentge of LCVs reported. However, QUASIMEME provides simple qulity criterion: when LCV/2 < (concentrtion corresponding to z = 3), then the LCV is supposed to be consistent with the ssigned vlue nd when LCV/2 > (concentrtion corresponding to z = 3), then LCV is supposed to be inconsistent with ssigned vlue (i.e. when the LCV reported by the lbortory is much higher thn the numericl vlues reported by other lbortories). The LCV vlues re not provided in this rticle but reported elsewhere [21]. 3. Results Of the 31 registered lbortories from Europe nd North- Americ, 29 submitted dt, but, since prticipting 207

5 Trends Trends in Anlyticl Chemistry, Vol. 43, 2013 lbortories were ble to choose between pckges of smples, the number of prticipting lbortories vried per smple. The detils re given in full report elsewhere [21]. For vegetbles, which were only vilble for prtners within the PERFOOD project, ll five prticipting lbortories submitted dt. All 29 lbortories reported on the PFAS concentrtions in the stndrd solution, which ws supplied to ll 31 prticipting lbortories. The results reported here re not on lbortory level but re the summry of the ILS performnce. The performnces of individul lbortories re reported elsewhere [21]. The submitted results hve been evluted sttisticlly nd, whenever the dt met the requirements (specified in Dt ssessments), n ssigned vlue ws estblished. Z-scores were clculted bsed on the ssigned vlue. The ssigned vlues nd the percentge of stisfctory to unstisfctory z-scores for ech determinnd nd mtrix re presented in Tbles 1 6. No ssessment of lbortory performnce is given where n indictive vlue is set or when vlues reported re LCVs. The between-lbortory CV ws clculted ccording the Cofino model (%). Depending on the determinnd, stisfctory z-scores (i.e. Z 6 2 ) were obtined from 50 71% of the lbortories tht submitted results for the drinking-wter smple, except for PFNA, PFDA nd PFOA. The performnce for the vegetble smple ws % depending on the determinnd. For the pig-liver smple, stisfctory z-scores could be clculted for only three compounds PFNA (29%), liner (L)-PFOS (50%), nd brnched (br)-pfos (57%). For fish-muscle-tissue smple A, stisfctory z-scores were obtined from 23 75% of the lbortories, depending on the determinnd. For fishmuscle-tissue smple B, the performnce ws % for lmost ll determinnds (except L-PFHxS nd L-PFDS). This difference in z-scores could be explined by the concentrtion difference of PFASs in the fish smples, which were 8 32-fold higher for most of the PFASs in fish-muscle-tissue smple B compred to fish-muscletissue smple A. 4. Discussion This is the first ILS including food smples (e.g., vegetbles nd pig liver). 21 prticipnts nlyzed the pig liver. Only seven PFASs could be given n ssigned vlue (Tble 1). PFBA (0.34 ng/g ww) nd PFPeA (0.29 ng/g ww) concentrtions were the highest reported, but the precisions were poor with CVs of 135% nd 246%, respectively. The L-PFOS determintion ws more successful; the ssigned vlue ws 0.24 ng/g ww, with 50% of the lbortories Tble 1. Assigned vlue nd the indictive vlue of the PFAS concentrtions (ng/g ww) in pig liver, percentge of dt received from prticipnts who subscribed for the smple, the percentge of stisfctory to unstisfctory z-scores for ech determinnd, the medin concentrtion clculted (ng/g ww), nd the percentge of the between-lbortory coefficient of vrition (CV). 21 lbortories prticipted in the nlysis of the pig liver PFAS Assigned vlue Indictive vlue Dt received Z < 2 3 > Z > 2 6 > Z > 3 Z > 6 Medin Lb CV (ng/g ww) (ng/g ww) (%) (%) (%) (%) (%) (ng/g ww) (%) PFBA NA PFPeA NA PFHxA NA PFHpA 0.07 NA PFOA NA PFNA 0.08 NA PFDA 0.08 NA PFUnDA NA PFDoDA NA PFTrDA NA NA PFTeDA NA NA L-PFBS NA L-PFHxS 0.07 NA L-PFHpS NA NA 20 NA L-PFOS 0.24 NA br-pfos 0.07 NA tot-pfos 0.27 NA L-PFDS NA FOSA NA NA NA 6:2 FTSA NA NA 47 NA NA not pplicble. Dt from fewer thn 3 prtners

6 Trends in Anlyticl Chemistry, Vol. 43, 2013 Trends Tble 2. Assigned vlue nd the indictive vlue of the PFAS concentrtions (ng/g ww) in the fish-muscle tissue A, percentge of dt received from prticipnts who subscribed for the smple, the percentge of stisfctory to unstisfctory z-scores for ech determinnd, the medin concentrtion clculted (ng/g ww) nd the percentge of the between lbortory coefficient of vrition (CV). 29 lbortories prticipted in the nlysis of the fish muscle tissue A PFAS Assigned vlue Indictive vlue Dt received Z < 2 3 > Z > 2 6 > Z > 3 Z > 6 Medin Lb CV (ng/g ww) (ng/g ww) (%) (%) (%) (%) (%) (ng/g ww) (%) PFBA NA PFPeA NA NA PFHxA NA PFHpA NA PFOA 0.07 NA PFNA 0.02 NA PFDA 0.10 NA PFUnDA 0.06 NA PFDoDA 0.04 NA PFTrDA 0.02 NA PFTeDA NA NA L-PFBS NA L-PFHxS 0.04 NA L-PFHpS 0.02 NA L-PFOS 1.9 NA br-pfos 0.35 NA tot-pfos 2.1 NA L-PFDS NA NA FOSA NA :2 FTSA NA NA 46 NA NA not pplicble. Dt from fewer thn 3 prtners. Tble 3. Assigned vlue nd the indictive vlue of the PFAS concentrtions (ng/g ww) in fish-muscle tissue B, percentge of dt received from prticipnts who subscribed for the smple, the percentge of stisfctory to unstisfctory z-scores for ech determinnd, the medin concentrtion clculted (ng/g ww) nd the percentge of the between lbortory coefficient of vrition (CV). 17 lbortories prticipted in the nlysis of the fish muscle tissue B PFAS Assigned vlue Indictive vlue Dt received Z < 2 3 > Z > 2 6 > Z > 3 Z > 6 Medin Lb CV (ng/g ww) (ng/g ww) (%) (%) (%) (%) (%) (ng/g ww) (%) PFBA NA NA 67 NA PFPeA NA NA PFHxA NA NA PFHpA NA NA PFOA NA PFNA 0.52 NA PFDA 2.5 NA PFUnDA 1.3 NA PFDoDA 0.31 NA PFTrDA 0.28 NA PFTeDA NA L-PFBS NA L-PFHxS 0.09 NA L-PFHpS NA NA L-PFOS 60 NA br-pfos 3.0 NA tot-pfos 62 NA L-PFDS 0.16 NA FOSA 1.1 NA :2 FTSA NA NA NA NA not pplicble. Dt from fewer thn 3 prtners

7 Trends Trends in Anlyticl Chemistry, Vol. 43, 2013 Tble 4. Assigned vlue nd the indictive vlue of the PFAS concentrtions (ng/l) in the drinking wter smple, percentge of dt received from prticipnts who subscribed for the smple, the percentge of stisfctory to unstisfctory z-scores for ech determinnd, the medin concentrtion clculted (ng/l) nd the percentge of the between lbortory coefficient of vrition (CV). 24 lbortories prticipted in the nlysis of the drinking wter PFAS Assigned vlue Indictive vlue Dt received Z < 2 3 > Z > 2 6 > Z > 3 Z > 6 Medin Lb CV (ng/l ww) (ng/l ww) (%) (%) (%) (%) (%) (ng/l ww) (%) PFBA 21 NA PFPeA 3.9 NA PFHxA 4.5 NA PFHpA 2.2 NA PFOA 4.9 NA PFNA 0.95 NA PFDA 0.68 NA PFUnDA NA PFDoDA NA PFTrDA NA NA 59 NA PFTeDA NA NA 59 NA L-PFBS 26 NA L-PFHxS 3.9 NA L-PFHpS NA L-PFOS 7.1 NA br-pfos 3.1 NA tot-pfos 10 NA L-PFDS NA FOSA NA NA :2 FTSA NA NA 64 NA NA not pplicble. Dt from fewer thn 3 prtners. Tble 5. Assigned vlue nd the indictive vlue of the PFAS concentrtions (ng/g ww) in the vegetble, percentge of dt received from prticipnts who subscribed for the smple, the percentge of stisfctory to unstisfctory z-scores for ech determinnd, the medin concentrtion clculted (ng/g ww) nd the percentge of the between lbortory coefficient of vrition (CV). 5 lbortories prticipted in the nlysis of the vegetble PFAS Assigned vlue Indictive vlue Dt received Z < 2 3 > Z > 2 6 > Z > 3 Z > 6 Medin Lb CV (ng/g ww) (ng/g ww) (%) (%) (%) (%) (%) (ng/g ww) (%) PFBA PFPeA NA PFHxA PFHpA PFOA NA PFNA NA PFDA NA PFUnDA NA PFDoDA NA PFTrDA NA PFTeDA NA 100 NA L-PFBS NA L-PFHxS L-PFHpS NA NA L-PFOS NA br-pfos tot-pfos NA L-PFDS NA 80 NA FOSA NA :2 FTSA NA 80 NA NA not pplicble. Dt from fewer thn 3 prtners

8 Trends in Anlyticl Chemistry, Vol. 43, 2013 Trends Tble 6. Assigned vlue nd the design vlue of the PFAS concentrtions (ng/ml) in the stndrd solution, percentge of dt received from prticipnts who subscribed for the smple, the percentge of stisfctory to unstisfctory z-scores for ech determinnd, the medin concentrtion clculted (ng/ml) nd the percentge of the between lbortory coefficient of vrition (CV). 29 lbortories prticipted in the nlysis of the stndrd solution PFAS Assigned vlue Design vlue Dt received Z < 2 3 > Z > 2 6 > Z > 3 Z > 6 Medin Lb CV (ng/ml) (ng/ml) (%) (%) (%) (%) (%) (ng/ml) (%) PFBA PFPeA PFHxA PFHpA PFOA PFNA PFDA PFUnDA PFDoDA PFTrDA PFTeDA NA L-PFBS L-PFHxS L-PFHpS NA L-PFOS 7.7 NA br-pfos 2.9 NA tot-pfos L-PFDS FOSA NA NA 6:2 FTSA NA NA NA not pplicble. Dt from fewer thn 3 prtners. receiving stisfctory z-score nd the between-lbortory CV ws 17%. The remining PFAS concentrtions reported were below 0.1 ng/g ww. Fish is one of the mjor contributors to PFAS vi food intke [12]. Fish A contined much lower levels (Tble 2) thn fish B (Tble 3). The results for fishmuscle tissue A showed tht PFDA (0.1 ng/g ww) nd L-PFOS (1.9 ng/g ww) were the mjor determinnds nd the between-lbortory CVs were 25% nd 19%. Fishmuscle-tissue B of the present study nd the fish smple of the study of 2009 [8] were identicl. Compring the z-scores shows n increse in percentge of stisfctory z-scores from 2009 to 2011 (Fig. 1A) nd the betweenlbortory CVs lower for ll determinnds except L-PFHxS (Fig. 1B). PFOA is not mjor contminnt in fish nd the between-lbortory CV ws eqully high in 2009 nd 2011 ILS. Fig. 2 compres the wter smple used in the 2009 study (freshwter tken from cnl close to Amsterdm) with the wter smple of the current study (tp wter). The percentge of stisfctory z-scores in 2011 (Tble 4) ws similr to tht of the 2009 study [8], despite the fct tht the tp wter from 2011 contined lower PFAS concentrtions compred to the 2009 wter smple, which contined elevted concentrtions of PFOA, PFHxS nd PFOS (17 ng/l, 25 ng/l nd 75 ng/l, respectively) fter contmintion from nerby loction [28]). We cn expect tht the nlyticl chllenge is lower with clener mtrix, such s tp wter, but s well tht the ccurcy is esier to perform with elevted concentrtions. The betweenlbortory CVs improved significntly between the two ILSs. The PFAS concentrtions in the vegetble mixture nlyzed in this ILS were below the ng/g ww level (Tble 5), lthough elevted levels could hve been expected from lettuce tht ws grown in spiked nutrient solutions in greenhouse experiment [20]. The betweenlbortory CV for perfluorocrboxylic cids (PFCAs), which were the most bundnt determinnds, were stisfctory (<25%), except PFPeA (54%) nd PFNA (35%). The CV for PFOS (L-, br nd tot-) were 42 82% nd this reflects the chllenge to nlyze levels below ng/g ww. The percentge of stisfctory z-scores ws high for the vegetble smple (80 100%), probbly due to the limited number of prticipnts. Only PER- FOOD members (n = 5) nlyzed the vegetble mtrix. One im of PERFOOD is to improve the nlyticl performnce of PFASs in food, so efforts were mde to identify sources of errors nd void them. The performnce for the stndrd solution ws less stisfctory thn in In the current ILS, 34 62% of the lbortories, depending on the determinnds, obtined stisfctory z-scores, with n verge of 47% (Tble 6). In the ILS of 2009, 40 86% with n verge of 74% obtined stisfctory z-scores (see Fig. 3A). Agin, the 7 16-fold higher PFAS concentrtion in the stndrd solution in the study of

9 Trends Trends in Anlyticl Chemistry, Vol. 43, 2013 Figure 1. Comprison of group performnce of ILS 2009 [8] nd ILS 2011 for the nlysis of PFASs in identicl fish-muscle tissue expressed s (A) percent stisfctory z-scores (i.e. Z 6 2 ) nd (B) in between-lbortory coefficient of vrition (CV, %). is resonble explntion for the decrese in lbortories obtining stisfctory z-scores in This could indicte tht nlyticl chllenges re still n issue regrding low concentrtions (ng/ml), lthough, for ll determinnds (except PFTeDA), between-lbortory CVs were below 25%, which is considered stisfctory (Fig. 3B). In summry, stisfctory between-lbortory CVs (<25%) were chieved for the mjor PFAS determinnds in the mtrices nlyzed here (Fig. 4)

10 Trends in Anlyticl Chemistry, Vol. 43, 2013 Trends Figure 2. Comprison of group performnce of ILS 2009 [8] nd ILS 2011for the nlysis of PFASs in wter (surfce wter nd tp wter) expressed s (A) percent stisfctory z-scores (i.e. Z 6 2 ) nd (B) in between-lbortory coefficient of vrition (CV, %). Similr levels were nlyzed in food in recent report on method developed with nlyticl performnce reching LODs below the ng/g level [17]. The method ws demonstrted on set of food smples prepred from mix of different food items nd nlyzed in triplicte. The PFASs determined in the vegetble smple rnged between non-detects to ng/g ww (most bundnt ws PFHxA), fish rnged between non-detects to 0.18 ng/ g ww (most bundnt ws PFOS) nd met between ng/g ww (most bundnt ws PFHxS)

11 Trends Trends in Anlyticl Chemistry, Vol. 43, 2013 Figure 3. Comprison of group performnce of ILS 2009 [8] nd ILS 2011 for the nlysis of PFASs in stndrd solutions expressed s (A) percent stisfctory z-scores (i.e. Z 6 2 ) nd (B) in between-lbortory coefficient of vrition (CV, %). Further, possible sources of ILS dt vrince were identified. Despite the request in 2009 to report perfluorolkyl sulfontes (PFSAs) on n nion bsis, severl lbortories reported PFSAs on slt bsis. In the current study, this vrince ws overcome by cler instructions nd ll prticipnts reported on n nion bse. Moreover, typiclly prticipnts quntify ginst stndrd consisting of only the liner isomer. The brnched isomers hve response fctors different from the liner isomers [29], which could bis the results observed. This problem is limited when the isomer profile in smple is dominted by the liner PFOS. To evlute 214

12 Trends in Anlyticl Chemistry, Vol. 43, 2013 Trends Figure 4. The between-lbortory coefficient of vrition (CV) is illustrted in color scheme where the lightest cells (green) correspond to stisfctory CVs (<25%), medium CVs (25 50%), nd drk cells (red) to non-stisfctory CVs (>50%). the mgnitude of this error, the prticipnts in the current study were sked to report both L-PFOS nd br-pfos nd to report the sum of liner nd brnched PFOS (tot-pfos). The brnched-isomer contribution differs between the nlyzed mtrices [i.e. in decresing order vegetbles (64%), drinking wter (44%), pig liver (29%), fish-muscle A (18%) nd fish-muscle B (5%)]. For the other PFSAs, prticipnts were sked to report the liner isomers only (L-PFBS, L-PFHxS, L-PFHpS, L-PFDS), which ws done by 63% of the prticipnts. The remining 37% of the prticipnts reported on the totl of brnched nd liner. The concentrtions reported by these lbortories were within two stndrd devitions (SDs) so no sttisticl difference could be seen. As biologicl smples cn contin the bile cid, TDCA, which co-elutes with PFOS on some chromtogrphic columns, TDCA ws dded (1.16 lg/g ww) to the fishmuscle-tissue A smple. To void co-elution, Fluoro- Sep-RP Octyl column (ES Industries, Germny) cn be used to seprte the bse-peks of the two compounds [18]. The mjority (76%) of the prticipting lbortories hd their system djusted for TDCA nd PFOS seprtion or they quntified PFOS on the m/z 499->99 trnsition. One of the prticipnts used high-resolution MS. The remining 24% of the prticipnts did not py specil ttention to differentition between PFOS nd TDCA nd only one of them noticed n interfering pek t the m/z 499->80 trnsition, which ws not observed in the m/z 499->99 trnsition. In this study, the TDCA interference hd minor influence on the reported vlues, but it should be noted tht possible error from TDCA interference cn be much higher in TDCA rich mterils (e.g., eggs) [18]. The lst source of error ws lredy identified in erlier ILSs but is still n issue (i.e. the usge of mss-lbeled ISs). All prticipnts used mss-lbeled ISs, but still not for ll trget compounds. Some lbortories used only one lbeled IS, while others used whole rnge. Quntifiction of PFAS with mss-lbeled stndrd of different PFAS determinnd cn influence the results, since not ll PFASs behve the sme wy through extrction nd clen-up. The ssigned vlue nd the resulting z-scores could be bised by the lck of use of mss-lbeled stndrds by some of the prticipnts, nd for some of the compounds. 5. Conclusions A lrge number of lbortories prticipted in this interntionl ILS, showing tht there is gret interest in nlysis of PFASs in food nd environmentl smples nd tht lbortories hve the intention to control nd to improve their dt. This study showed tht the overll performnce of lbortories prticipting in the nlysis of fish or wter smples hs improved compred to the ILS of The overll experience in PFAS nlysis in the present study is clerly greter thn in the previous ones. This cn be observed from the incresed frction of prticipnts using wider rnge of mss-lbeled ISs. Reporting errors for PFSAs were reduced compred to 2009 (by drwing ttention to this issue in the ILS guidelines). Sources tht hve contributed to the vrince in this study were identified. In conclusion, the different reporting methods of the PFSAs, the interference of TDCA to PFOS, nd the use of lbeled ISs cn ll hve influenced the results (e.g., the between-lbortories CVs, especilly when concentrtions of the compounds were close to or below LOQs. These spects should be ccounted for in future nlysis of PFASs to ensure high-qulity quntittive ssessment of contminnts in food nd environmentl smples. The concentrtions of the PFASs were often low (close to the LOQ) in the mtrices nlyzed in the current ILS. At these levels, the performnce of methods becomes less ccurte nd precise, which is reflected in the betweenlbortory vrince. Still, improvements were demonstrted, n indiction of step forwrd in nlyticl qulity nd control. As more knowledge is gined regrding the toxicity of these compounds, in combintion with the decresed 215

13 Trends Trends in Anlyticl Chemistry, Vol. 43, 2013 number of reported dt below LODs, there will be need to revise the TDI set by the EFSA [13]. In conclusion, this ILS reports improvements in the nlyticl performnce of PFASs in food nd environmentl smples. Relisticlly (low) concentrtions cn be determined when proper methods nd precutions re considered for the mjor PFASs. Acknowledgements The Europen Reserch Project PERFOOD (No. FP7- KBBE ) is grtefully cknowledged for the finncil support of this ILS. Sebstin Felizeter (UvA) is grtefully cknowledged for providing the lettuce for the vegetble smples. Kees Swrt, Rinne vn Dijk nd Robin vn Wissen (IVM) re grtefully cknowledged for preprtion of the smples. And, finlly, ll prticipnts in this ILS on PFASs in food nd environmentl smples re cknowledged for their prticiption nd their extensive reporting. Appendix A. Supplementry dt Supplementry dt ssocited with this rticle cn be found, in the online version, t /j.trc References [1] J.W. Mrtin et l., Environ. Sci. Technol. 38 (2004) 248A. [2] C. Lu, K. Anitole, C. Hodes, D. Li, A. Pfhles-Hutchens, J. Seed, Toxicol. Sci. 99 (2007) 366. [3] E. Mriussen, Arch. Toxicol. 86 (2012) [4] J.M. Weiss, P.L. Andersson, M.H. Lmoree, P.E.G. Leonrds, S.P.J. vn Leeuwen, T. Hmers, Toxicol. Sci. 109 (2009) 206. [5] C. Lu, J.R. Thibodeux, R.G. Hnson, J.M. Rogers, B.E. Grey, M.E. Stnton, J.L. Butenhoff, L.A. Stevenson, Toxicol. Sci. 74 (2003) 382. [6] D.J. Luebker, R.G. York, K.J. Hnsen, J.A. Moore, J.L. Butenhoff, Toxicology 215 (2005) 149. [7] S.P.J. Vn Leeuwen, A. Krrmn, B. Vn Bvel, J. De Boer, G. Lindström, Environ. Sci. Technol. 40 (2006) [8] S.P.J. Vn Leeuwen, M. Strub, W. Cofino, G. Lindström, B. vn Bvel, Report 11/04, Institute for Environmentl Studies (IVM), VU University, Amsterdm, The Netherlnds, [9] S.P.J. Vn Leeuwen, C.P. Swrt, I. Vn der Veen, J. De Boer, J. Chromtogr., A 1216 (2009) 401. [10] G. Lindström, A. Kärrmn, B. Vn Bvel, J. Chromtogr., A 1216 (2009) 394. [11] D. Trudel, L. Horowitz, M. Wormuth, M. Scheringer, I.T. Cousins, K. Hungerbühler, Risk Anlysis 28 (2008) 251. [12] R. Vestergren, U. Berger, A. Glynn, I.T. Cousins, Environ. Int. 49 (2012) 120. [13] EFSA, J. EFSA 653 (2008) 1. [14] EFSA, J. EFSA 10 (2012) [15] Europen Commission, Off. J. Eur. Union L 68 (2010) 22. [16] Food Stndrds Agency, UK, Report Sheet 05/09, [17] R. Vestergren, S. Ullh, I.T. Cousins, U. Berger, J. Chromtogr., A 1237 (2012) 64. [18] A. Bllesteros-Gomez, S. Rubio, S.P.J. Vn Leeuwen, J. Chromtogr., A 1217 (2010) [19] J. De Boer, Mr. Pollut. Bull. 35 (1997) 84. [20] S. Felizeter, M.S. McLchln, P. De Voogt, Environ. Sci. Technol. 46 (2012) [21] I. Vn der Veen, J. Weiss, S. Vn Leeuwen, W. Cofino, S. Crum, Report W-12/09, Institute for Environmentl Studies (IVM), VU University, Amsterdm, The Netherlnds, 2012, p [22] M. Thompson, S.L.R. Ellison, R. Wood, Pure Appl. Chem. 78 (2006) 145. [23] W.P. Cofino, D.E. Wells, F. Arise, I. Vn Stokkum, J.W. Wengener, R. Peerboom, Chemometrics. Intell. Lb. Syst. 53 (2000) 37. [24] W.P. Cofino, I.H.M. Vn Stokkum, J. Vn Steenwijk, D.E. Wells, Anl. Chim. Act 533 (2005) 31. [25] D.E. Wells, W.P. Cofino, J.A. Scurfield, Report 04/04, FRS Mrine Lbortory, 375 Victori Rod, Aberdeen AB11 9DB, UK, [26] D.E. Wells, J.A. Scurfield, Assessment rules for the evlution of the QUASIMEME lbortory performnce studies dt - version 2, FRS Mrine Lbortory, 375 Victori Rod, Aberdeen AB11 9DB, UK, [27] M. Thompson, R. Wood, J. AOAC Int. 76 (1993) 926. [28] S.P.J. Vn Leeuwen, Report 09/06, Institute for Environmentl Studies (IVM), VU University, Amsterdm, The Netherlnds, [29] J.P. Benskin, M. Btineh, J.W. Mrtin, Anl. Chem. 79 (2007)