Unequivocal identification and quantification of PAHs content in ternary synthetic mixtures and in smoked tuna by means of excitation-emission fluorescence spectroscopy coupled with PARAFAC

Jou nal P e-p oo Unequi ocal iden i ica ion and quan i ica ion o PAHs con en in e na y syn he ic mix u es and in smoked una by means o exci a ion-emission luo escence spec oscopy coupled wi h PARAFAC S. Ca ena In es iga ionW i ing - O iginal D a Supe ision , S. Sanllo en e In es iga ionSupe isionW i ing - Re iew & Edi ing , L.A. Sa abia Fo mal analysisSupe isionW i ing - Re iew & Edi ing , R. Boggia W i ing - O iginal D a , F. Tu ini W i ing - O iginal D a , M.C. O iz Concep ualiza ioSupe isionW i ing - Re iew & Edi ing PII: S0026-265X(19)33369-7 DOI: h ps://doi.o g/10.1016/j.mic oc.2019.104561 Re e ence: MICROC 104561 To appea in: Mic ochemical Jou nal Recei ed da e: 28 No embe 2019 Re ised da e: 19 Decembe 2019 Accep ed da e: 19 Decembe 2019 Please ci e his a icle as: S. Ca ena In es iga ionW i ing - O iginal D a Supe ision , S. Sanllo en e In es iga ionSupe isionW i ing - Re iew & Edi ing , L.A. Sa abia Fo mal analysisSupe isionW i ing - Re iew & Edi ing , R. Boggia W i ing - O iginal D a , F. Tu ini W i ing - O iginal D a , M.C. O iz Concep ualiza ioSupe isionW i ing - Re iew & Edi ing , Unequi ocal iden i ica ion and quan i ica ion o PAHs con en in e na y syn he ic mix u es and in smoked una by means o exci a ion-emission luo escence spec oscopy coupled wi h PARAFAC, Mic ochemical Jou nal (2019), doi: h ps://doi.o g/10.1016/j.mic oc.2019.104561 This is a PDF ile o an a icle ha has unde gone enhancemen s a e accep ance, such as he addi ion o a co e page and me ada a, and o ma ing o eadabili y, bu i is no ye he de ini i e e sion o eco d. This e sion will unde go addi ional copyedi ing, ypese ing and e iew be o e i is published in i s inal o m, bu we a e p o iding his e sion o gi e ea ly isibili y o he a icle. Please no e ha , du ing he p oduc ion p ocess, e o s may be disco e ed which could a ec he con en , and all legal disclaime s ha apply o he jou nal pe ain. ©2019 Published by Else ie B.V. 1 HIGHLIGHTS  PARAFAC a use ull ool coupled wi h exci a ion-emission luo escence spec oscopy  EEM-PARAFAC, a cheap, g een and use ull ool o de e mine PAHS in smoked una  EEM-PARAFAC succeeds in iden i ying unequi ocally and quan i ying h ee PAHs  Using a educed o hogonal design o ob ain mix u es o calib a ion s anda ds 2 Unequi ocal iden i ica ion and quan i ica ion o PAHs con en in e na y syn he ic mix u es and in smoked una by means o exci a ion-emission luo escence spec oscopy coupled wi h PARAFAC S. Ca ena (1), S. Sanllo en e (2), L.A. Sa abia (3), R. Boggia (1), F. Tu ini (1), M.C. O iz (2)* (1) Depa men o Pha macy, Uni e si y o Geno a, Viale Cemb ano 4, 16148 Geno a I aly. (2) Depa men o Chemis y, Facul y o Sciences, Uni e si y o Bu gos, Plaza Misael Bañuelos s/n, 09001 Bu gos, Spain (3) Depa men o Ma hema ics and Compu a ion, Facul y o Sciences, Uni e si y o Bu gos, Plaza Misael Bañuelos s/n, 09001 Bu gos, Spain. *Co esponding au ho . E-mail add ess: mco [email protected] 3 Abs ac I is well known ha se e al polycyclic a oma ic hyd oca bons (PAHs), p oduc s o incomple e py olysis o o ganic ma e ial, ha e p o ed o be ex emely oxic o humans. Food can be con amina ed by hese compounds in many di e en ways and die ep esen s nowadays he majo sou ce o exposu e o PAHs o non-smoke s popula ion. In he p esen s udy, h ee o he mos impo an ca cinogenic PAHs in oods, acco ding o he legisla ion cu en ly in o ce, i.e. benzo[a]py ene (BaP), benzo[a]an h acene (BaA) and ch ysene (Ch y), we e i s ly a anged in e na y mix u es, ollowing an expe imen al design. Then, an o ganic ex ac ion om a comme cial smoked una, po en ially a ec ed by PAHs, was pe o med o in es iga e he p esence o he h ee compounds unde s udy. A spec o luo ime ic me hod based on he second o de calib a ion o exci a ion-emission luo escence ma ices (EEMs) and pa allel ac o analysis (PARAFAC) decomposi ion was p oposed in his wo k as analy ical app oach o PAHs de ec ion. Bo h in he e na y mix u es and in he ood ma ix (smoked una), PAHs we e unequi ocally iden i ied and quan i ied wi h decision limi (CCα) and capabili y o de ec ion (CCβ) equal o 0.11 µg L-1 and 0.21 µg L-1 o BaP, 0.27 µg L-1 and 0.53 µg L-1 o BaA and 0.18 µg L-1 and 0.35 µg L- 1 o Ch y, espec i ely, when he p obabili ies o alse posi i e (α) and alse nega i e (β) we e ixed a 0.05. In he in es iga ed smoked una, de ec able le els o BaP we e ound, whe eas BaA and Ch y we e absen . Keywo ds Polycyclic a oma ic hyd oca bons; exci a ion-emission ma ix luo escence spec oscopy; pa allel ac o analysis; smoked una; unequi ocal iden i ica ion. Abb e ia ions 4 PAHs, Polycyclic a oma ic hyd oca bons; BaP, benzo[a]py ene; BaA, benzo[a]an h acene; Ch y, ch ysene; PARAFAC, pa allel ac o analysis; EEM, exci a ion– emission fluo escence ma ix; CCα, decision limi ; CCβ, capabili y o de ec ion; DMSO, dime hylsul oxide ; DMF, N, N-dime hyl o mamide; EPA, En i onmen al P o ec ion Agency; IARC, In e na ional Agency o Resea ch on Cance ; SCF, Scien i ic Commi ee on Food; EFSA, Eu opean Food Sa e y Au ho i y; AOAC, Associa ion o O icial Analy ical Chemis s; SPE, solid phase ex ac ion; SFE, supe c i ical fluid ex ac ion; ASE, accele a ed sol en ex ac ion; MAE, mic owa e assis ed ex ac ion; SPME, solid-phase mic oex ac ion; GPC, gel pe mea ion ch oma og aphy; HPLC, high pe o mance liquid ch oma og aphy; GC-MS, Gas Ch oma og aphy–Mass Spec ome y; HRGC-MS, high esolu ion GC-MS. 1. In oduc ion Polycyclic a oma ic hyd oca bons (PAHs) a e a g oup o o ganic compounds composed by mul iple a oma ics ings which a e widesp ead in he en i onmen as p oduc s o incomple e combus ion o py olysis o o ganic ma e ial, such as coal, pe oleum, wood and na u al gas [1,2]. 16 PAHs ha e been classi ied o decades as p io i y en i onmen al pollu an s by he Eu opean Union (EU) and he US En i onmen al P o ec ion Agency (EPA) [3]. Food can be con amina ed by PAHs ha a e p esen in wa e , ai , soil o packaging ma e ials, as well as hose ha a e o med du ing ood p ocessing o ce ain home cooking p ac ices, e.g. smoking, ba becuing, g illing, oas ing, oas ing, hea ing, d ying, baking, ying and ohmic-in a ed cooking [4]. Thus, he majo sou ce o PAHs exposu e, in non-ciga e e smoke s and non-occupa ionally exposed wo ke s, is die , esponsible o mo e han 90% o he o al exposu e o PAHs o popula ion in many coun ies o he wo ld [5]. 5 A subs an ial numbe o s udies showed ha PAHs a e ex emely oxic o lung, b eas , s omach, esophagus and panc eas [6,7]. Se e al o hese compounds, in ac , ha e been classi ied by he In e na ional Agency o Resea ch on Cance (IARC) as p obably ca cinogenic o humans (G oup 2A) o possibly ca cinogenic o humans (G oup 2B) and benzo[a]py ene (BaP) di ec ly as ca cinogenic o humans (G oup 1) [8]. In 2002, he Scien i ic Commi ee on Food (SCF) [9] s a ed ha benzo[a]py ene (BaP) migh be used as a ma ke o he occu ence o he ca cinogenic PAHs in ood p oduc s [10], bu in 2008 a epo o he Eu opean Food Sa e y Au ho i y (EFSA), showed ha BaP is no always de ec able, p o iding a nega i e esponse in abou 30% o all he es ing samples, e en i o he s PAHs, abo e all ch ysene, we e ound [11]. Thus, he EFSA „Panel on Con aminan s in he Food Chain‟ highligh ed ha benzo[a]py ene (BaP), ch ysene (Ch y), benzo[a]an h acene (BaA) and benzo[b] luo an hene (PAH4) plus ou addi ional PAHs, i.e. benzo[k] luo an hene, dibenzo[a,h]an h acene, benzo[g,h,i]pe ylene, indeno[1,2,3-cd]py ene (PAH8), a e cu en ly he only possible indica o s o he ca cinogenic po ency o PAHs in ood. This led o he Regula ion UE 835/2011, which se s he maximum le el o BaP in smoked mea and smoked ish p oduc s o 5 µg kg-1 oge he wi h he addi ional limi o he sum o PAH4 (30 µg kg-1) [12]. Smoked oods p esen signi ican a ia ions in PAH amoun s, e en wi hin he same kind o ood. This is due o he a iabili y o smoke composi ion, which is g ea ly a ec ed by p ocess en i onmen oxidizing powe , combus ion empe a u e, ype o gene a o , smoking ime, uel used, p esence o smoking la ou agen s and a con en in he ood [4]. T adi ional smoking echniques, in which he smoke p oduced by he incomple e combus ion o he wood comes in o di ec con ac wi h he p oduc , can lead o a high con amina ion by PAHs; o his eason, comme cial al e na i es a e used nowadays, like imme ging ood i ems in smoke ex ac ed solu ion ( he so called "liquid smoke") [13]. 6 The de e mina ion o PAHs in oods equi es a p elimina y ex ac ion ollowed by mul is ep clean-up p ocedu es [14], o isola e hese compounds, occu ing a he le el o µg kg-1 o ng kg-1, om all he in e e e s p esen in a complex a and p o ein con aining ood ma ix. S anda d me hods o PAHs de ec ion in ood ha e been published by Associa ion o O icial Analy ical Chemis s (AOAC) [15]. G imme and Böhnke‟s p ocedu e [16], in ol ing saponi ica ion, ex ac ion wi h cyclohexane, liquid-liquid pa i ion o cyclohexane ex ac wi h N, N-dime hyl o mamide (DMF), ollowed by silica gel column ch oma og aphy is s ill widely used. I s mos common modi ica ions conce n change o ex ac ion and pa i ion sol en s (e.g. dime hylsul oxide, speci ic sol en o PAHs which allows o sepa a ed hem om iglyce ides, ins ead o DMF [17] and use o p e-packed ca idges o solid phase ex ac ion (SPE) [18], which gua an ee ime and sol en sa ings as well as be e ep oducibili y, compa ed o ch oma og aphic columns. In addi ion o liquid–liquid ex ac ion, column ch oma og aphy and SPE, o he echniques like supe c i ical fluid ex ac ion (SFE) [19], mic owa e assis ed ex ac ion (MAE) [20], solid-phase mic oex ac ion (SPME) [21]. Fu he mo e, conce ning he analy ical me hods, Gas Ch oma og aphy–Mass Spec ome y (GC–MS) [22], high esolu ion GC-MS (HRGC–MS) [23] and high pe o mance liquid ch oma og aphy (HPLC) fluo escence de ec ion (FLD) o Diode A ay De ec o (DAD) [24,25] a e nowadays he mos applied echniques o quali a i e–quan i a i e analysis o PAHs in ood. In he p esen wo k, molecula luo escence spec oscopy has been used o he de e mina ion o BaP, BaA and Ch y in a comme cial smoked una. A sea ch wi h keywo ds “EEM” and (PAHs OR “polycyclic a oma ic hyd oca bons”) in Scopus ga e 276 pape s since 1980, none o hem deal wi h he de e mina ion o PAHs by using exci a ion- emission luo escence in smoked ood p oduc s. This echnique was selec ed as analy ical echnique because highly sensi i e, easy o use, as , non-des uc i e and low- cos . The in e p e a ion o luo escence spec al da a o he h ee compounds is complex 7 due o o e lapping signals o o he molecules o ions, since luo escence is a non- sepa a i e echnique; o his eason, he exci a ion-emission ma ices (EEM) need o be analyzed by means o h ee ways echniques in o de o ex ac use ul in o ma ion om a huge amoun o da a. PARAFAC decomposi ion o ilinea da a p o ides unique p o iles es ima ions o he di e en fluo opho es, when an app op ia e numbe o ac o s is chosen o i he model. This means ha i is possible o iden i y unequi ocally each ac o wi h each analy e, hus sepa a ing he signals o each luo opho e. Mo eo e , hanks o he second o de p ope y o PARAFAC, i is also possible o quan i y in p esence o uncalib a ed in e e e s. In his con ex , e na y mix u es o BaP, BaA and Ch y we e p epa ed and analysed wi h luo escence spec oscopy coupled wi h PARAFAC as chemome ic ool. Finally, he p ocedu e was used o de e mina ion and iden i ica ion o hese PAHs in smoked una. 2. Ma e ials and me hods 2.1. Chemical compounds and eagen s Benzo[a]py ene (CAS no. 50-32-8; 96% pu i y) was pu chased om Al a Aesa Gmbh (Ka ls uhe, Ge many), benzo[a]an h acene (CAS no. 56-55-3; 99% pu i y) om Ac os O ganics (Geel, Belgium) and ch ysene (CAS no. 218-01-9; 95% pu i y) om Me ck KGaA (Da ms ad , Ge many), n-Hexane (CAS no. 110-54-3; o liquid ch oma og aphy LiCh osol ®) was supplied by Me ck KGaA (Da ms ad , Ge many) and dime hyl sul oxide (CAS no. 67-68-5; 99% pu i y) by VWR In e na ional S.A.S. (Fon enay-sous-Bois, F ance). (Chemical compounds in able S1 in supplemen a y ma e ial). Deionised wa e was ob ained by using he Milli-Q g adien A10 wa e pu i ica ion sys em om Millipo e (Bed o d, MA, USA). Sep-Pak Silica Plus Long ca idge (690 mg So ben pe Ca idge, 55-105 µm Pa icle Size) and Sep-Pak Plus Sho C-18 ca idges (360 mg So ben pe Ca idge, 55-105 µm Pa icle Size) we e supplied by Wa e s (Mil o d, MA, USA). 2.2. S anda d solu ions o selec ed PAHs and ood ma ix 8 S ock solu ions o each polycyclic a oma ic hyd oca bon we e indi idually p epa ed in n- hexane a a concen a ion o 100 mg L−1. In e media e solu ions o each analy e a 100 µg L−1 we e p epa ed by dilu ion wi h n-hexane. Then, inal solu ions o he analysis we e p epa ed daily by u he dilu ion wi h n-hexane om he in e media e solu ions. All hese solu ions we e s o ed a low empe a u e (4°C) in ambe lasks. Smoked una was ob ained om a local supe ma ke in he a ea o Bu gos, Spain. 2.3. Ins umen s Fo he lyophiliza ion o smoked una, a eeze-d ie F eeZone 12 L Console F eeze-D y Sys em wi h d ying chambe , Labconco was used. Ul asound-assis ed ex ac ion (UAE) o lyophilized smoked una was ca ied ou in an ul asonic ba h (BandelinSono ex RK52, Be lin, Ge many) wi h 35 kHz equency, maximum powe o 240 wa s, and in e nal dimension o 150×140×100 mm. Sample p e-concen a ion was pe o med in a o a y e apo a o a p essu e o 335 mba (ILMVAC, Ilmenau, Ge many). The exci a ion-emission luo escence measu emen s we e pe o med a oom empe a u e on a Pe kinElme LS 50B Luminescence Spec ome e (Wal ham, MA, USA) equipped wi h a xenon discha ge lamp. A s anda d cell holde and a 10 mm qua z SUPRASIL® cell wi h cell olume o 3.5 mL by Pe kinElme (Wal ham, MA, USA) we e used. EEMs we e eco ded in he ollowing anges: emission wa eleng hs om 340 o 500.5 nm (each 0.5 nm) and exci a ion ones om 230 o 305 nm (each 5 nm). Exci a ion and emission monoch oma o sli wid hs we e bo h se o 10 nm and he scan speed was 1500 nm min−1. 2.4. So wa e 15 In he p esen wo k, he iden ifica ion o each analy e was pe o med by means o he co ela ion be ween i s emission and exci a ion e e ence spec a and he emission and exci a ion loadings es ima ed om he co esponding PARAFAC mode. The co ela ion coe ficien s o he emission and exci a ion p ofiles we e 0.992 and 0.995 o BaP, 0.980 and 0.963 o BaA and 0.996 and 0.991 o Ch y, espec i ely. A leas squa e eg ession be ween he sample loadings and he ue concen a ion o each compound in he mix u es was pe o med o he h ee PAHs. Then, he eg essions „p edic ed concen a ion‟ e sus „ ue concen a ion‟ [39]. Table 3 shows he pa ame e s o his eg ession line. The p ocedu e is unbiased, o all he analy es because he p- alues a e g ea e han 0.05 in bo h cases. 4.3. Valida ion and p edic ion o e na y mix u es’ model To alida e he model buil wi h he i e-le els expe imen al design, he 31 analysis we e pe o med again, bu , in his case, hey we e p eceded by he measu emen s o he single compounds in di e en concen a ions o ca y ou h ee new calib a ion lines, in he ange o he i e le els. The concen a ion o each PAHs a e shown in able 4. A new h ee-way a ay X5 (52x322x16) was buil wi h he composi ion o 31 samples in able 1 plus he i s 21 samples in able 4. Then, a PARAFAC decomposi ion wi h ou ac o s (wi h non-nega i i y cons ain s) was applied o X5, ob aining a CORCONDIA index o 99%, wi h explained a iance equal o 99.84%. In e nal alida ion was ca ied ou wi h he sample loadings o he PARAFAC model. Leas squa e eg essions be ween he sample loadings and he ue concen a ion we e pe o med using he i s 37 loadings o he model ha cons i u e he aining se . The las 15 sample loadings (co esponding o he second hal o he expe imen al design om able 1) we e used as es se . This accu acy lines in p edic ion was alida ed as in he p e ious case, being he mean o 16 ela i e e o s in absolu e alue in p edic ion o BaP, BaA and Ch y 6.8%, 3.6% and 2.5%. 4.4 Smoked una ex ac ion As al eady desc ibed in sec ion 2.7, wo po ions o 5 g lyophilized smoked una we e ex ac ed as such (named ex ac A and B) and o he s wo (ex ac C and D) we e added wi h 300 µL o BaP, BaA and Ch y in e media e solu ions. A e he ex ac ion p ocedu e, he ou ex ac s we e dissol ed in di e en amoun o n- hexane o be analysed, based on he di e en in ensi y o each luo escence signals: ex ac s B and C we e dissol ed in 30 mL o n-hexane, ex ac A in 20 mL and D in 10 mL. Then, all o hem we e dilu ed h ee imes, hus analysing each ex ac in ou di e en dilu ions. As in he case o he alida ion o e na y mix u es (sec ion 4.3), new calib a ion lines o BaP, BaA and Ch y we e buil and analysed in he same day o he ex ac s measu emen s. The da a o his s udy was buil by combining he luo escence spec a o he ex ac s A, B, C and D (16 samples) wi h hose o he new calib a ion models again acco ding o able 4 (26 samples) and o he 31 expe imen s ha made up he e na y mix u es design. So, he h ee-way da a enso X6 (73x317x15). I can be seen om he dimension o he enso X6 ha he emission and exci a ion anges ha e been educed by some wa eleng h, o a oid signal sa u a ion p oblems. The PARAFAC decomposi ion o his esul ing enso needed i e ac o s and showed a CORCONDIA index equal o 88% and explained a iance equal o 99.82%. Fig. 3 shows sample (3a), emission (3b) and exci a ion (3c) p o iles. Again, o he iden ifica ion o he PAHs, he co ela ion be ween he PARAFAC spec al loadings and he e e ence spec a ha e been used. Fig. 4 shows he compa ison 17 be ween he emission e e ence spec a (in ed) and he emission loadings o he PARAFAC ob ained a e smoked una ex ac ion (in blue). I mus be highligh ed ha he alues ha e been no malized in his figu e o compa e hem. The co ela ion coe ficien s o he emission and exci a ion p ofiles we e 0.975 and 0.982 o BaP, 0.993 and 0.996 o BaA and 0.997 and 0.993 o Ch y, espec i ely. Once he decomposi ion has been done by using he loadings co esponding o he sample p ofile, i is possible o conclude ha only BaP is p esen in he smoked una in es iga ed (see samples om numbe 58 o numbe 65, blue diamonds in Fig. 3 a). The a e age concen a ion o BaP in ex ac s A and B, bea ing in mind he dilu ion ac o s o each measu emen , was 5.42 µg kg-1, calcula ed o kg o we smoked una (be o e lyophiliza ion), since he we o m, no he d y one, is wha you ea . This BaP concen a ion is jus in acco dance wi h he maximum allowed by law; howe e , i mus be speci ied ha he amoun o BaP calcula ed by PARAFAC, be o e he modi ica ions ela ed o he dilu ion ac o s and o he di e ence be ween we weigh and d y weigh , was ound o be 0.1 µg L-1, which is jus a he limi o CCα (see able 2). The concen a ions o BaA and Ch y, in bo h ex ac s A and B, we e no signi ican ly di e en om ze o. Meanwhile, analysing he wo ex ac s C and D ob ained wi h he s anda d addi ion me hod, he a e age amoun (n = 8) o each compound in bo h ex ac s esul ed o be 2.23 µg L-1, 1.21 µg L-1, 1.94 µg L-1 o BaP, BaA and Ch y, espec i ely. Thei espec i e 95% con idence in e als, exp essed in µg L-1 a e [1.84, 2.62], [0.71, 1.72], and [1.68, 2.19]. The h ee PAHs we e eco e ed wi h a pe cen age o 72.3 (BaP), 40.5 (BaA) and 64.6 (Ch y). 5. Conclusions An analy ical me hod based on EEM luo escence spec oscopy associa ed wi h PARAFAC o de ec polycyclic a oma ic hyd oca bons (PAHs) has been se up and uned by a i e-le el expe imen al design. This p ocedu e has been pe o med o he i s ime 18 o de ec h ee cance ogenic PAHs in a ood ma ix (smoked una ish). Te na y mix u es o BaP, BaA and Ch y we e p epa ed and analysed by he EEM/PARAFAC me hod o e alua ing i s easibili y in de ec ing and quan i ying he h ee PAHs. Thanks o he „second o de p ope y‟ o PARAFAC, when mix u es we e analized, BaP, Ch y and BaA we e unequi ocally iden i ied by means o he co ela ion be ween he pu e spec a and he PARAFAC exci a ion and emission spec al loadings, being hese co ela ions coe icien s: 0.992 and 0.995 o BaP, 0.980 and 0.963 o BaA and 0.996 and 0.991 o Ch y. Mo eo e , he accu a e p ocedu e o he h ee PAHs has been assu ed, being hei capabili y o de ec ion 0.21, 0.53 and 0.35 µg L-1 o BaP, BaA and Ch y espec i ely in he case o indi idual calib a ion and 0.39, 0.25 and 0.29 µg L-1 o BaP, BaA and Ch y in he e na y mix u es (in all case he p obabili ies o alse posi i e and alse nega i e we e ixed a 0.05). In smoked una, only BaP was iden i ied and quan i ied in a concen a ion jus wi hin he limi allowed by legisla ion; BaA and Ch y we e no de ec ed. Sample CRediT au ho s a emen : S. Ca ena: In es iga ion , W i ing - O iginal D a , Supe ision S. Sanllo en e: In es iga ion ,Supe ision, W i ing - Re iew & Edi ing L.A. Sa abia Fo mal analysis, Supe ision, W i ing - Re iew & Edi ing R. Boggia: W i ing - O iginal D a F. Tu ini: W i ing - O iginal D a M.C. O iz: Concep ualiza io, Supe ision, W i ing - Re iew & Edi ing Funding: The au ho s hank he inancial suppo om Spanish MINECO (AEI/FEDER, UE) and Jun a de Cas illa y León h ough p ojec s CTQ2017-88894-R and BU012P17 espec i ely (all co- inanced wi h FEDER unds). Decla a ion o compe ing in e es : The e a e no con lic s o in e es o decla e. 19 Re e ences [1] B. Zhao, S. Zhang, Y. Zhou, D. He, J. Fang, Cha ac e iza ion and quan i ica ion o PAH a mosphe ic pollu ion om a la ge pe ochemical complex in Guangzhou: GC– MS/MS analysis, Mic ochem. J. 119 (2015) 140-144. h ps://doi.o g/10.1016/j.mic oc.2014.12.004 [2] C.Y. dos S. Siquei a, M.V.P. Lemos, B.C. da C. A aujo, R. da R.P.E. de Oli ei a, R.A. da S. San Gil, F.R. de Aquino Ne o, A mosphe ic dis ibu ion o o ganic compounds om u ban a eas nea Olympic games si es in Rio de Janei o, B azil, Mic ochem. J. 133 (2017) 638-644 h ps://doi.o g/10.1016/j.mic oc.2017.04.027 [3] Z. Zelinko a and T. 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[39] In e na ional O ganiza ion o S anda diza ion, ISO 5725–2, Accu acy (T ueness and P ecision) o Measu emen Me hods and Resul s, Pa 2: Basic Me hod o he De e mina ion o Repea abili y and Rep oducibili y o a S anda d Measu emen Me hod, Genè e, Swi ze land (1994). 31 Table 4 Dis ibu ion o concen a ions o he h ee PAHs unde s udy used o pe o m he calib a ion Sample BaA (μg L−1) BaP (μg L−1) Ch y (μg L−1) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 0 0.5 1 1 3 5 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 6 3 0 0 0 0 0 0 0 0 0 0.5 1 1 3 5 7 0 0 0 0 0 0 0 0 3 6 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.5 1 1 3 5 7 0 6 3 6 0 Decla a ion o in e es s ☒ The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape .