Amino Acid d15N Can Detect Diet Effects on Pollution Risks for Yellow-Legged Gulls Overlooked by Trophic Position

ma s-08-657131 May 13, 2021 Time: 13:57 # 1 BRIEF RESEARCH REPORT published: 14 May 2021 doi: 10.3389/ ma s.2021.657131 Edi ed by: Kyung-Hoon Shin, Hanyang Uni e si y, Sou h Ko ea Re iewed by: Ra ik Bal i, Uni e si y o Jendouba, Tunisia Tieyu Wang, Shan ou Uni e si y, China *Co espondence: An onio Bode [email p o ec ed] Special y sec ion: This a icle was submi ed o Ma ine Pollu ion, a sec ion o he jou nal F on ie s in Ma ine Science Recei ed: 22 Janua y 2021 Accep ed: 19 Ap il 2021 Published: 14 May 2021 Ci a ion: Bode A, Besada V, Pé ez-Fe nández B and Viñas L (2021) Amino Acid δ15N Can De ec Die E ec s on Pollu ion Risks o Yellow-Legged Gulls O e looked by T ophic Posi ion. F on . Ma . Sci. 8:657131. doi: 10.3389/ ma s.2021.657131 Amino Acid δ15N Can De ec Die E ec s on Pollu ion Risks o Yellow-Legged Gulls O e looked by T ophic Posi ion An onio Bode1*, Vic o ia Besada2, Begoña Pé ez-Fe nández2and Lucía Viñas2 1Ins i u o Español de Oceanog a ía, Cen o Oceanog á ico de A Co uña, A Co uña, Spain, 2Ins i u o Español de Oceanog a ía, Cen o Oceanog á ico de Vigo, Vigo, Spain The use o op-consume s as bioindica o s o he heal h o ood webs is hampe ed by unce ain ies in hei e ec i e use o esou ces. In his s udy, he abundance o s able ni ogen iso opes in amino acids om homogenised eggs o he Yellow-legged Gull (La us michahellis) allowed o iden i y a ia ions in ophic esou ce exploi a ion be ween geog aphically adjacen nes ing colonies in he Ria de Vigo (NW Spain) ha exhibi ed ma ked di e ences in pollu an s. Eggs om nes s in he Cíes Islands (loca ed in a Na ional Pa k) showed a la ge a iabili y in s able ca bon and ni ogen iso opes in bulk egg con en encompassing ha o eggs om Vigo ci y (a majo ishing ha bou ). Howe e , bo h colonies di e ed in he ela i e concen a ion and abundance o ni ogen iso opes o lysine, an essen ial amino acid p esen in ma ine p ey, bu also ex ensi ely used in eed s ocks o poul y and swine. No wi hs anding he simila i y in ophic posi ion o bo h colonies, gulls om Cíes Islands may ha e acqui ed a subs an ial ac ion o lysine om ga bage dump si es, while hose o he u ban colony elied on ish disca ds. This unexpec ed conclusion is pa ly suppo ed by he la ge a iabili y epo ed o gull’s die in his egion and calls o de ailed es ima ions o die when assessing he conse a ion s a us and pollu ion isks o ma ine ecosys ems. Keywo ds: bioindica o , s able iso opes, amino acids, seagull eggs, ood web INTRODUCTION Top consume s om oceanic ood webs a e good indica o s o he biogeochemis y and conse a ion s a us o ma ine ecosys ems. They in eg a e p ocesses ope a ing a lowe ood web le els and hus ep esen he esul o ecosys em unc ioning a la ge spa ial and empo al scales (Hebe e al., 2016;Ruiz-Cooley e al., 2017). Es ima ions o hei ophic posi ion (TP) a e key o de e mine ecosys em p ope ies as ood-chain leng h (Vande Zanden and Fe ze , 2007;Reum e al., 2015) o he biomagni ica ion o pollu an s (S o -Hansen e al., 1995;Die z e al., 2000; Kelly e al., 2008). Fo ins ance, changes in TP a e indica i e o al e a ions in ood web s uc u e (Jenkins and Da o en, 2020) o in nu ien inpu s (Ruiz-Cooley e al., 2017). Pa icula ly, seabi ds ha e been ex ensi ely used as biomoni o s o pe sis en o ganic and ace me al con aminan s (e.g., OSPAR, 1999), wi h biomagni ica ion e ec s c i ically dependen on hei TP (Ramos e al., 2013; F on ie s in Ma ine Science | www. on ie sin.o g 1May 2021 | Volume 8 | A icle 657131 ma s-08-657131 May 13, 2021 Time: 13:57 # 2 Bode e al. Amino Acid δ15N in Gulls Ga e al., 2020). Me hodological unce ain ies, howe e , di icul he accu a e de e mina ion o TP in hese o ganisms. Di ec quan i ica ion o die s canno be achie ed o mos ield popula ions, as hey may be inaccessible o long pe iods (e.g., Ma ias and Ca y, 2010;Ga e al., 2020) and he analysis o s omach con en s, pelle s o egu gi a es canno iden i y all he consumed p ey (González-Solís, 2003;Abdennadhe e al., 2010; Mo eno e al., 2010;Calado e al., 2018;Méndez e al., 2020). Al e na i e de e mina ions based on he p og essi e en ichmen in hea y ni ogen iso opes (exp essed as δ15N) h ough he ood web allow o es ima ions o TP and die composi ion in eg a ed in space and ime, bu a e highly dependen on ac o s as he app op ia e selec ion o e e ence baselines (Pos , 2002), he assump ions o he en ichmen model (Hussey e al., 2014; Jennings and an de Molen, 2015) and he issue analysed (Cau e al., 2009;Hebe e al., 2016). Ne e heless, hese limi a ions ha e no impeded he ex ensi e use o δ15N in bulk issues o in e TP and biomagni ica ion o pollu an s in many s udies (e.g., Abdennadhe e al., 2010;Ramos e al., 2013). The applica ion o compound-speci ic iso opic analyses (e.g., δ15N in amino acids) ha e educed some o he unce ain ies in TP es ima ions, because he changes in he nu ien sou ces a he base o he ood web can be accoun ed o Chika aishi e al. (2009). E en when he e a e s ill unce ain ies in he a iabili y o he iso opic en ichmen along he ood web (McMahon and McCa hy, 2016; Ohkouchi e al., 2017;Whi eman e al., 2019) he new es ima ions based on amino acids a e being inc easingly applied o ma ine bi ds (McMahon e al., 2015;Wu e al., 2018;Ga e al., 2020). Eggs o he Yellow-legged Gull (La us michahellis) ha e been used o moni o o ganic and ace me al pollu an s (Abdennadhe e al., 2010;Ramos e al., 2013;O e o e al., 2018; Viñas e al., 2020). The la ge eeding plas ici y epo ed o his species, howe e , di icul he iden i ica ion o he sou ces o pollu an s. Fo ins ance, gull colonies nea u ban a eas ha e been shown o consume mo e e es ial and an h opogenic esou ces han ma ine p ey (A izaga e al., 2013;Méndez e al., 2020; Zo ozua e al., 2020). A p e ious s udy o wo colonies only 10 km apa in he Ria de Vigo (NW Spain) e ealed signi ican di e ences in he load o pollu an s in hei eggs, poin ing o a apid adap a ion o local eeding esou ces (Viñas e al., 2020). The colony nes ing on he Cíes Islands (loca ed in a ma ine p o ec ed a ea) showed lowe concen a ions o pollu an s and a wide ophic niche han he colony nes ing in he indus ialised Vigo ci y. Fu he mo e, he es ima ed die composi ion in he egion, including colonies in he Cíes Islands, showed ma ked yea o yea a ia ions ela ed o he a ailabili y o ma ine s. an h opogenic esou ces, he la e including mainly bee , po k, and chicken emains (Mo eno e al., 2010;Calado e al., 2020). These indings sugges ha majo changes in he die o he Yellow-legged Gull may a ec TP and he e o e TP can be used as an indica o o changes in he ophic s uc u e o he ood web. Al e na i ely, pollu ion loads could be caused by he di e en ial exploi a ion o ophic esou ces wi hou a subs an ial change in TP. The objec i e o his s udy is o de e mine he ophic posi ion o Yellow-legged Gull in wo colonies di e ing in he accumula ion o pollu an s (Viñas e al., 2020). We applied o he i s ime es ima ions de i ed om he amino acid s able iso ope composi ion o eggs, allowing o in e di e ences in ophic posi ion and die be ween he gulls in he wo colonies. These es ima ions aimed o assis in he in e p e a ion o he di e en ial impac o con aminan s in hese colonies by e ealing he sensi i i y o TP es ima ions o die a iabili y. MATERIALS AND METHODS Sampling Yellow-legged Gull eggs we e collec ed om nes s in he Cíes Islands (11 eggs) and in he ci y o Vigo (7 eggs) du ing he b eeding season o 2011 (Ap il o ea ly Augus ). Fi s laid eggs in each nes we e selec ed o analysis, weighed and measu ed (leng h and wid h) be o e opening (Supplemen a y Table 1). Emb yona ed eggs we e disca ded and he emaining we e indi idually homogenised, s o ed ozen (−20◦C), and eeze-d ied be o e analysis. Fu he de ails on sampling we e p o ided in Viñas e al. (2020). Analysis Two ypes o iso opic de e mina ions we e made. Fi s , C:N a ios and na u al abundances o ca bon (δ13C) and ni ogen (δ15N) s able iso opes we e de e mined in bulk egg samples using an iso ope a io mass spec ome e coupled o an elemen al analyze . Egg samples we e analysed in iplica e wi h p ecision <0.05h o bo h iso opes. Due o he high lipid con en o egg samples, δ13C alues we e co ec ed using he C:N a io o each sample TABLE 1 | Mean and sd ophic posi ion alues (TP) es ima ed o gull species using s able iso opes in his s udy and in he li e a u e. Species Mean SD nZone Re e ences La us michahellis13.96 0.67 18 Galicia (NE A lan ic) This s udy La us michahellis23.86 0.47 18 Galicia (NE A lan ic) This s udy La us michahellis33.70 0.42 60 Tunis (Medi e anean Sea) Abdennadhe e al., 2010 La us michahellis43.91 1.56 30 S Po ugal (NE A lan ic) Lopezosa e al., 2019 La us a lan icus33.20 0.32 11 Pa agonia (SW A lan ic) Fo e o e al., 2004 La us dominicanus33.80 0.14 229 Pa agonia (SW A lan ic) Fo e o e al., 2004 La us a gen a us33.93 0.51 187 G ea Lakes Hebe e al., 1999 La us a gen a us33.29 0.25 16 Bal ic Sea Sø mo e al., 2011 La us a gen a us53.60 0.40 4 Lake Hu on Hebe e al., 2016 N, numbe o da a. De ails on he es ima ion models a e gi en in he sec ion “Ma e ials and Me hods.” 1δ15Nbulk in eggs, one baseline, SD = p opaga ed e o . 2δ15NAA in eggs, 2 TDF, SD = p opaga ed e o . 3δ15Nbulk in ea he s, one baseline; SD = epo ed SD. 4Calcula ed om o iginal δ15Nbulk in ea he s and die da a, 3 baselines (pelagic ish, deme sal ish, e es ial sou ces), SD = p opaga ed e o . 5δ15NAA in muscle, 2 TDF ( e es ial, aqua ic), SD = epo ed SD. F on ie s in Ma ine Science | www. on ie sin.o g 2May 2021 | Volume 8 | A icle 657131 ma s-08-657131 May 13, 2021 Time: 13:57 # 3 Bode e al. Amino Acid δ15N in Gulls (Ellio and Ellio , 2016). These analyses we e desc ibed in mo e de ail in Viñas e al. (2020). Second, 10 mg aliquo s o he homogenised eggs we e also used o de e mina ions o δ15N in indi idual amino acids (Chika aishi e al., 2009). Samples we e hyd olysed wi h 6N HCl, es e i ied wi h ace yl chlo ide:2-p opanol, and ea ed wi h a mix u e o 3:1 diclome hane: i luo ace ic anhyd ide (McCa hy e al., 2013;Mompeán e al., 2016). De i a ised amino acids we e pu i ied by sol en ex ac ion in 1:2 chlo o o m:phospha e bu e and cen i uga ion (Loick e al., 2019), e apo a ed a oom empe a u e unde N2, and s o ed a −20◦C in 3:1 diclome hane: i luo ace ic anhyd ide o up o 6 mon hs un il iso ope analysis. The indi idual amino acids we e sepa a ed in a ch oma og aphy column (T aceGOLD TG-5MS, 60 m, 0.32 mm ID, 1.0 µm ilm) using a gas ch oma og aph (T ace1310GC, The mo Fishe Scien i ic), and subsequen ly injec ed in o a mass spec ome e (Del aV Ad an age, The mo Fishe Scien i ic) ia a con inuous low in e ace and combus ion module (GC Isolink, The mo Fishe Scien i ic). Amino acid δ15N alues we e calib a ed wi h he alues ob ained o isola ed s anda ds (Shoko Science) analysed by combus ion as desc ibed o bulk analysis, and u he co ec ed using an in e nal L-no leucine s anda d (SIGMA) o known iso opic composi ion added o each sample. The mola ac ion o each amino acid (% mola ) was also de e mined in he same analy ical un by calib a ion o he spec ome ic signals wi h amino acid s anda ds (McCa hy e al., 2013). P ecision (±SE) o iplica e samples ( wo injec ions pe sample) was <0.6hpe indi idual amino acid. Values o δ15N and %mola we e ob ained o ophic and sou ce amino acids (McClelland and Mon oya, 2002;McCa hy e al., 2013;McMahon and McCa hy, 2016). T ophic amino acids included alanine (Ala), leucine (Leu), isoleucine (Ile), p oline (P o), aline (Val), and he mix u es o glu amine (Gln) and glu amic acid (Glu), and o aspa amine (Asn) and aspa ic acid (Asp). The la e mix u es we e caused by he acid hyd olysis and we e e med as Glx and Asx, espec i ely. Sou ce amino acids included glycine (Gly), h eonine (Th ), se ine (Se ), me hionine (Me ), phenylalanine (Phe), and lysine FIGURE 1 | Box plo o (A) δ15Nbulk,(B) lipid-co ec ed δ13Cbulk ,(C) δ15N p, and (D) δ15Ns c o Yellow-legged Gull eggs sampled in Cíes Islands (black) and Vigo ( ed). Each box encompasses he 25–75% pe cen iles, he median is indica ed by he ho izon al line, and he whiske s indica e he ange excluding ou lie s exceeding 1.5 (ci cles) o 3 imes (as e isks) he in e qua ile ange. F on ie s in Ma ine Science | www. on ie sin.o g 3May 2021 | Volume 8 | A icle 657131 ma s-08-657131 May 13, 2021 Time: 13:57 # 4 Bode e al. Amino Acid δ15N in Gulls (Lys). The ep esen a ion o hese amino acids in he bulk p o ein was assessed by co ela ion o hei mola -weigh ed a e age δ15N (δ15NTHAA) wi h δ15N alues o bulk samples (δ15Nbulk). Simila ly, he co ela ions be ween mola -weigh ed a e ages o ophic (δ15N p) o sou ce amino acids (δ15Ns c) wi h δ15Nbulk we e used o de e mine he ela i e impo ance o a ia ions in ophic s. ni ogen sou ce ac o s o he in e p e a ion o δ15Nbulk. Die Es ima ions The po en ial con ibu ion o ma ine and an h opogenic ood sou ces o he die o he gulls o each colony was es ima ed om he δ15N signa u e o sou ce amino acids. Re e ence alues o ma ine sou ces we e p o ided by sa dines (Sa dina pilcha dus) and ancho ies (Eng aulis enc asicholus) collec ed in sp ing (Ap il 2017 and Ap il 2019) nea he Ria de Vigo as pa o a egula ish su ey (see Bode e al., 2018). An h opogenic sou ces we e p o ided by samples o bee , po k and chicken mea ob ained om local ma ke s. Fou indi iduals o each ish species and h ee eplica es o each mea ype we e analysed as desc ibed o he gull eggs. The po en ial con ibu ion o hese sou ces o he gulls die we e es ima ed using he Mix SIAR Bayesian mixing model (SIAR 4.1.3) o S ock and Semmens (2013) by using only he δ15N alues o indi idual amino acids ha a ied signi ican ly be ween colonies and assuming no iso opic ac iona ion be ween sou ces and eggs. This simpli ied app oach was in ended only o highligh he po en ial implica ions o he ma ked di e ences in δ15N among he sou ces. T ophic Posi ion Es ima ions o he ophic posi ion o Yellow-legged Gull indi iduals we e made using he δ15N alues o ep esen a i e ophic (Glx) and sou ce (Phe) amino acids (TPAA) and a model conside ing wo ophic disc imina ion ac o s (McMahon and McCa hy, 2016): TPAA =2+(δ15NGlx−δ15NPhe−TDFp−β TDFb )(1) Whe e, βis he di e ence be ween Glx and Phe in p ima y p oduce s, and TDFp and TDFb a e he ophic disc imina ion ac o s o plank on and ma ine bi ds, espec i ely. Mean (±SE) alues we e 3.4 ±0.9h o β(Chika aishi e al., 2009), FIGURE 2 | Mean ±SE (A) δ15N (h) and (B) mola pe cen o ophic and sou ce amino acids o Yellow-legged Gull eggs sampled in Cíes Islands (black do s) and Vigo ( ed do s). The a ows indica e signi ican ly di e en medians (K uskal-Wallis, P<0.05). F on ie s in Ma ine Science | www. on ie sin.o g 4May 2021 | Volume 8 | A icle 657131 ma s-08-657131 May 13, 2021 Time: 13:57 # 5 Bode e al. Amino Acid δ15N in Gulls 7.6 ±1.2h o TDFp and 3.5 ±0.4h o TDFb (McMahon e al., 2015;Wu e al., 2018;Ga e al., 2020). As he e was iso opic ac iona ion be ween he di e en issues, inc emen s o 2.5h and 0.9hwe e applied o he measu ed alues o δ15NGlx and δ15NPhe, espec i ely, o p oduce TP es ima ions equi alen o hose ob ained o he muscle o adul gulls (Hebe e al., 2016). Fo compa a i e pu poses, addi ional TP es ima ions we e made using he measu ed δ15Nbulk in eggs using he classical model (Pos , 2002): TPbulk =2+(δ15Nbulk−δ15Nzoo TDFz ) whe e, δ15Nzoo was he alue o zooplank on samples collec ed in he nea by shel du ing sp ing 2011 (mean ±se = 5.97 ±0.92h,n= 14) as desc ibed in Bode e al. (2018), and TDFz= 3.4h(Pos , 2002). The e o o all TP es ima ions was compu ed by e o p opaga ion by aking in o accoun he analy ical e o s in δ15N measu emen s in bulk o ophic and sou ce amino acids, as well as he e o s epo ed o TDF and β alues employed (Pos , 2002;Ohkouchi e al., 2017). Fu he compa isons o TP we e made wi h hose epo ed in he li e a u e o Yellow-legged Gull and simila species (Table 1). In he case o da a epo ed in Lopezosa e al. (2019),TP was es ima ed om he epo ed δ15Nbulk in body ea he s and die da a using he model desc ibed in Hebe e al. (2016) by conside ing he consump ion o pelagic ish, deme sal ish, and ga bage. S a is ics Di e ences in iso opic measu emen s be ween eggs om Cíes and Vigo colonies we e analysed by means o non-pa ame ic ANOVA (K uskal-Wallis). Reg essions be ween a iables we e compu ed using educed majo axis eg ession, as he e we e la ge di e ences in he measu emen e o o he a iables. S a is ical analyses we e pe o med using SPSS 17.0 (SPSS Inc.) and Pas 4.0 (Hamme e al., 2001). RESULTS The e we e no signi ican di e ences in median bulk δ15N o δ13C be ween bo h colonies, no in a e aged δ15N alues o ophic o sou ce amino acids (K uskal-Wallis es , P>0.05, Figu e 1). Howe e , he weigh ed means o δ15N o o al amino acids (δ15NTHAA) and ophic amino acids (δ15N p) we e linea ly co ela ed wi h bulk δ15N (P<0.001, n= 16), while he means o sou ce amino acids showed no co ela ion (P>0.05, Supplemen a y Figu e 1). Values o δ15N alues o mos amino acids we e simila o he wo colonies (Figu e 2A), excep in he case o lysine ha showed lowe alues o he Vigo colony (K uskal-Wallis es , P<0.05). Mean ±SD δ15N alues o lysine in eggs om he Cíes Islands we e 1.50 ±1.18h(n= 9) while in hose om Vigo we e 4.68 ±0.77h(n= 7). E en when adjus ed o a ia ions in he δ15N o he canonical sou ce amino acid phenylalanine, he di e ences in iso opic composi ion o Lys be ween colonies pe sis ed, and we e indica i e o di e ences in he use o ma ine FIGURE 3 | Box plo o ophic posi ion o Yellow-legged Gull es ima ed om (A) bulk δ15No (B) amino acid δ15N o samples om Cíes Islands (black) and Vigo ( ed). Boxes and symbols as in Figu e 1. esou ces (Supplemen a y Table 2). The eggs om he Cíes Islands had also lowe mean ela i e mola concen a ion o lysine and highe concen a ion o alanine han hose om Vigo (K uskal-Wallis es s, P<0.05, Figu e 2B). As o gull eggs, he e we e also signi ican di e ences in Lys δ15N among ood sou ces (Mann-Whi ney es , P<0.05, Supplemen a y Figu e 2A). Using hese alues, he es ima ions o die sugges ed ha , no wi hs anding ma ine sou ces had a majo con ibu ion po en ial o bo h colonies, he gulls om Cies had a highe con ibu ion o an h opogenic sou ces han hose om Vigo (Supplemen a y Figu e 2B). The es ima ions o TP (Table 1) indica ed ha he s udied Yellow-legged Gulls we e almos e ia y consume s (i.e., TP ca. 3) and e ealed no signi ican di e ences using ei he bulk o amino acid δ15N (K uskal-Wallis es , P>0.05). Besides, he e we e no signi ican di e ences in TP be ween colonies (Figu e 3, K uskal-Wallis es , P>0.05). F on ie s in Ma ine Science | www. on ie sin.o g 5May 2021 | Volume 8 | A icle 657131 ma s-08-657131 May 13, 2021 Time: 13:57 # 6 Bode e al. Amino Acid δ15N in Gulls DISCUSSION The esul s o his s udy ep esen he i s es ima ion o Yellow-legged Gulls TP ob ained wi h amino acid δ15N. These es ima ions a e compa able o hose ob ained using bulk δ15N in his and p e ious s udies in he same and ela ed gull species (Table 1), and in o he ma ine bi ds (Ga e al., 2020). Fu he mo e, he signi ican co ela ion be ween δ15N o ophic amino acids and bulk δ15N (and he lack o co ela ion o δ15N in sou ce amino acids) suppo s p e ious assump ions using bulk δ15N as an index o TP (Abdennadhe e al., 2010;Muñoz-A nanz e al., 2012;Ped o e al., 2013;Calado e al., 2018). Howe e , he de e mina ion o δ15N in indi idual amino acids p o ided addi ional in o ma ion on he a iabili y o eeding among indi iduals ha complemen s TP es ima ions. Fo ins ance, he simila i y in TP be ween he s udied colonies suppo s ha he di e ences in pollu ion loads epo ed in Viñas e al. (2020) we e caused by changes in die un ela ed o TP, e en when de ailed die composi ion was no a ailable o he indi iduals s udied. Simila conclusions we e eached in o he s udies whe e, die and TP could be de e mined (Muñoz-A nanz e al., 2012; Ramos e al., 2013). The low δ15N in lysine ound in eggs om he Cíes Island colony sugges he consump ion o non-ma ine p ey. Fi s , because he mean di e ence be ween δ15N o Lys and Phe o he Cíes eggs was signi ican ly lowe han alues ypical o phy oplank on (Nielsen e al., 2015). Second, because he es ima ed con ibu ions o an h opogenic sou ces o he die in Cíes we e gene ally highe han hose in Vigo (Supplemen a y Figu e 2). This is an unexpec ed esul , as he gulls om he islands would ha e, in p inciple, mo e access o ma ine p ey han hose nes ing in he ci y. Lysine is an essen ial amino acid in he die o animals and is one o he majo addi i es o eed s ocks, no ably o poul y and swine aising (To ide, 2002). I is p oduced indus ially h ough e men a ion wi h he addi ion o suga s and ino ganic ni ogen (Ikeda, 2017). Indus ial lysine is likely o ha e low δ15N, since ammonium de i ed om a mosphe ic ni ogen ep esen s he mos economic sou ce o e ilize s (Smil, 2001). Fo ins ance, he alues o Lys δ15N measu ed in bee , po k and chicken mea in his s udy we e lowe han hose measu ed in ish. These esul s ag ee wi h hose epo ed o bulk δ15N om chick gull egu gi a es in he Bay o Biscay (A izaga e al., 2013). The e o e, a subs an ial consump ion o ea ing animals emains by gulls om he Cíes Island colony canno be disca ded, as no ed by p e ious s udies (Mo eno e al., 2010). Gi en he la ge a iabili y epo ed in he die o he Yellow- legged Gull, i would be no su p ising ha he Cíes Islands colony pa ly elied on ga bage, a leas du ing he ime ame conside ed in his s udy. This is suppo ed by he yea - o- yea educ ion in he consump ion o ish by his species along he NW Spanish coas and a ibu ed o he decline in sa dine popula ions (Calado e al., 2020). Fu he mo e, he epo ed mean composi ion o he gull die in his egion indica ed he dominance o an h opogenic o e ma ine ood i ems du ing 2010, an excep ional con ibu ion compa ed o he se ies o he whole eco ding pe iod (Supplemen a y Table 3). In his ega d, i mus be no ed ha he iso opic composi ion o eggs is conside ed ep esen a i e o bo h ecen (ca. 1 mon h) and pas (ca. 1 yea ) die o he b eeding adul emales (Hebe e al., 2016). The high consump ion o ma ine esou ces in Vigo ag ees wi h he gene al pa e n epo ed o he egion and could be a ou ed by he p oximi y o he ishing ha bou , one o he la ges in Eu ope wi h 84,000 me ic ons o esh ma ine p oduc s landed in 2011 (Po Au ho i y o Vigo, 2012). Fish o al would be a a ou ed esou ce o gulls nes ing in Vigo, as epo ed o simila species elsewhe e (Hebe e al., 1999;González-Solís, 2003; Calado e al., 2018). Di e ences in die , hus, we e he likely cause o he high le els o pollu an s in eggs om he Vigo colony (Viñas e al., 2020). Ma ine ishes o high TP we e epo ed o bioaccumula e o ganic and ino ganic pollu an s. Fo ins ance, Hg compounds in una (e.g., Méndez e al., 2001) can each concen a ions equi alen o hose ound in he gull eggs om Vigo (Viñas e al., 2020). These esul s imply ha biomagni ica ion does no only depend on TP bu also on he die o he consume species. The e o e, u u e assessmen s o pollu ion isks in ood webs would equi e coupled s udies o die and TP. Gull die can be econs uc ed om obse a ions o eeding emains o s omach con en s, bu also om ins umen al echniques as he analysis o ma ke a y acids (Hebe e al., 2016). The la e can be e y sensi i e when coupled o s able iso ope de e mina ions (Twining e al., 2020). The applica ion o hese echniques will help o de e mine he app op ia e biomagni ica ion ac o s when he moni o ed species, as he Yellow-legged Gull, eeds on di e en habi a s o ood webs. Conclusion Ou s udy shows ha eliable TP es ima ions o he yellow- legged gull can be ob ained using δ15N ei he in bulk homogenised eggs o in indi idual sou ce and ophic amino acids. Howe e , he la e echnique would be p e e ed because da a o assump ions abou he baseline e e ence alues om o he species a e no equi ed, and because i can p o ide aluable in o ma ion on he a iabili y in die among indi iduals. Indeed, die , a he han TP, appea s as he dominan ac o a ec ing he bioaccumula ion o pollu an s in he Yellow- legged Gull. DATA AVAILABILITY STATEMENT The o iginal con ibu ions p esen ed in he s udy a e included in he a icle/Supplemen a y Ma e ial, u he inqui ies can be di ec ed o he co esponding au ho . ETHICS STATEMENT E hical e iew and app o al was no equi ed o he animal s udy because gull eggs o his s udy we e collec ed as pa o he F on ie s in Ma ine Science | www. on ie sin.o g 6May 2021 | Volume 8 | A icle 657131 ma s-08-657131 May 13, 2021 Time: 13:57 # 7 Bode e al. Amino Acid δ15N in Gulls pollu ion moni o ing p og am o he IEO and he Minis e io pa a la T ansición Ecológica (Spain). AUTHOR CONTRIBUTIONS LV and VB designed he p ojec . AB designed he esea ch epo ed he e, analysed he da a, and w o e he manusc ip wi h commen s om coau ho s. LV, BP-F, and AB analysed he samples. All au ho s con ibu ed o he a icle and app o ed he submi ed e sion. FUNDING This s udy was unded in pa by a Fund Managemen Ag eemen be ween he IEO and he Minis e io pa a la T ansición Ecológica (Spain) and by g an numbe IN607A2018/2 o he Axencia Galega de Inno ación (GAIN, Xun a de Galicia, Spain). ACKNOWLEDGMENTS We acknowledge he Na ional Pa k o he A lan ic Islands (Spain) o acili a ing he sampling, and Ma ia Lema (Se icio de Análisis Ins umen al, Uni e sidad de A Co uña, Spain) o he iso opic de e mina ions. SUPPLEMENTARY MATERIAL The Supplemen a y Ma e ial o his a icle can be ound online a : h ps://www. on ie sin.o g/a icles/10.3389/ ma s. 2021.657131/ ull#supplemen a y-ma e ial REFERENCES Abdennadhe , A., Rami ez, F., Romdhane, M. S., Ruiz, X., Jo e , L., and Sanpe a, C. (2010). Biomoni o ing o coas al a eas in Tunisia: S able iso ope and ace elemen analysis in he yellow-legged gull. Ma . Pollu . Bull. 60, 440–447. doi: 10.1016/j.ma polbul.2009.10.003 A izaga, J., Jo e , L., Aldalu , A., Cuad ado, J. F., He e o, A., and Sanpe a, C. (2013). T ophic ecology o a esiden yellow-legged gull (La us michahellis) popula ion in he Bay o Biscay. Ma . En i on. Res. 87-88, 19–25. doi: 10.1016/ j.ma en es.2013.02.016 Bode, A., Ca e a, P., González-Nue o, G., Noguei a, E., Ri ei o, I., and San os, M. B. (2018). A ophic index o sa dine (Sa dina pilcha dus) and i s ela ionship o popula ion abundance in he sou he n Bay o Biscay and adjacen wa e s o he NE A lan ic. P og. 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