Evidence of ingested plastics in stranded loggerhead sea turtles along the Greek coastline, East Med

Evidenceofingestedplasticsinstrandedloggerheadseaturtlesalong theGreekcoastline,EastMediterraneanSea*

NikolettaDigka a, *,LauraBray a,CatherineTsangaris a,KonstantinaAndreanidou b , EiriniKasimati c,EvangeliaKofidou d,AnastasiaKomnenou d,HelenKaberi a

a HellenicCentreforMarineResearch,46.7kmAthens-SounionAve.Anavyssos,Attiki,19013,Greece

b MediterraneanAssociationtoSavetheSeaTurtles(MEDASSET),Likavittou1,Athens,Greece

c ARCHELON,TheSeaTurtleProtectionSocietyofGreece,Solomou57,10432,Athens,Greece

d SchoolofVeterinaryMedicine,FacultyofHealthSciences,AristotleUniversityofThessaloniki,St.VoutyraStr,Thessaloniki,54627,Greece

articleinfo

Articlehistory:

Received4December2019

Receivedinrevisedform

13April2020

Accepted13April2020

Availableonline14April2020

Keywords:

Anthropogenicdebris

Plasticsingestion

Carettacaretta

MediterraneanSea

Greece abstract

1.Introduction

Debrisfoundinthemarineenvironmentistheresultofvarious anthropogenicactivitiesandhasrecentlybecomeanimportant environmentalconcern.Marinedebrisconsistsofavarietyofmaterials(metal,paper,industrialwood),however,plasticrepresents 80%ofmarinedebrisfoundglobally(UNEP,2016).In2017,worldwideplasticproductionreached350milliontonnes (PlasticsEurope,2018),fromwhichlargequantitiesendupintothe oceans(Lebretonetal.,2017).This,combinedwiththelimited wastemanagementinfrastructuresandthedurabilityofplasticsin themarineenvironment,reinforcestheestimationthattheaccumulationofplasticintheoceanswillescalate(Jambecketal.,2015). Onceinthemarineenvironment,plasticdebrisissubjectedto fragmentationduetowind,air,andsolaractivity.Plasticdebrishas beendetectedfromthemosturbanised,andindustrialised,areasto themostremote,andpristine(Laversetal.,2019).Duetovariations inpolymertypesanddensities,plasticscaneither floatorsink;

https://doi.org/10.1016/j.envpol.2020.114596 0269-7491/© 2020ElsevierLtd.Allrightsreserved. EnvironmentalPollution263(2020)114596

Plasticdebrishasbecomeamajorthreattothemarineenvironmentandwildlife.Seaturtlesare particularlyvulnerable,andareknowntoingestplasticdebrisglobally;however,informationfromGreek watersisstillabsent.Inthisstudy,36strandeddeadloggerheadturtles(Carettacaretta)werecollected fromtheGreekcoastlinearea,andtheirgastrointestinalcontentwasanalysedforingestedplasticdebris. Twenty-sixindividuals(72%)werefoundtohaveingestedplastic,withanaverageof7.94 ± 3.85(SE) plasticitemsperturtle.Intotal,286plasticitemswerecountedandcategorisedbysize,shape,colour, andpolymertype.FourierTransformInfraredSpectrometryrevealedthatpolypropyleneandpolyethylenewerethedominantpolymerplastictypesfound.Resultsindicatedavariationinplastic ingestionamongstlifestagesoftheloggerheadspecimens.Thisstudyprovidesevidenceofplastic ingestionbyloggerheadturtlesinGreekwaters.

© 2020ElsevierLtd.Allrightsreserved.

* ThispaperhasbeenrecommendedforacceptancebyMariaCristinaFossi.

* Correspondingauthor.

E-mailaddress: ndigka@hcmr.gr (N.Digka).

therebyeitheraccumulatingattheseasurface,throughoutthe watercolumn,orevenindeep-seasediments(Choyetal.,2019; Woodalletal.,2014).

Entanglementinmarinedebrisisthemostvisibleeffectofplastic pollutiononmarineorganisms,whichhasbeenreportedtoaffect morethan344marinespecies(Kühn,2015).Lesseasilydetectedare theplasticsingestedbymarineorganisms;anissueobservedwith increasinginterestbythescientificcommunity(Digkaetal.,2018a; Jamiesonetal.,2019; Lusheretal.,2018; Matiddietal.,2017; Nelms etal.,2016).Intotal,morethan331differentmarinespecieshave beendocumentedashavingingesteddebris(Kühn,2015).Insea turtles,ingestionofplasticdebrishasbeenreportedforall7species (Kühn,2015),albeitwithaninter-speciesvariationforthequantity andtypeofplasticdebrisingestedduetodifferencesinhabitatsand feedingpreferences(Schuyleretal.,2014).Variationsinplastic ingestionmayalsobeapparentinseaturtlesofthesamespeciesat differentlifecyclestages.Mostseaturtlespeciesexploittwoormore differentforagingareasthroughouttheirlifehistory,whichmay suggestthatdifferentlifestagesareexposedindifferentwaysto plasticdebrisinthemarineenvironment(Schuyleretal.,2014).For example,early-stagejuvenileloggerheadseaturtles(Carettacaretta) liveandfeedprimarilyinopenoceanpelagichabitats(Bolten,2003).

Duringtheirpost-hatchingpelagicphase,theymigrateforlong distances,where floatingdebrisislikelytobefound(Phametal., 2017; Schuyleretal.,2014).Early-stagejuvenileloggerheadsare oftenfoundpassivelydriftinginoceanicgyresandencounterlarge quantitiesofaccumulateddebris(Lawetal.,2010; Nelmsetal., 2016).Late-stagejuvenilesandadultloggerheadseaturtlesswitch tocoastalwatersbuttheymayalsomigratetooceaniczonesfor breeding(Bolten,2003).Late-stagejuvenilesandadultloggerheads, improvetheirbenthicforagingcapacityandmaydivetobenthic habitatsforfeeding(Casaleetal.,2018; Schuyleretal.,2014).

Inturtles,itislikelythatplasticingestionoccursmainlyeither byaccident,whenplasticdebrisismixedwithnaturalfood(Di BenedittoandAwabdi,2014)orduetothesimilarityofplastic itemswiththeirnaturalprey(Hoarauetal.,2014; Tomasetal., 2002).Formostseaturtlespecies,therearereportedcasesoflethalorsublethaleffectsduetoplasticingestion,butthesearerare (Hoarauetal.,2014; McCauleyandBjorndal,1999; Wilcoxetal., 2018).Plasticdebris,eveninsmallamounts,canblockorharm seaturtles’ gastrointestinaltract,reducetheirdigestiveand swimmingcapacity,orpotentiallytransporttoxicsubstancesto theirbodytissues(Bjorndaletal.,1994; Cameddaetal.,2014; Di Belloetal.,2013; DiBenedittoandAwabdi,2014; Santosetal., 2015).

Theloggerheadseaturtle C.caretta hasaworldwidedistribution,nestingoverthebroadestgeographicalrangeofanyother turtlespecies.ItinhabitstheAtlantic,Indian,andPacificOceans andtheMediterraneanSea.TheMediterraneanSeaisnotonlya nurseryforjuvenilesbutalsoacommonplaceforadultloggerheads inthespringandsummermonths(Spotila,2004; Wallaceetal., 2010).Greeceisthemostpopularnestingloggerheadaggregation intheMediterranean,withmorethan3000nestsperyear (Margaritoulisetal.,2018; Spotila,2004).Constructiondevelopment,tourist,and fishingactivitiesaremajorthreats,thatloggerheadsfaceinGreece(Margaritoulisetal.,2018),togetherwith marinelitterwhichisnowadaysbecomingofmajorconcernaswell (Digkaetal.,2018b).Datafrom2010until2016,showthatthe annualaveragestrandingswere359 ± 67(mean ± SE)forthewhole ofGreece(Brayetal.,2017).IntheMediterraneanbasin,loggerhead interactionwithmarinedebrishasbeenreported(Casaleetal., 2008; Domenechetal.,2019; Duncanetal.,2019; Matiddietal., 2017),however,noinformationonplasticdebrisingestionbysea turtlesisavailableforGreekwaters.

Theloggerheadseaturtle(C.caretta)isgloballyconsideredasa vulnerablespecies,withadecreasingpopulationtrendworldwide (IUCN,2015).Duetotheirwidedistributionandtheirpropensityto plasticingestion,theloggerheadseaturtlehasbeenrecentlyproposedasasuitableindicatorspeciesformonitoringthepresence andimpactofmacroplasticsatabasinlevelintheMediterranean Sea(Fossietal.,2018; Galganietal.,2013),justasthenorthern fulmar(Fulmarusglacialis) isusedintheNorthSea(OSPAR,2015). FortheMarineStrategyFrameworkDirective(MSFD)(2008/56/EC), marinelitterisaddressedasoneofthe11DescriptorsoftheGood EnvironmentalStatus(GES)setbytheEuropeanUnion.Plastic ingestionbymarineorganismsisamongtheelementstobeused fortheassessmentofGESforMSFDDescriptor10 MarineLitter (Decision2017/848/EU),accordingtotheMSFDcriterionD10C3 “Theamountoflitterandmicro-litteringestedbymarineanimalsis atalevelthatdoesnotadverselyaffectthehealthofthespecies concerned” .

Thisstudyaimsto:i)identifytowhatextentstrandedloggerheadseaturtlesinGreekcoastlinehaveingestedplasticandii) describethesize,shape,colourandpolymertypeoftheingested plasticdebrisandtheirrelationtotheturtle’slife-stage.Theresults ofthisstudycancontributetotheestablishmentofbaselinelevels ofingestedplasticdebrisbyseaturtles,andthedefinitionofGESin

theMediterraneanregionforMSFDcriteriononlitterandmicrolitteringestedbymarineanimals.

2.Materialsandmethods

2.1.Samplecollection

Atotalof36loggerheadseaturtles, C.caretta,werenecropsied betweenNovember2017andMay2019.Ofthese,28werefound strandeddeadalongtheGreekcoastline(Fig.1),andeightwere foundinjuredanddiedlaterintherescuecentre.Aprotocolforthe strandedanimalswasfollowedinaccordancewithGreeklaw.Alive animalsweretransferredforrehabilitationatthefacilitiesofthe Non-GovernmentalOrganizationARCHELON,anddiedinlessthan 11daysshowingnosignsoffeedingorfaecalactivity.Afullnecropsywasperformedinallturtles(n ¼ 36),bytrainedveterinarians,andtheirgastrointestinaltract(GI)wasdissected,dividedinto threedifferentsections(oesophagus,stomach,intestine),and storedat 20 C.FrozenGIsectionswerethentransferredtothe HCMRlaboratoriesforfurtheranalysis.Priortonecropsy,various biometricdatai.e.curvedcarapacelength(CCL),straightcarapace length(SCL),curvedplastronlength(CPL),curvedcarapacewidth (CCW),agestage,sexandweightwererecorded.Accordingtotheir CCLsize,allturtlesanalysedwerecategorisedintothreelifestage groups:early-stagejuveniles,late-stagejuveniles,oradultspecimens,assumingthatloggerheadsleavetheoceanicfortheneritic phaseatapproximatelythesize(CCL)of40cm,andthatonaverage theymatureatthesize(CCL)of70cm(Bjorndal,1997; Casaleetal., 2011; Fricketal.,2009; Margaritoulisetal.,2003).SamplecollectionandanalysiswereperformedaccordingtotheINDICITprotocol (INDICITconsortium,2018; Matiddietal.,2019).

2.2.Plasticanalysisandidentification

Thegastrointestinalcontentsofthedeadseaturtleswereanalysedforingestedplasticitems.EachGIsectionwasindividually measured(weight,length,width,fullness)anditscontentwas emptiedandwashedontopof3stackedmetallicsievesofdifferent meshsize(5mm,1mmand300 mm).Eachsievewasthoroughly washedandinspectedforplastic.Allplasticitemswerecounted, weighed,andcategorisedbysize,shape,colour,andpolymertype. Morespecifically,foreachsection,thenumberofplasticitems, alongwiththeirdryweight,volume,(maximum)lengthandwidth wererecorded.Plasticswerecategorisedbyshape(sheets,threads, foams,fragmentsandothers)accordingtotheMSFDMarineLitter WorkingGroupsuggestions “GuidanceonMonitoringofMarine LitterinEuropeanSeas” (Galganietal.,2013).Plasticswereseparatedintothreecolourgroups(white/transparent,dark,coloured) andthreesizecategories:microplastics(300 mm-5mm),mesoplastics(5 25mm),andmacroplastics(>25mm).Microplastics smallerthan300 mmwerenotcounted,inordertopreventmiscountingduetoairborneorothertypesofcontamination(in particular fibres).Inspectionofplasticitemswasperformedvisuallyandnodigestionprocesstookplace,soanunderestimationin theamountofsmallmicroplasticparticles(<1mm)ispossible. FourierTransformInfraredSpectroscopy(FT-IR)wasusedprimarily toconfirmthesyntheticpolymeroriginofalltheextracteditems andthenusedtoidentifytheirexactpolymertype.PriortoFT-IR analysisplasticsurfaceswerecleanedwithdistilledwater,inordertoachieveprominentspectraandaccurateresults(Jungetal., 2018).FT-IRanalysiswascarriedoutonanAgilentCary630FTIR spectrometerusingaself-generatedpolymerlibrary(i.e.spectraof referencepolymertypesprovidedbyindustry).Thelevelofcertainty,whencomparingsamplespectrumtothatoftheselfgeneratedlibrarydatabase,wassetto80%.

2.3.Dataanalysis

Theresultsarepresentedasmean ± standarderrorofthemean (SE).Asdatadidnotcomplywiththeassumptionsofnormalityand homogeneityofvariance(Shapiro-WilktestandLevene’stest, respectively),theMann-Whitney U testwasappliedtodetermine differencesinplasticnumbersamongagestages.Frequencyof occurrence,e.g.thepercentageofanimalswithplasticdebrisin theirgastrointestinaltract,wascalculatedforallindividualsand eachagestageseparately.Theaveragenumberofplasticsanddry weightofplasticitemsperturtlewerecalculatedbothforallindividualsandfortheindividualswithingestedplastics.ThelikelihoodratioChi-squaretestwasusedtocomparetypesofingested plastics(sizeclasses,shapes,coloursandsinkingversus floating plastics)amongstagestages.Relationsbetweenturtlesizeand plasticsnumber,plasticsweightorplasticssize,weretestedusing theSpearman’srankcorrelation.Significancelevelwassetat p < 0.05.StatisticalanalyseswereperformedwithSPSSStatistics Version20andwithRversion3.5.2.(RCoreTeam,2018).

3.Results

3.1.Turtlesbiometrics

TurtleCCLrangedfrom22.5to80cm,withameanof 57.44 ± 3.16cm.Outofthe36turtlesanalysed,eightwereearly-

stagejuveniles,18werelate-stagejuveniles,and10wereadult specimens(Table1).Turtlespresentedameanweightof 28.14 ± 5.51kg,rangingbetween1.2and78kg.Themeanweight fortheearly-stagejuveniles,thelate-stagejuveniles,andadults was8.31 ± 4.66kg,29.10 ± 6.44kg,and60.43 ± 9.95kg,respectively(Table1).Outofallturtlescollected,fourspecimenswere foundactivelyentangledinmarinedebris:oneearly-stagejuvenile, twolatejuvenilesandoneadultspecimen.

3.2.Occurrence

26outof36turtlesanalysed(72%)weredetectedashaving ingestedplasticinoneormoreoftheGIsections.Frequencyof plasticoccurrencewashighineachlife-stage,i.e.88%inearlyjuveniles,67%inlatejuvenilesand70%inadults(Table1).Atotalof 286plasticitemsweredetectedwithatotaldryweightof7.5g,and awetvolumeof80.28ml(Table1).Ofthese,81%werefoundinthe intestine(n ¼ 232,dryweight ¼ 7.06g),15%werefoundinthe stomach(n ¼ 44,dryweight ¼ 0.39g)and3%intheoesophagus (n ¼ 10,dryweight ¼ 0.09g).

3.3.Averagenumberofingestedplasticitems

Theaveragenumberofingestedplasticitemsperturtle, consideringallexaminedindividuals,was7.94 ± 3.85,anditwas significantlyhigherinearly-stagejuveniles(24.88 ± 16.32)when

Fig.1. HeatmapofdeadandaliveseaturtlestrandingsalongtheGreekcoastlineanalysedinthisstudy(coloursshowthedensitiesofthestrandingsineachlocation).(For interpretationofthereferencestocolourinthis figurelegend,thereaderisreferredtotheWebversionofthisarticle.)

N.Digkaetal./EnvironmentalPollution263(2020)114596

Table1

Biometricdata(mean ± SE),totalnumberanddryweightofingestedplasticitems,frequencyofoccurrenceofingestedplastics(%ofindividualscontainingplastics)and averages(±SE)ofthenumberandthedryweightofplasticitemsinloggerheadseaturtles(C.caretta)atthreeagestages(earlyjuveniles,latejuvenilesandadults).Averagesare calculatedinallindividualsaswellasinindividualswithingestedplastics.

AgestagesEarlyjuveniles(CCL < 40cm)Latejuveniles(CCL40 70cm) Adults(CCL >70cm) Allturtles

Numberofseaturtles8181036

MeanCCL(cm)29.91 ± 1.7859.73

Meanweight(g)8.31

Frequencyofplasticoccurrence(%)88677072

Totalnumberofplasticitems1996225286

Totaldryweightofplastics(g)3.212.751.657.61

Totalwetvolumeofplastics(ml)40.635.883.880.28

Meannumberofplasticitemsperturtle(allindividuals)24.88 ± 16.32a a 3.44 ± 1.59a a 2.50 ± 0.757.94 ± 3.85

Meannumberofplasticsperturtle(onlyinindividualswithplastics)28.43 ± 18.39a,a 5.17 ± 2.25a,a 3.57 ± 0.7511 ± 5.24

Meandryweightofplasticsperturtle(inallindividuals)(g)0.40 ± 0.170.15 ± 0.090.16 ± 0.080.21 ± 0.06

Meandryweightofplasticsperturtle(onlyinindividualswithplastics)(g)0.46 ± 0.180.23 ± 0.120.24 ± 0.111.29 ± 0.08 a Indicatessignificantdifferencebetweenearlyandlatejuvenileseaturtles(Mann-Whitney U test).

comparedtolate-stagejuveniles(3.44 ± 1.59)(Mann-Whitney U test,U ¼ 32,p < 0.05).Early-stagejuvenileplasticingestionwas alsohigherwhencomparedtoadultindividuals(2.5 ± 0.75), althoughnotsignificantly(Mann-Whitney U test,U ¼ 18.5, p > 0.05)(Table1).Themaximumnumberofplastics,(137items), wasfoundintheGIofanearly-stagejuvenile.Therewasnocorrelationbetweenturtlesize(CCL)andthenumberofplasticitems (Spearmanrho ¼ 0.3;p > 0.05).

3.4.Averageweightofingestedplasticitems

Theaverageweightofingestedplasticitemsperturtle, consideringalltheexaminedindividuals,was0.21 ± 0.06g,butno significantdifferencesweredetectedbetweenearly-stagejuveniles (0.4 ± 0.17g)comparedtolate-stagejuveniles(0.15 ± 0.09g) (Mann-Whitney U test,U ¼ 37,p > 0.05),andadults(0.16 ± 0.08g) (Mann-Whitney U test,U ¼ 23.5,p > 0.05)(Table1).Therewasno correlationbetweenturtlesize(CCL)andweightofplasticitems (Spearmanrho ¼ 0.1;p > 0.05).

Theaveragenumberofplasticsanddryweightofplasticitems perturtle,presentedin Table1,werecalculatedbytakingintoaccountallindividualsaswellasonlytheindividualswithingested plasticstofacilitatecomparisonwithotherstudies(Kühn,2015).

3.5.Plasticitemssize

Themaximumlengthofplasticitemsrangedfrom0.4mmto 550mm,andtheaveragemaximumlengthwas47 ± 3.6mm (mean ± SE).ThemeanlengthofplasticitemsperGIsectionwas lowerinthestomach(25 ± 6.1mm),incomparisontothe oesophagus(47 ± 14.5mm)andintestine(50 ± 4.2mm),butnot significantly(Mann-Whitney U test,U ¼ 35,p > 0.05).Nocorrelationbetweenplasticlengthandturtlesize(CCL)wasdetected (Spearmanrho ¼ 0.1;p > 0.05).

Amongstallplasticitemsrecoveredfromtheturtles,macroplasticswerethemostabundantsizecategory(53%),followedby mesoplastics(31%),andmicroplastics(16%)(Fig.2a).Themajority ofthemesoplasticandmacroplasticitemswerefoundintheintestine(84%and90%,respectively),whilefewerwerefoundinthe stomach(10%and7%,respectively)andtheoesophagus(6%and3%, respectively)(Fig.3a).Ontheotherhand,microplasticswerepresentinequalquantitiesinthestomach(52%)andintestine(48%), whilenomicroplasticswerefoundintheoesophagus(Fig.3a).

3.6.Plasticshapeandcolour

Intermsofplasticshape,amongstthewholeturtlesample

analysed,threads(45%)werethemostcommonwith128items, followedbysheets(36%)with103items,fragments(16%)with46 items,foam(2%)withsixitems,andthreeunclassifieditems(1%) (Fig.2b).Noturtlewasfoundtohaveingestedindustrialplastics (i.e.pellets).Incontrast,plasticsheetswerethemostdominant shapeinlate-stagejuveniles(66%)andadults(44%)showing significantlyhigherpercentagedistributionsthaninearly-stage juveniles(likelihoodratioChi-squaretest,p < 0.05)(Fig.2b). Foamwaspresentinearlyandlate-stagejuveniles,butnotinadult loggerheads.Fragmentswerethemostabundanttypeofmicroplastic(76%),followedbysheets(11%),foam(9%)andthreads(4%) (Fig.3b).Mesoplasticsweremainlysheets(60%),followedby threads(25%),fragments(13%)andfoam(2%)(Fig.3b).Macroplasticswererepresentedbythreads(68%),sheets(30%)andother shapes(2%),whilemacrofragmentsorfoamwerenotdetected (Fig.3b).

Amongallplasticsdetected,theamountofwhite/transparent plastics(n ¼ 138)wassignificantlyhigherthanthedark(n ¼ 76) andthecoloured(n ¼ 72)plastics(Mann-Whitney U test, U ¼ 446.5,p < 0.05).Similarcolourpatternswereobservedinall agestages(Fig.2c).

3.7.Plastictype

Outofallplasticitemsextracted(286),themostcommon polymersidentifiedinloggerheadswerepolypropylene(PP)(56%), followedbypolyethylene(PE)(29%),nylon(7%),polyvinyl(PVA) (2%),polystyrene(PS)(2%)andpolyethyleneterephthalate(PET) (1%).Ontheotherhand,inlate-stagejuvenilesandadults,PEwas themostabundantpolymertype(42%and40%,respectively), althoughsignificantdifferencesinnumberofPEitemscomparedto theotherpolymertypeswerenotdetected(Mann-Whitney U test, U ¼ 65,p > 0.05).Interestingly,thepercentageofsinkingplastics (i.e.PET,Nylon)wassignificantlyhigherinadultscomparedtoearly andlate-stagejuveniles(likelihoodratioChi-squaretest,p < 0.05) (Fig.2d).

4.Discussion

Theresultsofthepresentstudyshowclearevidenceofplastic ingestionbystrandedloggerheadseaturtlesalongtheGreek coastline.Thewholegastrointestinalcontentofstrandedindividualswasusedtoquantifytheingesteditems,awidelyused methodalthoughlimitedtodeadanimals(Schuyleretal.,2014).In thisstudy,allplasticslargerthan300 mmwerecounted,independentlyoftheirresemblance.However,severalstudiesexclude small-sizeditems(<1mm, <5mmor <10mm)foundinseaturtles’

Fig.2. Sizeclass(a),shape(b),colour(c)andpolymerbuoyancy(d)distributionofplasticitems(%)ingestedby Carettacaretta collectedinthisstudyalongwithagestages.(For interpretationofthereferencestocolourinthis figurelegend,thereaderisreferredtotheWebversionofthisarticle.)

Fig.3. GIsection(a)andshape(b)distributionofplasticitems(%)ingestedby Caretta caretta collectedinthisstudyalongwithplasticsizeclasses(micro,mesoand macroplastics).

gastrointestinaltracts,consideringthemtobefragmentedpiecesof largeringesteditems(Nicolauetal.,2016; Phametal.,2017; Tomas etal.,2002).FTIRspectra(Fig.4)ofvisuallyresemblingplastic itemsprovidevaluableinformation,astheymayindicateacommonoriginofthedegradedplastics(Corcoranetal.,2009),leading toanimportantreductioninthenumberofrecordedingested items.Thisambiguityhighlightstheneedforstandardisationofthe methodsusedforquantificationofingesteditems.Currently,there isnoconsensusonwhetherwhencountingingestedplastic,one shouldconsider,ornot,visuallysimilarplasticsasoneitem. Includingthenumberofeverysingleplasticitemisessentialto increaseconsistencyamonglaboratories.

Thefrequencyofoccurrenceofingestedplasticsofthisstudy

Fig.4. Absorbancespectraofsixvisuallysimilarplasticitems(bluelines)overlapped withthereferencepolymerspectrum(redline).Peaksotherthanthoseofthereferencepolymershowthepossibilityofacommonoriginofthesesixplasticitems (yellowarrows).(Forinterpretationofthereferencestocolourinthis figurelegend,the readerisreferredtotheWebversionofthisarticle.)

(72%)isatsimilarlevelsofplasticoccurrenceinstrandedoraccidentallycaughtloggerheadsdocumentedby Domenechetal. (2019) (69%)andby Tomasetal.(2002) (75.9%)intheWestern MediterraneanSea,andby Campanietal.(2013) (71%)inthe CentralMediterraneanSea.Theoccurrenceofingestedplasticswas higherinourstudycomparedtothosepresentedby Cameddaetal. (2014) (13.2%), Casaleetal.(2008) (48.1%)and Gramentz(1988) (6%)intheCentralMediterraneanSea,andby LazarandGracan (2011) (24%)intheAdriaticSea,butlowercomparedto Matiddi etal.(2017) (83%)intheWesternMediterraneanSea.Inthecase of Matiddietal.(2017),frequencyofoccurrencerefernotonlyto plasticsbuttoallanthropogenicmarinelitter(e.g.paper,wood, metal),whichmayexplainthedifferencetoourresults.Thelimited informationonplasticingestionbyseaturtlesintheEastern MediterraneanSeashowslowplasticoccurrence(5%)alongthe Turkishcoast(Kaskaetal.,2004),while Duncanetal.(2019) recordedmicroplasticingestionin100%oftheseaturtlesanalysedinNorthernCyprus.Ingeneral,occurrencevariationsamong studiescanbeduetodifferencesinsamplingandplasticsdetection methodology,theoriginofthesamples(bycatchorstranded),the sizeofthedebrisconsidered,theorganssampledandthelifehistorystages,whichincludesvariationsamongthesizerangeandage stagesofthespecimens(Casaleetal.,2016; Nelmsetal.,2016; Phametal.,2017).Resultsonthemeannumberofplasticsand meandryweightofplastics,determinedinthisstudy,fallinthe rangeofvaluesreportedinloggerheadsworldwide(Cameddaetal., 2014; Campanietal.,2013; LazarandGracan,2011; Matiddietal., 2017; Nicolauetal.,2016; Phametal.,2017; Tomasetal.,2002). Despitetheglobaltendenciesofloggerheadsbeinglessproneto plasticingestioncomparedtootherspecies(Schuyleretal.,2014), thefrequencyofoccurrenceofplasticingestionrecordedinloggerheadsbythepresentstudy(72%)iscomparabletootherspecies intheMediterraneanSea(DiBenedittoandAwabdi,2014; Yaghmouretal.,2018).Interspeciesvariationonplasticingestion, possiblyrelatedtothefeedingpreferencesofeachspecies,hasbeen reported:greenturtles,mainlyherbivoresshowagreater

preferencetowardssoftorganismswhicharesimilartosoftplastics (Bjorndal,1997),leatherbacks(gelatinovores)aremorelikelyto ingestplasticsduetotheirresemblancetogelatinousorganisms (Schuyleretal.,2014)andhawksbills(omnivores)feedonavariety ofitems,whichcouldincludeplastics.Also,accordingto Bugoni etal.(2001),late-stagejuvenileandadultloggerheadseaturtles havealargergastrointestinaltractdiameterthangreenturtlesofa similarage,whichmaymakeiteasierforplasticstopassthrough andbeexcreted.

Variationofplasticingestionamongdifferentloggerheadlife stagespresentedinourstudymaybeduetotheirdifferenthabitats andfeedingecology.Seaturtleslivinginoceanicorcoastalenvironments,thatfeedinpelagicorbenthichabitats,mayencounter verydifferentdensitiesandtypesofmarinedebris,andmay, therefore,havedifferentprobabilitiesofdebrisingestion(Schuyler etal.,2014).Smalloceanicturtlesareconsideredmorelikelyto ingestdebris,thanolderbenthic-feedingturtles(PlotkinandAmos, 1990; Schuyleretal.,2012).Thisisinagreementwithourresults,as moreearly-stagejuvenileloggerheadswerefoundtoingestplastics (88%)comparedtolate-stagejuvenile(66%)andadultspecimens (70%).

Accordingly, Casaleetal.(2008) reportedahigheroccurrenceof plasticingestionbyloggerheadscaughtinoceanicwaters(64%) thaninbenthicwaters(22%)bytrawl fishers. Phametal.(2017) foundsimilarlevelsofplasticingestionoccurrence(83%)tothose ofthepresentstudyinoceanic-stageloggerheadsoftheNorth Atlantic,while Cameddaetal.(2014) foundlitterin25%ofthesmall juvenileloggerheads(CCL < 40cm)andin11.8%ofthelargerneritic adults(CCL > 40cm)intheSardiniansea.Consideringthevariationsinthenumberofingestedplasticitemsamongstlifestagesof theloggerheadsinthepresentstudy,themeannumberofplastic itemsperturtleinearly-stagejuveniles(24.88 ± 16.32)was significantlyhigherthaninthelate-stagejuveniles(3.44 ± 1.59), althoughdifferencesarenotsignificantwhentheearly-stagejuvenileindividualthathadingested137plasticitemsisexcluded; thustheseresultsshouldbeconsideredwithcaution.Toour

Fig.5. Examplesofingestedplasticitemsfoundinloggerheadseaturtles:(a)thread-likeplasticitemsrelatedto fishingactivity,(b)sheet-likeplasticitemsoriginatedfromfood wrapsandshoppingbags,(c)fragmentsofplasticitemsofunknownorigin,(d)otherundeterminedplasticitems.

N.Digkaetal./EnvironmentalPollution263(2020)114596

knowledge,informationonthevariationofthemeannumberof plasticitemsperturtleamongstagestagesisabsent,socomparisonswerenotpossible.

Inthisstudythemajorityoftheplasticitems,intermsof numberanddryweight,weredetectedintheintestine;acommon findinginotherstudies(Matiddietal.,2017; Nicolauetal.,2016; Phametal.,2017).Theintestine,duetoitslength,islikelytoactas anaccumulationareaforingestedplasticdebrisinloggerheads (Matiddietal.,2017).Thisstronglysuggeststhattoachieveaccurateresultsonplasticabundancewheninvestigatingplastic ingestioninloggerheads,intestineexaminationcannotbe excluded.Furthermore,theseresultsimplythatmostoftheplastic debrismanagestopassthroughloggerheads’ digestivetractandis closetobeingexcretedagaininthemarineenvironment,indicating lowvulnerabilitytoplasticingestion(Hoarauetal.,2014; Nelms etal.,2016).

The86%oftheingestedplasticsdetectedintheloggerheadsare inclinedto floatinseawater(PE,PP,PVA,PS),whiletheresttendto sink(PET,nylon)(Bergmannetal.,2015).Late-stagejuvenilesand adultindividualsingestedhigherpercentagesofsinkingplastics (15%and24%,respectively),incomparisontoearly-stagejuveniles (4%).Theabovecanberelatedtothedivingcapacityofthebenthicfeedingadults,incontrasttothenear-surfacefeedingbehaviourof theearly-stagejuvenileloggerheads(Bolten,2003; Schuyleretal., 2014).Amongallplasticitemsdetected,themostcommonshape wasthreads(45%)mostlyrepresentedbypiecesof fishingnetsand fishinglines(Fig.5a),followedbysheets(36%),whichweremainly piecesofplasticbagsandfoodwraps(Fig.5b).Plasticfragmentsof largerplasticitemswerealsofoundmainlyinthecategoryof microplastics(Fig.5c),whileotherundeterminedshapeswerealso detected(Fig.5d).Threadswerelesscommoninlate-stagejuvenilesandadults(19%and20%,respectively),comparedtotheearlystagejuveniles(56%).Thehighpercentageofthreadsislikely relatedtotheinteractionbetweenthenear-surfaceearly-stagesea turtlesandthelongline fisheriesinthesurfacewaters,aspreviouslysuggestedby Parkeretal.(2005) intheNorthPacific,since fisheryandaquacultureactivityiswidelyspreadinGreekwaters (Fisheries,2019).Additionally,ourresultsindicateaprevalenceof white/transparentplastics(48%)inloggerheadseaturtlesinalllife stages,inaccordancetopreviousstudies(Cameddaetal.,2014; Casaleetal.,2016; Fukuokaetal.,2016; Phametal.,2017).One possibleexplanationisthatlightcoloureddebrisislessconspicuousinthewaterandcouldbeingestedbymistakebytheforaging seaturtle(Santosetal.,2015).Otherssuggestthatseaturtles, includingloggerheads,haveshowncolourselectivityfortheirprey (Swimmeretal.,2005),whichmayexplainlightercoloureddebris prevalenceduetotheirresemblancetojellyfish(Hoarauetal., 2014; Schuyleretal.,2014; UNEP,2009).However,higherproportionsoflightercoloureddebrisinthemarineenvironment(Pan etal.,2019; Ryanetal.,2009),couldalsoexplainthehigherfrequencyofthesecoloursinseaturtleGI.

5.Conclusion

Thisisthe firststudytoshowclearevidenceofplasticsingestion byloggerheadseaturtles(C.caretta)inGreekwaters.Ourresults indicateahighoccurrenceofplasticingestionwhichmayvary throughoutseaturtles’ lifestages.Our findingsaddvaluableinformationonplasticingestionbyloggerheadsandfurthersupport theirpotentialuseasbio-indicatorspeciesforthemonitoringof litterandmicro-litterinthemarineenvironmentatabasinscale. Onceindicatorspeciesformonitoringmarinelitterareestablished, baselinevaluesofingestedlittercanbeusedtosetthresholdvalues fortheassessmentofGESfortheMSFDcriteriononlitteringestion D10C3 “Theamountoflitterandmicro-litteringestedbymarine

animalsisatalevelthatdoesnotadverselyaffectthehealthofthe speciesconcerned”.Thedataofthisstudywillcontributetothe definitionofbaselinelevelsofplasticingestedbyseaturtlesinthe Mediterraneanregion.However,researcheffortsforthestandardisationofmethodologiesdesignedtoquantifylevelsofingested debrisareessential,aswellas,furtherinvestigationsintothe impactandthepotentialadverseeffectsofplasticingestiononthe healthstatusofseaturtles.

Declarationofcompetinginterest

Theauthorsdeclarethattheyhavenoknowncompeting financialinterestsorpersonalrelationshipsthatcouldhave appearedtoinfluencetheworkreportedinthispaper.

CRediTauthorshipcontributionstatement

NikolettaDigka: Investigation,Formalanalysis,Writingoriginaldraft. LauraBray: Investigation,Writing-review & editing. CatherineTsangaris: Supervision. KonstantinaAndreanidou: Investigation. EiriniKasimati: Investigation. EvangeliaKofidou: Investigation. AnastasiaKomnenou: Investigation,Supervision. HelenKaberi: Supervision,Resources.

Acknowledgements

TheauthorswouldliketothankI.Kalliasforhisvaluable assistancewiththesampleanalysis.Thisstudywasfundedbythe INTERREGMediterranean-BiodiversityProtectionprojectMEDSEALITTERMediterraneanINTERREGproject[grantnumber310 1MED15_3.2_M12_334]andINDICITEuropeanDG-ENVprojectGA No.11.0661/2016/748064/SUB/ENV.C2.

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