Validation of an optimised protocol for quantification of microplastics in heterogenous samples: a case study using green turtle chyme

Method

Article

Validation

of

an

optimised

protocol

for

quanti

fi

cation

of

microplastics

in

heterogenous

samples:

A

case

study

using

green

turtle

chyme

Alexandra

G.M.

Caron

a,b,c

,

Colette

R.

Thomas

b,d

,

Kathryn

L.E.

Berry

a,c

,

Cherie

A.

Motti

a,

*

,

Ellen

Ariel

e

,

Jon

E.

Brodie

f

a

AustralianInstituteofMarineSciencePM3,TownsvilleMC,QLD4810,Australia

b

CentreforTropicalWaterandAquaticEcosystemResearch(TropWATER),JamesCookUniversity,Townsville 4811,Australia

c

AIMS@JCU,AustralianInstituteofMarineScienceandJamesCookUniversity,Townsville,Australia

d

SEEDScience,Australia

e_{CollegeofPublicHealth,MedicalandVeterinarySciences,JamesCookUniversity,Townsville4811,Australia} f_{ARCCentreofExcellenceforCoralReefStudies,JamesCookUniversity,Townsville4811,Australia}

ABSTRACT

Quantifyingtheextentofmicroplastic(<5mm)contaminationinthemarineenvironmentisanemergingfieldof

study.Reliableextractionofmicroplasticsfromthegastro-intestinalcontentofmarineorganismsiscrucialto

evaluatemicroplasticcontaminationinmarinefauna.Extractionprotocolsandvariationsthereofhavebeen

reported,however,thesehavemostlyfocussedonrelativelyhomogenoussamples(i.e.water,sediment,etc.).

Here,wepresentamicroplasticextractionprotocolforexamininggreenturtle(Cheloniamydas)chyme(i.e.

ingestedmaterialanddigestivetractfluid),whichisaheterogeneouscompositeofvariousorganicdietaryitems

(e.g.seagrass,jellyfish)andincidentally-ingestedinorganicmaterials(sediment).Establishedextractionmethods

weremodifiedandcombined.Thisprotocolconsistsofaciddigestionoforganicmatter,emulsificationofresidual

fat,densityseparationfromsediment,andchemicalidentificationbyFouriertransform-infraredspectroscopy.

Thisprotocolenablestheextractionofthemostcommonmicroplasticcontaminants>100mm:polyethylene,

high-density polyethylene, (aminoethyl) polystyrene, polypropylene, and polyvinyl chloride, with 100%

efficiency.Thisvalidatedprotocolwillenableresearchersworldwidetoquantifymicroplasticcontamination

inturtlesinareliableandcomparableway.

Abbreviations:SLS,sodiumlaurylsulphate;PE,polyethylene;HDPE,highdensitypolyethylene;PVC,polyvinylchloride;PP, polypropylene;PET,poly(ethyleneterephthalate);AM-PS,(aminoethyl)polystyrene;RO,reverseosmosis;ATR-FTIR,attenuated totalreflectanceFouriertransform-infraredspectroscopy.

*Correspondingauthorat:AustralianInstituteofMarineSciencePM3,TownsvilleMC,QLD4810,Australia.

E-mailaddresses:[email protected](A.G.M. Caron),[email protected](C.R. Thomas),

[email protected](K.L.E.Berry),[email protected](C.A. Motti),[email protected](E.Ariel),

[email protected](J.E. Brodie).

https://doi.org/10.1016/j.mex.2018.07.009

2215-0161/©2018TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http:// creativecommons.org/licenses/by/4.0/).

ContentslistsavailableatScienceDirect

MethodsX

Optimization ofmicroplasticextraction from multifarioustissues byapplyingestablished methodsin a

sequentialmanner.

Effectiveforheterogenoussamplescomprisingorganicandinorganicmaterial.

©2018TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://

creativecommons.org/licenses/by/4.0/).

ARTICLE INFO

Methodname:Sequentialprotocolforextractingmicroplasticsfromheterogenoussamplematrices

Keywords:Plasticingestion,Marinedebris,Plasticcontamination,Extractiontechnique,Chemicaldigestion,Densityseparation, Fouriertransform-infraredspectroscopy

Articlehistory:Received28March2018;Accepted11July2018;Availableonlinexxx

SpecificationsTable

Subjectarea EnvironmentalScience

Morespecificsubjectarea Marinepollution

Methodname Sequentialprotocolforextractingmicroplasticsfromheterogenoussamplematrices

Nameandreferenceof originalmethod

Nitricaciddigestion:Claessens,M.,VanCauwenberghe,L.,Vandegehuchte,M.B.,Janssen,C. R.,2013.Newtechniquesforthedetectionofmicroplasticsinsedimentsandfieldcollected organisms.Mar.Pollut.Bull.70,227–233.doi:https://doi.org/10.1016/j.

marpolbul.2013.03.009

Densityseparation:Hidalgo-Ruz,V.,Gutow,L.,Thompson,R.C.,Thiel,M.,2012.Microplastics inthemarineenvironment:Areviewofthemethodsusedforidentificationand quantification.Environ.Sci.Technol.46,3060–3075.doi:https://doi.org/10.1021/es2031505

FTIRspectroscopy:Hidalgo-Ruz,V.,Gutow,L.,Thompson,R.C.,Thiel,M.,2012.Microplastics inthemarineenvironment:Areviewofthemethodsusedforidentificationand quantification.Environ.Sci.Technol.46,3060–3075.doi:https://doi.org/10.1021/es2031505

Kroon,F.,Motti,C.,Talbot,S.,Sobral,P.,Puotinen,M.,2018.Aworkflowforimproving estimatesofmicroplasticcontaminationinmarinewaters:Acasestudyfrom North-WesternAustralia.EnvironmentalPollution,238,26–38.

Thompson,R.C.,Olsen,Y.,Mitchell,R.P.,Davis,A.,Rowland,S.J.,John,A.W.G.,McGonigle,D., Russell,A.E.,2004.Lostatsea:Whereisalltheplastic?Science304,838.doi:https://doi. org/10.1126/science.1094559

Jung,M.R.,Horgen,F.D.,Orski,S.V,C,V.R.,Beers,K.L.,Balazs,G.H.,Jones,T.T.,Work,T.M., Brignac,K.C.,Royer,S.,Hyrenbach,K.D.,Jensen,B.A.,Lynch,J.M.,2018.ValidationofATR FT-IRtoidentifypolymersofplasticmarinedebris,includingthoseingestedbymarine organisms.Mar.Pollut.Bull.127,704–716.doi:https://doi.org/10.1016/j.

marpolbul.2017.12.061

Resourceavailability Hardware: Extraction:

Aluminiumtraysandfoil

Blockheater(80_{C,modelAIM500,SEALAnalytical)}

Branson12200UltrasonicClearer

Coveredglassdishes(shallow,flatbottomed,5cmdiameter) Freezer( 20C)

Fridge(4_C)

Glassbeakers(200mL) GlassPasteurpipette Glassstirringrod Glasstesttubes(50mL)

GlassVacuum-filterapparatus(glassfritBuchnerfunnelwithmetalclampandvacuum Erlenmeyerflask;Millipore)

Hotplate(50_{C,StuartCB160)}

Lintfreetissue

Maggylamp(2magnification) Metalbucket

Metalneedles Metaltweezers Metalteaspoon

Oven(60C) Paperclip

Pliablesteelfabricwithameshsizeof100mm Steelmeshsieve(100mm,20cmdiameter) Analysis:

PerkinElmerSpectrum100InfraredSpectrometerwithAttenuatedTotalReflectance Software:

PerkinElmerSpectrum(V.10.5.4);NICODOMIRlibraries(PolymersandAdditives, Coatings,Fibres,DyesandPigments,PetrochemicalsFullVersion;NICODOMLtd.,Czech Republic)

Reagents:

Chemicaldigestion:Nitricacid(HNO3,69.5%,Scharlau)

Densityseparation:NaClsolution,1.2g/cm3

,SigmaAldrich

Emulsification:sodiumlaurylsulphatepellets(SLS,AcrosOrganics,CASnumber 151-21-3)

Fouriertransform-infraredspectroscopy:methanol Reverseosmosiswater

Methoddetails

Protocolbackground

Plasticsareubiquitousandwidespreadinthemarineenvironment;pollutingtheoceansurface, watercolumn, and benthos [1–3]. Theyare broadly divided intotwo categories; macroplastics (>5mm)andmicroplastics(<5mm,[4,5]),andmainlyimpactmarinelifethroughtheprocessesof entanglementandingestion[6].Allseventurtlespeciesareknowntobeaffectedbyplasticdebris globally[7–10].Unlikeothermarineorganisms,theherbivorousfeedingstrategiesofgreenturtles make them highly susceptible to ingestion of marine debris [11,12]. Plastic debris can become entangled among green turtle food sources such as seagrass leaves and macroalgae [13]. The backward-facing oesophageal papillae of turtles inhibits regurgitation and facilitates particle accumulationinthegut[14–16].Chyme(i.e.ingestedmaterialanddigestivetractfluid)from non-pelagicgreenturtlesisexpectedtoberelativelycomplex,comprisingarangeoforganic(plantand animalmaterial)andinorganic(mineralandsediment)matrices,astheirbroaddietincludesjellyfish, sponges,seagrass,algaeandassociatedsedimentsandepibionts[37].Therefore,aprotocolcapableof efficientlyextractingmicroplasticsfromallmatricesisrequiredtoaccuratelyestablishcontamination levels.

Methodsforextractingmicroplasticshavebeendevelopedforarangeofrelativelyhomogeneous samplematricesandinclude:visualassessmentusingmicroscopy[17],densityseparationusing hypersaline solutions [18–21] and chemical digestion [18,22–24]. Many of these methods are suitableandefficientforeitherhomogenouslyorganicorhomogenouslyinorganicsamplematrices; however,eachofthemalonearenotsuitableformicroplasticextractionfromgreenturtlechyme. Herewetestseveralmicroplasticextractionmethodsandreagentvariationsincombinationusinga processofeliminationtoidentifytheoptimalstepsnecessarytoestablisharobustandreliableway toextractandquantifymicroplasticcontaminationinavarietyofheterogeneousmarineorganism tissues.

Materialandmethods

AsummaryofthemicroplasticsequentialextractionprotocolispresentedinFig.1.

Samplepreparation

Materials.

Aluminiumtraysandfoil

Freezer( 20C)

Fridge(4_C)

Largeglassbeakerormetalbucket,tofitchyme

Largemetalspoonorladle

ROwater

Steelmeshsieve(100

m

m,20cmdiameter)

Procedure.

Greenturtlesthathadbeeneitherfounddeadorrequiredeuthanizingwerekindlymadeavailable byQueenslandParks and WildlifeService (QPWS).Necropsies wereundertaken bya qualified veterinary pathologist. Foreguts (including oesophagus, stomach, and small intestine) were necropsied,therestofthedigestivetractbeingrequiredforadifferentstudy.Anteriorandposterior extremitiesofthetractswereclosedwithnon-plasticstringspriortoremoval.Tractswereextracted fromturtlecavitiesandstoredinaluminiumtraysandfoil,andfrozenat 20Cwithinafewhours. Tractswerethawedinafridgeat4Cfor48hpriortoanalysis.

Collectchymefromgreenturtledigestivetractandtransferitintoaglassormetalcontainer.Mostof thechymecanbecollectedusingametalspoonorladle.Tocollectchymeadheredtothe gastro-intestinaltract,rinsetractwithROwaterovera100

m

msteelmeshsieveandaddretainedresidueto thechyme.Thefiltrate,being<100

m

misdiscarded.

Visuallyinspectthechymeandremoveanyvisiblemacro-debris(i.e.rope,_fishingline,bags,bottle lids,etc.).RinsethemwithROwaterover100

m

msteelmeshsieveandaddretainedresiduetothe chymetoassureminimallossofpotentialmicroplastics.Thefiltrate,being<100

m

misdiscarded.

Homogenisethechymebymanualstirringforoneminutewithametalspoon(excessivemechanical stirringorsonicationshouldbeavoidedtoreducethelikelihoodoflargerbrittleplasticitemsfurther fragmenting).Thoroughhomogenisationofthechymeismandatoryespeciallyifanalysingonlya

[image:4.468.56.415.63.258.2]

subsampleofthewholechyme,asplasticdebristendtoaccumulateinsomepartsofgreenturtle digestivetract[7,25].

WeighthechymesampletodeterminethenumberoftesttubesandvolumeofHNO3required(see

Aciddigestionoforganicmaterials).

Aciddigestionoforganicmaterials

Materials.

Aluminiumfoil

Blockheater(80C,modelAIM500,SEALAnalytical)

Glassbeaker(500mL,ortofitvolumeofsamplesandHNO3)

Glassstirringrod

Glasstesttubes(50mL)

Metalteaspoon

Nitricacid(HNO3,69.5%,Scharlau)

Paperclip

Pliablesteelfabricwithameshsizeof100

m

m

ROwater

Procedure.

Put6gwetweight(w/w)ofhomogenisedchymeper50mLtesttubeusingametalteaspoon. Wherechymefromanindividualis>6g,thesampleshouldbesplitandmultipletesttubesused.

Add18mLofHNO3toeachsample(ratio3:1,HNO3mL:chymegw/w).

Placethetesttubesintotheblockheater.Coverthetesttubeswithaluminiumfoilandletthemsit overnightatroomtemperature(20C).

Uncoverthetesttubesandheatsamplesfortwohoursat80Cintheblockheater.Testtubesshould beclosely monitored.Theycanbecautiouslystirredwitha glassstirringrodtomake sureall materialsremainimmersedandtominimisefoaming.

Filtersampleswhilestillwarmoverthe100

m

msteelmeshsieve.Tofilter,foldthe100

m

mmesh intoaconicalshapeandsecurewithapapercliptoasuitably-sizedglassbeaker.Allowretained materialtogatheratthebottomoftheconeforeaseofrinsingandcollection.

Discardtheacidsolutiondownthesinkwithtentimesitsvolumeoftapwatertoneutralizeit.

RinsecollectedmaterialswithROwateroverthesinkanddiscard_filtrate.Whilestillinthesieve, visuallyinspectthedigestedsampleforundigestedfatresidueand sediment.Iffatresiduesare observedproceedwiththeemulsificationmethod(Fig.1;seeEmulsificationoffats).Ifnofatresidues areobservedbutsediment ispresent,proceedwiththedensityseparationmethod(seeDensity separationfromsediments).Ifnofatresiduesorsedimentarepresent,proceedwithfiltration(see Filtration).

Note. Chyme fromdifferentturtlescan have differentreactivity toHNO3,especially during the

heatingstep.Veryreactivesamplescanproduceanunmanageableamountofhazardousfoaming. Runningatestpriortoapplyingthismethodtoalargenumberofsamplesisrecommended.Reduce thequantityofsampleinthetesttubeifthechymereactstoostronglytoHNO3.

Depending on the composition and complexity of the samples, multiple samples from one individualcanbecombinedatthefiltrationstep.Actaccordingtotheamountofresidueretained. Combiningtheresidue reducesthenumberof samplestobeprocessed insubsequentstepsand streamlinestheoverallprocess.

Emulsificationoffats

Materials.

Glassbeaker(200mL)

GlassPasteurpipette

Glassstirringrod

Hotplate(50C,StuartCB160)

Metalteaspoon

Paperclip

Pliablesteelfabricwithameshsizeof100

m

m

ROwater

Sodiumlaurylsulphatepellets(SLS,100%,AcrosOrganics,CASnumber151-21-3)

Ultrasonicbath(47kHz6%,Branson12200UltrasonicClearer)

Procedure.

Prepareawarm1g/LSLSsolutionbyaddingSLSpelletstoROwaterona50Chotplate.Usethe glassstirringrodtostiruntilwelldissolvedandkeepat50Conthehotplate.

Re-suspendallretainedresidue(i.e.collectedonthe100

m

msieve,seeAciddigestionoforganic materials)remainingfromtheaciddigestioninthewarmSLSsolution.Todoso,placethesievemesh upside-downovera200mLglassbeakerandrinseitusingaglasspipetteandthewarmSLSsolution (50mLorenoughtoremoveallresidue).Onceallmaterialsaretransferredintotheglassbeaker, addextraSLSsolutiontogiveafinalvolumeof200mLifrequired.

Sonicatefor 30sin an ultrasonic bath(47kHz6%). Avoidextended sonication toreducethe likelihoodoflargerbrittleplasticitemsfurtherfragmenting.

Filter samples while still warm over the100

m

msieve(as doneinAciddigestionof organic materials;fold the100

m

mmeshintoaconicalshapeandsecurewithapapercliptoasuitably-sizedglassbeaker).

DiscardSLSsolutiondownthesinkwithtentimesitsvolumeoftapwatertoneutralizeit.

Repeattheoperationuntilnofatresidueisobservedonthesieve.

RinseretainedmaterialswithROwateroverthesinkanddiscardfiltrate.Whilestillinthesieve, visuallyinspectretainedmaterialsforsediment.Ifsedimentisobserved,proceedwiththedensity separationmethod(Fig. 1,seeDensityseparationfromsediments).Ifnosedimentisobserved,proceed withfiltration(seeFiltration).

[image:6.468.130.345.64.268.2]

Note. Thisemulsificationmethodwassuccessfulindissolvingdropletsoffats.Iftheamountoffat presentinthesamplesislarge,suchaschunks,orformsathicklayer,furtherhomogenizationmaybe necessary,ordifferentdigestionscouldbetested.ThiscouldincludesampledigestionusingKOH (needingfromafewdaystoafewweeksprocessing)whichhasbeensuccessfullyappliedtostomach contentof_fish[22,26].Ifcombiningsamples(fromthesameindividual)afteraciddigestion,make suretovisuallyinspecttheretainedmaterialsonthesieve;itisimportanttomaintainamanageable amountoffatfortheemulsificationmethod.

Densityseparationfromsediments

Proceedwithdensityseparationifsedimentispresentafterfiltration(i.e.eitherafteraciddigestion orafteremulsification;Fig.3).

Materials.

Aluminiumfoil

Glassbeakers(200mL)

GlassPasteurpipette

Glassstirringrod

NaCl(SigmaAldrich)

ROwater

Procedure.

Prepareahypersalinebrine(NaCl,1.2g/cm3_{)bysaturatingROwaterwithNaCl(watersolubilityat}

20_{C=357g/L).NaClmustbeaddedwhilecontinuouslystirringuntilitnolongerdissolves.} Re-suspendallretainedmaterialsfromthepreviousmethodused(eitheremulsificationoracid

digestion)in200mLofthehypersalinebrine.Todoso,placethesievemeshupside-downovera 200mLglassbeakerandrinseitusingaglasspipetteandthehypersalinebrine(50mLorenough toremoveall retainedmaterials).Once transferred,addextra hypersalinebrinetogivea final volumeof200mL.

Stirthesolutionwiththeglassstirringrodfor30s.Rinsethestirringrodwiththebrineintothe beakercontainingthesample.

[image:7.468.123.340.405.606.2]

Coverthebeakerwithaluminiumfoilandallowtosettlefor1h.

Collectthetop20mLofthesolutionusingaglassPasteurpipetteandtransferittoaclean200mL glassbeaker.

RinsethepipettethreetimeswithROwaterintotheclean 200mLglassbeakertoremoveany particles that may have adhered tothe internal or external walls of the pipette. Discard the remaining180mLofbrineandsediment.

Note. MakesureNaClis freeofplasticcontamination;wherepossibleusescientificgradeNaCl. NaClinsoftplasticbagsorinplasticmeshbagscanbecomecontaminatedwithmicroplasticsasbags age and degrade.In addition, ensure the salt is stored in a covered, clean container, to avoid microfibercontaminationwhilethesaltisexposedtothelabair.Regardless,preparedNaClsolutions should bechecked for contamination priorto undertakingdensityseparation. If necessary, the solution should be filtered through cellulose nitrate/acetate 0.45

m

m pore membrane filters (MilliporeHA).

Filtration

Materials.

Coveredglassdishes(shallow,flatbottomed,5cmdiameter).Adaptedsizetomatchthefilters.

GlassPasteurpipette

GlassVacuum-filterapparatus(glassfritBuchnerfunnelwithmetalclampandvacuumErlenmeyer

flask;Millipore)

Metaltweezers

MilliporeHAcellulosenitrate/acetate0.45

m

mporemembranefilters,larger(>100

m

m)poresizes canbeusedtoselectivelyrecovermicroplasticsindifferentsizeranges

Oven(60C)

ROwater

Procedure.

Filterthecollected(lowdensity)supernatantfromthedensityseparationmethodundervacuum onto0.45

m

mporemembranefilters.WithROwater,rinsethewallofthe200mLglassbeakerinto thevacuum-_filterapparatus a minimum of threetimes (oras much asneeded) torecoverall particles.If thedensity separation methodwas not used(i.e.no sediment was presentin the samples),materialscollectedonthe100

m

msieveafterHNO3 digestionorafteremulsification

shouldbevacuum-filteredasabove.

Rinsethewallofthevacuum-filterfunnelthreetimeswithROwaterusingapipettetoensureall particlesareonthefilter.

Carefullyplaceeachfilterinacoveredglassdishusingmetaltweezers.

Oven-dryat60Cfor4h.

Visuallyinspectfiltersformicroplastics[17].CharacteriseparticlesusingFTIRspectroscopy(see Polymercharacterisation).

Polymercharacterisation

Polymer composition of recovered microplastics can be determined using FTIR spectroscopy [21,22,27–30].

Instrumentandsoftware.

PerkinElmerSpectrum 100 infraredspectrometer with attenuated total reflectance (ATR-FTIR) operatedwithPerkinElmerSpectrum(V.10.5.4)software

Materials.

Lintfreetissue(forcleaning)

Maggylamp(2magni_fication)

Metaltweezersandneedles

Methanol(forcleaning)

Procedure.

Noadditionaltreatmentofthemicro-particlesisnecessaryprior toFTIRanalysis.Macroplastics recovered duringthevisualinspectionofthechyme(see Samplepreparation)shouldberinsed thoroughlywithROwaterandoven-driedat60Cfor4hbeforeanalysisbyATR-FTIR.

PlaceindividualitemsontheATR diamondusingtweezersorneedles.Useapressureclampto ensuregoodcontactwiththesample.

AcquireFTIRspectraintransmissionmodeonaPerkinElmerSpectrum100FTIRSpectrometerusing anattenuated totalreflectance(ATR)accessoryas perKroonetal. [29]. UsetheData Tune-up commandtosmoothandperformabaselinecorrectionforallspectrausingdefaultPerkinElmer parameters.Inthisstudyspectraldatawereaccumulatedfor64scansat4cm 1_{resolutionwitha}

wavenumber range of 4000–400cm 1 _{[22,27] using Euclidian distance against commercially}

availableNICODOMIRlibrariesandapercentmatchbetweenthereferencespectraandthesample obtained.

AsperKroonetal.[29]sampleswithapercentmatchof<60%wereconsideredalowmatch,60–

<70%anintermediatematchand70–100%ahighmatch.Allspectramustbefurtherinspectedand anyunexplainedbandsinvestigatedbyreviewingthelowerpercentmatchesandtheliteratureto confirmpolymeridentification.

Protocolvalidationandsuitability

Protocolvalidation

The extractionefficiency of the sequential protocol was assessed by spiking three 6g (w/w) homogenisedchymesamples(seeSamplepreparation)withfivemicro-beads(high-density polyethyl-ene(HDPE);19248

m

m).Spikedsampleswerethenprocessedwiththesequentialprotocol.Since bothfatandsedimentswerepresentinthesesamples,allthreeextractionmethods:aciddigestion, emulsification of fats, and density separation, wereused. Every micro-bead was recovered; the extractionefficiencyoftheextractionprotocolwas100%forthe>100

m

mHDPEmicro-beads.

Toassessproceduralcontamination,threeblankscomprising6mLROwaterwereprocessedin accordancewiththesequentialextractionprotocol.Afteroven-drying,filterswerevisuallyinspected underadissectingmicroscopeandanyparticleorfibrepresentonthefilterswastreatedasprocedural contamination.Proceduralcontaminationblanksrevealedthepresenceofhair-likefibresandvery

finedarkparticles<100

m

moneachfilteraspreviouslyreportedbyFoekemaetal.[22].Becausethese particlesweresmallerthan100

m

m,theydidn’tinterferewiththeextractionofmicroplasticssized >100

m

mconductedwiththisprotocol.Ifthisprotocolisbeingadaptedtoinvestigatemicroplastics <100

m

m, greater care must be taken to prevent procedural contamination. This could include wearingsynthetic-freenaturalfibreclothing[31]orworkinginasealeddamp-wipedroom[32].

Protocolsuitability

Seventargetpolymerswereusedtotestthesuitabilityofthesequentialextractionprotocol:HDPE, polyethylene(PE), poly(ethyleneterephthalate) (PET),nylon (polyamide, PA),vinyl chloride/vinyl acetate/vinylterpolymer(PVC),(aminoethyl)polystyrene(AM-PS)andpolypropylene(PP).Tenpieces (1cmlong,cutusingascalpel)and20particles(<1mm2_{,grated)werepreparedfromeachpolymer}

1Theareaofeachofthetenpieces(1cmlong)perpolymertypewasmeasuredusingthesoftware ImageJ1beforeandafteraciddigestionfollowedbyemulsification.AStudent'st-test(two-tailed, pairedsamples)wasrunwithanullhypothesisofnochangeintheareaofthepiecesbeforeand afterthedigestionandemulsificationmethods.

2Twentyparticles(_<1mm2_{)perpolymertypewerealsosubjectedtoaciddigestionfollowedby}

emulsification.Particleswerethenmanuallysortedandvisuallycounted.

3Ten pieces (1cm long) of the target polymers that were recovered after acid digestion and emulsi_fication(i.e.notcompletelydegraded)weresubjectedtodensityseparation(NaCl, 1.2g/cm3_).

Thecapacityofeachtreatedpolymertofloatorsinkwasrecorded.

4ATR-FTIRspectroscopywasusedtodeterminewhetherthesequentialextractionprotocolaffected polymer identification.The polymer composition of each plastic piece was measured prior to treatmentusingFTIRspectroscopy.Pieceswerethentreatedwiththeaciddigestionfollowedby emulsification.PiecesthatwererecoveredafterthesetwomethodswererinsedwithROwaterand oven-driedat60Cfor4hbeforerepeatingthechemicalanalysisusingFTIRspectroscopy.Apercent similarity,calculatedbycomparingtheFTIRspectraofthetreatedpolymersagainstthoseofthe untreatedpolymers(PerkinElmerCOMPAREalgorithm),wasusedtoassesschemicaldegradation.

HNO3digestioncompletelydissolvedallpiecesandallparticlesofPETandPA.Theprotocoldidnot

affectPE,HDPE,PVC,AM-PSandPP;100%recoverywasachievedandnochangeinsizeorchangein polymeridentificationwasobservedafterexposureofthesefivepolymertypestoaciddigestionand emulsi_fication.

Thedensityseparationmethodissuitableforpolymerswithadensitylowerthanthehypersaline brine,i.e.

r

<1.2g/cm3_{.OfallpolymersinerttoHNO}

3digestion(i.e.PE,HDPE,PVC,AM-PSandPP),

onlyPVCwasnotrecoveredbydensityseparation,whichwasexpectedasitsdensityishigherthanthe hypersalinebrine(PVC

r

=1.16–1.58g/cm3_{,hypersalineNaCl}

r

_=1.2g/cm3_{).Torecoverawiderrangeof}

high-density polymers including PVC, solutions with a density >1.2g/cm3_{, such as sodium}

polytungstate(1.4g/cm3[33]),orsodiumiodide(1.6g/m3[31])couldbeused.

AlimitationofusingHNO3digestionasaseparationmethodisanotablediscolourationofthe

target polymers tested. Thismakes comparison withother studies difficult, as moststudies on microplasticingestionbymarinebiotarelyonvisualinspectionusingmicroscopyastheprimary identification technique [1]. For this reason, werecommend using FTIRspectroscopy [19,30,34], whichallowscomparableresultsonpolymeridentificationandisgainingtractionasitbecomesless expensiveandmoresensitive.

Results

Thissequentialextractionprotocolallowstheextractionofmicroplasticsfromgreenturtlechyme with100%efficiencyfor>100

m

mmicroplasticsoffivecommonplasticpollutants:PE,HDPE,AM-PS, PPandPVC(PVCisonlyextractableifdensityseparationisnotused,i.e.ifsamplesdon’tcomprise sediment).Fourofthepolymertypesdeemedsuitableforthisextractionprotocolrepresent70%ofthe plasticsproducedgloballyin2007:HDPE=21%,PP=24%,PS=6%,PVC=19%[35].

Usingthissequentialextractionprotocol,twomacroplasticsandsevenmicroplastics;twopaint chipsandfivemixed-yarnsyntheticplasticparticles,werefoundintheforegutoftwogreenturtles fromAustralia’sGreatBarrierReef[36].Theseresultshighlighttheneedforanalysisofanincreased samplesizeinordertoimproveourknowledgeofthemicroplasticloadofseaturtlesfromtheGreat BarrierReefWorldHeritageAreaandaroundtheworld.Thesequentialextractionprotocolvalidatedin thisstudywillenableresearchersworldwidetoquantifygreenturtlesmicroplasticcontaminationina reliableandcomparableway.

Acknowledgements

AppendixA.Supplementarydata

Supplementarymaterialrelatedtothisarticlecanbefound,intheonlineversion,atdoi:https://doi. org/10.1016/j.mex.2018.07.009.

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