Effects of chronic dietary petroleum exposure on reproductive development in polar cod (Boreogadus saida)

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Aquatic Toxicology

jou rn al h om ep a ge :w w w . e l s e v i e r . c o m / l o c a t e / a q u a t o x

Effects of chronic dietary petroleum exposure on reproductive development in polar cod (Boreogadus saida)

Morgan Lizabeth Bender

^a,∗

, Marianne Frantzen

^b

, Ireen Vieweg

^a

, Inger-Britt Falk-Petersen

^a

, Helge Kreutzer Johnsen

^a

, Geir Rudolfsen

^a

, Knut Erik Tollefsen

^c

, Paul Dubourg

^a

, Jasmine Nahrgang

^a

aDepartmentofArcticandMarineBiology,UiT-TheArcticUniversityofNorway,9037Tromsø,Norway

bAkvaplan-niva,FramCentre,9296Tromsø,Norway

cNorwegianInstituteforWaterResearch(NIVA),0349Oslo,Norway

a r t i c l e i n f o

Articlehistory:

Received15June2016 Receivedinrevisedform 14September2016 Accepted1October2016 Availableonline4October2016

Keywords:

Polarcod(Boreogadussaida) Reproductivedevelopment Gonadalhistology Sexsteroidhormones Spermmotility

Polycyclicaromatichydrocarbons(PAH)

a b s t r a c t

IncreasinghumanactivitiesintheArcticraisetheriskofpetroleumpollution,thusposinganelevated riskforArcticorganismstobechronicallyexposedtopetroleumcompounds.Theendocrinedisrupting propertiesofsomeofthesecompounds(i.e.polycyclicaromatichydrocarbons[PAHs])presentincrude oilmayhavenegativeeffectsonthelongandenergyintensivereproductivedevelopmentofpolarcod (Boreogadussaida),anArctickeystonespecies.Inthepresentstudy,selectedreproductiveparameters wereexaminedinferalpolarcodexposedtocrudeoilviaanaturaldiet(0.11,0.57and1.14␮gcrude oil/gfish/day[correspondingtolow,mediumandhightreatments,respectively])for31weekspriorto spawning.Fishmaturinginthecurrentreproductiveperiodmadeup92%oftheexperimentalpopulation while5%wereimmatureand3%wereidentifiedasrestingfish.PhaseImetabolismofPAHs,indicatedby ethoxyresorufin-O-deethylase(EROD)activity,showedadose-dependentincreaseinhighandmedium crudeoiltreatmentsatweek6and22,respectively.DecreasingERODactivityandincreasingPAHbile metaboliteconcentrationsovertheexperimentalperiodmaybeexplainedbyreproductivematurity stage.Significantalterationsinspermmotilitywereobservedincrudeoilexposedmalescomparedtothe controls.Theinvestigatedsomaticindices(gonadandhepatic),germcelldevelopmentandplasmasteroid levels(estradiol-17␤[females],testosterone[malesandfemales]and11-ketotestosterone[males])were notsignificantlyalteredbychronicdietaryexposuretocrudeoil.Theenvironmentallyrealisticdosespolar codwerechronicallyexposedtointhisstudywerelikelynothighenoughtoinduceadverseeffectsinthis ecologicallyimportantfishspecies.Thisstudyelucidatedmanybaselineaspectsofpolarcodreproductive physiologyandemphasizedtheinfluenceofmaturationstateonbiomarkersofPAHbiotransformation (ERODandPAHbilemetabolites).

1. Introduction

RapidenvironmentalchangeintheArctic(Barberetal.,2015) isenablingoilandgasexplorationandexploitationinthis area, shippingacrosstheArcticshelfseas,andtourismactivities(AMAP, 2009; Eguíluz et al.,2016).The remoteness ofthe Arcticcom- binedwithinclementweather,unpredictableseaiceconditions, limitedavailabilityofbathymetricdata,fewports,andageneral lackofprecedenteventsmakeoperationsin thisareachalleng- ing(Harsemetal.,2011).Thesefactorsmayincreasetheriskof

∗Correspondingauthor.

E-mailaddress:[email protected](M.L.Bender).

petroleumpollution,reduceenvironmentalmonitoringpossibili- ties,andcomplicateorprolongthecleanupandrecoveryeffortsin theeventofanaccident.

The primary toxic components in petroleum, polycyclic aromatichydrocarbons(PAHs),areubiquitousinthemarineenvi- ronment(Meador,2006).PAHsare readilytakenup byaquatic organisms. However, ﬁsh have the ability to metabolize and eliminatethesecompounds(Meadoretal.,1995)bydifferentbio- transformationpathwaysincludingthoseofthecytochromeP450 enzymesystem.Evenatconcentrationsinthelowerpartsperbil- lion(Kime,1995),PAHshavebeenfoundtobetoxictoﬁsh,inducing carcinogenic, genotoxic, and physiological impairment (Meador etal.,2006;Vignetetal.,2014).Furthermore,PAHshavebeenfound todisrupttheendocrinesystemandaffectreproductivefunction

http://dx.doi.org/10.1016/j.aquatox.2016.10.005

0/).

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andgrowthoffish(Horngetal.,2010).ExposuretoPAHsinfish hasbeenlinkedtoreducedinvestmentingonadaltissues(Booc etal.,2014)andinterferencewithsteroidmetabolism(Monteiro etal.,2000).Inmaturingfemalefish,exposuretoPAHshasbeen foundtoimpairoocytedevelopment,increaseprevalenceofatre- sia,anddecreasesteroidplasmalevelsofestradiol-17␤(E₂)and testosterone(T)(ArukweandGoksøyr,2003).Inmales,PAHexpo- surehasbeenfoundtosuppressspermatogenesisinclams(Frouin etal.,2007)andincreasetestosterone(T)productioningoldfish (Carassius auratus)and rainbowtrout(Oncorhynchus mykiss)by promotingtesticularsteroidogenesis(EvansonandVanDerKraak, 2001).

Polarcod(Boreogadussaida)isakeystonespecieswithaPan- Arcticdistribution(BradstreetandCross,1982)thathasbeenused asanindicatorspeciesinArcticenvironmentstounderstandthe effectsandmechanismsofoilpollution(ChristiansenandGeorge, 1995;Jonssonetal.,2010;Andersenetal.,2015;Nahrgangetal., 2010a,b).However,effects ofPAHcompoundsonthereproduc- tive development of this species have never been thoroughly investigated (Geraudie et al., 2014).Polar cod undertakes syn- chronousspawningundertheseaiceinthewintermonthsbetween Decemberand MarchacrosstheArctic(Rass,1968;Craig etal., 1982).Thereareapparentdifferencesinreproductivedevelopment betweenmalesandfemales,suchastimingofreproductivedevel- opment,investmentingonadaltissue,andfrequencyofspawning (Nahrgangetal.,2014).Malesreachmaturityatasmallersizeand anearlierage(Craigetal.,1982;Nahrgangetal.,2014)andgonadal investmentsstartearlierintheseason(Hopetal.,1995).

Thestudyofreproductivephysiologycanprovideanintegra- tive measure of the effects of pollutants on whole organisms andimportantinsightsintothepotentialriskstopopulations.It istherefore criticaltostudythelong-termeffects ofpetroleum compoundsatecologicallyrelevantdoses,whichmayaffectphys- iologicalprocessesinorganisms,furtherpropagatingtochangesat thepopulationandecosystemlevels.Thepresentstudyexplores thelong-termphysiologicaleffectsofchronicdietaryexposureto environmentallyrelevant levelsof crude oilonreproductionin polarcod.Todeterminethepotentialeffectsofdietarypetroleum exposureonreproductivedevelopmentinpolarcod,wildﬁshwere exposedtofourdosesofcrudeoilfor31weeks,startinginJune atanearlygonadalmaturityphaseandendinginearlyFeburary.

Gonadosomaticindex,gonadhistology,plasmasteroidconcentrations,andspermmotilityweremeasured.Exposureindiceswere measuredintermsofbiliaryPAHmetabolitesandhepaticethoxyre- soruﬁnO-deethylase(EROD)activity.Wehypothesizedthatdietary exposuretoenvironmentallyrealisticconcentrationsofpetroleum compoundswillalterphysiologicalresponsesrelatedtoPAHexpo- sureandnegativelyaffectthereproductivedevelopmentofpolar cod.

2. Methods

2.1. Fishcollectionandhusbandry

PolarcodwerecollectedinRijpfjorden,Billefjorden,andKongs- fjorden(Svalbard,Norway)inJanuary2014duringacruiseonRV HelmerHanssen. Fishweretrawledat200mdepthusingalive fishbox(HolstandMcDonald,2000).Fishwerekeptondeckin 500L flow-throughtanksfor twoweeks and treateddaily with Halamid^® (1:500)disinfectant,whileundertransporttoTromsø, Norway. On the 29th of January, fish were transferred to the TromsøAquacultureResearchStationinKårvika.Fishwerekeptin a4000LacclimationtankatKongsfjorden,Svalbardseawatertem- peratures(1.5–3^◦C)tothenearest0.5^◦Castakenfrommooring data(Nahrgangetal.,2014)andalightregimeof79^◦N.Duringthis

period,ﬁshwerefeddailyuntilsatiationonadietofthawedCalanus sp.copepodsfromLofoten,Norway(purchasedfromCalanusAS).

On the 5th and 6th of June, 535 fish were selected based on length(13–17cmforklength)andweight(11–24gtotalweight) forparticipationintheexperiment.Fishwereanaesthetizeduntil lossofequilibrium,using5mg/LFinquel^® (TricaineMethanesul- fonate)dissolvedinseawater.Forklength(±0.1cm)andtotalwet weight(±0.1g)wererecordedforeachfish.Fishwerethencare- fullytaggedwithapassiveintegratedtranspondertag(Biomark^®) insertedintraperitoneally,beforebeingplacedrandomlyinoneof eightexposuretanks(n=67fishpertank).

2.2. Experimentaldesign

Polarcodwereexposedtodietarycrudeoilovertheperiodof gonadaldevelopmentstartingonthe30thofJune2014andending onthe3rdofFebruary2015.Exposuretanksconsistedof300Lﬂow- through systemsdistributed randomlywithin theexperimental room.Theexposuresetupconsistedoffourdietarycrudeoiltreat- mentswithtwotankspertreatment.FishreceivedCalanussp.food spikedwithKobbecrudeoil(BarentsSea)atnominalconcentra- tionsof0,20,100,and200␮gcrudeoil/gCalanussp.,corresponding tocontrol,low,medium,andhighdoses,respectively.Treatment foodwaspreparedinlargebatchespriortotheexposureperiodby mixing500gCalanussp.,250mLdistilledwaterand50ggelatin, andeitherno(control)oroneofthreedifferentnominalconcen- trationsofcrudeoil.Thismixturewasfrozen,cutintosmallpellets, anddistributed intoindividualbags,correspondingto2%ofthe totalﬁshweightofeachindividualtank,andfrozenat−20^◦Cuntil use.

Fishineachtankwerefedasagroupfivetimesaweekwitha totalrationequalto4%bodyweightperfeeding(Christiansenand George,1995).Calanussp.wasdistributedthroughoutthetankto reducefeedinghierarchies,althoughtheexistenceofsomehier- archiesin thetanks wasobserved.Onthefirst andfifthdayof theweek,fishwereexposedtothedietarycrudeoilbyreceiving 2%bodyweight/feedingoftreatmentfoodfollowedby2%body weight/feeding raw Calanus food.The three otherfeedings per weekconsistedofrawCalanussp.amountingto4%bodyweight.

Withthisfeedingregime,theeffectivedosewas0,0.8,4and8␮g crudeoil/gfish/week(Table1).Theamountoffooddistributedin eachtankwasadjustedfourtimesoverthecourseoftheexperi- ment(September,October,NovemberandJanuary)toaccountfor changesintotalfishweightandfishremovalduetosamplingand mortality.

Fivesamplingevents(n=8fishpertank,i.e.16fishpertreat- ment)were performed0, 6, 17, 22 and 31 weeksafter dietary exposurebeganinJune,August,October,DecemberandJanuary, respectively.Onthe30thofJune(week0),onlycontrolfish(n=16) weredissected,andonthe3rdofFebruary(week31)theremaining fishfromalltreatments(control[n=12],low[n=12],medium[n=8]

andhigh[n=9])weredissected.Eachﬁshwasanaesthetizedand bloodwascollectedfromthecaudalveinusinga2mLheparinized vacuumtube(BDVacutainer^®)thatwaspromptlysetoniceuntil centrifugationfor30minat4^◦Cand3500rpm(SorvallRC5BPlus centrifuge).Theplasmasupernatantwasseparatedoutandstored at−80^◦Cuntilsteroidhormoneanalysis.Followingbloodsampling, theﬁshweregivenasharpblowtotheheadbeforewetweight(g) andforklength(cm)weremeasuredandliverandgonadswere removedand weighed.Gonadosomatic index(GSI) and hepato- somaticindex (HSI)were calculatedaccording tothefollowing equations:

GSI =(gonadweight/somaticweight)×100 HSI= (liverweight/somaticweight)×100

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Table1

Summaryofnominalcrudeoil(␮g/gwwt),measured

26polycyclicaromatichydrocarbon(PAH)concentrations(mean±SD,ng/gwwt)intheCalanussp.diet,estimated dailydosesofcrudeoil(␮g/gﬁsh/day),and

26PAHs(ng/gfish/day)infishpertreatment.Nisthenumberoffishsampledineachtreatmentgroup.Estimateddosesof

26PAHs(ng/gﬁsh/day)arebasedoninitialmeanﬁshtotalweightatthestartoftheexperiment(week0).

Concentrationsindiet Dosesinﬁsh

Treatment N Initialﬁshtotal

wetweight(g)

Nominalconc.ofcrude oil(␮g/gwwt)

Measuredconc.

of

26PAHs (ng/gwwt)

Estimateddosesof crudeoil(␮g/g ﬁsh/day)

Estimateddoses

of

26PAHs (ng/gﬁsh/day)

Control 76 16.7±3.4 0 141.5±103.3 0 0.8±0.3

Low 60 17.8±3.5 20 313.2±88.0 0.11 1.8±0.4

Medium 56 17.6±3.7 100 1058.5±237.4 0.57 7.0±1.7

High 57 17.4±3.4 200 2288.2±214.4 1.14 13.1±4.0

Themiddlesectionofthegonadwaspreservedin4%neutral bufferedformalinforhistologicalanalysis.Theanteriorsectionof theliverandthegallbladderweresnapfrozeninliquidnitrogenand storedat−80^◦CuntilanalysesofERODactivityandbilemetabo- liteswereundertaken.Remaininginternalorganswereremoved andsomaticweightofthecarcasswasrecorded.After31weeksof exposure(3rdofFebruary),maleﬁsh(n=21)werestrippedformilt forspermmotilityanalysisbygentlymassagingtheabdomenand takingcaretoavoidcontaminationbyurineorblood.

2.3. DeterminationofPAHdosesinthediet

Samplesfromeachtreatmentwereanalyzedforthe16Environ- mentalProtectionAgencypriorityPAHsand10furtheralkylated naphthalenes,phenanthrenesanddibenzothiophenescompounds atAkvaplan-nivaAS(Tromsø,Norway).Briefly,aninternalstandard containinglabeleddeuteratedPAHswasaddedtothesamplesthat wereextractedbysaponificationwithmethanol/KOHfollowedby extractionwithpentane.Theextractwascleanedongelperme- ationchromatographyandfurtherpurifiedbyfiltrationonasilica columnwithpentaneanddichloromethaneaseluents.Thefinal extractwasanalyzedbygaschromatography/massspectrometry.

Triplicateswererunforeachtreatmentdiet.

2.4. AnalysisofPAHmetabolitesinbile

BilaryPAHmetabolites,1-OH-phenanthrene,1-OH-pyreneand 3-OH-benzo[a]pyrene,wereanalyzedforpolarcodsampledinJune, August,andFebruary(week0,6and31,respectively).Preparation ofhydrolysedbilesampleswasperformedasdescribedbyKrahn etal.(1992).Brieﬂy,bile(1–20␮L)wasmixedwithaninternal standard(triphenylamine)anddilutedwithdemineralisedwater (10–50␮L) and hydrolysed with b-glucuronidasearylsulphatase (20␮L,1hat37^◦C).Methanol(75–200␮L)wasaddedandthesam- plewasmixedthoroughlybeforecentrifugation.Thesupernatant wasthentransferredtovialsandanalyzed.Highpressureliquid chromatography(Waters2695SeparationsModule)wasusedto separatehydroxylPAHsinaWatersPAHC18column(4.6×250mm, 5␮mparticlesize).Themobilephaseconsistedofagradientfrom 40:60acetonitrile:ammoniumacetate(0.05M,pH4.1)to100%ace- tonitrileataﬂowof1mL/min,andthecolumnwasheatedto35^◦C.

A2475Fluorescencedetectormeasuredﬂuorescenceattheopti- mumforeachanalyte(excitation/emissions:1-OH-phenanthrene 256/380;1-OH-pyrene346/384;triphenylamine300/360;3-OH- benzo[a]pyrene380/430).25␮Lofextractwasinjectedforeach analysis.Theresultswerecalculatedbyuseoftheinternalstan- dardmethod(Grungetal.,2009).Thecalibrationstandardsutilized wereobtainedfromChironAS,Trondheim,Norway,andwereinthe range0.2–200ng/g.

2.5. EthoxyresoruﬁnO-deethylase(EROD)activity

Liversampleswerehomogenizedat4^◦CwithaPrecellys24type homogenizerina phosphatebuffer(pH7.4)containing150mM KCL,100mMKH₂PO₄,100mMK₂HPO₄,1mMdithiothreitoland5%

glycerol.Homogenateswerecentrifuged(9000×g,4^◦C)for30min.

Supernatantsweresubsequentlycentrifuged(50,000×g,4^◦C)for 2hforextractionofthemicrosomalfraction.Pellets(microsomes) weredissolvedinphosphatebuffer(pH7.4)containing20%glycerol andstoredat−80^◦Cuntilfurtheranalysis.ERODactivitymeasure- mentswereperformedasdescribedbyNahrgangetal.(2010b).

Briefly,fluorescencewasmeasuredinafinalreactionmixturecon- tainingthemicrosomalfractionfromhomogenizedliver(10␮L), thesubstrateethoxyresorufin(2␮M)andNADPH(0.25mM),which startedthedeethylationreactionof7-ethoxyresorufintoresorufin.

Fluorescenceofresorufinwasmeasuredinfourreplicatesintheflu- orimetricplatereaderSynergyH1(BioTek^®,Winooski,U.S.)atthe wavelengthpair540/600nm(excitation/emission)everyminute for20min.Foreachplate,aresorufinstandardcurve(0–0.025␮M) wasincluded.ERODactivitywasnormalizedtothetotalprotein content of the microsomal fraction. Total protein content was determinedaccordingtoBradford(1976),usingbovineserumalbu- min,(0–8␮g/mL)asastandard.

2.6. Histologicalanalysis

Gonadsamplesfixedinbufferedformalinwererinsed,dehy- dratedinaseriesof70%ethanolbathsandembeddedinparaffin wax (Aldrich, USA) overnight using Histo-clear^® as a clearing agentinaShandonCitadel1000(MicronAS,Moss,Norway).Tis- sueswerethenembeddedintoaparaffinblockandsectionedat 5–7␮mthickness,usingaLeitzRM2255microtome,andstained withhaematoxylin/eosin.EachslidewasexaminedunderaLabor- Lux11Leitzmicroscopeequippedwithacamera(WildLeitzAS, Oslo, Norway). Female gonadal maturity stages were based on the stage of themost advanced cohort of oocytes observed in the slices. Oocytes were categorized based on Brown-Peterson et al.(2011) intoone ofthefollowing stages (including oocyte diameter measurements of each stage): primary growth (PG, 73–221␮m),cortical alveolar(CA, 268–320␮m), primary vitel- logenic(VtgI,312–400␮m),andsecondaryvitellogenicaandb (VtgIIa,375–500␮m,VtgIIb,500–855␮m).OocytesinthePGstage wereidentifiedby thepresenceof aprominent nucleus,multi- plenucleoli,andscantcytoplasm(FigureA.1A).CAstageoocytes weredistinguishedbythepresenceofcorticalalveolivesiclesand visiblefollicularcelllayersurroundingtheoocyte(FigureA.1B).

Vitellogenicoocyteshad increasingeggshell- andfolliclethick- ness,andwerefurtherdistinguishedbasedonthepresenceofyolk globulesandthedifferenceinareaofcytoplasmﬁlledwithyolk.

VtgIoocyteshadlessthanhalfofthecytoplasmﬁlledbyyolkglob- ules(FigureA.1C).VtgIIaoocyteswerelargerthanVtgIoocytesand hadbetweenhalfand2/3ofthecytoplasmﬁlledbyyolkglobules,

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whileVtgIIbhadover2/3ofthecytoplasmﬁlledandacentrally placednucleus,asthenucleusmigrationtowardtheanimalpole not yet started (FigureA.1D).Post-ovulatory folliclecomplexes (POFs)wereidentiﬁedbythepresenceofemptyandcollapsedfol- liclesremainingintheovaryafterspawning(FigureA.1E).Atretic oocyteswerecharacterizedbythedisintegrationofthenucleus andbreakdownoftheoocyteenvelope(FigureA.1F).InOctober, December,andFebruary,restingandimmaturefemalesweredis- tinguishedfrommaturingfemalesasthosehavingonlyPGoocytes andPOFs(restingfemales)orhavingonlyPGoocytes(immature females).

Maleswereseparatedintofourprogressivegonadalmaturity stagesbasedonNú ñezandDuponchelle(2009):earlymaturing, maturing,spawning,andspent.Severalmaleswerealsoobserved withportionsoftestisspentandearlymaturingandthusclassified assuch.Earlymaturingmalesweredeterminedbythepresenceof earlystagespermatocytes(Fig.A.2AandBofSupplementaryinfor- mation)whilelatestagespermatocyteswithstrongerbasophilic stainingwerefoundinmaturingmales(Fig.A.2CandDofSup- plementaryinformation).Spawningmaleshadspermatozoainthe lumenoflobules,andmiltranwhenpressurewasappliedtothe abdomen(Fig. A.2EandFofSupplementaryinformation).Spent maleshademptylobuleswhilespent/earlymaturingmaleswere clearlyspent(emptylobules),butearlystagespermatocyteswere alsoprominent(Fig.A2.GandHofSupplementaryinformation).In October,December,andJanuary,furtherdivisioninmaturitystatus weremadebetweenmaturingandnon-maturingmales.Immature maleswereidentifiedbytestiswithonlyearlystagespermato- cytesandalowGSI,andrestingmaleshadspenttestiscontaining portionsofearlystagespermatocytesandalowGSI.

2.7. Steroidhormoneanalysis

Plasmaconcentrationsofestradiol-17␤(E2,femalesonly),11- ketotestosterone (11KT, males only) and testosterone (T, both sexes) were measured using radioimmunoassay, according to Schulz(1985).Aplasmapoolcomposedofmale(n=43)andfemale (n=35)wildpolarcodwasusedasaninternalreference.Thecross reactivityoftheE₂andTantiserumisgivenbyFrantzenetal.(2004) and11KTcrossreactivityisgivenbyJohnsenetal.(2013)(forsum- maryseeTableA1ofSupplementaryinformation).Valuesthatfell belowthelevelofdetection(LOD)wereassignedazerovaluefor calculations(E₂LODis0.66,11KT0.72,andT0.82ng/mLplasma).

2.8. Spermquality

Sperm motilitywasexamined following theprotocol setby Rudolfsenetal.(2005).Brieﬂy,spermmotilityanalysiswascon- ductedusinganaliquot(<0.12␮L)offreshundilutedmiltplaced ona 4^◦C20␮mstandardcountslide(Leja,Art.No.SC20-01-C, The Netherlands)and sperm activationwas inducedby adding 4.5␮Lchilledseawater.Avideo camera(SonyXC-ST50CE,Sony, Tokyo,Japan)mountedonanegativephase-contrastmicroscope (OlympusCH30,Olympus,Tokyo,Japan)(×10objective)wasused torecordsperm activityfromeach male (n=21).Sperm swim- mingactivitywasrecordedfora 90speriodwithtworeplicate trialsforeachmale.Spermmotilitywasexaminedusingcomputer- assistedspermanalysis,anobjectivetoolforquantitativeanalysis ofﬁshspermquality(Kimeetal.,2001).Spermcelltrajectories wereanalyzedusinganHTM-CEROSspermtracker(CEROSversion 12;HamiltonThorneResearch,Beverly,MA,USA).Thespermana- lyzerwassetasfollows:framerate50Hz;numberofframes25;

minimumcontrast9;andminimumcellsize8pixels.Fivemotil- ityparameters wereassessed inthe presentstudy: (1) average pathvelocity(VAP,␮m/s),whichisthevelocityofthespermhead alongitsspatialaveragetrajectory;(2)straight-linevelocity(VSL,

␮m/s),whichisthevelocityofthespermheadalongitslineartrack betweenitsinitialandﬁnalpositions;(3)meancurvilinearvelocity (VCL,␮m/s),whichisthevelocityofthespermheadalongitsreal curvilineartrack;(4)percentagemotilesperm;and(5)percentage progressivesperm(progressivespermcellsweredeﬁnedashaving straightness>80%andVAP>100␮m/s).Toremovethepotential effectofdrift,cellshavingVAP<20␮m/sandvelocitystraightline

<10␮m/swereconsideredtobestaticandwereexcludedfromthe motilityanalysis.Allrecordingswereanalyzed30s,60s,and90s afteractivationandweredoneblindinrespecttotreatment.

Spermatocrit was measured as a proxy for sperm density (Rakitinetal.,1999).Miltwascollectfromthestrippedmaleﬁsh usinghematocrittubes(n=2/male).Tubes,blockedbyclayatone end,werespundowninacentrifugefor5minat4500g(Eppendorf centrifuge5415C).Thelengthoftheentiremiltsampleand the lengthofthepackedspermcellsweremeasuredtocreatearatioof spermcellstotheseminalﬂuidinthemilt.

2.9. Statisticalanalysis

Allstatistical analyseswere conductedwith R3.1.1 (RCore Team,2014).Aftersatisfyingtheassumptionsofnormaldistribu- tionandequalvariance,aone-wayanalysisofvariance(ANOVA) wasusedtotestfirstlyfordifferencebetweensexesandsecondly fordifferencesbetweentreatmentsonthecontinuousfactorsof ERODactivity,PAHbilemetaboliteconcentrations,GSIandHSI,and plasmasexsteroidlevelsfollowedbyasubsequentposthoctest ondifferencesbetweenmeans(Tukey‘shonestsignificantdiffer- encetest).Variablesthatviolatedtheassumptionofnormalityand homogenousvarianceweretestedusingtheKruskal-Wallistestby ranks.Pearson’scorrelationtestwasusedtoexploretherelation- shipbetweenparameters.Mature,andimmature/restingfishwere treatedseparatelyforallanalyses.Comparisonswereconsidered significantlydifferentthancontrolwhenp≤0.05level.Valuesare reportedasmean±standarddeviation(SD).

Distributionofmaturitystagesandfrequenciesofatresiaand POFswereanalyzedusingaFishersexacttestwiththenullhypoth- esisthattreatmentgroupshavethesamefrequencyofmaturity stages,atresiaandPOFsatagiventime.Alinearmixedeffectmodel wascreatedforeachspermmotilityparameterwiththeRpackage nlme(Pinheiroetal.,2016).Fixedeffectsweretreatmentandtime afteractivation.Theinteractionbetweentreatmentandtimeafter activationwasalsotestedinthemodel.Randomeffectsincluded fishidentitynestedwithintrialtocontrolforvariancewithinthe sameindividualstestedacrossdifferenttimesandtrials.Thefull modelwascomparedagainstamodelexcludingthe2-wayinter- actionandathirdmodelfurtherexcludingthetreatmentfactor.All significanttermsweremaintainedinthemodelsandcompeting modelswereselectedbasedontheirAICvalues.Eachmodelsat- isfiedtheassumptionsofparametricanalysisandautocorrelation waschecked.Whenafixedfactorindicatedsignificantdifferences, pairwisecomparisonsusingt-testswereassessedbetweentreat- mentsandthecontrolfollowingthelinearmodeloutputinR.

3. Results

3.1. Morphometricdata

Atthestartoftheexperimentonthe30thofJune,themeanfork lengthandtotalweightoffishwas14.7±0.9cmand17.3±3.5g, respectively(n=535).Nosignificantdifferenceswerefoundamong tanks or treatments with regard to initial length and weight (p=0.73and0.16,respectively).Fishmortalitywasnotsignificantly differentamongstthetreatmentgroupswithameanfrequencyof 56±3%.Fishthatperished(n=301)hadlowerconditionindices

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Table2

Meanconcentration(ng/gfood±SD)of26PAHsand26PAHsforeachtreatment(control,low,medium,andhightreatment).Fourreplicateswereanalyzedforeach treatmentgroupexceptforcontrol.Valuesunderlimitofdetection(LOD)arenotincludedin26PAHcalculations.

PAHcomposition Control Low Medium High

Naphthalene 10.33±3.14 13.65±2.10 39.97±8.06 97.05±5.12

C1-Naphthalene 22.97±7.00 33.61±5.71 108.53±38.30 258.83±4.54

C2-Naphthalene 29.95±11.15 62.24±13.17 207.47±28.65 446.74±12.25

C3-Naphthalene 67.12±36.22 147.81±42.0 533.03±156.04 1140.99±63.83

Phenanthrene <3.05 5.78±1.31 13.95±1.21 34.69±4.70

Antracene <0.33 <0.33 <0.33 0.43

C1-Phenanthrene/anthracene 7.29±5.21 9.81 23.47±3.76 53.57±16.79

C2-Phenanthrene/anthracene 20.01±9.25 25.33±12.56 61.60±14.00 154.10±29.49

C3-Phenanthrene/anthracene 15.68±8.93 14.04±5.49 27.85±5.12 64.22±9.41

Dibenzothiophene <0.51 1 1.74±0.27 5.07±1.07

C1-Dibenzothiophene 1.63 1.80±0.09 4.89±0.99 12.9±4.06

C2-dibenzothiophene 6.19±0.12 9.35 11.34±3.03 31.86±6.48

C3-dibenzothiophene 6.39±0.42 6.21±2.93 10.76±1.99 25.39±3.88

Acenaphthylene <0.15 <0.15 <0.15 <0.15

Acenaphthene <1.06 1.18 1.74±0.50 3.08±0.14

Fluorine 1.13±0.30 2.49±1.01 6.85±1.82 14.06±2.52

Fluoranthene <1.98 2.14±0.09 2.99±0.82 4.51±0.93

Pyrene <3.32 <3.32 <3.32 3.52

Benzo(a)anthracene <0.51 <0.51 <0.51 0.57

Chrysene 1.01±0.22 2.35±0.55 2.15±0.44 3.54±0.36

Benzo(b)ﬂuoranthene <0.71 <0.71 <0.71 0.76

Benzo(k)ﬂuoranthene <0.23 <0.23 0.37 0.34±0.07

Benzo(a)pyrene <0.34 <0.34 <0.34 0.35

Indeno(1,2,3-cd)pyrene <0.73 <0.73 <0.73 0.95

Benzo(ghi)perylene <0.61 <0.61 <0.61 0.91

Dibenzo(a,h)anthracene <0.26 <0.26 <0.26 0.31

SUM26PAHs,ng/g: 141.5±103.3 313.2±88.0 1058.5±237.4 2288.2±214.4

(somaticweight,forklength,andHSI)comparedtoﬁshsampled intheexperiment(Fig.A3ofSupplementaryinformation).Thesex ratioofthesampledspecimenswasunbalancedoverall,with68 femalesand181males(TableA2ofSupplementaryinformation).

Somaticweightandforklengthofmaturingpolarcoddidnotdif- fersignificantlybetweencrudeoilexposedfishandcontrolfish orbetweensexesatanytimeovertheexposureperiod(TableA2 ofSupplementaryinformation).ThemeansomaticweightinJune was14.1±2.7g(n=64)and,byFebruary,themeansomaticweight hadincreasedto23.4±5.2g(n=48),ameanincreaseof58.8±24%

(p<0.01).ThemeanlengthinFebruarywas16.9±1.1cm,asigniﬁ- cantincreaseof6.8±5%fromthestartofthestudyinJune(p<0.01).

Thesomaticweightofimmaturefish(n=13,5%ofsampledfish) was30.5±7%lowerthaninmaturingfish.Restingfish(n=7,3%of sampledfish)hadalowersomaticweight(27.9±9%less)compared tomaturingfish(notshown).

3.2. PAHdosesandbiomarkersofbiotransformation 3.2.1. Dietdoses

Fishwereobservedduringfeedingandthepresenceoffoodwas conﬁrmedintheirstomachsateachsamplingpoint,thereforethe doseofcrudeoilmixedintheCalanussp.pelletswasconsideredthe administereddose(Table1).Assumingpolarcodineachtankwere feedingproportionallytotheirbodyweight,theingesteddosescor- respondedto1.8,6.0,and13.1ng

26PAHs/gﬁsh/dayinthelow, medium,andhighcrudeoiltreatments,respectively(Table1).The relationshipbetweentheamountofcrudeoiladdedtofoodand measured

26PAHslevelswaspositivelylinear(R²=0.99).The

26PAHs accountedfor∼1%wetweight(wwt)ofcrude oilin alltreatments.ThePAHcompositionwassimilarinallcrudeoil treatmentswithalkylatednaphthalenes(e.g.C1–C3-naphthalene) accountingforapproximately80%oftheoverallPAHload(Table2) andnostatisticaldifferencesintheratioofparentPAHstotheir alkylatedhomologuesacrosstreatmentswereobserved (results notshown).

Fig.1. EthoxyresoruﬁnO-deethylase(EROD)activity(pmolmin/mg/proteins)inthe liverofpolarcodexposedtolow,mediumandhighdosesofdietarycrudeoil andcontrols.Fish(n=16/treatment)weresampledat0,6,17,22,and31weeks ofexposure.Boxplotsrepresentthemedian(horizontalline),1stand3rdquartile (box),non-outlierrange(whisker),outliers(pointsoutsidewhiskers)ofthedata.

Eachpointrepresentsamaturingpolarcodwhileimmatureandrestingﬁsh(grey triangles)areexcludedfromtheboxplots.Treatments(onlymaturespecimens)sig- niﬁcantlydifferent(p<0.05)fromoneanotherareindicatedbydifferentlowercase letters.

3.2.2. EthoxyresoruﬁnO-deethylase(EROD)activity

OverallERODactivityintheliverwaslowanddecreasedsignif- icantlyfromJunetoFebruaryincrudeoilexposedtreatmentsand controlgroups(Fig.1).ERODactivityinmaturefishwasnegatively correlatedwithincreasingGSI(p<0.01).Nosignificantdifference inERODactivitywasobservedincrudeoilexposedgroupscom- paredtocontrolwiththeexceptionofweek22whenthemedium treatment(n=16)hadsignificantlyhigheractivitycomparedtothe control(n=16)andlowtreatment(n=15).Atweek6,increased ERODactivity wasseen in thehightreatment compared toall othergroups (n=16 ineachtreatment),howeverthis difference

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Fig.2. PAHbilemetabolitesmeasuredas(A)1-OH-phenanthrenemetabolitesand(B)1-OH-pyrenemetabolitesincrudeoilexposedandcontrolpolarcodsampledat0,6, and31weeksofexposure.BoxplotsareasinFig.1.Eachpointrepresentsamaturingpolarcodwhileimmatureandrestingﬁsh(greytriangles)appearinweek31andare excludedfromtheboxplotsandanalysis.Treatmentssigniﬁcantlydifferent(p<0.05)fromoneanotherareindicatedbydifferentlowercaseletters.

wasnotstatisticallysigniﬁcant.Immatureandrestingﬁshhada doserelatedincreaseinERODactivityatweek22;however,statis- ticalanalysiswaslimitedbythelowsamplesizeinthetreatment groups(n=5,2,2and3incontrol,low,mediumandhightreatment groups,respectively).

3.2.3. PAHmetabolitesinbile

Concentrationsof1-OH-pyreneand 1-OH-phenanthrene bile metabolites increased significantly over time in all crude oil exposed and control groups (Fig. 2). Concentrations of 1-OH- phenanthrene were significantly higher in the high exposure group (n=14) compared to control (n=16) in August, after 6 weeks of exposure. At this time point there also wasa strong correlationbetweendietphenanthreneconcentrationand1-OH- phenanthrene bile metabolite concentration (R²=0.97; average per treatment group). Concentrations of 3-OH-benzo[a]pyrene metabolitesonlyexceededthelevelofdetectioninasingularfishin themediumtreatmentinAugust(datanotshown).Maturefishhad significantlyhigherconcentrationsof1-OH-pyrenebilemetabo- lites(65.0±38.2ng/g,n=46)comparedtoimmatureandresting fishatweek31(26.5±13.3ng/g,n=6,p=0.02).

3.3. Effectsonreproduction 3.3.1. Somaticanalysis

Thegonadosomaticindexwassigniﬁcantlydifferentbetween sexesandincreasedsigniﬁcantlyovertime(Fig.3).Femalesstarted withaGSIof2.2±0.3%inJunethatremainedlowuntilFebruary whenGSIincreasedto12.8±6.5%.TheGSIinmaleswas0.9±0.2%

inJune,begantoincreaseinOctoberandhighestGSIvalueswere measuredinFebruary(20.1±8.6%).In October(week 17),male GSIin medium treatment(n=11)wassignificantlyhigher than GSIinthehighcrudeoiltreatment(n=10).However,nodiffer- encewasfoundin female GSIamong treatments. In December (week22),theGSIofbothsexesbegantodivergeintotwosignif- icantlydifferentgroups,oneofwhichidentifiedthematuringfish cohortwithanincreasingGSItowardsspawning,andtheotheras non-maturing,(immatureandresting)fishmaintainedalowGSI (2.0±2.3%)regardlessofsexandtime.Themostadvancedgonadal developmentwasobservedinFebruarywithonemalereachinga GSIof34.7%andonefemalereachingaGSIof20.9%.Thehepatoso-

maticindexdidnotshowsignificantdifferencesamongtreatments, withanexceptionofmalesinOctober(week17)showingsignifi- cantlyhigherHSIincontrol(9.5±2.6%,n=9)comparedtothehigh (6.8±1.4%,n=10)treatmentgroup(p=0.04,datanotshown).HSI wassignificantlyhigherinfemalefishcomparedtomales(p<0.01) andincreasedsignificantlyovertime(p<0.01)withhighestHSIval- uesmeasuredinFebruary,reaching13.3±2.2%infemales(n=14) and11.1±3.7%inmales(n=27).GSIandHSIvaluesformaturefish werepositivelycorrelated(R=0.58,p<0.01).Immaturefishand restingfishhadsimilarHSIvaluestomaturefish.

3.3.2. Histologicalanalysisofgonads

Nodifferenceswerefoundamong treatmentswhencompar- ingthefemalegonadalmaturationstagedistributions(Fig.4A).In June, ovarieswerein theﬁrststageof oogenesis,PG; themost advanced oocyte cohorts reached CA stage by August, VtgI by October,andVtgIIbbyDecember.InFebruary,allmaturingfemales had VtgIIb oocytes present in ovaries regardless of treatment.

Immaturegonadswerefoundin17.6%ofcontrolfemalesand21.4%

offemalesinthelowexposuregroupwhileonlyasingularresting femalewasidentifiedinthehightreatmentgroup.Theexperiment wasterminatedbeforeanyfemalesreachedthefinalstageofvitel- logenesis,finaloocytematurationandovulation.Nodifferencein prevalenceofatreticoocytesor POFswasobservedincrude oil exposed femalescompared tocontrol(datanot shown).Atretic oocyteswereobservedinsomefemalessampledinOctober(n=7) andFebruary(n=2)whilePOFs(n=16)werefoundatallsampling pointsandinallmaturitystages.

Nodifferencewasfoundamongtreatmentswhencomparing themalegonadalmaturationstagedistributions(Fig.4B).InJune andAugust,maleswereeitherspentwithemptylobulesorinthe earlymaturingstagewithearlystagespermatocytes.ByOctober andDecember,mostmales(89%)wereinamaturingstagewith latestagespermatocytes.InFebruary,75%ofmaturingmalescould bestrippedformiltandspermatozoawasobservedinthesperm ductsinthehistologicalpreparationsofactivelyspawningmales.

Immaturemales(n=8)weresampledinDecemberwithearlystage spermatocytesintestisandalowGSI(2.19±2.35%).Restingmales (n=6)weresampledinOctober,December,andFebruarywitha lowGSI(1.09±0.65%).

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Fig.3. GonadosomaticIndexofmaturingfemalepolarcod(toppanel)andmalepolarcod(bottompanel)indifferentcrudeoiltreatmentgroupssampledafter0,6,17,22, and31weeksofexposure.BoxplotsareasinFig.1wheredifferenttreatmentgroupsaredistinguishedbycolor,eachpointrepresentationasingularﬁshandsigniﬁcant differencesbetweentreatmentgroupsandcontrolareindicatedbydifferentlowercaseletters(p<0.05).Fortreatmentgroupswheren<5onlythemedianlineisshown.

Fig.4.Histologicalanalysisofgonadalmaturitystagesinmaturing(A)femaleand(B)malepolarcodoveraperiodofgonadaldevelopmentfromJunetoFebruary.The frequencyofoccurrenceofeachmaturitystage,representedbydifferentcolors,ineachtreatmentgroupafter0,6,17,22,and31weeksofexposuretodietarycrudeoil.

Numberoffishineachsexsampledfromeachtreatmentisnotedatthebaseofeverycolumn.Nosignificantdifferencewasfoundbetweencrudeoiltreatmentgroupsand controlwithregardtogonadalmaturitystagefrequencyofoccurence.Immatureandrestingarenotincludedinthisfigure.

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Fig.5.Plasmaconcentrationsdisplayedbymean±standarderrorof(A)estradiol-17␤inmaturingfemalepolarcod;(B)11-ketotestosteroneinmaturingmalepolarcod;(C) testosteroneinmaturingfemalepolarcod;(D)testosteroneinmaturingmalepolarcodafter0,6,17,22and31weeksofexposuretodifferentcrudeoildosesandcontrols.

Differenttreatmentgroupsaredistinguishedbycolorwithimmatureﬁshandrestingﬁsh(triangles)excludedfromtrendlines.Samplesizeisdisplayedaboveeachmean.

Nosigniﬁcantdifferenceswerefoundbetweencrudeoiltreatmentgroupsandcontrolwithregardtoconcentrationsofsexsteroidsinbloodplasma.

3.3.3. Plasmasteroidconcentrations

3.3.3.1. Estradiol-17ˇ. Estradiol-17␤(E₂)levelswerenotsigniﬁ- cantlydifferentamongtreatmentsneitherforanytimepointnor withinasingulargonadalmaturitystagealthoughgreatvariation wasfoundinVtgIIbfemales(Fig.5A).InAugust,plasmalevelsof E₂waslow(1.37ng/mL,n=1).Asigniﬁcantincreaseoccurredover timeandwithprogressivegonadalmaturitystages,andmaximum E2levelswerereachedinFebruary(8.32±6.43ng/mL,n=39).The femalewiththehighestE₂ level (25ng/mL)exhibitedthemost advancedoocytematuritystage(VtgIIb)inFebruary(week31).

ThetemporalchangesinE2weresigniﬁcantlyandpositivelycor- relatedwiththechangesinGSI(R=0.59),HSI(R=0.46),andsomatic weight(R=0.31).TheimmatureandrestingfemaleshadE₂levels atorslightlyabovetheLOD(0.66ng/mL,n=4).

3.3.3.2. 11-ketotestosterone. 11-ketotestosterone(11KT)levelsin maleswerenotsigniﬁcantlydifferentamongtreatments(Fig.5B).

Plasma levels of 11KT in males rose steadily with advancing maturity stage throughout the exposure period with low val- uesinJune(0.08±0.27ng/mL)andmaximumvaluesinFebruary (3.38±1.87ng/mL).InFebruary,maturingmalesinthehightreat- ment(n=3) had11KT levels55% lowerthan thecontrolgroup (n=7),althoughnotstatisticallysignﬁciant.Immaturemalesand thoseinearlymaturingandspentstageshadgenerallylow11KT levels(<1.0ng/mL,n=9),buthigherlevelswerefoundincontrol malesthan in theexposed males(<5.0ng/mL, n=4).Increasing plasma11KTlevelsweresigniﬁcantlycorrelatedwithincreasing GSI(R=0.59).

3.3.3.3. Testosterone. Testosterone(T)levelsinfemaleswerenot significantlydifferentamongtreatments(Fig.5C).Plasmalevels ofTinfemalefishwerelowinAugust(2.6ng/mL).Testosterone levels remainedlow until February when an increase occurred inmaturingfemales(mean8.7±5.5ng/mL)correspondingtothe entryintoVtgIIgonadalmaturitystage.Thetemporalchangesin TweresignificantlycorrelatedwiththeincreaseinGSI(R=0.62).

Immature females had T levels close to LOD (0.6±0.3ng/mL, n=4). PlasmaT levelsin maleswere not significantly different amongtreatments(Fig.5D).Maturingmaleshadgreaterlevelsof Tatalltimepointscomparedtomaturingfemales.Inmaturing males,plasma Tlevelswere low(0.62±0.6ng/mL) inJune and roseto6.2±3.9ng/mLinDecemberandcontinuedincreasingto 11.7±7.1ng/mL in February.Testosteronelevels inmales were significantlycorrelatedwithGSI(R=0.54),HSI(R=0.31),somatic weight(R=0.40)andmaturitystage.Immatureandrestingmale fishhadlowTlevels(2.6±3.0ng/mL,n=3).

3.3.4. Spermquality

Crudeoilexposurenegatively affectedspermcellcurvilinear pathvelocity(VCL)(F=2.9,p=0.051)(Fig.6A).TheVCLofsperm inthemediumtreatmentwassigniﬁcantlyreducedcomparedto thecontrol treatment(p=0.038). Percentagemotile sperm was alsoaffectedbycrudeoilexposure(F=2.5,p=0.074),althoughnot signiﬁcantlyat the5%threshold.A higherpercentage ofmotile spermwasmeasuredinthelowcrude oilexposuregroupcom- paredtocontrolgroup(p=0.018)(Fig.6B).Thepercentageofmotile spermwasonaverageover80%inallgroups.Althoughnotsignif- icantatthe5%threshold,malesincrudeoiltreatmentshadlower

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Fig.6. Spermmotilitymeasurementsat30,60,and90safteractivationfrommaleﬁshstrippedinweek31(February3rd)displayedbymean±standarderror:(A)Curvilinear velocity;(B)Percentagemotilesperm;(C)Averagepathvelocityofsperm;(D)Straight-linevelocity;(E)Percentageprogressivesperm.Signiﬁcantdifferencesfromthecontrol groupacrossalltimesafteractivationaredenotedbydashedlineforthedistinguishedgroup(p<0.05).

VAP(F=2.3, p=0.09)and lowerVSL (F=1.3, p=0.28) compared tocontrolmales(Fig.6CandD).Contrarytoobservationsofthe percentageofmotilesperm,thepercentageofprogressivesperm fellonaverageby30±2.6%inallcrudeoiltreatmentscompared tothecontroltreatment(20.5±3.03%)(F=1.25,p=0.3)(Fig.6E).

Timeafteractivation(30,60and90s)wassigniﬁcantinallmodels (F>3.7,p<0.05).Nointeractionbetweenthetimeafteractivation andtreatmentwasfoundforanyspermmotilityparameter.

Spermatocritwasmeasuredfor16maleﬁshinFebruary.The meanspermatocritwas0.97±0.02andnosigniﬁcantdifferences werefoundbetweentreatments(datanotshown).

4. Discussion

4.1. Effectonexposureindices

Inthecurrentstudy,maturingpolarcodwereexposedfor31 weekstofourdifferentdietarydosesofcrudeoilthroughanatu- raldiet(measuredconcentrationof26PAHswereat141,313, 1058and2288ng/gindiet).ThesePAHdosesareconsideredenvi- ronmentallyrealisticandrepresentconcentrationsplanktivorous ﬁshmayencounterinzooplanktoncommunitiesafteranoilspill (Salasetal.,2006)orinareaswithchronicoilpollution(Carlsetal., 2006).Zooplankton communities sampled sixmonths afterthe PrestigefueloilspillofftheNorthwestcoastofSpainhadconcentra- tions(4.2–152ng/g14PAHs)(Salasetal.,2006)withintherange measuredinthepresentstudy(24.9–152.6ng/g14PAHs).The concentrationof26PAHsinthecontrol(141±103ng/g)andlow dose(313±88ng/g)ofthepresentstudyresembledconcentrations foundinnaturalcopepodassemblages(PAHs=120–256ng/g)in theoilshippingportofValdez,Alaska(Carlsetal.,2006).Thepres- enceofPAHsinthecontrolfoodisevidencefortheubiquityof

thesecompoundseveninrelativelypristinemarineenvironments likeLofoten,Norway,wheretheCalanussp.forthisexperimentwas collected(Greenetal.,2013).

Thedietaryroutemayplayasignificantroleintheexposureof marinefishestolipophiliccontaminants(i.e.PAHs)(Georgeetal., 1995;Meadoretal.,2006;Nahrgangetal.,2010b),especiallyfor polarcod,alargelydemersalfish(Geoffroyetal.,2016)withahigh assimilationefficiency(Hopetal.,1997).Otherstudiesthathave exploredtheeffectsofdietarycrudeoilexposureonpolarcodhave useddosesexceedingthoseofthepresentstudy(calculatedexpo- sure0,0.11,0.57,and1.14␮gcrudeoil/gfish/dayequatingto0.8, 1.8,7and13.1ng

26PAHs/gfish/dayinthecontrol,low,medium andhightreatments,respectively).PolarcodexposedtoNorthSea crudeoilatdosestwoordersofmagnitudehigher(571–1285ng 26PAHs/gfish/day)revealedsubstantialresponsesofexposure biomarkers(hepaticERODactivityandPAH metabolitesinbile) after4weeks(Nahrgangetal.,2010b).Polarcodexposedtoacrude oildosethreetimeshigherthaninthepresentstudy(averagedose 3.8␮gcrudeoil/gfish/day)for53daysduringgonadaldevelopment hadreducedgrowth(ChristiansenandGeorge,1995)andelevated ERODactivitycomparedtocontrolfish(Georgeetal.,1995).Ina comparablestudywithregardtotheduration(sevenmonthexpo- sure)anddose(2.8␮gcrudeoilinfood/gfish/day),maturerainbow troutshowednoeffectsoftreatmentontimingofspawning,fertil- izationorhatchingsuccess(Hodginsetal.,1977).Arecentstudyby Bakkeetal.(2016)foundthatmaturingpolarcod,whenexposedto asingledoseofradioactivelylabeledB[a]P(1.15±0.36␮g/gfish) orphenanthrene(0.4±0.12␮g/gfish),absorbedcompoundsinto intestines,liver,andbilewithintwodaysfollowingadministration andthecompoundsremainedinthefishtissueforover30days, thusexhibitingalongtermbioavailabilityofingestedPAHsinpolar cod.

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ERODactivityisasensitivebiomarkerfortheexposuretoPAHs infishandisusedtoassesstheactivityofthephaseIcytochrome P4501A1(CYP1A1),animportantenzymeinPAHbiotransforma- tion(StegemanandLech,1991).ThelowoverallERODactivityin thisstudymaybeanindicationofa loweffectivedoseofPAHs receivedbythepolarcodliver.However,increasedERODactivity wasseenincrudeoil-exposedfishcomparedtocontrolatgiven timepoints(week6and22),indicatingtheinductionofPAHbio- transformationuponcrudeoilexposureatthesetimepoints.Low molecularweightPAHssuchasnapthalenes,phenanthrenes,and fluoranthene,whichmadeupthemajorityofPAHsmeasuredin this study,havebeenfoundtocauseeither noeffect orinhibit ERODactivityinNiletilapia(Oreochromisniloticus)(Pathiratneand Hemachandra,2010)andinCaliforniahalibut(Paralichthyscalifor- nicus)(Serutoetal.,2005).TheoveralllowamountsofPAHsinthe exposuredietand thedominationoflowerweightPAHswitha potentialinhibitoryactiononERODactivitymayhavelimitedthe inductionofresponsesatthedosesusedinthepresentstudy.Fur- thermore,inclusionofintestinalERODactivitytoidentifypotential metabolismoccurringpriortosystemicuptakeofthePAHsand alkylatedPAHswouldhaveprovidedadditionalinformationonthe dietaryexposure,especiallyatlowlevels(Jamesetal.,1997;Van Veldetal.,1990).Suchcomplementaryanalysesmayallowthe comparisonoftissue-specificbiotransformationcapacityandhas previouslybeenfoundrelevantforpolarcodexposedtodietary crudeoil(Nahrgangetal.,2010b).

ThedecreasingERODactivityovertimewasnegativelycorre- latedwithmaturity andsimilardeclineshavebeenobservedin numerousotherfishspeciesduringsexualmaturation(Arukweand Goksøyr,1997;Whyteetal.,2000).Furthermore,crudeoil-exposed immatureandrestingfishhadhigherERODactivitycomparedto controland maturing fish, althoughthe number ofresting and immaturefishwastoolowtosecurearobuststatisticalcompar- ison.ThesuppressionoftheCYP1A1enzymeactivityinmaturing fishofbothsexesmaybeanadaptiveresponsetomaintainhigh steroidhormonelevelsnecessaryforendocrineregulationofrepro- ductive development(Arukwe etal.,2008; Förlin andHansson, 1982).Forinstance,E2hasbeenshowntohaveasuppressiveaction onCYP1Acatalyticactivitythroughcompetitionforbindingsites aswellasatpre-translationallevelsofCYP1A(NavasandSegner, 2001).Also, cross-talk between thesignaling pathways involv- ingthearylhydrocarbonreceptor(AhR),whichregulatesCYP1A expression,andtheestrogenreceptor(ER),whichregulatesvitel- logeninexpression,hasbeenexploredinthepastdecadealthough mechanismsarestillunclear(Bemanianetal.,2004;Gränsetal., 2010;Mortensenetal.,2007;MortensenandArukwe2007;Kirby etal.,2007).InhibitionofERODcatalyticactivitymaythusrepre- sentapossiblemechanismtoexplainlowERODactivitymeasured inmaturingcrudeoilexposedfishinthepresentstudy.

AhigherconcentrationofOH-phenanthrenebile metabolites inthehightreatmentgroupcomparedtocontrolinAugust,and a strong correlation between phenanthrene diet concentration and 1-OH-phenanthrene bile metabolite concentration, veriﬁes thatPAHs accumulatedafterdietaryexposuretocrude oil(Aas etal.,2000;Nahrgangetal.,2010b).Thelackofdose-dependent responsesinbilemetabolitesconcentrationsatothertimepoints maybeduetothelimitationsofbilemetabolitesasalong-term responseindicator(CollierandVaranasi,1991).Dietaryexposure tocrudeoilcomparedtowaterborneexposureinpolarcodresulted inlessconcentratedPAHbilemetabolites(Nahrgangetal.,2010b) duetoreducedsystemicavailabilityofPAHs(Ingebrigtsenetal., 2000).Furthermore,biotransformationandaccumulationofPAH metabolitesinbilemayhavebeenlimitedbyagenerallowactivity ofCYP1A1(ERODactivity),mostlikelyassociatedwiththematu- rationprocesses.Additionally,acontinuous(daily)feedingregime inducesaregularemptyingofthegallbladder,leadingtoapotential

lackofsigniﬁcantPAHmetabolitebioaccumulationinthegallblad- derovertimeandthuslowmetabolitelevels.

PyreneandphenanthrenePAHmetaboliteswerequantiﬁedin thebileofcontrolﬁsh,possiblyduetothebackgroundPAHsfound in the natural Calanus sp. diet (141±103ng 26PAHs/gfood).

Interestingly,matureﬁshinalltreatmentsshowedanincreased concentrationofOH-phenanthreneandOH-pyrenebilemetabo- litesfromAugusttoFebruary,whilelevelsinimmaturespecimens remainedlow.Althoughmaturation-associatedendogenouscom- poundswithstructuralfeaturesresemblingﬂuorescentPAHs(e.g.

steroids)arealsoexcretedinthebileandmaypotentiallyinter- fere with HPLC analysis (Honour, 2006), the specificity of the HPLCmethodused hereinhaslikelylimited suchartifacts.It is morelikelythatlife-stage/maturation-associateddifferencesinthe accumulationofPAHsorchangesintotalPhaseIorPhase-IIbio- transformationinpolarcod(Nahrgangetal.,2010b)havecaused thisapparentdiscrepancy.However,therapidandsubstantialbio- transformationofPAHsinfish(Meadoretal.,1995)suggeststhata combinationofanalysesofPAH-metabolitesandalargerassembly ofbiotransformationenzymeswouldlikelybethebeststrategyto decipherthesematuration-specificdifferences.

4.2. Effectsonreproductiveparameters

Gonadaldevelopmentwasnotsignificantlyaffectedbyexpo- suretodietarycrudeoilinpolarcodinthisstudyandappeared normal compared to histological studies from wild specimens (Nahrgangetal.,2016a)andlevelsofatresiawerelowcomparedto thoseobservedbyGeraudieetal.(2014).Thetemporaloccurrence ofatresiamayreflectaperiodofoogenesiswherefemalesfinetune theenergyresourcesusedinreproductionasatresiaallowsforthe reabsorptionofenergyrichoocytes(Hardardottiretal.,2001).The presenceofPOFsinFebruaryfromthepreviousspawningseason (12–13monthsprior)isquiteremarkablecomparedtootherfish species,althoughcoldtemperaturesmayprolongdegradationof POFssuchasseeninAtlanticherring(Clupeaharengus)andDover sole(Microstomuspacificus)(Hunteretal.,1992;Brown-Peterson etal.,2011).

Thepresentstudyisthefirsttopresentdynamicchangesofsex steroidlevelsinmaturingpolarcod.Theincreasingconcentrations inallsexsteroidhormonesmeasuredovertimematchedexpected profilesofmaturingfishwithincreasingGSIandadvancingooge- nesisandspermatogenesis.Previousstudiesquantifyingpolarcod sexsteroidhormonesfoundlevelsthatwereanorderofmagnitude lowerthaninthepresentstudy.Theselowlevelsmaybeexplained bythematuritystageof thefishasHopetal. (1995)lookedat fishactivelyorveryclosetospawningandGeraudieetal.(2014) mostprobablymeasuredlevelsinprevitellogenicpolarcod.Hop etal.(1995)foundlowerE₂ levelsinactivelyspawningfemales and maturefemales whohad notreachedovulation (0.272and 0.831ng/mL)thanwhatthepresentstudyreportedinfemalesat allgonadalmaturitystages(0.43–25ng/mLforPGthroughVtgII maturitystages,respectively).PlasmaE₂ andTlevelsareshown todropbacktobasallevelsatorimmediatelyafterspawningin Atlanticcod(Norbergetal.,2004)andArcticchar(Frantzenetal., 2004).Contrary totheplasmaprofilesmeasuredinthepresent study,Geraudieetal.(2014)foundnoincreaseinpolarcodTlevels overtimeinmales(0.023–0.149ng/mL)orinE₂levelsinfemales (0.015–0.187ng/mL).Theplasmaprofilesofimmaturefishinthe presentstudywerecomparabletothosemeasuredinGeraudieetal.

(2014).

TheabsenceoftreatmenteffectsonGSI,HSIandsexsteroidlev- elsinthisstudymaybeduetoinsufﬁcientdosesnecessarytoelicit anendocrinedisruptingeffect.Indeed,deviationinGSIisavalu- ablemeasureoflongtermexposureandmayresultfromsmaller, lessmatureoocytesandspermatocyteswithahigherfrequency

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ofatresiaintheovariantissue andlesionswithinthetesticular tissue (Kime,1995).However, somaticindices maynot always beverysensitiveendpointsforassessingeffectsassociatedwith gonadaldevelopment.Forinstance,noeffectonGSIwasobserved inAtlantic cod exposed for12 weeks toproduced water (PW), whichcontainsendocrinedisruptingalkylphenols,althoughthere weresigniﬁcantdecreasesinplasmaE₂levelsandanincreaseinthe frequencyofatresiainexposedfemalecod(SundtandBjörkblom, 2011).Inthepresentstudy,ﬁnalmaturationandspawningwasnot achievedwithintheexperimentalperiod.Fishwouldmostlikely havereachedspawninginearlyMarchaswasseeninferalpolar codnotincludedinthisexperimentbuttakenfromthesametrawl andheldunderthesamelaboratoryconditions(BenderMLetal.

unpublished).Signiﬁcantdifferencesinsexsteroidlevelsbetween crudeoiltreatmentgroupsandthecontrolsatalatermaturitystage cannotberuledout,andpossibleeffectsofcrudeoilmaytherefore havebeenmissed.OneyearaftercrudeoilexposurefromtheExxon Valdezoilspill,plasmaE2 levelswerestillreducedinwilddolly varden(Salvelinusmalma)andyellowﬁnsole(Limandaaspera)(Sol etal.,2000).

Certainparametersofspermmotilitywerenegativelyaffected bydietarycrude oilexposure. Inexposed males,relativelyhigh percentagesofmotilesperm(>87%)andlowpercentagesofpro- gressivesperm(<15%)weremeasuredillustratingthatspermwas inmotionbutconservativelyso,whichcouldpotentiallyaffectfer- tilizationsuccess. InAtlanticcod,percentage progressivesperm wasshown tobethemostindicativespermmotilityparameter offertilizationsuccess(Rudolfsenetal.,2008).Adverseeffectsof PAHsand other petroleum-related compoundsonmale gonads andsperm qualityhave beenreportedpreviously.For instance, spermmotilitydecreasedinspottailshiners(Notropishudsonius) naturallyexposed topolluted waters around Montreal,Canada (Aravindakshanetal.,2004),whitesucker(Catostomuscommer- sonii)exposedtobleachedkraftmilleffluentcontainingPAHsand PCBcompounds(McMasteretal.,1992)andinPacificoysters(Cras- sostreagigas)exposedtoPAHs(Jeongand Cho,2005).Exposure ofpolar cod toPW during reproductivedevelopment(28days) resultedinreducedspermatogenesisandincreasedprevalenceof histopathology(Geraudieet al.,2014).However,in thepresent studynoalterationinspermatogenesisorobvioushistopathologies wereobservedinthemaletestesinFebruarytoexplainthereduced spermmotilityincrudeoilexposedpolarcod.Polarcodusedinthis studyweremostlikelytospawnamonthlaterinMarch;therefore, finalcapacitationofspermatozoamayhavebeenincompleteand thusnotrepresentativeofmotilityofspermatthetruespawn- ingtime.Observeddifferencesinspermmotilityseenincrudeoil exposedmalescouldbeduetoadelayincapacitationnotobserved atthehistologicallevelorthroughpossibleendocrine-mediated effectsofPAHsonspermatozoadevelopment(Aravindakshanetal., 2004;Abdelrahimetal.,2006).OxidativestresselicitedbyPAHs (Hannametal.,2010)mayalsoprovideapossiblemechanismto explainreducedspermmotility(Kaoetal.,2008).Spermatozoaare susceptibletooxygen-induceddamageduetolargequantitiesof polyunsaturatedfattyacidsintheplasmamembranesofspermato- zoa(AlvarezandStorey,1995)andlowcytoplasmicconcentrations of antioxidantenzymes necessaryto repair damage (Saleh and Agarwal,2002).

Reproductionin captive polar cod has beenfound tobe an extremeenergyinvestmentwith87%ofinitialenergyinliverused for reproductive costsunder gonadal development(Hop et al., 1995).Thus,post-spawningmortalitymaybesubstantial.Thisis confirmedby field observations ofpolar cod perishingin large numberafterspawning(Moskalenko,1964).Inthepresentstudy, histologicalanalysesrevealediteroparousfemaleandmaleindi- viduals,afindingsupportedbypreviousstudies(GrahamandHop, 1995;Nahrgangetal.,2016a).In theseindividuals,ovariescon-

tainedbothvitellogenicoocytesandPOFsandtestescontainedboth spentandearlymaturingfractionsindicatingpreviousspawning andintentiontospawnin anupcomingseason.Hence,therel- ativelyhighfishmortalityinthepresentstudy(∼56%)couldbe relatedtothereducedconditionofpost-spawningfishandthecost ofreproductioninapreviousseason.Generally,reducedsomatic indices(GSIandHSI)inperishedfishmayindicatethatadeficiency inenergyreservescouldexplainthereducedsurvivalofpolarcod inthisexperiment.Analysisoftheperishedpolarcodrevealedthat thelikelihoodofsurvivalwasnotrelatedtocrudeoilexposure,sex, ortheinitialweightofthefish(unpublisheddata).Thepresenceof endoparasitesinthefishbodycavity(nematodes)wascorrelated withahigherstochasticalriskofmortality,suggestingthatfishwith lowerbodyindicesmayhavehadacompromisedimmunedefense.

4.3. Conclusionandoutlook

The investigated endpoints of weight and length, somatic indices,timingingonadaldevelopment,andsexsteroidhormone levelswerenotsignificantlyalteredbychronicdietaryexposure tocrude oil.However,alteredspermmotilitywasseenin mea- suresofspermvelocity.Theecologicallyrealisticdosesusedinthis chronicexposurestudy,exhibitedbylowERODactivityandPAH bilemetaboliteconcentrations,maynothavebeenhighenough toinduceadverseeffectsontheinvestigatedparametersofrepro- duction. Furthermore,the utility of widely-used PAH exposure biomarkers(EROD activityand bilemetabolitesconcentrations) maybereducedwhenpolarcodarereachingthefinalstagesof reproductionandthesematuration-specificinteractions needto befurtherinvestigated.

Pollutants may manifest effects on reproduction through endocrine disruption or by altering energy investment, which mayimpedeaﬁsh’sabilitytoovercomeothernaturalphysiolog- icalstresses(Petersonetal.,2003).Thisisespeciallyrelevantfor polarcod,aspeciesalreadyexperiencingenvironmentalchanges insea-icecover(Stroeveetal.,2007),risingseasurfacetempera- tures(Belkin,2009),polewardmovingcompetitors(Renaudetal., 2012;HopandGjøsæter,2013),andincreasedfreshwaterdischarge (Peterson et al., 2006), all of which may have the potentialto alterthetimingandsuccessofreproduction(BouchardandFortier, 2011).Thetoleranceof polarcod reproductivedevelopmentto crudeoilexposurehasbeenexploredinthisstudybutmanyaspects areyettobeinvestigatedsuchasﬁnalmaturationstages,fecun- dityandfertilizationsuccess,maternalandpaternaleffects,energy investment,andsurvivalofearlylifestages(Nahrgangetal.,2016b).

Ethicsstatement

PermissiontocarryoutthisexperimentwasgrantedbytheNor- wegianAnimalWelfareAuthorityin2014(ID6571).Permissionto carryoutworkwithradioactivematerialswasgivenbytheNor- wegianRadioactiveRegulatorybody(Strålevern,projectnumber 2014-13).

Acknowledgements

ThisstudywasﬁnancedbytheNorwegian ResearchCouncil (projectsnr214184and195160).WethankEniNorgeforproviding Akvaplan-nivawiththeKobbecrudeoilusedinthisstudy.Theﬁsh werecollectedwiththehelpofthecrewontheRVHelmerHanssen andtakencareofbytheteamattheUiTBiologicalstationinKårvika.

AdditionalsamplinghelpfromApollineLaengerandEmmaKäll- grenwasgreatlyappreciated.Authorsacknowledgecontribution fromKatharinaBjarnarLøkenandMereteGrung(NIVA)inanalysis