Effects of chlordecone on 20-hydroxyecdysone concentration and chitobiase activity in a decapod crustacean, Macrobrachium rosenbergii

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ContentslistsavailableatScienceDirect

Aquatic

Toxicology

jo u r n al h om ep ag e :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

chlordecone

on

20-hydroxyecdysone

concentration

and

chitobiase

activity

in

a

decapod

crustacean,

Macrobrachium

rosenbergii

Anne

Lafontaine

a,∗

_,

_Eric

_Gismondi

a

_,

_Céline

_{Boulangé-Lecomte}

b

_,

_Perrine

_Geraudie

c

_,

Nathalie

Dodet

a

_,

_Fanny

_Caupos

d,e

_,

_Soazig

_Lemoine

d

_,

_Laurent

_Lagadic

e

_,

Jean-Pierre

Thomé

a

_,

_Joëlle

_Forget-Leray

b

a_University_of_Liège,_Laboratory_of_Animal_Ecology_and_{Ecotoxicology}_(LEAE),_Centre_of_Analytical_Research_and_Technology_(CART),₁₅_Allée_du_Six_Aout,

B-4000Liège,Belgium

b_Normandie_University,_ULH,_UMR_I-02,_{Environmental}_Stresses_and_{Biomonitoring}_of_Aquatic_Ecosystems_(SEBIO)—FR_CNRS₃₇₃₀_SCALE,₂₅_rue_Philippe

Lebon,F-76600LeHavre,France

c_{Akvaplan-Niva}_(Norwegian_Institute_of_Water_Research)_AS,_Fram_Centre,₉₂₉₆_Tromsoe,_Norway

d_DYNECAR-UMR_BOREA_(MNHN/CNRS_{7208/IRD207/UPMC),}_University_of_the_French_West_Indies_and_Guiana,_Campus_de_Fouillole,_{Pointe-à-Pitre,}

GuadeloupeF-97110,France

e_INRA,_UMR0985_Ecology_and_Ecosystem_Health_Research_Unit,_{Ecotoxicology}_and_Quality_of_Aquatic_Environments_Research_Group,₆₅_rue_de_Saint_Brieuc,

F-35042Rennes,France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received8December2015 Receivedinrevisedform1April2016 Accepted5April2016

Availableonline6April2016

Keywords: Chlordecone Macrobrachiumrosenbergii 20-Hydroxyecdysone Chitobiase Endocrinedisruptors

a

b

s

t

r

a

c

t

Chlordecone(CLD)isanorganochlorineinsecticideabundantinaquaticenvironmentoftheFrenchWest Indies.However,fewstudieshaveinvestigateditsimpactonfreshwaterinvertebrates.WhereasCLDis suspectedofinducingendocrinedisruption,thisworkaimedtostudytheeffectsofenvironmentally relevantconcentrationsofCLDonthe20-hydroxyecdysone(20-HE)hormoneconcentrationandonthe chitobiaseactivity,bothhavingkeyrolesinthemoltingprocessofcrustaceans.Inaddition,the bioaccu-mulationofCLDwasmeasuredinthemuscletissueofMacrobrachiumrosenbergiitounderlinepotential dose-responserelationship.TheresultshaveshownthatCLDwasbioaccumulatedinexposedorganisms accordingtoatrendtoadose-responserelationship.Moreover,itwasobservedthatCLDdecreasedthe 20-HEconcentrationinexposedprawnswhencomparedtocontrol,whateverthedurationofexposure, aswellasitinhibitedthechitobiaseactivityafter30daysofexposure.Thepresentstudyindicatesthat CLDcouldinterferewithmoltingprocessofM.rosenbergiibydisturbingthe20-HEconcentrationand theactivityofchitobiase,suggestingconsequencesatthelongtermontheshrimpdevelopment.This studyalsoconﬁrmedthatCLDcouldbeanendocrinedisruptorindecapodcrustaceans,asitwasalready observedinvertebrates.

1. Introduction

Endocrinedisrupting compounds(EDCs) areexogenous

sub-stancesormixturesabletointerferewiththeendocrine system

ofexposedorganisms.Theycouldaffectthehormonalsignaling

pathwaysthroughseveralmechanisms,for example by

inhibit-ingthesynthesisofhormonesordecreasingthehormonerelease

byendocrinecells(Craigetal.,2011;TabbandBlumberg,2006;

∗ Correspondingauthorat:LaboratoryofAnimalEcologyandEcotoxicology (LEAE),UniversityofLiège,Bât.B6C,15,Alléedu6Aout,B-4000Liège,Belgium.

E-mailaddress:Anne.Lafontaine@ulg.ac.be(A.Lafontaine).

Rodríguezetal.,2007).EDCscanalsodisrupthormone-receptor

interactions and act as an agonist or antagonist by binding to

thehormone-receptorcomplex(Rodríguezetal.,2007;Tabband

Blumberg,2006).Aquaticenvironmentsareconsideredthemain

sinkforcontaminants(Kloasetal.,2009;Meyer-ReilandKöster,

2000),andthusaquaticorganismsarethereforemajorpotential

targetsforEDCs(Kloasetal.,2009).

SeveralstudiesonthebiologicaleffectsofEDCsinaquatic

ver-tebrateshaveledtothedevelopmentofbiomarkersin orderto

measure thebiological responsestowardsanthropogenic

pollu-tions,suchaschangesinthereproductivefunction.Forexample,

Jobling and Sumpter (1993) introduced the concept that the

productionofvitellogenin(Vg)inmaleﬁshindicatesanexposure

http://dx.doi.org/10.1016/j.aquatox.2016.04.006

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toestrogeniccompoundandVgisnowawidelyusedbiomarkerof

xenoestrogenexposureinvertebrates.AlthoughEDCeffectshave

beenextensively described in aquaticvertebrates (Kortenkamp

etal.,2011;LeBlanc,2007),onlyfewstudieshaveexaminedthe

effectsofEDCsininvertebrates,althoughtheyrepresentthemajor

partoftheanimalkingdom(DeFuretal.,1999).Thislackcould

bepartiallyduetothefactthattheendocrinesystemof

inverte-bratesisstillobscure(Tillmannetal.,2001).Ithasnevertheless

beenobservedthat EDCs canalsodisrupt invertebrate

physiol-ogy.Forexample,Giustietal.(2013)haveshownthattributyltin

affectedtheproductionofyolkferritin(i.e.Vgequivalent)in

Lym-naeastagnalis,andXuerebetal.(2011)observedthatnonylphenol

(estrogeniccompound)impactedtheexpressionofa

vitellogenin-likegene in theamphipod Gammarusfossarum. It appears that

vitellogenin-likeproteinsininvertebratescouldserveas

biomark-ersofEDCexposure,butmechanismsinvolvedinVgproductionin

invertebratesarestillunclear(e.g.hormonalreceptor).

EDCscancomefromdifferentsourcessuchassynthetic

hor-mones(e.g.ethinylestradiol),syntheticsubstances(e.g.plasticizers

suchasphthalates,herbicides)andpesticidesoftenusedto

eradi-cateinsects.IntheFrenchWestIndies(FWI),thetropicalclimate

promotestherapiddevelopmentofpestswhichexertssigniﬁcant

pressureoncropsleadingtotheuseofconsiderableamountsof

pesticidesintheseregions(BocquenéandFranco,2005).Theuse

oforganochlorinepesticidesﬁrststartedinthe1950’sandledto

widespreadcontaminationoftheenvironment(Coatetal.,2006).

Becauseoftheirphysicochemicalproperties,organochlorine

pes-ticidesarepersistent in theenvironment and are knowntobe

accumulatedalongfoodchains,resultinginpronounced

ecotoxico-logicaldamage(Cailleaudetal.,2009;Matsumura,1975).Themost

worryingorganochlorinepesticideresidueinGuadeloupe(FWI)is

chlordecone(CLD)(Coatet al.,2011).CLDisaninsecticide that

wascommonlyemployedtocontrolthebananaweevil

Cosmopo-litessordidusfrom1972to1993underthetradenameKepone®_or

Curlone®₍_Cabidoche_et_al.,_2006,₂₀₀₉_)._A_few_years_after_the_use_of

CLD,widespreadpollutionofsoils,rivers,wildanimalsandaquatic

organismswasreported(Cavelier,1980;Snegaroff,1977),which

ledtotheprohibitionoftheuseofthispesticideinGuadeloupe

in1993.Moreover,in2009,CLDwasincludedintheStockholm

ConventiononPOPs(PersistentOrganicPollutants)andits

produc-tionandusewerebannedworldwide(ZaldívarandBaraibar,2011).

Nowadays,CLDisstillpresentinsoils,especiallyinthedensely

cul-tivatedareasfromsouthofBasse-Terre(Guadeloupe)(Cabidoche

etal.,2009).Although,Fernández-Bayoetal.(2013)showedthe

existenceofCLDdegradingorganismsinatropicalsoil(andosol)

microcosmunder aerobicconditions, CLDundergoesno

signiﬁ-cantorquick bioticorabioticdegradation(Dolﬁngetal.,2012;

Levillainetal.,2012).Drivenbythewatercycle,CLDinsoilsis

pro-gressivelytransferredtoaquaticecosystems(Coatetal.,2011)and

alsointhefoodwebbecauseofitshighKoc(SoilOrganicCarbon

WaterPartitioningCoefﬁcient)(15849L/Kg),Kow(Octanol-Water

PartitionCoefﬁcient)(4.5–6.0)anditsafﬁnityforlipids(Cabidoche

andLesueur-Jannoyer,2012;Clostreetal.,2013;SterrettandBoss, 1977;UNEP,2005;US-EPA,2008).Humancontaminationhasalso

beendetectedinFWIpopulationmainlyresultingfrom

consump-tionofcontaminatedfood,seafoodandrootvegetables(Dubuisson

et al., 2007; Gaumeet al., 2014; Guldner et al., 2010). Fishing

becameprohibitedwhenmostaquaticspecieshaveexceededthe

Europeanlegalmaximalresiduallimit(LMR)of20␮gofCLDper

kgwetweight(determinedbyNationalordinance,Anon.,2008).

Until 2008, one of the most important aquaculture resources

in Guadeloupe was the farms of the tropical giant freshwater

prawnMacrobrachiumrosenbergii.Severalstudieshaveunderlined

effects of pesticides onthis species (Revathi and Munuswamy,

2010;Satapornvanitetal.,2009),butveryfewinvestigationswere

carriedontheCLDimpactsonM.rosenbergii.However,Gaumeetal.

(2014)have observedthat theCLDexposurecaused the

induc-tionofgenesinvolvedindefensemechanismsagainstoxidative

stress(e.g.catalaseandglutathioneperoxidase),orinvolvedinthe

biotransformationprocess(i.e.cytochromeP450and

glutathione-S-transferase).

AsCLDissuspectedofbeinganendocrinedisruptor(Newhouse

etal.,2009)andasM.rosenbergiimoltingishormonallycontrolled,

thepresentstudyaimstoinvestigatetheCLDimpactonthe

molt-ingprocessbyinvestigatingtheeffectsofCLDontheconcentration

ofthe20-Hydroxyecdysone(20-HE)hormoneandonchitobiase

activityintissuesofM.rosenbergii.The20-HEhormoneisan

ecdys-teroidhormone, secretedbytheY-organ(Mykles,2011), which

initiatesmanyphysiological processes,suchasovarian

matura-tion,growth,molting,andreproductionincrustaceans(LeBlanc,

2007).Afewstudieshavebeendesignedtoinvestigatetheeffectsof

exposuretoenvironmentalEDCsontheendocrinesystemof

crus-taceansthroughecdysteroidconcentration,ortoinvestigatethe

endocrinesystemofinvertebrates(Oberdörsteretal.,1999;Palma

etal.,2009;Soetaertetal.,2007),butnostudieshaveusedthe

20-HEconcentrationtoinvestigatetheeffectsofchlordecone.

Chitobiaseisachitinolyticenzymeinvolved inthe

exoskele-tondegradationinarthropodsandthusplaysanimportantrole

in themoltingand growthof crustaceans (Duchet et al.,2011;

ZouandFingerman, 1999a).Severalstudieshave demonstrated

thatchitinolyticenzymesareinducedbythe20-HEhormone(Zou

andFingerman,1999b).However,manypollutantshavebeenalso

showntoinducethechitobiaseenzyme.Indeed,ZouandFingerman

(1999c) observed that someestrogenic agents, suchas Aroclor

1242,diethylstilbestrolorendosulfan,inhibitedchitobiase

activ-ityofthecrustaceanUcapugilator.Similarly,GismondiandThomé

(2014)notedthatsomepollutants,suspectedofbeingEDCs(i.e.

polybromodiphenylethers),coulddisturbthechitobiaseactivity

oftheamphipodGammaruspulex.

Finally,inparallelwiththesetwoparameters,the

bioaccumu-lationofCLDinM.rosenbergiiwasevaluatedtounderlinepotential

concentration-responserelationshipsbetweentheCLD

concentra-tionintissues,the20-HEconcentrationandthechitobiaseactivity.

Thesemeasuresensure assessingthemoltingdisruption,called

theinvisibleendocrinedisruption,becauseofthedisruptionofthe

crustaceanmoltingwhichisnotreadilyseeninthewild(Zou,2005).

2. Materialsandmethods

2.1. Testedorganisms

The3-month-oldpost-larvaeofM.rosenbergii(approximately

2g,7cmlengthandsexuallyundifferentiated)comingfromthe

same berried female, were provided by an aquaculture farm

(OCEAN-SA)locatedatPointe-Noire(Guadeloupe,FWI)ina

geo-graphicareafreeofCLDcontamination.Pretestshavepreviously

beencarriedout toevaluatethepresenceor absenceofCLDin

tissuesofprawnsfromPointe-Noireandresultshaveshown no

contamination(concentrations below detectionlimit) (datanot

shown).Prawnswerethentransferredtothelaboratory(DYNECAR,

UniversityoftheFrenchWestIndiesandGuiana,Guadeloupe),and

acclimatedforoneweekinglassaquariaﬁlledwith28Loftapwater

preﬁlteredonactivatedcarbon.Aquariawereunderconstant

aera-tionwitha12hlight/darkphotoperiod.Duringacclimation,prawns

werefedoncedailywithartiﬁcialshrimppellets(completefood

forrearing,LeGouessant,France)atonepelletperindividual.A

constantwatertemperatureof27.6±0.2◦Cwasmaintained,and

pHremainedat7.57_±0.03throughouttheexperiment.These

val-uesare inaccordance withoptimalwater temperature andpH

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96.3±3.8%throughouttheexperiment,regardlessof the condi-tions.

2.2. Experimentaldesign

The10mgmL−1stocksolutionofCLD(100%,Riedel-de-Haën,

Sigma-Aldrich,USA)waspreparedinacetoneaswerealsoitsthree

differentdilutions:10␮gmL−1,100␮gmL−1and1mgmL−1.A

vol-umeof56␮Lofeachdilutionandofthestocksolutionwasdiluted

intothe28Lofaquariumwaterinordertoobtainthefour

nomi-nalconcentrationsofCLDinwater:0.02␮gL−1,0.2␮gL−1,2␮gL−1

and20␮gL−1.Theseconcentrationswerechosenbecauseoftheir

environmentalrelevance.AccordingtotheGuadeloupeanDIREN

(RegionalDepartmentfortheEnvironment),Guadeloupeanrivers

arecontaminatedbyCLDatconcentrationsthatrangefrom0.2to

4␮gL−1withamaximumof8.6␮gL−1 measuredintheGrande

AnseRiverin2003(GREPP,2004;InVS-Inserm,2009).

Inthisexperimentaldesign,twodifferentcontrolswereused.

Theﬁrst,called“watercontrol”,wastapwaterpreﬁlteredthrough

activatedcarbon;and the second,called “solvent control”,was

obtainedbyspiking28Lofpreﬁlteredtapwaterwith56␮Lof

ace-tone.Tenaquariawereusedforeachconditionofexposure.During

the30daysofexposure,M.rosenbergiiwerefeddailywithartiﬁcial

shrimppellets(completefoodforrearing,LeGouessant,France),

withonepelletperindividual.Wastewasremovedeverydayprior

tofeeding.Basedontheresultsofapre-testdesigned to

evalu-atetheconcentrationofCLDinwateraccordingtotheduration

ofexposure,itwasdecidedtorenewtheexposuremediumevery

96h,overatotalexposuretimeof30days.Thisprocessallowed

tomaintainconstantconcentrationsofCLDduringthe30daysof

exposure.

Atotal of360post-larvaeofM. rosenbergiicomingfromthe

sameberriedfemaleandhavingthesameage(seeSection2.1),

wereexposed (i.e.36prawnsper condition)for 30days, which

is the duration of two molt cycles (Ross, 2001). At the

begin-ning of the exposure (T0), prawns of M. rosenbergii were in

premoltstage,conﬁrmedbythemeasurementofthe20-HE

con-centrationin12prawnsrandomlysampledbeforeexposure(i.e.

2058.61±371.66pgg−1). Thisconcentration corresponds tothe

peakreachedbythe20-HEhormoneduringthemoltcycle(see

belowtheconﬁrmationbyresultsof20-HEconcentrationinboth

controlsfrom1 to15daysand from15to30days,

correspond-ingtoonemoltcycleeach).FifteenM.rosenbergiiwererandomly

sampledforeachcondition,afterfourdurationsofexposure:1,4,

15and30days.Fiveprawns(correspondingto5replicates)were

usedfortheCLDbioconcentrationassessments,andtenprawns

(correspondingto10replicates)wereusedtomeasurethe20-HE

concentrationaswellasthechitobiaseactivity.Aftersampling,the

prawnswereimmediatelyfrozeninliquidnitrogenandstoredat

−80◦_C_until_analyses._Before_analyses,_body_length_was_measured

andnosigniﬁcantdifference(two-wayANOVAtest,p>0.05)was

observedbetweengroups(Supplementarymaterial1).

2.3. ChlordeconeconcentrationinexposurewaterandM.

rosenbergii

2.3.1. Chlordeconeextractionfromwater

The CLDconcentration was analyzed in aquarium water by

liquid-liquid extraction. A volume from 10mL to 1L of water

(accordingtotheCLDconcentration)wascollectedand 5mLof

dichloromethane(Biosolve-Chimie,France)wasadded.Then,50␮L

of acetonic solution of PCB112 (100pg␮L−1₎ _(Dr._{Ehrenstorfer,}

Germany),usedassurrogateinternalstandard,wereaddedbefore

the extraction procedure. This internal surrogate was used in

ordertoquantifypossiblelossofCLDduringtheextractionand

puriﬁcationprocedures.Theorganicphasewasrecoveredandthen

apuriﬁcationprocedurewasperformed(seeSection2.3.3).

2.3.2. Chlordeconeextractionfrommuscletissue

TheCLDconcentrationwasanalyzedinabdominalmuscleof

M.rosenbergiiaccordingtoamethodderivedfromDebieretal.

(2003),Multigneretal.(2010)andLagarrigueetal.(2014).Prawns

were thawed, and approximately 500mg of muscletissue was

freeze-driedduring20hwithaBenchtop3LSentryLyophilisator

(VirTis,USA).Thelyophilizedsampleswereweighedinorderto

determinewatercontent.TheextractionofCLDwasperformed

withasolventmixtureofn-hexane:dichloromethane(90:10;v:v;

Biosolve-Chimie, France)usingan AcceleratedSolventExtractor

(ASE200) (Dionex,ThermoScientiﬁc,USA) at80◦Cand undera

pressureof1500Psi.Beforetheextraction,100␮Lofahexanic

solu-tionofPCBcongener112(Dr.Ehrenstorfer,Germany)wasadded

tothesamplesasasurrogateinternalstandardtoobtainaﬁnal

concentrationof50pg␮L−1_._Then,₅₀₀_mg_of_anhydrous_sodium

sulfatewereaddedtoavoidanywatertraceintheextract.The

sol-vent,containingtheextractedfat,wascollectedinpre-weighed

vialsandevaporatedat40◦Cunderagentlenitrogenﬂowusinga

TurbovapLV(Zymarck,USA)untilaconstantweightofresidues.

Then,thelipidcontentwasdeterminedgravimetrically.

2.3.3. Samplepuriﬁcations

The puriﬁcationstep wasthesamefor water and biological

samples. The residues from the two different extraction were

resuspendedinto2mLofn-hexane(Biosolve-Chimie,France)and

transferredintoatesttubetocarryoutanacidclean-up.Avolume

of2mLof98%sulfuricacid(Merck,Germany)wasaddedinthe

extractsinordertoremoveorganicmatter(e.g.lipids,lipoproteins,

carbohydrates).Themixturewashomogenizedbyvortexingfor

1minwithaVibramax110(Heidolph,Germany),andcentrifuged

for3minat2160g,10◦C.Theorganicphasewascollectedintoa

newtubeandthesulfuricacidlayerwasextractedinthesameway

withanother3mLofn-hexanetoensureanoptimalrecovery.The

tworesultingorganiclayerswerepooledand5␮Lofnonanewere

addedasakeeper.Thisextractwasevaporatedunderagentle

nitro-gen streamusing a Visidryevaporator(Supelco, Sigma-Aldrich,

USA)beforebeingresuspendedwith45␮Lofn-hexaneand50␮L

ofasolutionofPCB209(100pg␮L−1_in_n-hexane)_as_an_injection

volumeinternalstandard(Dr.Ehrenstorfer,Germany).

Inparallelwithsampleextractions,aproceduralblankanda

QualityControl(QC)werecarriedout.Theproceduralblank

con-sistedoftheASEextractionwithoutbiologicalmatrix,allowingto

controltheextractionandtheclean-upprocedure.TheQCwas

per-formedtoassesstheCLDrecoverybyusingaCLD-freewateror

biologicalmatrix(here,freeze-driedmuscleofthedecapodPenaeus

monodon)spikedwithanacetonicsolutionofCLDinordertoobtain

a ﬁnal concentration of 2.5ngL−1 for water and 2.5ngg−1 wet

weightforbiologicalsample.MuscletissueofP.monodonwasused

hereasbiologicalmatrixofdecapodbecausetheycamefrom

geo-graphicareafreeofCLDcontamination.

2.3.4. Chromatographyanalysis

Thepuriﬁedextracts,proceduralblankandQCwereanalyzed

by high-resolution gas chromatography using a Thermo Quest

Trace2000gaschromatographequippedwitha63_Ni_ECD

detec-tor(ThermoScientiﬁc,USA)and anauto-samplerThermoQuest

AS2000(ThermoScientiﬁc,USA).Theextractwasinjectedinthe

on-columnmodeat60◦C.CLDwasseparatedona30m×0.25mm

(0.25␮mﬁlm)DB-XLBcapillarycolumn(J&WScientiﬁc,USA).The

otheranalyticalparametersweredescribedelsewhere(Lagarrigue

etal.,2014;Multigneretal.,2010).Datawererecordedwith

Chrom-card 2.8 (Fisons Instruments,Italy) softwarefor Windows. CLD

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Table1

Chlordeconeconcentration(mean±S.D.)measuredinwaterofexposure.LOQmeanslimitofquantiﬁcation(0.01ngL−1_).

Conditions: Watercontrol Solventcontrol 0.02␮gL−1 0.2␮gL−1 2␮gL−1 20␮gL−1

Nominalconcentration(␮gL−1₎ ₀ ₀ _0.020 _0.20 _2.00 _20.00

Measuredconcentration(␮gL−1₎ _<LOQ _<LOQ _0.019_±_0.002 _0.20_±_0.03 _1.80_±_0.13 _20.43_±_2.56

Table2

CLDbioconcentrations(mean±S.D.)measuredinthemuscletissueofMacrobrachiumrosenbergiiexposedatfourconcentrationsofCLDandsampledatfourtimesofexposure. LODmeanslimitofdetection(0.02ngg−1_wet_weight)_and_LOQ_means_limit_of_{quantiﬁcation}_(0.06_ng_g−1_wet_weight)._Different_letters_indicate_{signiﬁcantly}_different_values

foreachCLDconcentrationofexposure.Tukey’sHSDtestwasperformedwiththelog-transformedCLDconcentration(p-values<0.05).

NominalConcentration(␮gL−1₎ _Time_(days) _Chlordecone_{concentration}_in_muscle_tissue_(ng_g−1₎ _BCF

Control 1 <LOD /

4 <LOQ /

15 <LOQ /

30 <LOQ /

SolventControl 1 <LOD /

4 <LOQ / 15 <LOQ / 30 <LOQ / 0.02 1 1.58±1.09a ₇₉ 4 4.20±3.34b ₂₁₀ 15 5.08±1.13b ₂₅₄ 30 30.80±10.37c ₁₅₄₀ 0.2 1 10.56±1.64a ₅₃ 4 7.89±1.68a ₃₉ 15 38.12±13.48b ₁₉₁ 30 45.96±34.09b ₂₃₀ 2 1 36.06±11.90a ₃₂ 4 27.66±5.01a ₁₄ 15 241.52±127.38b ₁₂₁ 30 845.15±374.31c ₄₂₃ 20 1 292.75±133.11a ₁₅ 4 547.36±228.74a ₂₇ 15 26637.37±12411.53b ₁₃₃₂ 30 5312.04±2683.82c ₂₆₆

withalinearcalibrationcurve(1.5to200pg␮L−1₎_established_with

certiﬁedsolutionsofCLD(Riedel-deHaën,Germany).TheCLD

con-centrationsineachsampleandintheQCwerecorrectedwiththe

percentagerecoveryofthesurrogatePCB112.Therecovery

efﬁ-ciencybasedontheCLDrecoveryinQC andontherecoveryof

thesurrogateinternalstandard(PCB112)rangedfrom88±4%to

115±5%,which waswithinthelimitsrecommendedbySANCO

(i.e.range from60 to140%–DocumentNoSANCO/12495/2011

EuropeanUnion,2011).The limit of detection(LOD) wasﬁxed

atthree times the backgroundnoise of thechromatogram (i.e.

0.005ngL−1forwatersampleand0.02ngg−1wetweightfor

mus-cletissue).Thelimitofquantiﬁcation(LOQ)wasdeterminedwith

CLD-freewaterorfreeze-driedprawnmusclespikedwithvarious

concentrationsofCLDandwasestablishedat0.01ngL−1forwater

sampleand0.06ngg−1wetweightformuscletissue,

correspond-ingtoabouttentimesthebackgroundnoiseofthechromatogram.

TheCLDconcentrationsinwaterandinmuscletissueofM.

rosen-bergiiweremeasuredinﬁvereplicatesperconditionandthemean

calculated.TheCLDconcentrationsexpressedin␮gL−1forwater

samplearepresentedinTable1andinngg−1wetweightfor

bio-logicalsamplearepresentedinTable2.Thebioconcentrationfactor

(BCF)wascalculatedforeachconditionusingthemethodofTaylor

(1983)describedinDuquesneetal.(2000)(Table2).BCFistheratio

betweenCLDconcentrationinmuscletissuesofprawnsandCLD

concentrationintankwater.

2.4. 20-Hydroxyecdysoneconcentration

Theconcentrationsofthe20-HEhormoneanditsderivatives

(called “20-HE” in the following text, ﬁgures and tables) were

measured by following the manufacturer’s instructions of the

EnzymeImmunoAssay(EIA)kit(CaymanChemicalCompany,USA)

whichwereadaptedtoourbiologicalorganism(e.g.weightof

tis-sue,solventofhomogenization,standardcurve).Muscletissuewas

chosenasbiologicalmatrixinordertobeconsistentwiththetissue

usedfortheCLDconcentrationassessment.Inaddition,apretest,

which investigated the20HE concentrationin different organs,

showedthatthemuscletissuehasahigh20-HEconcentration(see

Supplementarymaterial2).Aweightof250mgoffrozenmuscle

tissueofM.rosenbergiiwashomogenizedin2:1(w:v)of

methanol-water(4:1,v:v)(i.e.500␮Lofmethanol-waterto250mgoftissue)

usingaPrecellyshomogenizer(BertinTechnologies,France).

Sam-pleswereincubatedovernightat−20◦_C_and_centrifuged_for₁₀_min

at10,000gand4◦C.Theresultingsupernatantwastransferredinto

aglasstubeandthesolventwasevaporatedwithaCentriVap

Cen-trifugalVacuumConcentratorcombinedwithaCentriVapColdTrap

(Labconco,USA) usingcentrifugal force,vacuumandcontrolled

temperature.Afterevaporation,sampleswereresuspendedinEIA

Buffer(1Mphosphate,containing1%BSA,4Msodiumchloride,

10mMEDTA,and0.1%sodiumazide).

A 96-well microplate precoated with mouse anti-rabbit IgG

(Cayman Chemical Company, USA) was used. Each microplate

contained blanks (i.e. appropriated substrate solution), diluted

samples in EIA Buffer in duplicate, and an eight point 20-HE

(Sigma-Aldrich, USA) standard curve (of 7.8125–1000pgmL−1).

Eachsamplereceivedasolutionof20-HEEIAAntiserum(Cayman

ChemicalCompany,USA)containinganti-20-HErabbitIgG.Then,

asolutionof20-HEAcetylcholinesterase(AChE)EIATracer

(Cay-manChemicalCompany,USA)containingacovalentconjugateof

(5)

overnightincubation at 4◦C, the platewas washedwith Wash

Buffer(4Mphosphate,pH7.4).Next,Ellman’sReagent(Cayman

ChemicalCompany,USA)wasaddedandtheplatewasincubated

for2hinthedarkwithgentleshaking.Theabsorbancewas

mea-suredat420nmandtheaverageofduplicatesofeachsamplewas

calculated.The20-HEconcentrationswereanalyzedin10

repli-catespercondition(i.e.10samplespercondition),andtheresults

wereexpressedinpgg−1offreshweightofM.rosenbergiimuscle

tissue.

2.5. Chitobiaseactivity

A weightof 50mg of frozenmuscletissue of M.rosenbergii

washomogenizedin500␮Lofcitratephosphatebuffer(0.15M,

pH 5.5) using a Precellys homogeneizer (Bertin technologies,

France). Samples were centrifuged for 10min at 10 000g and

4◦C, and the resulting supernatants were recovered and used

toanalyzethe chitobiase activity.Chitobiaseactivitywas

mea-sured using the method described by Espie and Roff (1996).

The chitobiase reaction used

4-methylumbelliferyl-N-acetyl-ß-D-glucosaminide (MUFNAG) as a substrate and generated the

ﬂuorescent4-methylumbelliferone(MUF),whichwasevaluated.

Measurementswerecarriedoutin96-wellmicroplates

contain-ingablank(i.e.appropriatedsubstratesolution), aneight-point

MUF(Sigma-Aldrich,USA) standard curve(0–20␮M),and

sam-plesin duplicate.Everysample wasincubatedin thedarkwith

thesubstrateMUFNAG(Sigma-Aldrich,USA)for30minat25◦C.

Thereactionwasstoppedbyadditionof0.25NNaOH(pH14.1).

TheliberatedMUFwasmeasuredﬂuorometricallyatthe

excita-tionwavelengthof360nmandemissionwavelengthof450nm.

Thechitobiaseactivitywasmeasuredin10replicatesper

condi-tions.Eachreplicatewasanalyzedinduplicateandthemeanwas

calculated.Ingeneral,enzymeactivitiesarenormalizedagainstthe

totalproteincontentinsampleextracts.However,asnatural

vari-ationofproteincontents relatedtophysiologicalchanges could

constituteasourceofvariability,severalstudiesrecommendedto

expressenzymeactivityin␮molh−1(Owenetal.,2002;Radenac

etal.,2008;Xuerebetal.,2009).Therefore,thechitobiaseactivity

wasexpressedherein␮molofMUFformedperhour−1.

2.6. Statisticalanalysis

Afteralog-transformationoftheCLDconcentrationsin

mus-cle tissue, alldatamet normalityand homogeneityof variance

assumptions(ShapiroandBartletttests,p>0.05).Foreach

mea-suredparameter(i.eCLDbioaccumulation,20-HEconcentration

andchitobiaseactivity),comparisonswereperformedbytwo-way

ANOVAfollowedbyTukeyHSDpost-hoctests.Aprobabilityvalue

oflessthan0.05wasregardedassigniﬁcant.

Thecorrelationsbetweenmeasuredparameterswereanalyzed

usingthePearsoncorrelationcoefﬁcient.Alltestswereperformed

withSTATISTICA10Software(StatSoft,2012,Belgium).

3. Results

3.1. ChlordeconeconcentrationsinmuscletissueofM.rosenbergii

TheCLDconcentrationsinthemuscletissueofM.rosenbergii

weresigniﬁcantlyinﬂuencedbytheCLDconcentrationsof

expo-sure,durationofexposureandtheirinteraction(p<0.001).Results

shownthattheCLDconcentrationswerebelowthelimitof

quan-tiﬁcationinprawnsfromcontrolandsolventcontrolconditions.

Moreover,theCLDbioconcentrationinprawnswas

concentration-dependent,sinceprawnsexposedtohigherconcentrationsofCLD

accumulatedgreateramounts ofCLD(Table2).The

bioaccumu-lationofCLDinM.rosenbergiiwasalsotime-dependent.Indeed,

Fig.1.PositivecorrelationbetweenCLDbioconcentrationsmeasuredinthemuscle tissueofMacrobrachiumrosenbergiiandCLDconcentrationsinwaterofexposure (p<0.001,r=0.49,n=100),takingintoaccountalldurationofexposure.

thelongertheprawnswereexposed,thehigherwastheCLD

bio-concentration.These observations wereunderlined by a strong

signiﬁcantpositivecorrelationbetweenconcentrationsofCLDin

waterandtheCLDbioconcentrationinmuscletissueofM.

rosen-bergii(p<0.001,r=0.49,n=100;Fig.1).

Generally,whatevertheCLDconcentrationofexposure,results

showedthata bioconcentrationof CLDwasmeasuredfromthe

ﬁrst day of exposure (Table 2), following a trend towards a

concentration-responserelationship.However,nosigniﬁcant

dif-ferenceswereobservedintheCLDbioconcentrationsafter1and

4daysofexposure,exceptforexposureto0.02␮gL−1.Inaddition,

foreachconditionofexposure,thehighestCLDbioconcentration

measuredwasobservedafter30daysofexposure;exceptfor

expo-sure to20␮gL−1 where the highest CLDbioconcentration was

measuredafter15daysofexposure(Table2).

Thebioconcentrationfactor(BCF)wascalculatedforthefour

CLDconcentrationsandresultsshowedthatBCFincreasedwith

the duration of exposure. Indeed, BCF were higher in prawns

exposedfor30daysthatthoseexposedfor1or4day.Moreover,

BCFappearedhigherinM.rosenbergiiexposedtolowestCLD

con-centration(i.e.0.02␮gL−1)thantotheotherCLDconcentrations.

3.2. 20-Hydroxyecdysoneconcentration

Resultshighlightedanaturalvariationofthe20-HE

concentra-tionincontrolprawnsaccordingtothetime.Indeed,the20-HE

concentrationdecreasedbetweenthe1standthe4thdayof

expo-sure,andincreasedbetweenthe4thandthe15thday(Fig.2).In

addition,nosigniﬁcantdifferenceswereobservedbetween20-HE

concentrationsmeasuredinwatercontrolandinsolventcontrol

(p>0.05)regardlessofthedurationoftheexposure.

Results revealed that 20-HE concentration was signiﬁcantly

inﬂuencedbytheCLDconcentration,thedurationofexposureand

theirinteraction.Generally,lower20-HEconcentrationswereseen

inexposedprawnscomparedtotherespectivecontrol,regardless

ofthedurationofexposure,exceptafter4daysofexposure.Results

alsoshowedthat20-HEconcentrationsseemedgenerallylower

after4daysofexposurecomparedtoexposureslastingfor1,15or

30days.Foreachdurationofexposure,thelargestdecreaseof

20-HEconcentrationwasmeasuredinprawnsexposedto0.2␮gL−1

(exceptafter30daysofexposurewhereitwasthesecondlowest).

Indeed,the20-HEconcentrationwasonaveragetwicelowerinthis

CLDconcentrationthanintherespectivecontrolsforeachduration

(6)

Fig.2.20-HEconcentrations(pgg−1_;_mean_±_S.D.)_in_the_muscle_tissue_of_{Macrobrachium}_rosenbergii_exposed_at_four_{concentrations}_of_CLD_and_sampled_at_four_times_of

exposure.Differentlettersabovethebarsindicatesigniﬁcantlydifferentvaluesforeachsamplingtime(Tukey’sHSDtest,p-values<0.05).

Fig.3.Chitobiaseactivity(␮molMUFhydrolysedh−1_;_mean_±_S.D.)_in_the_muscle_tissue_of_{Macrobrachium}_rosenbergii_exposed_at_four_{concentrations}_of_CLD_and_sampled_at

fourtimesofexposure.Differentlettersabovethebarsindicatesigniﬁcantlydifferentvaluesforeachsamplingtime(Tukey’sHSDtest,p-values<0.05).

After1dayofexposure,the20-HEconcentrationwas

signiﬁ-cantlyfrom1.5-foldto1.8-foldlowerinM.rosenbergiiexposedto

0.2,2and20␮gL−1ofCLDcomparedtothecontrol.After4daysof

exposure,althoughthe20-HEconcentrationsseemedtobelower

inexposed prawns compared tothecontrol,nosigniﬁcant

dif-ferenceswereobserved.However,asigniﬁcantreductionofthe

20-HEconcentration(onaverage1.8-foldlower)wasmeasured

after15daysofexposureinallexposureconditionscomparedto

thecontrol,exceptfor20␮gL−1.Finally,after30daysofexposure,

prawnsexposedto0.2and20␮gL−1CLDhadtwicesmaller20-HE

concentrationthanthecontrol.

Moreover, a signiﬁcant negative correlation between20-HE

concentration and the log-transformed CLD bioconcentrations

wasobservedregardlessofthedurationoftheexposure(1day:

p<0.001, r=−0.87, n=19; 4days: p<0.001, r=−0.83, n=20;

15days: p<0.001,r=−0.76, n=24; 30days: p<0.001, r=−0.72,

n=21)(Fig.4).

3.3. Chitobiaseactivity

TheactivityofchitobiaseinmuscletissueofM.rosenbergiiwas

signiﬁcantlyinﬂuencedbytheCLDconcentration,thedurationof

(7)

Fig.4. Logarithmiccorrelationbetweenlog-transformedCLDbioconcentrationsandthe20-HEconcentrationrepresentedbycircles,aswellasthecorrelationbetweenthe log-transformedCLDbioconcentrationsandchitobiaseactivityrepresentedbytrianglesinthemuscletissueofMacrobrachiumrosenbergiisampledafter1day(A),4days(B), 15days(C)and30days(D)ofexposure.

activitymeasuredinwatercontrolandsolventcontrolwerenot

signiﬁcantlydifferent(p>0.05)regardlessoftheduration ofthe

exposure(Fig.3).Moreover,nosigniﬁcantdifferencewasobserved

inthechitobiaseactivitymeasuredinprawnsexposedtoCLDfor

1and4days,regardlessoftheCLDconcentrationcomparedtothe

respectivecontrols(Fig.3).After15daysofCLDexposure,a

sig-niﬁcantincrease inthechitobiase activitywasonlyobservedin

prawnsexposedto0.2␮gL−1comparedtothecontrol.The

chito-biaseactivitywas1.5-foldhigherthaninthecontrolprawns.On

thecontrary,asigniﬁcantinhibitionofthechitobiaseactivitywas

observedafter30daysofexposure,whatevertheCLD

concentra-tion.Indeed,thechitobiaseactivitywasinaverage1.8-foldlower

inexposedprawnsthatinthecontrol.Nocorrelationwasobserved

betweenthechitobiaseactivityandthelog-transformedCLD

bio-concentrationinprawns,expectforinprawnsexposedtoCLDfor

30days(1day:p=0.38;4day:p=0.85;15days:p=0.65;30days:

p<0.001,r=−0.74,n=25)(Fig.4).

4. Discussion

ThisworkaimedtostudytheimpactsofCLDinthecrustacean

M.rosenbergiibymeasuringCLDeffectsonthe20-HEhormone

con-centrationandthechitobiaseactivity,bothinvolvedinthemolting

processandthusinthegrowthandthedevelopment,aswellasthe

CLDbioaccumulationinthemuscletissueofprawns.

4.1. Chlordeconebioaccumulation

Thisstudyhighlightedthat chlordeconewasaccumulatedin

tissuesofM.rosenbergiifollowingatrendtowardsa

concentration-response relationship. The prawn survival was not affected by

theCLDexposure,indicatingthatexposureconcentrationswere

belowlethalconcentrations.Indeed,SchimmelandWilson(1977)

reportedthatthe96-hLC50ofCLDwas121␮gL−1forgrassshrimp

Palaemonetespugioandnosigniﬁcantmortalityinbluecrabs

Call-inectessapidusatmeasuredconcentrationsashighas210␮gL−1

CLDwasobserved.

Thebioaccumulationobserved inM.rosenbergiiis consistent

withthefactthatCLDhasahighpotentialofbioaccumulationin

aquaticorganisms,basedonitsphysicochemicalproperties(ATSDR

etal.,1995).Thisresultisinagreementwithpreviousstudies

high-lightingtheCLDconcentrationindifferentorganisms.Indeed,Van

Veldetal.(1984)demonstratedthatCLDcouldbeaccumulatedin

thechannelcatﬁshwhenexposedfor30days,andCoatetal.(2011)

observedtheCLDbioaccumulationintropicalfoodwebincluding

variousspeciesofmussels,shrimpsandﬁshes.TheCLD

concentra-tionsmeasuredinmuscletissueofM.rosenbergiiweresomewhat

lowerthanthosemeasuredinotherdecapodsexposedtosimilar

CLDconcentrations(Bahneretal.,1977;Gaumeetal.,2014).This

observationcouldbeexplainedbythedistributionofCLDintissues

ofprawns.Indeed,becauseofitsstructureandbindingproperties,

CLDbindspreferentiallytoserumproteinssuchaslipoproteinsor

albuminandtherefore,itsdistributioninthewholeorganismis

quitedifferentcomparedtootherorganochlorines(ATSDRetal.,

1995;Newhouseetal.,2009).For example,theaccumulationof

CLDis highestin theliver(Newhouseetal.,2009)while other

organochlorinepesticidessuchasHCBorp,p-DDEare

preferen-tiallydistributedintheadiposetissue(Gomez-Catalanetal.,1991;

Soineetal.,1982).Inaddition,Roberts(1981)observedhigherCLD

residualsinreproductivetissuesthaninthehepatopancreasofthe

bluecrabsC.sapidus.

OurresultssuggestthatanaccumulationofCLDwasobserved

fromthe1stdayofexposureand thatthis increaseofCLD

con-centrationsinprawnswastime-dependent fromthe4th dayof

exposure,whatevertheCLDconcentrationofexposure.However,

inthehighestCLDcondition(i.e.20␮gL−1),theaccumulationof

CLDdecreasedbetweenthe15thand30thdayofexposure.This

(8)

whichtheaccumulationisnolongerexponentialatthis

concen-trationofexposure.Severalstudieshavehighlightedtheabilityof

crustaceanstodepurateCLDslowly(Bahneretal.,1977;Roberts,

1981;Schimmeletal.,1979).ThisdepurationofCLDcouldexplain

thedifferenceofCLDconcentrationsinM.rosenbergiibetween15

and30daysofexposure.Thisdecreasecouldalsobeexplainedbya

toxicityofCLDontheorganismphysiology,resultinginlowerCLD

concentrationinprawns(e.g.gilldestructionreducingexchange

betweentheorganismand itsenvironment Hebel etal., 1997).

Indeed,onceintheorganism,asmostpesticides,chlordeconecan

exertadversetoxiceffects(Sandersetal.,1981).Severalstudies

havedemonstratedthatCLDcouldimpactthecrustacean

devel-opmentandgrowththroughdisturbanceofthemoltingprocess.

Indeed,Schimmeletal.(1979)showedthatCLDinterfereswith

themoltingof C. sapidus.Similarly, Bookhout etal. (1980) and

OberdörsterandCheek(2001)haveshownthatCLDimpactedthe

moltingandthemetamorphosisofP.pugioandRhithropanopeus

harrisii,respectively.Inaddition,Nimmoetal.(1977)andSanders

etal.(1981)reportedthatthegrowthofMysidopisbahiaand

Gam-maruspseudolimnaeus,respectively,closelylinkedtothemolting

process,wasreduced byexposuretoCLD.CLDcouldalsoaffect

thereproductionprocess(e.g.durationofembryonicdevelopment,

thejuvenileperiod,thereproductiveandpost-reproductive

peri-ods,etc.)oftherotifersofBrachionuscalyciﬂorusasobservedby

Zhaetal.(2007).AlltheseresultssuggestthatCLDaccumulatedin

organismscoulddisturbcriticalphysiologicalprocessescontrolled

bythehormonalsystem.

BCFresultsshowedthatthebioconcentrationofCLDwasmore

efﬁcientwithlowestconcentrationinwater.Prawnsexposedto

0.02␮gL−1hadhigherBCFthanprawnsexposedtootherCLD

con-centrations.Here,wecanhypothesizethatthehigherBCFobserved

inthelowestCLDconcentrationcouldbeexplainedbythe

poten-tialEDC effectof CLD.Indeed, it is wellknown that endocrine

disruptionoccurswithlow EDCconcentrationsbytheirbinding

tohormonalreceptors(Vandenbergetal.,2012).Atanexposure

concentrationof0.02␮gL−1,CLDcouldbesequesteredintissues

asmanypollutantsbutinadditionCLDcouldbesequesteredby

receptors, resulting in higher CLD concentration in organisms.

Moreover,BCFresultsindicatedthatnoplateaulevelwasreached

after 30days of exposure, especially when M. rosenbergii were

exposedto0.02␮gL−1,suggestingastrongaccumulationofCLDin

organismsexposedonthelongterm,whichcouldhavedeleterious

effects.

4.2. Chlordeconeeffectsonthe20-HEconcentration

Incontrolconditions (i.e.unexposedprawns), resultsshown

lower20-HEconcentrationafter4daysofexposurecomparedto

theothercontrols(i.e.1,15and30days).Thisdifferencemaybe

explainedbythepremoltstageofprawnsatthebeginningofthe

experimentandthemoltcycleoccurringduringtheexperiment.

Indeed,it wasestablished that themoltcycle of M.rosenbergii

lastsanaverageof15days(Ross,2001).Moreover,severalstudies

havehighlightedthevariationofthe20-HEconcentrationduring

thedifferentstagesofthemoltcycleofcrustaceans(Hyne,2011;

OkumuraandAida,2000;ZouandBonvillain,2004;Zou, 2005).

Thevariationof20-HEconcentrationobservedherecouldbedue

tovariationsofecdysteroidsynthesisaccordingtothe

developmen-talstageofprawns.Incrustaceans,theendocrinesystemregulates

manyprocessesthatdifferentiatesmalesfromfemales,larvaefrom

juveniles,andreproductivelyactivefromreproductivelysenescent

organisms(LeBlanc,2007).ChangandBruce(1980)attestedthat

ecdysteroidcompositionisaffectedbythedevelopmentalstageas

wellasthephysiologicalandreproductivestatusoftheorganism.

Indeed,theauthorsshowedthatthetotalecdysteroid

concentra-tioninhemolymphof Homarusamericanuscanﬂuctuateduring

themoltcyclefromlessthan10pg␮L−1_in_postmolt_to_more_than

350pg␮L−1_in_premolt._Generally,_ecdysteroid_{concentration}_is_low

duringintermoltandpostmolt,risingduringpremoltand

reach-ingapeakshortlybeforemolting(Mykles,2011).Thisincreasein

ecdysteroidconcentrationisnotonlyrelatedtoecdysteroid

synthe-sisbutcouldalsobeexplainedbyincreasedconversionofecdysone

orinactiveecdysteroidstotheactiveformofecdysteroids(i.e.

20-HE)(Mykles,2011).

Afterexposure toCLD, resultsrevealed an effect of CLD on

the20-HEconcentration.Indeed,adecreaseof20-HEinM.

rosen-bergiiexposedtoCLDwasseenregardlessoftheconcentrationand

thedurationofexposure(exceptforprawnsexposedto20␮gL−1

ofCLDfor15days).Thisobservationwassupportedbythefact

thatthe20-HEconcentrationandtheCLDbioconcentrationwere

signiﬁcantly negatively correlated regardless ofthe duration of

theexposure.The20-HEdisruptioncorroboratesthehypothesis

thatCLDcouldbeendocrinedisruptorininvertebratesasalready

observed in vertebrates. Indeed, several previous studies have

demonstratedthat CLDcouldbeanendocrine disruptorin

ver-tebratessuchas ﬁsh (Ankley etal., 1998; Donohoeand Curtis,

1996;NimrodandBenson,1997;SumpterandJobling,1995),birds

andmammals(Eroschenko,1981),byhavingestrogenicproperties

throughinteractionwiththeestrogen-receptorsystem(Donohoe

andCurtis,1996;Hammondetal.,1979;Rodríguezetal.,2007).In

crustaceans,variousestrogeniccompoundsandestrogenreceptor

agonists(e.g.bisphenolA;nonylphenol)havebeenshowntoact

asecdysteroidsynthesisinhibitorsorecdysteroidreceptor(EcR)

antagonists(LeBlanc,2007),leadingtoapossibledecreaseof

ecdys-teroid concentrations (Forget-Lerayet al., 2005; LeBlanc,2007;

Rodríguezetal.,2007).Thus,itcouldbehypothesizedthat CLD

decreasedthe20-HEconcentrationbybindingdirectlyto

ecdys-teroidreceptor,asaconsequenceofitsanti-ecdysteroidalactivity

(ZouandFingerman,1999d;Zou,2005).However,asotherEDCs,

CLDcanalsointerferetheendocrinesystemindirectlybyseveral

mechanisms,atanystepofthetransductionpathwayofthe

hor-mones(Hyne,2011;Rodríguezetal.,2007).The20-HEdecrease

observedherecouldalsobeexplainedbya modiﬁcationofthe

excretionrateofthehormoneoraninactivationoftheenzyme

involvedintheecdysteroidbiotransformation.Becauseofthefact

thatCLDimpactsthe20-HEconcentration,itcouldthereforeinthe

long-termdisturbthemoltingoforganismswhichisinducedby

thishormone(Hyne,2011;Rodríguezetal.,2007).Thishypothesis

issupportedbythestudiesofHiranoetal.(2009)whichhaveshown

thatlow 20-HEconcentrationintheshrimpAmericamysisbahia

exposedtononylphenol(alsoknowntobeaninhibitorof

ecdys-teroidactivity)affectedthegrowthoforganisms.Insimilarway,a

decreaseofthe20-HEconcentrationsinjuvenilesofDaphniamagna

exposedtothefungicidefenarimolwasassociatedwithabnormal

development(LeBlanc,2007;MuandLeblanc,2002).Snyderand

Mulder(2001)havealsosuggestedthatthedelayinthebeginning

ofmoltingoflarvaeinthelobsterH.americanuswhenexposedto

heptachlor,probablycorrelatedtoadropintheconcentrationsof

circulatingecdysteroids.Moreover,knowingthatcrustacean

repro-ductionislinkedtothemoltcycle(Hyne,2011),disruptioninthe

moltingprocesscouldalsocausedisturbancesinreproductionas

hypothesizedbyGismondiandThomé(2014)intheamphipod,G.

pulex.Indeed,itwasobservedthattheanti-ecdysteroidalactivity

ofnonylphenolcaused areductioninfecundityofmany insects

(LeBlancetal.,2000).

Results also highlighted stronger decrease (or tendency to

strongerdecrease)ofthe20-HEconcentrationinprawnsexposed

to0.2␮gL−1ofCLDwithtime(exceptafter30daysofexposure),

whilethisdecreasewaslessobviousinprawnsexposedtohigh

CLDconcentrations(i.e.2and20␮gL−1).Thisobservationcould

(9)

mayexertitseffects,werealreadyoccupiedbyCLDmoleculesat

theexposureconcentrationof0.2␮gL−1.WithhigherCLD

concen-trationsrequiredtoincreasetheresponse,noadditionalbinding

canoccur,andthus,themaximaleffectwasobservedinprawns

exposedto0.2␮gL−1(Welshonsetal.,2003).Moreover,itisnow

wellestablishedthatEDCsact atlow levels(Vandenberg etal.,

2012).Therefore,itcanalsobehypothesizedthat0.02␮gL−1ofCLD

istooweaktocausesigniﬁcantdisturbances,whilethe0.2␮gL−1

seemsto be thelowest sufﬁcient CLDconcentration to induce

effectsonendocrinehormones.

4.3. Chlordeconeeffectsonchitobiaseactivity

Withregardtochitobiaseactivity,resultshighlightedno

signiﬁ-canteffectsofCLDinprawnsexposedfor1or4days.However,after

15daysofexposure,asigniﬁcantincreaseofthechitobiaseactivity

wasobserved,butonlyforprawnsexposedto0.2␮gL−1ofCLD,

whileasigniﬁcantinhibitionwasobservedinallCLDconditions

after30daysofexposure.Inaddition,unlike20-HEresults,

chito-biaseactivitywasnotstronglycorrelatedwiththeCLDexposure.

Thisresultissurprisingduetothefactthatchitinolyticenzymes

areregulatedbyecdysteroidsincrustaceans(ZouandFingerman,

1999d). Indeed, Zou and Fingerman (1999b) underlined that a

decreaseinthechitobiaseactivityintheﬁddlercrab,U.pugilator,

wascorrelatedtoareduction ofecdysteroidconcentration

dur-ingthe moltingcycle, suggesting thatthe chitobiase activityis

regulatedatleastinpartby20-HE.Inthepresentstudy,based

onthedecreaseofthe20-HEconcentration,aninhibitionofthe

chitobiaseactivitywasthereforeexpected.However,theprecise

mechanismslinkingthe20-HEconcentrationandthechitobiase

activityarenotwellestablishedyet.CLDmayaffectthechitobiase

activitynotthroughtheeffecton20-HEconcentrationbutmaybe

bydisruptingseveralpathwaysandinteractionswitha fewkey

receptors.ThisresultisinlinewiththoseofSnyderandMulder

(2001).Indeed,althoughSnyder(1998)reported,inthelobsterH.

americanus,thatalthoughanincreasein20-HEconcentrationcan

inducetheexpressionofcytochromeP450-dependentdetoxifying

enzymes(CYP45),anupregulationoftheexpressionofthisenzyme

inlarvaeofH.americanusexposedtoheptachlor(organochlorine

pesticide)occurredtogetherwithadecreaseofthe20-HE

concen-tration.Inthepresentstudy,themajorabsenceofmodiﬁcation

ofthechitobiaseactivity,whileadecreaseofthe20-HE

concen-trationwasobserved,couldbeexplainedbyadirecteffectofCLD

onchitobiaseactivitythroughotherreceptorsashypothesizedby

Rodríguezetal.(2007).Indeed,itcouldbehypothesizedthatthe

inhibitionofthechitobiaseactivityduetothedecreaseofthe

20-HEconcentrationcouldbeoffsetbyaninductionofthechitobiase

activitythroughadirecteffectofCLD.Thus,thesetwoopposite

effectscouldresultinalackofthechitobiaseactivitydisruption.

However,resultsshowedadecreaseofthechitobiaseactivityafter

30daysof exposure.Thisobservationcouldbeunderstoodbya

differenceinthetimeofresponse.Indeed,asexplainedabove,a

decreaseofthe20-HEconcentrationwasobservedafter15daysof

exposure.20-HEisknowntoinducethemoltcycle,whichinvolves

thechitobiaseenzymetobreakdowntheoldcuticle(Hyne,2011).

Nevertheless,noinformation isavailableonthedurationofthe

moltcycleandthedifferentpathwaycascadesinM.rosenbergii(i.e.

from20-HEsynthesistochitobiaseactivityinduction).Therefore,it

couldbehypothesizedthattheconsequenceofthe20-HEdecrease

canappearlateronthechitobiaseactivity(here,after30daysof

exposure).

Thedisturbanceofthechitobiaseactivityobservedhere

sug-gestsadisruptionofthemoltcycleofM.rosenbergiiwhichcould

have seriousconsequences ontheindividual development,due

tothe fact that thedecapod growthis strongly linked tomolt

(LeBlanc,2007).Itcouldbehypothesizedthatchlordeconecould

affecttheembryonicandjuveniledevelopmentofM.rosenbergii,as

hasbeenobservedbyZhaetal.(2007)intherotiferiaB.calyciﬂorus

whereCLDcouldaffectthereproductionprocess(e.g.durationof

embryonicdevelopment,thejuvenileperiod,thereproductiveand

post-reproductiveperiods).

5. Conclusion

ThisstudyistheﬁrsttohighlighttheimpactsofaCLDexposure

onthehormonalsystemofthecommercialprawn,M.rosenbergii,

bymeasuringthe20-HEconcentrationandthechitobiase

activ-ity. The resultsunderscored that CLD is bioaccumulated in M.

rosenbergii causing thedecreaseof the 20-HEconcentration.In

addition, aninhibition ofthe chitobiase activitywas measured

after30daysofexposure.Althoughithasbeendocumentedafew

timesthatCLDcandisruptthemoltingprocessofinvertebrates,the

mechanismsinvolvedinthis effectremainunknown.Thiswork

suggeststhatCLDcoulddisruptthemoltingprocessthroughthe

disturbanceof the20-HEconcentration,as wellasthe

chitobi-aseactivity.ResultsallowedustoconcludethatCLDcouldhave

ananti-ecdysteroidalactivity,assuggestedfor other

estrogeno-mimeticEDCs.Nevertheless,furtherstudiesareneededtobetter

understandthemechanisminvolvedinthisendocrinedisruption

and todescribethechlordecone effectsonM.rosenbergii

popu-lation.Itcouldbeespeciallyinterestingtofocusresearchonthe

synthesisofthe20-HEandtheecdysteroidmetabolismpathways,

aswellastheeffectsofCLDonecdysteroidreceptorstoconﬁrm

theanti-ecdysteroidalactivityofCLD,andthedesignationofCLD

asanEDCininvertebrates.Finally,thelackofinformationonthe

endocrinesystemofinvertebratesmakesitdifﬁculttoexplainthe

resultsandtheirexpectedconsequences.Therefore,future

inves-tigations shouldfocus ontheinvertebrate endocrine systemto

reﬁneitsunderstanding,andtoimprovetheassessmentofeffectsof

endocrinedisruptors.Moreover,futurestudiesmightalsoinclude

anassessmentoftheinﬂuenceofmoltstageontheresponseof

organismstoxenobioticexposure.

Acknowledgments

The presentstudy wasﬁnanciallysupportedby grants from

the NationalResearch Agency (MACHLOMA, ANR-10-CESA-014,

France) and a FNRS-F.R.I.A. grant (Fonds pour la Formation à

la Recherche dans l’Industrie et dans l’Agriculture, FC 89232,

Belgium).TheauthorsthankPatrickBoucherandFranc¸oisHerman

(OCEAN-SA)fortheirhelpintheestablishmentoftheexperiment

andCatherineAdamforhertechnicalassistance.Theauthorsare

gratefultothereviewerswhohelpedtoimprovethemanuscript.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,athttp://dx.doi.org/10.1016/j.aquatox.2016.04.

006.

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