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Ecological
Indicators
jou rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / e c o l i n d
Original
article
The
effect
of
size,
weight,
body
compartment,
sex
and
reproductive
status
on
the
bioaccumulation
of
19
trace
elements
in
rope-grown
Mytilus
galloprovincialis
J.
Richir
∗,
S.
Gobert
MARECentre,LaboratoryofOceanology,UniversityofLiège,Sart-Tilman,B6c,4000Liège,Belgium
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received12February2013
Receivedinrevisedform20June2013 Accepted24June2013 Keywords: Traceelement Mytilusgalloprovincialis Size Weight Sex Gametogenesis Bodycompartmentalization
a
b
s
t
r
a
c
t
Numeroustraceelements(TEs)canbeconsideredaspotentialpollutantsoftheenvironment,theirmining productionsandindustrialusesincreasingworldwide.Theirmonitoringcanbeachievedthroughtheuse ofbioindicatorspecies,suchastheMediterraneanmusselMytilusgalloprovincialis(Lamarck,1819).That specieshasbeenwidelyusedtomonitorthechemicalpollutionofcoastalecosystemsbyCr,Ni,Cu,Zn, Cd,Pb,As,AgandV.Conversely,environmentallevelsofBe,Al,Fe,Mn,Co,Se,Mo,Sn,SbandBihavebeen littleornotmonitoredsofarinmusselwatchprograms.Bioaccumulationprocessesofthese19TEsin rope-grownM.galloprovincialispurchasedfromasaltpondwithgoodchemicalwaterqualitywerethus investigatedinthepresentstudy.
Musselsefficientlyaccumulatedthe19studiedTEs.Bioaccumulationprocessesweredrivenby numer-ousmutuallydependentbiologicalparameterssuchasthemusselsizeandfleshweight,thesexandthe reproductivestatusandthebodycompartmentconsidered.TEbioaccumulationwasapowerfunction ofthemusselsoftbodydryweight;totalcontentslinearlyincreasedwiththeshelllength.Small-size musselsoverallconcentratedmoreTEs,withahighinter-individualvariability,consequently influenc-ingthemodellingoftheirbioaccumulationinthewholeropepopulation.Althoughalargerangeof rope-grownM.galloprovincialissizescanbeusedformonitoringpurposes,onewillthustakecarenot touseextremesizeindividuals.TheinfluenceofgametogenesisindeterminingfemalebodyhigherTE concentrationspriortospawningcouldnotbeneglectedandvarieddependingontheelement.TEswere preferentiallyaccumulatedinthehepatopancreas,exceptforZn,Se,CdandMo,moreconcentratedin gills.GametogenesisdidnotinfluenceTEdistributionbetweenbodycompartments,butlikelydiluted theirconcentrationsasadirectconsequenceofmassivereproductivetissueproduction.
So,resultsfromthepresentstudyunderlinethepotentialuseofM.galloprovincialisinthebiomonitoring ofnumerouslittlestudiedTEsandgivesomeinsightsintothedecisiveroleplayedbysomerelevant biologicalparametersinbioaccumulationprocessesofthe19investigatedTEsinrope-grownmussels.
©2013ElsevierLtd.Allrightsreserved.
1. Introduction
Intheocean,traceelement(TE)biogeochemicalbalancevaries spatially and withdepth depending onwater masses physico-chemicalparameters(salinity,pH,temperatureetc.)andbiological processes(Brulandand Lohan,2003).Naturalcontinentalrunoff andatmospheric depositionofTEs totheoceans(Nriagu,1989, 1990;BrulandandLohan,2003)canbedramaticallyincreasedin coastalandestuarinewatersduetoanthropogenicactivitiessuch asurbanization, industry, agricultureor mining(Laubier,2005; Fernandezetal.,2007;Benedictoetal.,2011).TEsaremoreover consideredas non-degradablepollutants (Navratiland Minarik, 2011;PanandWang,2012);thispersistentcharactercanthusalter,
∗ Correspondingauthor.Tel.:+3243663329;fax:+3243665147. E-mailaddress:jonathan.richir@alumni.ulg.ac.be(J.Richir).
sometimesquitestrongly,theirnaturalbiogeochemicalbalancein contaminatedenvironments(Doney,2010).
Since the mid-70ies, when Goldberg (1975) proposed the mussel-watch concept to record the quality of marine waters, mussels from the genusMytilus have been used worldwide in biomonitoringsurveys(e.g.Andraletal.,2004;Kimbroughetal., 2008).Tobiomonitortheextendandtheimportanceofthecoastal pollutionbyTEs,someresearchersresorttoindigenouspopulations ofwildorcultivatedmussels(passivebiomonitoring),while oth-ersrelyupontransplantingindividualsfromareferencesite(active biomonitoring).Indeed,theactivebiomonitoringusingcagedraft musselsallowstobypassthenaturalvariability ofinternal bio-logical factors (size, sex, sexual maturity, reproduction stages, seasonalgrowthcycles)governingTEuptakeanddepuration pro-cessesin mussels (Casas and Bacher, 2006;Casas etal., 2008), and to solvethe scarcity of mussel stocksalong certain coasts (Andraletal.,2004).
1470-160X/$–seefrontmatter©2013ElsevierLtd.Allrightsreserved. http://dx.doi.org/10.1016/j.ecolind.2013.06.021
Musselwatchprogramshavehistoricallyfocussedonalimited numberofmetalssuchasPb,ZnorCu.Nowadays,thedevelopment ofverysensitiveequipment(GFAAS,ICP-MS,NAAetc.)allows sci-entistsandenvironmentmanagerstomeasuresomeTEsfoundat verylowenvironmentallevels(e.g.Be;Hsuetal.,2004).Inparallel, recenttechnologicaldevelopmentsleadtoanincreaseofmining extractionsandindustrialrefinementsofTEsofpreviouslittle con-cern(e.g.Sb;Filellaetal.,2002).Environmentalquantificationof certainlessstudiedpotentialpollutantsishenceforthnowpossible andrelevant(Luyetal.,2012).
Thefirstpurposeofthispaperwastomeasureconcentrations ofnumerousTEs(Be,Al,Fe,Mn,Co,Se,Mo,Sn,SbandBi)thathave beenlittleorneverstudiedintheMediterraneanmusselMytilus galloprovincialis(Lamarck,1819),inadditiontoTEsclassically mon-itoredwiththatspecies(Cr,Ni,Cu,Zn,Cd,Pb,As,AgandV),andto evaluatethecontaminationstatusoftheCorsican(France)shellfish pondtheywerepurchasedfrom.Astheinfluenceofthemusselsize onsofttissueTElevelsstillappearstobeambiguous(Przytarska etal.,2010),thesecondobjectiveofthisstudywastomodelthe effectofthemusselshelllengthanddryfleshweighton concentra-tionsandcontentsofthe19investigatedTEs.Knowingthatthe accumulationofreservesduringthesexualdormancyandtheir subsequentmobilizationduringgonadaldevelopmentinfluenceTE levelsandbodycompartmentalizationinMytilusspp.(Cossa,1989), thelastobjectiveofthisworkwastostudytheimportanceofthese physiologicalprocessesinM.galloprovincialis.
2. Materialsandmethods
Onlymaterialsinceramic,plasticandglasswereusedforsample treatmentandstorage.Allmaterialswerepreviously decontam-inated in HCl 5% of analytical grade for a minimum of 48h. Powder-freenitrilgloveswereused,anddissectionmaterialand labbenchesweresystematicallycleanedwithHCl2%ofanalytical grade.
2.1. Siteandbiologicalmaterial
Shellfishfarmingactivitiesoccurin2Corsicansaltpondsofits easterncoast,theUrbinopondandtheDianepond.Theoyster Cras-sostreagigasisrope-growninbothsites,whiletheMediterranean musselM. galloprovincialisisnowadays onlyrope-grown inthe Dianepond(Orsonietal.,2001;Ramsar,2008;Bouchouchaetal., 2012).TheDianepondisthethirdlargest(surfacearea:570ha)and thedeepest(maximaldepth:11m;meandepth:6m)Corsicansalt pond.FreshwaterinputsarefromtheArenastream(andthetwo smallerPietroniandRonsignesestreams)andfromthewaterrunoff frompartofthe69km2catchmentbasinoftheBravonariver.The pondisconnectedtotheopenseathroughasmall“grau”,a chan-nelsilteduppartoftheyear.Thelowwaterrenewingofthepond takesseveralmonths(totalvolume:30.2millionm3),whichmakes itsensitivetoanthropisation(Longereetal.,1972;Orsonietal., 2001;OrsoniandLaugier,2004).Althoughthepondreceivessome effluents(mainlyagricultural)fromthecatchmentbasin,the gen-eralchemicaldiagnosticofitswaterqualityremainsgood(Orsoni andLaugier,2004;Bouchouchaetal.,2012).Thehigh phytoplank-tonproductivityofthisrelativelystablesaltpond,mainlysustained bytherecyclingofnutrientsstoredinsediments,isarealadvantage fortheshellfishfarmingindustry(OrsoniandLaugier,2004).
The 3rdMarch 2010 (after mussel spawning) and the11th February2011 (beforemussel spawning), rope-grown M. gallo-provincialis werepurchased fromthe shellfishfarm SARL Etang deDiane (42◦0745.00N, 9◦3101.00E). Musselswere carefully detached from ropes with a ceramic scalpel. 74 mussels sam-pledinFebruary 2011wereusedtostudythebioaccumulation behaviourof the10 TEs littleor never studied in that species, and to biomonitor the chemical contamination of the Diane
pondbythe19investigatedTEs.Theywerealsousedtomodel relationshipsbetweenthemusselfleshdryweight(from0.17to 3.36g)orshelllength(from43.40to86.41mm)andTElevels.In Mytilusspp.,itispossibletotellthesexofindividualsbeforethey spawnbythecolour oftheirgonadal folliclesdeveloped inthe mantle(pinktoorangeforfemalesandcreamy-whitetoyellowfor males,Mikhailovetal.,1995).Onthebasisofthiscolourpattern, the74musselsweresegregatedaccordingtotheirsextostudy differences betweenmaleand female TEaccumulation priorto spawning.40supplementarylarge-size(70–80mmshelllength) mussels purchased in March 2010 (n=20) and February 2011 (n=20)wereusedforbody compartmentalizationanalysisafter andbeforespawning,respectively.Collectedmusselswerestored at−28◦C,withoutpreviousdefecationoftheirdigestivetract. 2.2. Samplepreparation
Inthelaboratory, musselsweremeasuredwithanelectronic calliper(0.01mm).Softtissues(byssusexcluded)werecarefully removedfromshellswithaceramic scalpel.Forthebody com-partmentalizationstudy,musselsweredissectedandtissueswere sorted as follows: gills, hepatopancreas,mantle and remaining softtissues. Allsamples(body compartmentsorwhole individ-uals)werefreezedried(BenchTop3L, VirTisCompanyInc.)and weighed.Samplesweighingmorethan300mgwerefurtherground withliquidnitrogeninanagatemortarandthenre-lyophilizedto eliminatecondensed ambientwatervapour. Driedpowdersand ungroundsamplesweremineralizedinTeflonbombsinaclosed microwavedigestionlabstation(EthosD,MilestoneInc.).The diges-tion procedureperformed wasa nitricacid-hydrogen peroxide mineralization(HNO3/H2O2;suprapuregrade,Merck).Digestates weredilutedtoanappropriatevolumeof50mlpriortobeing ana-lysed.Musselshellswereovendried(48hat60◦C)andweighedto calculateindividualconditionindices(Andraletal.,2004). 2.3. TEanalysis
TElevelsweredeterminedbyinductivelycoupledplasmamass spectrometry(ICP-MS)usingdynamicreactioncell(DRC) technol-ogy(ICP-MSELANDRCII,PerkinElmerInc.).Analyticalaccuracywas checkedbyanalysingCertifiedReferenceMaterials:BCR278 (mus-seltissue;n=3)fromtheBelgianInstituteforReferenceMaterials andMeasurements,andNIST1566b(oystertissue;n=10)andNIST 2976(musseltissue;n=10)fromtheAmericanNationalInstituteof StandardsandTechnology.MeanTErecoveries,all3CRMstogether, rangedfrom86%forSbto108±9%forSe;onlyCrmeanrecovery waslower(meanrecovery:72±3%).Theglobalmeanrecovery,all elementstogether,was95±9%(nocertifiedorindicativevaluefor BeandBi).ForeachTE,detectiondecision(LC),detectionlimit(LD) andquantificationlimit(LQ)werecalculatedaccordingtoCurrie (1999)orGrinzaidetal.(1977),dependingontheirspecificblank distribution(normalornot).DatawereanalysedasTE concentra-tionsortotalcontentsonadryweightbasisandareexpressedin gg−1DWoring,respectively.
2.4. Mathematicalandstatisticalanalysis
StatisticalanalyseswereperformedwithSTATISTICA10 (Stat-SoftInc.)andGraphPadPrism5(GraphPadSoftwareInc.)softwares. 2.4.1. Pairwiseandmultiplecomparisontestsofmeans
TEs measured in the 4 body compartments of each mus-sel were added to calculate individual TE total contents, and weighted by their respective dry weight to calculate individ-ualTEconcentrations. Significantdifferences betweenmeanTE levels (concentrations and contents) measured in the 4 body
compartmentsandinwholemussels(foreachreproductive sta-tus,respectively)werehighlightedthroughone-wayanalysisof variance (one-way ANOVA) followed by Tukey HSD pairwise comparison test of means with equal n’s (p<0.05), after test-ingfor normalityorhomogeneityof variances(Levenetest) on raworlog-transformeddata.Non-parametricanalysisofvariance (Kruskal–Wallistest)wasperformedwhenassumptions priorto ANOVAs(normalityand/orhomoscedasticity)werenotachieved, followedbyDunnpairwisecomparisontestofmeans(p<0.05).An equivalentstatisticalprocedure(forunequaln’s)wasperformed tocompareTEconcentrationsbetweenmusselsseparatedinto4 equivalentsize-classesofabout10mm(43–54,55–64,65–74and 75–87mm;Saavedraetal.,2004).
TE levels (concentrations and contents) in each body com-partment and in wholemussels were then compared between reproductivestatuses,usingStudentpairwisecomparisontestof meanswithequal n’s(p<0.05), aftertesting for normalityand homogeneityofvariances(Levenetest)onraworlog-transformed data.Non-parametricpairwisecomparisontestofmeans(testUof Mann–Whitney,p<0.05)wasperformedwhenassumptionsprior to Studenttests (normality and/or homoscedasticity) were not achieved.Anequivalentstatisticalprocedure(forunequaln’s)was performedto compare TEconcentrations between females and malespriortospawning.
2.4.2. Conditionindexcalculation
Musselconditionindex(CI=theratioofdryfleshweighttodry shellweight)wascalculatedaccordingtoAndraletal.(2004),to verifythatthis physiologicalindexdidnotstatistically(p>0.05) evolvewiththesizeofmusselssampledpriortospawning.There was no significant linear relationship (p=0.7818) between the CIand theshelllength.Themussel gametematurationcoupled togetherwiththeimportantfoodavailabilityinlatewinter–early springplanktonicbloomresultedintherelativelyhighmeanCI (0.226±0.053) calculated(Casas,2005).Asmusselsshowedthe samephysiologicalstatusindependentlyoftheirsize,theycould beallusedformodellingtheeffectofthemusselweightandsize onTElevels(concentrationsandcontents).
2.4.3. Principalcomponentanalysisandclusteranalysis
Datamatrices,whererowsaretheobjects(musselsamples)and columnsarethevariables(Cr,Ni,Cu,Zn,Cd,Pb,As,Ag,V,Be,Al,Fe, Mn,Co,Se,Mo,Sn,SbandBiconcentrations),werebuilt.Thedata pre-treatmentusedinthisstudywasthehalf-rangecentralvalue transformation(Moreda-Pineiroetal.,2001).Unsupervisedpattern recognitiontechniquesusedwereprincipalcomponentanalysis (PCA)andclusteranalysis(CA).Sampleswereclusteredusingthe Ward’smethod(Euclideandistancebetweenobjectsasmeasureof similarity).PCAandCAwerecarriedoutonafirstdataset com-posedofmusselssampledpriortospawning(n=74),withsizes rangingfrom43.40to86.41mm.PCAandCAwerealsocarried outona2nddatasetcomposedofbodycompartments(gills, hep-atopancreas,mantleandremainingsofttissues)andwholemussels (n=200)sampledatbothreproductivestatuses,i.e.beforeandafter spawning.
2.4.4. Datamodelling
TheeffectofthemusselweightandsizeonmeasuredTElevels inthe74individuals sampledpriortospawningweremodelled usingapowerfunction,accordingtopreviousstudies(Lobeletal., 1992;Saavedraetal.,2004;Mubianaetal.,2006):TEconcentration (M,ingg−1DW)isapowerfunctionofthemusselsofttissuedry weight(W),andtotalTEcontent(M,ing)isapowerfunctionof themusselshelllength(L):
M=aWb (1)
M=aLb (2)
RelationshipsofEqs.(1)and(2) weredouble logarithmically transformedtoyield2linearfunctions:
log10M=log10a+blog10W (3)
log10M=log10a+blog10L (4)
TheeffectofthemusselweightandsizeonTElevelswasalso modelledusing alinear regressionmodel, TEconcentration(M, ingg−1DW)andtotalTEcontent(M,ing)beingalinear func-tionofthemusselsofttissuedryweight(W)orshelllength(L), respectively:
M=bW+a (5)
M=bL+a (6)
bistheslopeoflinearfunctions(3)–(6);log10aandaarethe Y-intercepts.Toselectthemostadequatemodel(powerfunction orlinearregression)thatthebestdescribedtheeffectofthe mus-selweightandsizeonTElevels(concentrationsandcontents),an Akaikeinformationcriterion(AIC)analysiswasperformed.In par-ticular,thesecondorderinformationcriterion,oftencalledAICc, which takes into account sample sizewas used (Burnham and Anderson, 2002). An equivalentmodelling procedurewas per-formedfor mussels largerthan 55mm only (n=55). Thelinear regressionmodelwasfurtherusedtomodelrelationshipsbetween themussel shell lengthand TEconcentrations, allmussel sizes together(n=74)orlimitedtomusselslargerthan55mm(n=55), segregatedornotaccordingtotheirsex.
3. Resultsanddiscussion
3.1. ChemicalstatusoftheDianepond
TEconcentrationsdiscussedin followingSections3.1.1–3.1.4 areallexpressedingg−1DW(Tables1and2).
3.1.1. Cr,Ni,Cu,Zn,Cd,AsandPb
Cr, Ni,Cu, Zn, Cd, As and Pb concentrations in rope-grown mussels fromthe Dianepond werewithin therange of values measuredintheMediterranean,notablybyFrenchmusselwatch programs(RINBIO,2001;Andraletal.,2004;RNO,2006;Benedicto etal.,2011).For example,Cd(0.374±0.131),Pb (0.336±0.192) and Ni(1.41±0.54)concentrations werevery low tomoderate whencomparedto123sitesinthewesternMediterraneanbasin (meanlevelsforCd=1.33,Pb=1.40,Ni=1.10)surveyedwithcaged musselsbetweenyears 2004and 2006(Benedictoet al.,2011). Cr(0.554±0.320), CdandPbwerein thelow rangeof concen-trationswhen compared to97 mussel cages immersed in year 2000alongtheFrenchMediterraneancoasts(meanlevelsforCr=1, Cd=0.9,Pb=1andAs=20,Ni=2,Cu=4.1)andintheintermediate rangeforAs(31.2±6.1),Ni(1.41±0.54)andCu(4.82±1.50);Zn (72.6±33.6)concentrationswerehalfthemeanstablevalue mea-suredinthisarea(Zn=148.3;RINBIO,2001;Andraletal.,2004). SinceCr,Ni,Cu,Zn,Cd,AsandPbconcentrationsmeasuredinthe presentstudyrangedfromverylowtomediumwhencompared toprevious largespatialscale monitoringsurveys in the west-ernMediterranean(RINBIO,2001;Andraletal.,2004;RNO,2006; Benedictoetal.,2011),thechemicalstatusoftheDianepondfor these7elementscanthusbeconsideredasgood;Cr,Ni,Cu,Zn, Cd,AsandPbconcentrationsweremoreoversimilarto concentra-tionspreviouslyreportedinmusselsfromtheDianepond(RINBIO, 2001;Andraletal.,2004;RNO,2006).Finally,CdandPb concentra-tionswereunderthemaximalvalueof1gg−1ofbivalvemollusc freshweightfixedbytheEuropeanLegislationECNo.466-2001 (EC,2001).
J. Richir, S. Gobert / Ecological Indicators 36 (2014) 33–47 Table1
Traceelementmeanconcentrations,rangesofmeanconcentrationsorrangesofindividual(asterisks,*)concentrations(gg−1DW)inMytilusgalloprovincialisandothermolluscspecies.Numbersbetweenbracketsrepresentthe
numberofsampledsitesineachstudy,respectively(notavailableforref.18).
Reference Studiedarea Species Al V Fe Cr Mn
1. Benedictoetal.(2011) WesternMediterraneanbassin(123) M.galloprovincialis
2. RINBIO(2001)andAndraletal.(2004) FrenchMediterraneancoasts(93) M.galloprovincialis 1
3. Bartoloméetal.(2010) AtlanticcoastofnorthwesternSpain(10) M.galloprovincialis 1.7–7.1 2.6–6.1 5.6–55.3
4. Desiderietal.(2009) CentralAdriaticSea(7) M.galloprovincialis 45–755 73–83
5. C¸eviketal.(2008) EasternBlackSea(5) M.galloprovincialis 1150–4030 1–3 41–59
6. Favrettoetal.(1997) MuggiaBay,northAdriaticSea(1) M.galloprovincialis* 15.5–64.8 0.70–2.42
7. Thisstudy Dianepond,eastCorsica(1) M.galloprovincialis* 39–877 2.3–12.5 66–656 0.19–2.17 3.4-20.6
8. RNO(2006) FrenchMed.andEnglishChannel-Atlanticcoasts(83) Mytilusspp. 0.43–15.4 0.12–9.21
9. Giltrapetal.(2013) Irishcoasts(4) M.edulis 40–201 0.95–1.59 30–261 0.71–1.51 4.6–13.4
10. Gobertetal.(1992) Belgiancoast(4) M.edulis 166–247 0.9–1.2
11. BrooksandRumsby(1965) TasmanBay,NewZealand(1) M.edulisaoteanus* 2–8 960–2640 9–24 12–38
12. WatlingandWatling(1976) SaldanhaBay,SouthAfrica(3) C.meridionalis 9–11
13. Giltrapetal.(2013) Irishcoasts(4) C.gigas 81–148 0.88–1.29 175–254 0.79–1.70 21.3–47.1
14. Hsuetal.(2004) ChiguLagoon,southwesternTaiwan(1) C.gigas
15. WatlingandWatling(1976) LangebaanLagoon,SouthAfrica(1) C.gigas 12
16. BrooksandRumsby(1965) TasmanBay,NewZealand(1) O.sinuata* 2–4 630–750 2–6 1–11
17. BrooksandRumsby(1965) TasmanBay,NewZealand(1) P.novae-zelandiae* 5–14 1140–6900 3–23 12–306
18. Papadopoulu(1973)inEisler(2010) Greekcoasts A.noae
Co Ni Cu Zn Se Ag Cd Sn Sb As Mo Be Pb Bi 1. 0.1–3.4 0.46–2.89 0.5–8.3 2. 2 2.9–9.2 116–203 0.1–5.9 20 0.5–5.4 3. 0.4–69.3 0.8–15.4 6.9–59.9 192–301 5.4–9.3 0.4–2.3 0.1–1.4 14–32 1.1–13.3 4. 1.3–7.6 18–156 61–190 0.6–1.0 0.6–3.9 2.0–9.0 5. 1–6 90–260 180–630 2–4 5–21 6. 0.06–0.53 0.41–2.30 0.77–2.23 6.9–29.6 0.12–0.38 0.48–1.79 7. 0.37–1.37 0.71–3.39 2.8–7.4 35–224 1.5–4.3 0.005–0.033 0.21–1.03 0.015–0.089 0.007–0.028 17–46 4.8–33.1 0.005–0.037 0.14–1.10 0.005–0.021 8. 0.45–8.41 3.8–67.0 36–409 0.01–7.75 0.17–10.00 0.1–27.7 9. 0.54–0.88 0.88–1.56 5.8–14.1 118–251 0.67–1.77 0.25–0.39 6.7–17.4 1.00–2.64 1.01–4.24 10. 5.6–6.2 107–142 0.5–1.2 0.7–2.3 11. 1–17 5–11 50–180 0.1–0.3 <10 0.1–1.0 3–25 12. 2–3 2–3 7–14 73–113 1–8 2–5 5–6 13. 0.46–0.52 0.81–1.58 21–282 673–1466 1.14–1.49 0.27–0.28 9.5–20.7 0.83–1.85 0.74–1.65 14. 0.0247 15. 1 1 33 424 9 1 4 16. 1–3 21–53 850–1500 4.5–7.3 10–43 0.1–0.4 6–14 17. 2–17 2–14 195–368 0.2–2.3 210–299 0.1–2.3 10–23 18. 88.0
Table2
Traceelement(TE)concentrationsinrope-grownMytilusgalloprovincialisfromtheDianepond(eastCorsica),sampledinFebruary2011beforetheyspawned.Concentrations aregivenforallmusselstogether,independentlyoftheirsex(leftsideofthetable;mean±SDandmedian,ingg−1DW;n=74),ordistinctlyforfemales(n=29)andmales
(n=45;rightsideofthetable;mean±SD,ingg−1DW).Asterisks(*)representsignificantdifferences(p<0.05)betweensexes.
TEconcentrationsforallmusselstogether MeanTEconcentrationsbysex
Means Medians Females Males
Al 200±150 152 204±143 197±156 V 5.35±2.02 5.07 *6.55±2.40 *4.57±1.24 Fe 177±97 149 186±89 172±102 Cr 0.554±0.320 0.459 0.581±0.288 0.537±0.341 Mn 9.86±3.87 9.19 *12.18±3.48 *8.36±3.37 Co 0.634±0.205 0.589 *0.707±0.215 *0.587±0.185 Ni 1.41±0.54 1.28 *1.68±0.59 *1.24±0.43 Cu 4.82±1.50 4.03 *6.50±0.67 *3.74±0.61 Zn 72.6±33.6 64.3 *86.3±36.4 *63.7±28.8 Se 2.70±0.78 2.45 *3.48±0.34 *2.21±0.54 Ag 0.0123±0.0054 0.0111 *0.0151±0.0064 *0.0104±0.0038 Cd 0.374±0.131 0.336 *0.397±0.106 *0.358±0.144 Sn 0.0318±0.0167 0.0259 0.0323±0.0160 0.0314±0.0174 Sb 0.0126±0.0042 0.0115 *0.0140±0.0048 *0.0118±0.0036 As 31.2±6.1 31.3 *36.3±4.3 *27.8±4.6 Mo 17.1±5.8 17.1 *20.7±5.8 *14.8±4.5 Be 0.0135±0.0056 0.0122 0.0127±0.0056 0.0140±0.0057 Pb 0.336±0.192 0.279 0.378±0.211 0.309±0.175 Bi 0.0087±0.0032 0.0078 *0.0097±0.0034 *0.0080±0.0028 3.1.2. AgandV
TheAg(0.0123±0.0054) meanconcentrationwassimilarto mussel(Mytilusspp.)Agconcentrationsfromthecleaneststations of83sitessampledalongtheEnglishChannel-Atlanticand Mediter-raneanFrenchcoasts(rangeofAgconcentrations=0.01–7.75;RNO, 2006). Unlike Ag, the V mean concentration (5.35±2.02) was highcomparedtotherangeofavailabledatafortheDianepond (0.92–3.62;www.ifremer.fr/envlit/)andtomedianvaluesforthe EnglishChannel-Atlantic(1.62)andMediterranean(1.40)French coastsingeneral(RNO,2006).Itcanresultfromthecombinedeffect ofthepondconfinement,theimportantaquacultureactivitywith regardtothepondsize,inputsfromthewaterrunofffrompart ofthecatchmentbasinofBravonaandfromthe3smallstreams feedingthepond(importantprecipitationsineasternCorsicain lateJanuary2011;www.meteofrance.com)andreleasesfrom sed-iments(Orsonietal.,2001;Amiardetal.,2008;Santos-Echeandia etal.,2009;Abi-Ghanemetal.,2013).
3.1.3. Fe,Mn,Co,SeandSn
Availabledata fromtheliterature for Fe,Mn,Co, Seand Sn in mollusc bivalves are more scarce. Bartolomé et al. (2010) studied seasonal and pluriannual variations of Co, Mn, Se and Sn concentrations in wildM. galloprovincialisfrom theAtlantic coastofnorthwesternSpain(medianconcentrationsforCo=0.8, Mn=21.6,Se=8.1and Sn=0.4).Concentrationsmeasuredinthe Diane pond (Co=0.634±0.205, Mn=9.86±3.87, Se=2.70±0.78 andSn=0.0318±0.0167)weresimilarorlowerthan concentra-tionsreportedbytheseauthors.Moreover,meanSn(1.3)andMn (77.5)concentrationsmeasuredinwildandraftM.galloprovincialis fromthecentralAdriaticSeawereabout40and9timeshigherthan intheDianepond,respectively(Desiderietal.,2009).Conversely, meanlevelsofMn(1.29)andCo(0.27)measuredinraftM. gallo-provincialisfromtheMuggiaBay,northAdriaticSea,werelower thanintheDianepond(Favrettoetal.,1997).Feconcentrations (177±97)weresimilartomeanconcentrationsrecordedinMytilus spp.fromtheBelgiancoast(217;Gobertetal.,1992)andthe cen-tralAdriaticSea(199;Desiderietal.,2009),butlowerthaninthe contaminatedeasternBlackSea(2462;C¸eviketal.,2008). 3.1.4. Al,Sb,Mo,BeandBi
ForAl,Sb,Mo,BeandBi,littledatahavebeenpublished; fur-thermore,atleast3oftheseTEs(Sb,MoandBi)wereidentifiedas coastalpollutants(Luyetal.,2012).Giltrapetal.(2013)recently
monitoredAlandSbinIrishcoastalwaterswithcagedmussels Mytilusedulis(meanlevelsforAl=118,Sb=0.28)andoysters Cras-sostreagigas(meanlevelsforAl=117,Sb=0.32).Concentrations reportedbytheseauthorswerelowerthanthemeanAl concen-tration(200±150)measuredintheDianepond;thisterrigenous TEwasmoreconcentratedandbioavailableinthissemi-enclosed saltpondcatchingpartofstreamingwatersfromthecatchment basinofBravona.Conversely,Sbconcentrations(0.0126±0.0042) wereverybelow concentrationsmeasuredbytheseauthors.Be concentrationsinmarineorganismsarepoorlydocumentedmainly because they are often below detection limits of instrumental techniques employed. Nonetheless, Be concentrations in rope-grownmusselsfromtheDianepond(0.0135±0.0056)weresimilar to concentrations measuredby Hsu et al. (2004) in oysters C. gigas (0.0247) harvested from theChigu Lagoon (southwestern Taiwan).TheMomeanconcentration(17.1±5.8)wasin-between low concentrations measuredbyBrooks and Rumsby (1965)in threebivalves(meanMoconcentrationsinthemussel M.edulis aoteanus=0.6, in the oyster Ostrea sinuata=0.3, in the scallop Pecten novae-zelandiae=0.9) collected in theTasman Bay(New Zealand)andthehigherconcentrationreportedbyPapadopoulu (1973,inEisler,2010)forthebivalveArcanoae(88.0)fromGreek coastal waters. Finally, the only available Bi concentrations in bivalvesreportedbyWatlingandWatling(1976)forthemussel Choromytilusmeridionalis(5–6)andtheoysterC.gigas(4)werehigh whencomparedtoBiconcentrationsinmusselsfromtheDiane pond(0.0087±0.0032).
3.2. TEbioaccumulationinrope-grownmusselspriortospawning 3.2.1. TEconcentrationsasafunctionofthemusselweightvs.TE contentsasafunctionofthemusselshelllength
Thepresentsectioninvestigateswhichofthepowerfunction orthelinearregressionbettermodelsrelationshipsbetweenthe musselweightandTEconcentrationsorthemusselshelllength andTEcontents.
Tissueconcentrationsofthe19studiedTEsdecreasedwhenthe musselfleshdryweightincreased;theirtissuecontentsincreased whenthemusselshelllengthincreased(slopebinTable3A).This isconsistentwithpreviousstudiesmodellingtheeffectofthe mus-selweightandsizeonTEbioaccumulation(e.g.Lobeletal.,1991; Martincicetal.,1992).Significantregressionswerefoundforall
J. Richir, S. Gobert / Ecological Indicators 36 (2014) 33–47 Table3
Comparisonoflinearregressionsanddoublelogtransformedpowerfunctionsmodellingrelationshipsbetweentraceelement(TE)concentrationsinrope-grownMytilusgalloprovincialisfromtheDianepond(eastCorsica), sampledinFebruary2011beforetheyspawned,andtheirfleshdryweightandrelationshipsbetweentotalTEcontentsandthemusselshelllength.Relationshipsbetweenthemusseldryweightandtheirshelllengtharealso modelled.(A)Allmusselstogether(n=74).(B)Musselslargerthan55mm(n=55).bistheslopeoflinearfunctions;aandlog10aaretheY-intercepts.Fittingparametersarealsoindicated(r2;p-levels;deviation(dev.)fromthe
model:s.=significant,n.s.=non-significant;AICc).
A. RelationshipsbetweenTEconcentrationsandthemusselfleshdryweight—allmussels RelationshipsbetweenTEcontentsandthemusselshelllength—allmussels
Linearregressions Doublelogtransformedpowerfunctions Linearregressions Doublelogtransformedpowerfunctions
b a r2 p dev. AICc b log10a r2 p dev. AICc b a r2 p dev. AICc b log10a r2 p dev. AICc
Length 13.02 41.74 0.781 <0.001 n.s. 93.72% 0.289 1.752 0.764 <0.001 n.s. 6.28% Weight 0.0600 −2.134 0.781 <0.001 n.s. 92.16% 2.278 −3.903 0.766 <0.001 n.s. 7.84% Al −92.47 355.3 0.263 <0.001 n.s. <0.01% −0.672 2.349 0.475 <0.001 n.s. >99.99% 6.7880 −158.9 0.273 <0.001 n.s. 37.89% 1.734 −0.703 0.282 <0.001 n.s. 62.11% V −1.029 7.076 0.180 <0.001 n.s. 94.85% −0.172 0.752 0.113 <0.01 n.s. 5.15% 0.2320 −6.475 0.560 <0.001 n.s. 88.76% 1.669 −2.099 0.534 <0.001 n.s. 11.24% Fe −64.00 285.1 0.300 <0.001 n.s. <0.01% −0.531 2.298 0.507 <0.001 n.s. >99.99% 6.9590 −188.0 0.520 <0.001 n.s. 40.93% 1.812 −0.874 0.525 <0.001 n.s. 59.07% Cr −0.205 0.898 0.283 <0.001 n.s. <0.01% −0.565 −0.208 0.514 <0.001 n.s. >99.99% 0.0224 −0.632 0.520 <0.001 n.s. 34.62% 1.900 −3.541 0.528 <0.001 n.s. 65.38% Mn −3.055 14.99 0.430 <0.001 n.s. 72.89% −0.336 1.035 0.414 <0.001 n.s. 27.11% 0.3317 −6.611 0.425 <0.001 n.s. 67.03% 1.385 −1.340 0.413 <0.001 n.s. 32.97% Co −0.112 0.822 0.206 <0.001 n.s. 13.12% −0.230 −0.168 0.245 <0.001 n.s. 86.88% 0.0340 −1.174 0.820 <0.001 s. 97.18% 2.176 −3.946 0.801 <0.001 s. 2.82% Ni −0.429 2.136 0.436 <0.001 n.s. 0.16% −0.375 0.194 0.527 <0.001 n.s. 99.84% 0.0591 −1.675 0.752 <0.001 n.s. 87.57% 1.788 −2.914 0.739 <0.001 n.s. 12.43% Cu −0.815 6.195 0.205 <0.001 n.s. 96.69% −0.154 0.705 0.129 <0.01 s. 3.31% 0.1974 −5.002 0.443 <0.001 n.s. 76.62% 1.557 −1.934 0.425 <0.001 n.s. 23.38% Zn −12.60 93.76 0.097 <0.01 n.s. 22.23% −0.240 1.892 0.127 <0.01 n.s. 77.77% 3.9500 −137.9 0.578 <0.001 n.s. 65.17% 2.246 −2.016 0.571 <0.001 n.s. 34.83% Se −0.559 3.645 0.356 <0.001 n.s. 90.27% −0.224 0.462 0.316 <0.001 n.s. 9.73% 0.1145 −3.116 0.548 <0.001 n.s. 79.45% 1.685 −2.427 0.531 <0.001 n.s. 20.55% Ag −0.004 0.019 0.348 <0.001 n.s. 76.21% −0.331 −1.870 0.327 <0.001 n.s. 23.79% 0.0004 −0.008 0.376 <0.001 n.s. 66.52% 1.362 −4.204 0.365 <0.001 n.s. 33.48% Cd −0.059 0.472 0.138 <0.01 n.s. 2.75% −0.246 −0.397 0.217 <0.001 n.s. 97.25% 0.0194 −0.648 0.751 <0.001 n.s. 99.12% 2.004 −3.856 0.718 <0.001 s. 0.88% Sn −0.009 0.047 0.191 <0.001 n.s. 0.31% −0.411 −1.454 0.308 <0.001 n.s. 99.69% 0.0014 −0.040 0.374 <0.001 n.s. 60.03% 1.836 −4.646 0.367 <0.001 n.s. 39.97% Sb −0.003 0.018 0.368 <0.001 n.s. 2.73% −0.302 −1.861 0.426 <0.001 n.s. 97.27% 0.0006 −0.018 0.772 <0.001 n.s. 92.35% 1.923 −5.199 0.756 <0.001 s. 7.65% As −2.950 36.13 0.162 <0.001 n.s. 98.48% −0.069 1.504 0.062 <0.05 s. 1.52% 1.5280 −46.84 0.697 <0.001 n.s. 96.25% 1.844 −1.633 0.669 <0.001 n.s. 3.75% Mo −2.006 20.49 0.083 <0.05 s. 93.09% −0.059 1.242 0.016 n.s. s. 6.91% 0.7283 −18.92 0.466 <0.001 s. 88.66% 1.500 −1.270 0.435 <0.001 s. 11.34% Be −0.003 0.018 0.191 <0.001 n.s. 0.03% −0.360 −1.830 0.350 <0.001 n.s. 99.97% 0.0007 −0.023 0.646 <0.001 n.s. 51.21% 2.181 −5.638 0.646 <0.001 n.s. 48.79% Pb −0.077 0.465 0.111 <0.01 n.s. 12.10% −0.322 −0.435 0.157 <0.001 n.s. 87.90% 0.0197 −0.741 0.521 <0.001 n.s. 6.00% 2.809 −5.398 0.556 <0.001 n.s. 94.00% Bi −0.002 0.012 0.294 <0.001 n.s. 3.41% −0.306 −2.024 0.355 <0.001 n.s. 96.59% 0.0004 −0.011 0.672 <0.001 s. 93.65% 1.764 −5.069 0.648 <0.001 s. 6.35% Mean 0.244 35.76% 0.294 64.24% 0.564 69.95% 0.553 30.05% SD 0.109 42.51% 0.161 42.51% 0.153 26.03% 0.148 26.03% Median 0.206 12.10% 0.316 87.90% 0.548 76.62% 0.534 23.38%
B. RelationshipsbetweenTEconcentrationsandthemusselfleshdryweight—mussels>55mm RelationshipsbetweenTEcontentsandthemusselshelllenght—mussels>55mm
Linearregressions Doublelogtransformedpowerfunctions Linearregressions Doublelogtransformedpowerfunctions
b a r2 p dev. AICc b log10a r2 p dev. AICc b a r2 p dev. AICc b log10a r2 p dev. AICc
Length 9.389 50.32 0.529 <0.001 n.s. 95.67% 0.255 1.767 0.473 <0.001 n.s. 4.33% Weight 0.0563 −1.874 0.529 <0.001 n.s. 40.04% 1.927 −3.245 0.536 <0.001 n.s. 59.96% Al −38.36 235.4 0.088 <0.05 n.s. 42.49% −0.465 2.325 0.098 <0.05 n.s. 57.51% 9.1460 −329.6 0.211 <0.001 n.s. 43.54% 2.146 −1.476 0.218 <0.001 n.s. 56.46% V −1.807 8.875 0.383 <0.001 n.s. 0.05% −0.743 0.915 0.531 <0.001 n.s. 99.95% 0.1358 0.454 0.154 <0.01 n.s. 49.92% 0.949 −0.753 0.155 <0.01 n.s. 50.08% Fe −36.16 224.3 0.181 <0.01 n.s. 28.08% −0.468 2.307 0.208 <0.001 n.s. 71.92% 7.6150 −235.8 0.336 <0.001 n.s. 44.10% 1.829 −0.906 0.342 <0.001 n.s. 55.90% Cr −0.111 0.696 0.165 <0.01 n.s. 23.32% −0.479 −0.197 0.200 <0.001 n.s. 76.68% 0.0258 −0.880 0.359 <0.001 n.s. 42.88% 1.988 −3.707 0.365 <0.001 n.s. 57.12% Mn −3.028 14.99 0.340 <0.001 n.s. 39.82% −0.606 1.111 0.350 <0.001 n.s. 60.18% 0.1805 4.275 0.078 <0.05 n.s. 50.13% 0.751 −0.156 0.078 <0.05 n.s. 49.87% Co −0.163 0.948 0.308 <0.001 n.s. 1.66% −0.550 −0.060 0.403 <0.001 n.s. 98.34% 0.0321 −1.036 0.597 <0.001 n.s. 49.64% 1.842 −3.319 0.597 <0.001 n.s. 50.36% Ni −0.423 2.139 0.406 <0.001 n.s. 0.16% −0.678 0.290 0.530 <0.001 n.s. 99.84% 0.0537 −1.286 0.494 <0.001 n.s. 51.40% 1.507 −2.390 0.493 <0.001 n.s. 48.60% Cu −1.099 6.818 0.222 <0.001 s. 45.66% −0.433 0.781 0.227 <0.001 n.s. 54.34% 0.1121 1.123 0.093 <0.05 n.s. 49.87% 0.871 −0.655 0.093 <0.05 n.s. 50.13% Zn −17.46 105.7 0.115 <0.05 n.s. 26.28% −0.511 1.986 0.148 <0.01 n.s. 73.72% 3.9500 −138.1 0.317 <0.001 n.s. 47.38% 2.002 −1.559 0.320 <0.001 n.s. 52.62% Se −0.636 3.818 0.291 <0.001 s. 23.47% −0.475 0.533 0.321 <0.001 n.s. 76.53% 0.0850 −1.007 0.208 <0.001 n.s. 49.76% 1.207 −1.534 0.208 <0.001 n.s. 50.24% Ag −0.004 0.019 0.355 <0.001 n.s. 2.37% −0.728 −1.760 0.436 <0.001 n.s. 97.63% 0.0002 0.009 0.042 n.s. n.s. 50.11% 0.599 −2.780 0.042 n.s. n.s. 49.89% Cd −0.094 0.560 0.356 <0.001 n.s. 4.89% −0.506 −0.295 0.422 <0.001 n.s. 95.11% 0.0145 −0.290 0.388 <0.001 n.s. 50.22% 1.394 −2.715 0.388 <0.001 n.s. 49.78% Sn −0.006 0.041 0.082 <0.05 n.s. 33.90% −0.442 −1.424 0.104 <0.05 n.s. 66.10% 0.0013 −0.034 0.150 <0.01 n.s. 51.19% 1.557 −4.123 0.148 <0.01 n.s. 48.81% Sb −0.003 0.019 0.359 <0.001 n.s. 2.59% −0.566 −1.775 0.438 <0.001 n.s. 97.41% 0.0005 −0.014 0.517 <0.001 n.s. 51.82% 1.604 −4.601 0.516 <0.001 n.s. 48.18% As −5.407 41.68 0.339 <0.001 n.s. 29.66% −0.331 1.579 0.359 <0.001 n.s. 70.34% 1.0950 −15.57 0.322 <0.001 n.s. 50.05% 1.252 −0.525 0.322 <0.001 n.s. 49.95% Mo −6.253 30.17 0.458 <0.001 n.s. 7.82% −0.661 1.421 0.504 <0.001 n.s. 92.18% 0.1684 21.42 0.023 n.s. n.s. 48.73% 0.363 0.852 0.025 n.s. n.s. 51.27% Be −0.002 0.016 0.072 <0.05 n.s. 50.10% −0.238 −1.842 0.072 <0.05 n.s. 49.90% 0.0007 −0.027 0.432 <0.001 n.s. 51.34% 2.059 −5.409 0.430 <0.001 n.s. 48.66% Pb −0.078 0.473 0.089 <0.05 n.s. 24.45% −0.539 −0.355 0.126 <0.01 n.s. 75.55% 0.0264 −1.221 0.437 <0.001 n.s. 35.13% 3.003 −5.764 0.449 <0.001 n.s. 64.87% Bi −0.003 0.014 0.394 <0.001 n.s. 0.55% −0.676 −1.901 0.498 <0.001 n.s. 99.45% 0.0003 −0.003 0.294 <0.001 n.s. 49.92% 1.179 −3.976 0.294 <0.001 n.s. 50.08% Mean 0.263 20.39% 0.315 79.61% 0.287 48.27% 0.289 51.73% SD 0.129 17.31% 0.160 17.31% 0.169 4.14% 0.170 4.14% Median 0.308 23.47% 0.350 76.53% 0.317 49.92% 0.320 50.08%
Fig.1.Doublelogtransformedpowerfunctionsandlinearregressionsmodellingrelationshipsbetween(aandb)thesofttissuedryweightandSbconcentrations(gg−1DW)
andbetween(candd)theshelllengthandSbtotalcontents(g)ofrope-grownMytilusgalloprovincialis(n=74)fromtheDianepond(eastCorsica),sampledinFebruary 2011beforetheyspawned.Linearequationsandtheircorrespondingfittingparameters(r2;p-levels;deviation(dev.)fromthemodel:s.=significant,n.s.=non-significant;
AICc)arereportedongraphs.
TEs(p<0.05),exceptfortherelationshipbetweentheweightand Moconcentrationsinthedoublelogtransformedpowerfunction model.Meangoodnessoffitwashigherforrelationshipsbetween themusselshelllengthandTEcontents(meanr2=0.564or0.553 forthelinearregressionorthepowerfunction,respectively); lin-ear equationsdeviated less often fromthe trend ofdata when modellingtheeffectofthemusseldryweightonTE concentra-tions(4significantdeviations–p<0.05–forMo,CuandAs,both modelstogether).TheAICcshowedthatpowerfunctionsbetter describedrelationshipsbetweenthemusseldryweightandTE con-centrations(meanAICc=64.24%),whilelinearregressionsbetter describedrelationshipsbetweenthemusselshelllengthandTE contents(meanAICc=69.95%).TheexamplegiveninFig.1forSb properlyillustratesstatisticalconsiderationsofTable3A.
Resultsofregressionfittingparameterswereinconsistentwith observationsmadebySaavedraetal.(2004)whoelectedthepower functiontomodeltheeffectofraftM.galloprovincialissize,ranging from52to87mm,onTEcontents.Inorder toworkwith simi-lardatasets,bothmodelswererunagainformusselslargerthan 55mmonly(Table3B).MeancalculatedAICcwerethenequalto 48.27%and51.73%forlinearandpowerrelationshipsbetweenTE contentsandthemusselshelllength,respectively.Asthedifference inlikelihoodwasverysmall(i.e.similarmeanAICc),onemayhave concludedthatbothmodelswerecorrect,asdidSaavedraetal. (2004).Moreover,the7relationshipsbetweenV,Mn,Cu,Se,Ag, AsandMoconcentrationsandthemusseldryweight,previously bettermodelledbylinearregressions(AICcofTable3A),werethen bettermodelledbypowerfunctions(AICcofTable3B),in agree-mentwithLobelet al.(1989, 1991) and Mubianaet al.(2006). Overallfittingparametersofpowerfunctionswerealsoimproved: themeanAICcincreasedfrom64.24%to79.61%,andthemeanr2 from0.294to0.315.TheexamplegiveninFig.2forBiproperly illustratesstatisticalconsiderationsofTable3B.
Itcanbeconcludedthatsmall-sizerope-grownM. galloprovin-cialishadanantagonisteffectonthemodellingof relationships
betweenthemusselsizeandweightandTElevels:theydroveto electlinearregressionstomodelrelationshipsbetweenthe mus-selshelllengthandTEcontents,butdiminishedthesignificanceof powerfunctionsmodellingrelationshipsbetweenthemusseldry weightandsomeTEconcentrations.Graphicalrepresentationsof powerfunctionsmodellingrelationshipsbetweenthemusseldry weightandTEconcentrations(e.g.Figs.1aand2a)orlinear regres-sionsmodellingrelationshipsbetweenthemusselshelllengthand TEcontents(e.g.Figs.1dand2d)aregiveninAnnexAforthe19 studiedTEs.Forcomparisonpurposes,inAnnexA,pairedgraphs modelrelationships(powerfunctionsorlinearregressions)forall musselsorforindividualslargerthan55mm,respectively. 3.2.2. TEconcentrationsasafunctionofthemusselshelllength
The present section investigates if there is a relationship betweenTEconcentrationsandthemusselshelllength,orifthesize hasnosignificant(p>0.05)importanceonTEconcentrations bioac-cumulatedinrope-grownM.galloprovincialisandcanconsequently beovercomeinbiomonitoringsurveys.
Resultsshowedthatconcentrationsof14outofthe19 stud-iedTEs (Al, V, Fe,Cr, Mn,Ni, Cu,Se,Ag, Sn,Sb,As,Be and Bi) werecorrelatedwiththeshelllength(p<0.05;Table4).Thisis par-tiallyinconsistentwithresultsobtainedbySaavedraetal.(2004). TheseauthorsmodelledrelationshipsbetweenTEconcentrations andtheshelllengthforraftM.galloprovincialismeasuringfrom 52to87mm,andseparatedinto4size-classes;theyobservedno differencebetweenCd,Pb,Cr,Ni,As,CuandZnconcentrationsat differentshelllengths,asshownbythelackofsignificance(p>0.05) ofcorrespondinglinearregressions.
Whenthelinearregressionmodelwasrunagainformussels largerthan55mm(i.e.excludingsmall-sizeindividuals),only7 TEs(V,Mn,Cu,Se,Ag,AsandMo)werestillcorrelated(p<0.05) withtheshelllengthinsteadoftheprevious14(Table4).Mean TE concentrations calculated for mussels separated into the 4 size-classes(43–54,55–64,65–74and75–87mm)weremoreover
Fig.2.Doublelogtransformedpowerfunctionsandlinearregressionsmodellingrelationshipsbetween(aandb)thesofttissuedryweightandBiconcentrations(gg−1DW)
andbetween(candd)theshelllengthandBitotalcontents(g)ofrope-grownMytilusgalloprovincialisfromtheDianepond(eastCorsica)largerthan55mm(n=55), sampledinFebruary2011beforetheyspawned.Linearequationsandtheircorrespondingfittingparameters(r2;p-levels;deviation(dev.)fromthemodel:s.=significant,
n.s.=non-significant;AICc)arereportedongraphs.
alwayshigherforsmall-sizemussels(43–54mm),exceptforMo, AsandCd;standarddeviationswerealsogenerallylarger(Table4). Henceforth,ifsmall-sizeindividuals accumulatedproportionally more TEs, they also presented an important inter-individual variability.TheexamplegiveninFig.3forCrproperlyillustrates these results, as the significant linear relationship (p=0.0007; Fig.3a)existingbetweenthemusselshelllengthandCr concen-trationsdisappeared(p=0.9116; Fig.3b)when musselssmaller than 55mm were not taken into account. Small-size mussels werefoundinside theraft,closetothefixingrope,mixedwith
broken shells, covered by bigger individuals. Their shell also presentedsometimesdeformitiesduetotheconfinement.These unfavourableconditions,slowingdowntheirgrowth,ledtohigher TE concentrations and to bigger variabilities observed within this size-class. Graphical representations of linear regressions modellingrelationshipsbetweenthemusselshelllengthandTE concentrations(e.g.Fig.3)aregiveninAnnexBforthe19studied TEs.For comparisonpurposes,inAnnexB, pairedgraphsmodel relationshipsforallmusselsorforindividualslargerthan55mm, respectively.
Table4
Leftpartofthetable:p-valuesoflinearregressionsmodellingrelationshipsbetweentraceelement(TE)concentrationsandtheshelllengthofrope-grownMytilus gallo-provincialisfromtheDianepond(eastCorsica),sampledinFebruary2011beforetheyspawned,allmusselsizestogether(n=74)orlimitedtomusselslargerthan55mm (n=55).Significantp-values<0.05areinbold.RestoftheTable:TEconcentrations(mean±SD,ingg−1DW)inmusselsseparatedinto4equivalentsize-classesofabout
10mm(n=19,21,20and14forsize-classescl.1,2,3and4,respectively).Lettersrepresentsignificantdifferences(p<0.05)betweensize-classes. p-valuesoflinearregressions Meanconcentrationsbysize-class
Allmussels >55mm cl.1:43-54mm cl.2:55–64mm cl.3:65–74mm cl.4:75–87mm Al 0.0003 0.9637 323±223a 162±63ab 150±95b 160±88ab V 0.0310 0.0229 5.80±2.50ab 5.67±1.84a 5.43±1.67ab 4.11±1.68b Fe 0.0003 0.6747 255±146a 156±43ab 148±63b 146±55b Cr 0.0007 0.9116 0.803±0.489 0.477±0.146 0.462±0.197 0.465±0.181 Mn <0.0001 0.0081 12.89±4.03a 9.84±2.84b 9.07±3.62b 6.88±2.51b Co 0.2192 0.8275 0.688±0.255 0.605±0.135 0.626±0.235 0.616±0.173 Ni 0.0002 0.3083 1.81±0.66a 1.30±0.37b 1.34±0.50b 1.16±0.34b Cu 0.0004 0.0063 5.55±1.40a 4.86±1.47a 4.98±1.55a 3.56±0.69b Zn 0.4092 0.9809 79.7±37.8 66.2±22.2 75.7±43.8 67.9±25.2 Se <0.0001 0.0307 3.24±0.66a 2.64±0.76ab 2.64±0.81b 2.17±0.48b Ag <0.0001 0.0062 0.0157±0.0068a 0.0124±0.0040a 0.0116±0.0048ab 0.0083±0.0027b Cd 0.2599 0.1196 0.390±0.200 0.389±0.100 0.357±0.089 0.352±0.111 Sn 0.0048 0.4958 0.0413±0.0222 0.0282±0.0117 0.0312±0.0170 0.0248±0.0073 Sb 0.0026 0.4752 0.0152±0.0052 0.0119±0.0028 0.0119±0.0040 0.0113±0.0038 As 0.0096 0.0048 32.7±6.8a 31.7±5.5ab 32.3±5.9ab 26.9±4.5b Mo 0.1741 0.0002 16.3±6.0ab 19.5±4.9a 18.8±5.6a 12.3±4.2b Be 0.0173 0.5205 0.0169±0.0085 0.0122±0.0030 0.0121±0.0042 0.0128±0.0040 Pb 0.4056 0.1768 0.400±0.253 0.268±0.138 0.324±0.185 0.369±0.154 Bi 0.0064 0.0673 0.0100±0.0039 0.0089±0.0032 0.0082±0.0026 0.0073±0.0021
Fig.3.LinearregressionsmodellingrelationshipsbetweentheshelllengthandCrorSeconcentrations(gg−1DW),allmusselstogether(aandc;n=74)orlimitedtomussels
largerthan55mm(bandd;n=55),ofrope-grownMytilusgalloprovincialisfromtheDianepond(eastCorsica),sampledinFebruary2011beforetheyspawned.♀and ♂symbolizefemalesandmales,respectively.Thecommon(co.)regressionto♀and♂forCr,onlysignificant(p<0.05)whenconsideringallmussels(a),underlinesthe determiningeffectplayedbysmall-sizemussels.Thecommonregression(co.)to♀and♂forSe,significant(p<0.05)whenconsideringallindividuals(c)oronlymussels largerthan55mm(d),underlinesthedifferenceinSeaccumulationlinkedtosexpriortospawning,superimposedonthesizeeffect;regressionsspecifictoeachsex(fe., ma.)aremoreovernotsignificant(p>0.05).Probabilitiesoflinearslopestodeviate(dev.)from0(s.=significant,n.s.=non-significant)aregivenongraphs.
Resultsfurthershowedthatformid-tolarge-sizeM. galloprovin-cialisgrownonropes,thesizedidnotinfluencebodyconcentrations of most TEs (Table 4; little significant differences – p<0.05 – betweenmeanTEconcentrationsofsize-classescl.2–4).Astheir culturestartssynchronicallyandaccordingly,allmusselsonarope havethesameage(Saavedraetal.,2004),butmaydifferinsize. Then, when sorting mussels for monitoringpurposes, carewill betakennottousesmall-(restrainedgrowth)butalsolarge-size (rapidgrowth)mussels,theseindividualsbeingnotrepresentative oftheropepopulation.Thisconsiderationhastobeminimizedas small(<50mm)andlarge(>80mm)musselsonlyrepresentaround 5%oftotalindividualsona10kgrope.
3.2.3. Patternrecognitionsamongmusselssampledpriorto spawningusingPCAandCA
78%ofthetotalvarianceofthedatasetbuiltwiththe74mussels sampledpriortospawningwasexplainedbythethreefirst prin-cipalcomponents(PCs;eigenvalueshigherthan1)resultingfrom thePCA.Moreprecisely,concentrationsof11outofthe19studied TEs(Al,Fe,Cr,Mn,Co,Ni,Cd,Sn,Sb,BeandBi)werethe dominat-ingfeaturesinthefirstPC(PC1)andexplainedtogether51%ofthe totalvarianceofthedataset.Cu,AsandMoconcentrationsshowed thehighestweightsinthesecondPC(PC2),explaining19%ofthe totalvariance.InthethirdPC(PC3),onlyZnweightwascloseto butlowerthan0.700,followedbyVandSe;PC3explained8%of thetotalvariance.Table5liststheloadingvariablesalongthefirst threePCs.
ThedendrogramresultingfromtheCAonthedatasetbuiltwith the74musselssampledpriortospawningshowed4clustersat alinkagedistanceof10(Fig.4a).Thesmallestcentral1stcluster
wasformedby6musselswithsignificantly(p<0.05)highermean Mn,Ni,Cdand/orAl,Fe,Cr,Co,Sn,Sb,BeandBiconcentrations thanclusters3(leftsideofthedendrogram)and4(rightsideof thedendrogram);the2ndclusterwasformedby14musselswith significantly(p<0.05)highermeanMn,Ni,Sb,Biand/orAl,Fe,Cr, SnandBethanclusters3and4,respectively(results–notshown –ofsupplementarymultiplecomparisontestsofmeansbetween meantransformedconcentrationsofthe4clusters),althoughthese differenceswereproportionallylessimportantthanforcluster1. Examiningthethree-dimensional(3D)scoresplotofthe74mussels sampledpriortospawning(Fig.4b),musselsofcluster1(darkgrey patches)andhalfthemusselsofcluster2(lightgreypatches), plot-tedaccordingtoPC1,distinctlyemergedfromthemainscatterplot. Ofthe20musselsformingthese2clusters,9weresmall-size indi-viduals(<55mm)ofbothsexesand6werelargemales(>69mm). PCAandCAgroupingmethodsthusgaveresultsinagreementwith thelinearmodellingofrelationshipsbetweenTEconcentrations andthemusselshelllength(Fig.3;Table4)andconfirmedthe sig-nificant(p<0.05)effectofextremesizesonTEbioaccumulationin rope-grownM.galloprovincialis.
Oftheremaining2clustersatalinkagedistanceof10(Fig.4a), the3rdcluster(leftsideofthedendrogram)wasformedby32ofthe 45malemusselslargerthan55mm(exceptindividualsM1.2and M1.7;seeFig.4legendforlabelmeaning)andthe4thcluster(right sideofthedendrogram)wasformedby21ofthe29femalemussels (andmaleM1.6).The3Dscatterplotclearlyseparatedindividuals ofbothsexesaccordingtoPC2(withdominatingfeaturesCu,Asand Mo)andPC3(withdominatingfeaturesZn,VandSe).This supple-mentaryeffectofthesexonthebioaccumulationofTEsin rope-grownM.galloprovincialispriortospawning,superimposedonthe sizeeffect,ismodelledanddiscussedinthenextSection3.2.4.
Table5
Factorloadingsforthefirstthreeprincipalcomponents(PC1,PC2,PC3)resultingfromprincipalcomponentanalysiswhenusingtraceelementconcentrationsofrope-grown MytilusgalloprovincialisfromtheDianepond(eastCorsica).Thefirstdataset(n=74;leftpartofthetable)analysediscomposedofwholemusselssampledinFebruary 2011beforetheyspawned,withsizesrangingfrom43.40to86.41mm.Theseconddataset(n=200;rightpartofthetable)analysediscomposedofbodycompartments (gills,hepatopancreas,mantleandremainingsofttissues)andwholemusselssampledinFebruary2011beforetheyspawnedandinMarch2010aftertheyspawned.Factor loadingsequalorgreaterthan0.700inabsolutetermsareinbold.
Factorloadings
Dataset1 Dataset2
PC1 PC2 PC3 PC1 PC2 PC3 Al −0.835 −0.459 −0.092 −0.880 −0.417 −0.116 V −0.499 0.653 −0.447 −0.636 0.581 −0.230 Fe −0.886 −0.390 −0.099 −0.905 −0.366 −0.104 Cr −0.889 −0.402 −0.062 −0.895 −0.383 −0.084 Mn −0.872 0.105 −0.016 −0.794 −0.526 0.017 Co −0.821 0.107 0.195 −0.927 0.109 0.143 Ni −0.888 0.174 −0.172 −0.960 −0.043 0.024 Cu −0.365 0.702 0.322 −0.946 −0.002 −0.007 Zn −0.573 0.073 0.645 −0.409 −0.029 0.645 Se −0.579 0.559 0.418 −0.373 0.436 0.622 Ag −0.618 0.396 −0.183 −0.555 0.668 −0.142 Cd −0.721 −0.106 0.288 −0.478 0.337 0.622 Sn −0.770 −0.362 −0.105 −0.920 −0.241 −0.132 Sb −0.922 0.025 −0.126 −0.917 0.297 −0.154 As −0.196 0.863 −0.076 −0.617 0.637 −0.165 Mo −0.278 0.745 −0.296 −0.335 −0.317 0.716 Be −0.780 −0.445 −0.270 −0.924 −0.195 −0.119 Pb −0.612 −0.192 0.415 −0.775 0.340 −0.130 Bi −0.857 −0.022 −0.144 −0.940 −0.076 −0.131
3.2.4. TEbioaccumulationaccordingtothesex
Evenwhenconsideringonlymusselswitha sizelargerthan 55mm, sampled in February 2011 before their spawned, V, Mn,Cu, Se,Ag, Asand Moconcentrations were stillcorrelated (0.05<p<0.0001)withtheshelllength(Table4).Thisobservation resultedfromtheunequalaccumulationofcertainTEsbetween femalesandmalesduringgametogenesis,asraisedfromPCAand CAresults(Fig.4)andshownforSeinFig.3:thesignificant rela-tionshipbetweenSeconcentrationsandthemusselshelllength (Fig.3c)partlyreliedonsmall-sizemussels(asforCr;Fig.3a),but
thiseffectwasoverlappedbydifferencesbetweenfemaleandmale Seaccumulationpriortospawning(Fig.3d).Thissupplementary effectofthesexonthemodellingoflinearrelationshipsbetween themusselshelllengthandTEconcentrations(e.g.Figs.3bandd), formusselslargerthan55mm,isgraphicallyrepresentedforthe 19studiedTEsinAnnexB.
MeanTEconcentrationsinfemaleandmaleM.galloprovincialis dryflesh(Table2)differedsignificantlyformostofthe19 stud-iedTEs(p<0.05)andwerehigherinfemales(from3%forAlup to74%forCu),exceptforBe.Thisisconsistentwithobservations
Fig.4.(a)Dendrographicclassificationafterclusteranalysis(Euclideandistanceasmeasureofsimilarity)usingtraceelement(TE)concentrationsand(b)three-dimensional (3D)scoresplotafterprincipalcomponentanalysisaccordingtoTEconcentrationsofrope-grownMytilusgalloprovincialis(n=74)fromtheDianepond(eastCorsica),sampled inFebruary2011beforetheyspawned,withsizesrangingfrom43.40to86.41mm.Toidentifysamplesonthedendrogramandonthe3Dscoresplot,musselsarelabelled asfollows:(i)toeachindividualisgiventhecapitalletterofitsgender,i.e.“M”formalesor“F”forfemales;(ii)the1stnumberisrelatedtothesize-classthemusselbelongs to,i.e“1”forthesize-class43–54mm,“2”forthesize-class55–64mm,“3”forthesize-class65–74mmand“4”forthesize-class75-87mm;(iii)the2ndnumberisrelated totheplaceofeachindividualwithinitssize-classinascendingorder.Thedendrogramshows4clustersatalinkagedistanceof10(thickblackline).Onthe3Dscoresplot, malemusselsformingtheextremeleftclusteraregroupedwithinthefullellipse,femalemusselsformingtheextremerightclusteraregroupedwithinthedottedellipse, andthe6and14musselsformingthetwosmallercentralclustersarehighlightedwithdarkorlightgreypatches,respectively.
Table6
Dryweightsofthe4bodycompartmentsandwholemussels(mean±SD,ing),andcorrespondingTEconcentrations(mean±SD,ingg−1DW),ofrope-grownMytilus
galloprovincialisfromtheDianepond(eastCorsica),sampled(A)inFebruary2011beforetheyspawned(n=20)and(B)inMarch2010aftertheyspawned(n=20).Letters representsignificantdifferences(p<0.05)betweenthe4bodycompartmentsandwholemusselsofasamegametogenicstatus(i.e.multiplecomparisontestsofmeans); asterisks(*)representsignificantdifferences(p<0.05)forasamebodycompartmentorbetweenmusselsbeforeorafterspawning(i.e.pairwisecomparisontestsofmeans), respectively.
A
Beforespawning Gills Hepatopancreas Mantle Remainingsofttissues Wholemussel Dryweight 0.110±0.031 0.259±0.136 0.178±0.066 0.435±0.135 0.981±0.348 Al 8.8±7.2a* 40.3±27.7b* 3.7±2.4c* 8.5±6.8a* 14.9±7.3d* V 1.8±0.3ad* 11.6±5.3b* 0.8±0.3c 1.0±0.3ac* 3.7±1.3bd* Fe 50±11a* 125±46b* 33±7c* 30±8c* 57±15ab* Cr 0.239±0.060a* 0.420±0.140b* 0.131±0.071c* 0.158±0.056ac* 0.227±0.060a* Mn 2.68±1.41* 2.54±0.75* 2.18±1.04* 2.39±1.07* 2.45±0.98* Co 1.01±0.26ab 1.44±0.63b* 0.37±0.17c* 0.46±0.13c* 0.75±0.22a* Ni 1.19±0.45a* 2.64±1.38a* 0.46±0.28b* 0.55±0.23b* 1.12±0.45a* Cu 3.52±0.43a* 5.79±1.43b* 3.02±1.03ac* 2.48±0.54c* 3.51±0.65a* Zn 113±42a 79±25ab* 61±13b* 65±18b 74±20ab Se 4.62±0.61a 3.71±0.72b 3.15±0.78bc 2.96±0.46c 3.33±0.48bc Ag 0.0471±0.0142ab* 0.0672±0.0202b* 0.0286±0.0137c* 0.0295±0.0113c* 0.0405±0.0119ac* Cd 1.09±0.24a 0.91±0.26a 0.49±0.17b* 0.61±0.13bc 0.72±0.16c Sn 0.0193±0.0035ac* 0.0618±0.0237b* 0.0132±0.0054cd* 0.0127±0.0034d* 0.0252±0.0057ab* Sb 0.0213±0.0024a* 0.0551±0.0156b 0.0123±0.0049c* 0.0102±0.0022c 0.0229±0.0043a As 20.5±2.6a* 32.9±4.5b* 18.2±2.0c* 15.2±1.4d* 20.7±2.0a* Mo 13.8±5.0a* 10.7±4.1a* 4.5±1.5b* 4.2±0.9b* 7.1±2.2c* Be 0.0145±0.0078ab 0.0250±0.0093b* 0.0041±0.0021c* 0.0082±0.0035ac* 0.0125±0.0042a* Pb 1.74±0.39ab* 2.38±1.16b 0.74±0.24c* 0.98±0.28cd* 1.32±0.34ad Bi 0.0168±0.0037a 0.0419±0.0126b* 0.0064±0.0032c* 0.0076±0.0024c* 0.0170±0.0038a* B
Afterspawning Gills Hepatopancreas Mantle Remainingsofttissues Wholemussel Dryweight 0.102±0.025 0.176±0.054 0.136±0.052 0.326±0.091 0.740±0.182 Al 136.9±68.4a* 1493.2±774.4b* 39.2±21.8c* 187.6±110.3a* 468.4±190.2d* V 1.2±0.2a* 6.6±2.5b* 0.6±0.2c 0.9±0.3c* 2.2±0.6a* Fe 153±35ac* 1061±563b* 89±23c* 180±66a* 361±130b* Cr 0.599±0.119a* 3.421±1.851b* 0.324±0.117c* 0.742±0.325ad* 1.254±0.449bd* Mn 7.41±1.75a* 23.44±8.97b* 6.45±1.83a* 8.46±3.10ac* 11.38±3.80c* Co 1.06±0.28a 2.53±0.99b* 0.62±0.18c* 0.57±0.16c* 1.11±0.35a* Ni 1.70±0.64ad* 5.91±2.51b* 0.89±0.45c* 1.09±0.42ac* 2.26±0.86d* Cu 4.27±0.63a* 9.89±2.45b* 4.04±0.96ac* 3.19±0.57c* 5.07±0.95a* Zn 133±59a 112±47ab* 98±34b* 84±30b 93±38ab Se 4.23±0.64a 3.56±0.70b 3.08±0.50bc 2.71±0.38c 3.19±0.45b Ag 0.0212±0.0063ac* 0.0509±0.0210b* 0.0172±0.0073c* 0.0165±0.0049c* 0.0252±0.0079a* Cd 1.08±0.18a 1.03±0.29a 0.66±0.23b* 0.60±0.12b 0.75±0.15b Sn 0.0503±0.0208ad* 0.2346±0.1015b* 0.0197±0.0041c* 0.0373±0.0153d* 0.0763±0.0222ab* Sb 0.0200±0.0065ad* 0.0659±0.0207b 0.0092±0.0024c* 0.0112±00021cd 0.0258±0.0069ab As 18.4±3.2a* 27.2±2.7b* 16.1±1.5ac* 13.1±1.7c* 17.7±1.4a* Mo 93.8±29.2a* 43.0±14.1b* 29.5±8.7c* 21.5±7.2c* 38.6±12.4b* Be 0.0155±0.0054a 0.0835±0.0311b* 0.0075±0.0043a* 0.0150±0.0053a* 0.0295±0.0088b* Pb 0.96±0.24ad* 3.01±0.95b 0.52±0.17c* 0.76±0.18ac* 1.27±0.34bd Bi 0.0207±0.0049ad 0.1107±0.0310b* 0.0128±0.0048c* 0.0157±0.0037ac* 0.0381±0.0115bd*
madebyLobeletal.(1991)for Mn,Cu,AsandSemeasuredin wildM.edulisfromNewfoundlandsampledinsummer.Watling andWatling(1976)measuredhigherconcentrationsinwildfemale ChoromytilusmeridionalisfromSouthAfricaforZn,Cu,MnandFe, similarin bothsexes for Fe,Cd,Ag,Cr, Coand Ni,and slightly higherinmalesforPb,andBi.Afterthemainbreedingperiod,no differencewasfoundbetweensexes(Orrenetal.,1980), suggest-ingareproduction-relatedmetalaccumulation.InwildPernaperna fromtheGulfofAdencollectedinsummer,Cd,Cu,Mn,PbandZn weremoreconcentratedinfemalesthaninmales,andinversely forFe(Sokolowskietal.,2004).Hellouetal.(2003)collectedmale andfemaleM.edulisinNovaScotia,priorandduringthe spawn-ingperiod(betweenAprilandJuly),andmeasuredsystematically higherconcentrationsofCd,Pb,Hg,Ag,As,Cr,Cu,SnandZnin femalesofttissues.Theinfluenceofgametogenesisindetermining bodyTEconcentrationscanthusnotbeneglected,evenifitsrole isnotclearyetandneedsfurtherstudies.Wecanreasonably sug-gestthattheclosestmusselsarefromspawning(additionalmussels fromthepresentstudyeffectivelyspawned2–4weekslater;Richir etal.,unpubl.data),themostdifferencesinTEconcentrationsrise.
Sokolowskietal.(2004),fromobservationsmadebyLobeland Wright(1982)andPietersetal.(1980)onM.edulis,suggestedthat theformationofreservesduringtheprespawningperiod,more pronouncedin females,couldfavourthefasteraccumulationof TEs in this sex.However, Suárez etal. (2005) showedthat the contributionofreproductivetissuestothetotal bodyweightof raft M. galloprovincialis was systematically higher in males all yearround,thisdifferencebeingevenmorepronouncedinwinter and spring,duringgametogenesis.Thehypothesis suggestedby Sokolowskietal.(2004)mustthenbepartiallyrejected,atleastin thecaseofrope-grownM.galloprovincialis,sincethecontribution offemalereproductivetissuestotheirtotalbodyweightdoesnot exceedthatinmales(datanotshown).Someevidencessuggest thatdifferencesbetweenfemaleandmaleTEconcentrationsprior tospawningcouldrelyonafunctionalroleplayedby metalloth-ioneins(MTs),asalreadysuggestedbyLatoucheandMix(1981) intheearly80ies.Akberalietal.(1985,inEarnshawetal.,1986) experimentallyshowedthatCuandZnuptakeswerehigherinto spermthanintoeggsofM.edulis.Fitzpatricketal.(2008)observed noinfluenceofincreasingCuconcentrationsonM.trossulusegg
Fig.5. (aandb)Cr,(candd)Ag,(eandf)Seand(gandh)Culevelsingills“G”,hepatopancreas“H”,mantle“M”,remainingsofttissues“ST”andwholemussels“Mus.”of rope-grownMytilusgalloprovincialisfromtheDianepond(eastCorsica),sampledinFebruary2011beforetheyspawned(lightgray;n=20)andinMarch2010afterthey spawned(darkgrey;n=20).Levelsareexpressedasconcentrations(gg−1DW–4leftgraphs)ortotalcontents(g—4rightgraphs).Lettersrepresentsignificantdifferences
(p<0.05)betweenthe4bodycompartmentsandwholemusselsofasamegametogenicstatus(i.e.multiplecomparisontestsofmeans);asterisks(*)representsignificant differences(p<0.05)forasamebodycompartmentorbetweenmusselsbeforeorafterspawning(i.e.pairwisecomparisontestsofmeans),respectively.
viabilityandfertilization rates,while spermmotilityand fertil-izationratesdecreased,andMeistertzheimetal.(2009)recently measured a more important increase of MT concentrations in femalegonads of C.gigas than in malesduring gametogenesis. ThishypotheticalroleplayedbyMTsinM.galloprovincialisduring gametogenesisthereforeneedsfurtherinvestigations.
3.3. TEaccumulationandtissuecompartmentalizationaccording tothereproductivestatus
All40musselsanalysedfortissuecompartmentalization mea-suredbetween70and80mm.Meanbodyandgonad(i.e.follicles developed in the mantle) dry weights of mussels sampled in February2011(beforespawning)were0.981and0.178g, respec-tively,andmeanbodyandgonaddryweightsofmusselssampledin March2010(afterspawning)were0.740and0.136g,respectively.
Gonadweightsdifferedby31%;thiswassimilartothe33%whole bodyweightdifferencebetweenbothreproductivestatuses.Inthe genusMytilus,upto40%ofsofttissueweightcanbelostduring spawning,whichshowstheimportanceofgametogenesisintheir physiologicalcycle(Cossa,1989).
TEscouldbedividedinto4groups,dependingontheir concen-trationsandcontentsbeforeandafterspawning(Table6).Fig.5 givesonegraphicalexampleforeachgroup;histogramsofthe tis-suecompartmentalizationofthe19studiedTEsaregiveninAnnex C.ThefirstgroupwasmadeupofAl,Fe,Cr(Figs.5aandb),Mn,Ni, Sn,Mo,BeandBi:theywerelessconcentratedandlessabundant priortospawning,duetothecombinedeffectofatissuedilution duringthegametogenesisbodyweightincrease(lower concentra-tions)andanenvironmentaldiminutionofavailableTEsbetween thetwosampledyears(lowercontents).Inversely,asecondgroup wasmadeupofVandAg(Figs.5candd),moreconcentratedand
Fig.6.(a)Dendrographicclassificationafterclusteranalysis(Euclideandistanceasmeasureofsimilarity)usingtraceelement(TE)concentrationsand(b)three-dimensional (3D)scoresplotafterprincipalcomponentanalysisaccordingtoTEconcentrationsofbodycompartmentsandwholerope-grownMytilusgalloprovincialis(n=200)fromthe Dianepond(eastCorsica),sampledinFebruary2011beforetheyspawnedandinMarch2010aftertheyspawned.Toidentifysamplesonthedendrogramandonthe3D scoresplot,bodycompartmentsandwholemusselsarelabelledasfollows:(i)gills“G”,remainingsofttissues“ST”,hepatopancreas“H”,mantle“M”andwholemussels “Mus.”,(ii)withtheletter“b”or“a”referringtothereproductivestatus,i.e.beforeorafterspawning,respectively.Thedendrogramshows5clustersatalinkagedistanceof 15(thickblackline).Onthe3Dscoresplot,hepatopancreassamplesaregroupedwithinrectanglesandgillsamplesaregroupedwithinellipses,drawneitherwithfullor dottedlinesformusselssampledbeforeorafterspawning,respectively.Forclaritypurpose,oneinthreesamplelabelisreportedonthedendrogram,aswellasoneinthree M,STandMus.labelonthe3Dscoresplot.
moreabundantin2011priortospawning;these2TEswere suf-ficientlyabundantthatyeartomaskthedilutioneffectduetothe gametogenesis.
TheDianepondisasmall-sizesaltpond,highlyinfluencedbythe runoffoffreshwaterfrompartofthecatchmentbasinofBravona, bringingterrigenousTEssuchasAlorFe,andbythediscontinuous connectivitywiththeopenseathroughasmall“grau”,achannel silteduppartoftheyear(Longereetal.,1972).Theseparticular physicalcharacteristicsinfluencethephysicochemicalproperties ofthatwater body,andsothebioavailability ofTEs.Data com-piledfrom2musselcagingcampaignsinspring2010and2011in theCalviBay,northwesternCorsica,withmusselsfromthesame ropesthattheonesusedinthisstudyconfirmedthisconfinement hypothesis:TEconcentrationsweresimilarbetweenthe2 moni-toredyearsintheCalviBay(Richiretal.,unpubl.data),evenfor TEshowingdifferencesinthisstudyasimportantasforAlorFe (Table6;AnnexC).
Se(Figs. 5eand f), Cd, Sb,Asand Pb formeda third group, showingsimilartissue concentrationsat bothreproductive sta-tusesbutwithcontentsalittlehigherin2011priortospawning. These5TEs,moreaccumulatedinmusseltissuesin2011priorto spawning(Fig.5f),displayedsimilarconcentrationswithmussels havingspawned(Fig.5e)astheyweredilutedduring gametogene-sis.Finally,thefourthgroupwasmadeupofCo,Cu(Figs.5gandh) andZn:these3essentialsTEsdisplayedlowerconcentrationsprior tospawningandsimilarcontentsatbothphysiologicalstatuses. TheevidentroleofMTsinZnandCuregulationprobablyaccounts forstablecontentsobservedbetweenyears(BebiannoandSerafim, 2003;Strogyloudietal.,2012).
85%ofthetotalvarianceofthedatasetcomposedofbody com-partmentsandwholemussels(n=200)sampledbeforeandafter spawningwasexplainedbythethreefirstPCs(eigenvalueshigher than1)resultingfromthePCA.Moreprecisely,concentrationsof 12outofthe19studiedTEs(Al,Fe,Cr,Mn,Co,Ni,Cu,Sn,Sb,Be, PbandBi)werethedominatingfeaturesinPC1,whichexplained 60%ofthetotalvarianceofthedataset.InPC2,onlyAg,AsandV weightswereclosetobutlowerthan0.700;PC2explained14%of
thetotalvariance.Moconcentrationsshowedthehighestweight inPC3(followedbyZn,SeandCdwithweightsclosetobutlower than0.700),whichexplained10%ofthetotalvariance.Table5lists theloadingvariablesalongthefirstthreePCs.
ThedendrogramresultingfromtheCAonthedatasetcomposed of bodycompartments andwholemussels(n=200)sampledat bothreproductivestatusesshowed5clustersatalinkagedistance of15(Fig.6a).Hepatopancreassamplesformed2distinctclusters ontheleftsideofthedendrogram.The2clustersontherightside ofthedendrogramwereformedbywholemusselssampledbefore orafterspawning,theirassociatedgillsamplesandmostmantle samples.Theleftcentral5thclusterwasformedbysofttissuesand remainingmantlesamplesofindistinctlybothreproductive sta-tuses.Examiningthe3Dscatterplotofsamplesinthespacedefined bythethreefirstPCs,5maingroupscouldbeobserved(Fig.6b),in agreementwiththedendrographicclassification.Hepatopancreas samples,plottedaccordingtoPC1(dominatingfeaturesofPC1:Al, Fe,Cr,Mn,Co,Ni,Cu,Sn,Sb,Be,PbandBi),distinctlyemergedfrom themainscatterplotcomposedofmantle,remainingsofttissue andwholemusselsamples.EnvironmentalAgandV,sufficiently abundantin2011(beforespawning)tomaskthedilutioneffect duetothegametogenesis(e.g.AginFig.5c),separatedsamples plottedaccordingtoPC2(althoughthatseparationbetweenboth reproductivestatuseswaslessobviousthanonthedendrogram). Gillsamples,plottedaccordingtoPC3(dominatingfeaturesofPC3: Mo,followedbyZn,SeandCd)formedtwomoredistinctgroups oppositetohepatopancreasgroups.
PCA and CAgave complementaryinformation in agreement withresultsofpairwiseandmultiplecomparisontestsofmeans betweensamplingyears(i.e.beforeorafterspawning)andbetween bodycompartmentsandwholemusselsforagivenreproductive status (Table 6; Fig. 5; Annex C). So, most TEs were preferen-tiallyconcentratedinthehepatopancreas,exceptforZn,Se,Cdand Mo,moreconcentrated ingills,andtheirconcentrations signifi-cantly(p<0.05)differedbetweensamplingyears(i.e.beforeand afterspawning).Whenexpressedastotalcontentsineachbody compartment,the19investigatedTEsweresystematicallymore
accumulatedinthehepatopancreas(Fig.5;AnnexC).Sincethe hep-atopancreaspreferentiallyconcentratedmostofthe19studiedTEs, thisparticularorgancouldbeprivilegedinmonitoringsurveys,as previouslysuggestedbymanyauthorsforclassicallyinvestigated tracemetals(e.g.Adamietal.,2002;GuptaandSingh,2011).TE distributionbetweentissuesfurtherdisplayedthesamepattern, independentlyofthesamplingyearorthereproductivestatus. Dur-ingtheprespawningperiod,musselsaccumulatereserves(Pieters etal.,1980):glycogenis largelyaccumulatedandstored inthe mantle,andproteinsandlipidsinnon-mantletissues(Gabbotand Bayne,1973,inGabbott,1975).TEaccumulationismainlylimited tothehepatopancreas,kidneysandgills(LobelandWright,1982; Cossa,1989;thisstudy);ifthemantlecanalsotakeupsolubleTEs,it hastobeconsideredasatransitionaltissue,TEsbeingrapidly trans-portedtonongonadaltissues(LobelandWright,1982).Energyfor vitellogenesis,thefinalstageofgametogenesis,issuppliedfrom glycogenreserves,fromlipidreservesstoredinadipogranularcells inthemantle,andfromfreshlyingestedfoodmaterials(Newell, 1989).Followingtheconversionofthestoredglycogentothelipid reserveofdevelopingeggs(LatoucheandMix,1981),amovement ofnutrientsfromthedigestiveglandtothemantleoccurs(Gabbott, 1975),leadingtoaconsecutiveredistributionofTEswithinmussel tissues.So,thesimilarpatternofTEdistributionobservedbetween tissuesbeforeandafterspawningwascloselyrelatedtothemussel reproductivecycle.
4. Conclusion
Rope-grownM.galloprovincialisfromtheDianepondefficiently accumulatedthe10littlestudiedTEs(Be,Al,Fe,Mn,Co,Se,Mo,Sn, Sb,Bi)inadditiontothe9TEsclassicallyinvestigated(Cr,Ni,Cu, Zn,Cd,Pb,As,AgandV)inthatspecies.However,theimportant variabilityofTEconcentrationssometimesrecordedatregionalor evenlocalscalerequiresacoordinationofmonitoringprograms betweenneighbouringcountries,inordertodefinesensible refer-enceconditionstoproperlymonitorthechemicalpollutionofthe coastalenvironment.LowconcentrationsmeasuredintheDiane pondcouldserveasbaselinelevelsforcomparisonwithulterior surveys,atleastforthenorthwesternMediterranean.
Relationships between TE concentrations and the body dry weightorbetweenTEcontentsandthemusselshelllengthwere bettermodelledbypowerfunctionsorlinearregressions, respec-tively; conversely, relationships between TE contents and the musselshelllengthwereproperlymodelledbybothlinear regres-sions and power functions when only considering individuals largerthan 55mm.Small-sizeM.galloprovincialis(<55mm)had anantagonisteffect:theydrovetoelectthelinearregressionto model relationshipsbetween the mussel size and TE contents, butdiminishedthesignificanceofthepowerfunctionmodelling relationshipsbetweenthemusselweightandsomeTE concentra-tions.Small-sizemusselsfurthermoreconcentratedmoreTEs,and showedanimportantinter-individualvariabilityoftheir concen-trations.Althoughalargerangeofrope-grownM.galloprovincialis sizescanbeusedformonitoringpurposes,onewillthustakecare nottouseverysmallorlargemussels.
Manydifferenceswereobservedbetweensexespriorto spawn-ing. Although the influence of gametogenesis in determining femalebodyhigherTEconcentrationsisnotclearyetandneeds furtherstudies,theroleplayedbyMTscouldbefundamental.The hepatopancreaspreferentiallyaccumulatedTEs,exceptforZn,Se, CdandMo,moreconcentratedingills.Bodycompartmentalization wasnotinfluencedbythereproductivestatus,butgametogenesis dilutedTEsduetotheimportanttissueproductionpriorto spawn-ing.Basedontheseobservations,itappearsjudicioustomonitorthe 19studiedTEsduringmusselsexualdormancy.However,temporal
variationsofenvironmentalTEconcentrationswithintheconfined Dianepondmostlyhidthisgametogenicdilutioneffect.
Acknowledgments
FundingwasprovidedbytheFRS-FNRS(FRFC2.4.502.08)and bytheFrenchCommunityofBelgium(ARCRace05/10-333).This study is part of the STARECAPMED (Station of Reference and ResearchonChangeoflocalandglobalAnthropogenicPressures onMediterraneanEcosystemsDrifts)projectfundedbythe Ter-ritorial CollectivityofCorsica and byTheFrenchWaterAgency (PACA-Corsica).AuthorsaregratefultoMr.B.Pantalacci,manager oftheshellfishfarmSARLEtangdeDiane,whofurnishedmussel ropes,toM.FassinandA.Deraikemwhoprocessedsamplesduring theirmasterthesis,toR.BiondowhoperformedICP-MSanalyses andtoA.Nackerswhoimprovedthelanguageofthemanuscript. ThispublicationhastheMAREpublicationnumberMARE247.
AppendixA. Supplementarydata
Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.ecolind.2013.06.021.
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