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1-s2.0-S0166445X12000227-main.pdf_seabarents

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Pleasecitethisarticleinpressas:Nahrgang,J.,etal.,Seasonalvariationinbiomarkersinbluemussel(
Mytilusedulis
),Icelandicscallop(
Chlamysislandica
)andAtlanticcod(
Gadusmorhua
)—ImplicationsforenvironmentalmonitoringintheBarentsSea.Aquat.Toxicol.(2012),doi:10.1016/j.aquatox.2012.01.009
ARTICLE IN PRESS
GModelAQTOX-3209;No.ofPages15AquaticToxicology
ContentslistsavailableatSciVerseScienceDirect
Aquatic
 
Toxicology
Seasonal
 
variation
 
in
 
biomarkers
 
in
 
blue
 
mussel
 
(
Mytilus
 
edulis
),
 
Icelandic
 
scallop(
Chlamys
 
islandica
)
 
and
 
Atlantic
 
cod
 
(
Gadus
 
morhua
)—Implications
 
forenvironmental
 
monitoring
 
in
 
the
 
Barents
 
Sea
 J.
 
Nahrgang
a
,
,
 
S.J.
 
Brooks
b
,
 
A.
 
Evenset
a
,
 
L.
 
Camus
a
,
c
,
 
M.
 
 Jonsson
a
,
 
T.J.
 
Smith
a
,
 
 J.
 
Lukina
a
,
d
,M.Frantzen
a
,
 
E.
 
Giarratano
e
,
 
P.E.
 
Renaud
a
,
c
a
 Akvaplan-niva,FRAMCentre,NO-9296Tromsø,Norway
b
NorwegianInstituteforWaterResearch(NIVA),NO-0349Oslo,Norway
c
UniversityCentreInSvalbard(UNIS),NO-9171Longyearbyen,Norway
d
UniversityofTromsø,FacultyofBiosciences,FisheriesandEconomics,DepartmentofArcticandMarineBiosciences,NO-9037Tromsø,Norway
e
CentroNacionalPatagónico(CENPAT-CONICET),AR-9120PuertoMadryn,Argentina
a
 
r
 
t
 
i
 
c
 
l
 
e
 
i
 
n
 
f
 
o
 Articlehistory:
Received21October2011Receivedinrevisedform20December2011Accepted13January2012
Keywords:
BiomarkersBiomonitoringBarentsSeaBaselinelevelsSeasonalityPAH
a
 
b
 
s
 
t
 
r
 
a
 
c
 
t
In
 
the
 
Barents
 
Sea,
 
the
 
limited
 
data
 
onbiological
 
relevant
 
indicators
 
and
 
their
 
responses
 
to
 
various
 
anthro-pogenicstressors
 
have
 
hindered
 
the
 
development
 
of 
 
aconsistent
 
scientific
 
basis
 
for
 
selecting
 
indicatorspecies
 
and
 
developing
 
practical
 
procedures
 
for
 
environmental
 
monitoring.
 
Accordingly,
 
the
 
main
 
aimofthe
 
present
 
study
 
was
 
to
 
develop
 
acommon
 
set
 
of 
 
baseline
 
values
 
for
 
contaminants
 
and
 
biomark-ers
 
in
 
three
 
species,
 
and
 
to
 
identify
 
their
 
strengths
 
and
 
limitations
 
in
 
monitoring
 
of 
 
the
 
Barents
 
Sea.Blue
 
mussel
 
(
Mytilus
 
edulis
),
 
Icelandic
 
scallop
 
(
Chlamys
 
islandica
)
 
and
 
Atlantic
 
cod
 
(
Gadus
 
morhua
)
 
weresampled
 
from
 
anorth
 
Norwegian
 
fjord
 
in
 
March,
 
 June,
 
September
 
and
 
December
 
2010.
 
Digestive
 
glandsfromthe
 
bivalve
 
species
 
and
 
liver
 
from
 
Atlantic
 
cod
 
were
 
analysed
 
for
 
biomarkers
 
of 
 
oxidative
 
stress(catalase
 
[CAT],
 
glutathione
 
peroxidase
 
[GPX],
 
glutathione-S-transferase
 
activities
 
[GST],
 
lipid
 
peroxi-dation
 
as
 
thiobarbituric
 
reactive
 
substances
 
[TBARS]
 
and
 
total
 
oxyradical
 
scavenging
 
capacity
 
[TOSC]),biotransformation
 
(ethoxyresorufine-O-deethylase
 
activity
 
[EROD])
 
and
 
general
 
stress
 
(lysosomal
 
mem-branestability
 
[LMS]).
 
Concentrations
 
of 
 
polycyclic
 
aromatic
 
hydrocarbons
 
(PAHs)
 
and
 
metals
 
in
 
thebivalves
 
and
 
PAH
 
metabolites
 
in
 
fish
 
bilewere
 
quantified.
 
Finally,
 
energy
 
reserves
 
(total
 
lipids,
 
proteinsand
 
carbohydrates)
 
andelectron
 
transport
 
system
 
(ETS)
 
activity
 
in
 
the
 
digestive
 
gland
 
of 
 
the
 
bivalves
 
andliverof 
 
Atlantic
 
cod
 
provided
 
background
 
information
 
for
 
reproductive
 
cycle
 
and
 
general
 
physiologicalstatus
 
of 
 
the
 
organisms.
 
Blue
 
mussel
 
and
 
Icelandic
 
scallop
 
showed
 
very
 
similar
 
trends
 
inbiological
 
cycle,biomarker
 
expression
 
and
 
seasonality.
 
Biomarker
 
baselines
 
in
 
Atlantic
 
codshowed
 
weaker
 
seasonal
 
vari-ability.
 
However,
 
important
 
biological
 
events
 
may
 
have
 
been
 
undetected
 
due
 
to
 
the
 
large
 
time
 
intervalsbetween
 
sampling
 
occasions.
 
Physiological
 
biomarkers
 
such
 
as
 
energy
 
reserves
 
and
 
ETS
 
activity
 
wererecommended
 
as
 
complementary
 
parameters
 
to
 
the
 
commonly
 
used
 
stress
 
biomarkers,
 
asthey
 
providedvaluable
 
information
 
onthe
 
physiological
 
status
 
of 
 
the
 
studied
 
organisms.
 
Interpretation
 
of 
 
the
 
seasonal-ityin
 
oxidative
 
stress
 
biomarkers
 
wasin
 
general
 
difficult
 
but
 
TOSC
 
and
 
lipid
 
peroxidation
 
were
 
preferredoverthe
 
antioxidant
 
enzyme
 
activities.
 
This
 
study
 
is
 
the
 
first
 
reporting
 
seasonal
 
baseline
 
in
 
these
 
threespecies
 
in
 
asub-Arctic
 
location.
 
Overall,
 
the
 
Icelandic
 
scallop
 
was
 
considered
 
the
 
most
 
adequate
 
organismfor
 
environmental
 
monitoring
 
in
 
the
 
Barents
 
Sea
 
due
 
to
 
the
 
interpretability
 
of 
 
the
 
biomarker
 
dataas
 
wellasitsabundance,
 
ease
 
to
 
handle
 
and
 
wide
 
distribution
 
from
 
the
 
southern
 
Barents
 
Sea
 
to
 
Svalbard.
© 2012 Elsevier B.V. All rights reserved.
Correspondingauthor.Presentaddress:UniversityofTromsø,FacultyofBio-sciences,FisheriesandEconomics,DepartmentofArcticandMarineBiosciences,NO-9037Tromsø,Norway.Tel.:+4777645896.
E-mailaddress:
1.Introduction
Biologicallyrelevantindicatorsarerequiredtomonitorecosys-temstatusinrelationtodiffuseanthropogenicpollutantsoracuteaccidentaldischarges.Responsesandtolerancesofindividualtaxatovariousanthropogenicstressorshavebeenextensivelyinvesti-gatedintemperatesystems(CollierandVaranasi,1991;AasandKlungsøyr,1998;Baussantetal.,2009;Brooksetal.,2009),butfewdataexistfortheArcticandsub-Arcticregions(Chapmanand
0166-445X/$seefrontmatter
© 2012 Elsevier B.V. All rights reserved.
 
Pleasecitethisarticleinpressas:Nahrgang,J.,etal.,Seasonalvariationinbiomarkersinbluemussel(
Mytilusedulis
),Icelandicscallop(
Chlamysislandica
)andAtlanticcod(
Gadusmorhua
)—ImplicationsforenvironmentalmonitoringintheBarentsSea.Aquat.Toxicol.(2012),doi:10.1016/j.aquatox.2012.01.009
ARTICLE IN PRESS
GModelAQTOX-3209;No.ofPages15
2
J.Nahrgangetal./AquaticToxicology
 xxx (2012) xxx–xxx
Riddle,2005;Nahrgangetal.,2010a,b).FortheBarentsSeainpar-ticular,verylimitedlong-termroutinemonitoringofcontaminantlevelsandtheirbiologicaleffectshavebeencarriedout.Thishashinderedthedevelopmentofaclearandconsistentscientificbasisforselectingindicatorspeciesandendpointsanddevelopingprac-ticalproceduresfortheirapplicationinmanagement(Anon.,2006).Nevertheless,increasingindustrialactivitiesinthehighNorth,includingshippingactivitiesalongthecoastofRussiaandNorway(BambulyakandFrantzen,2009),oilandgasfieldsintheBarents SeasuchastheGoliatoilfieldclosetotheNorwegiancoast(Øienetal.,2011),presentanincreasingrisktotheenvironment.There-fore,theimplementationofmonitoringprogrammesisneededintheBarentsSea(Hasleetal.,2009).Biomarkerscomplementcontaminantanalysisinaquaticorgan-ismsbyprovidingrstbiologicalsignalsofexposure(VanderOostetal.,2003).Severalbiomarkersarecommonlyusedininter-nationalandnationalmonitoringprogrammestohelpidentifyadverseeffectsofxenobioticsinfishandbluemussels(OSPAR,2007;Hyllandetal.,2008;Brooksetal.,2011).Themostwidelyacceptedandusedbiomarkersinfisharetheethoxyresorufine-O-deethylase(EROD)andglutathione-S-transferase(GST)activitiesofphasesIandIImetabolism,respectively(Hyllandetal.,2008;Nahrgangetal.,2010b).
Furthermore,thequantificationofPAHmetabolitesinfishbilehasbeenshowntoprovidearelevantindica-tionofthePAHbioavailableexposureinfish(Arieseetal.,1993;Aasetal.,1998,2000;Nahrgangetal.,2010b).Biomarkersofoxidativestress,suchascatalase(CAT),glutathioneperoxidase(GPX)activ-ities,andlipidperoxidationhavebeenusedforbothbivalveandfishspecies,althoughtheirinterpretationisusuallydifficultduetotheirimplicationinawiderangeofbiologicalfunctions(VanderOostetal.,2003).Thetotaloxyradicalscavengingcapacity(TOSC)assayisahelpfulcomplementtoregularoxidativestressbiomark-ersasitprovidesadirectassessmentofanorganism’sabilitytoresistoxidativestress(RegoliandWinston,1998).Finally,biomark- ersofstress,suchasthelysosomalmembranestability(LMS)assay,areconsideredgeneralindicatorsoforganismhealth(Mooreetal.,2008)andarecentralparametersinintegratedmonitoringschemesdevelopedforbothfishandbivalves(Hyllandetal.,2008).Despitetheconsiderableunderstandingoftheirlinkswithcon-taminantexposure,theuseofbiomarkersisoftenlimitedbytheirstrongvariabilityduetonaturalbiologicalandenvironmentalcycles(Shawetal.,2004;Depledge,2009;Nahrgangetal.,2010a).Realisticinterpretationsofbiomarkerresponsesinenvironmen-talmonitoringthusrelyonextensiveknowledgeoftheorganismsbiologicalcyclesandthebiomarkers’seasonalbaselinelevels.Thepresentstudyaimedtocharacterisebaselinelevelsincontaminantburdenandresponsesinbiomarkerscommonlyusedorrecom-mendedforbiomonitoring(ICES,2010)inthreepotentialindicator speciescollectedoverfourseasons,andtoidentifytheirstrengthsandlimitationsforapplicationtoenvironmentalmonitoringpro-grammesintheBarentsSea.Bluemussel(
Mytilusedulis
),
 
Icelandicscallop(
Chlamysislandica
)andAtlanticcod(
Gadusmorhua
)wereselectedaspotentialindica-torspeciesfortheBarentsSeaduetotheirabundanceandwidedistribution.BluemusselandAtlanticcodhaveatemperatetoborealdistributionandarealreadyusedinseveralmonitoringpro-grammes(Hyllandetal.,2008;Brooksetal.,2011),andbothspecies havebeenrecordedasfarnorthasSvalbard(Bergeetal.,2005;Renaudetal.,inpress).Icelandicscallop,whichisasub-ArcticspeciesdistributedasfarnorthastheSvalbardarchipelago(Brand,2006),hasbeenlessstudiedthanbluemusselandAtlanticcodinecotoxicologyalthoughsomestudieshavereportedbiomarkerresponsesfollowingexperimentalcontaminationtopetroleum-relatedcompounds(Baussantetal.,2009;Hannametal.,2009,2010).Therehasbeennostudyofseasonalvariabilityinlevelsof biomarkersinanyofthesespecies,exceptforbluemussel(Viarengo
Fig.1.
Map
 
ofsamplingarea.
etal.,1989;Hardingetal.,2004;Haggeretal.,2010),norhavebiomarkersusedintemperatespeciesbeenvalidatedforsub-ArcticandArcticenvironments.Herein,wepresentbaselinelevelsoftheabove-mentionedbiomarkersandcontaminant(PAHsandmetals)bodyburdeninbluemussel,IcelandicscallopsandAtlanticcodcollectedinMarch, June,SeptemberandDecember2010.Energyreserves(totallipids,totalcarbohydratesandtotalproteins)andthepotentialmetabolicactivitymeasuredastheelectrontransportsystem(ETS)activity(Fanslowetal.,2001)servedasgeneralindicatorsofphysiological statusoftheorganisms.Theresultswillcontributetothedevel-opmentofanenvironmentalmonitoringprogrammebasedonrelevantbio-indicatorsandeffect-levelsasanthropogenicactivitiesandrisksforpollutionincreaseinArcticandsub-Arcticenviron-ments.
2.Methods
 2.1.Samplecollection
Allorganismswerecollectedinasub-Arcticlocation,nearTromsø,NorthernNorway(70
N),inMarch,June,SeptemberandDecember2010(Fig.1).Organismsweretypicallyprocessedwithin 2h(LMSassay),4h(allotherbiomarkers)and6h(bodyburden)followingcollection.Submergedbluemussels(
n
=60persamplingseason)werecollectedbyhandduringlowtideandtransportedtothelaboratoryfacilitiesinbucketscontainingseawatersoakedpapertowelstoprovideacoolandmoistenvironment.Icelandicscallop(
n
=60persamplingseason,except
n
=35inMarch)werecollectedbytriangulardredgefromRV
Hyas
(UniversityofTromsø),maintainedintankswithrunningseawateruntiltransportationtothelaboratoryandthereaftermaintainedinoxygenatedseawa-teruntildissection.Bluemusselhaemolymphwascollectedfrom20individualsfortheassessmentofLMS
 
bythemethodofneu-tralredretention(NRR,Section2.2.1).Totalwetweightincluding shell(
±
0.1gwwt),lengthandwidth(
±
0.1cm)
 
wererecordedfrombluemusselandIcelandicscallop.Digestiveglandswereremoved,
 
Pleasecitethisarticleinpressas:Nahrgang,J.,etal.,Seasonalvariationinbiomarkersinbluemussel(
Mytilusedulis
),Icelandicscallop(
Chlamysislandica
)andAtlanticcod(
Gadusmorhua
)—ImplicationsforenvironmentalmonitoringintheBarentsSea.Aquat.Toxicol.(2012),doi:10.1016/j.aquatox.2012.01.009
ARTICLE IN PRESS
GModelAQTOX-3209;No.ofPages15
 J.Nahrgangetal./AquaticToxicology
 xxx (2012) xxx–xxx
3
weighed(
±
0.01gwwt),snapfrozeninliquidnitrogenandstoredat
80
Cuntilanalyses.AtlanticcodwassampledonboardRV
Hyas
withbottomtrawl(
n
=21and20inMarchandJune,respectively)andshingrod(
n
=16and13inSeptemberandDecember,respectively),andkeptaliveintankswithrunningseawateruntiltransportationtothelaboratoryandthereaftermaintainedinoxygenatedseawateruntildissection.Fishweresacrificedbyasharpblowtothehead.Totallength(
±
0.1cm),totalandsomaticweights(
±
0.1gwwt),liverandgonadweights(
±
0.01gwwt)andgenderwererecorded.Liverslicesandbilewereplacedintoseparatecryovials,snapfrozeninliquidnitrogen,andstoredat
80
Cpriortoanalysis.Thegonado-somaticindex(GSI),thehepato-somaticindex(HSI)andtheFulton’sconditionfactor(
)weredeterminedforAtlanticcodaccordingtotheequations:GSI
=
100
×
totalgonadwetweightsWHSI
=
100totalliverwetweightsW
=
100
×
sW
L
3
wheresWisthesomaticweight(g)and
L
theforklength(cm).Duetolimitedamountoftissueforbiomarkeranalyses,diges-tiveglandsfromthebivalvesweresplitinto4batchesof15samplespersamplingpointthatwereusedfor(1)energyreservesandETSactivity,(2)antioxidantdefenceenzymesandlipidperoxidation,(3)TOSCand(4)GSTactivityrespectively.Eachbiomarkeranalysisisthusrepresentativeof15individualspersamplingmonth.ForAtlanticcod,allanalyseswereperformedonliverslicesfromeachsampledfish.
 2.2.Analyses 2.2.1.Determinationofcontaminantburdenintotalsofttissuesofbivalves
Contaminantanalyseswerecarriedoutbylaboratoriesaccred-itedforthemethodsused(UnilabAnalyse,PAH,andALSlaboratorygroup,metals).Forbluemussel,2poolsof10–15individualsfromeachsamplingpointwereanalysed,while3pools,eachconsistingof15Icelandicscallopsfromeachsamplingpoint,wereanalysed.ForPAHconcentrations,eachsampleofpooledsofttissue(10–20gwwt)wasthoroughlygroundandhomogenisedpriortoanalyses.Sampleswereweighedandapotassiumhydroxide-methanolsolu-tionandaninternalstandard-mixofdeuteratedPAHswereadded.Thesolutionwasboiledwithrefluxfor4h(saponification),beforefiltrationandextractionwithpentane.Sampleswerepurifiedusinggelpermeationchromatography(GPC),withdichloromethaneasmobilephase.Sampleswerefiltratedandfurtherpurifiedbysolidphaseextraction(SPE).AnalyseswereperformedusingaGC-MSD(Agilent7890GCwithsplit/splitlessinjector,Aglient7683andAgilent5975C,massspectrometerwithEIionsource).Blindsam-pleswererunparalleltoallsamples,andproficiencytestsamples(Quasimeme,Netherlands)wereusedascontrolsamples.Thelimitofdetection(LOD)wasdeterminedfromanalysesofaseriesoblanksamples,processedalongwithrealsamples,andcalculatedas:LOD=(blankaverage)+3
×
(blankstandarddeviation).Ascon-centrationsofPAHsinbivalvetissueswereingeneralclosetoorundertheLOD,resultswerepresentedasmin-maxrangeofcon-centrationsintheanalysedpoolsinsteadofmeansandstandarddeviations(Table1).Furthermore,forthecalculationofthesum ofNPDs(naphthalene,anthracene/phenanthrene,dibenzothion-pheneandtheiralkylatedhomologues)and16EPA-PAHs,halfof thedetectionlimitvaluewasusedforcompoundsthatwerebelowLOD.Totallipidcontentinthebivalvesofttissuewas
 
quantifiedaccordingtoFolchetal.(1957)inordertorelateeventualseasonal variationsofPAHstothelipidcontent.Homogenisedsampleswereweighed;methanolwas
 
added,followedbychloroform.Afterfil-tration,anaqueoussolutionofpotassiumchloridewasaddedforpurification,beforetheextractswerecentrifugedandtheaque-ousphaseremoved.Theremainingorganicphasewasevaporatedunderaowofnitrogengasuntildryness.Thetubewasthenweighed,andtheamountofextractedlipidwas
 
calculatedaswetweight%oftheinitialsample.Formetalanalyses,stillfrozensofttissueswerehomogenisedandfreeze-dried.Thedriedsamplesweredissolvedinconcentratednitricacidandhydrogenperoxidebymicrowavedigestion(170
C,30min)insealedTeflonvessels.Cooledsamplesweretransferredtotesttubesanddilutedto10ml.
 
Thesampleswereanalysedbyinductivelycoupledplasmasectorfieldmassspectrometry.Analyt-icalqualitywas
 
confirmedthroughanalysesofcertifiedreferencematerialbovinemusclepowder(NIST8414).
 2.2.2.PAHmetabolitesinAtlanticcodbile
PreparationofhydrolysedbilesampleswasperformedasdescribedbyKrahnetal.(1992).
Briey,bile(20
l)wasmixedwithinternalstandard(triphenylamine)anddilutedwithde-mineralisedwater(50
l)andhydrolysedwith
-glucuronidase/arylsulphatase(20
l,1hat37
C).Methanol(200
l)wasaddedandthesamplewascentrifugedandthesuper-natantwas
 
analysed.TheHPLCusedwas
 
aWaters2695SeparationsModulewitha2475fluorescencedetectorattached.Thecolumnwas
 
aWatersPAHC18(4.6mm
×
250mm)
 
with5
mparticles.Themobilephaseconsistedofagradientfrom40:60acetonitrile:ammoniumacetate(0.05M,
 
pH4.1)to100%acetonitrileataflowof1ml/min,andthecolumnwasheatedto35
C.Fluorescencewas
 
measuredattheoptimumforeachanalyte.25
lofextractwas
 
injectedforeachanalysis.
 2.2.3.Lysosomalmembranestability
Lysosomalmembranestabilitywas
 
measuredinhaemocytesof20bluemusselspersamplingperiod,usingtheNRRproce-dureadaptedfromLoweandPipe(1994).
Approximately0.1mlhaemolymphwasremovedfromtheadductormuscleofthebluemusselswithasyringecontainingapproximately0.1ml
 
physiolog-icalsaline(pH7.3).Thehaemolymph/salinesolutionwasplacedinamicrocentrifugetube,fromwhicha40
lsamplewasremovedandpipettedontothecenterofamicroscopeslide.Theslidewasleftinadarkhumidchamberfor15min
 
toallowadhesionofthecellstotheslide.Excessliquidwas
 
removedfromtheslideafterthistimeand40
lofneutralredsolutionwasadded.Theneutralredsolutionwas
 
takenupinsidethehaemocytesandstoredwithinthelysosomes.Theabilityofthelysosometoretaintheneutralredsolutionwas
 
checkedevery15min
 
bylightmicroscopy(
×
40).Thetestwasterminatedandthetimerecordedwhenmorethan50%ofthehaemocytesleakedtheneutralreddyeoutofthelysosomeintothecytosol.
 2.2.4.Energyreserves
Totallipid,carbohydrateandproteincontentsweredeterminedindigestiveglandsofbluemusselandIcelandicscallopandliverofAtlanticcodhomogenisedina0.1MTrizmaHCl/basebufferpH7.5containing,0.4MMgSO
4
,15%polyvinylpyrrolidoneand0.2%(w/v)TritonX-100andcentrifugedfor10min(3000
×
 g 
,4
C).LipidswereextractedfollowingBlighandDyer(1959).Briefly,thehomogenates(200
l)weremixedwith500
lchlo-roform,500
lmethanoland250
ldH
2
Oandcentrifugedat10,000
×
 g 
for5min.A100
lsampleofthechloroformphasewasthenpipettedintoglassreagenttubes,500
lofH
2
SO
4
(95–97%)

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