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Aquatic Toxicology

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

Mortality and deformities in European lobster (Homarus gammarus) juveniles exposed to the anti-parasitic drug teﬂubenzuron ^夽

Ole B. Samuelsen

^a,∗

, Bjørn T. Lunestad

^b

, Eva Farestveit

^a

, Ellen S. Grefsrud

^a

, Rita Hannisdal

^b

, Bjarte Holmelid

^b

, Tore Tjensvoll

^b

, Ann-Lisbeth Agnalt

^a

aInstituteofMarineResearch,P.O.Box1870Nordnes,N-5817Bergen,Norway

bNationalInstituteofNutritionandSeafoodResearch,P.O.Box2029Nordnes,N-5817Bergen,Norway

a r t i c l e i n f o

Articlehistory:

Received4November2013

Receivedinrevisedform10January2014 Accepted24January2014

Availableonline4February2014

Keywords:

Teﬂubenzuron

Europeanlobsterjuveniles Mortality

Deformities

a b s t r a c t

Thisstudydescribesexperimentscarriedouttoexamineeffectsoftheantiparasiticdrugteﬂubenzuron, usedindelousingfarmedsalmon,onanon-targetspecies,theEuropeanlobster(Homarusgammarus).

Juvenilelobsterswerefedtwodosesofteflubenzuron,10and20mg/kgsuccessivelyfor7dayscorre- spondingtoastandardmedicationofthefish(10mg/kgday)andtwicethestandarddose(20mg/kg day).Monitoringlasted3monthstoincludeatleastonemoultingperiodforallindividuals.Cumula- tivemortalitywashigherinallreplicatesgivenmedicatedfeedcomparedwiththecontrolgroup.Mean cumulativemortalityforeachdosingwas41±13%for10mg/kgand38±8%for20mg/kg,i.e.nodiffer- ence.Drugresiduewasanalysedinalljuvenilesthatdied,inadditionto12juvenilesatday8andthefirst 12survivinglobsters.Adeclineinconcentrationofteflubenzuronfromover8000ng/g(day5)to14ng/g (day70)wasobservedinthejuvenilesthatdiedduringtheexperiment.Twelveindividualsthatdiedcon- tained82ng/gorlesswhereasthemeanconcentrationinthefirst12lobstersthatsurvivedmoultingwas 152ng/g.Followingasingleoraladministration,thehalf-lifeofteflubenzuroninlobsterwasestimated to3.4daysandtheinitialconcentration(C0)to515ng/gattimet0.Attheendofthestudyaconsiderable numberofjuvenilelobsterswereobservedwithdeformitiesinvariousorgans;carapace,walkinglegs, cheliped,tailfan,abdomenandantenna.Theoccurrenceofobserveddeformitiesvariedfrom0to15%

intreatedreplicatesandwillmostlikelyaffectabilitytolocateandconsumefood(antenna,clawand walkinglegs),respiration(carapace)andabilitytomove/swim(walkinglegs,tailfanandabdomen).In total,themortalityandsenescentdamageswerecloseto50%inallreplicates.Juvenilesthatsurvived medicationwithoutdeformitieshowever,moultedandincreasedinsizeateachmoultequallywellas theunmedicatedcontrols.

1. Introduction

Salmonlice(Lepeophtheirus salmonis,Krøyer1837) aresmall marine ectoparasites that feed on mucous, blood and skin on salmonandtroutandifpresentinsufficientnumbers,maycause mortalitybyosmoticstressandsecondaryinfectionsbybacterial andviralpathogens.Infestationwithsealiceisthereforeconsid- eredamajorproblemfortheAtlanticsalmon(Salmosalar)industry inEuropeandNorthAmerica(PikeandWadsworth,1999;Costello, 2006;Burkaetal.,2012).Inadditiontoreducingthegeneralwel- fareofthefarmedfish,salmonlicecauseasignificanteconomicloss

夽Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCom- monsAttribution-NonCommercial-NoDerivativeWorksLicense,whichpermits non-commercialuse,distribution,andreproductioninanymedium,providedthe originalauthorandsourcearecredited.

∗Correspondingauthor.Tel.:+4797076918.

E-mailaddress:ole.samuelsen@imr.no(O.B.Samuelsen).

duetoreducedgrowth,increasedmortality,downgradingofﬂesh qualityandthecostofdelousingtreatments(MacKinnon,1997).

Excessivenumbersof adultlicein a salmonfarm alsoincrease thenumberof freeswimminglarvae inthesurroundingwater, hencegeneratinganegativeimpactonwildpopulationsofseatrout (Salmotrutta)andmigratingwildpost-smoltsofAtlanticsalmon (Wagneretal.,2008;Costello,2009).

Severalantiparasitic agents are currently in use for delous- ing farmed fish and the compounds are either dissolved in water and used for bath treatment or administered orally via the feed. In Norway, teflubenzuron [1-(3,5-dichloro-2,4- difluorophenyl)-3-(2,6-difluorobenzoyl)urea] and diflubenzuron [1-(4-chloro-phenyl)-3-(2,6-difluorobenzoyl)urea]) were fre- quently in use from late 1990s and until 2001. From 2002, emamectinandthepyrethroids,cypermethrinanddeltamethrin were the drugs of choice but due to instances with reduced sensitivity for these drugs, flubenzurons were reintroduced in 2009. Consumption of teflubenzuron was 2028, 1018, 26 and 751kg active compound in the years 2009–2012 respectively 0166-445X/$–seefrontmatter©2014TheAuthors.PublishedbyElsevierB.V.Allrightsreserved.

http://dx.doi.org/10.1016/j.aquatox.2014.01.019

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(NorwegianInstituteofPublicHealth,Oslo,Norway,www.fhi.no).

Flubenzuronsareorallyadministeredagentsthatactbyinterfering withthesynthesisofchitininthesalmonlice.Theyareeffective againstallstagesofsealicethatundergomoulting,includingthe larval and pre-adultstages (Bransonet al.,2000; Ritchieet al., 2002;Campbelletal.,2006a,b).Teflubenzuronisadministeredvia medicatedpelletscontaining2gteflubenzuronperkgfeedandthe recommendeddosingregimenis10mg/kgfishdailyfor7days.The excretionpathwayofteflubenzuroninAtlanticsalmonismainly viatheliverandbiletotheintestineandtheeliminationistemper- aturedependantwithhalf-livesinmusclebetween2and6days (Anon, 1999).SincethebioavailabilityinAtlanticsalmonislow (approximately10%)andthemetabolismisminimal,mostofthe drugwillbereleasedfromthefishasparentcompoundviafaeces intheperiod ofmedicationand immediatelyfollowing a treatment(Anon,1999).Apreliminarystudyanalysingfaecalmaterial fromAtlanticsalmonundergoingmedicationwithteflubenzuron showedconcentrationsmorethantwicetheinitialconcentration inthemedicatedfeed(Samuelsen, unpublisheddata). Solubility ofteflubenzuroninwaterislow(0.0094mg/Lat20^◦C),andthe substanceassociatereadilywithparticlesrichinorganiccontent (Marsellaetal.,2000).Itisthereforereasonabletobelievethatonce pellets and faecal material containing teflubenzuron reachthe marinesediment,thecompounddoesnotreadilydisappear.Based onresultsfromaScottishstudy,ahalf-lifeofteflubenzuronof115 daysinamarinesedimentcouldbecalculated(Langford,2011).

Ithasbeenshown thattheorganicwasteintheformofexcess pelletsandfaecalmaterialfromﬁshfarmsundergoingmedication containshighconcentrationsofﬂubenzurons(Selviketal.,2002).

However, concentrations found in sediment samples collected inthevicinityofﬁveinvestigated farmsweremuch lower.The highestconcentrationwas40mg/kgsediment(wetweight)(Selvik et al., 2002; Langford, 2011; Samuelsen, unpublished data). In contrast,usingaremotelyoperatedvehicle(ROV)pelletsandlarge faecalparticleswereregisteredonthebottomin thevicinityof ﬁshfarms(Kutti,2008).Itisthereforereasonabletoexpectthat theaccesstodrugcontainingorganicwastefornon-targetspecies willvaryconsiderableevenwithinasmallarea.

Sinceamajorpartoftheteflubenzuronadministeredviafish feedwillendupinthesurroundingenvironment,therehavebeen concernsaboutthepossibletoxiceffectsonnon-targetmarinebiota (Burridgeetal.,2010; Eisler,1992;Fisherand Hall,1992;Haya etal.,2005).Sinceteflubenzuronreduceschitinsynthesis,thedrug isrelativelynon-toxictomarinespecieslikefish,algaeandmol- luscs,butispotentiallyhighlytoxictoanyspeciesthatundergo moultingwithintheirlifecycle,includingcommerciallyimportant specieslikelobster,crabsandshrimps.Astudyoftheeffectoforally administeredflubenzuronsonnon-targetcrustaceanis,however, complicatedbythefactthattheinvestigationmustincludeamoult- ingphaseandtheexperimentalset-upbedesignedtoassurelow mortalityofnon-medicatedcontrolgroups.Furthermore,sinceitis likelythatnon-targetcrustaceansmayingestteflubenzuronfrom contaminatedsediments,faeces,excessfoodpelletsorbyeating contaminateddepositfeederslikepolychaetesitisdifficulttopre- dictadosingregimenthatinfullrepresentstheconditionundera fishfarm.Inordertohaveeffect,adefinedconcentrationofthedrug mustprobablybepresentinanindividualataspecifictimeperiod duringthemoultingcycle.Theconcentrationrangethatinduces mortality following moulting in marine non-target crustaceans is,however,notknown.Furthermore,sinceteflubenzuronaffects theformationofthenewexoskeletonitisapossibilitythatnon- fataldamagemayoccur.Copepodeggs(Acartiatonsa)exposedto diflubenzurondissolvedinwaterat1and10␮g/Lshowedreduced hatchingabilityandthosethathatchedwereabnormallyshaped andfailedtomoultatthenextstageofdevelopment(Testerand Costlow,1981).

For drugslike teﬂubenzurongivingnoimmediate andacute effect, knowledge of the uptake and elimination kinetics in a speciﬁcspecieswillprovideimportantinformationindetermining theperiodoftimeafteranexposurewherethedrugisexpectedto haveeffect.

Theaimofthisstudywastoexaminetheeffectsoftefluben- zurononjuvenilelobsterwhenoffereddosessimilartothedose administeredtothefarmedfishandtwicethisdosecorresponding totheconsumption ofdrugcontainingfaecal particles.Further- more,theuptakeandeliminationofteflubenzuroninlobsterwas studiedfollowingasingleoraladministration.

2. Materialsandmethods

2.1. Animalsandfeed

This study was approved by the National Animal Research Authority(NARA)inNorwayandhasbeencarriedoutinaccordance withTheCodeofEthicsoftheWorldMedicalAssociationforanimal experimentshttp://europa.eu.int/scadplus/leg/en/s23000.htm.

Aseriesofexperimentswereruntoassesstheeffectsoforally administeredteﬂubenzurononEuropeanlobster(Homarusgam- marus).Juvenilestagesoflobsterwerechosenastheymoultmore frequentlythansubadultsandadults.Thejuvenileswereproduced attheInstituteofMarineResearch(IMR)ﬁeldstationatParisvatnet locatedoutsideBergen(60^◦3775N,4^◦4811E)inlateJuly/August 2010.Broodstockwerecollectedfromthesurroundingareaclose by,inØygarden.Sizesofthejuvenileswererecordedascarapace length(CL)measuredasthedistancefromtheposteriorrimofthe eyesockettotheposterioredgeofcarapaceandtotallength(TL) measuredasthedistancefromtheanteriortipofrostrumtothe endoftelson.CLandTLwererecordedwithacallipertotheclosest 0.1mmbelow.Wetbodyweight(BW)wasrecordedonaTE2101 Sartoriusscaletothenearest0.1g.

Twodosesofteflubenzuronwerechosen,10and20mg/kg.The lowerdosecorrespondstothedoseadministeredtothesalmon whereasthehighdoseissimilartotheconcentrationfoundinfae- calmaterialfromsalmonundergoingmedication.Thelobsterswere fedspecialcommerciallyproducedpellets(3mm)patentedbyNor- wegianLobsterFarm(www.norwegian-lobster-farm.com/en/)and producedbyNofima(www.nofima.no/en).Thepelletshadanaver- ageweightof 50±5mg.The medicatedfeeds werecustomised to a 3g individual and madeby homogeneously mixing 60 or 120mgteflubenzuronwith1gglucoseandusingafewdropsof herring oil,and by gentleshakingthe drugwascoatedontwo batchesof100gpellets.Assumingevendistribution,this corre- spondstoconcentrationsof30␮g(lowdose)or60␮g(highdose) drugperpellet.Analysisof10 pelletsfromeach prepareddose revealed an average concentration of 36±6␮g/pellets for low doseand72±13␮g/pelletsforthehighdose(analyticprocedures describedinSection2.4.1)providingdosesof12mg/kg(lowdose) and24mg/kg(highdose)fora3globster.

2.2. Eliminationofteﬂubenzuron

The purpose of this experiment was to assess the elimina- tionrateofthedrugfromlobsterand calculatetheelimination half-life.TheexperimentwasconductedattheIMRlaboratoryin Bergen,Norwayintheperiod13thFebruaryto5thMarsin2012.

Atotalof36 lobsterjuvenileswithameanweightof4.3±1.0g wereused.Juvenileswerekeptseparatelyin18cm×16cm×13cm ﬂow-throughplasticcontainersstackedintwostandseachhous- ingamaximumof25containers(5×5).Seawaterwasobtained from120mdepthclosetotheIMR-Bergen facility,heated toa temperatureof15.0±0.5^◦Candaddedtotheexperimentalcon- tainersataﬂowofabout1L/min.Thejuvenileswerestarvedfor

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3dayspriortoadministrationofonehigh-dosepelletcontaining 72±13␮gteﬂubenzuron.All36juvenilesconsumedtheirpellet andsixindividualswererandomlysampledatday2,4,7,11,16 and20postmedicationandanalysedfordrugresidues.

2.3. Simulationofa7-daymedicationperiod–followedby monitoringfor3months

Themainfocusofthisexperimentwastosimulateamedica- tionperiodof7days,similartotheprescribedmedicationregime atasalmonfarm.Theobjectivewastoassess long-termeffects overa3-monthperiodinmortality,growthandconcentrationof teﬂubenzuroninlobsterjuveniles.Theexperimentwasconducted attheIMRﬁeldstationatParisvatnetoutsideBergen,Norwayin theperiodAugust22toNovember23in2011,atotalof93days.

Theexperimentalperiodwaschosentoincludeatleastonemoult- ingforallindividuals.Seawater wasobtainedfrom25mdepth andthe temperaturein thetanksdecreased duringthe experi- mental period from14 to9^◦C. A total of 286lobsterjuveniles weredividedintothreemajorgroups,twofedmedicatedpellets (highandlowdose)andonecontrolgroupfedun-medicatedfeed.

Eachmedicatedgroupwasdivided intothreereplicatesreceiv- inglow dose:C (n=38),F(n=38)and G(n=48)andhighdose:

A(n=38),D(n=38)andE(n=48).Numberofjuvenilesinthecon- trolgroupwas38.ReplicateF inlowdoseandcontrolwasrun outdoor,whiletheotherreplicateswererunindoor.Anetcovered theoutdoorreplicates.Thejuvenileswereheldseparatelyinwhite PVC plastic compartments of 170mL (7.0cm×3.5cm×7.0cm).

The bottom of the compartments was perforated with 2.5mm diameterroundholestoensurewaterflow.Eachreplicateandthe controlconsistedof8×5(or10×5)compartments,termedasa tray,heldtogetherbyplasticstrips.Eachtraywasplacedinsep- arate600L(1m×1m×0.6m)flow-throughfibreglasstankswith awaterflowof10–15L/min.Waterlevelinthetankswasabout 0.5mandflowwassetto30–40L/min.

Priortotheexperiment,thelobsterswerestarvedfor3days.The medicationperiodlastedfromday1to7andthepelletgivenatday 7wasleftforanother3daysbeforebeingremovedifnotconsumed.

Basedonpreviousknowledgeoffeedconsumption,lobsterswith aweightoflessthan2.0g(n=135includingthecontrol)werefed everysecondday(intotal4medicatedpellets)whereaslobsters withaweightequaltoorhigherthan2.0g(n=151includingthe control)werefeddaily(intotal7medicatedpellets).Eachindivid- ualwasinspecteddailyduringthemedicationperiod,anduneaten pelletsregistered.Mortalityandmoultingwasrecordeddaily.At day8,thedayafterthelastmedication,twoindividualsfromeach ofthesixmedicatedreplicatesweresampledandanalysedfordrug residues.Likewise,thetwoﬁrstjuvenilesineachmedicatedrepli- catethatsurvivedmoultingweresampledandanalysedfordrug residues.Cumulativemortalityperdaywascalculatedsubtracting samplingatday8andsamplingoftheﬁrsttwojuvenilessurviv- ingmoultingineachreplicate.Likewise,cumulativemoultingwas calculatedsubtractingsamplednumbers,includingmortality.At theendoftheexperiment(day93),allsurvivorswerecheckedfor deformitiesandCL,TLandBWrecorded.Juvenileswithdeformi- tiesweremonitoredoverseveralmoultingperiodstostudythe permanencyofthedamage.

2.4. Analyticalprocedures

Teﬂubenzuron(analyticalstandard),diﬂubenzuron(analytical standard),acetonitrile,heptane,diethyletherandacetone(allHPLC grade)werepurchasedfromSigma–Aldrich(Steinheim,Germany).

Tetrahydrofuran(HPLCgrade)andammoniumhydroxide(25%)(PA grade)werepurchased from Merck(Darmstadt, Germany). The waterusedwaspuriﬁedwithaMilli-Qwaterpuriﬁcationsystem

fromMillipore.Stocksolutionsofteﬂubenzuronanddiﬂubenzuron werepreparedataconcentrationof1mg/mLintetrahydrofuran andstoredat4^◦C.Workingstandardswerepreparedbydilutionof stocksolutionswithamixofacetonitrile:water(50:50,v/v).

2.4.1. Concentrationsinfeedpellets

Eachpelletwasaddedtoa10mLtubecontaining2mLacetoni- trile,shakenfor10minfollowedbytransfer(1mL)toanEppendorf tubeandcentrifugationfor5minatapproximately14,500×gusing aBiofugeAtablecentrifuge(HeraeusSepatech,OsterodeamHartz, Germany).Acalibrationcurvefor teflubenzuronrangingfrom5 to40␮g/mLwasmadeintriplicatebydilutionofstocksolution withacetonitrile:water(50:50,v/v).Analysisoffivenon-medicated pelletswasincludedinordertoconfirmtheabsenceoftefluben- zuron. Thesamples wereanalysed by High-PerformanceLiquid Chromatographywithultravioletdetection(HPLC-UV) usingthe equipmentandanalyticalprocedureasdescribedinSelviketal.

(2002).

2.4.2. Concentrationsinlobsterjuveniles

Each individual lobster was weighed, placed in a 25mL centrifugetubeandthoroughlycrushedusingaglassrod.Diﬂuben- zuron (50␮L, 0.5␮g/mL) wasadded tothesamplesas internal standard.Acetone(5–10mLdependingonsamplesize)wasadded andthemixturewashomogenisedbyawhirlmixerandplaced inanultrasonicbath(40kHz)for10minbeforethesampleswere centrifugedfor3minat2500×g (EppendorfCentrifuge5810R, Eppendorf AG, Hamburg, Germany). The resulting extract was transferred toa 10mLcentrifuge tube. Toremove fat, heptane wasadded(1mL)andthesolutionwasshakenbeforecentrifuga- tionat1250×gfor2min.Fromtheresultingtwo-phasemixture theupperheptanephasewasremovedanddischarged.Thisstep wasrepeatedonce.Theacetoneextractwasevaporatedtodry- ness under nitrogen at 40^◦C, theresidue dissolvedin heptane (5mL)andcleaned-upbysolidphaseextractionusingaGX-271 ASPEC (Automated Solid Phase Extraction)system from Gilson (Middleton,USA).Thesolid phaseextractioncolumn,BondElut Si,500mg,3mL(AgilentTechnologies,Boeblingen,Germany)was conditionedwith2.5mLheptanepriortoloadingofthesample.

Afterloading,thecolumnwaswashedwith3mLofheptane,5mL heptane/diethylether(95:5,v/v)and5mLheptane/diethylether (90:10v/v).Theanalyteandinternalstandardwereelutedwith 5mLheptane/diethylether(60:40,v/v).Theeluatewasevaporated todrynessusingnitrogenat40^◦Candre-dissolvedin250␮Lofa solutionofacetonitrile:water(40:60v/v).Thesamplewasfiltered througha0.45␮msyringefilterandwasreadyforanalysis.Forthe calibrationcurves,blanksampleswerespikedwithteflubenzuron atconcentrationsof15–500and500–8000ng/gandpreparedas describedabove.Thestandardcurveswerepreparedintriplicate.

The samples were analysed using a HP 1100 LC-system (Hewlett-Packard, Waldbronn, Germany) coupledto an Agilent MSDquadrupolemassspectrometer(AgilentTechnologies,Wald- bronn, Germany). ChemStation software (Agilent Technologies, Waldbronn,Germany)wasusedforinstrumentcontrolanddata analysis.TheanalyticalcolumnusedwasaShodexAsahipakODP- 504D4.0mm×125mm,4␮m(ShowaDenko,Munich,Germany).

The injectionvolumewas 10␮l.The mobilephase was a mix- ture of acetonitrileand aqueousammonium hydroxide (75:25, v/v)atanisocraticflowrateof0.7mL/minat25^◦C.Theretention timewas4.8minfordiflubenzuronand5.3minforteflubenzuron.

The analyte and internal standard were analysed by negative electrosprayionisation(ESI)anddetectedastheirdeprotonated molecular anions by selected ion monitoring (SIM). The mass to charge (m/z) ratio was 378.9 (qual.) and 359.0 (quant.) for teﬂubenzuronand309.0(qual.)fordiﬂubenzuron.Thefollowing experimentalparameterswereused:nebuliserpressure,40psig;

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dryinggastemperature,350^◦C;dryinggasﬂow,8L/min;needle voltage,3600V;desolvatingchambertemperature,300^◦C;frag- mentorvoltage,70V.Thelimitofdetection(LOD)wasdetermined to5ng/gandthemethodwaslinear(r=0.98)overtherangesstud- ied(15–500ng/g,500–8000ng/g).Inter-runprecisionrangedfrom 7.5to10.0%.

2.5. Statisticalanalysis

Forestimationofeliminationhalf-life(t_½ˇ)alinearregression analysiswasmadeonlogarithmically(ln)transformeddrugcon- centrationsversustime usingtheformulat½=ln2/k,where kis theslopeoftheregressionline.Theinitialconcentration(C_o)was determinedbyextrapolationoftheregressionlinetotimezero(to).

Analysisofvariance(ANOVA)wasmadeonsizescomparingrepli- catesandtreatments.Toanalysedifferencesbetweenlowandhigh doseconcentrations,analysisofcovariance(ANCOVA)wasapplied tolog-transformedconcentrations.

3. Results

3.1. Eliminationofteﬂubenzuron

Followingintakeofone pelletwithhighdoseteflubenzuron (72␮g) the concentrations in thelobsters decreased with time (Fig.1).Thehighestconcentration,602ng/g,wasmeasuredatthe firstsampling,i.e.2daysafterintake.Thelowestconcentrationat thatsamplingwas215ng/ggivinganindicationoftheindividual variation.FromthedatapresentedinFig.1aneliminationhalflife (t_½ˇ)of3.4daysandaninitialconcentration(Co)515ng/gattime tocouldbedetermined.

3.2. Simulationofa7-daymedicationperiod-followedby monitoringfor3months

3.2.1. Mortality

Overall41%(n=46)ofthejuvenilesadministeredlowdoseof teflubenzurondied.Mortalitywas30%,39%and56%inthedifferentreplicates.Mortalitycommencedwithonejuvenileatday4and of15juvenilesthatmoultedduringthemedicationperiod,10died immediatelyandonediedwhenmoultingforasecondtimeatday 51containing15ng/gteflubenzuron.Theremainingfivejuveniles didnotmoultasecondtimeandwerestillaliveattheendofthe experiment.Ofthejuvenilesexposedtoahighdoseoftefluben- zuron38%died(n=42)andmortalityvariedbetween32%,44%and 47%inthedifferentreplicates.Themortalitycommencedatday5 (threejuveniles)andoffthesevenindividualsthatmoulteddur- ingthemedicationperiodsixdied.Theonlysurvivoralsomoulted

y = 515e^-0.20^x R² = 0.99

0 100 200 300 400 500 600

0 2 4 6 8 10 12 14 16 18 20

Mean concentration ng/g

Days in experiment

Fig.1.Meanconcentrationofteflubenzuron(n=6ateachsamplingtime)vstime profileinjuvenileEuropeanlobster(Homarusgammarus)followingasingleoral administrationof72␮gteflubenzuron.Standarddeviationaregivenasverticalbars.

0 10 20 30 40 50 60

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Cumulative mortality (%)

Days in experiment Low dose

High dose Control

Fig.2.Cumulativemortality(%)inEuropeanlobster(Homarusgammarus)juveniles givenpelletswithloworhighdoseofteﬂubenzuronfromday1to7comparedwith controlwithnomedicationinthefeed.Thenumbersforlowandhighdosesarethe meansofthreereplicates.

successfullyasecondtimeatday50andwasaliveattheendof theexperiment.Forbothlowandhighdose,morethan80%ofthe mortalityhadtakenplacebeforeday30.Inthecontrolgrouptotal mortalityamountedtoﬁveindividuals(13%),alldiedwithinthe ﬁrst23days.Nodifferencewasfoundinmeancumulativemor- talitycomparinghigh-andlow-dosetreatmentsattheendofthe studywhereasbothtreatmentshadhighercumulativemortality comparedtothecontrolgroup(Fig.2).Someofthejuvenilesdied whileinamoultingprocessasshowninFig.3.

Successfulmoultingafterthelastdoseofmedicinestartedat aroundday30,inbothtreatments.Notallmoultswerehowever recordedasthejuvenilescaneattheirexoskeletoninafewhours andmoultingscanthereforebemissed.Asaconsequence,only58%

ofthejuvenilesinthelow-dosetreatmentwererecordedtohave moultedcomparedto79%inthehigh-dosetreatmentand72%in thecontrol.However,intheendoftheexperiment,measurements ofCLandTLshowedthatallsurvivingjuvenileshadmoultedatleast oncebutrecordingsofexactdatesweremissing.Fourjuveniles wererecordedtohavemoultedtwice.

3.2.2. Growth

Whentheexperimentwasinitiatedthemeanjuvenilesizewas 16.0±2.1mmCL;45.4±6.3mmTLand2.2±0.9gBWandnosig- niﬁcantdifference(ANOVA,p>0.05)wereregisteredinCL,TLor BWbetweenreplicatesortreatmentgroups.Intheexperimental periodthelobsterincreasedinweightandsizeequallyinallgroups andreplicates.Attheendoftheexperiment(day93)nosigniﬁcant differencewerefoundinneitherCL,TLorBW(Table1)(ANOVA, p>0.05).

3.2.3. Concentrationofteﬂubenzuroninthejuveniles

Duetovariable appetitetheprearranged dosewasnot con- sumedbyallindividualsduringthemedicationperiod.However, asthenumberofpelletseatenbyeachjuvenilewasrecordeditwas

Fig.3. Europeanlobsterjuvenile(nr.A06)hadconsumedthreepelletswithhigh doseofteﬂubenzuronbeforeitdiedduringthemoultingprocess,atday7.Thenew exoskeletonisvisibleundertheoldone.

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Table1

Summaryofcarapacelength(CL),totallength(TL)andbodyweight(BW)ofEuro- peanlobster(Homarusgammarus)atday93.

Lowdose Highdose Control Carapacelength(mm) 18.6±7.7 18.3±2.3 18.0±2.2 Totallength(mm) 52.9±5.7 52.7±6.9 52.8±6.5 Bodyweight(g) 3.7±1.2 3.7±1.6 3.6±1.4

Number 66 68 32

4 7

4 4

7 7

4 4

4

4 4

4

0 1000 2000 3000 4000 5000 6000

C02C03 F02F03G03G06A01A03D01D03E01E02

Low dose High dose

Concentration (ng/g)

Fig.4.Concentrationofteﬂubenzuron(ng/g)atday8injuvenileEuropeanlobster (Homarusgammarus)thathadconsumedtheprescribednumberofpellets(marked aboveeachcolumn)withloworhighdoseofteﬂubenzuronfromday1to7.

possibletocalculatethegivendoseforeachindividual.Inthelow- dosegroupthejuvenilesconsumedonaverage79␮g/g,compared with173␮g/ginthehigh-dosegroup.Allthe12lobsterssampled onedayaftertheendofmedication(day8)consumedtheappointed numberofpellets.Themeanconcentrationsforlowandhighdoses were1715±770and2338±1727ng/g,respectively(Fig.4).How- ever,sinceoneindividual(A03)inthehigh-dosetreatmentwasa majorcontributortothedifference,themeanvalueforthisgroup wasreducedto1666±588ng/gifA03wasexcluded,givingnosig- niﬁcantdifferenceinmeanconcentrationbetweenlowandhigh doses.Theﬁrstsixjuvenilessurvivinglowdosemoultedfromday 20to42,and thosesurviving highdoseatday23 (allsix).The concentrationsinthesejuvenileswereonaverage174±103ng/g and130±115ng/ginlow-andhigh-dosetreatment,respectively.

Therewasalargevariationbetweenthejuveniles,e.g.atday23 (highdose)theconcentrationvariedfrom26to351ng/g.

A decline in concentration of teﬂubenzuron withtime was observed in those juveniles that died during the experiment (Fig. 5).The highest concentrations weredetectedin two low- dosed juveniles with 8176 and 6843ng/g, respectively. In the high-dose treatment, the highest concentration detected was 4450ng/g.Overall,therewerenosigniﬁcantdifferencesinconcen- trationswhencomparinglowandhigh-dosedjuveniles(ANCOVA,

0 1 000 2 000 3 000 4 000 5 000 6 000 7 000 8 000 9 000

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Concentration (ng/g)

Days in experiment

Low dose High dose

Fig.5. Concentrationofteﬂubenzuron(ng/g)inthejuvenileEuropeanlobster (Homarusgammarus)thatdiedduringtheexperiment.Teﬂubenzuronwasadmin- isteredfromday1to7.

0 20 40 60 80 100

B C F G A D E

Control Low dose High dose

Percent (%)

Senescent damages Mortality

Fig.6. Ratioof%cumulativemortalityandsenescentdamagesbetweenreplicates ofEuropeanlobster(Homarusgammarus)juvenilesatday93whentheexperiment wasterminated.

p=0.178).Mortalityalsooccurredinindividualswithlowconcen- trationofthedrug,andevenin individualsmoulting laterthan 60daysaftermedicationwasended.Elevenindividualsthatdied contained82ng/gorlessandthelowestconcentrationwas14ng/g.

3.2.4. Deformities

Whentheexperimentwasterminated(day93),thesurviving juvenileswereinspectedfordeformities.Sincedeformitieswere absentamongjuvenilesinthecontrolgroupitwasassumedthat thedeformitiesweresenescentdamagesduetotheexposurefor teﬂubenzuron.Inoverall,29ofthe126survivingjuvenileswere deformedbutnodifferenceinnumberbetweenlow-andhigh-dose treatmentwasfound(n=14and15,respectively).Deformitiesvar- iedbetweenreplicatesfrom0to15%,andthereplicateswithlowest mortalityhadhighestnumberofsurvivorswithsenescentdamages (Fig.6).Deformitieswerefoundinthecarapace,walkinglegs,cheliped,tailfan,abdomenandtheantennas(Table2andFig.7b–d).

Ninejuvenileshaddevelopedmultipledeformitiesandwhereone juvenilehadthree(swollencarapace,stiffwalkinglegs,damagesto thetailfan),whiletheothereighthadtwo.Themostcommonco- occurringdeformitiesweredamagestothetailfanandstiffwalking legs(33%).

Affected juveniles were monitored over several moulting periodsandstiffwalkinglegs(Fig.7b)werereplacedinonemoult whereasseveredamagestothechelipeds(Fig.7d)neededthree moultingstoappearnormal.Damagestothetailfanseemedper- manentasthiswasnotrepairedthroughmoultings.

4. Discussion

Thechitinsynthesisinhibitorteflubenzuroniswidelyusedas anorallyadministeredagentagainstectoparasiticsalmonlicein marinesalmonfarming.Duetolowbioavailabilityandmetabolism inthefishandhighaffinityfororganicparticlesamajorpartofthe drugwillendupinthesurroundingenvironmentassociatedwith faecesoruneatenpellets.Concernshavebeenraisedaboutpossible effectsofvariousantiparasiticdrugsusedinfishfarmingonnon- targetmarinespecies.Inthisstudy,theeffectofteflubenzuronwas testedonEuropeanlobsterjuveniles.Twodoseswereused.One representedthedoseinmedicatedfeedprescribedforthesalmon, lowdose.Thesecond,highdose,representedthehigherconcentra- tionfoundinfaecesofmedicatedsalmon.Thestudyshowedthat lobsterswereaffected,eitherbymortalityorsenescentdamagesas observed3monthsaftermedication,givingatotaleffect(mortal- ityandsenescentdamages)ofingestingteflubenzuroncloseto50%, independentofwhetherloworhighdosewasadministered.The

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Table2

ClassiﬁcationofdeformitiesfoundinjuvenilestagesofEuropeanlobster(Homarusgammarus)affectedbyteﬂubenzuroninthefeedfromday1to7.Observationsweremade atday93.

Organaffected Category Description Occurrence(%)

Carapace Puffy Carapacepuffy/swollen,orupfoldedononesideoftenleavingsomepartsofthegillsexposed 15 2ndto5thpereopod Stiff/twisted Thejointswerefusedtogetherasifovercalciﬁcationmadethejointsgrowntogether.Theentire

pereopodlegwaslikeonestiffpiece,sometimes“frozen”inanarbitrary/twistedposition

20

1stpereopod/cheliped Miss-shaped Variousshapesofthechelipeddeviatingfromnormal 15

Uropod Damages Damagestopartsofthetailfan,orevenlackingoneorbothofthetailfans 23 Abdomen Stiff Abnormalshapeoftheabdomenasifsomeofthesegmentswereoncebrokenandthengrown

backinawrongshape

18 2ndantenna Stiff Segmentsoftheantennawerefused,almostasifovercalciﬁcationmadethejointsgrown

together.Felt“stiff”whentouching.Difﬁculttoobservewhenanimalwasoutofwater

10

Fig.7. SenescentdamageinjuvenileEuropeanlobster(Homarusgammarus)observedatday93aftermultipleadministrationoftheantiparasiticdrugteﬂubenzuronfrom day1to7.(a)Normallobsterwithnosenescentdamages,(b)damagesinthewalkinglegs,(c)swollencarapace,(d)deformedcheliped.

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lackofdifferenceinmeancumulativemortalitybetweenhighand lowdosedgroupsisreﬂectedinthelackofsigniﬁcantdifference inmeanconcentrationswhencomparingdeadjuvenilesfromeach doseandintheconcentrationsmeasuredin12individualsafterlast medication.

Simulating a 7-day medication period does not necessarily mimicthenaturalconditionsunderaﬁshfarmsincelobstersinthe wildhavethepossibilitytochoosefromavarietyoffoodsources.

However,nopreferenceswerefoundinastudygivingEuropean lobsterjuvenilesthe option tochoose betweenmedicated pellets(teﬂubenzuron),non-medicatedpelletsorArtemiasp.(Jelmert, unpublisheddata).Inourstudyonlydrug-containingpelletswere offeredtothelobsters,andthedosingregimencouldberegardedas aworst-casescenario.Ontheotherhand,incontrolledexperiments itwasshownthatjuvenilesareabletocollectandstorefoodine.g.

substrateand/orshelter.Ifthisisthecaseinthewild,thenum- berofstoredandconsumedmedicatedpelletsmayevenexceed whatwasadministratedinthisexperiment.However,asoneofthe aimsofthestudywastogaininformationconcerningwhichcon- centrationsofteﬂubenzuroninducedmortalityinjuvenilelobster, wefoundtheexperimentalset-uplegitimatetouse.

Eliminationhalﬂife(t_½ˇ)ofteﬂubenzuronafterasingledose administrationof72␮gwas3.4daysata temperatureof15^◦C.

Noeliminationdataareavailableforteflubenzuronincrustaceans butin Atlanticsalmonmuscle,t½ ˇvalues of4.7 and2.6 days, respectively,werefoundfollowingasingleoraladministrationof 10mg/kgofteflubenzuronormultipleadministrationsof10mg/kg dailyfor7days(Anon,1999).Thesalmonwereheldat10^◦C.At6^◦C thet_½ˇvaluefollowingamultiple-dosestudywas3.8days(Anon, 1999).Theeliminationinlobsterjuvenileswasthereforefoundto beinthesamerangeasinAtlanticsalmonmuscle.However,asthe drugcouldbedetected60days(approximately18half-lives)after medicationinlobster,itispossiblethatteflubenzuronisdistributed accordingtoatwo-compartmentmodelandthat,duetotheexper- imentalperiodofonly20days,asecondandslowerelimination pathwaywasnotrevealed.

Whencomparingresultsfromthesingleandmultipleadminis- trationsinthisstudyitisclearthatmultipleadministrationscaused abuildupofthedruginthelobsterbody.However,thediffer- encein meanconcentrations after8days comparinglow-with high-dosetreatmentwasnon-signiﬁcantwhichmayindicatelower palatabilityadministeringthehighdosepelletsand/ordecreased absorptionratewithincreasingdose,processesthatpreviouslyhas beendescribedforantibacterialagentsinﬁsh(Hustvedtetal.,1991;

Rigosetal.,1999).Furthermore,lobsters thissizewillbreakup thepelletinsmallerpieceswhileeatingandtheamountofspill isdifﬁculttomeasure,whichmayaccountforthelargeindividual variationsinconcentrationsthatwasobserved.

Thereareveryfewdataavailabledescribingthemortaleffectof flubenzurons,particularlyonnon-targetmarinecrustaceansand afteroraladministrationofthedrug.WhileLC50valueshavebeen establishedforanumberofterrestrialinsectsandalsosomeaquatic crustaceanswhenexposedtothedrugsdissolvedinwater(summ- arisedin Langford, 2011), studiesinvolving theeffect of orally administereddrugsaresparse.ScottishEnvironmentalProtection Agency(SEPA)haveconsideredtheecotoxicityofteflubenzuron when usedas an anti sea-liceagentin aquaculture and setan EnvironmentalQualityStandard(EQS)valueforteflubenzuronin marinesedimentto2ng/g(dryweight).ThedefinedEQSvaluewas basedonaNoObservableEffectConcentration(NOEC)valueforthe amphipodCorophiumvolutatorafterachronicexposurefor28days andasafetymarginof10(SEPA,1999).Unfortunatelynodetailed informationwasgivenabouttheexperimentalset-upandfindings, excludingadirectcomparisonofresults.

Thisstudyshowedthatindividualssurvivedmoulting3weeks afterthe last medicationand witha concentration of 350ng/g

teflubenzuron. In contrast,11 individuals died withconcentra- tionsof82ng/gorless,thelowest14ng/g.Thisdemonstratesthe variationinsensitivitybetweenindividualsandmadeitdifficult todetermine a break point, where higher concentrations most likelywouldinducemortalityandlowerconcentrationsmostlikely wouldnot.Ontheotherhand,thestudyrevealsthelowercon- centration range that induces mortalityin lobster juvenile and consequentlyindicatestheconcentrationrangethatmighthave effecton othernon-target crustaceans, providing similarsensi- tivity. In two field studies the concentrations of teflubenzuron weremeasuredincrustaceanstrappedinthevicinityoffishfarms duringandshortlyaftermedication(Langford,2011;Samuelsen, unpublishedresults).Langford(2011)foundminorresiduesofthe drug(between0.2and11.3ng/g)indeepwaterprawns(Pandalus borealis)collectedbytrawl1–5km awayfrom thefarmsand a maximumof185.7ng/ginbrowncrab(Cancerpagurus)caughtby trapsatadistanceof100–300mfromthefarms.Inthestudyby Samuelsen(unpublishedresults)crustaceansweretrappedwithin anareaof 300mfromacommercialfarmundertreatmentand drugresidueswerefoundinallexaminedspecies.Themaximum concentrationswere200ng/gin deepwaterprawns,319ng/g in Norwaylobster (Nephrops norvegicus), 393ng/g in squatlobster (Munida sp.) and 865ng/g in King crab(Lithodes maja) indicat- ingthattheconcentrationsofteflubenzuronindefinedindividuals werehighenoughtoinducemortalityifmoultingwasimpending.

Lobsteriscoveredbyanexoskeletonthatneedstobeshedin ordertogrow.Sinceteﬂubenzuronreduceschitinsynthesis,itis assumedthatmortalityinallthemedicatedindividualswascon- nectedwithecdysis.Theexoskeletonisdividedintothreelayersall consistingofchitin,protein,calciumandmagnesiumsalts,phos- phatesandafewothercomponents,butwherethecomposition variesinthedifferentlayers(Bobelmannetal.,2007;Romanoetal., 2007;Al-Sawalmihetal.,2008;Sachsetal.,2008;Fabritiusetal., 2009;Kunkeletal.,2012).Moultinginlobsterisacomplexprocess anditmightbethatchitinisformedduringashortperiodduringthe moultingprocess,andvulnerabilityishigherduringthisstage.The workingmechanismofﬂubenzuronsinblockingthechitinsynthe- siswillaffectformationofthenewexoskeletonleadingtoathinner andweaker structure. Thecauseofdeathmaybedisruptionof bloodvesselsintheprocessofliberationfromtheoldexoskeleton.

Noexaminationofthethicknessandstratiﬁcationoftheexoskele- tonwere,however,performedinthisstudybutanongoingstudy aimsatrecordingthechangesthattakeplaceintheexoskeleton inlobsterjuvenilesduringamoultingperiod,andrevealinghow teﬂubenzuronisaffectingtheprocess.

Thisstudyisthefirsttoreportaconsiderablenumberofjuve- nilelobsterswithdifferentabnormalitiesfollowingadministration ofteflubenzuron.Neverthelessasimilareffectwasreportedalready in1981onthecopepod,A.tonsabyTesterandCostlow(1981)who foundthatthehatchingviabilitywasless than50%aftera12-h exposureto1␮g/Lofdiflubenzuronandlessthan5%after24-h exposureto10␮g/L.Thosethathatchwereabnormallyshapedand failedtomoultatthenextstageofdevelopment.Incontrast,juve- nilelobsterswithabnormalitiesinourstudywereabletomoult successfullyandstartregeneratingthedamagedparts.However, sincethedeformitiesmostlikelywillaffecttheabilitytolocate andconsumefood(antenna,clawandwalkinglegs),respiration (carapace)andability tomove/swim(walking legs, tailfan and abdomen)thepossibilitythattheywillsurviveinthewildissmall.

Interestingly,ourstudyrevealednonegativeeffectongrowthof juvenilesthatsurvivedmedicationwithoutdeformities,compared withcontrols.

Ithasbeenshownthatﬂubenzuronsarepresentinthesediment andsedimentdwellingorganismsforanextendedtimeaftermed- icationofafarm(Langford,2011;Selviketal.,2002,Samuelsen, unpublishedresults). Furtherresearch shouldtherefore include

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long-termexposureofjuvenilelobsterstolowerdrugconcentra- tionsandfurthermoretoincludeotherimportantcrustaceanlike crabs,shrimpsand crayﬁsh.Suchdataaredeemednecessaryin ordertoevaluatetheenvironmentaleffectsofﬂubenzuronswhen usedinaquaculture.

Acknowledgements

ThetechnicalassistanceofthestaffatParisvannetFieldStation ishighlyappreciated.TheprojectwasfundedbyNorwegianMin- istryofFisheriesandCoastalAffairs,NationalInstituteofNutrition and Seafood Research, Bergen, Norway and Instituteof Marine Research,Bergen,Norway.

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