Radiation impacts on morbidity in Crustaceans

Morbiditycanbebroadlydefinedas“Alossoffunctional capac-itiesgenerallymanifestedasreducedfitness,whichmayrender organismslesscompetitiveandmoresusceptibletootherstressors, thusreducingtheirlifespan”(Copplestoneetal.,2008).Definition ofthetermmorbidityvariesbetweenauthorsandencompassesa vastnumberofendpointsincludingperturbationstogrowthrates, behaviouralalterationsandimmunesystemeffects(Copplestone etal.,2004).Inordertomaintainrelevancebothecologicallyand forenvironmentalprotection,anendpointshouldbeamenableto

N.Fulleretal./AquaticToxicology167(2015)55–6759 Table2

Numericalbenchmarkvaluesin␮Gy/hr⁻¹proposedbyanumberofdifferentorganisationsanddirectivesfortheprotectionofpopulationsofarangeofbiota.USDOE=UnitedStatesDepartmentofEnergy.NCRP=NationalCouncil onRadiationProtectionandMeasurements.IAEA=InternationalAtomicEnergyAgency.-=Nodataprovided.AdaptedfromAnderssonetal.,(2008).

DoseLevel(␮Gy/h⁻¹)

USDOE(1990) NCRP(1990) IAEA(1992) Environment Canada(2003)

FASSET(2003) Larsson,(2004)

ERICA(2007) Beresfordetal.

(2007)

ICRP(2008) UNSCEAR(2008) PROTECT(2009) Andersson,(2008)

FreshwaterOrganisms 400 400 400 – 100 10 – 400 10

Algae – – – 100 – – – – –

Macrophytes – – – 100 – – – – –

BenthicInvertebrates – – – 200 – – – – –

Fish – – – 20 – – – – –

ReferenceTrout – – – – – – 40–400 – –

ReferenceFrog – – – – – – 4–40 – –

MarineOrganisms 400 400 – – 100 10 – 400 –

MarineMammals – – – – – – – – –

DeepOceanOrganisms – – 1000 – – 10 – – –

ReferenceCrab – – – – – – 400–4000 – –

ReferenceFlatfish – – – – – – 40–400 – –

ReferenceBrownSeaweed – – – – – 40–400 – –

TerrestrialOrganisms – – – 100 100 10 – 100 10

Plants – – 400 – – – – – –

ReferencePineTree – – – – – – 4–40 – –

ReferenceWildGrass – – – – – – 40–400 – –

Animals – – 40 – – – – – –

Invertebrates – – – 200 – – – – –

ReferenceBee – – – – – – 400–4000 – –

ReferenceEarthworm – – – – – – 400–4000 – –

Mammals – – – 100 – – – – –

ReferenceDeer – – – – – – 4–40 – –

ReferenceRat – – – – – – 4–40 – –

Birds – – – – – – 4–40 – –

ReferenceDuck – – – – – – 4–40 – –

Table3

SummaryofmorbiditystudiesinCrustacea.HTOrepresentsTritiatedWater.Acuteexposuresaredefinedhereasthoselastinglessthan24h,withchronicexposureslasting overaperiodoftheorganismslifespanandgreaterthan24h

Species DoseRate/Total Dose

LowestObserved EffectDose/Dose Rate(LOEDR)

RadiationSource ExposureDuration ExposureType Conclusion Reference

Pollicipespolymerus7.9,62.5nGy/hr⁻¹, 0.625,6.25and 62.5␮Gy/hr⁻¹

0.000625mGy/h⁻¹ HTO 32Days Chronic Alteredmoulting

patterns

Abbott&Mix, (1979)

Daphniamagna 0.02,0.11and 0.99mGy/hr⁻¹

0.11mGy/h^{−1 241}Am 23Days Chronic Reductioninbody

mass,Increased

Daphniamagna 0.3,1.5and 15mGy/hr⁻¹

0.3mGy/h^{−1 241}Am 70Days Chronic Increasedoxygen

consumption,

Artemiasalina 100,200,400and 800Gy

200Gy ^6◦Co ∼30–220Minutes Acute Decreaseinrespiration rate

Angelovic&Engel (1968)

Callinectessapidus 40,80,160,320 and640Gy

40Gy ^6◦Co ∼11–175Minutes Acute Behaviouralchanges;

reductioninirritability, catatonicstateathigh doses

Engel,(1967)

Daphniamagna 0.41,4.2and 31mGy/hr-1

31mGy/hr^{−1 137}Cs 23Days Chronic Decreasein

mass-specific

0.5and5Gy 0.5Gy ^6◦Co <5Minutes Acute Morphological

aberrations;

Daphniamagna 0.007,0.07,0.65, 4.7and 35.4mGy/hr¹

4.7mGy/hr^{−1 137}Cs 75Days Chronic Reductionsinbody

lengthandVon

9.1Gy ^6◦Co 0–20Minutes Acute Alterationstomoulting

patterns

5.6Gy X-Ray 1Minute Acute Behaviouralchanges;

detectionand

3mGy ^6◦Co ∼0–10Minutes Acute Behaviouralchanges;

alterationsto

3mGy ^6◦Co ∼0–10Minutes Acute Morphological deformations, Decreased hepatosomaticindex

Stalinetal.(2013b)

measurement,specifictothehazardinquestionandappropriate forextrapolationtohigherlevelsofbiologicalorganisation(Ankley etal.,2010;Suter,1990).Thepracticalityofusingmorbidityasan endpointforradiationexposuremaythereforebelimitedduetothe lackofspecificityandmultitudeofeffectsitincludes.Thisis exem-plifiedwithinthecrustaceansubphylum,withadiversearrayof endpoints(SeeTable3forsummary)usedtoassessmorbidity.

3.1. Radiation-inducedimpactsongrowth&respiration

Alonzo et al., (2006, 2008a) investigated the effects of chronic internal exposure to the alpha emitting radionuclide,

241Americium,onthegrowth dynamicsof Daphniamagna. The authorsrecordedasignificantlylowerdrymassandbodylength ofirradiatedspecimensatdosesof∼1.5mGy/hr⁻¹infirst gener-ationorganisms(F0),withsignificantincreasesintheseverityof effectsovergenerations.Forexample,individualsoftheF2 genera-tiondisplayeda15%reductionindrymassatdosesof0.3mGy/hr⁻¹

(Alonzo et al., 2008a). A recent study furtherunderpinned the potential of ionisingradiations to perturb growth dynamics in daphnids(Parisotetal.,2015),withreductionsof5and13 %in thegrowth rateof F2generation daphnidsexposed to 4.7 and 35.4mGy/hr⁻¹ofgammaradiation,respectively.Experimental evi-dencesuggeststhatlargerdaphnidshaveenhanced competitive and resourceexploitation abilityrelative tosmallerindividuals, leadingtoelevatedmortalityin thoseindividuals withreduced competitioncapacity(KreutzerandLampert,1999).Thefinding thatradionuclideexposuremayperturbgrowthdynamics there-forehasimportantimplicationsfornaturalcrustaceanpopulation dynamics.

Inthepreviousstudy(Alonzoetal.,2008a),oxygen consump-tionofD.magnawaselevatedabovecontrolsatalldoses,suggesting anincreaseinmetabolicexpenditureinducedbyradiationstress.

Exposureoforganisms tostressorsand adverseconditionsmay resultinreallocationofmetabolicenergytowardsmaintenanceand leadtoreducedenergyinvestmentperoffspring(Baillieuletal.,

N.Fulleretal./AquaticToxicology167(2015)55–67 61

2005).Thiswasreflected by a reduced resistancetostarvation recordedinneonatesderivedfrom0.02mGy/hr⁻¹ exposedadult daphnids(Alonzoetal.,2006).Itisofnotethatthisdoseratefalls belowthevalueof∼0.4mGy/hr⁻¹providedbyanumberof orga-nisationsbelowwhichnodeleteriouspopulationleveleffectsare predictedtooccurinaquaticorganisms(SeeTable2)byanorderof magnitude.Arecentstudy(SarapultsevaandGorski,2013)further suggesteddeleteriousimpactsonneonatesrelatingtometabolic perturbations.Followingparentalexposuretoacutegammadoses of100and1000mGyfromCobalt-60,a∼20%decreaseinthemean lifespanofnon-exposedfirstgenerationD.magnaoffspringwas demonstrated.

Anotherstudyof Daphniamagnaexposed tochronicgamma irradiationfrom¹³⁷Csreportedcontrastingresultstothe afore-mentioned study (Alonzo et al., 2008a) of decreased oxygen consumptionwithincreasingdose(Gilbinetal.,2008).D.magna receivinggammadoseratesof 31mGy/hr⁻¹ displayeda signifi-cantlylowermass-specificrespirationrate,comparedwithdose ratesof0.3,1.5and15mGy/hr⁻¹allelicitinganincreasein respira-tionratefollowingAmericium-241(analphaemitter)exposurein thestudyofAlonzoetal.,(2008a).Whilstthelownumberof repli-cates(n=6)recognisedbytheauthorinthestudyofGilbinetal., (2008)maypreventcomparisonacrossstudies,thisunderpinsthe importanceofaccountingfordifferingradiationsourcesandthe correspondingvariabilityinrelativebiologicaleffectiveness(RBE).

ThetermRBEwascoinedin1931(FaillaandHenshaw,1931)to accountforthevariabilityinbiologicaleffectobservedwithdose, doserateandtypeofradiation(Valentin,2003).RBEincreasesas afunctionofLETwithhighlinearenergytransfer(LET)sourcesof radiation,e.g.,alphaemitters,typicallymoreeffectiveateliciting biologicaldamageinexperimentalsystemsthanlowLETradiation, i.e.,gammaandbetaraysreachingamaximumat∼100keV/␮m (HallandHei,2003;UNSCEAR,1996).Thismaybeusedtoaccount forthedifferentresponsesofD.magnainthesetwostudies.

Thevariabilityinbiologicaleffectrelatingtothegivenradiation sourceisexemplifiedbyastudyofmorbidityinthegoosebarnacle, Pollicipespolymerus,whichrecordedalteredmoultingpatternsat extremelylowbetadosesof0.62␮Gy/hr⁻¹(AbbottandMix,1979).

Theradiationsourceemployedinthepreviousstudywastritiated water(HTO),aradionuclidethatisdischargedintogroundwater systemsfromnuclearoperations(Jaeschkeetal.,2011;Jhaetal., 2005).Despitetherelativelylowenergy emissionofbeta parti-clesfromHTO(average betaenergy of5.73±0.03keV(Pillinger et al.,1961), thenature and behaviourof this radiation source withinorganismshasledtosignificantconcernovertheRBEof theradionuclide(Bridges,2008;LittleandLambert,2008).Ithas beendemonstratedthatHTOmaybeirreversiblyincorporatedinto organiccompoundswithinorganisms(TakedaandKasida,1979) andthereforemayproducea biologicaleffectdisparatewithits emissioncharacteristics.Inaddition,theauthorsofthe aforemen-tionedstudyonPollicipespolymerus(AbbotandMix,1979)stated thatcalculateddoseswereexclusiveofbackgroundradiationwhich wasnotquantified.Thishighlightstheimportanceofrobust quan-tificationofreceiveddoseinradiobiologystudies(Pentreath,2009).

3.2. Theeffectsofionisingradiationonthebehaviour&

histopathologyofCrustaceanspecies

Ionisingradiationhasbeendemonstratedtoinducebehavioural changesinanumberofcrustaceanspeciesincludingcrabs(Engel, 1967), prawns (Stalin et al., 2013a) and crayfish (Rodriguez and Kimeldorf, 1976). Alterations to behavioural patterns are fundamental in environmental risk assessments since these perturbationsmayariseasanadaptivemechanismtochronic con-taminantexposureandhavethepotentialtoalterspecies–species interactions(Dell’Omo,2002).Theavailableliteratureregarding

behaviouralimpactsofradiationinvolvesmostlyacuteexposures tohighdosesofradiation(Engel,1967;RodriguezandKimeldorf, 1976),withthemagnitudeofbehaviouralchangescorrelatingwith doselevels.Forexample,Engel(1967)assessedtheimpactofboth chronic and acuteradiation exposures onthebehaviour of the bluecrab,Callinectessapidus,ahighlyaggressiveandcannibalistic species(Bushmann,1999).Areductioninaggressivenessof Call-inectessapidusspecimenssubjecttosingleacuteirradiationswith

6^◦Codosesfrom40to640Gywasobserved,whilsthigherdoses induceda catatonicstate.Continuousexposurestolowerdoses (0.72,1.64&6.53Gy/d⁻¹)for70daysinducedcessationof feed-ingandabnormalbehaviouralpatternsdeviatingfromthenormal pugnacious natureofC. sapidus,withtheextentofbehavioural effects relating to dose. Whilst the receiveddose remains sig-nificantly higher than estimates of the highest external doses infreshwatersystemsimmediatelyaftertheChernobylaccident ([4.2–8.3mGy/hr⁻¹frombottomsediments]Kryshevetal.,2005), the findingthat prolongedexposures may perturb behavioural patterns has implications for contaminated areas where radia-tionlevels remainelevatedoverlong time scales.Furthermore, limited datasuggestsinduction of behaviouraleffects atlower, environmentallyrelevantdoses.Stalinetal.,(2013a)demonstrated behaviouralchangesincludingalterationstoswimmingpatterns inthegiantfreshwaterprawn,Macrobrachiumrosenbergiiatacute gammadosesof3mGy.

Fewstudieshaveconsideredtheimpactsofionisingradiation onmorphologicalandhistologicalparametersincrustaceans.Stalin etal.,(2013a,b)demonstratedinductionofhistologicaland mor-phologicalaberrationsincludingswollenandnecroticlamellaein the gill,deformations of the uropod, and discolouration of the abdomeninM.rosenbergiioveradoserangeof3–3000mGy(Stalin etal.,2013a),withthemagnitudeofeffectsrelatingtodose.Iwasaki (1973)adoptedahistologicalapproachtoassessgamma radiation-inducedeffectsinoogoniaandoocytesofthebrineshrimp,Artemia salina. A dose-dependent increase in cellulardeformations and thenumber ofpyknoticcells (celldegradationcharacterisedby chromatincondensation)wasrecordedoverahighdoserangeof 250–3000GyfromCobalt-60.Furthermore,Mothersilletal.,(2001) recordedperturbations tocytoplasmicorganellesin hematopoi-etic cultures of Nephrops norvegicus at gamma doses of 0.5Gy.

Deformationsincludedabnormalmitochondrial-rough endoplas-mic reticulum complexes at 0.5Gy, progressing to complete disintegration ofthecellular cytoplasmatdoses of 5Gy. Struc-turalperturbationstothegilllamellaeofcrustaceanshavebeen recorded in response toa number of toxicants (Li et al.,2007;

SaravanaBhavanandGeraldine,2000)andmayultimatelyimpair gillfunctioning(Tamseetal.,1995)leadingtoasphyxia.Future studiesshouldconsiderhistologicalimpactsonthecrustaceangill usingchronic,environmentallyrelevantradiationdosesinorder tocorroboratethisfinding.Adecreaseinthehepatosomaticindex ofM.rosenbergiiwasalsoobservedasaconsequenceofradiation exposure(Stalinetal.,2013b)whichmayprovidefurtherevidence thatradiationelicitsalterationstoenergybudgetssincechangesto theHSImayreflectmobilizationandutilizationofenergyreserves (Sánchez-Pazetal.,2007).

Behaviouralanalysisofcrustaceanspeciesexposedtoionising radiationhasreliedlargelyuponanecdotalvisualobservationsover adefinedtimeperiod(Stalinetal.,2013a).Thisapproachis sub-jecttoanumberoflimitationsincludingalackofteststandards (Kaneetal.,2004),alowsensitivitycomparablewithvideo-based behaviouralanalysersandthepotentialfor individualbias. Fur-thermore, the available studies have employed acute radiation exposureswhichmayinducedifferentbehaviouraleffectsto equiv-alentdosesdeliveredoverlongertimescales(Solomonetal.,2009).

Futurestudies shouldcouple chronic,environmentally relevant exposuredurationswithahigh-throughputbehaviouraltracking

system.Suchsystemsminimisebiasbyprovidingsensitive,reliable recordingsofsmallanimalbehaviourundercontrolledconditions.

4. Theeffectofionisingradiationonreproductionin