ANABSTRACTOFTHETHESISOF
GeorgeR.Tuttleforthedegree ofMasterofScienceinToxicologypresentationonOctober
30,2012.
TITLE:SizeandSurfaceAreaDependentToxicityofSilverNanoparticlesinZebrafish
Embryos(Daniorerio)
Abstractapproved:
StaceyL.Harper
Manystudiesaddressingthetoxicityofsilvernanomaterialshavefoundthatsmallersized
silvernanoparticlesareusuallymoretoxictoorganismsandincellculturethanparticlesof
largersizesyetitisnotentirelyclearwhy.Weinvestigatedthesizedependenttoxicityof
silvernanoparticlesbymeasuringthe responseofembryoniczebrafish(Daniorerio)
followingexposuretoalibraryofthirteendistinctsilvernanoparticlesizedistributions
withmeandiametersbetween8.9nmand112.6nm.Dataanalysisusingdose‐response
modelingrevealedthatsilvernanoparticles(AgNP)inducedembryotoxicitythatis
dependenton thetotalsurfaceareaandnotonthemassorparticlenumberinsolution.
Includedinthisstudyisacomparisonbetweenembryotoxicityinducedbysilvernitrate
(AgNO3)andAgNPsforcardiovascularendpoints,aswellasaninvestigationintothe
influenceofthechoriononAgNPtoxicity.Thisstudydemonstratestheimportanceofusing
alternativedosemetricsinnanotoxicology,andhighlightsthevalueofusingtheembryonic
zebrafishtoexplorenanomaterialstructureactivityrelationships.
©CopyrightbyGeorgeR.Tuttle
October30,2012
AllRightsReserved
SizeandSurfaceAreaDependentToxicityofSilverNanoparticlesinZebrafishEmbryos
(Daniorerio)
by
GeorgeR.Tuttle
ATHESIS
Submittedto
OregonState University
inpartial fulfillmentof
therequirementforthe
degreeof
MasterofScience
PresentedOctober30,2012
CommencementJune2013
MasterofSciencethesisofGeorgeR.TuttlepresentationonOctober302012.
APPROVED:
MajorProfessor,RepresentingToxicology
HeadoftheDepartmentofEnvironmentalandMolecularToxicology
DeanoftheGraduateSchool
Iunderstandthatmy thesiswillbecomepartofthepermanentcollectionoftheOregon
StateUniversityLibraries.Mysignaturebelowauthorizesreleaseofmythesistoanyreader
uponrequest.
GeorgeR.Tuttle,Author
ACKNOWLEGMENTS
TheauthorexpressesheartfeltappreciationtohismentorandadvisorDr.Stacey L.
Harper,hiscommitteemembers,researchgroup,collaborators,friendsandlovingfamily
foralloftheiramazingsupportandencouragement.
TABLEOFCONTENTS
Page
Chapter1:Introduction…………………………………………………………………………………………………..2
1.1:NanosilverBackground……………….………………………………………………………………...2
1.2:LiteratureReview…………………………………………………………………………………………2
1.2.1InvitroAgNPToxicity………………………………………………….............................2
1.2.2SizeDependentInvitroToxicity…………………………….............………………..3
1.2.3AgNPOxidativeDissolution…………………………….……............…………………4
1.2.4SilverIonMediatedToxicity……………….…………….............……………………..4
1.2.5InvivoAgNPToxicity……………………….…………….............………………………..5
1.2.6EmbryonicZebrafish…………………..………………….............………………………5
1.2.7AgNPandAg+MediatedToxicityinZebrafish……...........……..……..…….......5
1.2.8AgNPMediatedToxicityinAdultandLarvalZebrafish…..........……...…….6
1.2.9PrimaryMechanismofAgNPToxicity……………………............…….…………..6
1.2.10AlternativeMechanismsofAgNPToxicity...............…….................................7
1.2.11ZebrafishEmbryoChorionsPlayaRoleinENMToxicity…..….....……….8
1.2.13SizeDependentEmbryoToxicityofAgNPs………………………….....………. 8
1.3StudyDesign………………………………………………………………….……........………..9
1.3.1SimilaritiesBetween AgNO3 andAgNPToxicity……..…….……........……...…9
1.3.2EstimatingtheEffectoftheChorion………………………….……...........………...9
1.3.3DoseModelingandAlternativeDoseMetricsAnalysis……….……........…..9
Chapter2:MaterialsandMethods………………………………………………………………………………….11
2.1TestMaterials…………………………………………………………………………………………….11
2.2NanomaterialCharacterization………………………………………………………………...…12
2.3ZebrafishHusbandryandEmbryoCollection…..……………………………….……..…...12
2.4EmbryoDechorionation……………………………………………………….………………..……13
2.5ExposureMethod………………………………………………………………….…………………….13
2.6ZebrafishEmbryoEvaluation………………………………………..……………………………..14
2.7DataanalysisStatisticalEvaluation………………………………..……………………………..14
Chapter3:Results……………….………………………………………………………………………………………...16
3.1DoseModelingandAgNO3Toxicity….………………………………………………………..….16
3.2AlternativeDoseMetricsAnalysis……………………………………………………………..….16
3.3EffectoftheChoriononEmbryoToxicity……………………………………………………...21
TABLEOFCONTENTS (Continued)
Chapter4:Discussion………………………………..…………………………………….…………………………….26
4.1OverallFindings……………………………………………………..............................................…….26
4.2OverallCharacterizationofAgNPandAgNO3Toxicity............................................…….26
4.3PresenceoftheChorionEffectsAgNPToxicity………………………………….…………..29
4.4DoseMetrics:ConsiderationsforAgNPs..………..……………………………………....……30
Chapter5:Conclusions………………………………………………………………………………………….………33
References……………....……………………………………………………………………………………………………37
LISTOFFIGURES
FigurePage
1. EstimatedLC50valuesusingthreedosemetrics.Datapointsrepresentthepercent
survivalplottedasafunctionof,(A)massconcentration,(B)particleconcentration,and
(C)surfaceareaconcentration……..………............…………………………………………………………17
2. EstimatedLC50valuesplottedagainstmeanprimaryparticlediameterforthreedose
metrics.LC50valuesareplottedasafunctionofmeanprimaryparticlediameterfor (A)
massconcentration,(B)particle concentration,and (C)surfacearea
concentration…………………………………………….……………………………………………………………19
3. PercentembryomortalityforCh(+)andCh(‐)embryoswhenexposedtoAgNO3,8.9nm
AgNP,or99.4nmAgNP.The threepanelsshowthesameembryoresponsedataplotted
asafunctionofconcentrationfor(A)massconcentration,(B)particleconcentration,
and(C)surfaceareaconcentration………….......................................................................................... 23
4. Percentofembryossurvivingat120hpfwithPEforCh(+) andCh(‐)embryoswhen
exposedtoAgNO3,8.9nmAgNP,or99.4nmAgNP.Thethreepanelsshowthesame
embryoresponsedataplottedas afunctionofconcentrationfor(A)massconcentration,
(B)particleconcentration,and(C)surfaceareaconcentration………………..………………..24
5. Differenceinheartratefromcontrolembryosat48hpf forCh(+)andCh(‐)embryos
followingexposureto AgNO3,8.9nmAgNP,or99.4nmAgNP.The threepanelsshowthe
sameembryoresponsedataplottedasafunctionofconcentrationfor(A)mass
concentration,(B)particleconcentration,and(C)surfaceareaconcentration...…………25
LISTOFFIGURES(Continued)
FigurePage
6. Conceptualdiagramsummarizingtheimportanceofsurfaceareaasadosemetricfor
nanomaterials.Relationshipsobservedbetweenembryotoxicityand(A)massbased
concentration,(B)particlenumberbasedconcentration,and(C) surfaceareabased
concentration………………………………………………………………………………………………………….34
LISTOFTABLES
TablesPage
1. MeasuredandcalculatedAgNPscharacteristics.Thistabledisplaysthe(a)
measurementsprovidedbythemanufactureand(b)theparticlecharacteristics
calculatedasoutlinedinthemethodsandmaterialssectionforeachmaterialusedin
thisstudy………………………………………………………………….…………………………………………….12
SizeandSurfaceAreaDependentToxicityofSilverNanoparticlesinZebrafishEmbryos
(Daniorerio)
2
Chapter1:Introduction
1.1NanosilverBackground
Silver,initsvariousinorganicforms,hasbeeninuselongbeforetheadventofengineered
nanomaterials(ENMs).Silvernanomaterials(nanosilver)havebecomeoneoftheleading
nanomaterialsusedtocreatenanoenabledconsumerproducts.Silvernanoparticles
(AgNPs)arecharacteristicallysphericalinshapeandrepresent oneformofnanosilver.
WhileAgNPshaveseveralpotentialapplicationsduetotheiruniqueopticalproperties,the
primaryapplicationforAgNPsexploitstheirpotentanti‐microbialproperties(Chen and
Schluesener,2008;Hwangetal.,2008;Liuetal.,2010;Sotiriouetal.,2011;Raietal.,2012).
Nanosilverhasbeenincorporatedintoconsumergoodsincludingtextiles,contraceptives,
cosmetics,children’stoys,medicalequipment,airfilters,waterfilters,andresidential
washingmachines(Stensbergetal.,2011).Becauseofthegrowingutilizationofnanosilver
incommonconsumerproducts,andthegrowinginterestinnanosilverapplications,
concernsarebeingraisedoverthe unforeseenandpotentiallyadverseeffectsthatthese
materialscouldposetohumansandenvironment(Bennetal.,2010).Bennetal.,(2010)
foundthatnanosilverhadbeenincorporatedinto severalconsumerproductsincludingan
athleticshirt,amedicalmask,medicalcloth,toothpaste,shampoo,detergent,atowel,atoy
teddybearandtwohumidifiersatconcentrationsrangingfrom1.4μg/gto270mg/g.Benn
etal.,(2010)alsofoundthattheseproductsleachedupto45μgofsilverpergramof
productwhenwashedwithtapwater.Althoughtheanti‐microbialpropertiesofnanosilver
aretypicallyattributedtothereleaseofAg+ ionsasaresult ofparticledissolution,research
suggeststhatnanomaterialsmayelicittoxicitythatisnotfullyattributedtothereleaseof
Ag+ions.
1.2LiteratureReview:
1.2.1InvitroAgNPToxicity
Thetoxicityofnanosilvertolivingsystemshasbeeninvestigatedextensivelyusing
bothinvitroandinvivosystems.Numerousresultsfrominvitro studiesreportthatsilver
nanomaterialsinducedcytotoxicityandreactiveoxygenspecies(ROS)generation(Carlson
etal.,2008;AshaRanietal.,2009;Parketal.,2011a;b;Piaoetal.,2011).Carlsonetal.,
(2008)showedasignificantdose‐dependentandsize‐dependentdecreaseinmitochondrial
function(MTTassay),mitochondrialmembraneintegrity(LDHassay),ROSgeneration
(DCFH‐DAassay),releaseofinflammatorycytokines(TNF‐α,MIP‐2,andIL‐1β)and
3
glutathionedepletioninratalveolarmacrophages.AshaRanietal.,(2009) reported
increasedROSgenerationandexcessoxidativestress,resultingfromtheintercellular
productionofhydrogenperoxideandsuperoxideinAgNPtreatedcells.AshaRanietal.,
(2009)alsoshoweddecreasedmetabolicactivityassociatedwithareductioninATP
productionintreatedcells,suggestingmitochondrialdysfunction.Theinvitrofindings
demonstratedthatexposuretoAgNPscanresultingenotoxicityfromincreasedoxidative
stress(AshaRanietal.,2009;Parketal.,2011b;Piaoetal.,2011).StarchstabilizedAgNPs
inducedDNAdamage,increasedchromosomalaberrations,andcausedG2/Mcellcycle
arrestincancerousandnon‐canceroushumancelllines(AshaRanietal.,2009).Several
studiesusedelectronmicroscopytoshowtranslocationofAgNPsintocellular
compartmentsincludingthecytosol,nucleus,mitochondria,andendosomes(AshaRaniet
al.,2009;Carlsonetal.,2008).Althoughtheresultsoftheseinvestigationsalludetothe
natureofthemechanismsinvolvedinAgNPmediatedtoxicity,theexactmechanisms
remainelusiveandimportantquestionsregardingthewayinwhichspecificnanomaterial
propertiesplayaroleinmodulatingtoxicityremainunanswered.
1.2.2SizeDependentInvitro Toxicity
ManyinvitrostudiesdescribingAgNPtoxicityhaveprovidedevidenceforthe
relationshipbetweenparticlesizeandthedegreeoftoxicity(Carlsonetal.,2008;Parketal.,
2011a;b).Particlesizehasbeen implicatedasanimportantphysicalcharacteristicof
nanomaterialsthatisoftenpredictiveoftoxicity(Oberdörsteretal.,2005;Nel,2006;Nelet
al.,2012).Manynanomaterialpropertiesvaryas afunctionofparticlesizeincluding
particlemass,volume,surfacearea,particlenumber,andthepercentageofatomsatthe
particlesurface.Toxicitycould potentiallybeafunctionofparticlesizedueto its
relationshiptodissolutionrate,amountofreactivesurfacearea,orbioavailability.Primary
physicalcharacteristics,suchasshape,surfacearea,surfacechemistry,chemical
composition,surfacecharge,andcrystallinitymayinfluenceparticletoxicity(Neletal.,
2012).Manysecondarycharacteristicsincludingparticlesolubility,polydispersity,
agglomeratesize,aggregatesize,rateofdissolution,hydrophobicity,etc.mayalso
contributetoparticletoxicity. Understandinghow differencesinnanomaterial
characteristicsleadtochangesintoxicityisanimportantaspectofnanotoxicologyandone
thatrequirestheuseofalternativedosemetrics.
4
1.2.3AgNPOxidativeDissolution
Dissolutiondescribesthedecompositionofmetaloxideandzerovalentmetal
nanomaterialspecies(ex:Ag0)intotheirconstituentmetalions.Studiesdesignedto
investigatetheenvironmentalfate,transformationandstabilityof silvernanomaterials
showthatsilvernanomaterialsundergooxidativedissolutioninsystemswhereoxygenand
protonsarepresent,resultingin the releaseofsilverAg+ ionsinsolutionovertime(Zhang
etal.,2011;Kittleretal.,2010; LiuandHurt,2010;Maetal.,2012;Loketal.,2007).Liuand
Hurt,(2010)showedthatdissolvedoxygenandprotonsarenecessaryfordissolutionto
occur.LiuandHurt,(2010)alsoshowedthat therateofoxidativedissolutionofAgNPs
increaseswithincreasingtemperature,time,andconcentration,butdecreaseswith
increasingpH,salinity,andfollowingadditionofnaturalorganicmatter(humicorfulvic
acids).SeveralstudiesclearlydemonstratethattheoxidativedissolutionofAgNPsis
stronglydependentonparticlesize,wheresmallerparticlesizesreleasemoresilverions
duetotheirgreatersurfaceareatomassratio(Zhangetal.,2011;Maetal.,2012).
1.2.4SilverIonMediatedToxicity
AcentralquestionregardingAgNPtoxicityiswhethertheobservedtoxicity is
mediatedsolelybyionicsilverreleasedbytheoxidativedissolutionofnanosilverorifthere
aremechanismsoftoxicityinherentto theNPitself.ThetoxicityofmanyENMsare
associatedwiththereleaseofmetalions,andthathigherratesofdissolution areoften
associatedwithincreasedtoxicity(Loketal.,2007;Kittleretal.,2010).Loketal.,(2007)
specificallyshowedthatsurfaceoxidationofAgNPsisnecessarytoproduceantibacterial
activityandthatparticlesizecandeterminetherateofparticledissolution.Furthermore,
theoxidativedissolutionofAgNPproduceshydrogenperoxideanddepletesbothdissolved
oxygenandprotons(LiuandHurt,2010),whichcouldcontributetotheintercellularROS
generation.Although Ag+ionscontributesignificantlytothetoxicityofAgNPs,itisdifficult
toknowforcertainwhetherthetoxicityassociatedwithnanomaterialsisduesolelyto the
releaseofmetalionsorifanalternativemechanismofAgNPtoxicityexists.Thepotential
thatAgNPsmaybeassociatedwithsomealternativemechanism of toxicitybringsinto
questionconcernsregardinghumanhealthandwhetherAgNPscanberegulatedunderthe
sameguidelinesas otherformsofsilver.Determiningifalternativemechanismsexistand
whichphysicochemicalpropertiesresultinincreasedordecreasedtoxicityarenecessary
stepsinassessingthepotentialrisksposedbythesenovelmaterials.
5
1.2.5Invivo AgNPToxicity
Silverisacutelytoxictomanyformsofaquaticandterrestriallifeandthetoxicityof
AgNPsoftenexceedsthatofothermetalandmetaloxidenanoparticles,includingAu,Pt,
SiO2,Al2O3,CuO,NiO,ZnO,Co3O4,NiandTiO2(Georgeetal.,2011;Linetal.,2011; Griffitt et
al.,2008b).FindingsfromadiversearrayofinvivoanimalmodelsincludingCaenorhabditis
elelgans(Rohetal.,2009;Meyeretal.,2010;Yangetal.,2012),Drosophilamelanogaster
(Ahamedetal.,2010;Websteretal.,2011),Daphniamagna(Stensbergetal.,2011;Römer
etal.,2011;Leeetal.,2012;Georgantzopoulouetal.,2012),Chlamydomonasrinehardii
(Navarroetal.,2008),andseveralteleostsmodels(ShawandHandy,2011)showthat both
Ag+ionsandAgNPsareacutelytoxicandelicitavarietyofsub‐lethalresponses,including
physiological,neurological,behavioralandbiochemicalendpoints.
1.2.6EmbryonicZebrafish(Daniorerio)
OneanimalmodelbeingusedextensivelytoaddressthetoxicityofAgNPsandother
ENMsisthezebrafish(Daniorerio).Zebrafisharerapidlygainingareputationasan
importantanimalmodelforconductingnanomaterialshazardassessments.Zebrafish
embryosareidealforthisapplicationbecauseoftheirsmallsize,highreproductive
capacity,shortdevelopmentalperiod,andeaseofculture.Becauseofthesequalities,
embryoniczebrafishareparticularlyvaluableforconductingmediumtohighthroughput
studiesandsystematictoxicityevaluationsacrossmultiplematerialswhichcanthenbe
usedtogeneratestructuralactivityrelationships(Harperetal.,2008b;a,2011;Georgeet
al.,2011; Linetal.,2011;Bar‐Ilanetal.,2009).
1.2.7AgNPandAg+MediatedToxicityinZebrafish
Embryoniczebrafishhavebeenusedinmanystudiestoinvestigatethetoxicityof
AgNPs(Leeetal.,2007;Asharanietal.,2008;YeoandKang,2008;Min‐KyeongYeoandJae‐
WonYoon,2009;Bar‐Ilanetal.,2009;Powersetal.,2010;Georgeetal.,2012;Bowmanet
al.,2012).Ofthesepreviousstudies,manyreportsimilarobservationsregardingthetype
andmagnitudeoftheresponse.Specifically,Asharanietal.,(2008)foundthatstarchand
BSAstabilizedAgNPscauseddosedependentembryomortality,severe dismorphology
(bentspineandcloudyappearanceofchorionicfluid),cardiovasculardefects,including
pericardialedema,anddepressedheartrateaswellasdecreasedhatchingrate.Bar‐Ilanet
6
al.,(2009)confirmedthedosedependenteffectsreportedbyAsharanietal.,(2008)and
alsodemonstratedsizedependenteffectsinwhichsmallerAgNPweremoretoxicthan
largerparticles.Georgeetal.,(2011and2012)reportedsimilarmorpholigical
malformations(bentaxis,opaqueyolk,andstunted growth),pericardialedema,heartrate
depression,andreducedhatchratefollowingexposuretopolyvinylpyrrolidone(PVP)
stabilizedAgNPs.
1.2.8AgNPMediatedToxicityinAdultandLarvalZebrafish
InadultzebrafishtreatedwithAgNPs,Choietal.,(2010)observedheightened
oxidativestressresponse,inducedapoptosis,increasedglutathionelevels,increasedlipid
peroxidation,andtranslocationofAgNPintothecytosolandnuclearmembrane.Increased
levelsofγ‐H2AX,amarkerofdoublestrandedDNAbreaks,andp53,animportanttumor
suppressorprotein,wereobservedinAgNPtreatedzebrafishaswellasincreased
expressionofp53targetgenes(Bax,Noxa,andp21)intheliverofadultfishindicatingDNA
damage(Choietal.,2010).Unfortunately,inthestudybyChoietal.(2010),nofishwere
treatedwith AgNO3andthereforegeneexpressionchangeswerenotdirectlycompared
betweenAgNPsandadirectsourceofsolubleAg+ions.Anotherstudyfoundthat,adult
zebrafishexposedtolowconcentrationsofAgNPshadsignificantlyhighertissueburden
levelsingilltissuethaninothertreatmentsincludingAgNO3,thedissolvedsilverion
fractionfromAgNPsuspensions,coppernanoparticlesordissolvedcopperions.AgNPsdid
not,however,producesignificantgillthickeningaswasobservedwithdissolvedsilverand
copperions(Griffittetal.,2008a).Powersetal.,(2010)discoveredthatAgNO3 affects
neurobehavioralendpointsinlarvalzebrafish,suchasswimmingperformanceanddistance
atsub‐lethalconcentrationsthatdidnotelicitchangesinmorphology.Differencesin
neurobehavioraleffectsforexposuretoAg+ions,10nmcitratestabilizedAgNPs,10nmPVP
stabilizedAgNPs,and50nmPVPstabilizedAgNPswerelaterdescribedbyPowersetal.
(2011)usingavisualacuitytest.Thestudydemonstratedthatfishmovementwasaltered in
treatedfishfollowingalternatinglightanddarkperiodsatconcentrationsthatdidnotelicit
malformations(Powersetal.,2011).
1.2.9PrimaryMechanismofAgNPToxicity
OfthestudiesthatsuggestreleaseofAg+ionsasthesolesourceoftoxicityelicited
fromAgNPexposures,mosthavefocusedonthecorrelationbetweentheconcentrationof
7
Ag+insolutionandtoxicityendpoints.Choietal.(2010)show thatremovaloftheions
throughpurificationoftheparticlesreducesoreveneliminatesthetoxicityoftheparticles.
OtherstudieshaveshownthatusingligandsthatbindfreeionicsilveralsoreducesAgNP
toxicity(Georgeetal.,2012).Bar‐Ilanetal.(2009)showedthatalthoughbothsilverand
goldnanoparticlesaretakenupbyzebrafishembryos,onlysilverinducedtoxicityleading
theauthorstosuggestamechanismofinvivoparticledestabilizationresultinginthe
releaseofsilverions.Furthermore,Bar‐Ilanandcolleagues(2009)notedthat
malformationsinducedbyexposureto AgNO3intheirstudywereverysimilartothatthose
inducedbyAgNPs.Itisimportanttohighlightthatotherstudiestestingtheeffectsofboth
AgNPsandAgNO3(Min‐Kyeong YeoandJae‐WonYoon, 2009)alsotendtoshowasimilar
spectrumandfrequencyofmalformationsindicatingasimilarmodeoftoxicity.Further
evidencefromChoietal.,(2010)showedthatremovalofsilverionsfromexposuresolution
usingionexchangeresinresultedinadoseresponseexpressionofmetallothionein 2 (mt2)
inductioninzebrafishlivertissuesuggestingthereleaseoffreesilverinvivo.
1.2.10AlternativeMechanismsofMediatedEmbryoToxicity
ThemajorityofstudiesshowthatAgNPtoxicityisoverwhelminglydrivenby
generationofAg+ionsthroughparticlesdissolution(Loketal.,2007).Thecontroversy
surroundingAgNPtoxicity resultsfromabodyofliteraturethatsuggeststhe toxicity
inducedbyexposuretoAgNPsisnotfullyexplainedbythedissolutionofAgNPs,andthat
someparticle‐mediatedmechanismis likelytoexist(Griffittetal.,2008a,Griffitt,etal.,
2008b;Asharanietal.,2008;Georgeetal.,2012).Asanexample,aparticle‐mediated
mechanismreferredtoastheTrojan‐horsemechanismcouldallownanoparticlestogain
accesstocellinteriors,etherforciblybydisruptingcellsurfaces,orthroughpreferential
uptakebyreceptorsorendocytosiswheretheycouldsubsequentlyreleasetoxicions or
reactwithcellinteriorswheremetalionsorbulkmetalswouldotherwisebeexcluded(Park
etal.,2010).Somestudieshave directlycomparedAgNPtoxicitywithtoxicityfromAgNO3
andhavecometosimilarconclusions(Asharanietal.,2008).Griffitt,etal.,(2008)found
thattheamountofsolublemetalleachedfromnano‐silverandnano‐copperinmoderately
hardwaterovera48hexposureperiodwasbelowthenoeffectlevelforthe corresponding
solubleformofthemetal,yetAgNPswereassociatedwithsignificanttoxicityto~24hpf‐
48hpfjuvenilezebrafish.Perhapsthemostconvincingevidencesuggestiveofaseparate
mechanismindependentofthe toxicityinducedbyfreeAg+ions,wasarecentpublicationby
8
Georgeetal.(2012).Thisworkdemonstratedthatnano‐silverplatesinducedtoxicityand
increasedROSgenerationinembryoniczebrafishthatwereassociatedwith defectsonthe
platesurface(Georgeetal.,2012).Theauthorssuggestedthatamorethoroughand
systematic investigationofAgNPstructureactivityrelationshipsiswarrantedand
necessarytodeterminethepropertiesofAgNPsthatcontributetotheparticle‐mediated
toxicityofAgNPs(Georgeetal.,2012).
1.2.11ZebrafishEmbryoChorionsPlayaRoleinENMToxicity
Manystudiesutilizingembryoniczebrafishremovethechorionusingaprotease
enzymeormanualremoval(Truongetal.,2011).Zebrafishchorionshavenumerouspores
ranginginsizebetween0.5and0.7μm(Rawsonetal.,2000;Leeetal.,2007).Becausethe
chorionisthoughttoserveasanaturalprotectivebarriertoparticulatesand some
chemicals,removalofthechorionmayallowforgreaterexposureandwilleliminatethe
chorionasapossibleconfoundingfactor.Somestudieshaveshownthatthechorions may
actuallyincreasenanoparticletoxicity.Linetal.,(2011)investigatedthephysiological
abilityofthechoriontoconcentratemetalsintheperivitellinefluidusingmetal‐sensitive
dyesandICP‐MS,andshowedthatmetalionsfromCuO,ZnO,NiO,andCo3O4metaloxide
nanoparticlesconcentratewithinthechorion.KingHeidenetal.evaluatedtheeffectofthe
chorionduringexposureswithpolyamidoamine(PAMAM)dendrimersandobserved
greatertoxicityinembryospossessinganintactchorionincomparisontothosethathad
theirchorionremovedpriortoexposure(KingHeidenetal.,2007).
1.2.13SizeDependentEmbryoToxicityofAgNPs
Thissize‐dependenttoxicityofAgNPswithrespecttozebrafishembryosholdstrue
formanystudiesthatutilizemultiplesizesofAgNPs(Bar‐Ilanetal.,2009;Georgeetal.,
2012).Bar‐Ilanetal.,(2009)comparedthemedianlethalconcentration (LC50)profilesfrom
3nm,10nm,50nm,and100nmcolloidalsilvernanoparticles,andobservedclearsize‐
dependentanddose‐dependentresponsesfromzebrafishembryos.Bar‐Ilanetal.,(2009)
alsoshowedevidenceforatime‐dependentresponseusingasinglehighconcentration(250
μM),causingsignificantlydifferentsizedependentmortalityat24hpfwhichbecameless
significantlaterduringtheexposure.Thiscouldindicatethattherateofdissolutionof
smallerparticlescausedhighermortalitysoonerthanlargerparticles.Basedonthe
availableliteratureandonourinitialobservationsofAgNPtoxicity,AgNPembryotoxicityis
9
dependentupontheprimaryparticlediameter(Bar‐Ilanetal.,2009).AlthoughAgNP
toxicityseemstobesizedependent,thisdoesnotspecificallyexplainwhytheyexhibitsize
dependenttoxicity.InthisstudyweinvestigatewhyAgNPtoxicityissizedependentby
lookingatthreephysicalparametersthatallscalewithparticle size;mass,particlenumber,
andsurfaceareatodeterminethephysicalparameterthatexplainsthephenomenon.
1.3StudyDesign
1.3.1SimilaritiesBetween AgNO3 andAgNPToxicity
Inthepresentstudy,exposuretoAgNO3wasinvestigatedasadirectsourceof
solublesilverions.Wecomparedthesimilaritiesanddifferencesintoxicityresultingfrom
exposuretoAgNO3withanindirectsourceofionsfromAgNPstodetermineifthesourceof
silverionsaffectedthemagnitudeandpresentationofthetoxicityendpointsandwhether
differencesmightsuggestanalternativemechanismfortheparticles.
1.3.2EstimatingtheEffectof theChorion
ThisstudyalsoinvestigatedtheinfluenceofthechoriononAgNPtoxicity.Limited
researchhasbeenperformedtodirectlycomparenanomaterialtoxicityinembryoswith or
withoutachorion,yetitispossiblethatthechorionmaybeaconfoundingfactorconcerning
ENMtoxicity.Toaccomplishthis,theeffectofthepresence(Ch(+))orabsence(Ch(‐))of
thechorionwasexaminedfollowingexposuretoAgNO3,10nmAgNPs,or100nmAgNPs.
Weassessedmortalityaswellastwocardiovascularendpointsfoundtobesensitive during
rangefindingexperiments:decreasedembryoheartrateat48hpfasameasurementof a
physiologicalresponse,andthefrequencyofpericardialedema(PE)inembryossurviving
at120hpf.Theembryotoxicitybetween thethreematerialswith Ch(+)orCh(‐)embryos
wereanalyzedusingthreealternativedosemetrics:massconcentration,particle
concentration,surfaceareaconcentration.
1.3.3DoseModelingandAlternativeDoseMetricsAnalysis
Theoverarchingaimofthisstudywastoaddressthereasonthatsize‐dependenceis
observedinotherAgNPtoxicitystudies.Thisaimwasaccomplishedbyconstructinga
structureactivityrelationship(SAR)usingsize‐dependent,andconcentration‐dependent
embryoniclethalityfollowingexposuretothirteendiscreteAgNPsizedistributions,with
meanprimaryparticlediametersrangingfrom8.9nmto112.6nm.Theresultsfromthese
experimentswereanalyzedtodeterminehowembryomortalityisaffectedinasize‐
10
dependentmannerandwhichparticlepropertiesthatscalewithprimaryparticlediameter
maybethemostrelevantinpredictingtoxicity.Embryomortalitywasmodeledforeach
particlesizetoestimateLC50valuesforthreedifferentmeasuresofconcentration:themass
oftheparticlesinsolution(massconcentration),thetotalnumberofparticlesinsolution
(particleconcentration),andthetotal surfaceareaoftheparticlesinsolution(surfacearea
concentration).Ourhypothesiswasthatparticlesurfaceareathatscalesgeometricallywith
particlediameteristheultimatedriverofAgNPtoxicity.Weshowsupportforthis
hypothesisbyanalyzingstudyresultsanddirectlycomparingalternativedosemetrics.
11
Chapter2:MaterialsandMethods
2.1TestMaterials
Citratestabilized1.0mg/mLBioPureTM silvernanoparticlespurchasedfrom
NanoComposix,Inc.(SanDiego,CA)in10mLvolumeswereusedthroughoutthe study
withmean(±1SD)primaryparticlediametersof8.9±1.3nmand99.4±7nm.Elevenother
AgNPsuspensionsBioPureTMSilvernanoparticleswerealsopurchasedfrom
NanoComposix,Inc.in1mLvolumes.Thesesamplesconsistedofonecitratestabilized
AgNPsuspension,withameanprimaryparticlediameter10.2±1.7nm,andtenphosphate
stabilizedAgNPsuspensionswithmeanprimaryparticlediametersof20.3±1.9nm,
34.4±3.4nm,41.9±3.6nm,53.1±4.1nm,61.2±5.3nm,67.3±5.4nm,79.8±5.1nm,90.8±7.3
nm,102.3±9.4nm,and112.6±7.8nm.AgNO3ACS,99.9%waspurchasedfromAlfaAesar
(WardHill,MA).
2.2NanomaterialCharacterization
AgNPcharacteristicsforeachmaterialarelistedinTable1.Themanufacturer
providedmeanprimaryparticlediameterandprimaryparticlesizedistributionsmeasured
usingaJEOL1010TransmissionElectronMicroscope(TEM)Table1.Allparticle
suspensionswereassociatedwithnarrowsizedistributionsandwerereportedasthe
coefficientofvariation(CV).Massconcentration(g/mL)providedbythemanufacturerwas
measuredusingaThermoFisherXSeries2ICP‐MS.Meanprimaryparticlesurfacearea
(nm2/particle)andmeanprimaryparticlevolume(nm^3/particle(s))werecalculatedusing
thegeometricequationsforsurfacearea,4*π*1/2d2wheredisthemeanprimary particle
diametermeasuredbyTEM,and[4/3]*π*r3isthevolume ofauniformspherewithradius
(r)whichishalfthemeanprimaryparticlediametermeasuredbyTEM.Theoreticalparticle
concentration(particle(s)/mL)wascalculatedbymultiplyingthemassconcentration
(g/mL)bythedensityofsilver(0.001g/cm3)tofindthevolumeofsilverpermL(cm3/mL).
ThevolumeofsilverpermL(cm3/mL)wasthendividedbymeanprimaryparticlevolume
(nm^3/particle)todetermine thetheoreticalparticleconcentration(particles/mL).
Theoreticalsurfaceareaconcentration(mm^2/mL)wascalculatedbyfirstdividingthe
meanprimary particlesurfacearea(nm2/particle(s))by1*1012withappropriateunits.
Meanprimary particlesurfacearea(mm2/particle(s))wasthenmultipliedbythe
theoreticalparticleconcentration(particle(s)/mL)toyieldtheoreticalsurfacearea
12
concentration(mm2/mL).Thesecalculationsareinaccordancewithpreviousstudies(Bar‐
Ilanetal.,2009;Georgeetal.,2012;Bowmanetal.,2012)thatutilizetheTEMderived
diametertodeterminetheoreticalparticleconcentrationandtheoreticalsurfacearea
concentration.
Table1.MeasuredandcalculatedAgNPscharacteristics.Thistabledisplaysthe(a)
measurementsprovidedbythemanufacturerand(b)theparticlecharacteristicscalculated
asoutlinedin themethodsandmaterialssectionforeachmaterialusedinthisstudy.The
valuesthatrefertoconcentrationarereferringtothestocksolutionsfromwhichindividual
treatmentlevelsweremadeinequalproportions.
2.3ZebrafishHusbandryandEmbryoCollection
Fishwater(FW)withaconductivityof450‐510μswasmadebydissolving0.3g/L
InstantOceansalts(AquaticEcosystems,Apopka,FL)inreverseosmosiswaterand
adjustingpHto7.0‐7.4withsodiumbicarbonate(MacronchemicalsPhillipsburg,NJ).Adult
13
zebrafishDaniorerio(TropicalD5strain)wererearedinstandardlaboratoryconditionsof
28°CwithapHof7±0.2ona14‐hlight/10‐hdarkphotoperiodattheSinnhuberAquatic
ResearchLaboratoryatOregonStateUniversity (Truongetal.,2011).Newlyfertilizedeggs
werecollected,rinsed,andplacedinfreshFWina150‐mmplasticpetridish.Viable
embryoswerehousedinanincubatorat28°CinFWona16hlight/8hdarkphotoperiodin
thelaboratoryuntil6hourpostfertilization(hpf).
2.4EmbryoDechorionation
ThedechorionationprocedurewasadaptedfromTruongetal.(2011).Pronase
(proteinasesisolatedfromStreptomycesgriseus)waspurchasedfromSigma‐Aldrich(St
Louis,MO,USA).Embryosbetween6‐7hpfweredechorionatedusing64.3mg/mLprotease
enzyme.Embryoswere incubatedfor4‐6minuteswithgentlyswirling,afterwhichthe
enzymewasremovedbyrinsingseveraltimes(approximately5minutes)withfreshFW.
Healthyviableembryosbetween7‐8hpfwerethentransferredto a newpetridishwith
freshFWjustpriortoexposure.
2.5ExposureMethod
AgNPstocksolutionconcentrationsarelistedasmassconcentrationinTable1.For
eachAgNPsuspension,stocksolutionsweredilutedinFWbya4‐folddilutionfollowedby
six5‐foldserialdilutionstogeneratetheseventestconcentrations(0.061‐250μg/mL)for
thedosemodelingstudies.A250mg/LAgNO3stocksolutionwaspreparedinFWonthe
dayofexposureanddilutedusingsix5‐foldserialdilutions.AgNPsandAgNO3 suspensions
wereloadedinto96‐wellplates(150μlperwell).Twelveindividualembryoswereexposed
ateachexposureconcentration(oneperwell)foratotalof96embryos,includingthe
controls,foreachofthethirteendiscreteparticlesizes.
Toinvestigate theeffectsofthechoriononembryotoxicity,exposuresolutionswere
preparedbydilutingthe8.9nmAgNPsand99.4nmAgNPsstocksolutions(1000mg/L)2‐
foldinFWfor finalconcentrationsbetween5‐40μg/mL.TheAgNO3stocksolution(250
mg/L)wasdilutedinFW4‐fold(0.0098‐40μg/mL).Exposureswereperformedforeach
material(AgNO3,8.9nmAgNPs,and99.4nmAgNPs)forbothCh(+)andCh(‐)embryos.
Fourexperimentalreplicateswereperformedforeachexposuretreatmentforatotalof48
individualembryospertreatment(n=48).
Theorderoftherowswasrandomizedforeachexperimentalreplicatetominimize
14
potentialplateeffects.Embryoexposureswereinitiatedfollowingdechorionation,oronce
themeanageoftheembryosreached~7hpf(between 6hpfand8hpf)forembryosthat
werenotdechorionated.EmbryosexposedtoFWalonewereincludedoneveryplateto
provideacontrolforembryoviability.Embryoswereindividuallyexposed tostatic,
nonrenewaltreatments,andincubatedat28°Cwitha16h‐light/8hdarkphotoperiodfor
thedurationoftheexposure.Allexperimentswereconcludedat120hpf.
2.6ZebrafishEmbryoEvaluation
Theembryotoxicityevaluationprocedureusedinthisstudyassessestwentythree
distinctendpointscorrespondingtoembryomortality,dysmorphology,andbehavior,which
wereevaluatedatspecifiedtimepointsduringdevelopmentbasedonthosepreviously
describedinTruongetal.(2011).Assessmentswereconductedforeveryembryo.For
exposuresdesignedtoinvestigatetheeffectofthechoriononembryotoxicity,heartrateat
48hpf,pericardialedema(PE)at120hpf,andmortalityat120hpfwereassesseddueto
theirprevalenceduringrangefindingexperiments.Embryoheartratewasrecordedat48
hpfforeachembryobycountingthenumberofbeatsthatoccurredovera10‐second
interval.Thepresence ofPEwasevaluatedandrecordedonlyforembryosthatwerestill
aliveat120hpf.Embryoswereanesthetizedduringthe120hpfevaluationswith0.5mg/mL
of3‐aminobenzoateethylestermethanesulfonatesalt(tricaine) purchasedfromSigma‐
Aldrich(StLouis,MO,USA).Theembryoswerehumanelyeuthanizedwith2.5mg/mL
tricaineatthecompletionofthe120hpfobservations.
2.7DataAnalysisandStatisticalEvaluation
Statisticalanalysesandconcentrationresponsemodelingwereconductedusing
SigmaPlotTMversion12(SanJose,CA).Concentrationresponsedataweremodeledusinga
three‐parameterLogisticfunction,usingglobalcurvefittingandasharedmaximumvalueof
93(percentsurvival)tocalculateLC50values(Figure1).Thedatawerealsomodeledusing
afour‐parameterLogisticfunction,usingglobalcurvefittingandsharedmaximumand
minimumvalues.LC50valuesestimatedusingthefour‐parameterLogisticfunctionwere
onlyconsideredinregardstoSpearmanrankcorrelationdescribedbelow.The
concentrationresponsedatawerealsomodeledusingtheToxicityRelationshipAnalysis
Program(TRAP),availablefromtheU.SEPAMid‐ContinentEcologyDivision (TRAP|Mid‐
ContinentEcologyDivision|USEPA)tovalidatethemodelingresultsfromtheglobalcurve
15
fittingmodels.Thismodelingalsousedathree‐parameterLogisticfunction butdidnotuse
sharedvaluestocalculate LC50valuesandEC50values.Again,LC50valuesestimatedusing
theEPATRAPAnalysiswereonlyconsideredinregardstoSpearmanrankcorrelation
describedbelow.
TherelationshipsbetweenmeanprimaryparticlediameterandtheestimatedLC50
valuesweredescribedusinglinearregressionforeachdosemetric(Figure2)andthe
strengthoftherelationshipswereinvestigatedusingSpearmanrankcorrelation.Spearman
rankcorrelationisanonparametrictestthatmeasurestheassociationbetweenthetwo
variablesbasedontheorder,or“rank”inwhichtheestimatedLC50valuesoccurredfrom
mosttoxictoleasttoxicinrelationtomeanprimaryparticlesize.Therankforthe
dependentvariablewasassignedfromlowtohighwithincreasingparticlediameterandthe
rankforthe independentvariablewasassignedfromlowtohigh withincreasingLC50 value.
Therelationshipbetween the ranksofthedependent,andindependentvariablesare
statisticallysignificantwhenassociatedwithap‐value<0.05,andthecorrelationcoefficient
(ρ)isameasureofthestrengthoftheassociation.Correlationcoefficientapproaching1or‐
1indicatesastrongrelationship,avaluecloseto0indicatesnorelationship, andnegative
coefficientsindicateaninverserelationshipbetweenthetwovariables.TheSpearmanrank
correlationwasusedtoanalyzedthe LC50valuesestimatedusingthe3‐parameterglobal
curvefitting(SigmaStat),4‐parameterglobalcurvefitting(SigmaStat),andthe3‐parameter
individuallymodeledcurves(EPATRAPAnalysis).
MeanandstandarderrorforembryomortalityandPEfromwerecalculatedfrom
fourreplicatesconsistingoftwelveembryospertreatmentforatotalof48embryosper
treatmentgroup.Meanandstandarderrorwerecalculatedforheartratebytreatmentfrom
thecompletedataset(notbetweenindividualexperimentalreplicates)bysubtractingthe
meanofthecontrolanimalsfromeachindividualvalue fortreatedembryos,whichwere
thenaveragedtocalculatethedifferencefromcontrol.Oneway AnalysisofVariance
(ANOVA)wasusedtodetermine theoveralleffectsofexposureonthedependentvariables
(mortality,PE,andheartrate). Whereeffectswereshowntobesignificant(p<0.001),
pairwisemultiplecomparisontestswereperformedbetweenthemeansusingHolm‐Sidak
method.Multiplepairwisecomparisonswereusedtoidentifysignificantdifferencesfrom
control,differencebetweenCh (+)andCh(‐)embryosbetweeneachmaterial,andwithin
eachamaterial.
16
Chapter3:Results
3.1DoseModelingandAgNO3 Toxicity
Theeffectofparticlesizeonembryomortalitywasinvestigatedutilizingasuiteof
AgNPsconsistingofthirteendiscreteparticlesizesrangingbetween8.9nmand112.6nm.
TheoreticalparticlecharacteristicsforeachoftheparticleslistedinTable1arebasedon
theaverageprimaryparticlediametermeasuredbyTEMandthemassofAgNPsinthestock
solution.Embryomortalityat 120hpfwasanalyzedusingnonlinearregressionmodeling to
estimateaLC50 valueforeachparticlesize.Thethree‐parameterlogisticfunctionwasfound
tobethemostdescriptiveandintuitivemodelingtoolforestimatingLC50values.LC50 values
wereestimatedusingthreedifferentdosemetrics:massconcentration(μg/L),particle
concentration(particles/mL)andsurfaceareaconcentration (mm2/mL)(Figure1).The
LC50 valuefor AgNO3wasestimatedbymassconcentrationandresultedinaLC50 valuethat
wasapproximatelythirtytimeslowerthantheLC50ofthesmallestAgNP,0.257μg/mLas
comparedto7.72μg/mLrespectively.
3.2Size‐DependentDoseMetricsAnalysis
Whenthedosemodelingdatawasanalyzedtoassesstherelationshipbetween
embryosurvivalandmeanprimaryparticlesize,itwasobservedthattheorderingofthe
LC50valuesinFigure1,frommosttoxictolesttoxicwassignificantlydifferentbetweenthe
threedosemetrics,andthattherangeoverwhichtheLC50 valuesoccurredalsovaried
considerably.TherangeoftheLC50valuesestimatedbyparticleconcentrationcoveredthe
largestrangewherethesmallestLC50value (9.5x109particles/mL)wastwohundredand
seventyfivetimessmallerthan thelargestLC50value(2.6x1012 particles/mL).LC50 values
determinedbymassconcentrationcoveredthesecondlargestrangewhere thesmallest
LC50 value(8.4μg/mL)wassixteentimessmallerthanthelargestLC50value(136.1μg/mL).
TheLC50valuesforsurfaceareaconcentrationweredistributedoverthenarrowestrangeof
concentrations,wherethesmallestLC50value(160.3mm2/mL)wasonlysevenandahalf
timessmallerthanthelargestLC50value(1207.2mm2/mL).
17
Figure1.EstimatedLC50valuesusingthreedosemetrics.Datapointsrepresentthepercent
survivalplottedasafunctionof,(A)massconcentration,(B)particleconcentration,and (C)
surfaceareaconcentration.Thedose‐responsecurves(solidlines)weremodeledforeach
ofthethirteenparticlessizesforeachdosemetric.Horizontaldashedlinesindicate50%
survival.
18
Figure1.(Continued)
TheestimatedLC50valuesformassconcentration(LC50(μg/mL)),particle
concentration(LC50(particles/mL)),andsurfaceareaconcentration(LC50 (mm2/mL))were
plottedagainstthemeanprimaryparticlediameter(Figure2).Althoughlinearregression
analysiswashelpfulinvisuallydescribingtheassociationbetweentheLC50valuesand
particlesize,Spearmanrankcorrelationwasperformedtobetterunderstand the
relationshipbetweentheLC50valuesandmeanprimaryparticlediameter.TheSpearman
rankcorrelationcoefficientsandp‐valuesweresimilarbetweentheindividuallymodeled
curves(EPATRAPAnalysis)andtheglobalcurvefitting(SigmaStat,using3‐parameterand
4‐parameterLogisticregression)forbothmassconcentrationandparticleconcentration
butweredissimilarforsurfaceareaconcentration.
19
Figure2.EstimatedLC50valuesplottedagainstmeanprimaryparticlediameterforthree
dosemetrics.LC50valuesareplottedas afunctionofmeanprimaryparticlediameter for(A)
massconcentration,(B)particle concentration,and (C)surfaceareaconcentration.The
best‐fitlinesfromalinearregressionanalysisaredisplayed(solidlines)aswellastheir
associated95%CIs(dashedlines).
20
Figure2.(Continued)
Theorderofparticlesizes fromlowesttohighestLC50 (μg/mL)value,basedonmass
concentration,usingthe3‐parameterglobalcurvefittingLogisticregression(SigmaStat),
wereasfollows:8.9nm,10.2nm,67.3nm,20.3nm,90.8nm,53.2nm,79.8nm,41.9nm,
61.2nm,34.4nm,102.3nm,112.6nm,and99.4nm.Thecorrelationcoefficientbetween
LC50(μg/mL)andmeanprimaryparticlediameterwas(0.652)withap‐value(0.0143),
indicatingapositiverelationshipbetweenincreasingparticlediameterandincreasingLC50
values.TheLC50(μg/mL)valuesmodeledby4‐parameterglobalcurvefitting(SigmaStat)
andthe3‐parameterindividuallymodeledcurves(EPATRAPAnalysis)yieldedcomparable
correlationcoefficientsof(0.702)and(0.626)withp‐valuesof(0.00651)and(0.0207),
respectively.
Theorderofparticlesizes fromlowesttohighestLC50(particles/mL)value,based
onparticleconcentration,andmodeledusingthe3‐parameterglobalcurvefittingLogistic
regression (SigmaStat),wereasfollows:112.6nm,90.8nm,67.3nm,102.3nm,79.8nm,
99.4nm,61.2nm,53.1nm,41.9nm,34.4nm,20.3nm,8.9nm,and10.2nm.Therewasa
stronginversecorrelation(‐0.923)betweenmeanprimaryparticlediameterandLC50
(particles/mL)withap‐value(<0.001)forthe3‐parameterglobalcurvefitting(SigmaStat)
indicatingastrongrelationshipbetweenincreasingparticlediameteranddecreasingLC50
values.Thevaluesmodeledby4‐parameterglobal curvefitting(SigmaStat), andthe3‐
21
parameterindividuallymodeledcurves(EPATRAPAnalysis),yieldedverycomparable
correlationcoefficientsof(‐0.885)and(‐0.874)withp‐values of(<0.001).
Theorderofparticlesizes fromlowesttohighestLC50 (mm2/mL)value,basedon
surfaceareaconcentration,andusingthe3‐parameterglobalcurvefittingLogistic
regression (SigmaStat),wereasfollows:67.3nm,90.8nm,112.6nm,102.3nm,79.8nm,8.9
nm,61.2nm,20.3nm,53.1nm,99.4nm,10.2nm,41.9nm,and34.4nm.Spearmanrank
correlationdemonstratedaweakinversecorrelation(‐0.549)betweenparticlediameter
andLC50(surfacearea/mL),withasignificantcorrelation(p=0.049)forthe3‐parameter
globalcurvefitting(SigmaStat) indicatingarelationshipbetweenincreasingparticle
diameter,anddecreasingLC50values.Incontrast,thevaluesmodeledbythe4‐parameter
globalcurvefitting(SigmaStat)orthe3‐parameterindividuallymodeledcurves(EPATRAP
Analysis)generatedcoefficientsof‐0.0275(p =0.921)and‐0.390(p=0.179)respectively,
indicatingnosignificantcorrelation.
3.3EffectoftheChoriononEmbryoToxicity
Thepresenceofthechorionanditseffectonthetoxicityofnanomaterialshasbeen
demonstratedinpreviouswork(KingHeidenetal.,2007).KingHeidenetal.(2007)showed
thatembryospossessinganintactchorionweremoresensitivetoPAMAMdendrimersat
specifictimepointsandconcentrationsthanembryoslackingachorion.Weweretherefore
interestedintheabilityoftheCh(+)/Ch(‐)variabletoactasacontributorto thetoxicity
ofnanomaterialseitherthroughenhancementofthemechanismorthroughaseparate
mechanism.WeexposedCh(+)andCh(‐)embryosto10nmAgNPs,100nmAgNPs,and
AgNO3toexplorethedifferencesbetweennanoandsolublesilvertoxicity.Differences
between10nmAgNPsand100nmAgNPstoxicitywerecomparedusingalternativedoes
metrics.Resultswereanalyzedusingaone‐wayANOVAfollowedbytheHolm‐Sidak
multiplecomparisonsposthoctest.Significantdifferences(p<0.05)wereobserved
betweencontrolanimalsandtreatedanimalsandbetweenCh(+)andCh(‐)embryosfor
mortality,incidenceofPE,andheartratedepression.Formortality(Figure3A),increased
toxicitytoCh(+)embryoswasobservedfor10nmAgNPs,butnodifferenceswereobserved
betweenCh(+)andCh(‐)embryosfor100nmAgNPsorAgNO3 treatedembryos.ForPE
(Figure4A),increasedtoxicitytoCh(+)embryoswasobservedforboth10nmAgNPsand
100nmAgNPs,butnodifferenceswereobservedbetween Ch(+)andCh(‐)embryos
treatedwith AgNO3.Forheartrate(Figure5A),toxicitywasgreaterforCh(‐)embryos
22
followingexposureto10nmAgNPs,butnot100nmAgNPs.Concentrationdependent
differenceswereobservedbetweenCh(+)andCh(‐)embryosforheatratedepression
followingexposureto AgNO3(Figure5A).
23
Figure3.Percentembryomortality for Ch(+)andCh(‐)embryoswhenexposedtoAgNO3,
8.9nmAgNP,or99.4nmAgNP.Thethreepanelsshowthesameembryoresponsedata
plottedasafunctionofconcentrationfor(A)massconcentration,(B)particle
concentration,and(C)surfaceareaconcentration.Embryoresponsedata for AgNO3could
onlybeplottedinpanelA,becausesurfaceareaandparticlenumbercalculationsdonot
apply.Themeanoffourexperimentalreplicateswereplotted±SEM.Symbolsrepresent
significantdifferencesbetweentreatments:(a)treatedembryosandcontrolembryosfora
givenmaterialtype(p<0.05),and(b)Ch(+)andCh(‐)embryosforagivenmaterialtype
(p<0.05).
24
Figure4:Percentofembryossurvivingat120hpfwithPEforCh(+)andCh(‐)embryos
whenexposedtoAgNO3,8.9nmAgNP,or99.4nmAgNP.Thethreepanelsshowthesame
embryoresponsedataplottedas afunctionofconcentrationfor(A)massconcentration,(B)
particleconcentration,and(C)surfaceareaconcentration.EmbryoresponsedataforAgNO3
couldonlybeplottedinpanelA,becausesurfacearea andparticlenumbercalculations do
notapply.Themeanoffourexperimentalreplicateswereplotted±SEM.Symbolsrepresent
differencesbetweentreatments: (a)treatedembryosandcontrol embryosforagiven
materialtype(p<0.05),and(b)Ch(+)andCh(‐)embryosforagivenmaterialtype(p<
0.05).
25
Figure5.Differenceinheartratefromcontrolembryosat48hpfforCh(+)andCh(‐)
embryosfollowingexposureto AgNO3,8.9nmAgNP,or99.4nmAgNP.Thethreepanels
showthesameembryoresponsedata plottedas afunctionofconcentrationfor(A)mass
concentration,(B)particleconcentration,and(C)surfaceareaconcentration. Embryo
responsedataforAgNO3couldonlybeplottedinpanelA,becausesurfacearea andparticle
numbercalculationsdonotapply.Themeanofthecontrolanimalsweresubtractedfrom
valuesoftreatedembryostocalculatethedifferencefromcontrol.Individualmeanswere
thencalculatedforeachexperimentalreplicate.Themeanoffourexperimentalreplicates
wereplotted±SEM.Symbolsrepresentdifferencesbetweentreatments: (a)treated
embryosandcontrolembryosforagivenmaterialtype(p<0.05),and(b)Ch(+)andCh(‐)
embryosforagivenmaterialtype(p<0.05).
26
Chapter4:Discussion
4.1OverallFindings
Theoverallfindingsofthisstudysuggestthatthetoxicityobservedinzebrafish
embryosassociatedwithexposuretoAgNO3wasverysimilarto thetoxicityobserved
followingexposuretocitrateandphosphatestabilizedAgNPs.Themostsensitiveendpoints
observed for AgNO3andAgNPsweredecreasedheartrate(48hpf)andincidenceofPE(120
hpf),whichisconsistentwiththeliterature(Asharanietal.,2008;Bar‐Ilanetal.,2009).
Becauseofthestrikingsimilaritybetween AgNO3andAgNPtoxicity,AgNPinducedtoxicity
islikelyaconsequenceofAgNPoxidativedissolutionandthesubsequentreleaseofAg+ ions
eitherinsolution,orinvivo.Embryoswereconsistentlymoresensitiveto AgNO3thanto
AgNPs.TheLC50 valuefor AgNO3(8.4μg/mL) wasfoundtobeapproximatelythirtytimes
belowtheLC50valueofthemosttoxicAgNPsbymassconcentration.Wefoundthat LC50
valuesincreasedasthemeanoftheAgNPsizedistributionincreasedinapproximateorder
from thesmallestmeandiameter(8.9nm)tolargestmeandiameter(112.6nm)basedon
massconcentration.Uponconsiderationofthetwootherdosemetrics,particlenumber
concentrationandsurfaceareaconcentration,surfaceareaconcentrationwasfoundtobe
theexposuremetricthatbestexplainedtherelationshipbetweenembryo survivaland
meanprimaryparticlediameter.Wealsofoundthatthechoriondidincreasethetoxicityof
10nm and100nmAgNPs,butnotAgNO3(p<0.05).
4.2OverallCharacterizationofAgNPandAgNO3 Toxicity
Theembryotoxicityobservedinthis studyareconsistentwiththosereportedby
Asharanietal.andotherinvestigationsthathaveshownthatAgNPsincreasedmortality,
increasedtheoccurrenceofPE,anddecreasedheartrateinaconcentration‐dependent
manner(Asharanietal.,2008;Bar‐Ilanetal.,2009).Otherstudieshaveverifiedthatedema
andcardiovasculareffectsareamong themostsignificanteffectstozebrafishembryosfrom
exposuretoAgNPs(YeoandKang,2008).Inthecurrentstudy,thedifferenceinmagnitude
betweenAgNO3toxicity,andAgNPtoxicitywastheonlydifferenceobservedpertainingto
embryoresponsebetween thematerials.Thisisingeneralaccordancewithprevious
studiesthatshownosignificantdifferencesinthepresentationoftoxicitybetweenAgNO3
andAgNPotherthanpotency(Bar‐Ilanetal.,2009;Min‐KyeongYeoandJae‐WonYoon,
2009).Specifically,weshowedthatthemostsensitiveendpointsforAgNPsandAgNO3
includedecreasedembryoheartrateandPE,whichalongwithembryosurvival,all
27
displayedsimilarconcentrationresponserelationships.However,accordingtoamass
baseddosemetric,embryosdisplayedthegreatestsensitivitytoAgNO3,followedby10nm
AgNPs,andweretheleastsensitiveto100nmAgNPsforeachendpointmeasured.
TherearemanyplausiblereasonswhystudiescomparingAgNPparticle‐mediated
toxicityandthetoxicityassociatedwithexposuretoAg ionsareunabletomakea
distinctionbetweenthetwo.Onereasonmaysimplybethattheparticle‐mediatedtoxicity
issignificantlylowerthanthebackgroundtoxicityinducedbysilverionsandistherefore
difficulttodetect.EvidenceforthisexplanationwaspresentedinGeorgeetal.(2012)where
onlysilvernano‐plates,butnotsilvernano‐spheres,causedtoxicityabovewhatwas
expectedfromtheAg+ionsalone.Severalotherstudiesshowthatwhendissolvedsilver
ionsareremovedfromnanoparticlesuspensionsusingdialysisorcentrifugation
techniques,theresultingdecreaseinthemassofdissolvedsilverinsolutionwaslessthan
themassofsolublesilverions(AgNO3)foundtocauseanequivalentleveloftoxicity.These
findingshavepromptedseveralauthorstosuggestthatsolublesilveralonecouldnot
entirelyaccountforthedegreeoftoxicityobservedandthatsomeothermechanismof
toxicitymustthereforeexisttoaccountforthedifference(Griffittetal.,2008a,Griffitt,etal.,
2008b;Asharanietal.,2008;Georgeetal.,2012).
Wesuggestthattherearelikelytwopossiblereasonswhytheseauthorsfound
discrepanciesbetweentheobservedtoxicityandtheamountofsolublesilverinsolution.
Thefirstpracticalexplanationis thepresenceofsilverionssorbedtosurfaceligandsor
bareparticlesurfaces(Liu andHurt,2010;Liuetal.,2010). Ifsilverionsaresorbedtothe
surfaceofparticlesandnotfreelydissolved,theionsmaynot necessarilyberemovedunder
dialysisorcentrifugationconditions,butcouldremainbioavailable.Thesecondexplanation
isthatitisnotjustifiedtoassumethatthedissolvedfractionofsilverionsinsolutionisan
adequateindicationofthe doseofsilverionsatthesiteofactionandoverlooksthe
possibilitythatAgNPsareaslikelytoundergooxidativedissolutionreactionsinbiological
systemsastheyareinsimpleaqueousenvironments.Inasimpleaqueousenvironmental
system,dissolvedmolecularoxygenorhydrogenperoxideactsas theoxidizingagents,and
thezero‐valentsilveronthesurfaceoftheAgNPisoxidizedtoproduceAg+ ions(Liuand
Hurt,2010).WhenAgNPsundergooxidativedissolution,somesubstrate,usuallyanoxygen
containingmolecule,mustalsobereducedandgainanelectrontobalanceoutthe lossofan
electronfromthesilveratom(LiuandHurt,2010;Liuetal.,2010).Theprocessofoxidative
dissolutionresultsinthereleaseofAg+ ionsandsubsequentgenerationofreactiveoxygen
28
intermediateswhichcanleadtofurtheroxidativedissolution(LiuandHurt,2010).The
sameredoxreactionsthatgenerate Ag+ionsundersimpleaqueousconditions,could
thereforegenerateROSinbiologicalsystemsbyreactingwithoxygencontainingmolecules
andredoxsensitivesubstrateseitheronthesurfaceofcellsorinintercellularspaces.
IntercellularenvironmentshavebeensuggestedtoaccelerateAgNPsurfaceoxidationin
vivo(AshaRanietal.,2009;Bar‐Ilanetal.,2009).IninstanceswhereAgNPscouldenter
intercellularspaces,AgNPsarelikelytoexperienceincreasedratesofdissolutionin
biologicalsystemsascomparedtosimpleenvironmentalsystemsduetothehigh
concentrationsofreactiveintermediates.LiuandHurt(2010)showedthatlowpHalso
contributestoAgNPdissolutionandtherefore,specificintercellularspaceslikethe
lysosomeandmitochondriamayfurtherincreasedissolutionduetotheirlowpHandhigh
redoxpotentialrespectively.Invivodissolutionfollowinguptakeandbiodistributioncould
producerelativelyhighregionalconcentrationsofAg+ionsatthesiteofactionascompared
todissolutioninthemedia.InvivodissolutionofAgNPswould alsolead tointercellular
generationofROSasabyproductofdissolutionwhichwouldfurtherincreaseAgNP
mediatedtoxicity.Determiningwhether,andtowhatextent,AgNPsundergoaccelerated
oxidativedissolutioninvivocouldexplainhowstudiesreporting lowionconcentrationin
solutionstillresultinconsiderableAgNPtoxicity,whichexceedsthetoxicitythatcouldbe
attributedtothesilverionsalone(Griffittetal.,2008a;b;Asharanietal.,2008;Georgeet al.,
2012).ThisexplanationcouldalsospecificallyaddressthefindingsofGeorgeetal.(2012)
whofoundthatsurfacedefectsonAgnano‐platesproducedfargreatertoxicitythanAg
nano‐spheresifthecombinationofshapeandsurfacedefectscouldfurtheracceleratethe
rateofinvivodissolutionresultinginahigherdoseatthesiteofaction.
Despitethepresenceofmetalionsincolloidalandnanomaterialsuspensionsthat
undoubtedlycontributesignificantlytothetoxicityofmanyENMs,understandingthe
surface‐mediatedtoxicity, whichencompassesoxidativedissolution,isfundamentalto
understandingENMtoxicity.Inalllikelihood,AgNPassociatedtoxicityisduetothe
chemicalprocessofoxidativedissolution,whichgeneratesionsandROS.Liuetal.(2010)
providesome supportforthishypothesisbyshowingthattheantimicrobial activityof
nanosilvercanbeincreasedoreliminatedcompletelybydirectlycontrollingtherateof
dissolutionusingAgNPsurfacetreatment.Theinherentinstabilityandreactivityofmany
ENMsneedstobecarefullyevaluatedtoaccountforpotentialeffectsonlivingsystemsin
thecontextof metaliontoxicityanditsrelationshiptoreactivesurfacearea.Thetoxicityof
29
severalENMsislikelyaresultofthisinterconnectedprocess,linkingparticlemediated
surfacereactivityandionmediatedtoxicity.Thisrepresentsasunifiedmechanismsof
actioninwhichbothprocessesareoccurringsimultaneouslyasaresultofoxidative
dissolution,liberatingionsandgeneratingreactiveoxygenspeciesinasurfacearea
dependentmanner.Thishypothesiswillneedtobetesteddirectlywithfuturestudiesthat
addressthisquestioninamaterial‐specificmannerandonethatattemptstodistinguish
betweenthereactivityoftheparticle surfaceandtheinnatetoxicityoftheionsinvivo.
4.3PresenceoftheChorionEffectsAgNPToxicity
InpreliminaryworkinvestigatingthetoxicityofAgNPs,weobservedthatthe
toxicityof10nmand100nmAgNPswasgreatertoCh(+)embryosthanto Ch(‐)embryos
andnanoparticleswereobservedcoveringthesurfaceofthechorion.Wewantedtolearn
howthechorionmightactasabarriertoAgNPs,butstillresultinincreased toxicity.To
exploreifthe chorionenhancedtoxicityisspecifictoENMs,wetestedwhetherCh(+)and
Ch(‐)embryosexposedto AgNO3wouldalsoresultinasignificantdifferenceintoxicity.The
literaturesuggeststhat dissolvedAg+ionswouldmovefreelyinandoutofthechorion
whereasAgNPsmovementcouldberestrictedorhindered.Aswehadpreviouslyobserved,
thetoxicityof10nmand100nmAgNPswasfoundtobegreaterinCh(+)embryosthanin
Ch(‐)embryosatsometestconcentrations.IncontrasttotheAgNPs,andinsupportofour
hypothesis,wedidnotobserveasignificantincreaseintoxicityforCh(+)embryosexposed
toAgNO3versusCh(‐)embryos.Thissuggestsanadditionalnano‐mediatedeffectnot
observedinrelationtothefreelydissolvedmetal.
Insupportofthis,Leeetal.(2007)showedthatAgNPscanbecometrappedinthe
chorionicporespaceandthatporescanbeblockedbyagglomerates.Byblockingpores,
ENMscouldpotentiallydecreasegasexchangeacrossthechorionicsurfacebycloggingthe
chorioniccanalsandeffectivelycreatinghypoxicconditionsintheinnerchorionicspace
causingincreasedtoxicity.Thiscouldhelptoexplaintheincreaseintoxicityobservedin
chorinatedversusdechorionatedembryosfollowingexposuretoAgNPsand might also
accountforthelackofadifferenceintoxicityforAgNO3exposures(Figure3,4,5).Other
studieshaveshownthatthechorioncanactivelyaccumulatemetalionsintheperivitelline
fluidfollowingexposuretometaloxidenanoparticlesathigherconcentrationsthaninthe
soundingmedia(Xiaetal.,2011; Linetal.,2011).Thisisthoughttobedueto thepresence
ofseveralproteinswithin thechorionicfluidthatmaybindandsequestermetals(Xiaet al.,
30
2011; Linetal.,2011).Otherstudieshave shownthatsomeENMsincludingZnO
nanoparticlesmayinhibittheactivityofthezebrafishhatchingenzyme(ZHE1)whichcould
insomecasesleadtodecreasedsurvival(Xiaetal.,2011; Linetal.,2011).
Incombinationwithpreviouslypublishedliterature,thestudiespresentedhere
demonstratethecapacityofthechoriontoenhancetoxicityforspecificENMs.While
removingthe chorionmayincreasetoxicitybyallowingfordirectexposuretosome
materials,forothermaterialschorionremovalmayactuallyunderestimatethetoxic
potentialofsomeENMswhosemechanismofactionmaybeenhancedbyorrelyonthe
presenceofachorion.Moreworkisclearlyneededtodeterminethemechanismsbywhich
chorionsincreasethetoxicityofENMs.
4.4DoseMetricsConsiderationsforAgNPs
Innanotoxicology,alternatemetricsforexposureanddoseareneededtofully
understandtoxicityassociatedwithparticlecharacteristics(Oberdörsteretal.,2005;
Teeguardenetal.,2007).However,usingalternativedoseandexposuremetricschallenges
thetraditionalwaywemeasurethetoxicityofmaterialsandthewayweclassifyhazards.In
contrasttoconventionalsmallmolecules,wheremostexposurevariablesareclosely
proportionaltooneanother,exposurevariablesdonotusuallyscalelinearlyfor
nanomaterials,butmoreoftenscaleexponentiallyduetotheirthree‐dimensionalshape.
Thisstudyillustrateswhyalternativedosemetricsmatterwhencharacterizingthetoxicity
ofENMs.Thisstudyshowsthatthedosemetricschosenfornanotoxicologydataanalysis
canhaveadrasticinfluenceontheinterpretationofthestudyfindings.Weconcludethat
particleconcentrationandsurfaceareaconcentrationsaremorevaluableestimatorsfor
silvernanoparticletoxicitythanconventionalmassbaseddosemetricsandthatthe
observedsize‐dependenttoxicityofsphericalAgNPsisduespecificallytosurfacearea.
Specifically,ouranalysesillustratethatsmallerAgNPsizedistributionshavelower
LC50(μg/mL)valuesandthereforeappeartobemoretoxictoembryos thanlarger particles,
givenequivalentmasses.Incontrast,whenthetheoreticalnumberofparticlesinsolutionis
considered,largerAgNPsizedistributionsareassociatedwithlowerLC50 (particles/mL)
values,andthereforeappeartobefarmoretoxicthansmallerparticlesonaperparticle
basis.Whenthetoxicityisconsideredasafunctionofthecombinedsurfaceareaofthe
particlesinsolution,thedifferencein LC50(mm2/mL)valuesisminimizedandlarger
particlesappeartobelessthan afactoroftentimesmoretoxicthansmallerparticles.The
31
resultsoftheSpearmanrankcorrelationanalysisdemonstratedthatthecorrelation
betweenthemeanprimary particlesizeforbothmassconcentrationandparticle
concentrationappeartobestrongerthanforsurfaceareaconcentration.Wedetermined
thatthiswasbecausethecalculationforestimatingsurfaceareaconcentrationfactorsout
particlenumber.Factoringoutparticlenumbereffectivelyeliminatesthevariablethatis
mostinfluencedbydifferencesinprimaryparticlesizeandthevariablewiththegreatest
rangeofvalues.Incontrast,particlenumberisnotfactoredoutformassconcentrationand
particlenumberconcentration.Thisisthereasonthatmassconcentrationandparticle
numberconcentrationexhibitclearsize‐dependentrelationships.Withdecreasingparticle
diameter,particlenumberandsurfacearea perparticlebothvaryexponentiallybut in
oppositedirectionsandatdisproportionaterates;particlenumberwillincreaserapidlyas
diameterdecreasesandsurfaceareaperparticlewilldecreaseat aslowerrate.When
consideringequivalent masses,asmallerparticlesizedistributionwillhavean
exponentiallygreaterparticleconcentrationthantheparticleconcentrationforalargersize
distribution.Asaconsequenceofthedisproportionateratechangeforthesetwovariables,
smallerparticlesizedistributionwillalsohaveamuchgreatertotalsurfacearea.Thefact
thatsmallparticlesizedistributionwillhaveanexponentiallygreatersurfacearea
concentrationgivenanequivalentmasshelpstoexplainwhyour originalobservationsand
manyothernanotoxicologystudiesthatonlyreportmassbasedconcentration,alsoreport
thatsmallersizedistributionsaremoretoxictoorganismsthanlargersizedistributions.
Becauseparticlenumberincreasesmorequicklythansurfaceareadecreaseswith
decreasingparticlesize,ittakesasmallermassofsmallerparticlestoreachtheeffective
surfaceareadosethanitdoesforlargerparticles,despitethefactthatlarger particleshave
farmoresurfaceareaperparticlethansmallerparticles.This studyhasfoundthatwhen
particlenumberisnominalizedtoderivesurfaceareavalues,theresultisanarrowrangeof
surfaceareaconcentrationsthatcauseequivalentmortalityforAgNPsrangingfrom8.9nm
to112.6nm.Forthisreason,wesuggestthattotalsurfaceareaconcentrationbest
representsthephysicalfeatureoftheAgNPsthatisresponsibleforelicitingtoxicityin
embryoniczebrafish.
WorkbySotiriouetal.(2011)investigatingtheantimicrobialactivityofAg/silica
nanoparticlesutilizedthesamethreealternativedosemetrics(Agsurfacearea
concentration,Agmassconcentration,andAgparticlenumberconcentration)intheirstudy
andshowedthatantimicrobialactivitywasmoststronglycorrelatedwithAgspecific
32
surfacearea(R2=0.90).Sotiriouetal.,(2011)alsoobservedthattherelationshipbetween
antimicrobialactivityandmassconcentration(R2=0.56)providedtheweakestcorrelation.
Theauthorssuggestedthatthatrelyingonmassbaseddosemetricsimposeslimitationson
understandingtheantibacterialactivityofnanosilver(Sotiriouetal.,2011).Thisstudythe
andstudybySotiriouetal.,suggestthatsurfaceareaconcentrationisamoreaccurate
predictorofnanosilvermediatedtoxicity,andthatmassconcentrationisthemost
misleadingofthethreedosemetricsbecauseitinadequatelyrepresentsthethree
dimensionalnatureoftheparticles.However,ourcurrentparadigmfordoseresponseis
limitedtomassbasedmeasures.
Targeteddoseresponsemodelingwasnotconductedforeachmaterialbecausethe
dosemodelingdatausedinthisstudywasinitiallyintendedonlyasatier‐1hazard
identificationandrange findingstudy.Asaresult,theactualconcentrationresponseareaof
thecurvesoccurredoverthehighestfourconcentrationsforalloftheAgNPs tested,with
fewerthanfourconcentrationsrepresentedinthedoseresponseregionforthelargest
particlesizedistributions.Despitethislimitation,bysystematicallyvaryingparticle
diameteracrossalargenumberofdistinctparticlesuspensionswithnarrowsize
distributions,thedatafromthedosemodelingexperimentsclearlyshowsizedependent
toxicityofAgNPstozebrafishembryos.
Theimportanceofchoosingappropriatedosemetricsinnanotoxicologywasalso
illustratedby thedosemetricanalysesfortheCh(+)/Ch(‐)embryoresponsedata.The
concentrationstestedintheCh(+)/Ch(‐)exposureswereselectedtorepresentequivalent
massbasedconcentrations(μg/mL)andarethereforeoverlapping.Alternativedose
metricswere not,however,takenintoconsiderationatthe time theexposure
concentrationswereselected.Consequently,whentheembryoresponsewasplottedby
particleconcentration(particles/mL)orbysurfaceareaconcentration(mm2/mL),the
embryoresponsesnolongeroccuroverasharedrangeofconcentrationsandwere
thereforenolongerdirectlycomparable.Thisobservationhelpedtoqualitativelyverifythe
findingsfromthedosemetricanalysisfromthedosemodelingresults.Alternativedose
metricsareavaluabletoolformakingcomparisonsbetweentherelativepotencyof
differentENMsandwhetherobserveddifferences arereal,orartifactsofstudydesignand/
ordataanalysis.Althoughretrospectivealternativedosemetricsanalysesofnanotoxicology
dataispossible,itislesslikelytobe informative,reiteratingtheimportance ofdesigning
studieswithalternativedosemetricsin mind.
33
Chapter5:Conclusions
Inthisstudy,wehavecomparedthreeofthemostcommonalternativedose metrics
todescribenanomaterialtoxicity:surfacearea,particlenumber,andmass.Wefoundthat
concentrationbasedontotalsurfaceareawasthemostinformativeandthemost
appropriatedosemetricfordescribingAgNPmediatedembryotoxicity.Althoughthe
conceptthatsurfaceareaisadrivingfactorinnanoparticlemediatedtoxicityhas been
previouslyproposed(Oberdörsteretal.,2005),thedatapresentedhereprovidestrong
supportforthisgenerallyhypothesizedrelationshipbylinkingtheoretical nanoparticle
surfaceareatoaninvivobiologicalresponse.Theimportanceofsurfaceareaasadose
metricfortoxicologystudiesmaybeofequallygreatimportanceforbothmetal
nanomaterialsandsignificantlylargerbulkmaterialsinrelationtotheiraqueous
dissolution.Liuetal.demonstratedthisbycomparingthedissolutionratesbetween4.8nm
sphericalAgNPs,60nmtriangularAgNPsanda1mmmacroscopicsilverfoil whichall
releasedAg+ionsatasimilarrateonthebasisofsurfacearea,butnotonamassbasis(Liu
etal.,2010).Thisimpliesthat dissolutionisafunctionoftheelementalpropertiesofthe
materialitselfasopposedtoinherentnano‐scaleproperties.
Theimportanceofsurfaceareaastheprimarydeterminantandpredictoroftoxicity
inrelationtothedissolutionofnon‐nanoscalemetalsandmetalalloyshasalsobeen
previouslyobserved.Metals,includingmetalpowders,producedforcommercialpurposes
aresubjecttoregulationbasedonhazardidentificationandclassification.Onecomponent
ofmetalpowderhazardcharacterizationinvolvesastandardlaboratorytechniquereferred
toasatransformation/dissolutionprotocol(T/DP).Theprocedureisusedtomeasurethe
rateofdissolutionandthereleaseofionsandotherbioavailablemetalspeciesin
environmentallyrelevantaqueousmediumbysamplingthesoluble fractionover time
whichcanthenbecomparedtoacutetoxicitydatatocharacterizetheinherenthazard
(Skeaffetal.,2000,2009).Skeaffetal.(2009)explainsthataccordingtotheGlobal
HarmonizationSystem’shazardclassifacationsystem,metalcoumpoundswithecotoxicity
values(LC50orEC50)thatareabove100mg/Lwouldneednofurtherhazardclassification
butcompoundswithLC50 or(EC50)valuesbelow100mg/Lwouldrequireanalysisusinga
T/DPtoassessthereleaseofdisolvedmetal(Skeaffetal.,2009).Basedonthisclassification
system,particle/grainsizecouldsignificantlyimpactthehazardclassificationunless
surfaceareaisaccountedfor.Skeaffandcolleagueshavetherforedefinedthecritical
surfacearea(CSA)asthesurfaceareaperliter,ormetalalloythatwillresult inamassof
34
dissolvedmetalthatisequaltotheLC50 (orEC50)valueforthemetal’ssolublesaltand
estimatedusingarelevanttestorganismwithastandardtoxicityassay(Skeaffetal.,2000).
CSAcanbebasedoncalculatedormeasuredsurfaceareas(Skeaffetal.,2000).CSAderived
toxicityisdependentontheinherentpropertiesofthematerial,includingitsabilityto
undergodissolution,itstendencytoformofstableionicspeciesinsolution,andthetoxicity
oftheionicspecies.Becausethesurfaceareadirectlyinfluencesthedissolutionrateof
dissolutionperunitmass,itsubsequentlyinfluencestheapparenttoxicity.Wesuggestthat
thetoxicityassociatedwithsphericalAgNPsisalsodominatedbythereleaseofionicsilver
throughoxidativedissolutioninsolution.Figure7showsthreeschematicrepresentations
thatillustrate thedifferentdosemetricsandhowtheyrelateto embryotoxicityinthe
contextof thisstudyandtheconceptofCSA.
Figure6.Conceptualdiagramsummarizingthe importanceofsurfaceareaasadosemetric
fornanomaterials.Relationshipsobservedbetweenembryotoxicityand(A)massbased
concentration,(B)particlenumberbasedconcentration,and(C) surfaceareabased
concentration.Theflatlineindicatesverynarrowrangeofsurfaceareaconcentrationsthat
approximateasingleCSAconcentration.
Therelationshipbetweenembryotoxicityandmassconcentration (Figure 7A)
showsthatsmallerdiameterparticlesaremoretoxicduetoalargersurfaceareatomass
35
ratio.Smallerparticles,therefore,reachaCSAconcentrationatlowermassthanlarger
diameterparticles,whichhavelesssurfaceareapermass.Therelationshipbetween
embryotoxicityandparticleconcentration(Figure7B)showsthatlargerdiameterparticles
aremoretoxictoembryosbecauselargerparticleshavemoresurfaceareaperparticlethan
smallerparticles.Therefore,ittakesfewerlargerdiameterparticlestoreachtheCSA
concentration.Lastly,therelationshipbetweenembryotoxicityandsurfacearea
concentration(Figure7C)showsthatlargerparticlesarenotsignificantlymoreorlesstoxic
toembryosthansmallerparticles.Thisisbecauseembryomortalityisdependentupona
singleCSAconcentration.
Moreworkis requiredtounderstandtheroleofsurfaceareamediatedeffectsand
theirroleinAgNPtoxicity.Nanoparticlesurfaceareamediated toxicityis ofsignificant
importancefromaregulatoryperspectivebecauseofpotentialimplicationsregarding
humanexposureand ourcurrentmassbasedparadigmforhazardassessment.
UnderstandingthesurfaceareamediatedeffectsofENMsisimportantbecausemanyother
metallicnanomaterialsincludingcopper,aluminum,cobalt,andnickelundergo dissolution
(Griffittetal.,2008b).Nanomaterialdissolutionandinherentinstabilitycould constitutea
commonmechanismoftoxicityformetallicnanomaterials.Itremainsunknownifthe
toxicityofAgNPsisdueonlyto thesurfaceareadependentreleaseofsilverions,orifcell
surfacesandinteriorscan reactdirectlywithAgNPsurfaces,resultinginROSgeneration
anddamagetocellularcomponents.Verylikely,bothformsoftoxicityareoccurring
simultaneously.Evidencesupportingthishypothesisisbasedon theunderstandingthat
silverionsareacutelytoxictoaquaticlife.AdditionallyAgNPsareknownto undergo
considerableoxidativedissolutioninaquaticenvironmentsandoxidativedissolutionboth
generatesROSandis acceleratedbyROS.
Ourworkinghypothesisisthatitisnottheionsalonethatcausetoxicitybut rather,
theoxidativedissolutionoftheAgNPsthatgeneratesbothsilverionsandROS.Recent
studiessupportthishypothesis,showingasizedependentincreaseinintercellularROS
generationandgrowthinhibitioninnitrifyingbacteriaasparticlesizedistributions
(averagesize9‐21nm)wereshiftedtowardincreasingnumbersofsmaller(≤5nm)AgNPs
(ChoiandHu,2008).Althoughthereisstilllimitedknowledgeregardingpotentialinvivo
andintercellularROSgenerationfromtheoxidativedissolutionofAgNPs,itseemsplausible
thatsilverionsthemselvesareresponsibleforthemajorityofthetoxicityobserved in
aquaticanimalmodels.
36
NanomaterialSARsandalternativedosemetricshave thepotentialtouncover
importantchemicalandstructuralrelationshipsandinteractionsbetweennanomaterials
andbiologicalsystemsandtopredictthebehavioroffuturematerials.Thisworkreports on
asystematicevaluationof AgNPsbasedonsizeandhasyieldedaphysicochemicalSAR
betweenaverageAgNPsizeandembryomortality.Thisstudyunderscorestheimportance
ofusingasystematicapproachinnanotoxicologystudies.TheAgNPsusedinthisstudy
werewellcharacterized,welldispersed,andhadnarrowsizedistributions.Without
accuratecharacterizationinformationandtightcontrolovermaterialproperties,itis
difficulttomoveawayfromreportingresults,andmovetowardmaking informed
conclusionsabouttherelationshipsbetweennanomaterialstructuralpropertiesandthe
biologicalresponsetheyelicit.
Afundamentalobjectiveofnanotoxicologyistounderstandthephysicochemical
propertiesthatultimatelyleadtotoxicity.Forthisreason,acombination ofhighthroughput
assays,highqualitymaterialcharacterization,andmodelingstrategiesareneededto
advancethescienceofnanotoxicologyandclearlyunderstandthepotentialhazards,
inherentrisks,andenvironmentalfate ofENMs.It isimportantfornanotoxicologystudies
tomoveawayfromconventionaldosemetrics(i.e.massbaseddosemetrics)andtoutilize
dosemetricsthataccountforthethree‐dimensionalshapeofnanomaterials(i.e.surface
area,volume,andparticlenumberdosemetrics).AnalogoustotheworkdefiningtheCSA
formetalsandmetalalloysbySkeaffetal.(Skeaffetal.,2000)itwillbeinterestingtosee
howalternativedosemetricswillbeincorporatedintonanomaterialriskassessment
frameworksandhazardcharacterizationin thefuture.
37
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