The Toxicity of Carbon Nanotubes to Daphnia magna

Transcription

1 Clemson University TigerPrints All Dissertations Dissertations The Toxicity of Carbon Nanotubes to Daphnia magna Aaron Edgington Clemson University, Follow this and additional works at: Part of the Environmental Sciences Commons Recommended Citation Edgington, Aaron, "The Toxicity of Carbon Nanotubes to Daphnia magna" (2011). All Dissertations This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact

2 TOXICITYOFCARBONNANOTUBESTODAPHNIAMAGNA ADissertationPresented tothegraduateschoolof ClemsonUniversity InPartialFulfillmentofthe RequirementsfortheDegree DoctorofPhilosophy EnvironmentalToxicology By AaronJ.Edgington December2011 Acceptedby: Dr.StephenJ.Klaine,CommitteeChair Dr.AaronP.Roberts Dr.LisaBain Dr.AndrewMount Dr.ElizabethCarraway

3 ABSTRACT Aparticleisclassifiedasnanoinsizeifithasatleastonedimensionlessthan 100nmwhichgivesthemchemicalandphysicalcharacteristicsthataredifferent fromthebulkmaterial.overthelasttwentyyearsnanotechnologyhasexplodedas anindustry.ithasbeenestimatedthatthereare1000consumerproductsonthe markettodaythatcontainnanomaterialsand2millionjobsworldwidewillbe devoteddirectlytonanotechnologyresearch,development,andproduct manufacturingby2015.carbonnanotubes(cnts)canbevisualizedassheetsof graphenerolledintoacylinder.carbonnanotubessmalldiameter(nanometer scale)andlonglength(micronscale)givecntstheuniquecharacteristicofalarge aspectratio,hightensilestrength,andhighelectronconductancewhichhasmade themacommonlyusednanomaterialsintheindustry.despiteenvironmental regulationsimposingindustrialsafeguardschemicalcontaminantsfrom manufacturingprocessareoftendepositedintotheaquaticenvironment.the overallobjectiveofthisresearchwastodeterminethetoxicityandbiodistribution ofcarbonnanotubestodaphniamagna. Multi wallednanotubes(mwnts)suspendedinnomwereacutelytoxictod. magnaandtheaverage96hlc50valueforallthetestswithsr NOM( mg/ldoc)wasapproximately2mg/l.however,themwnt EdistoRiverNOM complexwaslesstoxic(lc504mg/l)thanmwnt NOMcomplexesproducedfrom theothertwonomsources(blackriverlc502mg/landsuwanneeriverlc50 2mg/L)andthisdifferenceintoxicitycouldnotbeexplainedfromresultsofeither theparticlesuspensioncharacterizationorthenomcharacterization.thisstudy ii

4 suggestedmwntsweretolargetoabsorbacrosstheguttractofd.magna,but single wallednanotubes(swnts)havetopotentialduetotheirsmallerdiameter. D.magnawereexposedfor96htohydroxylated,silcadioxide,poly aminobenzenesulfonicacid,andpolyethyleneglycolfunctionalizedswntsinstatic renewalbioassays.usingtransmissionelectronmicroscopy(tem),high resolution TEM,selectiveareadiffraction,andelectronemissionlossspectroscopywewere unabletodefinitivelydetectabsorptionofswntsacrosstheguttractofd.magna fortheseveraldifferentsurfacemodifiedtubestested.thissuggeststhat,while thereisnoabsorbanceofcnts,thematerialmayinterferewithnormalgut processes. DaphniamagnawereexposedtoMWNTsandSWNTsandresourcegene transcriptionwascomparedtoacontrolandastarvedtreatment.anadverse outcomepathwaywasformedusingthechangesinresourcegenestodescribe effectstonutrition,energy,growth,andmoltingthatleadstoanapicalendpointof reducedreproduction.thesestudiessuggestthatsuspendedcntexposurehasthe potentialtocausesomeadverseeffectstoexposedd.magna.moreresearchneeds tobedonetoadequatelydeterminepotentialeffectsatlowerconcentrationsto mimicpossibleenvironmentalconditions. iii

5 ACKNOWLEDGEMENTS Iwouldliketothankmyadvisor,Dr.StephenKlaine,fortakingachanceon meandhisconstantsupportandencouragementthroughoutmytenure.iwould alsoliketothanktherestofmycommitteefortheirsupportandcontributions,dr. AndyMount,Dr.LisaBain,Dr.ElizabethCarraway,andDr.AaronRoberts. Iwouldalsoliketoacknowledgeallofmylabmatespastandpresent;Sarah White,KatieSciera,KristenGaworecki,AnthonySowers,HollyZahner,AlanJones, JoeBisesi,BrandonSeda,SarahRobinson,BradGlenn,RossGarner,AustinWray, LaurenSweet,andKimNewton.Duringlabmeetingsalloftheirquestionsand commentsaboutmyresearchstrengthenedmyprojectsandmyabilityto communicatemyscience. Mostofall,Iwouldliketothankmyparents.Althoughtheynevergotthe chancetogotocollegetheymademanypersonalsacrificessothatimaygoand achievemydreams.itisbecauseoftheirselflessnessandconstantsupportthati havebeenabletogoasfarasidid.simplyput,thanksmomanddad! Last,butcertainlynotleast,IwouldliketothankErica.Asabestfriendand asapartneryouhavealwaysbeenthereforsupport.thankyouforyourpatience, understanding,andconstantpresenceinmylife. iv

6 TABLEOFCONTENTS Page TITLEPAGE...i ABSTRACT...ii ACKNOWLEDGEMENTS...iv LISTOFTABLES...x LISTOFFIGURES...xiv CHAPTER1:LITERATUREREVIEW...1 Nanomaterials...1 NanomaterialManufacturing...1 NanomaterialClassification...2 CarbonNanomaterialCharacteristics...4 CarbonNanotubeProduction...5 DualLaserMethod...5 ChemicalVaporDeposition...6 BallMilling...6 FlameSynthesis...7 CarbonNanotubeProducts...7 CarbonNanotubeToxicity...7 Inhalation...7 Cytotoxicity...13 AquaticToxicity...16 CarbonNanotubeAbsorptionAcrossCellMembranes...23 v

7 TableofContents(Continued) Page PhysiologicalEffectsofCarbonNanotubes...27 Conclusions...29 References...30 CHAPTER2:THEINFLUENCEOFNATURALORGANICMATTERONTHETOXICITY OFMULTIWALLEDNANOTUBES...42 Abstract...42 Introduction...43 MaterialsandMethods...45 Organisms...45 MWNTs...46 NOMSources...46 MWNTsSuspensions...47 Bioassays...48 MWNTandNOMCharacterization...50 DataAnalysis...52 Results...52 Bioassays...52 NanoparticleSuspensionCharacterization...53 Discussion...56 References...61 vi

8 TableofContents(Continued) Page CHAPTER3:ABSORPTIONOFFUNCTIONALIZEDSINGLE WALLEDNANOTUBESIN Daphniamagna...78 Abstract...78 Introduction...79 MaterialsandMethods...82 Organisms...82 SWNTs...82 NaturalColloids...83 SWNTSuspensions...83 ParticleCharacterization...84 Bioassays...85 RamanSpectroscopy...85 TransmissionElectronMicroscopy...86 HighResolutionTransmissionElectronMicroscopy, SelectiveAreaDiffraction,andElectron EmissionLossSpectroscopy...86 Results...87 ParticleCharacterization...87 Bioassays...87 RamanSpectroscopy...87 TransmissionElectronMicroscopy...88 vii

9 TableofContents(Continued) Page HighResolutionTransmissionElectronMicroscopy, SelectiveAreaDiffraction,andElectron EmissionLossSpectroscopy...90 Discussion...91 Conclusions...96 References...98 CHAPTER4:DIFFERENTIALTRANSCRIPTIONOFRESOURCE GENESINDaphniamagnaEXPOSEDTOCARBONNANOTUBES Abstract Introduction MaterialsandMethods Organisms CarbonNanomaterials NaturalOrganicMatter(NOM) CarbonNanotubeSuspensions Bioassays Primers Real TimePCRAnalysis Results ParticleCharacterization Real TimePCR viii

10 TableofContents(Continued) Page Discussion Conclusions References CONCLUSIONS Multi wallednanotubetoxicity NaturalOrganicMatterandMulti WalledNanotube ParticleCharacteristics Single WalledNanotubeAbsorption CarbonNanotubeAdverseOutcomePathway ix

11 LISTOFTABLES Table Page 2.1 MWNTparticlecharacterizationandtoxicityresults,(*) meansstatisticallysignificantlydifferentvalues (p<0.05) DaphniamagnaMWNTguttracteliminationdata Nuclearmagneticresonancespectroscopyanalysis resultsofnom Primersequencesandannealingtemperaturesofresource genes Single wallednanotubecharacteristics Gene sfunctionandtreatmentresponses.d.plucaria datafromdudychaetal(inpress)[28].(*)denotes statisticalsignificance(p<0.05).na=not Applicable,D.Regulated=DownRegulated x

12 LISTOFFIGURES Figure Page 2.1 Daphniamagnagrowth(mg)followingexposureto multiwallednanotube naturalorganicmatter particles(mwnt NOM)(NP;mg/L).Growthwas inhibitedatallexposureconcentrationsgreater than0.125mg/l(p<0.01;r 2 =0.574) Ceriodaphniadubiareproduction(%Control)during exposuretomwnt NOM(NP;mg/L).Significant decreaseswereobservedinallconcentrations >0.25mg/L.Lettersdenotestatisticalgroupings (p<0.05) C.dubiagrowth(mg)followingasevendayexposureto MWNT NOM(NP;mg/L).Adecreaseingrowthwas observed(p=0.045) Fluorescenceexcitation emissionmatrices;a)black RiverNOM,B)EdistoRiverNOM,andC)Suwannee RiverNOM.BR NOMpeakintensity(excitation 340,emission385),ER NOMpeakintensity (excitation340,emission380),andsr NOMpeak intensity(excitation355,emission400)suggestall sourcesaremorefulvic like Scanningelectronmicroscopy(SEM)micrographof MWNTsA)bulkmaterialB)sonicatedinmoderately hardwater(mhw)andc)sonicatedinsuwannee River NOMsolutions(2mg/LDOC) Infrared(IR)spectraofNOMsourceswithfunctional grouppeakslabled(thesamplelablednomis SuwanneeRiverNOM)andIRspectraofNOMand NOM MWNTcomplex.Identifiedpeakswereas follows: OHstretchofcarboxylicgroups; C Ostretchingofesters,ethersand phenols; CHdeformation(CH3); CHdeformation(aliphatic);1550and1 640C=O(ketonesandquinines); aliphatic C H,C H2,andC H3stretching;3400OHgroups...73 xi

13 Figures(Continued) Page 2.7 Transmissionelectronmicroscopy(TEM)micrographsof controld.magnaguttract,algaeandotherfood particlesarepresent(a)andimbeddedwithin microvili(b).theguttractofmwntexposedd. magnahasbeenimpactedwithmwnts,whichhave disaggregated(c),butthemicrovilliappeartoact asabarriertopreventabsorptionacrossthegut lumen(d) Thetopimageisthesampletobeanalyzed;thelefthand sideisthedorsalsurfaceofthesectioneddaphnia magnathebluesquareistheareaanalyzedby RamanSpectroscopy.TheG bandandd band signalsarestronginthegutandabsentattheedges indicatingahighconcentrationofingested OH SWNTswithinthegutbutnodetectablesignal inthetissues Thetopimageisareaofthegutchosenforanalysisandin thesecondimagetheareaanalyzedathigher magnificationishighlightedinblack.theresulting G bandsignalismappedoutandshowsahighlighted regionofhighoh SWNTconcentrationwithinthe gutofexposedd.magna,butthereisnodetectable signalwithinthetissues Transmissionelectronimagesofmaterialspostsonication; A)OH SWNTsinnaturalorganicmatter,B) SiO2 SWNTsinnaturalorganicmatter,C) PABS SWNTsinnaturalorganicmatter,D) PEG SWNTsinnaturalorganicmatter,andE) OH SWNTsinFBS Transmissionelectronmicroscopyimagesofthecross sectionofnomsuspendedoh SWNTexposedD. magna.imageashowsthegut,microvilli,and lumen.imagebandcshowoh SWTNsinthegut (squares)andsuspectedmaterialinassociatedwith themicrovilliandabsorbedinthelumen(circles) xii

14 Figures(Continued) Page 3.5 Transmissionelectronmicroscopyimagesofthecross sectionofnomsuspendedpeg SWNTexposed D.magna.ImageAshowsthegut,microvilli,and lumenwithpeg SWNTsinthegut(squares). ImageBandCshowssuspectedPEG SWNTs absorbedinthelumen(circles) Transmissionelectronmicroscopyimagesofthecross sectionofnomsuspendedpabs SWNTexposed D.magna.ImageAshowsthegutandlumenwith PABS SWNTsinthegut(squares)andsuspected PABS SWNTsabsorbedintothelumen.ImageB showssuspectedpabs SWNTsabsorbedinthe lumen(circle) TransmissionelectronmicroscopyimageofNOM suspendedsio2 SWNTsexposedD.magna.The imageshowsthematerialpresentinthegut (square)butnotassociatedwiththemicrovilli orabsorbedinthelumen Transmissionelectronmicroscopyimagesofthecross sectionoffbssuspendedoh SWNTexposedD. magna.imageashowsthemicrovilliandlumen withsuspectedoh SWNTsassociatedwiththe tissues(circles).imagebshowssuspectedoh SWNTs absorbedinthelumenandassociatedwiththe microvilliinmoredetail(circles) Transmissionelectronmicroscopyimagesinz contrast modeofthecrosssectionoffbsdispersed OH SWNTexposedD.magna.Electrondense materialappearswhitecomparedtotheless electrondensetissues(circles).inbandclong tube likematerialthatappearselectrondenseis foundwithinthelumen(circles) AandBareHRTEMimagesoftubularstructuresfound withintheguttractoffbsoh SWNTexposed D.magna.CisHRTEMimagewithSADdiffraction patternconfirmingtheabsenceofswnts.disthe energydispersionspectrumofstainedfbs OH SWNTsectionconfirmingthedarkareasare particlesofstainnotswnts xiii

15 Figures(Continued) Page 3.11 High resolutiontransmissionelectronmicrographsof PBS SWNTsimagedinexposedD.magnasections rangingfrom100nm(a)to10nm(c).swntscan beseenincircledregionofc Aisarepresentativeselectiveareadiffraction(SAD) patternofareasabsentofswntsandbisa representativesadofswnts(aandbareatthe samemagnification,scalebaris50nm).candd arerepresentativelow lossandcore loss (respectively)electronemissionlossspectra (EELS)ofSWNTs.Youcanobservethelargepeaks thatareindicativeofswntseelsspectra HRTEMinvestigationofapparentPEG SWNTsinDaphnia atlowermagnificationswiththecameraoutof focus(a)infocus(b)andathigherresolutions (CandD).TheinsetofpartDshowstheselective areadiffractionpatternindicativeofamorphous carbon A)TEMimageofanunstainedFBSOH SWNTexposed D.magnasection,B)TEMimageofastainedcontrol D.magnasection Transmissionelectronmicroscopyimagesofmult walled nanotubes(a)andsingle wallednanotubes(b) postsonicationinnaturalorganicmattersolutions GenetranscriptionforD.magnatreatments.Symbol(*) denotesstatisticalsignificance(p<0.05) Adverseoutcomepathwayfordemoicacidfrom Watanabeetal(2011) Conceptualflowchartforanadverseoutcomepathway forsuspendedcarbonnanotubesexposeddaphnia magna.(1)healthyorganismingests(2)suspended carbonnanotubesthatresultsin(3)impactedgut. Thisleadsto(4)decreasedfoodassimilationfrom xiv

16 physicalblockageofthegutthatcauses(5a D) poornutrition(asseenwithlipaseand chymotrypsindata),energyreduction(observed withptm2andatp/adptranslocasedata), decreasedgrowth(showninpreviousgrowthdata byedgingtonetal(2010)[16]andthepresent studiesdataonneuroparsinandepoxidehydrolase transcription,andreducedmolt(suggestedby NPC2andchitinasetranscription).Theresulting apicalendpointis(6)reducedreproduction xv

17 CHAPTER1:LITERATUREREVIEW NANOMATERIALS Aparticleisclassifiedasnanoifithasatleastonedimensionlessthan100 nm[1].althoughsomenanomaterialscanbenaturallycreated,largescale manufacturingofnanomaterialsbeganafterkrotoetal.(1985)[2]discoveredc60 fullerenesandiijima(1991)[3]reportedcarbonnanotubes.overthelasttwenty yearsnanotechnologyhasexplodedasanindustry.in2001theunitedstates launchedthenationalnanotechnologyinitiative(nni)tocoordinatefederal researchanddevelopmenttokeeptheu.s.competitiveinthegrowingfield[4].the NNIstatesworldwide2millionjobsbywillbedevoteddirectlytonanotechnology research,development,andproductmanufacturingby2015andiscurrentlya billiondollarindustry.theprojectonemergingnanotechnologies[5],awoodrow WilsonScholarsthinktank,hasadatabasethathasover1000consumerproducts onthemarkettodaythatcontainnanomaterials.theseproductsrangefrom medicalproductstoeverydaycosmetics.nanomaterialstrulyhavebeenintegrated intoeverydaylife. NANOMATERIALMANUFACTURING Therearetwoapproachestomanufacturingnanomaterials,top downor bottom upapproach[6].thetop downapproachismanufacturingthe nanomaterialsfromthebulkmaterial.thisinvolvesmechanicalmethodssuchas millingorphotolithography.thebottom upapproachinvolvesusingchemical reactions,nucleation,andgrowthprocessestoformmorecomplexparticles. 1

18 NANOMATERIALCLASSIFICATION Therearethreemainclassesofmanufacturednanomaterials:metal base, dendrimers,andcarbon based[7].metal basednanomaterialsincludegold,silver, copper,metaloxides,andquantumdots.silvernanomaterialsandmetaloxidesare themostcommonnanomaterialsbecauseoftheirapplicationinpersonalcare productsandconsumergoods[8 9].Silver snaturalantimicrobialabilityhasmade nanosilveracommonchemicaladditiontomanyconsumergoodsincludingclothing, washingmachines,andchildren stoystopreventthegrowthofbacteria.titanium dioxideandzincoxideoflongbeenusedinsunscreensbutareknowforcreatinga thinkwhitefilmontheskin.whenthenano sizedformsareusedtheproductisless viscousandblendsintotheskinwithoutthethickwhitefilmcreatingamore marketableproduct[10].awidevarietyofuseshavebeenfoundforgold nanoparticlesincludingcosmetics,conductiveink,anddrugcarriersforcancer therapy[11].coppernanoparticlesareoftenaddedtolubricantoilasanadditive becauseoftheireffectivereductioninfrictionandabilitytomendawarnsurface [12 13]. Dendrimersareauniqueclassofnanomaterials.Theyaresynthetic polymerswithamulti functionalcore,branchedunitseminatingfromthecore,and externalcapping groupsonthebranchedunits[14].thevariousclassesof dendrimersareglycodendrimersandpeptidedendrimers.dendrimershavebeen showingtheirpotentialinthemedicalfieldandbioengineering.theyhavebeen 2

19 designedfordrugdeliverysystems[15],medicaldevices[16],cellandtissue engineering[17],andbiosensors[18,19]. Therearetwovarietiesofcarbonnanomaterials:fullerenesandcarbon nanotubes.c60wasthefullerenediscoveredbykrotoetal.in1985[2].c60isa sphericalcagelikestructuremadeupof60sp 2 bondedcarbonatomsinpentagonal andhexagonalrings[20].c70issimilarbutthe70carbonatomsmakeaslightly elongatessphere,muchlikearugbyball.theuniquepropertiesoffullerenes, includingelectronconductance,strength to weightratio,andchemicalreactivity havemadethemtheobjectofanabundanceofresearch.theyhavebeenusedin manyareasofresearchandaddedtomanyfullerenecontainingmaterialswhich includechemicalsensors,datastoragedevices,hydrogenstoragedevices,in photovoltaiccells,intelecommunications,ascatalysts,andaddedtopolymersto increasestrength[21,22]. Carbonnanotubes(CNTs)canbevisualizedassheetsofgraphenerolledinto acylinderwithsp 2 hybridizedbondsbetweencarbonatoms[23,24].dependingon directionthegraphenesheetisrolledthefinalnanotubemayhaveoneofthree chiralities:zigzagstructure,armchairstructure,orchiralstructure[23].the chiralityofthenanotubesaffectstheoverallcharacteristicsthematerialwillhave; nanotubeswithdifferentchiralitieswillhavedifferencesinopticalactivity, mechanicalstrength,andelectricalconductivity[23]. Thereexistthreedifferenttypesofcarbonnanotubes,single walled nanotubes(swnts),double wallednanotubes(dwnts),andmulti walled nanotubes(mwnts).single wallednanotubesareasinglesheetofsp 2 bonded 3

20 graphenerolledintoacylinderwithendscappedwithstructuressimilartothe curvatureofafullerene[23,25].double wallednanotubesaretwosheetsof increasingsizerolledintoconcentriccylinders,andmwntsaremultiplecarbon tubesofincreasingsizeplacedconcentricallywithineachotherwithoutendcaps [3]. CARBONNANOMATERIALCHARACTERISTICS Carbonnanotubessmalldiameter(nanometerscale)andlonglength(micron scale)givecntstheuniquecharacteristicofalargeaspectratio[26].their structurealsogivesthemhightensilestrength,highelectronconductance,chemical reactivityandspecificopticalproperties[23 26].Thesmalldiameter,hightensile strength,andstiffnessofcntsmakethemattractiveforcompositematerials makingthecompositeastronger,butlightermaterial,andincreasingthelongevity oftheendproduct[27].thechemicalreactivityofcntsisadirectresultofthe curvatureofthetubesurface,whichcausesamismatchofpi orbitalbonds[28]. Therefore,thereisadifferencebetweenthechemicalreactivityofthesidewalland endcapsofcnts[23].throughthischemicalreactivitycovalentmodificationscan easilybedonetofunctionalizethesurfaceofcntstoachievedesired characteristics.mostfunctionalizationsaredonetoaltertheaqueousstabilityofthe CNTsormakethemmorecompatibleincomposites.Researchershaveused lysophospholipids[29,30],copolymers[31,32],andotherorganicmolecules[33, 34]asfunctionalgroupsonCNTs. 4

21 Becauseofthedifferencesinchirality,CNTscaneitherbemetallicorsemiconducting.Thedifferencesinelectronconductivityareduetothechangein molecularstructurewithchirality,andthusadifferenceinbandgap[23,35]. Therefore,CNTsaresemiconductingwhenthechiralitycreatesasmallerbandgap. CARONNANOTUBEPRODUCTION Krotoetalfirstbrieflydescribedtheproductionofcarbonnanomaterialsin 1985[2].Theyfoundthatvaporizingcarbonwithafocusedpulsedlaserinthe presenceofhigh densityheliumgascouldproducefullerenes.theheliumgas allowedaflowratethatwouldcarrytheparticlesdownstreamtoatime of flight massspectrometerforanalysis.thisresearchstimulatedintenseinterestand explorationinthemanufactureofcarbonstructures.usingthecarbonarcdischarge method,iijima(1991)[3]reportedtheproductionofcnts.thearcdischarge methodcreatescntsusingtwocarbonrodsplacedendtoendin furnace thatis filledwithhelium,oranotherinertgas,atlowpressure.adirectcurrentbetween 50to100Acreatesahightemperaturebetweenthecarbonelectrodes,thus vaporizingoneoftheelectrode ssurfacesandcreatingcntsdepositsonthe oppositeelectrode[36].althoughrelativelysimpletocomplete,thismethoddoes produceaproductthatneedsfurtherpurificationtoseparatecntsfromthesoot. ToachieveamorepureproductandgreatefficiencyotherwaysofCNT manufacturingweredeveloped,whichhavebeenoutlinedbywilsonetal2002[36]: DualLaserMethod:Samplesarepreparedbydual pulsedlaser vaporizationofgraphiterods.thesecondlaserpulsevaporizesthecarbonelectrode 5

22 moreuniformlydecreasingtheamountofcarbonsootproduced.a50:50catalyst mixtureofcobaltandnickelareusedat1200 Cinflowingargongasandan additionalheattreatmentat1000 Cinavacuumremovesanyfullerenesproduced. ThematerialproducedisprimarilySWNTsalongacommonaxiswithadiameterof 10 20nmandalengthupto100µm.OnedrawbackisthattheSWNTsareintight bundlesmixedwithamorphouscarbon,soot,andresidualcatalystmetalsmakingit difficultforpurification. ChemicalVaporDepositionMethod(CVD):LargeamountsofCNTsand othercarbonstructurescanbeproducedbyvaryingthecatalystandgasusedinthis method.usingacetylenegasandcobaltandironcatalystsonsilicaorzeolite substratesyieldslargequantitiesofmwnts.orethylenegasat545 Cusinga nickelcatalystalsoproducesmwnts.toremovepossiblecatalystimpuritiesone couldproducemwntsinethylenegasat900 Cwithoutacatalystmetal.High yieldsofswntsmaybeproducedbythecatalyticdecompositionofh2/ch4over metalparticlessuchascobalt,nickel,andirononamagnesiumoxidesubstrateat 1000 C.TheCVDmethodhasbeenusedextensivelyoverthepasttwentyyearsfor highyieldsofcnts. BallMilling:Thismethodisthesimplestmethodtomechanicallymake CNTs.Graphitepowderisplacedinastainlesssteelcontainerwithfoursteelballs. Thecontainerispurgedandthenfilledwithargonandmillingtakesplaceat ambienttemperaturesforupto150hrs.themechanismofcntgrowthisnot known,butisassumedthatduringtheballmillingprocessnanotubesnecleiare formed.aftertheballmillingthesubsequentproductisthenannealedunderan 6

23 inertgasflowat1400 Cforsixhrs.Thisannealingprocessisthenthoughtto activatenanotubegrowth. FlameSynthesis:Ahydrocarbongasisburnedtocreatethehigh temperaturesneededforcntproductionandtheremaininggasistherequired carbonsource.thisformofsynthesiscanbescalableforhigh volumeproductionas itisenergyefficientandalowcostprocess. CARBONNANOTUBEPRODUCTS Theeaseofmass producingcntsandtheiruniquecharacteristicshave madethemavailableforwidespreadapplicationandthis,inturn,willincreasethe probabilityofcntsenteringtheenvironment.despiteenvironmentalregulations imposingindustrialsafeguardschemicalcontaminantsfrommanufacturingprocess areoftendepositedintotheaquaticenvironmentanditisforeseeablethatcntswill aswell.thisconcernhasleadmanyenvironmentalresearcherstoexplorethe possibletoxiceffectstheseparticlesmayhaveonbiota. CARBONNANOTUBETOXICITY INHALATION Carbonnanotube ssmalldiameterandlonglengthresemblesasbestos fiberlikestructure.manyresearchersfocusedontheinhalationoftheseparticlesto determineiftheytoocauseddamagetothelungs. In2004Lametal[37]determinedtheeffectsofthreedifferenttypesofCNT productstomice7and90dayspostintratrachealinstillation.thethreecntsthey 7

24 usedwereswntsmadebydifferentmethodsandeachcontainingdifferenttypesor amountsofresidualcatalystmetals.theyhadraw(rt)andpurifiedironcontaininghigh pressurecarbonmonoxideproduced(pnt)swntsfromrice Universityandnickel containingelectric arcproductfromcarbolex(cnt).serum andcarbonblackservedasanegativecontrolandquartzservedasapositive control.theydosedwith0.1mgswntspermouseof0.5mgswntspermouseand determinedeffects7and90dayspostintratrachealinstillation.theyfoundthat5if the9miceinthe0.5mgcnttreatmentdied4 7dayspostexposure.Therewereno mortalitiesinanyofthe0.1mgand0.5mgrntandpnttreatments.thesurviving miceofthe0.5mgcnttreatmentsat7daysand90dayspostexposureallhadlarge aggregatesofparticlesinmacrophagesinthealveolarspaceofthelungs.someof theseaggregateswerealsoobservedwithintheinterstitium,whichleadto granulomaformationinthe0.5mgcnttreatedmice.the0.5mgrntandpnt dosedmiceshowedgranulomaslocatedbeneaththebronchialepithelium throughoutmostofthelung.thesegranulomasconsistedofmacrophagesladen withblackparticlesbutwithlittlelymphocytes,neutrophils,eosinophils,orother inflammatorycells.allofthe90day0.5mgrnt,pnt,andcnttreatedmice showedextensivegranulomasthatcontainedparticlefilledmacrophageswith necrosis,interstitialinflammationandperibronchialinflammation.theyconcluded thatallthreeofthenanotubeproducts,regardlessofthetypeandamountof residualcatalystmetalspresent,causeddose dependentlunglesionscharacterized byinterstitialgranulomaswhereasbothnegativecontrols(serumandcarbon black)andthepositivecontrol(quartz)failedtocauseanydosedependenteffects. 8

25 ThereforetheCNTsthemselveshavethepotentialtocausedetrimentaleffectstothe lung,whichshouldbeconsideredaseriousoccupationalhealthhazardtothose workinginanenvironmentwherecntdustispresent. Warheitetal(2004)[38]ranasimilarstudytodeterminewhetherSWNTs causetoxicitytothelungsofratscomparedtoreferenceparticles.theyuseda short termpulmonarybioassayusinginratrachealinstillationandlung histopathologyof1or5mg/kgofswnts,quartz,orcarbonyliron.animalswere assessed24hr,1week,1monthand3monthspostexposurebybronchoalveolar lavage.exposuretothe5mg/kgofswntscausedmortalityin15%ofratswithin 24hrpostinstillationsimilartoresultsseeninLametal2004[37],butthiswas contributedtothemechanicalblockageoftheairwayandnotduetoinherent pulmonarytoxicityofswnts.exposuretoseveralparticletypesproducedshorttermpulmonaryinflammatoryresponses(inductionofneutrophils)post24hr exposure,butonlythequartzexposedanimalsinthe1and5mg/kgtreatments sustainedpulmonaryinflammatoryresponsesthroughoutthe3 monthperiod. Histopathologicalevaluationsoflungtissuesfromquartzexposedanimalsrevealed adose dependentlunginflammatoryresponsecharacterizedbyneurtophilsandan alveolarmacrophageaccumulation.furthermore,thelungtissuewasthickenedasa preludetofibrosisdevelopment.histopathologicalevaluationsoflungsfromswnt exposedratswerecharacterizedbyanon dose dependentseriesofgranuloma production.thiswasfirstobservedat1 weekpostexposureandby1 monthpost exposuretheproductionofgranulomashadincreased,althoughtherewaslittleto noprogressionat3monthspostexposure.theysuggestthegranulomarresponse 9

26 representsanattempttosequesturethecntparticulatesandthiswasthereason therewasnofurtherprogressionofgranulomaspast1 monthpostexposure.they notedanincreaseintracheobronchialcellproliferationrates(measuredas percentageofimmunostainedcellstakingupbrdustain)inswnt5mg/kg24hr postexposuretreatmentsandquartz1and5mg/kg24hrto3monthpostexposure treatmentsbuttheincreaseswerenotstatisticallysignificant.theyrecongnized thatthefindingofgranulomasinanondosedependentmanner,intheabsenceof pulmonarybiomarkers,doesnotfollowthenormalpathwayofeffectsdetermined byquartz,asbestos,andsiliconcarbidewhiskers.thustheyconcludedthatto accuratelyassessthepulmonaryeffectsofcntsthematerialmustbedeliveredvia anaerosoltobetterrepresentexposurescenarios. TheinconclusivedatafrompreviousinhalationstudiespromptedMulleretal (2005)[39]toslightlymodifythenanotubesbeforeexposingthemtorats.They receivedmwntsfromthefacultesuniversitairesnotre DamedelaPaixinNamur, groundtheminanoscillatoryagateballmill,andthensonicatedthemina1%tween solution.previousstudiesbylametal(2004)[37]didminorshearingbefore sonicationina1%tweensolutionandwarheitetal(2004)[38]didneitherbefore placingswntsin1%tweensolution.mulleretalsolutionpreprepresentsan attemptatcreatingamuchmoredispersedsuspension.thissuspension,anda suspensionofungroundmwnts,wastheninjecteddirectlyintothelungsby inratrachealinstillationat0.5,2,or5mgmwnts/animalwithnegativecontrolsof salinesolutionandcarbonblackandasbestosservingasapositivecontrol. Inflammatoryresponsewasdeterminedatdays3and15postexposureviaseveral 10

27 parametersinbronchoalveolarlavage.fibroticdevelopmentwasdetermined biochemically(solublecollagenandhydroxyproline)andhistopathologicallyat60 dayspostexposure.persistenceofmwntwithinthelungswasdeterminedat60 dayspostexposuretoallowadirectcomparisonwithfibroticdevelopment.they determinedpersistencebymeasuringtotalcobaltconcentrationsatday0,28,and 60postexposure.CobaltisacatalystmetalthatremainstightlyboundtoCNTs,thus theyusedittodeterminethepersistenceofmwntsinthelungs.theydetermined thatmwntsgroundbyballmillingwereeliminatedgreater(36%recoveredafter 60days)thanMWNTsthathadnotundergonethemillingprocess(81.2%recovered after60days).theyconcludedthatthissuggestsmwntpersistencewithinthe lungisrelatedtotheirlength(groundmwntshadshorterlengths). Bronchoalveolarlavagefluid(BALfluid)determinedLDHactivity,a biomarkerofcelltoxicity,increasedafterthe2mg/ratasbestostreatment,butnot inthecarbonblacktreatments.boththegroundandungroundmwnttreatments producedadosedependentincreaseofldhactivity.theproteinconcentrationin thebalfluid,whichrepresntsalveolo capillarypermeabilityandalviolitis,wasalso increasedinthemwnttreatments.bothmwnttreatmentsalsoinducedthe productionofneutrophilsandeosinophilsleadingtotheoverallconclusionsthat MWNTsinduceaninflammatoryresponse,whichwasslightlyelevatedinthe groundmwnttreatments.lungcollagenproduction,measuredbyhydroxyproline (OH proline)andsolublecollagen1concentrations,determinedthedevelopmentof fibrosis.oh prolinelevelsweresignificantlyincreasedinadosedependentmanner aftertheungroundmwnttreatments,butonlythe5mg/ratgroundmwnt 11

28 treatmentsawasignificantincrease.theasbestostreatmentsawincreasedohprolinelevelswhilecarbonblacktreatmentsdidnot.asbestos,ungroundmwnts, andgroundmwntsallhadincreasedcollagen1concentrationswhilenoincrease wasseenincarbonblacktreatments.thesemeasurementsthereforeindicate MWNTscauseafibroticresponseinadosedependentmanneralthoughthe responseofthe5mg/ratgroundmwntstreatmentswasequivalenttothe2mg/rat treatments.thehistopathologicalexaminationsofmwnttreatedratsrevealedthe productionofcollagen richgranulomasblockedorpartiallyblockedthebronchial lumen.muchlikeinpreviousstudiesbylametal(2004)[37]andwarheitetal (2004)[38]thegranulomasweresurroundingMWNTmaterialandwereformedof macrophages,miltinulceargiantcells,andotherinflammatorycells.thehistology alsoshowedthatthegroundmwntswerebetterdispersedinthelungtissue.they concludedthatmwntsarenotrapidlyeliminatedandmaycauseinflammatoryand fibroticresponsesinlungtissues.furthermore,industrialapplicationsofcnts includeinstanceswherethematerialmaybegroundpriortouse.thereforetheir examinationofeffectscausedbygroundmwntsisrelevanttodeterminerealworld scenariosofexposure. Theseinhalationstudies,andothers,demonstratedthatCNTsposeaserious risktothosethatmaybeexposedtoairborneparticles.theirresultsshowedthe importanceofneedingindustrialhygienepracticesthatwouldprotectworkerswho wouldcomeintocontactwithcntsandasaresultsuchprogramshavebeen implementedtoensureworkersafety.butitalsosuggeststhatcntshavethe 12

29 potentialtocauseothereffectstobiotaifthereisanenvironmentalrelease.invitro studiesareoftenusedtoassessmechanismsofcontaminanttoxicity. CYTOTOXICITY TofurtherexplorepossibleeffectsofinhaledCNTsJiaetal(2005)[40] exposedalveolarmacrophages(am)toswnts,mwnts,andfullerenes.tofully determineeffectstoamtheymeasuretheinhibitionofmitochondrial dehydrogenaseactivity,thephagocyticresponsetolatexbeads,andtransmission electronmicroscopy(tem)examinationsforvisualobservationsofanystructural alterationspostexposure.nanomaterialsuspensionsweremadebymixingthe materialinculturemediumwithahomogenizer,thensonicationfor20min.swnts andfullerensweredosedat1.41,2.82,5.65,11.30,28.20,56.50,113.00,and ug/cm 2 andmwntsweredosedat1.41,2.82,5.65,11.30,and22.60ug/cm 2.Quartz servedasapositivecontrolinallexperiments.cytotoxicitywasdeterminedusing themttreductionassay,whichisbasedonmitochondrialdehydrogenaseactivity. SWNTsandMWNTscausedadose dependentcytotoxicresponse.thelowest SWNTdose,1.41ug/cm 2,caused>20%inhibitionofmitochondrialdehydrogenase whereasthehighestmwntdose,22.6ug/cm 2,caused14%inhibition.TheSWNT andmwnttoxicitywasgreaterthanthetoxicresponsefromquartz.thefullerene C60failedtocauseanycytotoxicresponse.ThemajorfunctionofAMsareto phagocytizeforeignparticlesinthelung,thereforephagocyticresponseto2um latexbeadscandetermineamhealth.phagocyticresponsetothelatexbeadstook place6hrspostexposure.swntssignificantlyimpairedamphagocytosisat

30 ug/cm 2,whereastheothernanoparticlesimpairedphagocytosisat3.06ug/cm 2.In alldosesswntscausedmoreamtobenonphagocyticcomparedtomwnts,c60, andquartz.transmissionelectronmicroscopyimagesshowedamexposedto0.76 ug/cm 2 SWNTscausedcondensedfoldsandtheformationofplywoodbody.The 3.06ug/cm 2 dosecausedtheswellingoftheendoplasmicreticulum,vacuolar changes,andthepresenceofphagosomes.alveolarmacrophagesexposedto0.76 ug/cm 2 hadlargephagosomesandinthe3.06ug/cm 2 treatmentshadnuclei degenerationandenlargementandrarefactionofthenuclearmatrix.inboththe 3.06ug/cm 2 SNWTandMWNTtreatmentschromatincondensationatthenuclear envelop,condensedorganelles,andtheformationofsurfaceprotrusionswere visible,alllikelytobecausedbytheapoptoticprocess.alloftheseeffectssuggest thatcarbonnanomaterialshavethepotentialtohavedetrimentaleffectstoamand thatswntsmayposeagreaterrisktothoseexposed. Dumortieretal(2006)[41]cametocontradictoryconclusionstestingthe toxicityoffunctionalizedswntstoimmunecellsisolatedfrommouselymphoid organs.pristinesnwtswerefunctionalizedviathe1,3 dipolarcycloaddion reactionorviatheoxidation/amidationmethodologyproducinganammonium functionalizedswntsandanoxidizedswntsrespectively.theoxidizedmaterial wasfurtherfunctionalizedwithpoly(ethyleneglycol)(peg).boththeammonium functionalizedandpegfunctionalizedmaterialwasfurthermodifiedwith fluoresceinisothiocyanate(fitc)tocreatefluorescentnanotubesforacellular uptakestudy.nanotubeuptakewasdeterminedbyexposingspleencellsto1 10 ug/mloffluorescentswntsthenexaminingthemonaconfocalmicroscope.the 14

31 24hr10ug/mLPEGfluorescentSWNTexposedcellshadthegreatestuptakewith observedswntsinthecytoplasmbutnothenucleus.differentiallycellularuptake wasdeterminedbyisolationblymphocytes,tlymphocytes,andmacrophagesfrom thespleen,lymphnodes,andperitonealcavityofmice.theisolatedcellswerethen exposedfor24hrtofitc labeledammoniumandpegswntsat10ug/ml.both typesoffitc labeledswntswerefoundinthecytoplasmofallthreecelltypes suggestingthatthereisnodifferentialuptakebetweenimmunecells.effectsofcell viabilityweredeterminedbyexposingblymphocytes,tlymphocytes,and macrophagestoammoniumandpegfunctionalizedswnts.theydidnotobserve anychangestocellviabilityinthethreecelltypesafterexposuretothetwodifferent SWNTsupto50ug/mLfor48hrs.Todetermineeffectstolymphocytefunctionality theydeterminedtheirabilitytoproliferatepostswntexposure.lymphocytes wereexposedupto50ug/mlandcellactivationwasassessed65 70hrslater. TheysawnodifferencebetweeneitherSWNTexposedtreatmentscomparedto controlwhenthecellswerestimulatedtoproliferate.furthermore,ammonium functionalizedswntexposedmacrophageswerenotsignificantlystimulatedbythe presenceofthematerial,butthepegfunctionalizedtreatmentsdid.the confoundingconclusionsbetweenthisstudyandjiaetal(2005)[40]demonstrated howcntsurfacechemistrymightbethedrivingfactordetermininguptakeand cytotoxicityofcnts. ToassessthepotentialcytotoxicityofCNTsoncellsnotassociatedwiththe lungsorimmuneresponsesrajaetal(2007)[42]exploredtheimpactofcntstorat aorticsmoothmusclecells.theypurifiedswntsproducedbythehigh pressure 15

32 carbonmonoxideprocessthroughacidtreatment.theywerethensuspendedin reverseosmosiswaterviasonication.theresearchersdidnotwanttosonicatethe SWNTsinthecellmediaaspreviousstudieshavedonebecauseitcanalterthe integrityofthemediabydenaturingproteins.therefore,theyusedaliquotsofthe stockconcentrationtodosethecellsfrom0.0to0.1mg/mlswntanda concentrationof0.1mg/mlofactivatedcarbonforanegativecontrol.itwas observedthatasssoonasthesesolutionswereaddedtothecellsintheirmediaboth theswntsandactivatedcarbonaggregatedandsettlednon uniformlyonthecells. Thequickaggregationwasduetothehighioncontentofthecellmedia.Regardless oftheaggregationstatethecellswereexposedandcellgrowthmonitoredfor3.5 days.therewasasignificantswntdose dependentdecreaseincellgrowthbyday 2.5upto0.05mg/mLSWNT,howevertherewasnosignificantdifferencebetween SWNTconcentrationsof0.05and0.1mg/mLafter3.5days.Furthermore,cell growthbegantostagnatebetween2.5and3.5daysresultingincellnumber reductions(comparedtothecontrol).concentrations0.01,0.05,and0.1mg/ml SWNTshadroughly30.3%,46.6%,and53.3%reductionrespectivelycomparedto control.todeterminethedifferencesbetweenaggregatedandstableswntsthey filteredthespikemediawith0.2umfiltertoremovetheaggregatedswnts.they wereleftwithaclearfiltratethatresembledthecontrolmediumbutassumedthat therewerestillstableswntspresent(theyfilteredthecontrolmediaaswelltorule outanyeffectsfromthefiltrationsprocess).therewasasignificantdose dependent decreasedincellgrowthbyday2.5forconcentrationsfrom0.01to0.1mg/ml SWNTsinthefilteredmedia.Thehighestfilteredconcentration(0.1.mg/mL 16

33 SWNTs)inhibitedcellgrowthtothesamedegreeastheunfilteredsolutions.To confirmthepresenceofswntsinthefilteredmediatheyconcentrateditroughly15 fold.ablackprecipitatewasformedwhichtheycollectedandwashedseveraltimes withroandimagedwithscanningelectronmicroscopy(sem).thematerial appearedtohavenumeroussphericalparticleswithadiameterof20 60nmand bundlesofswntswithdiameterslessthan5nm.thepresenceofswntswas confirmedviaramanspectroscopyandtheypositedthatthevisiblecoatingwas adsorbedproteinfromtheserum,whichaidedparticlestability.theconclusions fromthisstudyclearlydemonstratedthatswntsinanaggregatedstateanda stablestatebothhadadverseeffectsoncellgrowth.furthermore,whenthese studiesarecomparedtojiaetal(2005)[40]anddumortieretal(2006)[41]it becomesclearthatsurfacechemistrymaynotonlyplayaroleinuptakebutalso toxicity. TheSEMimagesfromRajaetal(2007)[42]suggestedthatSWNTsmaybe ableadsorbproteinsfromthecellmediaanditsbeenestablishedthatsurface chemistryplaysaroleinuptakeandtoxicity.davorenetal(2007)[43]evaluated thetoxicityofswntsonhumana549lungcellsinserumcontainingandserum free mediumtoelucidatetheinfluencethatproteinadsorptionhasoncytotoxicityand uptake.theobjectivesoftheirstudywastoconductathoroughexaminationof SWNTcytotoxicitytoA549cellsbydeterminingeffectstothemetabolic,lysosomal, andmitochondrialactivitiesofthecells.theydeterminedswntuptakeby visualizationusingtem.theyfoundthatswntshaveaverylowacutetoxicityon A549cells.Cellmetabolism,measuredbytheAlamarBlueassay,showedthemost 17

34 significanteffectswithacalculated24hrec50of800ug/mlinboththeserumand serum freemedia.howeverthisec50valuewasequaltothehighestconcentration tested.furthermore,celldetachmentassay(coomassieblueassay)and mitochondrialactivityassay(mttassay)calculated24hrec50valuesallexceeded theirhighesttestedconcentration(800ug/ml).buttheseresultsoflowacute toxicitycouldbeexplainedbytheobservationthattheswntswereinteractingwith thecolorimetricandfluorescentdyesusedintheassays.theadsorbingproperties oftheswntscausedadsorptionofthedyesandthusinterferencewiththe absorptionspectraofthedyesresultinginpossiblefalsereadings.althoughthey notedsomestructuralchangestocellmorphology,theycouldn tdetermineany uptakeofswntsinserumorserum freemediausingtem.thissuggeststhat proteinabsorptiononswntshasnoeffectoncelluptakeina549cells. Whiletherearemanymoreinvitrotoxicitytestsintheliterature,thefour discussedabovehelpillustratethedifficultiesindeterminingcntcytotoxicity. WhiletheuniquecharacteristicsofCNTshavemadethemamarvelinmany differentindustries,itisthesesameuniquecharacteristicsthathavemadethema challengetodeterminetheirbehaviorandeffectsininvitrotoxicitytests.these uniquecharacteristicsalsochallengeresearchersassessingtheaquatictoxicityof CNTstobiota. AQUATICTOXICITY OnesuchcharacteristicofSWNTs,durability,isthereasonFergusonetal (2006)[44]assessedthelifecyclechronictoxicitytoAmphiascustenuiremis (estuarinemeiobenthiccopepod)overa28dayperiod.the28dayperiodis 18

35 characterizedbythreelifestagesofa.tenuiremis;alarval naupilarstage,a copepoditestage,andanadultstage.theyincludedrawswntsproducedbythe arc dischargemethod,purifiedfractionsoftherawmaterial,andlowmolecularweightnanocarbonbyproductsintheirtestingregime.thepurifiedmaterialwas madebyoxidizingwithnitricacidtoremovemetallicandcarbonaceousimpurities. Thissuspensionwasthenseparatedintothreesizefractionsusingelectrophoretic separation;alargesizefractionrepresenting53wt%oftheswntsuspension,an intermediatefractionrepresenting37wt%oftheswntsuspension,andasmall fractionthataccountedfor10wt%oftheswntsuspension.therewasdose dependentmortalityintherawswnttreatmentswiththehighestmortalityof36 ±11%observedinthe10mg/Ltreatmentatthenaupliarstageandcumulativelifecycle.The10mg/LSWNTtreatmenthadreduceddevelopmentsuccessto51%for thenaupliustocopepoditestage,89%forethecopepoditetoadultstage,and34% overallforthenaupliustoadultperiodwhichwassignificantlydifferentfrom controls.themortalitydataforthepurifiedmaterialwasmuchdifferent.there was13±2,3±0,and0±0%mortalityinthenaupliar,copepodite,andadultstage respectively.furthermore,alllife cyclemortalitieswerewithin5%ofcontrolsand noexposuresshowedanysignificantmortality.therewerealsonosignificant effectsondevelopmentsuccessfornaupliar to copepodite,copepodite to adult,and naupliar to adultlife stages.thenauplius to adultstagehadmortalitiesinthetwo highestexposuresbutwasonlysignificantforthe10mg/ltreatment.averagefull life cylcemortalitieswere81±7%inthe10mg/ltreatmentwhichwasdrivenby thecopepoditestagespecificmortalites.therewasalsosignificantlyreduced 19

36 developmentsuccessinthe0.97,1.6,and10mg/ltreatments.theassessmentof thedifferentswntproductsdeterminedthattherawswntsdidnotcausechronic toxicityinconcentrationslessthan1.6mg/l.thepurifiedswnts(thehighest molecularfractionafterelectrophoreticseparation)causedtochroniceffectseven atthe10mg/ltreatment.however,thelowestmolecularweightfractionofthe purifiedswntscausedtoxicresponsessimilartotherawswnts.thissuggests thatthelowestmolecularweightfractionmaybedrivingthetoxicresponseinthe rawmaterial.furthermoreitsuggeststhatparticlesizemaybeacontributing factorintheaquatictoxicityofswnts. OneofthefirstcommonstandardacuteaquatictoxicitytestswithCNTswas donebyrobertsetal(2007)[45]ondaphniamagna,afilter feedingcrustacean oftenusedforregulatoryenvironmentaltoxicitytesting.theysurfacecoatedthe particleswithlysophophatidylcholine(lpc)bysonicatingswntsina1:5ratio (SWNTs:LPC).Dmagnawereexposedfor96hrswithdailyrenewalsin concentrationsof0,2.5,5,10,and20mg/lswnts.therewasnomortalityin concentrations0,2.5,and5mg/lswntsbut20%and100%mortalityin concentrationsof10and20mg/lrespectively.duringthetoxicitytestsitwas observedthatablackprecipitatewouldformatthebottomofthetestchambersbut theprecipitatewasnotvisibleinthelpc SWNTstocksolutions.Theyhypothesized thatthed.magnawereingestingthematerialandusingthelpccoatingasafood source.oncestrippedofthelpccoatingtheswntswouldbeexcretedand precipitateoutofsolutionbecausetheylackedaqueousstabilitywithoutthelpc coating.totestthishypothesistheycarriedoutanexperimentsimilartotheone 20

37 describedabovewiththeadditionofreplicateswithoutorganisms.absorbance(at 360nm)oftestsolutionsweremeasuredevery6hrovera48hrperiodand comparedtoastandardcurvetodeterminelpc SWNTconcentration.They determinedthatconcentrationsdecreasedby50%oftheoriginallpc SWNT concentrationwhenorganismsarepresentanddecrease20%withoutorganisms. ThisresearchdemonstratesthatSWNTsfunctionalizedtohaveaqueousstability cancauseacutetoxiceffects.furthermore,itsuggeststhattheexposedorganisms havethepotentialtobiomodifytheswntstherebychangingthesurfacechemistry andbehaviorinaquaticsystems. TofurtherexaminetherelationshipbetweenCNTingestionandelimination byd.magnapetersenetal(2009)[46]assessedthebioaccumulationpotentialof acidtreatedmwnts.daphniamagnawereexposedwith0,0.04,0.1,or0.4ug/ml ofswntsandinexposurevolumesof30,100,or200ml.organismsweresample after1,4,10,24,and48hrexposure.nanotubeuptakewasmeasuredby radioactivityafterexposedorganismsweredried,weighed,andmixedwithultima Goldcocktail.TheydeterminedthatMWNTuptakeincreasesfrom0to24hrs,but levelsofffrom24to48hrs.furthermore,uptakeincreasedasexposuresolution volumeincreasedwith0.1ug/mlmwnts,after24hrsroughly10ugmwnts/mg daphniadrymassin30mlexposurevolumecomparedto45ugmwnt/mg daphniadrymassin200mlexposurevolume.whensolutionvolumewasheld constantat200ml,uptakeincreasedwithincreasingconcentrationsofmwnts. After24hrsexposureto0.04ug/mLMWNTsuptakewasjustabove10ug MWNTs/mgdapniadrymassand0.4ug/mLMWNTexposureapproached70ug 21

38 MWNTs/mgdaphniadrymass.However,whenthisdataisnormalizedbythe suspendednanotubesconcentrationsthe48hrbodyburdendataisnotsignificantly differentbetweendifferentexposureconcentrationssuggestinguptakeisnotdriven byexposureconcentration.depurationofingestedmwntswasdeterminedin varietyofconditions,cleanwaterandwaterwithnaturalorganicmatter(asfiltered lakewater).regardlessoftheconditions,therewasnodecreaseinconcentrations ofingestedmwntsafter24hrdepurationperiod.however,iftheorganismswere allowedtodepurateinthepresenceoffoodbodyburdensdecreased50 85%during thefirstfewhoursbutwereultimatelyunabletocompletelycleartheirgutsafter48 hrs.theseresultsdemonstratethepotentialofsignificantnanotubeaccumulation withlimitedabilityatdepuration.thissuggestsmwntsmayhaveastrong interactionwithd.magnagutthatcouldbedrivingtoxicityanduptake. Fromotherstudiesthroughouttheliteraturetoxicitydatasuggestsrelatively highconcentrationsareneededtoelicitatoxicresponsetoaquaticbiota.kennedy etal(2008)[47]testedtheaquatictoxicityofseveraltypesofmwnts.they determinedthatthe48hrsurvivalforceriodubiadaphniain39.5mg/lraw MWNTs,120.2mg/LOH MWNTs,and88.9mg/LCOOH MWNTswas7±12%,80± 20%,and100±0%.EventhoughtherawMWNTscausedasignificantdeclinein survivalitoccurredatarelativelyhighconcentration,andevenmoresurprisingis thesurvivalinthetworemainingfunctionalizedmwnts.kennedyetal(2009)[48] repeatedasimilarstudytofurtherexploretherelationshipofmwnttoxicityand surfacechemistry.theytestedthe96hrtoxicityofrawmwnts,oh MWNTs,C8 MWNTs(functionalizedwithanalkylgroups),andNH2 MWNTs(functionalized 22

39 withaminegroups)toc.dubia.resultsindicatedgreatertoxicitywiththec8 MWNTswith0%survivalat15mg/LandthegreatesttoxicitywithNH2 MWNTs with0%survivalat2mg/l.therawmwntsandoh MWNTswereleasttoxicwith 25%survivalat26mg/Land93%survivalat48mg/Lrespectively.Thisagain clearlyindicatessurfacechemistrymaybethedrivingforcewithcntaquatic toxicity.yet,inanotherstudychengetal(2007)[49]determinedthatrawswnts causednodevelopmentaleffectstozebrafishembryosuptoahighconcentrationof 360mg/L.Theydeterminedthatthechorionsurroundingtheembryowasan effectivebarriertotheswntsandthelittledelayindevelopmenttheydidseewas contributedtocatalystmetalspresentintheswnts.thisclearlyshowsadifference incnttoxicityamongaquaticspeciesusedinstandardtoxicitytesting. Theaforementionedaquatictoxicitytestsdemonstratethatittakes considerableconcentrationsofsuspendedswntsormwntstocauseatoxic response.theirdataalsostronglysuggestthatparticlesizeandsurfacechemistry haveadirectinfluenceontoxicitytoaquaticspecies.however,lower concentrationsofsuspendedcntsmayhaveaneffectonaquaticbiota.thesmall diameterandlongsizeofcntsmayenablethemtoabsorbacrosscellmembranes orbetakenupbyphagocyticpathwaysleadingtoeffectsonthecellularorgenetic level. CARBONNANOTUBEABSORPTIONACROSSCELLMEMBRANES Carbonnanotubeshavebeenusedinmanybiomedicalapplications,most notablyasdrugdeliverysystems.theresmalldiameterandlonglengthhavemade 23

40 themanattractiveplatformfordelivering payloads ofdrugstotargetedcellsand tissues.pantarottoetal(2004)[50]functionalizedswntswithfluorescein isothiocyanateorfluorescentpeptide.theyimagedthematerialinthecytoplasm andnucleusof3t3or3t6fibroblastsusingconfocalmicroscopy.insimilarwork ShiKametal(2005)[51]functionalizedoxidizedSWNTswithfluorescentlylabeled proteins.theywereabletoimagethematerialwithinhl60andjurkat nonadherentcellsandhelaandnih 3T3adherentcellsusingconfocalmicroscopy. Theysuggestthatuptakewasviaanendocytoticpathwaybecausetheinternalized SWNTswerelocatedwithinendosomes.InconflictingstudiesPantarottoetal (2004)[52]andCaietal(2005)[53]suggestinternalizationisendocytosis independentafterimagingcntswithincellsinthepresenceofanendocytosis inhibitor.theysuggestthemechanismofuptakeispassivediffusionacrossthelipid bilayerwithoutperforatingthecellmembraneandcausingcelldeath[54].these studiesshowthepotentialcntshaveasdrugdeliverysystems.theycanbe functionalizedwithdifferentproteins,internalizedincells,andabletodeliver drugs.however,italsoshowsthepotentialforuptakewhenexposedinthe workplace,orifbiotacomesintocontactwithreleasedcnts. AssumingthatreleasedCNTscouldenterthebodyWangetal(2004)[55] andsinghetal(2006)[56]intravenouslyexposedmicetotwodifferent functionalizedswntstodeterminebodydistribution.theyfoundthatthesnwts passedthroughthekidneysandwereclearedthroughtheurinewithlittleuptake. Furthermore,Singhetal(2006)[56]determinedthatthefunctionalizedSWNTshad ahalf lifeinthebodyofjustover3.5hrs.inacontradictorystudyshipperetal 24

41 (2008)[57]determinedthatPEG SWNTsthatwereinjectedintomicewere sequesteredintheliverandspleenandhadaresidencetimeofuptofourmonths. ImagingCNTswithincellshasbeensuccessfullydonewithavarietyof techniques.confocalmicroscopy[50 53],transmissionelectronmicroscopy [58,59],andnear infraredfluorescencemincroscopy[60]haveallbeenusedto imagetheinternalizationofcntswithincells.however,ininvivomodels determiningtheabsorptionofcntshasproventobeachallenge. Robertsetal(2007)[29]analyzedLPC SWNTuptakeinD.magnausing Micro Ramanspectroscopy.Ramanspectroscopycandeterminechemical compositionandmolecularstructureofsamplesusinglaserexcitation.single wall nanotubeshaveacharacteristicallylargeband,termedg band,uponlaser excitation.theg bandrepresentsthegraphite relatedvibrationalmodesofthe carbonbonds[61].whilethistechniqueprovedsuccessfulindeterminedlpc SWNTuptakeinthegut,itlackedtheresolutiontodetermineifanyofthematerial wasabsorbedintothebodytissuesofexposedorganisms. InanaquatictoxicitystudybySmithetal(2007)[62]juvenilerainbowtrout wereexposedtoswnts.theydeterminedrespiratorytoxicity,tissuepathologies, andotherphysiologicaleffects.after10daysofexposurefishweresacrificedand tissuesweretakenforhistology.theintestinaltissueswereexaminedanditwas foundthatat0.1mg/lswntstheepitheliumwithintheintestinesshowedevidence oferosionandat0.5mg/lswntstheintestinalvilliwerefused.therewerevisual precipitatedswntsimbeddedinthegutlumen,howeveritwasnotdeterminedif theywereabsorbedintothetissuesusinglightmicroscopy. 25

42 InoneofthefewtoxicitystudiesusinganamphibianmodelMouchetetal (2008)[63]exposedAfricanclawedfrogs(Xenopuslaevis)todoublewalled nanotubes(dwnts).larvaewereexposedfor12daysandsurvivingorganisms weresacrificedtodetermineuptakeofdwnts.usingmicroramanspectroscopy theywereabletoconfirmthatsomeoftheblackmaterialwithinthegutcontained ingesteddwnts.tofurtherexploretheingestedmaterialtheysectionedintestinal tissuesfortemimaging.usinghigh resolutiontransmissionelectronmicroscopy theywereabletoshownodifferencesinintestinalmorphologybetweenexposed andcontrolorganisms,buttheywereabletofindisolateddwntswithinthe intestinallumenofdwntexposedlarvae. InanonaquatictoxicitystudyLeeuwetal(2007)[64]fedfruitflylarvae (Drosophilamelanogaster)SWNTsanddeterminedbiologicalfate.Afterthelarvae emergedasadultstheorganismsweresacrificedandindividualtissueswere removedandfixedfornear infrared(nir)fluorescencemicroscopy.theyfound highconcentrationsofswntfluorescenceassociatedwiththebrainlobes.further strongswntfluorescencewasfoundinthegutanddorsalvessel.allothertissues hadlowswntfluorescencebuttheyconfirmeditwasfromsinglenanotubes throughtwotests.firstitwasdeterminedthattheintensityofeachspot semission wasdependentonthepolarizationorientationoftheexcitationbeam,whichis characteristicofswnts.second,theemissionspectraoftheindividualspotshad strongpeaksthatarecharacteristicofsemiconductingsnwts.inthiscasenir microscopysuccessfullyidentifiedswntsthathadbeeningested,absorbedacross thegut,anddistributedthroughouttheorganisms body. 26

43 PHYSIOLOGICALEFFECTSOFCARBONNANOTUBES Nanomaterialshavebeenshowtocauseothereffectsotherthanmortality anddecreasesindevelopmentorgrowth.lovernetal(2007)[64]determinedthe effectsofc60,hydrogenatedfullerenes(c60hxc70hx),andtitaniumdioxideonthe importantsurvivalbehaviorsandheartrateofdaphniamagna.thehydrogenated fullerenesandc60increasedhoppingfrequencyby121hopsperminuteand113 hopsperminuterespectively.titaniumdioxidehadnoeffectscomparedto controls.theonlychangestoheartratewerecausedbyc60,increasingtheaverage rateby43.6beatsperminute.appendagemovementwasincreasedbyc60and hydrogenatedfullerenesby64.51cyclesperminuteand61.66cyclesperminute respectively.itgenerallytooktheexposedorganisms30mintocompletelyreturn totheratesmeasuredpriortonanomaterialsexposure.thisclearlydemonstrates thatnanomaterialscancauseshort termeffectstod.magnathatmayhavelasting ecologicalimplications. Nanomaterialshavebeenshowntocauseotherphysiologicaleffectsaswell. Klaperetal(2009)[65]examinedtheeffectsoffullereneexposureand functionalizationonglutathione S transferase(gst)andcatalase(cat)inductionin Daphniamagna.Theseenzymesarecommonlyusedbiomarkerstodeterminean organism sphysiologicalresponsetoreactiveoxygenspecies(ros)producedby contaminantexposure.theytestedc60suspendedbytetrahydrofuran(thf), stirredc60,hydrogenatedc60(hx C60),andhydroxilatedC60(OH C60).Thehighest inductionofgstwasobservedinthefullerenetreatmentthathadbeensuspended with5ppmthf,almostathreefoldincreaseabovecontrols.gstlevelswerealso 27

44 increasedinstirredc60andoh C60treatmentsat100and500ppm.CATwasalso increasedsignificantlyfromcontrolsinsomeofthehx C60andOH C60treatments butitwasnotdosedependent.theseresultsindicatethatfullerenessuspendedin differentwaysorwithdifferentfunctionalgroupscancausetheproductionofros andsubsequentinductionofrosquenchingenzymesind.magna. Inaquatictoxicitystudiesithasbeenobservedthatthegutofexposed organismshadbeensignificantlyimpactedwithingestednanomaterials[29,46 48,62,63].ThisisespeciallythecaseforthefilterfeedingDaphniaspecies.Ithas beenshownthatwheninvertebratesareexposedtocontaminantsthereare physiologicalchangeswithintheguttoreducepossibleeffects.theperitrophic membrane(pm)isalayerfoundinthegutofinvertebratesmadeofproteins,chitin, polysaccharides,andothernonlivingmaterialthatsurroundsingestedmaterial[66]. Waterhouse(1954)[67]foundthattheDDTresistantBlowflystrainproduced9 timesasmuchpmasthesusceptiblestrainwhenexposedtothepesticideallowing forfasterelimination.infactthepmhasbeendescribedasprovidingmechanical protectiontothemidgutepitheliuminmanyinsectspecies[68].changestopm productionanddigestiveenzymeshavebeenfoundindaphniaspeciesexposedto differentenvironmentalconditions.dudychaetal(inprep)[69]founddifferences inthetranscriptionofgenesassociatedwithdigestionandothergutprocesses, includingtwopmgenes,whendaphniplucariawereexposedtodifferentqualities offood.itispossiblethatdaphniaspeciesmayalsohavealteredgeneexpression associatedwithdigestiveenzymesandgutprocesseswhentheirgutshavebeen impactedwithcarbonnanomaterials. 28

45 CONCLUSIONS Ithasbeenshownthatnanomaterialscancauseavarietyofeffectstomany differentspecies.weareonlyjustbeginningtounderstandthebehaviorthese uniquematerialsexhibitintheenvironmentandwithinbiota.carbonnanomaterials aredurableandresistanttodegradationandhavethepotentialtoremaininthe environmentforyearsifreleased.moreresearchisneededtofurtherexamine sublethal,chronic,andphysiologicaleffectstoorganismssowecanbetter determinethepotentialimpacttoecosystems. 29

46 REFERENCES 1.BritishStandardsInstitution:Nanotechnology. 7, KrotoHW,HeathJR,O BrienSC,CurlRF,SmalleyRE.1985.C60: Buckminsterfullerene.Nature.318: IijimaS.1991.HelicalMicrotublesofGraphiticCarbon.Nature.354: NationalNanotechnologyInitiative. 5.TheProjectonEmergingNanotechnolgies. September7, Ju NamYandLeadJR.2008.ManufacturedNanoparticles:AnOverviewoftheir Chemistry,Interactions,andPotentialEnvironmentalImplications.Scienceofthe TotalEnvironment.400: U.S.EnvironmentalProtectionAgencySciencePolicyCouncil NanotechnologyWhitePaper.EPA100/B 07/001.Washington,DC. 30

47 8.KlaineSJ,AlvarezPJJ,BatleyGE,FernandesTF,HandyRD,LyonDY,MahendraS, McLaughlinM,LeadJR.2008.CriticalReviewNanomaterialsintheEnvironment: Behavior,Fate,Bioavailability,andEffects.EnvironmentalToxicologyand Chemistry.27(9): FabregaJ,LuomaSN,TylerCR,GallowayTS,LeadJR.2011.SilverNanoparticles: BehaviourandEffectsintheAquaticEnvironment.EnvironmentInternational.37: NewmanMD,StotlandM,EllisJI.2009.TheSafetyofNanosizedParticlesin TitaniumDioxide andzincoxide BasedSunscreens.JournaloftheAmerican AcademyofDermatology.61(4): BalasubramanianSK,JittiwatJ,ManikandanJ,OngCN,YuLE,OngWY BiodistributionofGoldNanoparticlesandGeneExpressionChangesintheLiverand SpleenafterIntravenousAdministrationinRats.Biomaterails.31(8): LiuG,LiX,QinB,XingD,GuoY,FanR.2004.InvestigationoftheMending EffectandMechanismofCopperNanoparticlesonTribologicallyStressedSurface. TribologyLetters.17(4): ChenZ,MengH,XingG,ChenC,ZhaoY,JiaG,WangT,YuanH,YeC,ZhaoF,Chai Z,ZhuC,FangX,MaB,WanL.2006.ToxicologyLetters.163:

48 14.OliveiraJM,SalgadoAJ,SousaN,ManoJF,Reis,RL.2010.Dendrimersand DerivativesasaPotentialTherapeuticToolinRegenerativeMedicineStrategies A Review.ProgressinPolymerScience.35(9): FahmyTM,SchneckJP,SaltzmanWM.2007.ANanoscopicMultivalentAntigen PresentingCarrierforSensitiveDetectionandDrugDeliverytoTCells. Nanomedicine.3: WienerEC,KondaSD,WangS,BrechbielM.2002.ImagingFolateBinding ProteinExpressionwithMRI.AcademicRadiology.9: DuanX,McLaughlinC,GriffithM,SheardownH.2007.Biofunctionalizationof CollagenforImprovedBiologicalResponse:ScaffoldsforCornealTissue Engineering.Biomaterials.28: YoonHC,LeeD,KimH S.2002.ReversibleAffinityInteractionsofAntibody MoleculesatFunctionalizedDendrimerMonolayer:Affinity SensingSurfacewith Reusability.AnalyticaChimicaActa.456:

49 19.SnejdarkovaM,SvobodovaL,EvtugynG,BudnikovaH,KaryakinA,NikolelisDP AcetylcholinesteraseSensorsBasedonGoldElectrodesModifiedwith DendrimerandPolyaniline:AComparativeResearch.AnalyticaChimicaActa.14: WilsonMA,PangLS,WillettGD,FisherKJ,DanceIG.1992.Fullerenes Preparation,Properties,andCarbonChemistry.Carbon.30(4): SchurerGandPeukertW.2005.PredicitonofThermodynamicPropertiesfrom PureCompoundInformation:CharacterizationofFullerenes.AppliedSurface Science.252: XieB,XuZ,GuoW,LiQ.2008.ImpactofNaturalOrganicMatteronthe PhysicochemicalPropertiesofAqueousC60Nanoparticles.EnvironmentalScience andtechnology.42: DaenenM,deFouwRD,HamersB,JanssenPGA,SchoutedenK,VeldMAJ WondersouWorldofCarbonNanotubes:AReviewofCurrentCarbonNanotube Technologies.EindhovenUniversityofTechnology. 24.HirlekarR,YamagarM,GarseH,VijM,KadamV.2009.CarbonNanotubesand itsapplications:areview.asianjournalofpharmaceuticalandclinicalresearch. 2(4):

50 25.McEuenPL.2000.CarbonNanotubes:Single WallCarbonNanotubes.Physics World.June: AvourisP.2002.CarbonNanotubeElectronics.ChemicalPhysics.281: BaughmanRH,ZakhidovAA,deHeerWA.CarbonNanotubes TheRoute TowardApplications.Science.297(2): NiyogiS,HamonMA,HuH,ZhaoB,BhowmikP,SenR,ItkisME,HaddonRC ChemistryofSingle WalledCarbonNanotubes.AccountsofChemical Research.35: RobertsAP,MountAS,SedaB,SoutherJ,QiaoR,LinS,KePC,RaoAM,KlaineSJ InvivoBiomodificationofLipid CoatedCarbonNanotubesbyDaphnia magna.environmentalscienceandtechnology.41: KePC,QiaoR.2007.CarbonNanomaterialsinBiologicalSystems.Journalof Physics:CondensedMatter.19:

51 31.ZhaoB,HuH,HaddonRC.2004.SynthesisandPropertiesofaWater Soluble Single WalledCarbonNanotube Poly(m AminobenzeneSulfonicAcid)Graft Copolymer.AdvancedFunctionalMaterials.14: ZhaoB,HuH,YuA,PereaD,HaddonRC.2005.SynthesisandCharacterization ofwatersolublesingle WalledCarbonNanotubeGraftCopolymers.Journalofthe AmericanChemicalSociety.127: YamaguchiI,YamamotoT.2004.SolubleSelf DopedSingle WalledCarbon Nanotube.MaterialsLetters.58: HuN,DangG,ZhouH,JingJ,ChenC.2007.EfficientDirectWaterDispersionof Multi WalledCarbonNanotubesbyFunctionalizationwithLysine.MaterialsLetters. 61: TansSJ,DevoretMH,DaiH,ThessA,SmalleyRE,GeerligsLJ,DekkerC IndividualSingle WallCarbonNanotubesasQuantumWires.Nature.386(3): WislonM,KannagaraK,SmithG,SimmonsM,RaguseB.2002.Nanotechnology: BasicScienceandEmergingTechnologies.ChapmanandHall/CRCPress.NewYork 271pgs. 35

52 37.LamC W,JamesJT,McCluskeyR,HunterRL.2004.PulmonaryToxicityof Single WallCarbonNanotubesinMice7and90DaysAfterIntratrachealInstillation. ToxicologicalSciences.77: WarheitDB,LaurenceBR,ReedKL,RoachDH,ReynoldsGAM,WebbTR ComparativePulmonaryToxicityAssessmentofSingle WallCarbonNanotubesin Rats.ToxicologicalSciences.77: MullerJ,MuauxF,MoreauN,MissonP,HeileirJ F,DelosM,ArrasM,Fonseca, NagyJB,LisonD.2005.RespiratoryToxicityofMulti WallCarbonNanotubes. ToxicologyandAppliedPharmacology.207: JiaG,HaifangW,YanL,WangX,PeiR,TaoY,ZhaoY,GuoX.2005.Cytotoxicity ofcarbonnanomaterials:single WallNanotube,Multi WallNanotube,and Fullerene.EnvironmentalScienceandTechnology.39: DumortierH,LacotteS,PastorinG,MaregaR,WuW,BonifaziD,BriandJ P, PratoM,MullerS,BiancoA.2006.FunctionalizedCarbonNanotubesAreNon CytotoxicandPreservetheFunctionalityofPrimaryImmuneCells.NanoLetters. 6(7):

53 42.RajaPMV,ConnolleyJ,GanesanGP,CiL,AjayanPM,NalamasuO,ThompsonDM ImpactofCarbonNanotubeExposure,Dosage,andAggregationonSmooth MuscleCells.ToxicologyLetters.169: DavorenM,HerzogE,CaseyA,CottineauB,ChambersG,ByrneHJ,LyngFM InvitroToxicityEvaluationofSingleWalledCarbonNanotubesonHuman A549LungCells.ToxicologyinVitro.21: TempletonRC,FergusonPL,WashburnKM,ScrivensWA,ChandlerGT Life CycleEffectsofSingle WalledCarbonNanotubes(SWNTs)onanEstuarine MeiobenthicCopepod.EnvironmentalScienceandTechnology.40: RobertsAP,MountAS,SedaB,SoutherJ,QiaoR,LinS,KePC,RaoAM,KlaineSJ InvivoBiomodificationofLipid CoatedCarbonNanotubesbyDaphnia magna.environmentalscienceandtechnology.41: PetersenEJ,AkkanenJ,KukkonenJVK,WeberWJ.2009.BiologicalUptakeand DepurationofCarbonNanotubesbyDaphniamagna.EnvironmentalScienceand Technology.43: KennedyAJ,HullMS,SteevensJA,DontsovaKM,ChappellMA,GunterJC,Weiss CA.2008.FactorsInfluencingthePartitioningandToxicityofNanotubesinthe AquaticEnvironment.EnvironmentalToxicologyandChemistry.27(9):

54 48.KennedyAJ,GunterJC,ChappellMA,GossJD,HullMS,KirganRA,SteevensJA InfluenceofNanotubePreparationinAquaticBioassays.Environmental ToxicologyandChemistry.28(9): ChengJ,FlahautE,ChengSH.2007.EffectofCarbonNanotuubesonDeveloping Zebrafish(DanioRerio)Embryos.EnvironmentalToxicologyandChemistry.26(4): PantarottoD,BriandJ P,PratoM,BiancoA.2004.TranslocationofBioactive PeptidesAcrossCellMembranesbyCarbonNanotubes.ChemistryCommunications ShiKamNWandDaiH.2005.CarbonNanotubesasIntracellularProtein Transporters:GeneralityandBiologicalFunctionality.JournaloftheAmerican ChemicalSociety.127: PantarottoD,SinghR,McCarthyD,ErhardtM,BrianJP,PratoM,KostarelosK, BiancoA.2004.FunctionalizedCarbonNanotubesforPlasmidDNAGeneDelivery. Angew.Chem.Int.Ed.43:

55 53.CaiD,HuangZ,CamahanD,MatarazaJM,ChilesTC,QinZH,HuangJ,KempaK, RenZ.2005.HighlyEfficientMolecularDeliveryinotMammalianCellsUsing CarbonNantoubeSpearing.Nat.Methods.2: KlumppC,KostarelosK,PratoM,Bianco.2006.FunctionalizedCarbon nanotubesasemergingnanovectorsforthedeliveryoftherapeutics.biochimicaet BiophysicaActa.1758: WangH,WangJ,DengX,SunH,ShiZ,GuZ,LiuY,ZhaocY BiodistributionofCarbonSingle WallCarbonNanotubesinMice.Journalfo NanoscienceandNanotechnology.4(8): SinghR,PantarottoD,LacerdaL,PastorinG,KlumppC,PratoM,BiancoA, KostarelosK.2006.TissueBiodistributionandBloodClearanceRatesof IntravenouslyAdministeredCarbonNanotubeRadiotracers.Proceedingsofthe NationalAcademyofSciences.103(9): SchipperM,Nakayama RatchfordN,DavisCR,ShiKamNW,ChuP,ZhuangL, SunX,DaiH,GambhirSS.2008.APilotToxicologyStudyofSinge WalledCarbon NanotubesinaSmallSampleofMice.NatureNanotechnology.3: BiancoA,KostarelosK,PratoM.2005.ApplicationsofCarbonNanotubesin DrugDelivery.CurrentOpinioninChemicalBiology.9:

56 59.PulskampK,DiabateS,KrugHF.2007.CarbonNanotubesShowNoSignof AcuteToxicitybutInduceIntracellularReactiveOxygenSpeciesinDependenceon Contaminants.ToxicologyLetters.168: CherukuriP,BachiloSM,LitovskySH,WeismanRB.2004.Near Infrared FluorescenceMicroscopyofSingle WalledCarbonNanotubesinPhagocyticCells. JournaloftheAmericanChemicalSociety.126: JuurioA,SouzaAG,DresselhausG,DresselhausMS,SwanAK,UnluMS,Goldberg BB,PimentaMA,HafnerJH,LieberCM,SaitoR.2002.G BandResonantRaman Studyof62IsolatedSingle WallCarbonnanotubes.PhysicsReviewB.65: SmithCJ,ShawBJ,HandRD.2007.ToxicityofSingleWalledCarbonNanotubes torainbowtrout,(oncorhynchusmykiss):respiratorytoxicity,organpathologies, andotherphysiologicaleffects.aquatictoxicology.82: MouchetF,LandoisP,SarremejeanE,BernardG,PuechP,PinelliE,FlahautE, GauthierL.2008.CharacterisationandInVivoEcotoxicityEvaluationofDouble WallCarbonNanotubesinLarvaeoftheAmphibianXenopuslaevis.Aquatic Toxicology.87:

57 64.LovernSB,SticklerJR,KlaperR.2007.BehavioralandPhysiologicalChangesin DaphniamagnaWhenExposedtoNanoparticleSuspensions(TitaniumDioxide, Nano C60,andC60HxC70Hx).EnvironmentalScienceandTechnology.41: KlaperR,CragoJ,BarrJ,ArndtD,SetyowatiK,ChenJ.2009.ToxicityBiomarker ExpressioninDaphnidsExposedtoManufacturedNanoparticles:Changesintoxicity withfunctionalization.environmentalpollution.157: LehanMJ.1997.PeritrophicMatrixStructureandFunction.AnnualReviewof Entomology.42: WaterhouseDF.1954.TheRateofProductionofthePeritorphicMembranein SomeInsects.Aust.J.BiolScie.7: SudhaPMandMuthuSP.1988.DamagetotheMidgutEpitheliumCausedby FoodintheAbsenceofPeritrophicMembrane.CurrScie.57: Dudycha,J.L.,Brandon,C.S.,Deitz,K.C.PopulationGenomicsofResource Exploitation:InsightsfromGeneExpressionProfilesofDaphniaFedAlternate Resources.InPrep. 41

58 CHAPTER2:THEINFLUENCEOFNATURALORGANICMATTERONTHE TOXICITYOFMULTIWALLEDCARBONNANOTUBES ABSTRACT Engineeredcarbonnanostructures,suchasmulti walledcarbonnanotubes (MWNTs),areinherentlyhydrophobicandarenotreadilystableinaqueousmedia. However,theaqueousstabilityandbioavailabilityofthesenanotubesmaybe influencedbythewaterqualityparameterssuchasionicstrength,phandnatural organicmatter(nom).nomadsorbsontothesurfaceofmwntseffectively coveringthehydrophobicsurfaceandresultinginincreasedaqueousstability.this enhancedstabilityislikelytoleadtoanincreasedresidencetimeinthewater columnandincreasedexposuretimesforpelagicorganisms. InthepresentstudyNOMfromthreedifferentriversystemsintheSoutheast USincreasedthestabilityofMWNTsuspensions.Theeffectsofthesesuspensions wereevaluatedusingacuteandchronicbioassayswithdaphniamagnaand Ceriodaphniadubia.The96hLC50forD.magnaexposedtoMWNTssuspendedin SuwanneeRiverNOMwasapproximately2.0mg/Landwasnotsignificantly influencedbynomconcentrationsrangingfrom mg/Ldissolvedorganic carbon(doc).however,thereweredifferencesin96hlc50valuesamong differentsourcesofnom(suwannee,black,andedistoriver).daphnidgrowthwas reducedinbothd.magnaandc.dubiawhilereproductionwasreducedinc.dubia. CharacterizationofthedifferentNOMsourcesandMWNTsuspensionswas conducted.visualinspectionusingtransmissionelectronmicroscopy(tem)and 42

59 guteliminationobservationssuggestedthatthetoxicitywasduetoingested MWNTscloggingtheguttractofD.magna.TheTEMmicrographsindicatedthat MWNTscandisaggregatewithintheguttractbutsingleMWNTsareunableto absorbacrossthegutlumen. INTRODUCTION Nanotechnologyisarapidlyevolvingfieldthatpotentiallyimpactsevery sectorofsociety.presently,thereareover800commerciallyavailableproducts containingsometypeofnanotechnology( industryisexpectedtoexceed$1trillionannuallyby2015[2].currentandfuture commercialusesincludetitaniumnanoparticlesforsunscreensandpaints,silica nanoparticlesassolidlubricants,carbonnanotubesinautomobilebumpersand tennisracquets,andaluminananoparticlesinshampoos,detergents,and antiperspirants[3]. Manufacturedcarbonnanomaterialsincludecarbondots,fullerenes, nanowires,nanocoils,andnanotubes.aspotentialusescontinuetogrowsowill production.duringapressreleaseinjune2009,cnano,announcedthatitsplantin Chinawascomingtofullcapacityproductionof500tonsofmultiwalled nanotubes/yr( environmentalregulations,chemicalcontaminantsfrommanufacturingprocesses areroutinelydepositedintoaquaticecosystems.frombiomedicaltomaterials scienceapplications,increasedproductionanduseofthesematerialswillresultin increasedprobabilityofnanoparticlesenteringtheaquaticenvironment[4]. 43

60 Carbonnanotubes(CNTs)arepurecarbonmacromoleculesformedfrom sheetsofcarbonatomscovalentlybondedtoformaone dimensionalhollow cylindricalshape[5].therearetwoclassesofcnts:singlewalled(swnts)and multiwallednanotubes(mwnts).mwntsarecomprisedofmultipleswntsthat areconcentricallyplacedwithineachotherakintoarussiandollclose configuration[6]. Engineeredcarbonnanoparticlesareinherentlyhydrophobicand,as produced,donotformstablesuspensionsinaquaticecosystems.however,recent researchhasfocusedonsurfacemodificationoftheseparticlestofacilitatetheiruse inaqueousapplicationsincludingbiomedicalimaginganddrugdelivery[7,8].by increasingthepolarityofthecarbonnanoparticlesurface,thelikelihoodof agglomerationandaggregationisdecreased.forexample,stablesuspensionsof fullerenesareproducedaftersurface modificationwithphenolicacids[9]and SWNTshavebeensurface modifiedwithlysophospholipids[10,11],copolymers [12,13],andotherorganicmolecules[14,15]toobtainstablesuspensions. Furthermore,asisthecasewithmanyaquaticcontaminants,theaqueousstability andbioavailabilityofthesecompoundsmaybeinfluencedbywaterchemistry parameterssuchasionicstrength,phandnaturalorganicmatter(nom). Engineeredcarbonnanotubeshavebeenshowntoresultininflammation, fibrosis,andoxidativestressininvivoandinvitromodels[16].robertsetal.[10] demonstrateddose relatedacutetoxicityofsurfacemodifiedswntstod.magna. SurfacemodifiedMWNTswerealsoshowntoincreasethebioavailabilityofCutoD. magna[17]. 44

61 NOMisacomplexmoleculethatresultsfromthedecompositionofvegetative materialinthewatershed;itvariesinmolecularweightandchemicalcharacteristics withbothhydrophilicandlipophiliccomponents.nomhasbeenshowntoincrease thesolubilityandtransportofsparinglysolubleorganics[18]andreducethe bioavailabilityofcopperandsilverions[19 21].ThelipophiliccomponentsofNOM driveadsorptionontomwntsinnaturalwaters[22].hyungetal.[23] demonstratedthatthestabilityofaqueousmwntsuspensionswasincreasedinthe presenceofnom.further,thestabilityofthissuspensionwasinfluencedby solutionphandionicstrength[24].chappelletal.[25]alsosuggestedthatmwnt stabilitymaydifferwithdifferentnomsources.thisincreasedstabilityislikelyto leadtoincreasedmwntconcentrationsandprolongedmwntresidencetimesin thewatercolumnand,thus,increasedriskofexposureandpotentialfordeleterious toxicologicaloutcomes. ThegoalofthepresentstudywastocharacterizetheinfluenceofNOMonthe acuteandchronictoxicityofmwntstod.magnaandc.dubia.specificattention wasgiventocharacterizingthenomsolutionsaswellasthemwntsuspensionsin anattempttoexplaintheobservedeffects. MATERIALSANDMETHODS Organisms D.magnawereobtainedfromculturesmaintainedinEPAmoderatelyhard reconstitutedwater(mhw)[26,27]attheinstituteofenvironmentaltoxicology, ClemsonUniversity(CU ENTOX)(Pendleton,SC,USA).Routine reference acute 45

62 toxicity tests have been performed with this culture to ensure consistent culture sensitivity to sodium chloride. Results are available from CU-ENTOX through the corresponding author. C.dubiawereobtainedfromanexistingcultureattheInstitute ofappliedsciences,universityofnorthtexas(denton,tx,usa)maintainedin MHW. MWNTs MWNTsweresynthesizedatClemsonUniversityusingthe thermalchemical vapordepositionmethodviathedecompositionofaferrocene xylenemixture[28]. AsproducedMWNTshadanapproximatediameterof25nm,lengthof approximately50µm,andanapproximatepurityof>95%(impuritiesare remainingcatalystmetalsandamorphouscarbon).mwntsuspensionswere characterizedasdescribedbelow. NOMSources ThisresearchusedthreedifferentsourcesofNOM.SuwanneeRiverNOM (SR NOM)waspurchasedfromtheInternationalHumicSubstancesSociety(St.Paul, MN,USA);thisproductwasconcentratedbyreverseosmosis(RO)toapowder. NOMfromtwocoastalSouthCarolinarivers,BlackRiver(BR NOM)andEdisto River(ER NOM),wereisolatedbyROtoproduceaconcentrate[29].ER NOMand BR NOMsolutionsweremadebydilutingtheROconcentratewithMilli Qwater (Millipore,Bedford,MA,USA)thenaddingsalts(MgSO4,CaSO4. 2H2O,NaHCO3and KCl;FisherScientific,Pittsburgh,PA,USA)toreachthedesiredhardnessand alkalinity.sr NOMsolutionsweremadebydissolvingthedrymaterialinMHW. 46

63 EachNOMsolutionwasfilteredwitha0.2µmcellulosemembranefilterpriortouse. TheorganiccarboncontentofeachNOMsolutionconcentrationswasdetermined byacidifying(ph1 2)a30 mlsampleandanalyzingitonatotalorganiccarbon (TOC)analyzer Vwithcorrespondingautosamplerautomaticsampleinjector (Shimadzu,Columbia,MD,USA). MWNTSuspensions MWNTsweresuspendedinNOMsolutionsusingthefollowingprocedure: (1)theywerefirstweighedonwaxedweighpaperandplacedina100mlglass centrifugetube;(2)25mlsofnomsolutionwereaddedtothecentrifugetubeand thesolutionwassonicatedwithafishermodel300sonicatorwitha1/8 microtip (FisherPittsburgh,PA,USA)for15minwithanoutputof300watts;(3)twenty five mlaliquotsofdilutionwater(containingnom)wereaddedandthesolution sonicatedforadditional15minintervalsaftereachadditionuntilthesolution reachedatotalvolumeof100mlwithacumulativesonicationtimeof1h;(4) solutionswereallowedtosettleforapproximately24handthesupernatant(stable suspensionfortesting)wasremovedwithaglasspipette;and(5)sedimented MWNTswerequantifiedgravimetrically.FinaltestconcentrationsofNOM MWNT wereachievedbysonicatingappropriatevolumesofnom MWNTsupernatantstock solutionindilutionwatercontainingnom.theamountofsedimentedmwntswas determinedgravimetricallyusingpre rinsed,oven dried,andweighed0.2µm cellulosemembrane.thesolutionremaininginthetesttubeafterthestablemwnt suspensionwasremoved,rinsedontopreweighedfilterwithmilli Qwater,and 47

64 weighed.thisweightwassubtractedfromtheinitialweighedofmwntsand dividedbythevolumeofthestablestocksolutiontodeterminetheinitialstock concentrationforbioassays.solutionswerepreparedfreshdailyandthe concentrationsfortestsetupandeachrenewalwereaveragedtodeterminean averageexposureconcentration. Bioassays D.magnaacute96hstaticrenewalbioassayswereperformedfollowingUS EPAmethods[26,27]withslightmodificationusingadilutionseriesofMWNTsin NOM.NOMsolution(withoutMWNTs)andMHWwereusedascontrols.Fifteenml ofeachtreatmentofcontrolsolutionwasaddedto30mlglassbeakertestchambers andfive<24holdorganismswereplacedineachtestchamber.eachbioassayhad atotalof5concentrationsreplicated3times.testsolutionswerereneweddaily, survivalmonitored,andorganismsfed0.25mlsofa4:3algae/ytcmixture2 4h priortotestsolutionrenewal.growthofd.magnaneonateswasmeasuredafter96 hasdryweightonakahnelectromicrobalance.theexperimentaldesignused nominaldocconcentrationsof15mg/lforblack,edisto,andsuwanneerivernom forthefirstsetoftests,andnominalconcentrationsof2,5,10,and20mg/lasdoc forsuwanneerivernominthesecondseriesoftests. Guteliminationassayswereconductedusing72holdD.magna.Organisms wereexposedtomwntsinsr NOM,BR NOM,andER NOMsolutions,preparedas describedabove,for24h.afterwhichtimetheywereallowedtoeliminatethe MWNTsinNOMorMHWsolutionsinthepresenceorabsenceoffoodtodetermine 48

65 theeffectsofnomandfoodontheeliminationofthematerialfromtheguttract. Organismswereimagedunderadissectingmicroscopeat2,4,8,12,24,and28h postexposuretodeterminetime to gut elimination. C.dubiaacuteandchronicbioassayswereperformedfollowingstandardUS EPAprocedures[26,27].Theexperimentaldesignfortheacutebioassayswas similartothatdescribedford.magnawithmortalityastheendpoint.c.dubia chronictoxicitywasmeasuredusingreproductionandgrowthasendpoints.asingle <24holdneonatewasplacedineachtestchambercontaining20mloftestsolution (MWNT NOM,NOM,orMHW;n=5replicatespertreatment).Organismswerefeda mixtureofalgae YTCandtestsolutionreneweddaily.Survivalandreproduction weremonitoredoveraseven daytestperiod.growthwasmeasuredasdryweight onakahnelectromicrobalance. Inallbioassays,dissolvedoxygen(DO),pH,andtemperatureweremeasured ineachtreatmentaftertherenewal.dowasmeasuredusingathermoorion4 STARmeterwithportableDOprobe(Waltham,MA,USA).MeasurementsofpH wereconductedusingathermoorionphprobeconnectedtoamultichannel ph/mvthermoorionmodel710a+meter(waltham,ma,usa).allprobeswere calibratedbeforemeasurementsweretaken.temperaturewastakenwithan alcoholthermometer.waterhardnessandalkalinityweremeasuredinthemhw controlandnomsolutioncontrolattestinitiation.waterhardnesswasmeasured bytitrationwith0.01nethylenediaminetetraacetate,andalkalinitywasmeasured bytitrationwith0.02nh2so4. 49

66 MWNTandNOMCharacterization HydrodynamicdiameteroftheMWNT NOMsolutionswereanalyzedby dynamiclightscattering(dls)onazetapalszetapotentialanalyzerwitha90plus BI MASmulti angleparticlesizingoption(brookhaveninstrumentscorp., Holtsville,NY,USA)usingarefractiveindexof1.12[30].DLSwasconductedin varioussr NOMconcentrationsandinBR NOMandER NOMtodeterminethe effectofnomconcentrationandsourceonmwntparticlesize. ZetapotentialwascalculatedusingaMalverninstrumentsZetaSizer (Worcestershire,U.K.)NanoSeriesmodelZSwithaDTS1060Ccleardisposablezeta cell.arefractiveindexof1.12andabsorbtioncoefficientof39.92wasusedto calculatedsurfacecharge[31]. NOMcompositionwascharacterizedbynuclearmagneticspectroscopy (NMR).SamplesofEdistoandBlackRiverwaterwerefreezedriedonaVirTis freezedryermodelnumber7.0ldbtes 55(Gardiner,NY,USA)toobtainsolid NOMforanalysis. NOMhydrophilicitywasdeterminedusingthespecificUV absorbance(suva) methoddescribedbymatilainenetal[32].asuvavalueisdeterminedby: (Absorbanceat254nm/DOC(mg/L))*100 ASUVAvalue>4indicatesmainlyhydrophobicmaterialwhileaSUVAvalue<3 indicatesmainlyhydrophilicmaterial.absorbancewasdeterminedusinga ShimadzuUV 2501PCspectrophotometer. TodeterminestructuralcompositionoftheNOMfluorescencewas determinedat5mg/ldocforallnomsourcesusingaptiphotontechnologiesinc 50

67 fluorescencespectrometer.a1cmcuvettepathlengthwasusedwithexcitationand emissionslitssetat2and4nmrespectively.excitationwasdeterminedfrom200 nmto520nmat5nmstepsandemissionwasdeterminedfrom230nmto550nm at1nmsteps.allsolutionshadaphof8.1andwereatambienttemperatureswhen analyzed.peakfluorescenceandtheratioofemissionintensitiesat450nmand500 nmwasdeterminedatexcitation370nmtocharacterizethehumic likeorfulviclikecharacteristicsandtodistinguishterrestrialversusautochthonoussources[33]. VisualexaminationofrawMWNTsandpostsonicatedMWNTswere conductedandimagedonahitachi4800scanningelectronmicroscope(sem)with electrondispersionspectroscopy(eds)at5kv.rawmwntswereplacedonasem stubcoveredindoubledbackedcarbontape.adropofmwnt NOMandMWNT MHWsolutionwasplacedonaSEMstubcoveredindoublebackedcarbontapeand allowedtodry.allsemsamplesweresputtercoatedfor80swithplatinumpriorto viewingonthemicroscope.asampleofcontrolsr NOMsolutionwasdriedona SEMstubcoveredindoublebackedcarbontapeandanalyzedwithMWNT MHW andmwntsr NOMsamplesusingEDSontheHitachi4800. SolutionsamplesforIRspectroscopy(MWNT NOMandMWNT MHW)were driedinaluminumweighboatsforanapproximate3mgsolidsample.thesolid samplesandapproximately3mgofrawmwntswerethenmixedwith50mgof KBrpowder,whichwerethenpressedintoa5mmdiameterpellet.Thesesamples wereanalyzedonabrukerfouriertransforminfraredspectrometermodelifs 66v/sequippedwithadeuteratedtriglycinesulfateddetector(Billerica,MA,USA)to collecttheinfraredabsorptionspectrumintherangeof cm 1. 51

68 D.magnaexposedtoMWNTsfor96hwerefixedina1.25%gluteraldehyde and1%paraformaldehydesolutionovernightand,afterdehydration,fixedin EMBED812Resin(ElectronMicroscopySciences,Hatfield,PA,USA).Aftertheresin driedfor72hat60 Ctheblocksweresectionedonanultramicrotome.Sectionsof theorganism sguttractwereplacedonformvarfilm200meshcoppertemgrids (ElectronMicroscopySciences,Hatfield,PA,USA).Thesectionswereexaminedand imagedonahitachi7600temat120kv. DataAnalysis AllstatisticalanalyseswereconductedusingSASsoftware(SASInstituteInc. Cary,NC).Differencesbetweentreatmentsingrowthdataweredeterminedusing AnalysisofVariance(ANOVA)followedbyaTukey spost hoctest(α=0.05). Concentration growthdatarelationshipswereanalyzedbyregressingmeanmass perreplicateagainstlog transformedmwntconcentration.lc50valuesforacute toxicitydataweredeterminedusingatrimmedspearmankarbertest.differences inlc50valuesweredeterminedusinganf testfollowedbypair wiset testsrunin Microsoft Excel asamicrosoft VisualBasicmacro(α=0.05). Results Bioassays Therewasdose dependentmortalityind.magnaexposedtomwnt suspendedinnom.however,96hlc50valuesdidnotvaryasafunctionofsr NOMconcentrationasmeasuredbyDOC(Table1).However,96hLC50valuesdid 52

69 varyamongnomsourcewithmwntsuspendediner NOMbeingsignificantlyless toxic(table1;p<0.05).d.magnagrowthover96hwasinverselyrelatedto MWNTconcentrations(Fig.1;r 2 =57%;p<0.01). Resultsoftheeliminationbioassayssuggestedthatguttracteliminationof MWNTsincreasedinthepresenceoffood,butthatthepresenceofNOMhadno effectontime to elimination.thoseorganismsthatwereexposedtomwntsinsr NOMandER NOMhadfastereliminationtimesinthepresenceoffoodthanthose organismsexposedtomwntsinbr NOMandallowedtoeliminateinthepresence offood(table2).interestingly,d.magnaexposedtomwntsinthepresenceofer NOMhadshortereliminationtimesthanthoseexposedinthepresenceofother NOMsourceswheneliminationoccurredinMHWintheabsenceoffood. MWNTswerenotlethaltoC.dubiaatthetestedconcentrations(0 1mg MWNT/L)duringthe7dexposure.Meansurvivalwasgreaterthan85%inall treatments.reproductionwassignificantlyreducedatallmwntconcentrationsby atleast20%(fig.2;p<0.05).furthermoretherewasadecreaseingrowthafterthe 7dexposure(Fig.3;p=0.045). NanoparticleSuspensionCharacterization MWNTssuspendedinBR NOMhadsignificantlysmallerhydrodynamic diametersthanthosesuspendedinsr NOMwhilethosesuspendedinER NOMhad significantlyhigherhydrodynamicdiameters(table1).however,withinthesame source(suwanneeriver)theconcentrationofnomdidnotinfluenceparticlesize withtheexceptionofthehighestconcentrationof18.8mg/lthatresultedin 53

70 significantlylargerhydrodynamicdiameters.therewasnotrendinzetapotential withincreasingconcentrationofsr NOM(Table1).Likewise,NOMsourcedidnot influencezetapotentialofthemwntsuspensions. NMRspectroscopyanalysisoftheNOMsourcesindicateddifferencesamong thethreesources(table3).however,thesedifferencesdidnotexplainthe differencesintheacutetoxicityvaluesobservedamongthenomsources.suva valueswere4.9,4.5,and5.1forblackriver,edistoriver,andsuwaneerivernom respectively.thisindicatesthatallthenomsourcesweremainlyhydrophobicwith thesuwaneeriversourcebeingslightlymorehydrophobic. Fluorescencemaximumpeakintensitieswithexcitationof nmand emissionof nmarecharacterizedasfulvic likeandmaximumpeak intensitieswithexcitationof nmandemissionof nmarehumiclike[33].BR NOMpeakintensity(excitation340,emission385),ER NOMpeak intensity(excitation340,emission380),andsr NOMpeakintensity(excitation 355,emission400)suggestallsourcesaremorefulvic like(figure4).aratioof emissionintensitiesat450and500nmof1.5orlowerindicatesterrestrialnom sourcewhilearatioabove1.5indicatesautochthonoussources.theratioforbr NOM,ER NOMandSR NOMwas1.67,1.73,and1.48respectively.Thissuggests thatbr NOMandER NOMarederivedfromautochthonoussourceswhileSR NOM isderivedfromterrestrialsources. TheSEMmicrographofrawbulkMWNTsshowedauniformedcompact material(fig.5a).semmicrographsofmwntssonicatedinmhwandsr NOM solutionshowedmwntsthatwerenolongerinabulkformandseemtohavebeen 54

71 brokenintoshorterlengthsfromsonication(fig.5bandc).duringsemimaging EDSanalysisofSR NOMsamplesandsamplesofMWNTssonicatedinSR NOM solutionwasconducted.theelementalcompositionsuggeststhatthematerialon themwntsasseeninfigure5cisadsorbedsr NOM. IRspectroscopywasconductedonNOMsamplesandMWNT NOM complexestodeterminechangesinnomfunctionalgroupconcentrationsafter adsorption.whilesomefunctionalgroupsinthenomsourceswereidentified(fig. 6),thismethodwasnotsensitiveenoughtodetermineconcentrationofthose functionalgroups.changesinpeakheightandwidthinirspectraformwnt NOM andnomalonesuggestqualitativechangesinnomfunctionalgroupconcentration afteradsorptionontomwnts. FixedorganismsfromcontrolandlowestMWNTtreatmentsweresectioned fortemanalysis.thepurposeofimagingsectionsoftheguttractsofthese organismswastovisuallycharacterizethefateofthematerialonceingestedbythe organism.controlorganismshadrelativelycleanguttractswithalgalcells(s. capricornutum)embeddedwithinthemicrovilliofthegutlumen(fig.7aandb). OrganismsexposedtoMWNTshadindividualtubebundleswithintheguttractand upagainstthelumenmicrovilli,butnotimbeddedwithinthemicrovilliorupagainst theguttractlumen(fig.7candd). Discussion MWNTssuspendedinNOMwereacutelytoxictoD.magnaandtheaverage 96hLC50valueforallthetestswithSR NOMwasapproximately2mg/L(Table1). 55

72 ThereweremixedconclusionsintheliteratureregardingMWNTtoxicity.Kennedy etal.[34]exposedc.dubiatomwntsstabilizedin100mg/lsr NOM.The solutionswerepreparedeitherbymagneticstirringorsonication.aftera96h exposureofc.dubiatothemwnt NOMsolutionstheydetermineadifferencein toxicitybetweenthestirredandsonicatedsolutions.thestirredmwntsolutions weremoretoxic(96hlc5017mg/lmwnts)thanthesonicatedmwntsolutions (96hLC5021mg/LMWNTs).However,thisrelationshipwasreversedwhen HyalellaaztecawasexposedtothepreparedMWNTsolutions.Fractalanalysisof MWNTTEMimagessuggestedthedifferencesintoxicitymaybeduetothe organisms responsetoenhancedsurfaceroughness,exfoliation,andfragmentation inthesonicatedmwnt NOMsolutions. Olasagastietal.[35]foundthat16mg/Lofcarboxyl functionalizedmwnts stabilizedbytween20(160mg/l)caused95%immobilizationofd.magnaafter48 h.althoughthetween20didnotcontributetoanyimmobilization(testedasa carriercontrol)therewasnodeterminationonitseffectstoparticlesize,surface chemistry,ortoxicitywhencombinedwithmwnts.theyalsofoundthatexposure of12mg/lmwntsstabilizedbytween20(160mg/l)causedupto45%mortality todaniorerioembryosafter48h.however,tween20controlcaused20% mortalityafter48haswell. TherewasnodifferenceinacutetoxicityamongMWNTsuspensionsinSR NOMconcentrationsrangingfrom mg/LDOC.Thissuggeststhatthe MWNTsurfacebecamesaturatedwithNOMatverylowconcentrationsofDOC. Further,zetapotentialdidnotchangesuggestingthattheadditionofmoreNOMin 56

73 thesolutionphasedidnotresultinachangeinsurfacecharge.therewasa significantincreaseinhydrodynamicdiameteratthehighestnomconcentration suggestingthatadditionalnomadsorptionontothemwntstookplaceinthis treatment.thetoxicityresultsalsowerenotinfluencednomconcentration increases. TheMWNT EdistoRiverNOMcomplexwaslesstoxicthanMWNT NOM complexesproducedfromtheothertwonomsources.unfortunately,this differenceinacutetoxicitycouldnotbeexplainedfromresultsofeithertheparticle suspensioncharacterizationorthenomcharacterization.hydrodynamicdiameter ofthemwntsiner NOMwassignificantlylargerthantheothertreatmentsat approximately15mg/ldoc.however,mwntssuspendedin18.8mg/lsr NOM hadsimilarhydrodynamicdiametersbutdidnothavesimilarreductionsintoxicity suggestingthatthisdidnotaccountforthetoxicitydifferencesamongthenom sources. VisualinspectionusingTEMsuggestedthattheacutetoxicityofMWNTswas aphysicaleffectduetocloggingofthedaphnidguttractsuggestinginterference withfoodprocessing.thisobservationwasreportedbyrobertsetal.[10]ford. magnaexposedtolysophospholipidcoatedswnts.thephysicaleffectsobserved arealsosimilartothosereportedford.magnaexposedtocolloidalclay[36].while Robinsonetal.[36]reportedgutclearancetimesofapproximately30min,D.magna requiredaslongas28htocleartheingestedmwntsinthepresentstudy.these observationssupportthehypothesisthattoxicityofingestednanoparticlesto daphnidsmaybeafunctionofinterferingwithfoodprocessingbythedaphnids. 57

74 Thishypothesisisfurthersupportedbytheobserveddifferencesingrowthratesin MWNTexposeddaphnids.Whilenopreviousresearchhasreportednanoparticle interferencewithfoodassimilation,kennedyetal.[37]reportedthattheingestion ofalgaewasnecessaryfortheeliminationofmwntsfromc.dubia.resultsofthe presentstudysupportthisconclusiondemonstratingthatthepresenceoffood decreaseseliminationtimeford.magnaexposedtomwnts.guttractelimination timeisacriticalparameterintherecoveryofaquaticorganismsfromparticle exposures[38].shorterparticleresidencetimesintheguttractwouldsuggestless interferencewithfoodassimilation.insimilarresearchusingterrestrialorganisms Petersenetal.[39]foundthataftera24hpurgingintervalEiseniafoetida (Earthworms)stillhadcarbonnanotubes(singlewalledandmultiwalled)intheir guts.thepresentclearancedata,whilesuggestive,arenotdefinitivesinceitwas onlypossibletodoonereplicateforeachtreatment.clearly,moreresearchis neededtofurtherquantifytheinfluenceofwaterqualitycharacteristics,suchas NOM,andthepresenceoffoodontheeliminationofMWNTsandotherparticles fromtheguttractofaquaticorganisms. ThepresentresearchhadasimilarD.magnaacutebioassaydesignasKimet al.[17]whoexploredacutetoxicityandpossibleroseffectscausedbymwnts stabilizedinnom.the96hlc50inthepresentstudydoesnotdiffergreatlyfrom theirdata.however,organismsexposedto0.5and1.0mg/lmwntsinsr NOM solutionsof21mgc/lshowednoincreaseinrosproductioncomparedtomhw andsr NOMcontrols.Thus,therolebetweenNOM,MWNTs,andROSproduction causingtoxiceffectsisstillunclear. 58

75 TEMdidnotrevealMWNTscrossingthegutlumen.Imagessuggestedthat themicrovilliofthegutkeptthelongstrandsofmwntsfrompenetratingthegut lumen.someinvitrostudies[40 42]usingMWNTsandanamphibianmodel[43] usingdoublewallednanotubessuggestedthatcarbonnanotubescrossedcell membranes.but,petersenetal.[38],usinglightmicroscopy,alsofoundthat MWNTsdidnotabsorbintoD.magnatissues. SEMimagesusingEDSanalysisconfirmedtheadsorptionofNOMand visualizedtheeffectsofsonicationonmwnts.thesonicationenergyhadbroken themwntsintoshorterlengthsbutseparatedthematerialfromabulkproductand aidednomadsorption.theaffectsofshorterlengthsontoxicityinunknown,butall ofoursamplesweresonicatedinequaldurationstoproducecomparablesamples. ThesampleprepforSEMsamplesinvolveddryingthesampleinanoven. DuringthisdryingprocesstheNOMandMWNTsmostlikelyconcentratedforming largerparticlesofmwntsandpossiblecrystallizationofnom,thisultimately createdascenarioforthemwntsandnomthatisnotcomparabletowhenthey wereinsolution.but,thesemimagesdidhelpvisualizewhathappenedtothe materialaftersonicationandthepossibleparticleconformation. TheMWNT NOMcomplexwascharacterizedusingSEM,DLS,andIR spectroscopy.thesemmicrographsshowedvisiblechangesinthemwnts suggestingacoatingbythenom.edsanalysisfurthersuggestedthatthiscoating wasnom.whileparticlesizefromsemmicroscopyisnotreliablebecauseofthe inherentaggregationasthesampleisdehydrated,dlscangiveusarelativeparticle sizebetweendifferentnomsolutions.however,dlsparticlesizeisnotthetrue 59

76 particlesize.itistheradiusofasphericalparticlethathasanequaldiffusion coefficientasthesample. Althoughallofourparticlecharacterizationmethodswereunableto determinethecauseofthedifferenceintoxicitybetweennomsources,theywere importantinelucidatingtherelationshipbetweenmwntsandnom.thenom characterizationmethodswerealsounabletodeterminethecauseofthedifference intoxicitybetweennomsources.whilesr NOMisaterrestrialbornNOM, indicatingmorearomaticgroups,andbr NOMandER NOMareautochthonous bornnomallsourcesarefluvic likewhichdoesnotindicatewhymwntsiner NOMarelesstoxic. ResultsofthepresentresearchsuggestthatstableNOM MWNTsuspensions aremoretoxicthanpreviouslyreportedandthatthistoxicitymightberelatedto cloggingoftheguttract.furtherresearchisneededtoquantifytherelationship betweenguttractresidencetimeandtoxicity.inaddition,theseresultsunderscore theneedforstable,well characterizednanoparticleexposuresinaquatictoxicity assessmentsinordertofacilitatecomparisonsamongotherresearch.ultimately,to properlydeterminelongtermeffectsofthesematerialsitwillbeimportanttoasses therolethatparticlecharacteristicshaveonbioavailability,absorption,distribution, excretion,andtoxicity[4]. 60

77 REFERENCES 1. CenterforScholars.. 2. NelA,XiaT,MadlerL,LiN.2006.Toxicpotentialofmaterialsatthe nanolevel.science311: LecoanetHF,BotteroJ,WiesnerMR.2004.LaboratoryAssessmentofthe MobilityofNanomaterialsinPorousMedia.EnvironSciTechnol38: KlaineSJ.2009.Considerationsforresearchontheenvironmentalfateand effectsofnanoparticles.environmentaltoxicologyandchemistry28: SmartSK,CassadyAI,LuGQ,MartinDJ.2006.Thebiocompatibilityofcarbon nanotubes.carbon44: IijimaS.1991.Helicalmicrotubulesofgraphiticcarbon.Nature354: BosiS,DaRosT,SpallutoG,PratoM.2003.Fullerenederivatives:an attractivetoolforbiologicalapplications.eurjmedchem38:

78 8. DonaldsonK,StoneV,TranCL,KreylingW,BormPJA.2004.Nanotoxicology. OccupationalandEnvironmentalMedicine61: QiaoR,RobertsAP,MountAS,KlaineSJ,KePC.2007.TranslocationofC60 anditsderivativesacrossalipidbilayer.nanoletters7: RobertsAP,MountAS,SedaB,SoutherJ,QiaoR,LinS,KePC,RaoAM,Klaine SJ.2007.InvivoBiomodificationofLipid CoatedCarbonNanotubesbyDaphnia magna.environscitechnol41: KePC,QiaoR.2007.Carbonnanomaterialsinbiologicalsystems.Journalof Physics:CondensedMatter19: ZhaoB,HuH,HaddonRC.2004.SynthesisandPropertiesofaWater Soluble Single WalledCarbonNanotube Poly(m aminobenzenesulfonicacid)graft Copolymer.AdvancedFunctionalMaterials14: ZhaoB,HuH,YuA,PereaD,HaddonRC.2005.Synthesisand CharacterizationofWaterSolubleSingle WalledCarbonNanotubeGraft Copolymers.JAmChemSoc127:

79 14. YamaguchiI,YamamotoT.2004.Solubleself dopedsingle walledcarbon nanotube.materlett58: HuN,DangG,ZhouH,JingJ,ChenC.2007.Efficientdirectwaterdispersionof multi walledcarbonnanotubesbyfunctionalizationwithlysine.materlett61: LamC,JamesJT,McCluskeyR,ArepalliS,HunterRL.2006.AReviewof CarbonNanotubeToxicityandAssessmentofPotentialOccupationaland EnvironmentalHealthRisks.CritRevToxicol36: KimK,EdgingtonAJ,KlaineSJ,ChoJ,KimSD.2009.InfluenceofMultiwalled CarbonNanotubesDispersedinNaturalOrganicMatteronSpeciationand BioavailabilityofCopper.EnvironSciTechnol43: Johnson LoganL,BroshearsRE,KlaineSJ.1992.Partitioningbehaviorand themobilityofchlordaneingroundwater.environscitechnol26: VanGenderenEJ,RyanAC,TomassoJR,KlaineSJ.2003.Influenceofdissolved organicmattersourceonsilvertoxicitytopimephalespromelas.environmental ToxicologyandChemistry22:

80 20. RyanAC,GenderenEJV,TomassoJR,KlaineSJ.2004.Influenceofnatural organicmattersourceoncoppertoxicitytolarvalfatheadminnows(pimephales promelas):implicationsforthebioticligandmodel.environmentaltoxicologyand Chemistry23: RyanAC,TomassoJR,KlaineSJ.2009.InfluenceofpH,Hardness,Dissolved OrganicCarbonConcentration,andDissolvedOrganicMatterSourceontheAcute ToxicityofCoppertoDaphniaMagnainSoftWaters:ImplicationsfortheBiotic LigandModel.EnvironmentalToxicologyandChemistry28: LuC,SuF.2007.Adsorptionofnaturalorganicmatterbycarbonnanotubes. SeparationandPurificationTechnology58: HyungH,FortnerJD,HughesJB,KimJ.2007.NaturalOrganicMatter StabilizesCarbonNanotubesintheAqueousPhase.EnvironSciTechnol41: HyungH,KimJ.2008.NaturalOrganicMatter(NOM)AdsorptiontoMulti WalledCarbonNanotubes:EffectofNOMCharacteristicsandWaterQuality Parameters.EnvironSciTechnol42:

81 25. ChappellMA,GeorgeAJ,DontsovaKM,PorterBE,PriceCL,ZhouP,Morikawa E,KennedyAJ,SteevensJA.2009.Surfactivestabilizationofmulti walledcarbon nanotubedispersionswithdissolvedhumicsubstances.environmentalpollution 157: EPAUS.1993.Methodsformeasuringtheacutetoxicityofeffluentsand receivingwaterstofreshwaterandmarineorganisms LewisP,KlemmD,LazorchakJ,Norber KingT,PeltietW,HeberM Short termmethodsforestimatingthechronictoxicityofeffluentsandreceiving waterstofreshwaterandmarineorganisms.epa600 /4 91/ AndrewsR,JacquesD,RaoAM,DerbyshireF,QianD,FanX,DickeyEC,Chen J.1999.Continuousproductionofalignedcarbonnanotubes:astepcloserto commercialrealization.chemicalphysicsletters303: ScieraKL,IselyJJ,TomassoJR,KlaineSJ.2004.Influenceofmultiplewaterqualitycharacteristicsoncoppertoxicitytofatheadminnows(Pimephales promelas).environmentaltoxicologyandchemistry23:

82 30. JonssonM,NerushevOA,CampbellEEB.2007.Insitugrowthrate measurementsduringplasma enhancedchemicalvapourdepositionofvertically alignedmultiwallcarbonnanotubefilms.nanotechnology18: LiZF,LuoGH,ZhouWP,WeiF,XiangR,LiuYP.2006.Thequantitative characterizationoftheconcentrationanddispersionofmulti walledcarbon nanotubesinsuspensionbyspectrophotometry.nanotechnology: MatilainenA,GjessingET,LahtinenT,HedL,BhatnagarA,Sillanpaa.2011.An OverviewoftheMethodsUsedintheCharacterisationofNaturalOrganicMatter (NOM)inRelationtoDrinkingWaterTreatment.Chemosphere.83: HurJ,WilliamsMA,SchlautmanMA.2006.EvaluatingSpectroscopicand ChromatographicTechniquestoResolveDissolvedOrganicMatterViaEndMember MixingAnalysis.Chemosphere.63: KennedyAJ,GunterJC,ChappellMA,GossJD,HullMS,KirganRA,SteevensJA InfluenceofnanotubepreparationinAquaticBioassays.Environmental ToxicologyandChemistry28:

83 35. OlasagastiM,AlvarezN,VeraC,RainieriS.2009.EvaluationofMWNTtoxic effectsondaphniaandzebrafishembryos.journalofphysics:conferenceseries : RobinsonSE,CapperNA,KlaineSJ.2010.Theeffectsofcontinuousand pulsedexposuresofsuspendedclayonthesurvival,growth,andreproductionof Daphniamagna.EnvironmentalToxicologyandChemistry29: AlanJ.Kennedy,HullMS,SteevensJA,DontsovaKM,ChappellMA,GunterJC, WeissCA,Jr.2008.Factorsinfluencingthepartitioningandtoxicityofnanotubesin theaquaticenvironment.environmentaltoxicologyandchemistry27: PetersenEJ,AkkanenJ,KukkonenJVK,WeberWJ.2009.BiologicalUptake anddepurationofcarbonnanotubesbydaphniamagna.environscitechnol 43: PetersenEJ,PintoRA,LandrumPF,WeberJ,WalterJ.2009.Influenceof CarbonNanotubesonPyreneBioaccumulationfromContaminatedSoilsby Earthworms.EnvironSciTechnol43:

84 40. MengJ,YangM,SongL,KongH,WangCY,WangR,WangC,XieSS,XuHY Concentrationcontrolofcarbonnanotubesinaqueoussolutionandits influenceonthegrowthbehavioroffibroblasts.colloidsandsurfacesb: Biointerfaces71: KostarelosK,LacerdaL,PastorinG,WuW,WieckowskiSebastien, LuangsivilayJ,GodefroyS,PantarottoD,BriandJ,MullerS,PratoM,BiancoA Cellularuptakeoffunctionalizedcarbonnanotubesisindependentoffunctional groupandcelltype.natnano2: DumortierH,LacotteS,PastorinG,MaregaR,WuW,BonifaziD,BriandJ, PratoM,MullerS,BiancoA.2006.FunctionalizedCarbonNanotubesAreNon CytotoxicandPreservetheFunctionalityofPrimaryImmuneCells.NanoLetters 6: MouchetF,LandoisP,SarremejeanE,BernardG,PuechP,PinelliE,FlahautE, GauthierL.2008.Characterisationandinvivoecotoxicityevaluationofdouble wall carbonnanotubesinlarvaeoftheamphibianxenopuslaevis.aquatictoxicology 87:

85 FIGURES Figure1. Daphniamagnagrowth(mg)followingexposuretomultiwalled nanotube naturalorganicmatterparticles(mwnt NOM)(NP;mg/L).Growthwas inhibitedatallexposureconcentrationsgreaterthan0.125mg/l(p<0.01;r 2 = 0.574). 69

86 Figure2.Ceriodaphniadubiareproduction(%Control)duringexposuretoMWNT NOM(NP;mg/L).Significantdecreaseswereobservedinallconcentrations>0.25 mg/l.lettersdenotestatisticalgroupings(p<0.05). Figure3.C.dubiagrowth(mg)followingasevendayexposuretoMWNT NOM(NP; mg/l).adecreaseingrowthwasobserved(p=0.045). 70

87 Figure 4. Fluorescence excitation emission matrices; A) Black River NOM, B) Edisto River NOM, and C) Suwannee River NOM. BR NOM peak intensity (excitation 340, emission 385), ER NOM peak intensity (excitation 340, emission 380), and SR NOM peak intensity (excitation 355, emission 400) suggest all sources are more fulvic like. 71

88 Figure5.Scanningelectronmicroscopy(SEM)micrographofMWNTsA)bulk materialb)sonicatedinmoderatelyhardwater(mhw)andc)sonicatedin SuwanneeRiver NOMsolutions(2mg/LDOC). 72

89 Figure6.Infrared(IR)spectraofNOMsourceswithfunctionalgrouppeakslabled (thesamplelablednomissuwanneerivernom)andirspectraofnomandnom MWNTcomplex.Identifiedpeakswereasfollows: OHstretchof carboxylicgroups; C Ostretchingofesters,ethersandphenols; CHdeformation(CH3); CHdeformation(aliphatic);1550and1640 C=O(ketonesandquinines); aliphaticC H,C H2,andC H3stretching; 3400OHgroups. 73

90 Figure7.Transmissionelectronmicroscopy(TEM)micrographsofcontrolD. magnaguttract,algaeandotherfoodparticlesarepresent(a)andimbeddedwithin microvili(b).theguttractofmwntexposedd.magnahasbeenimpactedwith MWNTs,whichhavedisaggregated(C),butthemicroviliappeartoactasabarrierto preventabsorbtionacrossthegutlumen(d). 74

91 Nanoparticle Suspension MWNTin18.8mg/L DOC(SR NOM) MWNTin15.2mg/L DOC(SR NOM) MWNTin10.4mg/L DOC(SR NOM) MWNTin5.1mg/L DOC(SR NOM) MWNTin2.0mg/L DOC(SR NOM) MWNTin15.1mg/L DOC(ER NOM) MWNTin15.7mg/L DOC(BR NOM) ph Zeta Potential Hydrodynamic Diameter LC50 (mg/l) 95% CI(mg/L) 8.08 NA 706.4±35.9* , ± ± , NA 655.7± , ± ± , ±4.6 NA , ± ±19.1* 4.09* 3.41, ± ±24.2* ,2.62 Table1.MWNTparticlecharacterizationandtoxicityresults,(*)meansstatistically significantlydifferentvalues(p<0.05). 75

92 ExposureSolution EliminationSolution Fed TimetoElimination SR NOM SR NOM No 24h SR NOM SR NOM Yes 2h SR NOM MHW No 24h SR NOM MHW Yes 4h ER NOM ER NOM No 28h ER NOM ER NOM Yes 2h ER NOM MHW No 12h ER NOM MHW Yes 2h BR NOM BR NOM No 28h BR NOM BR NOM Yes 4h BR NOM MHW No 24h BR NOM MHW Yes 4h Table2.D.magnaMWNTguttracteliminationdata. 76

93 Source %O alkyl %OCH 3orN alkyl %Nonpolaralkyl %Anomerics Suwannee River(USA) BlackRiver (USA) EdistoRiver (USA) %Ketones, Source quinines, %Arom.C OPhenols %Aromatics aldehydes %N C=O,COO Suwannee River(USA) BlackRiver (USA) EdistoRiver (USA) Table3.NuclearmagneticresonancespectroscopyanalysisresultsofNOM

94 CHAPTER3:ABSORPTIONOFFUNCTIONALIZEDSINGLE WALLEDNANTOBUES INDaphniamagna ABSTRACT Therearemanynaturallyoccurringcolloidsthatexistinaquaticecosystemsthat havethepotentialtoadsorbtocarbonnanomaterialsandinfluencetheiraqueous stabilityconsequentlyimpactingtheirfateandbioavailabilitytoaquaticorganisms. Naturalorganicmatter(NOM)isacomplexheterogeneousmoleculethatis ubiquitousinallsurfacewaters.itvariesinbothmolecularweightandchemical compositionhavingbothhydrophilicandlipophiliccomponents.proteinsare anothernaturaloccurringcolloid,withthemajorcontributioninsurfacewaters comingfromwastewatertreatmentplanteffluents. Theobjectivesofthisresearchweretodeterminetheabsorptionofsinglewallednanotubes(SWNTs)acrosstheguttractofDaphniamagnaandtodetermine ifabsorptionwasinfluencedbyswntsurfacefunctionalization..thisproject utilizedseveralmicroscopictechniquesincludingmicro Ramanspectroscopy,highresolutiontransmissionelectronmicroscopy(HRTEM),andanalyticalTEMwith electronenergylossspectroscopy(eels)andselectiveareadiffraction(sad).four differentfunctionalizedswntswereusedinthisstudy;hydroxylated(oh SWNTs), silicadioxide(sio2 SWNTs),polyaminobenzenesulfonicacid(PABS SWNTs)and polyethyleneglycol(peg SWNTs). Inthepresentstudy,RamanspectroscopywasabletodetectOH SWNTs withinthegutevenafterbeingsuspendedwithnomandinthepresenceofother 78

95 gutcontents.however,thetechniqueisunabletoworkatthescalethatisneededto investigatelowerconcentrationsofswntsthatmaybeabsorbingintobodytissues. ThereforewesectionedOH SWNT,SiO2 SWNT,PABS SWNT,andPEG SWNT exposedd.magnatoimagewithtem.althoughtemproducedimageswherethere areseveralareasofinterestwithintissuessuggestingabsorptionofswntsit becameclearwiththeanalyticaltemtechniques(hrtem,sad,andeels)that theseareasareartifactsofstainingorotherorganicstructures.thisisnotmeantto definitivelyindicatethatnoabsorptionoccurred,butratherthatwewereunableto detectasmuchusinganalyticaltem.asthefieldofnanotoxicologycontinuesto advanceitisimperativethatweusethebestandmostadvancedtechniquesto detectnanomaterialsinbiologicalmatrices. Introduction Single walledcarbonnanotubes(swnts)areanallotropeofcarbon characterizedbystrongcovalentbondingbetweencarbonatoms,ahighaspect ratio,hightensilestrength,andhighelectronconductance[1,2].these characteristicshavemadeswntsusefulforavarietyofapplicationsincluding electronics,computer,andaerospaceindustries[3].asthedemandforproducts containingnanomaterialsincreaseandthescaleofnanomaterialmanufacturing grows,thelikelihoodofenvironmentalreleaseofmanufacturednanomaterialswill alsoincrease[4].currentcalculationspredictannualcntproductionofover20 tonesintheus[5].anyenvironmentalreleaseofnanomaterialswillultimately resultinentryintoaquaticecosystems.therearemanynaturallyoccurringcolloids 79

96 thatexistinaquaticecosystemsthathavethepotentialtoadsorbtocarbon nanomaterialsandinfluencetheiraqueousstabilityconsequentlyimpactingtheir fateandbioavailabilitytoaquaticorganisms. Naturalorganicmatter(NOM)isacomplexheterogeneousmoleculethatis ubiquitousinallsurfacewaters.itvariesinbothmolecularweightandchemical compositionhavingbothhydrophilicandlipophiliccomponents[5].researchers haveshownthatnomcanadsorbontothesurfaceofcarbonnanotubesand influencetheiraqueousstability[6 10],andafewstudieshavealsoinvestigated interactionsbetweencntsandsoilsandpeat[10,11].ithasalsobeenshownthat waterqualitycharacteristics,suchasphandionicstrength,affectthestabilityof NOMnanomaterialsuspensions[8]anddifferentNOMsourcesmayhaveaneffect onstabilityandtoxicity[5,12]. Proteinsareanothernaturaloccurringcolloid,withthemajorcontributionin surfacewaterscomingfromwastewatertreatmentplanteffluents.raunkjaeretal [13]reportedproteinconcentrationsrangingfrom34 171mg/Lininfluentsinto fourdifferentwastewatertreatmentplantsindenmark.whileprotein concentrationsoftreatedeffluentmaynotbewithinthisrangefortreatmentplants, itisexpectedthatproteinsatelevatedconcentrationswillbepresentintheeffluent. Furthermore,researchershaveshownthatproteinshaveanaffinityfor nanomaterialsandwillbindtotheirsurfacecreatingaproteincoronaaroundthe nanomaterial[14,15].therelationshipbetweennanomaterialandproteinshas beenmodeledforfullerenes[16 17],andZhuetal.[18]determinedthat1mgof multi walledcarbonnanotubesiscapableofadsorbing0.47mgofserumproteins. 80

97 Therehasbeenanaturalprogressionofincreasinglymorecomplextoxicity studiestodeterminetheimpactofswnts.invitrostudieshaveshownthatswnts cancauseanumberofadverseeffectsinavarietyofcellslines[19 22].Inhalation studieswithmicehaveshownthatswntscancauseinflammationresponsesand grossmorphologicalabnormalitiesinlungtissues[23,24].templetonetal.[25] foundreducedsurvivalfortheestuarinemeiobenthiccrustaceancopepod Amphiascustenuiremisinconcentrationsrangingfrom0.97mg/Lto10mg/L SWNTsandreducedlifestagedevelopmentinthe10mg/Ltreatments.Smithetal. [26]sawanincreasedincidenceofoedemaandenlargedmucocytesinexposed rainbowtroutgills,butamorerecentstudybyfraserandcoworkersdidnotfind toxiceffectswithfishfedswnt spikedfoodataconcentrationof500mg/kgfood [27].Moreover,highconcentrationsofSWNTshasgenerallynotcausedsubstantial toxiceffectstosedimentandsoilorganisms[28 32].Biodistributionstudieshave beencontradictory.singhetal(2006)[33]haveshownthatmiceinjectedwith radiolableddiethylentriaminepetaacticfunctionalizedswntshaveabloodhalf life ofjustover3.5hrsandareeliminatedviaurine.but,shipperetal(2008)[34] concludedthatpegylatedswntsinjectedintomicearesequesteredintheliverand spleenuptofourmonths.instudieswithcarbonnanotubesaddedtosoilsand sediments,evidenceofpassageofcarbonnanotubesthroughtheguthasbeen observedbutevidenceofbioaccumulationhasnotbeenfound[28 31,35]. However,thesestudieshavenotusedadvancedimagingtechniquessuchaselectron microscopytoinvestigatecntuptake.furthermore,knowledgeregardingthe 81

98 absorptionandaccumulationofswntsinwholeorganismmodelsexposedtolower concentrationsinenvironmentallyrelevantconditionsislacking. TheobjectivesofthisresearchweretodeterminetheabsorptionofSWNTs acrosstheguttractofdaphniamagnaandtodetermineifabsorptionwas influencedbyswntsurfacefunctionalization.thisprojectutilizedseveral microscopictechniquesincludingmicro Ramanspectroscopy,high resolution transmissionelectronmicroscopy(hrtem),andanalyticaltemwithelectron energylossspectroscopy(eels)andselectiveareadiffraction(sad).anunderlying themeofthisresearchwasalsotoinvestigatethesuitabilityofvarious characterizationtechniquesforprobingthebiologicaluptakebehaviorofswnts. MaterialsandMethods Organisms DaphniamagnawereobtainedfromculturesmaintainedattheInstituteof EnvironmentalToxicology,ClemsonUniversity(Pendleton,SouthCarolina,USA). TheywereculturedinU.S.EnvironmentalProtectionAgency(U.S.EPA)moderately hardreconstitutedwater(mhw)at25 Cwitha16:8hrlight:darkcycle[37,38]. SWNTS FourdifferentfunctionalizedSWNTswereusedinthisstudy.Hydroxylated SWNTs(OH SWNTs)werepurchasedfromcheaptubes.com(Brattleboro,Vermont, USA).Accordingtothemanufacturer sspecifications,theswntswere1nm 2nm indiameter,10µm 30µminlength,>90%pure,andwasfunctionalized3.96%by 82

99 mass.asampleofthismaterialwasthenfurtherfunctionalizedbydr. Mukhopadhyay slaboratoryatwrightstateuniversity(dayton,ohio,usa)with silicadioxide(sio2 SWNTs).TwoadditionalmaterialswerepurchasedfromCarbon Solutions,Inc(Riverside,California,USA),polyaminobenzenesulfonicacid(PABS SWNTs)andpolyethyleneglycol(PEG SWNTs)functionalizedSWNTs.These materialswerebothcertifiedbythemanufacturertobe>90%purebutless informationwasprovidedregardingthelengthanddiameteroftheseswnts. NaturalColloids TheNOMsourceusedduringthisstudywasSuwanneeRiverNOM(SR NOM) thatwaspurchasedfromtheinternationalhumicsubstancessociety;thisproduct wasconcentratedbyreverseosmosistoapowder.naturalorganicmattersolutions weremadebydilutingsr NOMinMHWandthenfilteringitwitha0.2umcellulose membranefilterpriortotoxicitytests.alltestsanddilutionwaterwereusedat concentrationsofapproximately2.5mg/ldissolvedorganiccarbon(doc). TheproteinsourcewasHyclonestandardfetalbovineserum(FBS)(Logan,Utah, USA).Thisproductwasusedinsolutionformasis. SWNTSuspensions SinglewallednanotubesweresuspendedinSR NOMsolutionusingthe followingprocedure:thematerialwasfirstweighedonaluminumfoilandplacedin a100mlglasscentrifugetube100mlofsr NOMsolutionwasthenaddedtothe centrifugetube,andthesolutionwasprobesonicatedwithabransondigitalsonifier 83

100 (Danbury,Connecticut,USA)for20minat40%power.Thispoweroutputwas calculatedtobeapproximately85.2wattsusingthemethoddescribedbytaurozzi etal(2010)[36].thesolutionswereallowedtosettleforapproximately24hrs beforethesupernatantwasusedforbioassays.concentrationsofswntsin suspensionweredeterminedgravimetricallybyweighingtubesremainingafter withdrawalofthesupernatant(seebelow). HydroxylatedsinglewallednanotubesweresuspendedwithFBSusingthe followingprocedure:thematerialwasfirstweighedonaluminumfoilandplacedin aglasscentrifugetube;8mloffbssolutionwasaddedtothecentrifugetube,and thesolutionwasbathsonicated(fisherscientificmodelfs30)for10min (approximately100watts).theswnt FBSsolutionwasdilutedto100mLwith MHWandthenprobesonicatedforanadditional10min.Theproteinconcentration forthissolutionwasapproximately2.5mg/l.thesolutionswereallowedtosettle forapproximately24hrsbeforethesupernatantwasusedforbioassays. Thestablesolutionwasdecantedandconcentrationsweredeterminedby weighingtheremainingprecipitateonapre rinsed/dried0.2umcellulose membranefilter.thisweightwassubtractedfromtheinitialweightofswntsand dividedbythevolumeofthestablestocksolutiontodeterminetheinitialstock concentration.thestocksolutionwasfurtherdilutedwithnomsolutiontoachieve concentrationof1mg/lswntsforbioassays. 84

101 ParticleCharacterization AllmaterialswereimagedpostsonicationinNOMsolutionsusedin exposurestodetermineparticlesize.adropofstocksolutionwasallowedtodryon 200meshcoppergridsElectronMicroscopyScience,Hattield,Pennsylvania,USA). ImagesweretakenonaHitachi7600TEMat120kV.Particlesizewasdetermined qualitativelythroughexaminingtheimagedmaterial. Stocksolutionelectrophoreticmobility(EM)wasdeterminedusinga MalvernZetasizer(MalverInstruments,Worcestershire,UK).Solutionconductivity andphwerealsorecorded. Bioassays Daphniamagnaacute96hrstaticrenewalbioassayswereperformed followingu.s.epamethods[37,38]butonlyusingoneconcentration(1mg/l)and 3replicatesforeachSWNTandanNOMcontrolwith3replicates.Fifteenmilliliters ofswntsolutionorcontrolsolutionwasaddedto30mlglassbeakertest chambers.fiveorganisms,<24hrsold,wereplacedineachtestchamber.test solutionswerereneweddailyandfed0.25mlofa4:3algae:yeast/trout chow/cereal(ytc)mixture2hto4hbeforerenewal. Ramanspectroscopysamples After96hrsactiveD.magnawerefixedin4%gluteraldehydesolution overnightand,afterdehydrationstepswithethanolsolutions,embeddedin 85

102 Immunobedresinandsectionedonamicrotome.Lateralsections8umthickofthe wholeorganismwereplacedonglassslidesforramanspectroscopyanalysis. Transmissionelectronmicroscopysamples After96hactiveD.magnawerefixedin4%gluteraldehydesolution overnightand,afterdehydrationstepswithethanolsolutions,embeddedinlr Whiteresin.Thesampleswerepolymerizedat90 Covernightthensectionedonan ultramicrotome.sectionsoftheorganism smidgutwereplacedon200mesh coppergridsthathadalphabeticalcoordinates(electronmicroscopyscience, Hatfield,Pennsylvania,USA). HighResolutionTransmissionElectronMicroscopy(HRTEM),SelectiveArea Diffraction(SAD),andElectronEnergyLossSpectroscopy(EELS) Sampleswhichlow magnificationtemimagingsuggestedswntabsorption withind.magnatissuesweresubjectedtohr TEM,SAD,andEELStoconfirm particleidentity.thisdatawasacquiredusinganfeititan80 300TEM/SEM operationat300kvthatwasequippedwithagatantridiem865imagingenergyfilterforeelsanalysis.hr TEMimagingofSWNTswasconductedusingphase contrastduetothecrystallineordertheyexhibitrelativetothesurrounding amorphousorganicmatrix. Inaddition,thecrystallinityofSWNTsproducesadistinctSADpattern consistingofsharpspotscorrespondingtothebraggdiffractionpeaksforgraphenebasedstructures,whereastheamorphousorganicmatrixwillproducediffuserings 86

103 duetothelackofmedium andlong rangeorder.thistechniqueisuniquetoswnt identificationbecauseitwasfirstusedbyiijima(1991)[2]toconfirmthe productionofswntsfromthecarbon arcmethodusedinfullereneproduction. Electronenergy lossspectroscopymeasurestheenergydistributionofelectrons aftertheyhaveinteractedwithasampleandhavelostenergyduetotheinelastic scattering[39].single wallednanotubesgiveadistincteelsspectrum,thefine structureofthecarbonk edgeoftheeelsspectrapotentiallyoffersathird routfor thediagnosticdeterminationofthepresenceofswnts,duetothepresenceofa significantamountofsp 3 bondedcarbonatomsrelativetothatfoundinamorphous, organicmaterial. Results ParticleCharacterization TherewerenosignificantdifferencebetweenparticlesuspensionEM,all solutionswereapproximately 1.5µmcm/Vs.Therewerealsonosignificant differencesinsolutionconductivityandph. Bioassays TherewasnoobservedmortalityinanyofthecontrolsorSWNTtreatments overthe96hexposureperiod. 87

104 RamanSpectroscopy SamplesofrawOH SWNTswereanalyzedwithRamanSpectroscopyto determinethematerial sganddbandsignal.anoh SWNTexposedD.magnagut lateralsectionwasthenanalyzed.theareaswithinthissamplethathadthesameg anddbandsignalastherawoh SWNTsweremappedoutinthesectionedD. magna(figure1).themappedsectionshowsastrongganddbandwithinthegut andanabsentsignalwithinthetissuesofthed.magnasuggestingthegutis impactedwithswnts,butthematerialisnon detectedwithintheothertissues. Thismethodwasrepeatedonanotherareaofthesectionlowerinthegutandwith highermagnification.theresultingg bandsignalismappedoutandshowsa highlightedregionofhighoh SWNTconcentrationwithinthegutregionbutthere isnosignaloutsideofthegutregionsuggestingnoswntsabsorbedintoother tissues. TransmissionElectronMicroscopy TheTEMimagesofthepost sonicatedswntssuggestthatthesonication createsadispersedmaterialwithoutalteringthestructureoftheswnts(figure3). AlthoughsonicationofthePABSandPEGSWNTsinNOM(Figures3CandD respectively)resultedindispersalintosingleswnts,sonicationofohandsio2 SWNTsinNOM(Figures3AandB)orOHSWNTsinFBS(Figure3E)resultedin nanotubebundles.thismorecompletedispersionislikelyduetothemore hydrophilicpabsandpegfunctionalgroupsinteractingmorefavorablywithwater 88

105 molecules.however,itispossiblythataggregationcouldhaveoccurredduringthe dryingprocesstopreparethetemgrids. ThecrosssectionsofNOMdispersedOH SWNTexposedD.magnashowed incidencesofdarkmaterialassociatedwithinthegut,onthemicrovilli,and imbeddedinthelumen(figure4).whilethesizeofthesuspectedabsorbedoh SWNTsistoolargetobeindividualSWNTsitisconsistentwiththatexpectedfor largeraggregatesofswnts(figure4bandc).suspectedabsorbedmaterialis againseenincrosssectionsofnomdispersedpeg SWNTexposedD.magna(Figure 5).Thismaterialappearsmorefiber likeandembeddedwellwithinthelumen (Figure5BandCcomparedtoFigures4BandC).FurtherevidenceofSWNT absorptionisseeninfigure6.alongtubeshapedmaterialwithdimensions comparabletoaswntbundleisembeddedwithintheboundarylayerbetweenthe microvilliandthelumenofnomdispersedpabs SWNTexposedD.magna(Figure6 B).NoneofthissuspectedSWNTabsorptionispresentinNOMdispersedSiO2 SWNTexposedD.magna(Figure7). ThecrosssectionsofFBSdispersedOH SWNTexposedD.magnashows similarincidencesofdarkmaterialassociatedwiththemicrovilliandimbedded withinthelumen(figure8)comparedtonomdispersedoh SWNTexposedD. magna(figure4).figure8bshowsthematerialhasadarkcorewithalighter coating.thiscouldbeoh SWNTaggregateswhichwillappearblackduetothehigh electrondensityandthelightercoatingcouldbeaproteincoronaaroundthe aggregate. 89

106 TofurtherexaminethispotentialSWNTabsorptioncrosssectionsofFBS dispersedoh SWNTexposedD.magnawereexaminedonaHitachiHD2000 transmissionelectronmicroscopeinz contrastmode(figure9).thiswasdonein scanningtransmissionelectronmicroscopy(stem)modeusingahighangle annulardark fielddetector.inthismodeelectrondensematerialappearswhite comparedtothelowelectrondensetissues,whichappeardark.longelectron densetube likematerialwasfoundembeddedwithinthelumenofthesesamples (Figure9BandC)withdimensionscomparabletoSWNTbundles. HighResolutionTransmissionElectronMicroscopy(HRTEM),SelectiveArea Diffraction(SAD),andElectronEmissionLossSpectroscopy(EELS) TheD.magnaexposedtoFBS coatedoh SWNTssampleswerefurther investigatedtoidentifythedarkandtube likematerialfoundwithinthetissues usingtemandteminz contrastmode.surprisingly,wewereunabletofind SWNTsintheguttractsorintheorganism stissuesofthesesamples.infigure10a andb,weobservedapparenttubularstructures,butwhenweviewedthisareawith ahighermagnification(figure10c),wedidnotobserveswntsnoradiffraction patternindicativeofswnts.forthefbsoh SWNTexposedD.magnastained samples(figures8and9)therewerealargenumberofparticlespresent.using electrondispersionspectroscopy(eds)(figure10d),wedeterminedthatthe elementalcompositionoftheseparticleswasuraniumandlead,whichindicatesthat theseparticleswereformedduringthestainingprocess. 90

107 WealsosearchedforSWNTsintheguttractofthedaphniatreatedwith PABS SWNTs(Figure11).WewereabletofindSWNTsinnumerouslocationsand clearlyimagethematerialathighmagnification(figure11f).weconfirmedthese resultsusingselected areadiffraction(sad)andelectronenergy lossspectroscopy (EELS)(Figure12).WealsoacquiredanEELSspectrumandSADfromotherareas intheguttractnearbybutwithoutswntsandfoundadifferentsignalforeelsand didnotobservetheappropriatesadpatterns(seefigure12a).weusedelectron diffractiontomovearoundthetissuesoftheorganismstoidentifylocationswith SWNTsbutwereunabletoobserveanydiffractionpatternsindicativeofSWNTs. Wealsoinvestigatedseverallocationsthatcontainedtubularstructuresatlower magnificationsusinghigh resolutiontransmissionelectronmicroscopy(hrtem) butdidnotlocatenanotubesbasedonthephase contrastexhibitedbythehrtem imagesorthefeaturesobservedinthesadpatterns. AreasthatsuggesteduptakeofPEG SWNTswerealsoinvestigated(see Figure13).WhiletheredidappeartobeSWNTuptakewhenthemicroscopewas outoffocus(seeparta),thiswaslessevidentwhenthemicroscopewasinfocus (seepartb).whenthemagnificationwassubstantiallydecreased,itwasobserved thattheapparentswntswereactuallyamorphouscarbonanddidnothaveanyfine structureindicativeofswnts(seepartscandd).thisresultindicatesthe importanceofconfirmingapparentswntsusinghrtemandotherimaging techniqueswhenavailable. 91

108 Discussion Carbonnanotubecellularuptakehasbeenobservedininvitroresearch[40 44]yettheexactmechanismofuptakeisstillunderdebate.Pantarottoetal(2004) [41]foundCNTlabeledwithafluorescenttagwereeasilyabsorbedintothe cytoplasmandnucleusoffibroblasts.theydeterminedtheuptakeofthistypeof CNTwaspassiveandnotendocytosisdependentwhentheyincubatedthecellsin thepresenceofendocytosisinhibitorsandstillobserveduptakeusing epifluorescenceandconfocalmicroscopy.shikametal(2004,2005)[40,44] suggestedthatcntsfunctionalizedwithfluorescentlytaggedproteinsstreptavidin, bovineserumalbumin,proteina,andcytochromecweretakenupviaan endocytoticpathwaybecauseoftheirlocalizationwithinendosomesinavarietyof testedmammaliancells.whileitisclearthatusingsimpleinvitromodelsto determinetheuptakeoffluorescentlytaggedcntscanbeaccomplished,itisnot environmentalrelevantforaquaticspeciesbecausethosefluorescenttagsmay changetheenvironmentalbehaviorsofthecnts. Robertsetal(2007)[45]characterizedthebehavioroflipidcoatedSWNTs duringexposuretod.magna.usingstaticrenewaltoxicityteststheywereableto determinethatthemajorityoftheingestedswntswerebeingstrippedofthelipid coating(hypothesizedasapossiblefoodsource)beforebeingexcretedbythe organism.usingramanspectroscopyandconfocalfluorescentmicroscopythey wereabletodeterminethattheswntshadcompletelyimpactedthegut,butthey wereunabletodetermineabsorptionfromtheguttoothertissues. 92

109 Smithetal(2007)[26]determinedphysiologicaleffects,organpathologies, andbiochemicalendpointstojuvenilerainbowtroutexposedtoswnts.histology ontheintestinaltissuesdeterminedeffectstotheepithelialcellsandprecipitated SWNTswithinthelumen.Becausethehistologywasdoneonalightmicroscope theycouldnotdetermineiftherewasswntabsorptionacrossthelumen. Mouchetetal(2008)[46]conductedoneofthefewstudiesonanamphibious model,theafricanclawedfrogxenopuslaevis.theyexposedlarvalx.laevisto double wallednanotubes(dwnts)for12daystodetermineacutetoxicity, genotoxicityendpoints,anduptake.theyusedlightmicroscopy,tem,andraman spectrometrytolocatedwntswithinexposedlarvae.theyobserveddarkmasses withintheintestinaltissuesofexposedorganismsusinglightmicroscopyand confirmedwithramanspectroscopyandhighresolutiontem(hrtem)thatwithin thedarkmassesdwntsarepresent.furthermore,withhrtemtheywereable imagewhatappearedtobedwntsembeddedwithinthelumen.however,itisnot clearintheramanspectroscopyanalysisorwiththehrtemimagesifthematerial hasabsorbedacrosscellsmembranesorifitisjustembeddedwithintheintestinal tissue. Edgingtonetal(2010)[6]determinedtheacuteandchronictoxicityof MWNTstoD.magna.TocharacterizeMWNTsoncetheywereingestedbyD.magna theysectionedexposedorganismsandimagedthemidgutwithtem.thematerial wasclearlyseenwithinthegutandupagainstthemicrovillilayer,butwasnotseen withinthelumenorothertissues.theyhypothesizedthatbecauseofthelarger 93

110 diameterandlengthofthemwnttheywereunabletoabsorbacrossthecellular membranesofthelumen. Fromtheabovestudiesitisclearthatthemoreusefulmethodsforprobing theabsorptionbehaviorofswntsinaninvivomodelareramanspectroscopyand TEM.Inthepresentstudy,RamanspectroscopywasabletodetectOH SWNTs withinthegutevenafterbeingsuspendedwithnomandinthepresenceofother gutcontents.however,thetechniqueisunabletoworkatthescalethatisneededto investigatelowerconcentrationsofswntsthatmaybeabsorbingintobodytissues. ThereforewesectionedOH SWNT,SiO2 SWNT,PABS SWNT,andPEG SWNT exposedd.magnatoimagewithtem.althoughtemproducedimageswherethere areseveralareasofinterestwithintissuessuggestingabsorptionofswnts(figures 4,5,6,8,and9)itbecameclearwiththeanalyticalTEMtechniques(HRTEM,SAD, andeels)thattheseareasareartifactsofstainingorotherorganicsturctures.we wereunabletofindswntsinthetissuesbutwereabletolocatetheminthegut tractforthepabs SWNTsusinganalyticalTEMtechniques.Thisisnotmeantto definitivelyindicatethatnoabsorptionoccurred,butratherthatwewereunableto detectasmuchusinganalyticaltem.also,theorganismscouldprobablybedosed withlargerswntconcentrationsbecauseonlyasmallamountofswntswere detectedintheguttracts.apotentialfuturestepwouldbetoembedswntsinthe resinbyitself,andinvestigatehowtheeelssignalsmightchangeandhowmuch morechallengingitwouldbetoidentifytheswntscomparedtoswntsadded directlytoagrid.giventhelengthsoftheswntsinthemicrographsforthedried SWNTs(Figure3),weweresurprisedatthesmallsizeoftheSWNTsobservedinthe 94

111 guttract.theremayhavebeensubstantialnanotubedamageduringthesonication process,butweexpectedthenanotubestobemuchlarger.also,thethinsections usedinthetemimagingtypicallyrangedfrom nm(determinedusing EELS).Giventhatthenanotubesobservedinisolationweremuchlongerthan200 nm(figure3),itispossiblethatthesectioningmayhaveremovedsomeofthe longernanotubesviaapulloutmechanism.inotherwords,nanotubesnotdirectly alignedintheplaceofsectionmayhavebennphysicallyremovedbytheglassknife usedtopreparethesamples.however,wedidnotconsistentlyobservelarge numbersoftearsinthethinsectionsthatwouldlikelyhavebeenpresentifalarge numberofembeddednanotubeshadbeenremoved.inaddition,thelongest nanotubesarelesslikelytoenterintothedaphniatissues,sothesectioning approachisunlikelytohaveimpactedourultimateconclusions. Thestainingprocesscreatedartifactsthatwereinitiallythoughttobe absorbedswntsbecausetheleadcitratecounterstainmayprecipitateinthe presenceofco2creatingelectrondenseareaswithinsamples.duringthestaining processstepsaretakentoensurethattheleadcitratestainwillnotprecipitate.the stainingisdoneinacoveredcontaineronabedofnaohbeads,whichareintended toabsorbtheco2,andthestainisfilteredpriortouse.however,whenworkingat suchasmallscaleitisdifficulttodetermineeventheslightestprecipitationinthe stain.figure14isanexampleoftheartifactsofstaining.figure14aisanimageof FBSOH SWNTexposedorganismthatwasnotstainedcomparedtothesame organisminstainedsectionsinfigures8and9.figure14bisanimageofacontrol 95

112 organismwherestainingwasused.itbecomesclearhowthestainingprocedurecan producefalsepositivesduringimaging. However,theremaybeanalternativeconclusiontotheobservationof amorphouscarbonabsorbedinthelumen.ithasbeendemonstratedthatswnts canbebiodegradedwithenzymesandfluidsthatmimicsphagolysosomecontent [47 50].Staretal[47,50]foundthatcarboxylatedSWNTscouldbedegradedinthe presenceofhorseradishperoxidase(hrp)andlowconcentrationsofhydrogen peroxide.after10daysvirtuallyalltheswntsweredegraded.tofurtherexplore thebiodegradationofcntsrussieretal(2011)[51]assessedthedegradationof bothcarboxylatedswntsandmwnts.thecntswereaddedtophagolysosomal simulantfluid(psf)thatmimicstheenvironmentwithinphagolysosomeswhere nanomaterialsmaybelocatedafterphagocytosis.hydrogenperoxidewasadded weeklytofullysimulateoxidizingconditionswithinphagolysosomes.an experimentusinghrpandh2o2wasrunintandemtodeterminewhichconditions degradedcntsmorefavorably.dynamiclightscattering,tem,andirspectroscopy wasusedtodeterminecntdegradation.after30daysthecarboxylatedswnts weremostlydegradedinthepsfandafter60daysbothpsfandhrpenvironments degradedtheswnts.thecarboxylatedmwntsweresimilarlydegradedafter60 daysinbothenvironments.thisresearchdemonstratedthebothswntsand MWNTscanbebiodegraded.Whilethesestudieshaveshownthatitispossiblefor CNTstobedegradedinenvironmentsthatmimicphagosomes,nutriphils,and macrophagesitisstilluncertaintheextentthatthisdegradationmayoccurinwhole 96

113 organismsandiftheamorphouscarbonthatwasobservedinthisstudywasadirect resultfromthedegradationoftheswnts. Conclusions ThepresentstudywasunabletodefinitivelydetectabsorptionofSWNTs acrosstheguttractofd.magnafortheseveraldifferentsurfacemodifiedtubes tested.however,wewereabletodeterminewhattechniquesmightbeusefulin determininguptakeininvivostudies.whiletemseemsthemostlogicalbecauseof thesizeofthesturcutesinvolved,wehaveshownthatsamplepreparationmay causefalsepositivesresultingininaccurateconclusions.previousstudiesthathave shownsimilardarkmaterialintheirsectionsmayhaveinfactbeenreporting artifactsfromthestainingpreparationthatisstandardprotocolforproducing qualitytemimages,orotherunidentifiedorganicmaterial.wehaveshownitisnot onlynecessarytousetembutalsoadditionalanalyticaltechniques,suchaseelsor SAD,toconfirmthepresenceofembeddednanotubes.Asthefieldof nanotoxicologycontinuestoadvanceitisimperativethatweusethebestandmost advancedtechniquestodetectnanomaterialsinbiologicalmatrices. Acknowledgements:Certaincommercialequipmentormaterialsareidentifiedin thispaperinordertospecifyadequatelytheexperimentalprocedure.such identificationdoesnotimplyrecommendationorendorsementbythenational InstituteofStandardsandTechnology,nordoesitimplythatthematerialsor equipmentidentifiedarenecessarilythebestavailableforthepurpose. 97

114 References 1.NiyogiS,HamonMA,HuH,ZhaoB,BhowmikP,SenR,ItkisME,HaddonRC ChemistryofSingle WalledCarbonNanotubes.AccountsofChemicalResearch. 35(12): Iijima,S.1991.HelicalMicrotubulesofGraphiticCarbon.Nature.354: LamCW,JamesJT,McCluskeyR,HunterRL.2004.PulmonaryToxicityofSingle WallCarbonNanotubesinMice7and90DaysAfterIntratrachealInstillation. ToxicologicalSciences.77: MaynardA,AitkenR,ColvinV,DonaldsonK,OberdorsterG,PhilbertM,RyanJ, SeatonA,StoneV,TinkleS,TranL,WalkerN,WarheitD.2006.SafeHandlingof Nanotechnology.Nature.444: GottschalkF,SondererT,ScholzRW,NowackB.2009.ModeledEnvironmental ConcnetrationsofEngineeredNanomaterials(TiO2,ZnO,CNT,Fullerenes)for DifferentRegions.EnvironmentalScienceandTechnology.43: EdgingtonAJ,RobertsAP,TaylorLM,AlloyMM,ReppertJ,RaoAM,MaoJ,Klaine SJ.2010.TheInfluenceofNaturalOrganicMatterontheToxicityofMultwalled CarbonNanotubes.29(11):

115 7.LuC,SuF.2007.AdsorptionofNaturalOrganicMatterbycarbonnanotubes. SeparationandPurificationTechnology.58: HyungH,FortnerJD,HughesJB,KimJ.2007.NaturalOrganicMatterStabilizes CarbonNanotubesintheAqueousPhase.EnvironmentalScienceandTechnology. 41: HyungH,KimJ.2008.NaturalOrganicMatter(NOM)AdsorptiontoMulti WalledCarbonNanotubes:EffectofNOMCharacteristicsandWaterQuality Parameters.EnvironmentalScienceandTechnology.42: Zhang,L.,Petersen,E.J.,Huang,Q.PhaseDistributionof 14 C labeledmultiwalledcarbonnanotubesinaqueoussystemscontainingmodelsolids:peat. EnvironmentalScienceandTechnology.2011,44(4), Petersen,E.J.,Pinto,R.A.,Zheng,L.,Huang,Q.,Landrum,P.F.,Weber,W.J.Jr. EffectsofPolyethyleneimine MediatedFunctionalizationofMulti WalledCarbon NanotubesonEarthwormBioaccumulationandSorptionbysoils.Environmental ScienceandTechnology.2011,45(8),

116 12.ChappellMA,GeorgeAJ,DontsovaKM,PorterBE,PriceCL,ZhouP,MorkawaE, KennedyAJ,SteevensJA.2009.SurfactiveStabilizationofMulti WalledCarbon NanotubeDispersionswithDissolvedHumicSubstances.EnvironmentalPollution. 157: RaunkjaerK,Hvited JacobsenT,NielsenPH.1994.WaterResearch.28(2): CedervallT,LynchI,LindmanS,BerggardT,ThulinE,NilssonH,DawsonKA, LinseS.2007.UnderstandingtheNanopartilce ProteinCoronaUsingMethodsto QuanitifyExchangeRatesandAffinitiesofProteinsforNanoparticles.Proceedingof thenationalacademyofsciencesoftheusa.104(7): LundqvistM,StiglerJ,EliaG,LynchI,CedervallT,DawsonKA NanoparticleSizeandSurfacePropertiesDeterminetheProteinCoronawith PossibleImplicationsforBiologicalImpacts.ProceedingsoftheNationalAcademy ofsciencesoftheusa.105(38): BenyaminiH,Shulman PelegA,WolfsonHJ,BelgorodskyB,FadeevL,GozinM InteractionofC60 FullereneandCarboxyfullerenewithProteins:Dockingand BindingSiteAlignment.BioconjugateChemistry.17(2):

117 17.DeguchiS,YamazakiT,MukaiS,UsamiR,HorikoshiK.2007.StabilizationofC60 NanoparticlesbyProteinAdsorptionandItsImplicationsforToxicityStudies. ChemicalResearchinToxicology.20(6): ZhuY,LiW,LiQ,YuguoL,LiY,ZhangX,HuangQ.2009.EffectsofSerum ProteinsonIntracellularUptakeandCytotoxicityofCarbonNanoparticles.Carbon. 47: ChengWW,LinZQ,WeiBF,ZengQ,HanB,WeiCX,FanXJ,HuCL,LiuLH,Huang JH,YangX,XiAG.2011.Single WalledCarbonNanotubeInductionofRatAortic EndothelialCellApoptosis:ReactiveOxygenSpeciesareInvolvedinthe MitochondrialPathway.TheInternationalJournalofBiochemistryandCellBiology. 43: DavorenM,HerzogE,CaseyA,CottineauB,ChambersG,ByrneHJ,LyngFM InvitroToxicityEvaluationofSingleWalledCarbonNanotubesonHuman A549LungCells.ToxicologyinVitro.21: DiGiorgioML,DiBucchianicoS,RagnelliAM,AimolaP,SantucciS,PomaA EffectsofSingleandMultiWalledCarbonNanotubesonMacrophages:Cyto andgenotoxicityandelectronmicroscopy.mutationresearch.722:

118 22.WangJ,SunP,BaoY,LiuJ,AnL.2011.CytotoxicityofSingle WalledCarbon NanotubesonPC12Cells.ToxicologyinVitro.25: LamCW,JamesJT,McCluskeyR,HunterRL.2004.PulmonaryToxicityof Single WallCarbonNanotubesinMice7and90DaysAfterIntratrachealInstillation. ToxicologicalSciences.77: InoueKI,YanagisawaR,KoikeE,NishikawaM,TakanoH.2010.Repeated PulmonaryExposuretoSingle WalledCarbonNanotubesExacerbatesAllergic InflammationoftheAirway:PossibleRoleofOxidativeStress.FreeRadicalBiology andmedicine.48: TempletonRC,FergusonPL,WashburnKM,ScribensWA,ChandlerGT Life CylceEffectsofSingle WalledCarbonNanotubes(SWNTs)onanEstuarine MeiobenthicCopepod.EnvironmentalScienceandTechnology.40: SmithCJ,ShawBJ,HandyRD.2007.ToxicityofSingleWalledCarbon NanotubestoRainbowTrout,(Oncorhynchusmykiss):RespiratoryToxicity,Organ Pathologies,andOtherPhysiologicalEffects.AquaticToxicology.82: FraserTWK,ReinardyHC,ShawBJ,HenryTB,HandyRD.2011.Dietarytoxicity ofsinglewalledcarbonnanotubesandfullerenes(c60)inrainbowtrout (Oncorhynchusmykiss).Nanotoxicology5:

119 28.GallowayT,LewisC,DolciottiI,JohnstonBD,MogerJ,RegoliF.2010.Sublethal toxicityofnano titaniumdioxideandcarbonnanotubesinasedimentdwelling marinepolychaete.environpollut158: PetersenEJ,HuangQG,WeberWJ,Jr.2008.Bioaccumulationofradio labeled carbonnanotubesbyeiseniafoetida.environscitechnol42: FergusonPL,ChandlerGT,TempletonRC,DemarcoA,ScrivensWA,Englehart BA.2008.Influenceofsediment amendmentwithsingle walledcarbonnanotubes anddieselsootonbioaccumulationofhydrophobicorganiccontaminantsbybenthic invertebrates.environscitechnol42: PetersenEJ,HuangQG,WeberWJ,Jr.2008.Ecologicaluptakeanddepurationof carbonnanotubesbylumbriculusvariegatus.environhealthperspect116: PetersenEJ,PintoRA,LandrumPF,WeberWJ,Jr.2009.Influenceofcarbon nanotubesonpyrenebioaccumulationfromcontaminatedsoilsbyearthworms. EnvironSciTechnol43:

120 33.SinghR,PantarottoD,LacerdaL,PastorinG,KlumppC,PratoM,BiancoA, KostarelosK.2006.TissueBiodistributionandBloddClearanceRatesof IntravenouslyAdministeredCarbonNanotubeRadiotracers.Proceedingofthe NationalAcademyofSciences.103(9): SchipperM,Nakayama RatchfordN,DavisCR,ShiKamNW,ChuP,ZhuangL, SunX,DaiH,GambhirSS.2008.APilotToxicologyStudyofSinge WalledCarbon NanotubesinaSmallSampleofMice.NatureNanotechnology.3: PetersenEJ,HuangQG,WeberWJ,Jr.2010.Relevanceofoctanol water distributionmeasurementstothepotentialecologicaluptakeofmulti walledcarbon nanotubes.environtoxicolchem29: TaurozziJS,HackleyVA,WiesnerMR.2010.UltrasonicDispersionof NanoparticlesforEnvironmental,Health,andSafetyAssessment Issuesand Recommendations.Nanotoxicology.EarlyOnline: U.S.EnvironmentalProtectionAgency.1993.MethodsforMeasuringtheAcute ToxicityofEffluentsandReceivingWaterstoFreshwaterandMarineOrganisms. EPA/600/4 90/027F.Cincinnati,OH. 104

121 38.LewisP,KlemmD,LazorchakJ,Norber KingT,PeltietW,HeberM.1994.Short TermMethodsforEstimatingtheChronicToxicityofEffluentsandReceivingWaters tofreshwaterandmarineorgansims.epa600/4 91/002.U.S.Environmental ProtectionAgency,Cincinnati,OH. 39.EgertonRF.2009.ElectronEnergy LossSpectroscopyintheTEM.Reportson ProgressinPhysics.72: ShiKamNW,JessopTC,WenderPA,DaiH.2004.NanotubeMolecular Transporters:InternalizationofCarbonNanotube ProteinConjugatesinto MammalianCells.JournalofAmericanChemicalSociety.126: PantarottoD,BriandJP,MullerS,PratoM,BiancoA.2004.Translocationof BioactivePeptidesAcrossCellMembranesbyCarbonNanotubes.Chemical Communications.1: PantarottoD,SinghR,McCarthyD,ErhardtM,BriandJP,PratoM,KostarelosK, BiancoA.2004.FunctionalizedCarbonNanotubesforPlasmidDNAGeneDelivery. AngewandteChemieInternationalEdition.43: BiancoA,KostarelosK,PratoM.2005.ApplicationsofCarbonNanotubesin DrugDelivery.CurrentOpinioninChemicalBiology.9:

122 44.ShiKamNWandHongjieD.2005.CarbonNanotubesasIntracellularProtein Transporters:GeneralityandBiologicalFunctionality.JournaloftheAmerican ChemicalSociety.127: RobertsAP,MountAS,SedaB,SoutherJ,QiaoR,LinS,KePC,RaoAM,KlaineSJ InvivoBiomodificationofLipid CoatedCarbonNanotubesbyDaphnia magna.environmentalscienceandtechnology.41: MouchetF,LandoisP,SarremejeanE,BernardG,PuechP,PinelliE,FlahautE, GauthierL.2008.CharacterizationandinvivoEcotoxicityEvaluationofDouble WallCarbonNanotubesinLarvaeoftheAmphibianXenopuslaevis.Aquatic Toxicology.87: AllenBL,KichambarePD,GouP,VlasovaII,KapralovAA,KonduruN,KaganVE, StarA.2008.NanoLetters.8: AllenBL,KotcheyGP,ChenY,YanamalaNVK,Klein SeetharamanJ,KaganVE, StarA.2009.JournaloftheAmericanChemicalSociety.131: KaganVE,KonduruNV,FengW,AllenBL,ConroyJ,VolkovY,VlasovaII, BelikovaNA,YanamalaN,KapralovA,TyurinaYY,ShiJ,KisinER,MurrayAR,Franks J,StolzD,GouP,Klein SeetharamanJ,FadeelB,StarA,ShvedovaAA.2010.Natural Nanotechnology.5:

123 50.LiuX,HurtRH,KaneAB.2010.BiodurabilityofSingle WalledCarbon NanotubesDependsonSurfaceFunctionalization.Carbon.48: RussierJ,Menard MoyonC,VenturelliE,GravelE,MarcolongoG,MeneghettiM, DorisE,BiancoA.2011.OxidativeBiodegradationofSingle andmulti Walled CarbonNanotubes.Nanoscale.3:

124 Figures Figure1.Thetopimageisthesampletobeanalyzed;thelefthandsideisthedorsal surfaceofthesectioneddaphniamagnathebluesquareistheareaanalyzedby RamanSpectroscopy.TheG bandandd bandsignalsarestronginthegutand absentattheedgesindicatingahighconcentrationofingestedoh SWNTswithin thegutbutnodetectablesignalinthetissues. 108

125 Figure2.Thetopimageisareaofthegutchosenforanalysisandinthesecond imagetheareaanalyzedathighermagnificationishighlightedinblack.the resultingg bandsignalismappedoutandshowsahighlightedregionofhighoh SWNTconcentrationwithinthegutofexposedD.magna,butthereisnodetectable signalwithinthetissues. 109

126 Figure3.Transmissionelectronimagesofmaterialspostsonication;A)OH SWNTs innaturalorganicmatter,b)sio2 SWNTsinnaturalorganicmatter,C)PABS SWNTs innaturalorganicmatter,d)peg SWNTsinnaturalorganicmatter,andE)OH SWNTsinFBS. 110

127 Figure4.TransmissionelectronmicroscopyimagesofthecrosssectionofNOM suspendedoh SWNTexposedD.magna.ImageAshowsthegut,microvilli,and lumen.imagebandcshowoh SWTNsinthegut(squares)andsuspectedmaterial inassociatedwiththemicrovilliandabsorbedinthelumen(circles). 111

128 Figure5.TransmissionelectronmicroscopyimagesofthecrosssectionofNOM suspendedpeg SWNTexposedD.magna.ImageAshowsthegut,microvilli,and lumenwithpeg SWNTsinthegut(squares).ImageBandCshowssuspectedPEG SWNTsabsorbedinthelumen(circles). 112

129 Figure6.TransmissionelectronmicroscopyimagesofthecrosssectionofNOM suspendedpabs SWNTexposedD.magna.ImageAshowsthegutandlumenwith PABS SWNTsinthegut(squares)andsuspectedPABS SWNTsabsorbedintothe lumen.imagebshowssuspectedpabs SWNTsabsorbedinthelumen(circle). Figure7.TransmissionelectronmicroscopyimageofNOMsuspendedSiO2 SWNTs exposedd.magna.theimageshowsthematerialpresentinthegut(square)but notassociatedwiththemicrovilliorabsorbedinthelumen. 113

130 Figure8.TransmissionelectronmicroscopyimagesofthecrosssectionofFBS suspendedoh SWNTexposedD.magna.ImageAshowsthemicrovilliandlumen withsuspectedoh SWNTsassociatedwiththetissues(circles).ImageBshows suspectedoh SWNTsabsorbedinthelumenandassociatedwiththemicrovilliin moredetail(circles). 114

131 Figure9.Transmissionelectronmicroscopyimagesinz contrastmodeofthecross sectionoffbsdispersedoh SWNTexposedD.magna.Electrondensematerial appearswhitecomparedtothelesselectrondensetissues(circles).inbandclong tube likematerialthatappearselectrondenseisfoundwithinthelumen(circles). 115

132 Figure10.AandBareHRTEMimagesoftubularstructuresfoundwithinthegut tractoffbsoh SWNTexposedD.magna.CisHRTEMimagewithSADdiffraction patternconfirmingtheabsenceofswnts.distheenergydispersionspectrumof stainedfbsoh SWNTsectionconfirmingthedarkareasareparticlesofstainnot SWNTs. 116

133 Figure11.High resolutiontransmissionelectronmicrographsofpbs SWNTs imagedinexposedd.magnasectionsrangingfrom100nm(a)to10nm(c). SWNTscanbeseenincircledregionofC. 117

134 Figure12.Aisarepresentativeselectiveareadiffraction(SAD)patternofareas absentofswntsandbisarepresentativesadofswnts(aandbareatthesame magnification,scalebaris50nm).canddarerepresentativelow lossandcoreloss(respectively)electronemissionlossspectra(eels)ofswnts.youcan observethelargepeaksthatareindicativeofswntseelsspectra. 118

135 a b c d Figure 13. HRTEM investigation of apparent PEG SWNTs in Daphnia at lower magnifications with the camera out of focus (A) in focus (B) and at higher resolutions (C and D). The inset of part D shows the selective area diffraction pattern indicative of amorphous carbon. 119

136 Figure14.A)TEMimageofanunstainedFBSOH SWNTexposedD.magnasection, B)TEMimageofastainedcontrolD.magnasection. 120

137 CHAPTER4:DIFFERENTIALTRANSCIRPTIONOFRESOURCEGENESINDaphnia magnaexposedtocarbonnanotubes ABSTRACT Themanufactureofnanomaterialshasdramaticallyincreasedoverthelast decade.carbonnanotubes(cnts)haveexcellentthermalandelectrical conductivityandastrength to weightratiothatis460timethatofsteel.these characteristics,andothers,havemadethemusefulinavarietyofapplicationsfrom electronicstobikeframes.therearetwotypesofcarbonnanotubes,single walled nanotubes(swnts)andmulti wallednanotubes(mwnts)andtheyareoneofthe mostmanufacturednanomaterialstoday.suspendedcntshavebeenshownto causeadverseeffectssuchasreducedfertilizationandmoltinginanestuarine copepod,respiratorytoxicityinrainbowtrout,reducedgrowthandmortalityin DaphniamagnaandmortalityinXenopuslaevis.Ithasbeenobservedthatthegutof organismsexposedtonanomaterialshadbeensignificantlypackedwithingested material.itisforseeablethatnormaldigestiveprocesswillbeadverselyaffectedin theseexposurescenarios. Cellularleveleffectscanbeusedtoextrapolatetoapicalendpointson individuals.aconceptualframeworkthatlinksamolecular leveleffectwith adverseeffectsthatareusedtodetermineriskassessmenthasbeencalledan adverseoutcomepathway(aop).theobjectivesofthisstudyaretodeterminethe differentialexpressionofresourcegenesindaphniamagnaexposedtosuspended 121

138 MWNTsandSWNTstobeusedasanadverseoutcomepathwaytopredictapical endpointeffectstogrowthandreproduction. Thecurrentstudydeterminedgeneticmarkersthatcouldbeusedtoexplain thechangestoresourceallocationthatresultsinreducedgrowthandreproduction aftercntexposureandcomparethesetoastarvedtreatment.effectstonutrition wereexaminedusinglipaseandchymotrypsingenes.downstreameffectsof nutritionalterationwouldbeanalterationinothergutprocessandareductionin energystores.peritrophicmatrixprotein2(ptm2)transcriptiondetermined effectstoothergutprocessesandatp/adptranslocasedeterminedchangesin energycycling.reductionofgrowthwasexaminedusingneuroparsinandepoxide hydrolase.growtheffectswouldcausechangestothemoltingcycle.theniemann Picktype2(NPC2)andchitinasedatawereinconclusiveduetohighvariability.A conceptualaopwasdevelopedusingthetranscriptiondata.futureresearchcould elucidatebetterindicatorstochangesingrowthandmoltingandalsoconfirmthat ingestedcntscauseaphysicalblockagebytestingwithotherinertparticlessuchas clayorpolystyrenebeads. INTRODUCTION Themanufactureofnanomaterialshasdramaticallyincreasedoverthelast decade.theirsmallsizegivesthemalargesurfaceareaandhigherreactivity comparedtothebulkform[1].carbonnanotubes(cnts)haveexcellentthermal andelectricalconductivityandastrength to weightratiothatis460timethatof steel[2].forthisreasontheyhavebeenusedinplasticcomposites,catalysts, 122

139 batteryandfuelcellelectrodes,componentsinelectronics,aircraft,aerospace,and automotiveindustries[2].therearetwotypesofcarbonnanotubes,single walled nanotubes(swnts)andmulti wallednanotubes(mwnts).single walled nanotubesareasinglesheetofsp 2 bondedgraphenerolledintoacylinderwith endscappedwithstructuressimilartothecurvatureofafullerene[3].multi walled nanotubesaremultiplecarbontubesofincreasingsizeplacedconcentricallywithin eachotherwithoutendcaps[4].carbonnanotubesareoneofthemost manufacturednanomaterialswithacurrentglobalannualproductioncapacityfor MWNTsestimatedtobegreaterthan3,400tonsandexpectedtoreach9,400tons by2015[5].thehighlevelofcarbonnanotubeproductionanduseincreasestheir likelihoodofenvironmentalrelease.itisforthisreasonthatin2001president ClintonsignedabillestablishingtheNationalNanotechnologyInitiative(NNI)to coordinategovernmentfundingforu.s.researchinnanotechnology[6]. SincetheninvitrostudieshaveshownthatCNTscancausereducedgrowth, reactiveoxygenspeciesproduction,apoptosisinduction,andcelldeathinavariety ofcelllines[7 10].InhalationsstudieswithmicehaveshownCNTscancause inflammation,epitheloidgranulomaproduction,andperibronchialnecrosisinlung tissuesofexposedmice[11,12].furthermore,suspendedcntshavebeenshownto causeadverseeffectssuchasreducedfertilizationandmoltinginanestuarine copepod[13],respiratorytoxicityinrainbowtrout[14],reducedgrowthand mortalityindaphniamagna[15,16]andmortalityinxenopuslaevis[17]. Asidefromtheseapicalendpoints,carbonNanotubeshavealsobeenshown tocausephysiologicalchangesinaquaticorganismsaswell.forexample,lovernet 123

140 al(2007)[18]determinedthatc60andhydrogenatedfullerenes(c60hxc70hx) increasedhopping,appendagemovement,andheartratesinexposedorganisms. Thesephysiologicalchangeswouldincreaseanorganism sstressandreducetheir fitnessandabilitytoavoidpredation.klaperetal(2009)[19]determinedthatgst levelswereincreasedind.magnaexposedtostirredc60,c60(suspendedin tetrahydrofuran(thf C60)),andhydroxylatedC60(OH C60).Catalase(CAT)was increasedinhydrogenatedc60(hx C60)andOH C60treatments.Theseresults indicatethatsuspendedcarbonnanomaterialsmaycausephysiologicalchangesto exposedd.magna. Ithasbeenobservedthatthegutoforganismsexposedtonanomaterialshad beensignificantlypackedwithingestedmaterial[14 16,18,20 22].Itisforseeable thatnormaldigestiveprocesswillbeadverselyaffectedintheseexposurescenarios. Dudychaetal(inprep)[23]hasshowndifferencesresourceallocationgene transcriptionindaphniaplucariaexposedtodifferentqualitiesoffood.organisms thatwerefedalowerqualitydiethad10ofthe14genesofinterestdownregulated whileonly4wereupregulatedcomparedtothoseorganismsfedahigherquality food.wethereforehypothesizedthatdaphniamagnaexposedtocntswillhave generegulationsimilartostarvedorganismsbecauseofblockagetotheguttract causebyingestedcnts. Cellularleveleffectscanbeusedtoextrapolatetoapicalendpointson individuals.aconceptualframeworkthatlinksamolecular leveleffectwith adverseeffectsthatareusedtodetermineriskassessmenthasbeencalledan adverseoutcomepathway(aop)[24].theproposedaopinitiatingeventincnt 124

141 exposedd.magnaistheingestionofcntsthatleadstoanimpactedgutthatcauses poorfoodassimilation.this,inturn,mayleadtopoornutrition,whichcauses effectstogrowth,molting,andeventuallyreproduction. Theobjectivesofthisstudyaretodeterminethedifferentialexpressionof resourcegenesindaphniamagnaexposedtosuspendedmwntsandswntstobe usedasanadverseoutcomepathwaytopredictapicalendpointeffectstogrowth andreproduction. MATERIALSANDMETHODS Organisms DaphniamagnawereobtainedfromculturesmaintainedattheInstituteof EnvironmentalToxicology,ClemsonUniversity(Pendleton,SouthCarolina,USA). TheywereculturedinU.S.EnvironmentalProtectionAgency(U.S.EPA)moderately hardreconstitutedwater(mhw)at25 Cwitha16:8hrlight:darkcycle[25,26]. CarbonNanomaterials Multi wallednanotubesweresynthesizedatclemsonuniversityusingthe chemicalvapordepositionmethodviathedecompositionofaferrocene xylene mixture[27].themwntshadanapproximatediameterof50nm,lengthof approximately50um,andanpurityofgreaterthan95%.hydroxylatedswnts (OH SWNTs)werepurchasedfromcheaptubes.com(Brattleboro,Vermont,USA). Accordingtothemanufacturer sspecifications,theswntswere1nm 2nmin 125

142 diameter,10µm 30µminlength,>90%pure,andwasfunctionalized3.96%by mass. NaturalOrganicMatter(NOM) TheNOMsourceusedduringthisstudywasSuwanneeRiverNOM(SR NOM) thatwaspurchasedfromtheinternationalhumicsubstancessociety;thisproduct wasconcentratedbyreverseosmosistoapowder.naturalorganicmattersolutions weremadebydilutingsr NOMinMHWandthenfilteringitwitha0.2µmcellulose membranefilterpriortotoxicitytests.alltestsanddilutionwaterwereusedat concentrationsof2.5mg/ldissolvedorganiccarbon(doc). CarbonNanotubeSuspensions SWNTsandMWNTsweresuspendedinSR NOMsolutionusingthefollowing procedure:thematerialwasfirstweighedonaluminumfoilandplacedina200ml glasscentrifugetube200mlofsr NOMsolutionwasthenaddedtothecentrifuge tube,andthesolutionwasprobesonicatedwithabransondigitalsonifier(danbury, Connecticut,USA)for20minat40%power.Thispoweroutputwascalculatedtobe approximately85.2wattsusingthemethoddescribedbytaurozzietal(2010)[28]. Thesolutionswereallowedtosettleforapproximately24hrsbeforethe supernatantwasusedforbioassays.thestablesolutionwasdecantedand concentrationsweredeterminedbyweighingtheremainingprecipitateonaprerinsed/dried0.2umcellulosemembranefilter.thisweightwassubtractedfrom theinitialweightofswntsanddividedbythevolumeofthestablestocksolutionto 126

143 determinetheinitialstockconcentration.thestocksolutionwasfurtherdiluted withnomsolutiontoachieveconcentrationsof2mg/land0.5mg/lmwntsor SWNTsforbioassays. ZetapotentialofsuspendCNTswasanalyzedusingadisposablecapillarycell withamalvernzetasizer(malverinstruments,worcestershire,uk).lengthand diameterofthecntsinsolutionwasdeterminedusingtransmissionelectron microscorpy. Bioassays Daphniamagnaacute48hstaticrenewalbioassayswereperformedwith3 replicatesforeachmwnt,swnt,andnomcontrol.anadditionalnomcontrol, with3replicates,wasnotfedthroughoutthetesttosimulatestarvedconditions. Thirtyorganisms,3 4dold,wereplacedin250mLglassbeakersfilledwith150mL ofcntorcontrolsolution.testsolutionswerereneweddailyandfed(exceptthe starved control)2mlofa4:3algae:yeast/troutchow/cereal(ytc)mixture4h beforerenewalandorganismsampling. Primers Primersweredesignedfor8resourcegenes:PeritrophicMatixMembrane2 (PTM2),Chymostrypsin(CHY1),ATP/ADPTranslocase(ATP/ADP),Chitinase, Neuroparsin,EpoxideHydrolase(EpoxideH.),Niemann PickTypeC2(NPC2),and Lipase(Table1).Actinwasusedasahousekeepinggenetonormalizethedata (Table1).PeptidesequencesofthegeneswereobtainedfromDudychaetal(2011) 127

144 [23].UsingtheDaphniaspeciesgenomeonwFleaBasegenenucleotidesequences weredeterminedfromthepeptidesequencesusingtheblasttool.thegene sequenceswerereenteredintotheblasttooltoensurethecorrectgeneofinterest wasproduced.thegenesequencewasenteredintheprimerquesttoolfrom IntegratedDNATechnolgieswebsite( wereenteredintotheblasttoolfromwfleabasetoconfirmtheproductionofthe desiredgene.pcrreactionswererunusingthedesignedprimersandthepresence ofagenewasconfirmedbygelelectrophoresis.thepcrproductwaspurifiedusing Qiagen sqiaquickpcrpurificationkit. Real TimePCRAnalysis After48hofexposure,D.magnawerecollectedandRNAwasextracted usingtri Reagent(Sigma).cDNAwaspreparedusing2µgtotalRNAincubated with50ngrandomhexamers,rnasin,10mmdntpmix,and200umoloneymurine leukemiavirus(mmlv)reversetranscriptaseat37 o Cfor1hour.Standardcurves weremadebyrunningpcrreactionsforeachgeneusing1xbuffer,0.2µmdntp mix,0.2µmofeachprimer,1utaqpolymeraseand100ngofcompositecdna. Real timepcrreactionswereperformedusingbio Rad si Cycler(Hercules,CA) usingrt 2 SYBRGreen/FluoresceinqPCRmastermix(SABiosciences,Frederick, MD),andindividualprimersforthegeneofinterest.AllPCRreactionshada denaturingstepof95 Cfor15seconds,anannealing/extensionstepat60 o Cfor1 minuteforatotalof40cycles.meltcurveanalyseswereperformedeachtimeto ensuresingleproductformation.sampleswererunintriplicate,standardcurveto 128

145 determineefficiency,andfold changesfromthecontrolswereanalyzedfollowing thepfafflmethod[29].statisticaldifferencesweredeterminedusingat test (p<0.05)betweenindividualtreatmentresponsesandthemeancontrolresponse usingsassoftware(sasinstitute). RESULTS ParticleCharacterization TherewasnodifferenceinzetapotentialoftheMWNTandSWNTsolutions (Table2).ThissuggeststhesurfacechargeoftheCNTsareequivalentandtherewill benodifferenceinsurfacechargeeffectstogutepithelialcells.however,tem imagesdeterminedthatthemwntswereofmixedlengthsbetween1and10µm (duetoshearingfromthesonicationprocess)whiletheswntsweremore uniformedlengths(1to2µm)(table2,figure1).thissuggeststhatany differencesbetweentheparticlesmaybecontributedtodifferencesinsize. RealTimePCR Lipasewassignificantlyinducedinthestarvedtreatmentcomparedto controls(figure2).the0.5mg/land2mg/lswnttreatmentsweredown regulatedalthoughnotsignificantly,whilethe2mg/lmwnttreatmentwas significantlydownregulated(figure2).chymotrypsinwassignificantlydown regulatedinboththestarvedand2mg/lmwnttreatment.peritrohpicmatrix protein2(ptm2)wassignificantlydownregulatedinthe0.5mg/lswnttreatment anddownregulated,butnotsignificantly,inthestarved,2mg/lmwnt,and2mg/l 129

146 SWNTtreatments.ATP/ADPtranslocasewassignificantlyupregulatedinthe starvedtreatmentwhiletherewasnodifferencefromcontrolsinthecnt treatments.neuroparsinwasupregulatedinthestarvedtreatment,but significantlydownregulatedinthe2mg/lmwnttreatment.thestarved,2mg/l MWNT,0.5mg/Land2mg/LSWNTtreatmentshaddownregulationofepoxide hydrolase,althoughitwasnotsignificant.therewasalotofvariabilityinthe transcriptionofthisgeneinthe0.5mg/lmwntandswnttreatments.therewas significantdownregulationofniemann picktype2inthe2mg/lmwnt,0.5mg/l and2mg/lswnttreatments,whilethestarvedtreatmentwasdownregulatedit wasnotsignificant.therewashighvariabilityinthetranscriptionofchitinaseinall treatmentsresultinginnosignificantchanges. DISCUSSION Robinsonetal(2010)[30]demonstratedthatsuspendedclayreduced growthandreproductionind.magna.lightmicroscopyimagesrevealedthatthe ingestedclayimpactedtheguttractsofexposedorganismsdisruptingnormalfood assimilation.thisresultedinchangestoresourceallocationcausingareductionin growthandreproduction.resultsofthisresearchsuggestthatotherinertparticles thatcancausesimilarguttractblockagewillhavethesameapicalendpoint; reducedgrowthandreproduction.edgingtonetal(2010)[16]demonstrated similareffectstogrowthandreproductionafterd.magnaandceriodaphniadubia wereexposedtosuspendedmwnts.conclusionsfromguteliminationexperiments suggestedthatd.magnamayhavedifficultyineliminatingingestedmwntsfrom 130

147 theguttractandtransmissionelectronmicroscopyimagessuggestingestedmwnts mayinterferewithnormalgutprocess. Thecurrentstudydeterminedgeneticmarkersthatcouldbeusedtoexplain thechangestoresourceallocationthatresultsinreducedgrowthandreproduction aftercntexposure.effectstonutritionwereexaminedusinglipaseand chymotrypsingenes.lipaseisresponsibleforlipiddigestionwhilechymotrypsinis responsibleforproteindigestion.lipasewassignificantlyinducedinthestarved treatmentsuggestingtheorganismswererelyingonfatstoresforenergy.in contrast,organismsexposedtocntsshoweddownregulationoflipase(figure2), suggestingtheseorganismswerenotyetinatruestarvedstate.chymotrypsinwas significantlydownregulatedinthestarvedtreatmentimplyingthatastarved organismwouldnothaveproteinsfordigestionandwouldrelyonfatstoresfor energy(figure2).d.magnaexposedto2mg/lmwntalsohaddownregulated chymotrypsinsuggestingareducedneedforproteindigestionduetogutblockage byingestedcnts(figure2).theseresultssuggestthatproteinmetabolismmaybe downregulatedquickerthanlipidmetabolismisupregulated. Downstreameffectsofnutritionalterationwouldbeanalterationinother gutprocessandareductioninenergystores.peritrophicmatrixprotein2(ptm2) isresponsiblefortheformationoftheproteinandchitinlayerthatlinesthegutof invertebratesthataidsindigestion[31].thisgenewasdownregulatedinthe starvedorganismsaswellasorganismsexposedtocnts(figure2).thisdown regulationmaybeduetothelackofresourcestoallocatetotheproductionof proteinsandchitin.lackofresourcesalsoaffectsenergyproduction.evidenceof 131

148 changestoenergyproductioncanbeseeninthetranscriptionofatp/adp translocase.thisgeneisresponsiblefortransportingadpintothemitochondriaso itcanberephosphorylatedandrecycledtoatp[32].thestarvedtreatmenthada significantinductionofatp/adptranslocasesuggestingthestarvedorganismsare relyingontherecyclingofadptoatpbecauseofthelackofresourcesforthe productionofnewatp(figure2).incontrast,cnt exposedorganismsshowedno changestothetranscriptionofatp/adptranslocasesuggestingtheseorganisms werenotyetinatruestarvedstate(figure2).thisisconsistentwiththelipase data. Changesinnutritionandeffectstoenergyproductionwouldbeexpectedto resultinreducedgrowth.thiswasinvestigatedusingneuroparsinandepoxide hydrolase.neuroparsinisresponsibleforinhibitingthejuvenilehormone(jh)that regulatesthegrowthcyclewhileepoxidehydrolaseisresponsibleforbreaking downjh[23].neuroparsinwasonlyslightlyinducedinthestarvedtreatmentwhile epoxidehydrolasewasonlyslightlydownregulated(figure2).incontrast,cntexposedorganismsdownregulatedneuroparsinandepoxidehydrolase(figure2). WhilethisdataisinconclusiveitsupportsthelipaseandATP/ADPtranslocasedata thatthecntexposedorganismswerenotyetinastarvedstatetoseeeffectstothe JHpathway.Further,thesedataalsosuggestthatJHitselfmaybeabetterindicator ofchangesingrowth. Areductioningrowthwouldcausechangestothemoltingcycle.Niemann Picktype2(NPC2)regulatesmoltbycontrollingtheproductionofthesterol substrateformoltinghormonesandchitinaseisresponsibleforbreakingdownthe 132

149 oldexoskeletonpriortomolting[23].bothstarvedandcnt exposedorganisms showeddownregulationofnpc2(figure2).thissuggestsahaltingorreductionof themoltingprocessduetothelackofthesterolsubstrateformoltinghormones. Therewerenosignificantchangesinchitinaseregulationforanytreatment.(Figure 2).Highvariabilityinthesedatamaybeduetotheorganismsbeingindifferent stagesofmolt;futurestudiesmayrequireusingorganismsonlyafewhoursapartin agetoensuretheyareinthesamestagesofmoltpotentiallyreducingvariability. TheimplementationofAOPsreliesonunderstandinghowatoxicant interactswithtargetcellsorspecificreceptorbindingsites[33].wantanabeetal (2011)[34]developedanAOPfordemoicacid,anaminoacidproducedbyblue algaethatcanreachhighconcentrationsduringalgalblooms.theaopisinitiated whenbraintissueconcentrationsofdemoicacidarehighenoughtoactivatethe glutamatereceptorandfromthereitbeginsacascadeofeffectsthatultimately resultinimpactstopopulations(figure3).perkinsetal(2011)[35]similarly outlinedanaopusingtoxicitydatafromflutamidestudiesanddemonstratedthe apicalendpointeffectsofreducedreproductioninexposedfatheadminnowsbegin withthecompetitionofflutamidewithtestosteroneanddihydrotestosterone bindingtotheandrogenreceptor.krameretal(2011)[35]usedestablished mechanistictoxicitydatatodevelopaopsforcontaminantsresponsibleforthe inhibitionofvitellogenesis,activationofthearylhydrocarbonreceptor,inhibitionof acetylcholinesterase,inhibitionofcalcium adenosintriphsophatase,andthe initiationoftheretinoi X receptor. 133

150 InthecurrentstudyweattemptedtoreverseengineeranAOP.(Figure4). TheAOPbeginswith(1)ahealthyorganismthatconsumes(2)suspendedCNTsthat resultsin(3)andimpactedguttract.thisresultsin(4)decreasedfoodassimilation fromthephysicalblockageinthegutpreventingfoodfrombeingprocessedand assimilated.this,inturn,leadsto(5a D)poornutrition(lipaseand chymostrypsin),energyreduction(observedwithptm2andatp/adptranslocase data),decreasedgrowth[16](neuroparsinandepoxidehydrolasetranscription), andreducedmolting(npc2andchitinasetranscription).theresultingorganismal apicalendpointis(6)reducedreproductionandthiscouldleadto(7)population effectsand,ultimately,(8)ecologicaleffects. TherearetwomaindifferencesinourconceptualAOPfromothers previouslydiscussed.thefirstisthatthisaopisnotdevelopedaroundcnts interactingwithasinglereceptororbindingsite.insteadit sdevelopedarounda weightofevidenceapproachusingdatatodetermineeffectstonutrition,energy, growth,andmolting.effectstooneofthesepathwaysisnotenoughtoconcludean adverseoutcome,buttogetherthedataisevidentenoughtoconcludetheeffects cascadewillresultintheapicalendpointofreducedreproductionandpossible populationeffects. TheseconddifferenceinthisconceptualAOPfromothersisthatbecauseit isnotdevelopedaroundaspecificreceptororbindingsiteitcanbeextrapolatedto manydifferentspeciesofvertebratesorinvertebratesbecausetheinitiatingeventis animpactedguttract.thepossibledownstreameffectstopopulationsmayoccurif thereissignificantreductioninreproductionwithintheexposedpopulation. 134

151 Daphniamagnaareimportantfilterfeedingorganismswithinafoodchainandarea valuablefoodsourceforfishspecies.ifthisspeciesissignificantlyreducedwhole ecosystemeffectsmayincludealgalbloomsandreducedfoodforinsectivorousfish. CONCLUSIONS TheobjectivesofthisstudyweretodevelopanAOPusingdatafromresource genetranscriptiontodetermineanapicalendpointofreducereproduction.we hypothesizedthatcnt cloggedguttractswouldleadtoreducedfoodassimilation andthatresourcegenetranscriptionintheseorganismsorganismswouldbesimilar tostarvedorganisms.manybutnotallresultssupportthishypothesis.proposed AOPisdifferentthanotherAOPsthatarebasedonsinglereceptorandspecific bindingsites.theweightofevidenceoftheresultsofthisresearchissufficientto warrantfurtherinvestigation.futureresearchshouldinvestigateotherindicators ofpoornutrition,reducedenergy,reducedgrowth,andreducedmoltingtoconfirm thesepathwaysandstrengthentheaop.furthermore,otherinertparticlessuchas clayandpolystyrenebeadsshouldbetestedtofurtherprovetheinitiatingeventisa physicalblockageofthegut. 135

152 REFERENCES 1.StoneV,NowakB,BaunA,VandenBrinkN,VonderKammerF,DusinskaM, HandyR,HankinS,HassellovM,JonerE,FernandesTF.2010.Nanomateialsfor EnvironmentalStudies:Classification,ReferenceMaterialIssues,andStrategiesfor Physico ChemicalCharacterisation.ScienceoftheTotalEnvironment.408: KlaineSJ,AlvarezPJJ,BatleyGE,FernandesTF,HandyRD,LyonDY,MahendraS, McLaughlinMJ,LeadJR.2008.NanomaterialsintheEnvironment:Behavior,Fate, Bioavailability,andEffects.EnvironmentalToxicologyandChemistry.27(9): McEuenPL.2000.CarbonNanotubes:Single WallCarbonNanotubes.Physics World.June: IijimaS.1991.HelicalMicrotublesofGraphiticCarbon.Nature.354: PRNewsWire. adds production and application of carbon nanotubescarbon nanofibers fullerenes graphene and nanodiamonds a global technologysurvey and market analysis htmlApril28,

153 6.GuzmanKD,TaylorMR,BanfieldJF.2006.EnvironmentalRisksof Nanotechnology:NationalNanotechnologyInitiativeFunding, EnvironmentalScienceandTechnology.40: ChengWW,LinZQ,WeiBF,ZengQ,HanB,WeiCX,FanXJ,HuCL,LiuLH,Huang JH,YangX,XiAG.2011.Single WalledCarbonNanotubeInductionofRatAortic EndothelialCellApoptosis:ReactiveOxygenSpeciesareInvolvedinthe MitochondrialPathway.TheInternationalJournalofBiochemistryandCellBiology. 43: DavorenM,HerzogE,CaseyA,CottineauB,ChambersG,ByrneHJ,LyngFM InvitroToxicityEvaluationofSingleWalledCarbonNanotubesonHuman A549LungCells.ToxicologyinVitro.21: DiGiorgioML,DiBucchianicoS,RagnelliAM,AimolaP,SantucciS,PomaA EffectsofSingleandMultiWalledCarbonNanotubesonMacrophages:Cytoand GenotoxicityandElectronMicroscopy.MutationResearch.722: WangJ,SunP,BaoY,LiuJ,AnL.2011.CytotoxicityofSingle WalledCarbon NanotubesonPC12Cells.ToxicologyinVitro.25:

154 11.LamCW,JamesJT,McCluskeyR,HunterRL.2004.PulmonaryToxicityof Single WallCarbonNanotubesinMice7and90DaysAfterIntratrachealInstillation. ToxicologicalSciences.77: InoueKI,YanagisawaR,KoikeE,NishikawaM,TakanoH.2010.Repeated PulmonaryExposuretoSingle WalledCarbonNanotubesExacerbatesAllergic InflammationoftheAirway:PossibleRoleofOxidativeStress.FreeRadicalBiology andmedicine.48: TempletonRC,FergusonPL,WashburnKM,ScribensWA,ChandlerGT Life CylceEffectsofSingle WalledCarbonNanotubes(SWNTs)onanEstuarine MeiobenthicCopepod.EnvironmentalScienceandTechnology.40: SmithCJ,ShawBJ,HandyRD.2007.ToxicityofSingleWalledCarbon NanotubestoRainbowTrout,(Oncorhynchusmykiss):RespiratoryToxicity,Organ Pathologies,andOtherPhysiologicalEffects.AquaticToxicology.82: RobertsAP,MountAS,SedaB,SoutherJ,QiaoR,LinS,KePC,RaoAM,KlaineSJ InvivoBiomodificationofLipid CoatedCarbonNanotubesbyDaphnia magna.environmentalscienceandtechnology.41:

155 16.EdgingtonAJ,RobertsAP,TaylorLM,AlloyMM,ReppertJ,RaoAM,MaoJ,Klaine SJ.2010.TheInfluenceofNaturalOrganicMatterontheToxicityofMultwalled CarbonNanotubes.29(11): MouchetF,LandoisP,SarremejeanE,BernardG,PuechP,PinelliE,FlahautE, GauthierL.2008.CharacterizationandinvivoEcotoxicityEvaluationofDouble WallCarbonNanotubesinLarvaeoftheAmphibianXenopuslaevis.Aquatic Toxicology.87: LovernSB,SticklerJR,KlaperR.2007.BehavioralandPhysiologicalChangesin DaphniamagnaWhenExposedtoNanoparticleSuspensions(TitaniumDioxide, Nano C60,andC60HxC70Hx).EnvironmentalScienceandTechnology.41: KlaperR,CragoJ,BarrJ,ArndtD,SetyowatiK,ChenJ.2009.ToxicityBiomarker ExpressioninDaphnidsExposedtoManufacturedNanoparticles:Changesintoxicity withfunctionalization.environmentalpollution.157: PetersenEJ,AkkanenJ,KukkonenJVK,WeberWJ.2009.BiologicalUptakeand DepurationofCarbonNanotubesbyDaphniamagna.EnvironmentalScienceand Technology.43:

156 21.KennedyAJ,HullMS,SteevensJA,DontsovaKM,ChappellMA,GunterJC,Weiss CA.2008.FactorsInfluencingthePartitioningandToxicityofNanotubesinthe AquaticEnvironment.EnvironmentalToxicologyandChemistry.27(9): KennedyAJ,GunterJC,ChappellMA,GossJD,HullMS,KirganRA,SteevensJA InfluenceofNanotubePreparationinAquaticBioassays.Environmental ToxicologyandChemistry.28(9): DudychaJL,BrandonCS,DeitzKC.2011.PopulationGenomicsofResource Exsploitation:InsightsFromGeneExpressionProfilesoftwoDaphniaEcostypesFed AlternateResources.EcologyandEvolution.Inpress. 24.VilleneuveDL,Garcia ReyeroN.2011.AVisionandStrategyforPredictive EcotoxicologyTestinginthe21 st Century.EnvironmentalToxicologyand Chemistry.30: U.S.EnvironmentalProtectionAgency.1993.MethodsforMeasuringtheAcute ToxicityofEffluentsandReceivingWaterstoFreshwaterandMarineOrganisms. EPA/600/4 90/027F.Cincinnati,OH. 140

157 26.LewisP,KlemmD,LazorchakJ,Norber KingT,PeltietW,HeberM.1994.Short TermMethodsforEstimatingtheChronicToxicityofEffluentsandReceivingWaters tofreshwaterandmarineorgansims.epa600/4 91/002.U.S.Environmental ProtectionAgency,Cincinnati,OH. 27.AndrewsR,JacquesD,RaoAM,DerbyshireF,QianD,FanX,DickeyEC,ChenJ Continuousproductionofalignedcarbonnanotubes:Astepcloserto commercialrealization.chemicalphysicsletters.303: TaurozziJS,HackleyVA,WiesnerMR.2010.UltrasonicDispersionof NanoparticlesforEnvironmental,Health,andSafetyAssessment Issuesand Recommendations.Nanotoxicology.EarlyOnline: PfafflMW.2001.ANewMathematicalModelforRelativeQuantificationin Real TimeRT PCR.NucleicAcidsResearch.29(9): RobinsonSE,CapperNA,KlaineSJ.2010.TheEffectsofContinuousandPulsed ExposuresofSuspendedClayontheSurvival,Growth,andReproductionofDaphnia magna.environmentaltoxicologyandchemistry.29(1): LehaneMJ.1997.PeritrophicMatrixStructureandFunction.AnnualReviewof Entomology.42:

158 32.CasarettandDoull.2001.Toxicology:TheBasicScienceofPoisons.Sixth Edition.EdKlaassenCD.McGraw HillPublishing.NewYork. 33.VilleneuveDL,Garcia ReyeroN.2001.AVisionandStrategyforPridictive EcotoxicologyTestinginthe21 st Century.EnvironmentalToxicologyand Chemistry.30(1): WatanabeKH,AndersenME,BasuN,CarvanMJ,CroftonKM,KingKA,SunolC, Tiffany CastiglioniE,SchultzIR.2011.DefiningandModelingKnownAdverse OutcomePathways:DomoicAcidandNeuronalSignalingasaCaseStudy. EnvironmentalToxicologyandChemistry.30(1): PerkinsEJ,ChipmanJK,EdwardsS,HabibT,FalcianiF,TaylorR,VanAggelenG, BulpeC,AntczakP,LaguinovA.2011.ReverseEngineeringAdverseOutcome Pathways.EnvironmentalToxicologyandChemistry.30(1): KramerVJ,EttersonMA,HeckerM,MurphyCA,RoesijadiG,SpadeDJ, SprombergJA,WangM,AnkleyGT.2011.AdverseOutcomePathwaysand EcologicalRiskAssessment:BridgingtoPopulation LevelEffects.Environmental ToxicologyandChemistry.30(1):

159 Figures Figure1.Transmissionelectronmicroscopyimagesofmult wallednanotubes(a) andsingle wallednanotubes(b)postsonicationinnaturalorganicmattersolutions. 143

160 Figure2.GenetranscriptionforD.magnatreatments.Symbol(*)denotesstatistical significance(p<0.05). 144

161 Figure3.AdverseoutcomepathwayfordemoicacidfromWatanabeetal(2011)[32]. 145

162 Figure 4. Conceptual flow chart for an adverse outcome pathway for suspended carbon nanotubes exposed Daphnia magna. (1) Healthy organism ingests (2) suspended carbon nanotubes that results in (3) impacted gut. This leads to (4) decreased food assimilation from physical blockage of the gut that causes (5 A D) poor nutrition (as seen with lipase and chymostrypsin data), energy reduction (observed with PTM2 and ATP/ADP translocase data), decreased growth (shown in previous growth data by Edgington et al (2010) [16] and the present studies data on neuroparsin and epoxide hydrolase transcription, and reduced molt (suggested by NPC2 and chitinase transcription). The resulting apical endpoint is (6) reduced 146