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PolyhydroxyFullerenes(FullerolsorFullerenols): BeneficialEffectsonGrowthandLifespaninDiverse BiologicalModelsJieGao1,2* ,YihaiWang3,KevinM.Folta3,VijayKrishna1,WeiBai2,PaulIndeglia2,AngelinaGeorgieva1, HideyaNakamura1,BenKoopman1,2,BrijMoudgil1,4*1 ParticleEngineeringResearchCenter,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmerica, 2 DepartmentofEnvironmentalEngineeringSciences, UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmerica, 3 PlantMolecularandCellularBiologyProgram,HorticulturalSciencesDepartment,Universityof Florida,Gainesville,Florida,UnitedStatesofAmerica, 4 DepartmentofMaterialsScienceandEngineering,UniversityofFlorida,Gainesville,Florida,UnitedStatesof AmericaAbstractRecenttoxicologicalstudiesoncarbonnanomaterials,includingfullerenes,haveledtoconcernsabouttheirsafety. Functionalizedfullerenes,suchaspolyhydroxyfullerenes(PHF,fullerols,orfullerenols),haveattractedparticularattention duetotheirwatersolubilityandtoxicity.Here,wereportsurprisinglybeneficialand/orspecificeffectsofPHFonmodel organismsrepresentingfourkingdoms,includingthegreenalgae Pseudokirchneriellasubcapitata ,theplant Arabidopsis thaliana ,thefungus Aspergillusniger ,andtheinvertebrate Ceriodaphniadubia .TheresultsshowedthatPHFhadnoacuteor chronicnegativeeffectsonthefreshwaterorganisms.Conversely,PHFcouldsurprisinglyincreasethealgalculturedensity overcontrolsathigherconcentrations(i.e.,72%increaseby1and5mg/LofPHF)andextendthelifespanandstimulatethe reproductionof Daphnia (e.g.about38%by20mg/LofPHF).WealsoshowthatatcertainPHFconcentrationsfungal growthcanbeenhancedand Arabidopsisthaliana seedlingsexhibitlongerhypocotyls,whileothercomplexphysiological processesremainunaffected.ThesefindingsmayopennewresearchfieldsinthepotentialapplicationsofPHF,e.g.,in biofuelproductionandaquaculture.Theseresultswillformthebasisoffurtherresearchintothemechanismsofgrowth stimulationandlifeextensionbyPHF.Citation: GaoJ,WangY,FoltaKM,KrishnaV,BaiW,etal.(2011)PolyhydroxyFullerenes(FullerolsorFullerenols):BeneficialEffectsonGrowthandLifespani n DiverseBiologicalModels.PLoSONE6(5):e19976.doi:10.1371/journal.pone.0019976 Editor: RichardC.Willson,UniversityofHouston,UnitedStatesofAmerica Received December5,2010; Accepted April21,2011; Published May27,2011 Copyright: 2011Gaoetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisresearchwassupportedbyaresearchgrantfromParticleEngineeringResearchCenter(PERC)(perc.ufl.edu)andCenterforNano-BioSensors (CNBS,cnbs.centers.ufl.edu).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanu script. PublicationofthisarticlewasfundedinpartbytheUniversityofFloridaOpen-AccessPublishingFund. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:dencyl@ufl.edu(JG);bmoudgil@perc.ufl.edu(BM)IntroductionSincetheirdiscoveryin1985,C60fullereneshavebeenamong themostwidelystudiedandusedcarbon-basednanomaterials duetotheiruniquestructuralandelectronicpropertiesthat enablenumerousindustrial,electricalandmedicalapplications [1,2,3].Comparedwiththepristineform,functionalized fullereneshavemorepotentialapplicationsduetotheirenhanced watersolubility.Functionalizedfullereneshavebeenreportedto reduceoxidativestressbyscavengingreactiveoxygenspecies [4,5]andhavebeenexaminedfortheirantioxidantproperties. Conversely,C60fullereneanditswater-solublederivativeshave beenthesubjectofconcernbecauseoftheirrapidlygrowing productionandpotentialenvironmentalandhealthimplications [6].Oneheavilystudiedfunctionalizedfullereneisthepolyhydroxyfullerene(PHF,alsonamedfullerolorfullerenol,Figure1), whereinthefullerenesaredecoratedwith12–42hydroxylgroups permoleculeleadingtoenhancedsolubility.PHFhasbeen shown,forexample,toinducetheproductionofreactiveoxygen speciesandcausemembranedamageinratlivermicrosomes[7], andexhibitcytotoxicityandphototoxicitytohumanepithelial cells[8,9]. Astheglobalproductionoffullerenesandtheirderivativesis growingrapidly,fullerene-containingproductswillinevitablyenter variousenvironmentaltheatresdur ingtheirproduction,deployment anddisposal.Amongtheseenvironments,aquaticandsoilsystemsare likelytheultimatesinksforcarbon-basednanomaterials[6,10,11], thusexposingaquaticandsoilorganismstofullerenes,especiallythe watersolublederivatives.Therefore,itiscriticaltoidentifythe potentialimpactsofthisnanomaterialinecologicalsystems. TheobjectiveofthisstudywastoinvestigatetheeffectsofPHFon growth,development,andreproductioninrepresentativebiological systems.Thestudyexploitstheadaptivecapacities,sensoryspectra and/orreproductiveabilitiesofthesebiologicalsystemsthat ultimatelyserveaslivingsensorsofbio-activecompoundsintheir environments.Theirbehaviorsprovidevaluableinformationthat describeimpactsongrowthanddevelopment,andmayevendetail themechanismofPHFingressthatshapesbiologicalprocesses.The short-termorchronicgrowthand/orreproductiveeffectsofPHF areinvestigatedforamodelfreshwaterphytoplanktonicorganism, thegreenalgae Pseudokirchneriellasubcapitata ,amodelplant Arabidopsis thaliana ,amodelfungus Aspergillusniger ,andamodelzooplankter, theinvertebrate Ceriodaphniadubia PLoSONE|www.plosone.org1May2011|Volume6|Issue5|e19976

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ResultsThefirsttestswereperformedon Pseudokirchneriellasubcapitata ,a standardalgalspeciescommonlyusedtoassesstoxicityofatest substanceinanaqueoussystem.AllPHFconcentrationsexceptfor 0.001mg/Land0.1mg/Lshowedsignificanteffects(p 0.05).After fourdaysofgrowth,thenumberof P.subcapitata cellswas72%higher inthepresenceof1mg/Land5mg/LPHFrelativetothenoPHF controltreatment(Fig.2A).PHFconcentrationsof0.001mg/Land 0.1mg/Ldidnothavesignificanteffectsat a =0.05,eventhough meanvalueswereslightlyelevatedrelativetocontrols. Lighthasaprofoundeffectontheregulationofplantgrowth.The effectsaremostsalientinthedevelopingseedling.Discreteportionsof theelectromagneticspectrum,nutrients,toxinsandotherenvironmentalfactorsoftentimeshavepotenteffectsonhypocotyl(stem) growth.Thedevelopingseedlingisthereforeanexcellentbiosensorto describehowagivencompoundmaycontributetogrowthresponses. Duetoitsshortlifecycle,welldefinedphysiologyandsubstantial geneticresources, Arabidopsisthaliana isoneofthepopularplant modelsforplantbiologyandgeneticresearch.Inthisstudy,theroot andhypocotyllengthsofArabidopsisseedlingsweremeasuredafter fourdaysofgrowthonvariousPHFconcentrations.Theresults Figure1.Chemicalstructureofapolydroxyfullerene(PHF) molecule(x=12to42). doi:10.1371/journal.pone.0019976.g001 Figure2.Effectsofpolyhydroxyfullerens(PHF)ongrowthand/orlifespan. (A)Normalizedalgalgrowthcomparedtountreatedcontrols. Theculturesweregrowninthepresenceofthetreatmentfor4days(n=9).(B)Normalized Arabidopsis hypocotylgrowthagainstuntreatedcontrols. Datarepresentmeanofthreetrials,whereineachtrialcomprisesatleast25seedlingmeasurements.(C)Normalized Daphnia reproductionas measuredbythenumberofneonatesproducedoverthelifetimeof5maturedaphnids(n=4).(D)Lifespanoftesteddaphnidsatconcentrationsfrom 0.001to20mg/L(n=20).Errorbarsindicatestandarderrorofthemean.*:p 0.05;**:p 0.01. doi:10.1371/journal.pone.0019976.g002 BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org2May2011|Volume6|Issue5|e19976

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indicatethatseedlingsgerminatedinthepresenceof100and 200mg/LPHFhavesignificantly(p 0.01)longerhypocotyls comparedtolowerconcentrationsorcontrols(Fig.2B).Noeffects wereobservedinrootelongation(Fig.3)oronphototropicgrowth (notshown).Thedifferenceinhypocotylelongationwasnotobserved indarknessormonochromaticlightconditions(Fig.4).Theresults indicatethattheeffectsonseedlinggrowtharespecific,dependon integrationofsignalsmultiplephotosensorypathways,andaffectonly discreteplantorgans. TheeffectsofPHFwereassessedonthe Aspergillusniger ,the causativeorganismofblackmold.Weobservedthatconcentrationsabove10mg/Lstimulatedgrowth.Treatmentof A.niger with PHFfor120hours(media:amendedRPMI1640)causedmore sporestoformcomparedtocontrols(Fig.5),indicatingthatPHF athigherconcentrationcanstimulatereproduction. ThegrowthandreproductiveeffectsofPHFwerealsotested on Ceriodaphniadubia ,anestablishedEPA-recommended freshwaterbiologicalinvertebratesystem.PHFtreatmentover thelifetimeofthedaphnidsincreasedthenumberofneonatesat 20mg/LPHFby38%(Figure2C).Thedaphnidswere monitoredforPHFeffectonlifespan.At20mg/LofPHFtheir lifespanincreased38%overcomparablenon-PHFtreated controls(p 0.05)(Figure2D).LowerPHFconcentrationshad nosignificanteffect.Figures6a-cshowsamplemicrographstaken forthedaphnidsinthecontrol,0.001mg/Land20mg/Lof PHFtreatmentsafter2-dayexposure.AccumulationofPHFwas clearlyobservedinthe Daphnia GItractexposedto20mg/Lof PHF(Figure6c;arrow).Duringthefirst8days,thebodylength ofeachtesteddaphnidwasmeasureddailyandthelengthwas determinedfromthebaseofthetailspinetothebaseofthehead [12](whitelinesinFigure6a–c).AsshowninFigure6d,there werenoobservableeffectsonthe Daphnia growthatconcentrationsof5mg/Lorlower.At20mg/LofPHF,thegrowthrate wasaffectedwithinthefirsthoursoftreatment,butwasmostly unaffectedthereafter,resultinginasmallerdaphnidsizeoverthe testperiodof8days( a =0.05).DiscussionAlthoughitisunclearhowPHFsaffectcellulargrowthor expandlifespan,theunexpectedobservationsmaybeattributed tothefactthatPHFisoneofthemostpotentROSscavengers andcouldreduceoxidativechallengeexperiencedbyorganisms duringagingandgrowth[13,14].Itwasfoundthatfullerene cagecanabsorbupto6electronsanddispersethemthroughthe 20benzeneringsoveritssurface[15].InastudybyQuicketal. [16],anothertypeoffunctionalizedfullerene,carboxyfullerene, whichisaknownantioxidant,hasbeenshowntoextendthe lifespanofmiceby5%,andtoreverseage-relatedcognitive impairment.However,noclearcorrelationhasbeenobserved betweenantioxidantsandlifespanintheliterature,sofar,and othermechanismsmayberesponsibleforthelifespaneffect [17]. Recentdiscoveriesthatcertainspeciesoffungicandecompose PHFandincorporatethemintobiomass[18]andthatPHFcan bemineralizedtootherinorganiccarbonbydirectphotolysis[19] shedlightonotherpossibilitiesthatalgal,fungalorplantcells maybeabletoutilizePHFmoleculesasanutritionsource.In addition,thefactthatstemgrowthisaffectedunderlight conditions,butrootgrowth,phototropiccurvatureanddarkstem growtharenotaffected(Figures2B,3and4),indicatesthatPHF effectsaremechanisticallydiscreteandnotsimplypleiotropic effectsofagenerallytoxiccompound.Theseresultsindicatethat PHFeffectsarespecificandareaffectingseedlinggrowthdueto interactionwithgrowthregulatoryprocesses,particularlythose associatedwithstemelongationinlight.Theprincipleeffectthat isseenisalongersteminPHFtreatedseedlings.Theeffectisnot light-qualityspecific,sotheeffectislikelyinresponsetolight, ratherthansensingoflight.Examplesofsuchresponsepathways Figure3.NormalizedrootlengthsofArabidopsisseedlingsinvariousPHFconcentrationsunderwhitelightcondition. No observabledifferenceonrootelongationwasdetectedinthePHFtreatedseedlingscomparedwithcontrolseedlings(p 0.05).Rootlengthswereall normalizedtocontrolwithineachexperimentalreplicate.Atleast25seedlingspertreatmentweremeasuredineachreplicate.Errorbarsrepresent s thestandarddeviationofthethreeindependenttrials. doi:10.1371/journal.pone.0019976.g003 BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org3May2011|Volume6|Issue5|e19976

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mayincludethegibberellicacidsensing/responsemechanism,a cellelongationsystemthatisinhibitedbylight.PHFmakes hypocotylslonger,suggestingthatthecompoundispossibly interactingwiththemechanismsthatadjuststemgrowthinthe lightenvironment,suchasahypersensitivityto,orproductionof, planthormones. Theslightreductionof Daphnia bodylengthinthe20mg/LPHF (Fig.6)treatmentindicatesapossibledelayinagingcauseddirectly [16]orindirectly(e.g.,dietaryrestrictionatthisconcentration, whichhasbeenknowntoextendlifespaninavarietyoforganisms [20,21,22]byahighdoseofPHF.Inaddition,theincreaseand improvementofalgalbiomassmayaddfavorablenutrientstothe Daphnia foodandthusincreasetheirlifespan. Frombacteriatomammaliancellstomammaliansystems, polyhydroxyfullereneshavegenerallybeendescribedastoxic [9,23,24,25].TheresultsobtainedfromthisstudyareunexpectedbecausePHFfunctionedoftenasagrowthstimulant,or atleastagrowthregulator.Ourfindingsalsosuggestthat applicationofPHFhasdiscreteb iologicalconsequencesthatare speciesandtissuespecific.Thus,itisimportanttonotethat PHF’seffectscanbegenerallyre gardedasbeneficial,withno evidenceofdeleteriouseffectsd escribedforothernanomaterials.Thesefindingsaresignif icantbecausetheyindicatethat PHFhasthepotentialtoserveasagrowthregulatoror modulatorofbiologicalprocesse sthatspanseveralkingdoms, openingthedoortonewapplicationsthatmaybenefitindustry andagriculture.Suchapplicati onsmayincludeinstanceswhere growthstimulationisdesired,f orinstance,inalgalproduction forbiofuelapplication,aquacu lture,orinmodulatingplant statureordevelopment.MaterialsandMethods 1.PreparationofPHFsolutionPHFusedinthealgaeand Daphnia studywerepurchasedfrom Nano-C,Inc.(Westwood,MA),andfromBuckyUSA(Houston, TX)forthefungalstudy,bothsampleswithassignedpossible compositionC60(OH)24–26.PHFwerealsosynthesizedthrough analkaliroute[26]andusedintheplantstudy.Briefly,inadry 250mlroundbottomflask160mgC60wasmixedwith55ml toluene(Aldrich)andstirredfor30mincoveredwithrubber septumandequippedwithNitrogenfilledballoon.Tothisdark purplesolutionwasadded0.3ml40%aqueoustetrabutyl ammoniumhydroxide(FisherSci)followedbyadditionof5ml Figure4.NormalizedrootorhypocotyllengthsofArabidopsisseedlingsunderbluelight(BL)orredlight(RL)conditions. PHF concentrationwas100mg/L.Nodifferenceonrootandhypocotylelongationwasobservedunderthosetwomonochromaticlightconditions (p 0.05).Rootandhypocotyllengthswereallnormalizedtocontrolthatgrownunder0.2mmolm2 2s2 1BLorRLwithineachexperimentalreplicate. Atleast25seedlingspertreatmentweremeasuredineachreplicate.Errorbarsrepresentthestandarddeviationofthethreeindependenttrials. doi:10.1371/journal.pone.0019976.g004 BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org4May2011|Volume6|Issue5|e19976

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50%aqueousNaOH(Acros).Reactionmixturewasstirredat roomtemperaturefor5daysuntilthepurplecolordisappeared. Thenflaskwasplacedinafreezer( 2 10 u C)andthecleartoluene layerwasdecantedoutofthefrozenmixture.Tothemixturewas added40mlnanopurewaterandmixturestirredatroom temperaturefor2days.Thedarkbrownmixturewasfreeze-dried toremovethewaterandtheremainingpowderwaswashedwith methanol5X20ml.Itwascentrifugedandmethanolwas decantedeachtime.Theresidualpowderdriedinvacuumoven atroomtemperature(340mg)andpurifiedonneutralSephadex sizeexclusioncolumnpreparedwithpurewaterand20g SephadexG-25(Sigma-Aldrich).Collectedwere25mlfractions, darkfractions1–3werecombinedandfreeze-driedtogive 200mgdarkbrownpowder,completelysolubleinwater.Priorto useinthisstudy,thePHFsolutionswerecharacterizedas describedelsewhere[27]. Astocksolutioncontaining1000mg/LofPHFwasprepared bydissolving10mgofPHFin10mLofNanopure H waterand theresultantsolutiondisplayedacharacteristicdarkbrownhue.2.96-hourPseudokirchneriellasubcapitataGrowthAssayMaintenanceofthealgalculturewasdescribedinGriffittetal. [28].Briefly,theculturemediumwaspreparedfromstock solutionsaccordingtoEPAstandardmethodforpreliminaryalgal assayprocedure[29].ThepHoftheculturemediumwasadjusted to7.5 6 0.1with0.1NNaOHor0.1NHClandthenfiltered througha0.45mmmembraneandsterilizedbyautoclaving.A purecultureof P.subcapitata wasobtainedfromHydrosphere Research(Alachua,FL)andgrowninPreliminaryAlgalAssay Procedure(PAAP)mediumwithEDTAat25 6 1 u C.Lightsource (86 6 8.6mEm2 2s2 1)andcontinuousaerationwereprovided 24hoursperday.Newcultureswerepreparedeveryweekunder sterileconditionsbytransferringapproximately20–30mLofthe matureculturesto1–2Loffreshsterilemedia. Thegrowthassaywasperformedinautoclaved125mL ErlenmeyerflasksaccordingtotheEPAprotocol[29].Allsample dilutions(i.e.culturemediaspikedwithincreasingconcentrationsof PHFfrom0.001to20mg/L)andnegativecontrolswererunin triplicatesandinoculatedwith1mLofa4to7-dayoldalgal cultures.Allflaskswereplacedunderthefluorescentlightsinthe samegrowthconditions.Thealgalgrowthafter96-hwas determinedbycellnumbercountbetween3.4and8mm[30]using aCoulterMultisizerIII(BeckmanCoulter,Inc.Brea,CA,USA).3. Arabidopsis GrowthExperimentsArabdopsisthaliana seeds(ecotypeColumbia-0)wereplacedontoa minimalmedium(1mMKCl,1mMCaCl2;solidifiedwith1% phytoagar)containing0–200mg/LPHFinsquarePetridishesand stratifiedat4 u Cfor48h.Theseedswerethentreatedwithwhite light(20mmolm2 2s2 1)for15minutestosynchronizegermination. Thentheplatesweremovedtoexperimentalconditionsinavertical positionsuchthatseedlingswouldgrowuprightontheagarsurface. Theexperimentalconditionsweredarkness,whitelight(coolwhite fluorescent;20mmolm2 2s2 1),blueorredLEDlight(various fluenceratestestedforblueandredlightat0.2,2or20mmolm2 2s2 1).Seedlingsweregrownfor96hinexperimentalconditions, imagedonaflatbedscanner,andthenmeasuredusingImageTool 3.0againstknownstandards.Atleast25seedlingsweremeasured pertreatmentinatleastthreeindependentexperimentalreplicates.4.FungiGrowthExperimentsFungigrowthexperimentswereconductedina96-wellplateby followingstandardmicro-dilutionprotocolwithtwodifferentgrowth media:RPMI1640with2%glucoseandRPMI1640withpotato dextrosebroth.TheprotocolinvolvespreparingaseriesofPHF dilutionsina96wellplate,inoculatingthewellswith A.niger ,andusing theabsorbanceat600nmasanindic atorofabiomassconcentration. Visiblelightabsorbancecorrelatestobiomasstoindicatefungal growthorinhibitionrelativetotheabsorbancemeasuredfroma controlwithoutPHFastaughtinSchwalbeetal.[31]. Pre-preparedRPMI-1640wasobtainedfromMediatechInc. (Manassas,VA).Potatodextrosebrothwaspreparedinthe laboratory.PHFstocksolutionswerepreparedintheculture mediaatconcentrationof2000mg/L,followedby10folddilution Figure5.Aphotographicreproductionofa96-wellplatetestafter120-hourincubation. Thecolumnoftestcellshaving A.niger exposed to1,000mg/LPHFshowssignificantlyhighergrowththancontrolorotherPHFquantities. doi:10.1371/journal.pone.0019976.g005 BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org5May2011|Volume6|Issue5|e19976

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withtherespectivemediatogiveconcentrationsof200,20and 2mg/L.AserialdilutionofthePHFstocksolutionwascarriedout in96wellplates.Eachplatecomprised12columnsand8rows. Eachcellwasidentifiedbyitsrowletter(A–H)andcolumn number(1–12).Thus,thetop,leftmostwellwouldbecellA1,and thebottom,rightmostcellwouldbecellH12.Afterdilution, inoculationof A.niger wascarriedoutfollowingthestandard protocoldescribedinSchwalbeetal.[31].Theplateswere coveredwithaluminumfoil,keptinanorbitalshakerwith temperatureat37 u Cand75RPM. Figure6.Microscopicimages(10Xmagnification)ofdaphnidsexposedtocontrolandpHtreatmentsafter2days. (A)Control,(B) treatmentwith0.001mg/LofPHF,and(C)treatmentwith20mg/LofPHF.(D)Meanbodylengthofdaphnidsoveradurationof8daysinresponse tovariousconcentrationsofPHF(n=2). doi:10.1371/journal.pone.0019976.g006 BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org6May2011|Volume6|Issue5|e19976

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5.SurvivalandReproductionAssayUsing Ceriodaphnia dubiaMaintenanceofthe Daphnia culturewasdescribedinGaoetal. [10]andGriffittetal.[28].Briefly,moderatelyhardwater(MHW) preparedfollowingtheEPAstandardmethod[29]wasusedas culturemediainthistest.Purecultureof C.dubia wasobtained fromHydrosphereResearch(Alachua,FL)andkeptin1L beakerscontaining500mLofMHWinaPervicalTMmodel # E30BXenvironmentalchamberat25 u Cwithconstantaeration. Thephotoperiodwas16-hrlight/8-hrdark. C.dubia wasfedwith concentrated P.subcapitata cellsandYCT(madefromyeast,cereal leavesandtroutchow).Thedaphnidswerefedeveryotherday with6.67mLYCTand6.67mLalgaesolution/Lculture. Neonatesoflessthan24hourswereseparatedfromadultsdaily andusedfortesting. Thesurvivalandreproductionassayswereperformedaccording toEPA’sprotocol[29].MHWservedasnegativecontrolandas thediluenttopreparemediawithincreasingconcentrationsof PHF.Neonateslessthan24hourswereseparatedfromadultsand fed2hourspriortoteststart.Foreachtest,groupsof5 Daphnia neonatesweretransferredinto30mlplasticcupscontaining20ml ofMHW(controls)orMHWplusPHFatvariousconcentrations (i.e.0.0001,0.001,0.01,0.1,1,5and20mg/L)for7daystoassess theeffectsofPHFongrowth,survivalandreproduction.The daphnidswereculturedat25 6 1 u Cwiththesamephotoperiod andfedwith40mLofYCTand P.subcapitata dailyperdaphnid. Theculturemediumwasrenewedthreetimesaweek.Everyday thesurvivalandnumberofnewbornswererecordedandthe offspringsproducedwereseparatedfromthetestcontainers.All sampledilutionsandnegativecontrolswereruninfourreplicates andeachtreatmenthadatotalof20daphnids.6.GrowtheffectsofPHFon CeriodaphniadubiaAteachconcentration,twoneonateswerekeptinseparatecups (i.e.,onepercup)atsameconditionsasothertestdaphnidsand observedbyopticalmicroscopy1hourafterfeedingeveryday. Themicrographstakenwereanalyzedforsize(i.e.corebody length)ofexposeddaphnidsanddistributionofPHFinthebody.7.EffectofPHFonthelifespanof CeriodaphniadubiaThesamedaphnidsusedinthesurvivalandreproductionassay werekeptandtestedfortheirlifespanwithandwithoutthe exposuretoPHF.Thedaphnidswereculturedandfedinthesame manneraspreviouslydescribed.Theculturemediumwasrenewed threetimesaweek.Everydaythesurvivalandnumberof newbornswererecordedandtheoffspringsproducedwere separateddailyuntilthedeathofalldaphnids.8.DataAnalysisAlltheexperimentswererunintriplicateorgreater,anddata wereillustratedasmean 6 standarderror(SE).Statisticalanalyses wereperformedusingone-wayanalysisofvariance(ANOVA) followedbyDunnett’stestwithanNCSS2004(Kaysville,Utah). 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30.HallJA,GoldingLA(1998)Freshwateralgae( Selenastrumcapricornutum ):chronic toxicitytestprotocol.Wellington,NewZealand:NIWAreportfortheMinistry fortheEnvironment.MFE80205Appendix4MFE80205Appendix4. 31.SchwalbeR,Steele-MooreL,GoodwinAC(2007)Antimicrobialsusceptibility testingprotocols.BocaRatonFL:CRCPress.BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org8May2011|Volume6|Issue5|e19976