Research Article Responses of Algal Cells to Engineered Nanoparticles Measured as Algal Cell ...

13 Pages·2014·1.86 MB·English

HindawiPublishingCorporation
JournalofNanotechnology
Volume2012,ArticleID237284,12pages
doi:10.1155/2012/237284
Research Article
Responses of Algal Cells to Engineered Nanoparticles Measured
as Algal Cell Population, Chlorophyll a, and Lipid Peroxidation:
Effect of Particle Size and Type
D.M.Metzler,1A.Erdem,2Y.H.Tseng,3andC.P.Huang1
1DepartmentofCivilandEnvironmentalEngineering,UniversityofDelware,Newark,DE19716,USA
2DepartmentofEnvironmentalEngineering,AkdenizUniversity,Akdeniz07058,Turkey
3DepartmentofChemicalEngineering,NationalTaiwanUniversityofScienceandTechnology,Taipei106,Taiwan
CorrespondenceshouldbeaddressedtoC.P.Huang,[email protected]
Received15May2012;Accepted24July2012
AcademicEditor:MallikarjunaNadagouda
Copyright©2012D.M.Metzleretal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Thispaperinvestigatedtoxicityofthreeengineerednanoparticles(ENP),namely,Al O ,SiO ,andTiO totheunicellulargreen
2 3 2 2
algae,exemplifiedbyPseudokirchneriellasubcapitatawithanemphasisonparticlesize.ThechangesinpH,cellcounts,chlorophyll
a,andlipidperoxidationwereusedtomeasuretheresponsesofthealgalspeciestoENP.ThemosttoxicparticlesizewasTiO
2
at42nmwithanEC20of5.2mg/LandAl O at14–18nmwithanEC20of5.1mg/L.SiO wastheleasttoxicwithanEC20of
2 3 2
318mg/L.ToxicitywaspositivelyrelatedtothesurfacechargeofbothENPandalgae.Thechlorophyllcontentofthealgalcells
wasinfluencedbythepresenceofENP,whichresultedinlimitedlightandavailabilityofnutrientsduetoincreaseinturbidity
andnutrientadsorptionontotheENPsurface,separately.Lipidperoxidationwasattributedtoreactiveoxygenspecies(ROS).Fast
reactionbetweenalgalcellsandROSduetodirectcontactbetweenTiO andalgalcellsisanimportantfactorforlipidperoxidation.
2
1.Introduction Reactiveoxygenspecies(ROS)aregeneratedinthethyl-
akoidmembraneduringphotosynthesis[7].Hydrogenper-
Nanomaterialshavebeenusedinindustrialapplicationsand oxidehasbeensuggestedtobethedominantspeciesofROS
commercialproductsatarapidpace.NewtypesofNMare inalgae.Thisisduetorelativelylongreactiontimeinvolving
beingdevelopedeveryday.Thesenewmaterialscanbemore hydrogenperoxides,approximately1millisecondcompared
hazardousthantheirbulkstatesduetonotonlysmallsizebut to nanoseconds of other ROS species. The ROS-protective
alsonumeralnovelproperties.Amongtheseparticles,TiO2, mechanisms of algae include superoxide dismutase, ascor-
SiO ,andAl O havebeenshowntohaveadverseeffectson bateperoxidase,catalase,andglutathioneperoxidase[7].The
2 2 3
thepulmonarysystems[1–3].Therehavebeenafewstudies photoactivityofTiO alsogeneratesROS.Thegenerationof
2
on the effect of these particles on algae. Van Hoecke et al. ROS has also been linked to particle size, crystalline struc-
[4] observed changes in the growth rate of P. subcapitata ture,andsurfacedefects[8].Theparticlesizeisanimportant
when exposed to SiO . Warheit et al. [5] reported growth variableinthatasthespecificsurfaceareaincreasesreversely
2
inhibition of P. subcapitata with a 72-hour EC50 of 87 and proportionally to particle diameter. The increase in specific
61mg/L for TiO at size of 38.5 and 100nm, respectively. surface area will result in the increase in specific quantum
2
Ji et al. [6] reported a 6-day survival EC50 of 120mg/L for yield of photocatalytic particles thus increasing the ROS
anatase with a size of 20–50nm. Literature results so far generation[9–11].Bakardjievaetal.[10]demonstratedthat
have indicated a clear dose-response relationship between asthespecificsurfaceareaofTiO increased,4-chlorophenol
2
microbial survival and ENP concentration. However, more degradationincreased.Tesngetal.[9]indicatedthat2-chlo-
studiesareneededtoillustratethemechanismsofmicrobial rophenoldegradationdecreasedwithincreasedprimarypar-
responsestoENP. ticle size. The size effect on photoctalytic capacity has been
2 JournalofNanotechnology
seen in other particles including CdS [12], HgSe and PbSe allowedtoequilibrateforonehour.Afteronehour,100mL
[13], ZnO [14], and SnO [15]. It was concluded that oftheCSTRalgalculturewasaddedtotheNPsuspensionto
2
particle size of quantum dots is one major factor affecting achieve an algal cell density of 1.4±0.9×106cell/mL. The
cytotoxicity [16]. Kashiwada [17] demonstrated that latex sampleswerethenplacedinoneofthreegrowthchambers.
nanoparticle was adsorbed to spawned ST II eggs at an Thedurationoftheexposuretestwas4days.
optimumparticlesizeof474nm.Adamsetal.[18]examined Each day during the course of exposure experiment the
the effect of particle size of TiO , SnO , and ZnO on B. sample pH was measured with a Corning pH/ion analyzer
2 2
subtilis and E. coli and observed some degree of toxicity 350 equipped with a Corning semimicro combo pH probe.
whethertheparticleswereunderlightornot.However,they Additionally,theminimumandthemaximumtemperatures
wereunabletodrawconclusionabouttheparticlesizeeffect ofthegrowthchamberswererecordedforeach24-hourtime
duetothechangeofparticlesizeonceinsuspension. period.Attheendofthe4-dayexposure,thesamplevolume
The objective of this study was to determine the tox- remaining was determined by measuring the difference in
icity of photocatalytic (e.g., TiO ) and non-photocatalytic volume before and after exposure runs. Forty milliliters of
2
nanoparticles (e.g., Al O and SiO ) over a wide range of samplewerecollectedin50mLFisherbrandcentrifugetubes
2 3 2
particle size by evaluating the effects on cell population, which were analyzed for chlorophyll a. Additionally, 12mL
chlorophyll, and lipid peroxidation on P. subcapitata. The were collected in 15 mL Fisherbrand centrifuge tubes and
modeoftoxicitywasalsoexamined. analyzedforcelldensityandlipidperoxidation.Thesamples
were stored at 4◦C in the dark (a maximum of 7 days)
2.MethodsandMaterials until analyzed. According to Weber et al. [21], no changes
inchlorophyllcontentshouldoccurofthistimeperiod.
2.1. Nanoparticles. Stock suspensions of 2g/L were made Cell densities were measured by direct cell counts. One
withaluminumoxide,silicadioxide,ortitaniumdioxidein milliliter of sample was diluted in 1mL of 0.5M lauryl
150–500mLofErlenmeyerflaskwithalgalgrowthmedium sulfate.ThiswasthenmixedonaDadevortexshakermodel
[19]. Table1 gives the information about the physical and S8223-1 for 2 minutes. An aliquot of sample was then
chemical properties of ENP materials studied. The stock added to a hemocytometer for counting on an Olympus
suspensionswereautoclavedfor20minutesat120◦Catleast AX70 microscope; samples were counted a minimum of 4
onedaypriortoexperimentandstoredatroomtemperature times.Celldensitieswereconvertedintototalcellpopulation
untiluse. by multiplying the cell counts by the volume of sample
remaining in the 1L test beakers at the end of the test
2.2. Algal Culture. Algal culture, P. subcapitata, was pur- duration.Thetotalcellpopulationswerethencorrectedfor
chased from Aquatic Biosystems (Fort Collins, CO). The theinitialpopulationandnormalizedbasedonthecontrol,
algalinoculumswerestoredat4◦Cinthedarkfornolonger usingthefollowingequation:
than 6 months. Culturing of the algae in 3L continually (cid:2) (cid:3)
Hstiyrdreradutlaicnkrerseidaecntocre(tCimSTeR()HfRoTll)owoefdthtehaCtSoTfRMwetazsle3r d[2a0y]s., Rs= (cid:2)ρρ××vv(cid:3)t, (1)
c
withaflowrateof50mL/h.
where Rs is the normalized growth, ρ is the cell density
2.3. Growth Chambers. Growth chambers were approxi- (cell/mL), v is the volume of sample at the end of the 4-
mately 80cm × 62cm × 68cm, wood or metal frame that day exposure period (mL), t is the duration a sample was
was covered in black cloth. A bank of 6 fluorescent lights exposed to a specific ENP, and c is the control sample with
wassuspendedoverheadontheframe.Lightsusedincluded notreatment.
model GE PL/AQ F12T20 (General Electric, Fairfield, CT), For the determination of chlorophyll a, the samples
Gro-luxF12T20/Gro/AQ,and2SylvaniaGro-luxwidespec- were concentrated to between 0.5–3mL, and then analyzed
trum F12T20/Gro/AQ/WS (Rutherford, NJ, USA). These accordingtoStandardMethodsfortheExaminationofwater
lightfixturessuppliedanaverageluxof1632±157,measured andWastewater[22].Inbrief,theconcentratedsampleswere
fromthetopofthetestbeakers.Amuffinfanorcentrifugal added to glass homogenization tubes. The 50 mL sample
miniblower removed excess heat generated by the lights. tubeswererinsedwithafewmillilitersofMgacetone(2.0g
Each fan or blower used was suspended on the outside of of(MgCO ) andMg(OH) ·5H Odilutedto200mLindis-
3 4 2 2
thechamberapproximately1inchbelowthelights,withan tilleddeionizedwaterthendiluted1:10inacetone).Thevol-
accessportcutintothecloth.Anorbitalplatformshakerheld umeinthehomogenizationtubewasbroughtto3mLwith
thebeakersandwassettooscillateat150rpm. Mg-acetone solution. The samples were then homogenized
for 1 minute at 2000rpm with a Glas-Col variable speed-
2.4.ExposureExperiments. AliquotsofstockNPwereadded reversible homogenizer. The samples were then steeped at
to a series of 1L beakers. Stock suspensions were continu- 4◦Cinthedarkfor2hoursin15mLplasticcentrifugetubes.
ously stirred with a magnetic stirring bar during this time. After the steep time, the samples were centrifuged for 30
Algalgrowthmediumwasthenaddedtothebeakerstobring minutes at 664×g. The optical density of the supernatant
thevolumeto100mL.Thesuspensionsweresonicatedfor2 wasmeasuredat750and664nm,respectively,onaHewlett-
minutesatapowerlevelof48WusingaCole-Palmer4710 Packard UV-Vis model 8525A. HCl (0.1mL of 0.1N) were
series ultrasonic homogenizer. The suspensions were then addedtothesamplesandtheoptical densitywasmeasured
JournalofNanotechnology 3
T.
cm) BE
-
Diameter(n 35.62614.39.6 67.5 17.6 15.8 204.1122.345.842.542 35.1 30.318.7 5.3 4.8 4.8 easuredbyN2
m
g)
ge 2m/
urfacecharb(pH)pzc 2.0 8.2–9.1 5.4–6.5 oftheNP(
S ea
ar
e
Surfacearea2/g)(m76.5105.1190.6284.8 24.1 92.3 102.9 7.5±0.9c12.633.636.236.6 ±44.02.9c 87.7 ±8.2c290.5 319.3 322.4 specificsurfac
e
a) th
cc is
ensity(g/ 2.22.22.22.2 3.69 3.69 3.69 3.93.93.93.93.9 3.9 3.93.9 3.9 3.9 3.9 Σ3m),and
sentstudy. IDD Aero90Aero130Aero200Aero300 StdAl Al1520 γαAl- 8-8409-7807-660SL110-600 P25 SL2Wo5 R5 R10 U1100 particle(g/c
e e
r h
p t
anoparticlesusedinthe Methodofproduction Continuousflamehydrolysis Proprietarycalcining Proprietarycalcining Temperatureannealing Sol-gelTemperatureannealingLiquid-gasphasesynthesisSol-gelReversemicelleWet-chemistryprecipitationWet-chemistryprecipitationSol-gel ρcle(nm),isthedensityof
Table1:N TypeNameSource Aerosil90DegussaCorp.Aerosil130DegussaCorp.Silicadioxide(SiO)2Aerosil200DegussaCorp.Aerosil300DegussaCorp. StandardaluminapowderAlliedHighTechProductsAluminumoxideAluminaO)(Al23Gammaaluminapowder,AlliedHighTechProductstypeN8–8409–7807–660UniversityofDelawareSL110–600TitaniumdioxideP25DegussaCorp.)(TiO2SL2UniversityofDelawareWo5Dongetal.[63]NanostructuredandReade5AmorphousMaterials,Inc.NanostructuredandReade10AmorphousMaterials,Inc.U100SachtlebenChemicals a:referencesforNPdensity:(TiO)[31],[63];(SiO)[64](AlO)[64,65].2223b:referencesforsurfacecharges(AlOandSiO)[30];(TiO)[66].2322r/ρΣr6=××c:diameterofNPcalculatedfrom3(10);whereisthediameteroftheparti±d:9–780,P25,andR5weremeasuredintriplicate1standarddeviationshown.
4 JournalofNanotechnology
again at 750 and 665nm after 90 seconds of equilibration. 1.5
Theconcentrationofchlorophylla([Chla]inmg-Chla/m3)
wascalculatedbythefollowingequation:
[Chla]=26.7(A −A )×V ×(V ×l)−1, (2) 1
664 665 1 2
whereA istheabsorbanceat664nmbeforeaddingacid,
665 s
A istheabsorbanceat665nmafteraddingacid,V isthe R
665 1
volume of extract (L), V is the sample volume (m3), and l 0.5
2
opticalpath(cm).Thespecificchlorophyllawascalculated
bythefollowingexpression:
y=[Chla]×ρ−1×10−6 , (3) 0
0 1 2 3
where y is the specific chlorophyll a (mg Chla/cell), [Chla] Cp, log (mg/L)
istheconcentrationofchlorophylla(mgChla/m3),andρis
the cell density (no. cell/mL). The specific chlorophyll a, y, Figure1:Normalizedspecificfour-daygrowth(Rs)dose-response
curve. Solid circles are Al O (17.6nm). Open squares are SiO
wasthennormalizedwithrespecttothecontrolaccordingto 2 3 2
(14.3nm). Solid triangles are TiO (18.7nm). Error bars are ±1
2
thefollowingequation: standarddeviation(n≥3).Linesrepresentfour-parameterlogistic
curves fitted to the data in SigmaPlot ver. 9.01. Experimental
Y = y×yc−1, (4) conditions:initialalgaeconcentration=1.4±0.9×106cell/mL,test
duration=4days,andinitialtestvolume=200mL.
whereY isthenormalizedspecificchlorophylla(dimension-
less), y isthespecificchlorophylla(mgChla/cell),and yc is
thespecificchlorophyllaofthecontrol(mgChla/cell).
total number of organisms that died during exposure, were
Finally,analysisofsampleforlipidperoxidationfollowed
notusedtodeterminethetoxicityofNPtoalgae.Therefore,
the method in Maness et al. [23]. In brief, 1mL of sample
ECwaschosen.ECvalueisdefinedasthedifferenceintotal
wasaddedto2mLof10%trichloroaceticacid.Thesamples
growth between control and test sample. EC20 and EC50
werethencentrifugedfor45minutesat11000×g.Thesuper-
are concentrations that limit growth to 20 and 50% of the
natant was then added to 3mL of 6.7g/L 2-thiobarbituric
acid,whichwaspreviouslyheatedto60◦Cinordertodissolve controlsamples.
the solid. The samples were then heated in boiling water
3.Results
for 10 minutes. After cooling, the absorbance of samples
was measured on a Hewlett-Packard UV-Vis model 8525A
The size and type are important attributes of ENP. The
at532and600nmwavelengths,respectively.Theabsorbance
major objective of the present research was to determine
at 532nm was corrected for the background at 600nm.
how particle size and type will affect the physiology and
Calibrationstandardsweremadefrom1,1,3,3-tetramethoxy-
propane.Thespecificlipidperoxidation(z)wascalculatedby biochemicalbehaviorofalgaeexemplifiedbyP.subcapitata.
ThealgalcellswereexposedtoTiO ,Al O ,andSiO for96h
thefollowingequation: 2 2 3 2
atvarioussizeclassesandconcentrations.Duringthetimeof
z=[MDA]×ν×(cid:2)ρ × ν(cid:3)−1×10−3, (5) exposure,theaveragetemperaturewas23.0±1.2◦Candthe
averagepHwas7.6±0.4.
where z has the unit of pmol MDA/cell, [MDA] is the
concentrationofmalondialdehyde-thiobarbituricacidcom- 3.1. Effect of Particle Concentration. In order to determine
plex measured (μM), ν is the volume of sample at the the effect of NP concentration on P. subcapitata, Al2O3
end of the 4 day exposure (mL), and ρ is the cell density (17.6nm),SiO2(14.3nm),andTiO2(18.7nm)wereselected
(no. cell/mL). The z value was then normalized against the due to their similarity in size. Figure1 shows the dose-
controlaccordingtothefollowingequation: response curves of Al2O3, SiO2, and TiO2 in terms of
normalizedspecificgrowth(Rs).Inallcases,asconcentration
Z =z×zc−1, (6) increasedfrom0to1000mg/L,theRsdecreased.EC20values
were calculated using SigmaPlot version 9.01 as follows:
whereZisrelativelipidperoxidation(dimensionless),zisthe Al O (17.6nm) = 5.14mg/L, TiO (18.7nm) = 129mg/L,
2 3 2
specific lipid peroxidation of the sample (pmol MDA/cell), andSiO (14.3nm)=318mg/L.
2
andzc isthespecificlipidperoxidationofthecontrol(pmol Chlorophyll content of algae can be used as a measure-
MDA/cell). ment of physiological health [22]. In order to determine if
Dose-responsecurveswereusedtocalculatetheeffective NPwasstressingthealgae,chlorophyllameasurementswere
concentration(EC).TheUSEPA[19]usesdailycelldensity conducted. Figure2 shows chlorophyll a content plotted
to calculate the growth inhibition concentration (IC). The againsttheconcentrationsoftherespectiveNP.Itcanbeseen
number of samples to be processed per day made this that there was a slight increase in the chlorophyll content
impractical.Lethalconcentration(LC)values,definedasthe astheAl O concentrationwasincreasedtoabout10mg/L.
2 3
JournalofNanotechnology 5
4 4
3
2
Y Z 2
1
0
0
0 1 2 3 0 1 2 3
Cp, log (mg/L) Cp, log (mg/L)
Figure2:Normalizedspecificchlorophylla(Y)asafunctionofthe Figure 3: Normalized specific lipid oxidation (Z) as a function
concentration of the NP. Solid circles are Al O (17.6nm). Open of the concentration NP. Solid circles are Al O (17.6nm). Open
2 3 2 3
squares are SiO (14.3nm). Solid triangles are TiO (18.7nm). squares are SiO (14.3nm). Solid triangles are TiO (18.7nm).
2 2 2 2
Errorbarsare±1standarddeviation(n≥3).Linesare2nd-order Error bars are ±1 standard deviation. Error bars were omitted
polynomialfitstothedata.Thedatawasfittedwithanempirical from SiO plot. Lines are 2nd-order polynomial fits to the data.
2
2nd-order polynomial from SigmaPlot ver. 9.01. Experimental Experimentalconditions:initialalgaeconcentration=1.4±0.9×
conditions:initialalgaeconcentration=1.4±0.9×106cell/mL,test 106cell/mL,testduration=4days,andinitialtestvolume=200mL.
duration=4days,andinitialtestvolume=200mL.
5
As the Al O (solid circles) concentration was increased
2 3
4
above10mg/L,thechlorophyllcontentdecreasedtoapprox-
imately 50% of the control at 1000mg/L of Al O . As
2 3
3Si0Om2g(/oLp,etnhesqCuharlaesi)nccorenacseendtrfartoiomn1watos i2n.c2r.eTasheedCfrholma t0hetno mg/L) 3
decreasedto1at1000mg/LofSiO2.TheNPofTiO2 (solid og ( 2
triangles) had the inverse effect of Al2O3 and SiO2. The α, l
Chladecreasedfrom1to0.63whenTiO concentrationwas
2
increasedfrom0to30mg/L.AtTiO concentrationgreater 1
2
than30mg/LtheChlaincreasedto0.83at1000mg/L.
Figure3 shows the normalized specific MDA, Z, as a 0
function of ENP concentration for SiO (open squares), 0 50 100 150 200 250
2
Al2O3 (solid circles), and TiO2 (solid triangles). The nor- d1(nm)
malized specific MDA increased with the log concentration EC50
ofSiO .ThemaximumnormalizedspecificMDA,3.1±1.3, EC20
2
occurredat500mg/LofSiO2.However,theSiO2 resultsare Figure 4: Critical specific growth (α) as a function of primary
not significantly different (α = 0.05) from those of TiO2 particle size of TiO2 in terms of EC20 and EC50. Lines are a
and Al2O3 due to large standard deviations in the data. five-parameter modified log normal empirical fitting (7) from
ErrorbarswerenotshowninFigure3forSiO2 duetolarge SigmaPlotver.9.01forEC20(dotted)andEC50(solid).Errorbars
values.Al O causedanincreasednormalizedspecificMDA are ±1 standard deviation. Experimental conditions: initial algae
2 3
onlyatconcentration>100mg/L.Themaximumnormalized concentration=1.4±0.9×106cell/mL,testduration=4days,and
specific MDA was 2.8 ± 2.7 at 1000mg/L of Al O . For initialtestvolume=200mL.
2 3
TiO (Figure3) normalized specific MDA decreased from
2
1 to 0.61 when the TiO concentration was increased from
2
0 to 30mg/L, respectively. At concentrations greater than EC20 and EC50 were calculated with Toxicity Relationship
30mg/L,thenormalizedspecificlipidperoxidationincreased Analysis Program ver. 1 (TRAP) [24] using the logistic
until a maximum of 1.8±1.4 at 600mg/L was reached for equation. Figure4 gives the EC20 and EC50 values as a
TiO2. The increase in normalized specific MDA was most function of the primary NP size (d1) exemplified by TiO2.
likelyduetophotocatalyticpropertiesoftheTiO2. As the ENP size increased from 5.3 to 41nm both, EC20
and EC50 decreased. A minimum EC20 of 5.2mg/L and
3.2. Particle Size Effect. In order to determine how particle a minimum EC50 of 25.5mg/L were observed at 42nm
sizeaffectsthetoxicityoftitaniumdioxide,algaewasexposed primary particle size. The EC50 and EC20 then increased
toTiO atparticlesizeintherangebetween5.3and204nm. at d > 42nm. Warheit et al. reported 72-h growth EC50
2 1
6 JournalofNanotechnology
7 20
6
16
5
−β1, (g/L)TiO2 1234 −ε1, (g/L)TiO2 128
0 4
−1
0 50 100 150 200 250
0
d1(nm) 0 50 100 150 200 250
EC20 d1(nm)
EC50 EC20
Figure5:Criticalspecificchlorophyllacontent(β)asafunctionof EC50
primaryparticlesizeofTiO2intermsofEC20andEC50.Linesare Figure 6: Critical specific lipid peroxidation (ε) as a function of
fitted3-parameterGaussianequation(7)inSigmaPlotver.9.01for primaryparticlesizeintermsofEC20andEC50.Lineisfitted3-
EC20 (dotted) and EC50 (solid). Experimental conditions: initial parameterGaussianequation(8)inSigmaPlotver.9.01forEC20
algaeconcentration=1.4±0.9×106cell/mL,testduration=4days, (dotted) and EC50 (solid). Experimental conditions: initial algae
andinitialtestvolume=200mL. concentration=1.4±0.9×106cell/mL,testduration=4days,and
initialtestvolume=200mL.
of 87 and 61mg/L for d of 38.5 and 100nm, respectively
1
[5]. Gonclaves et al. reported EC50 values varied between productionincreasedatEC20andEC50.Whentheprimary
241mg/L and 71.1mg/L for TiO2 sized between 10 and particle size was greater than ca. 150nm, the β20 and β50
300nm[25].Astheprimaryparticlesizeincreasedto204nm becamenegative,indicatingadecreaseinChla.Thedatawas
the EC50 and EC20 increased to 9761.3 and 103.7mg/L, empiricallyfittedtoathree-parameterGaussianequation(8)
respectively. The results were fitted with an empirical four- usingSigmaPlotversion9.01,thatis,
parameterlog-normalequation:
⎧ ⎡(cid:15) (cid:16)⎤ ⎫
α=d11+ a(cid:4) exp⎧⎨⎩−0.5(cid:7)ln(db1(cid:4)/d12)(cid:8)2⎫⎬⎭, (7) β=a(cid:4)(cid:4)exp⎪⎨⎪⎩−0.5⎣ d1b−(cid:4)(cid:4)d13 ⎦2⎪⎬⎪⎭. (8)
where α is the effect concentration at either 20 or 50%, a(cid:4) The variables in (8) are as follows: a and b are fitting
isafittedconstant,b istheconcentrationofthemosttoxic parameters, d is the ENP size (nm), and d3 is the NP size
1 1
ENPsize,d (nm)istheinitialdiameteroftheNP,d1 isthe (nm) with the largest β. Fitting the data to (8) resulted in
1 1
smallest d (nm), and d2 is the d in which the minimum a chlorophyll production maximum at 33nm of TiO . The
1 1 1 2
EC50 or EC20 occurs. Ther2 values of these regressions maximumβvalueswere5.4and5.9timesthecontrolforβ
20
were0.80and0.90forEC50andEC20,respectively.Within andβ ,respectively.Themodelfittedthedatawellwithanr2
50
therangeof30to60nm,ECvaluesdecreased.Theaverage valueof0.69and0.73forEC20andEC50data,respectively.
minimumECvalueoccurredat39.3±0.2nmforTiO .The Figure6showstheeffectofd onthelipidperoxidation.
2 1
calculatedminimumECvalueswere1.5mg/LforEC20and The slope of the Z plotted against TiO concentration at
2
21mg/LforEC50. theEC20andEC50ofTiO wasobtained.Thisvalueisthe
2
Figure5 shows the effect of d1 of nano-TiO2 on the critical specific lipid peroxidation (εx) where x is the EC20
chlorophyll a content of the algal cells. The ordinate (β) is or EC50. Here ε and ε were plotted as a function of d .
20 50 1
the slope of the Chla at the EC20 and EC50 for TiO or AsheENPsizeincreasedfrom4.8to30nm,therewaslittle
2
the critical specific chlorophyll (CSC) at EC20 or EC50, that changeintheε withvaluesbetween0.54±0.14L/gTiO .
20 2
is,β andβ ,respectively.Apositiveβ indicatesincrease The ε then increased to 1.78L/g TiO at d of 45.8nm.
20 50 20 20 2 1
in chla production per mass of TiO at EC20 compared to The ε then decreased to 0.22L/g TiO at d of 204.1nm.
2 20 2 1
thecontrolsample;anegativevalueimpliestheopposite.As The ε data showed a significant increase in the primary
50
the particle size increased from 4.8 to 30.3nm, the β and particle size range between 30 and 50nm. The maximum
20
β50 increased from about 0 to 5.5L/g TiO2. The βx reached ε50, 15.8, occurred at 42nm. At primary particle size <30
a maximum value at 30.3nm, then abruptly decreased as and>50nm,theaverageε valuewas0.70±0.23.Bothdata
50
the primary particle size increased. At NP size between 4.8 setsinFigure6werefittedto(8).Thefittedequationhadr2
and 18.7nm, there was little difference between the control value of 0.48 and 0.99, for ε and ε , respectively. Results
20 50
and exposure. Results clearly showed that over the primary showedthatregardlessofparticlesize,lipidperoxidationwas
particlesizerangestudied,forexample,19∼50nm,theChla increasedapproximatelyby70%atEC50.However,therewas
JournalofNanotechnology 7
1.4 12
1.2 10
1
8
0.8
6
s
R Y
0.6
4
0.4
2
0.2
0
0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
d1(nm) d1(nm)
Figure 7: Normalized specific growth (Rs) for three particle- Figure 8: Normalized specific chlorophyll (Y) for three-NP
types.CirclesareSiO trianglesareAl O ,andsquaresareTiO .
2, 2 3 2 samples.CirclesareSiO ,trianglesareAl O ,andsquaresareTiO .
Open symbols are 100mg/L of exposure dosage. Solid symbols 2 2 3 2
Solid symbols are 100mg/L of exposure dosage. Closed symbols
are1000mg/Lofexposuredosage.Experimentalconditions:initial
algaeconcentration=1.4±0.9×106cell/mL,testduration=4days, are1000mg/Lofexposuredosage.Experimentalconditions:initial
andinitialtestvolume=200mL. algaeconcentration=1.4±0.9×106cell/mL,testduration=4days,
andinitialtestvolume=200mL.
anincreaseinε between30and50nm,withamaximumof 18
50
16.6L/gTiO at40.8nm.
2 15
Figure7 shows the normalized specific cell growth (Rs)
in the presence of nanosized Al O , SiO , and TiO at 12
2 3 2 2
variousd valuesandtwoparticleconcentrationsof100and
1
1000mg/L.Astheprimaryparticlesizeincreasedfrom15.8 9
to 67.5nm for 100mg/L of Al O , the NSG increased from Z
0.57±0.13to1.20±0.09,respe2cti3vely.AsSiO sizeincreased 6
2
from 9.6 to 35.6nm at 100mg/L, the Rs increased from 3
0.78±0.23to0.95±0.05,respectively.Rsreachedamaximum
of 0.84 ± 0.18 at 30.3nm when TiO2 concentration was 0
100mg/L.TheRsthendecreasedfrom0.84±0.18to0.44±1.2
asparticlesizeincreasedfrom30.3to45.8nm.Forbothtype −3
0 20 40 60
eaffndecstizoen, TthiOe2Rasttchoannceantt1ra0t0iomngo/Lf,1e0x0c0epmtgf/oLrhSaidOa2 g2r6enatmer. d1(nm)
The Al2O3 was at 1000mg/L and the Rs was reduced from Figure 9: Normalized specific lipid oxidation (Z) for three types
0.44±1.2to0.36±0.05asAl2O3particlesizewasincreased ofNP.CirclesareSiO2,trianglesareAl2O3,andsquaresareTiO2.
from 45.8 to 67.5nm. The greatest impact on Rs by TiO2 Opensymbolsare100mg/L.Solidsymbolsare1000mg/L.Lineis
aTtre1a0t0m0emntg/wLitwhaSsiOat2da1to1f00406mnmg/LwictohnacnenRtrsaotifo0n.1c4au±se0d.0R9s. fiE10txt6pecederli4lm/-mpenaLrt,aatmlescetotdneurdriGatitaoiuonnsss:i=ainn4itdeiaaqlyusaa,ltgainaoednci(no9int)ciaeilnntteSrsaitgtvimoonlauP=mloe1t.=4ve2±r0.009.m9.0L1×..
to increase from 0.27 ± 0.01 to 1.1 ± 1.2 as particle size
increased from 9.6 to 26nm, respectively. The Rs decreased
from 1.1±0.12 at 26nm to 0.64±0.24 at 35.6nm, in the
presence of 1000mg/L of SiO2. The Rs responded similarly the control with respect to concentration or size. SiO2 at a
to 100mg/L of Al O and SiO . As both particles increased concentration of 100mg/L significantly increased the Y at
2 3 2
ind1,theRsincreased.Figure7showsnoapparentdifference alld1 valuescomparedtothecontrol,exceptat26nm.SiO2
intheresponsebetweenthetwotypesofNP. atconcentrationof1000mg/LsignificantlydecreasedtheY
Figure8 gives plots of the normalized specific chloro- by84±4%thecontrolatalld values.Asd valueofTiO
1 1 2
phyll a (Y) for Al O , SiO , and TiO at several primary at a concentration of 100mg/L increased, the Y increased;
2 3 2 2
particle sizesand twoparticle concentrations(e.g.,100 and however, the average Y at this treatment was not different
1000mg/L). Al O at concentration of 100mg/L increased fromthecontrol.TheTiO at1000mg/LincreasedtheYbya
2 3 2
the Y by an average of 71% between 15.8 and 17.6nm. factorof10overthecontrollevelastheparticlesizeincreased
The largest Al O at 100mg/L increased the Y to 3.25 ± from4.8to30.3nm.
2 3
2.16. Al O at 15.8nm and 1000mg/L decreased the Y to Figure9 is a plot of normalized specific MDA (Z) at
2 3
0.78±0.70;however,thiswasnotstatisticallydifferentfrom various d values for Al O , SiO , and TiO at two particle
1 2 3 2 2
the control. There was no difference between Al O and concentrations (e.g., 100 and 1000mg/L). The Al O at
2 3 2 3
8 JournalofNanotechnology
a concentration of 100mg/L resulted in an average Z of 8 and 10−9.5mol/L at pH 7. The concentration was several
1.39 ± 0.14. The maximum of 2.7 occurred for the TiO orders of magnitude smaller than what caused significant
2
concentration of 100mg/L at 46nm. The minimum of responses in other algal studies and were not considered
0.29 occurred for the Al O concentration of 1000mg/L at the cause of the adverse effects observed. Additionally,
2 3
67.5nm.The1000mg/Ltreatmentssignificantly(α = 0.05) Al O is not a photocatalyst. In Figures 3 and 9, the
2 3
increasednormalizedspecificMDAforallsamples,butSiO degreeoflipidperoxidationwasreducedcomparedtoSiO
2 2
at 26nm. SiO at 26nm decreased the normalized specific and TiO . The increase in Z (normalized specific MDA)
2 2
MDAto0.86fromcontrollevels.Al O ataconcentrationof for Al O (Figure3) is most likely due to measurements
2 3 2 3
100mg/Lsignificantly(α = 0.05)decreasedthenormalized approaching the detection limits of the method (≈0.2μM
specific MDA compared to the control. SiO and TiO as malondialdehyde-thiobarbituric acid complex (MDA-
2 2
at a concentration of 100mg/L significantly (α = 0.001) TBA)). In combination with low cell counts at high NP
increased the normalized specific MDA compared to the concentration, low MDA-TBA will give large variation in
control. The average normalized specific MDA for the normalizedspecificlipdperoxidation,Z.Thisindicatesthat
1000mg/L of Al O was 1.14±0.13. This was significantly Al O did not increase the ROS in the system. Therefore,
2 3 2 3
higherthanthecontrol(α=0.05).TheSiO andTiO were ROSwasalsonotconsideredafactorinAl O toxicity.
2 2 2 3
fittedwith(9),representedaslinesinFigure9: Al O NP did not affect the normalized specific lipid
2 3
⎡ ⎧(cid:15) (cid:16)⎫ ⎤ peroxidation, Z, or the chlorophyll a content of the P.
Z =d 1+a(cid:4)(cid:4)(cid:4)exp⎢⎣−0.5⎨ d1−d12 ⎬2⎥⎦, (9) soufbcthapeitaaltgaalcecllesl.lsH. oTwheisvedr,isAalg2rOee3dNwPitdhidJiaffeetctalt.he[6g]rowwhtho
1 ⎩ b(cid:4)(cid:4) ⎭ reportedthatnano-Al O didnothaveasignificanteffecton
2 3
Chlorellasp.SeveralfactorsthatgoverntheeffectofAl O on
2 3
where Z is the normalized specific lipid peroxidation, d algalgrowthincludedflocculation,lightavailability,nutrient
1
is the ENP size (nm), d 2 is the ENP size (nm) which availability, and primary particle size, d . First, flocculation
1 1
the minimum value of Z occurs, a(cid:4)(cid:4)(cid:4) and b(cid:4)(cid:4) are fitting was affected by the surface charge (i.e., pH ) of both the
pzc
parameters, and y (cid:4) is value at which the equation crosses algal cells and Al O [31, 32]. Al O has a pH of 8.2–
0 2 3 2 3 pzc
the y intercept. Al O was not included in model fitting 9.1 [30] and P. subcapitata has a pH of about 2 [31].
2 3 pzc
becausetherewerenotdifferencesin Z acrosstheENPsize The difference in pH between Al O and algal cells will
pzc 2 3
profile.ThiswasasexpectedasAl O isnon-photocatalytic. induceflocculationandincreasetheapparentdensityofthe
2 3
The correlation coefficient (r2) was 0.67 and 0.68 for 100 algae [31]. In turn, the ability of the algal cells to remain
and1000mg/L,respectively.Theaverageminimumvaluefor buoyantwilldecreaseasseenwithMicrocystisspp.andclay
thefittedequationoccursat22.1±0.3nmforboth100and aggregation[32].IntheabsenceofENP,asalgaecellssettlein
1000mg/Ltreatments. apondorriver,thelightandnutrientavailabilitywillchange
[33].Inmostcases,thesechangesdonotfavoralgalgrowth
4.Discussion [33]. Second, Al2O3 can influence the nutrient availability.
Al O can affect the nutrient availability due to adsorption
2 3
The discussion will be divided into sections of different [34]. Speohr and Milner [35] related the availability of
ENP materials. The sequence of the section will be from nutrientstothechlorophyllcontentinalgalcells(Figures2
NPwiththeleastnumberofinfluentialfactors(e.g.,Al O ) and 8). In this study the possible nutrient adsorption was
2 3
to that of the most number of influential factors (e.g., most prevalent when Al O concentration was greater than
2 3
TiO ). Factors such as water solubility, light availability, 100mg/L(Figure2).Finally,NPwhichhaslimitedsolubility
2
flocculation, photocatalytic properties, and ENP size that undertheexperimentalconditionswillincreasetheturbidity
mayimpactthealgae. of the test sample. This turbidity will cause a shading
effect which will decrease the available light to the algae
4.1. Aluminum Oxide. Aluminum oxide is the ENP with in photosynthesis [36]. Decreased available light has been
least number of influential factors in terms of the response shown to decrease chlorophyll content of algae in sponges
by the algae and NP properties. The solubility of Al O anddecreasegrowthrateofD.tertiolecta.Theshadingeffect
2 3
was not considered a factor in the toxicity seen in Figure1. wouldbefromtheAl O flocculatedwiththealgaeandthose
2 3
Unlike other NM such as CdSe that may bring about toxic NPparticleswhichremainunattached[31].Theflocculation
metals such as Cd2+ [26, 27], Al O would not generate between NP and the algae cells resulted in incomplete,
2 3
enough Al3+ to cause the decreased growth. Gensemer and multilayeredsurfacecoverage[31].
Playle [28] reported aluminum toxicity to P. subcapitata The effect of NP size on growth is seen in Figure7.
(formally known as S. capricornutum), with an EC value Increased growth occurred with increasing the primary
of 460μg/L based on the biomass and 1,320μg/L based on particle size of Al O , d . Smaller particles will increase the
2 3 1
reduction of cell counts, respectively. The pH range of the turbidityofthesuspensionmorethanlargerparticlesonthe
above study was 7 to 8.2, which was similar to this study. same mass basis, which will increase the effect of shading
Metalionsareconsideredthemostbioavailable/toxicspecies on the algal cells. The adsorption capacity of nutrients will
[29].UsingequilibriumconstantreportedbySparks[30],the begreateratsmallerd thanlargerd .Withmorenutrients
1 1
concentration of (Al3+) from solubility of γ–Al O (c) was being removed from the suspension, the algae will have
2 3
calculated to be in the range between 10−12.5mol/L at pH reducedgrowth.
JournalofNanotechnology 9
4.2. Silicon Dioxide. SiO NM has the same influential availability to the algae increased; the less chlorophyll a the
2
factors(nutrientlimiting,lightlimiting)asAl O buthasthe algae need to produce. The Chla was reduced significantly
2 3
additional effect on increasing the lipid peroxidation. Like at 1000mg/L SiO (Figure8). Manganese deficiency has
2
Al O ,thesolubilityofSiO NPwasnotconsideredafactor beenthecauseofdecreasedchlorophyllcontentinChlorella
2 3 2
affectingtheECvalue.VanHoeckeetal.[4]foundthatSiO fuscaandChlorellavulgaris[42].Nutrientlimitationdueto
2
atparticlesizeof12.5and27.0nmhada72hspecificgrowth the adsorption of nutrients on SiO may have caused the
2
rate EC20 of 20.0 ± 5.0 and 28.8 ± 3.2mg/L, respectively. decreasedChla.TheSiO hasapH about2,similartothat
2 pzc
OftheNPconcentrationsusedintheVanHoeckeetal.[4] ofalgae.Therehavebeenmanystudiesthatshowadsorption
study,thehighestconcentrationofdissolvedSiwas4.1mg/L ofcationstoNM[43–45].Inparticular,SiO hasshownto
2
atpH7.5.ThemeasureddissolvedSiatEC20inthepresent adsorbMg(II),Mn(II),Na(I),K(I),Cu(II),andZn(II)[30]
studywas234mg/L,whichwasadequatetoaffectthegrowth whichareallrequirednutrientsfromalgalcellhealth.
ofalgae.Therefore,dissolvedSiwasnotconsideredamajor
factorinSiO toxicity.ThedifferenceinECvaluesbetween 4.3.TitaniumDioxide. TiO istheNPwiththemostnumber
2 2
thisworkandthatofVanHoeckeetal.[4]couldbedueto of influential factors that affect the responses of P. subcap-
differentinitialalgaldensities.Weusedaninitialcelldensity itata. TiO with pH of 6.5 acted similarly to Al O by
2 pzc 2 3
of106 andVanHoeckeetal.[4]used105cell/mL.SiO did flocculating with algae, adsorbing nutrients, and limiting
2
notplayamajorroleinthegrowthofP.subcapitataoverthe lightavailability.TiO alsoactedsimilarlytoSiO byhaving
2 2
concentrationrangetestedinourwork,whichagreedwithJi aportionoftheNPremainfreeinsuspension,limitinglight
et al. [6] who reported little effect on Chlorella sp. Growth availabilityandaffectinglipidperoxidation.However,unlike
atSiO concentrationof1000mg/Linthesizerangeof20– SiO ,TiO isaknownphotocatalystthatgeneratesROS.
2 2 2
50nm. NP is soluble in water leading to release of metal ions
SiO causedanincreaseinlipidperoxidation(Figures3 (Mn+)ions,whicharetoxictoorganisms[28,29].Thiscould
2
and9).AnincreaseinSiO causedanincreasedaveragenor- notbeakeyfactorinthecaseofTiO .Thereislittleinfor-
2 2
malized specific lipid peroxidation, Z, (Figure3). Although mation regarding the toxicity of Ti4+. Thermodynamically,
not considered a photocatalyst, SiO has been observed to the formation of Ti4+ in aqueous solution will be limited
2
produce similar photosensitive effects, including increased due to the formation of dissolved TiO [46]. This will have
2
ROSlevelsandreducedglutathionelevels,asaphotocatalyst minimal effect on organisms because of the small percent
[1].Inthisstudy,theeffectofSiO NPonnormalizedspecific of total metal that is present as free metal ion. Therefore,
2
MDA, Z, could be due more to limit light availability than thesolubilityofTiO wasnotamajorfactorcontrollingthe
2
the production of ROS directly. Limited light availability responsesofalgaetothisNP.
has been shown to produce 1O under aerobic conditions Major factors affecting the algal responses include light
2
through back flow of electrons to excite P to 3P [37, availability, flocculation, particle size, and photoactivity.
680 680
38]. Here P is the reaction center containing chlorophyll Thesefactorsareinterrelated.WhenTiO andalgalcellsare
680 2
withinthethylakoidmembrane.TheROSgeneratedinthis introducedintothesuspension,flocculationtakesplaceright
reaction is responsible for increasing normalized specific away[31].LikeAl O ,TiO willaffecttheapparentdensityof
2 3 2
lipid peroxidation, Z. The decrease in normalized specific thealgalcells.FlocculationbetweenalgalcellsandTiO was
2
MDAastheconcentrationofSiO ENPincreasewasdueto observed and was consistent with Lin [31] which reported
2
turbidityincreaseandlightavailabilitydecreaseasaffectedby absorption of TiO to P. subcapitata. The flocculation with
2
theparticle size.Rayleightheoryoflight scatteringpredicts TiO hastheeffectofbringingthealgaeindirectcontactwith
2
thatlightscatteringisproportionaltotheratiobetweenthe theROS-generatingTiO (Figure3).Thelipidperoxidation
2
radiusofaparticleandagivenwavelengthoflightwithlight mayhavebeenaffectedbytheadsorptionofNPtothesurface
scatteringefficiencybeingproportionaltothefourthpower ofthealgalcells.ThedirecttransferofROSfromthepoint
of particle radius [36]. This would reduce the back flow of of generation to the surface of algal cells is important. This
electrons. The growth of cells at 1000mg/L (Figure7) was canbeexemplifiedbycalculatingthedistancehighlyreactive
inverselyproportionaltothelipidperoxidationat1000mg/L hydroxyl radical (OH•) will travel before it reaches the end
(Figure9).Thiswasalsoaffectedbythelackofflocculation of the first half-life. The half-life of ROS is on the order
between the algae and the SiO which resulted in evenly of milliseconds [47, 48]. Lee et al. [49] used the diffusion
2
distributedshadingeffectthroughoutthealgalcells. layer thickness of 800nm to calculate Ag+ diffusion to P.
SiO affected the chlorophyll content of the algal cells subcapitata.Thehydroxylradical(OH•)diffusioncoefficient
2
(Figures 2 and 8). Low NP concentration increased Chla, is2.3×10−9m2/s[50].FromFick’ssecondlawofdiffusion
whereashighNPconcentrationdecreasedit.Resultsshowed and assuming the reaction rate of OH• is on the order
that limited light availability can affect the chlorophyll of milliseconds, then the OH• would travel approximately
contentinalgae[39,40].Thereductioninlightavailability 18nm before one half of the OH• would react with other
would encourage the algae to produce more chlorophyll oxygenspeciesinthewater.TheconcentrationofOH•would
per cell [41]. At low concentrations the algae will attempt be reduced by >99% by the time and would diffuse to the
to overcome the decreased light availability. Following the surface of the algal cell. It is feasible that if the NP and
Rayleigh theory of light scattering, the turbidity of the algaearenotincontact,theROSwillnotbringaboutlipid
solutionsincreasedwithdecreasedNPsizeonthesamemass peroxidation.Thelipidperoxidationwasmaximizedatd of
1
basis(Figure8opencircles).WithincreasedNPsizethelight 42nm (Figure6) which correlates with the maximum size
10 JournalofNanotechnology
effect on growth (Figure4). The number of particles that playin toxicity? Does thetypeofNPplayaroleintoxicity,
flocculatewithanalgalcellmightbecorrelatedtod .Larger chlorophyllcontent,andlipidperoxidation?
1
d1mightdecreasethenumberofNPthatwillflocculatewith TheeffectsofphotocatalyticTiO2onalgaewascompared
algae. This will also affect available light. The incomplete, tothatofnon-photocatalyticnanoparticles(e.g.,Al O and
2 3
multilayered coverage of the algal cells, like Al2O3, may SiO2). Based on results, it can be concluded that TiO2 is
decreaselightavailabilitytothealgalcells.Asdiscussedwith “harmful-toxic”betweenad of30to60nmandnontoxicat
1
Al2O3 and SiO2, the decrease in light will affect the growth d1 <30nmandd1 >60nm.Thetermharmfulisdefinedas
andchlorophyllcontent. measurement endpoints from 10–100mg/L [62]. The term
The primary particle size of TiO2, d1, played a role non-toxic is defined as measurement endpoints >100mg/L
in algal growth, chlorophyll content of algal cells, and [62]. The most important factor was the surface charge of
lipid peroxidation of algal cells (Figures 4–6). All of these thematerialasitaffectedtheaggregationbetweenalgaeand
endpoints have maximum effect values at the same particle NP,whichinturnaffectedlipidperoxidation,chlorophylla
sizeof42nm.LikeAl2O3andSiO2,nutrientssuchasCu,Fe, content,andgrowth.
or N would be imitated due to the scavenged by NP, which If nanoparticles were released into the environment,
in turn could affect the chlorophyll content. It has been algaewouldbeflocculateddependentontheextentofsurface
suggested that NP can decrease bioavailability of nutrients, charge of the nanoparticles and the algal cells. The algae
which would contribute to toxicity [6]. The smaller the willattempttoovercometheshadingeffectbyincreasingthe
d1, the greater the specific surface area and the more chlorophyllcontentinordertooptimizethelightavailability.
nutrients would be adsorbed. Gao et al. [51] observed that With a high degree of light scattering, that is, limited
maximumadsorptiondensityofCd2+(Γmax)variedinversely light availability, lipid peroxidation may be generated by
proportionaltotheNPsize.Thatrelationshipwouldbetrue “back flow of electrons in the PSII from the semiquinone
fornutrientssuchasCu,Fe,orN,whichinturnwouldaffect acceptortotheS oxidationstatesofthedonorside”[37].
the chlorophylla production and algalgrowth [35]. The d1 Additionally,asth2,e3algaeareremovedfromthewatercolumn
primarilyaffectthecriticalspecificchlorophyl(Figure5).Lin
by an increased rate of settling, the light availability will
[31]studiedtheadsorptionofTiO ontoP.subcapitataand
2 continuetodecrease.Exposuretimeinthewatercolumnwill
reportedthatatpH7.3andinitialTiO of150mg/L,theTiO
2 2 belimited,inmostcases,duetoflocculationandsubsequent
uptake(no.TiO peralgalcell)wasconstantat3×105TiO
2 2 settlingoutofnanoparticlesinthewatercolumn.
particlesperalgalcell.AthigherconcentrationofTiO ,there
2 Lipid peroxidation was the only measurement used to
willbenoadditionaluptake.
determine reactive oxygen species activity. However, it is
The extent of lipid peroxidation has been related to the
suggested that several additional biochemical markers be
amountofreactiveoxygenspeciesinmanydifferentsystems
measuredinordertoobservehowthedefensivemechanism
includinghumanstofishtobacteria[43–55].Generationof
responseofalgaeinresponsetonanoparticles.Itistherefore
ROSbyNPphotocatalystsisrelatedtothesizeandthetype
suggested that subchronic endpoints be a measurement for
of NP [10, 56]. Size of the particles controls the band gap
future work, including, but not limited to, glutathione,
andsurfacearea[8,11].Thebandgapdeterminesthelevelof
superoxidedismutase,andvariousdirectROSassays.Addi-
lightenergythatisconvertedtoprotonsandthesurfacearea
tionally,theresponseofseveraladditionalspeciesofalgaeis
affectstherecombinationofelection-holepairs.Manystud-
recommendedforthepurposeofcomparison.Thiswilladd
ies have shown that optimum degradation of organic com-
inverifyingtheresultsofthisstudy.
poundsoccursofTiO between3.8and40nm[36,57–61].
2
These values are smaller than what is observed in Figure6.
Acknowledgments
The lipid peroxidation in this study may be influenced by
adsorption of NP to the surface of the algae. The half-life Dr. Y. L. Liao, a visiting scholar from Taiwan, synthesized
of ROS is on the order of microseconds, for example, the TiO nanoparticles of various sizes used in this study.
superoxideanion(•O2−)hasahalf-lifeof1μsandhydrogen This2 paper was funded by USEPA STAR Grant no. R-
peroxide (H2O2) has a half-life of 1ms [48, 49]. Due to 83172101.Dr.NoraSavagewastheprojectmanager.Results,
this time scale, it is feasible that if the NP and algae are discussions, and conclusions are those of the authors and
notincontact,theROSwillnotpromptlipidperoxidation. should not be interpret, as endorsement of the funding
However,noliteraturewasavailableregardinghowd1affects agency. Sources of nanoparticles studied came from the
the aggregation between algae and NP. Additionally, light following suppliers: Degussa Corp., Allied High Tech Prod-
availability is being optimized for ROS generation at d1 of ucts, Nanostructured and Amorphous Materials, Inc., and
ca. 42nm. At d1 smaller than 42nm, the surface density of Sachtleben Chemicals. The authors have no direct financial
particles increases, which decreases the light availability to interestinorrelationshipwithanyoftheabovecompanies.
theNPindirectcontactwiththesurfaceofthecells.
References
5.Conclusions
[1] W.Lin,Y.W.Huang,X.D.Zhou,andY.Ma,“Invitrotoxicity
This research was undertaken to answer the following ofsilicananoparticlesinhumanlungcancercells,”Toxicology
questions.HowtoxicistheTiO2toalgae?WhatdoesNPsize andAppliedPharmacology,vol.217,pp.252–259,2006.