Journal of Environmental Science and Health Part A (2009) 44, 1485–1495Copyright C© Taylor & Francis Group, LLCISSN: 1093-4529 (Print); 1532-4117 (Online)DOI: 10.1080/10934520903263231

Aggregation and toxicity of titanium dioxide nanoparticlesin aquatic environment—A Review

VIRENDER K. SHARMA

Chemistry Department, Florida Institute of Technology, Melbourne, Florida, USA

The use of nanoparticles—particles with size ∼1–100 nm is increasing worldwide. This is particularly the case for applicationsof titanium dioxide nanoparticles (nano-TiO2) in consumer products, which have expanded at a fast rate in the last decade. Theproperties of nano-TiO2 differ significantly from bulk-TiO2 of the same composition because of an increase in surface area. A releaseof nano-TiO2 from application sources to the aquatic environment may pose possible risks due to their bioavailability and toxicity.The aggregation of nano-TiO2 plays an important role in the environmental effects of nanoparticles because the size and shape ofnanoparticles will determine the magnitude of any potentially toxic effect. Aggregation is affected by pH, ionic strength, and ionicidentity (inorganic and organic) of aqueous suspensions and is reviewed in this paper. The current information on the evaluation ofecotoxicological hazards of nano-TiO2 to bacteria, algae, invertebrates, nematodes, and rainbow trout is also given.

Keywords: TiO2, aggregation, ph, ionic strength, organic matter, aquatic toxicity, reactive oxygen species, bacteria, invertebrates,algae.

Introduction

Nanoparticles are usually defined as particles smaller than100 nm in at least two dimensions. The physical, chemi-cal, and biological properties of nanoparticles are gener-ally different from the properties of the same materials ata larger scale. Nanomaterials have been shown to offer sig-nificant potential applications for providing solutions totechnological and environmental challenges in fields suchas medicine, solar energy conversion, catalysis, and waterpurification.[1–6] Because of such diverse applications ofnanomaterials in a wide range of industries, the productionof engineered nanomaterials is expected to increase from400 tons to 58,000 tons in 2011–2020.[7] In other words, thenanomaterials industry is growing with an estimated enter-prise of $1 trillion by 2015.[8] With this growth, synthesis ofnanomaterials requires the use of the 12 principles of greenchemistry in order to eliminate hazards. Figure 1 demon-strates the applications of these principles to nanoscience,which suggest the need of developing new synthetic andcharacterization tools.[9] A better understanding of bio/ecotechniques may also be needed to design safe novel mate-rials efficiently.[9] Furthermore, the interaction of nanoma-

Address correspondence to Virender K. Sharma, Chem-istry Department, Florida Institute of Technology, 150 WestUniversity Boulevard, Melbourne, Florida 32901, USA. E-mail:vsharma@fit.eduReceived June 25, 2009.

terials with DNA, lipids, cells, tissues, proteins, and bio-logical fluids needs to be considered for evaluation of theirsafety to humans and to the environment.[10–16] Moreover,nanoparticles in aquatic environments may influence envi-ronmental processes differently than the same materials oflarger size.[17] As shown in Figure 2, the reactivity of theparticle does not always increase with decreasing particlesize, but depends on the chemical reaction and the materialinvolved.[17] Four interrelated factors have been suggestedto cause changes in the reactivity at the nanoscale. Theseare: (i) the effect on the thermodynamics of chemical reac-tivity through the dependence of the surface free energy onthe particle size; (ii) increase in surface area as nanoparticlesget smaller and smaller; (iii) the variation in atomic struc-ture in terms of bond angles, bond lengths, and vacanciesand other defects near and on surfaces; and (iv) modifi-cation of electronic structure with size. The material andthe size range of the particles ultimately determine whetherone or all four of the factors contribute to size-dependentchemical reactivity.[17]

Of the various nanomaterials, titanium dioxide (TiO2)has been of great interest. There are natural and man madesources of TiO2 in the environment.[18–21] Natural sourcesof TiO2 are dominated by the minerals ilmenite (FeTiO3)and rutile (TiO2), which are commonly found in platonicand metamorphic rocks and also found as distinct mineralsin beach sands.[18] Because of the use of nano-TiO2 in paintsand coatings as self-cleaning, antimicrobial, and antifoul-ing agents and in cosmetics as a UV-absorber, it is produced

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Fig. 1. Translating the 12 green chemistry principles for application in the practice of green nanoscience. The principles are listed,in abbreviated form, along with the general approaches to designing greener nanomaterials and nanomaterial production methodsand specific examples of how these approaches are being implemented in green nanoscience. Within the figure, PX, where X = 1−12,indicates the applicable green chemistry principle. (Reproduced from [9] with the permission of the American Chemical Society)

in a large scale.[19–22] Although natural nano-TiO2 has beenfound in river water,[23] recently, direct evidence of the re-lease of synthetic nano-TiO2 from urban applications intothe aquatic environment has been documented.[24] It wasshown that nano-TiO2 leached out from the nano-TiO2containing painted house facades and entered into receiv-ing waters; giving concentrations in the range of a few µgL−1 in a small stream.[24]

Model calculations on the release of nano-TiO2 into theenvironment have been done using substance flow analy-

Fig. 2. Generalized trend for size-dependent reactivity change of amaterial as the particle transition from macroscopic (bulk-like) toatomic. (Reproduced from [17] with the permission of the RoyalChemical Society)

sis from products to air, soil, and water in Switzerland.[25]

The predicted environmental concentrations (PEC) un-der a realistic scenario (RE) and high emission scenario(HE) are given in Table 1. The PEC values for nano-TiO2 in water ranged from 0.7–16 µg L−1, which is nearlyequal to or higher than the predicted no effect concentra-tion (PNEC) of <1 µg L−1. These values gave risk quo-tients (PEC/PNEC) of nano-TiO2 as >0.73 µg L−1 and>15.83 µg L−1 in the RE and HE scenarios, respectively.This indicates a possible ecotoxicity of nano-TiO2 in wa-ter. It is clear that the behavior of nano-TiO2 needs to bestudied in the aquatic environment.

The toxicity of nanoparticles depends on both thesize and surface chemistry of nanoparticles in water.[26,27]

Aggregation of nanoparticles thus becomes critical in un-derstanding toxicity, because of the relationship betweenaggregation state, size, and observed effects.[28–31] This pa-per therefore first reviews the aggregation of nano-TiO2under natural conditions, followed by a discussion on tox-icity of nano-TiO2 in the aquatic environment.

Table 1. Predicted environmental concentrations (PEC) of nano-TiO2 in air, water, and soil. (Data were taken from[25]).

Unit RE HE

Air µg m−3 1.5×10−3 4.2×10−2

Water µg L−1 0.7 16Soil µg kg−1 0.4 4.8

RE: realistic scenario; HE: high emission scenario.

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Fig. 3. Modification of nanoparticles in the environment. (Reproduced from [32] with the permission of Springer)

Aggregation

Free nanoparticles usually tend to aggregate in the envi-ronment and then can possibly be eliminated or trappedthrough sedimentation (Fig. 3). Generally, aggregatednanoparticles are less mobile and can interact with filterfeeders and sediment-dwelling animals.[32] Agglomerationis thus important in understanding the fate and transportof nanoparticles in the natural environment. Though stud-ies have been published on the transport of small par-ticles, experiments often involve particles of an organicnature, such as polystryrene beads, fullerols, and carbonnanotubes.[33–36] Limited information on the transport ofnano-TiO2 is available.[37,38] The pH of experiments wasfound to be an important parameter in the aggregation ofnano-TiO2.

[37]

Figure 4 shows the effect of pH on aggregate size over thepH range of 1–12. As the pH approached the point of zerocharge (pHzpc), the aggregation size increased. This trendis similar to phenomena observed in the settling of parti-cles. It seems that electrorepulsion is fundamental in theunderstanding of the conditions under which aggregationof nanoparticles occurs. As the solution pH approachesthe pHzpc, the repulsion between nanoparticles of similarsurface potentials decreases, making it easier for particlesto aggregate (Fig. 4). Research has shown that more than80% of suspended particles and aggregates can be mobilizedover the pH range of 1–12, except when close to pHzpc.

[37]

It has been shown independently that the pHzpc of nano-TiO2 changes with size. The smallest particle sizes show thelowest pHzpc (pHzpc (3.6 nm) = 4.8) while largest particleshave a higher pHzpc (pHzpc (8.1 nm) = 6.2).[39]

It is therefore expected that the mobility of nano-TiO2in natural environments may be dependent on the size ofnanoparticles. Aggregation of nano-TiO2 may limit trans-port due to settling out of solution. Besides pH, ionicstrength and the nature of the electrolyte(s) in aqueoussuspensions influence the aggregation of nano-TiO2.

[40] AtpH ∼4.5 and in 0.0045 M NaCl suspensions, 4–5 nm TiO2readily aggregated to average diameter sized particles of 50–60 nm. If the ionic strength increased to 0.0165 M NaCl,the formation of micro-sized aggregates occurred within 15minutes. In increasing the pH to 5.8–8.2, the formation of

Fig. 4. Particle/aggregate size populations as a function of pHas measured by dynamic light scattering (DLS). The bars repre-sent the range. Reproduced from [37] with the permission of theAmerican Chemical Society)

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micro-sized aggregates happened within 5 minutes at evenlow ionic strength of 0.0084–0.0099 M NaCl.

Importantly, a significantly faster aggregation of nano-TiO2 took place in replacing NaCl with CaCl2 aqueoussuspensions at an ionic strength of 0.0128 M at pH 4.8.[40]

The results suggest that cations present in soils and surfacewaters may play an important role in the aggregation ofnano-TiO2.

The studies mentioned above do not account for the in-fluence of the presence of natural organic matter (NOM)such as microbial and humic substances, which are presentin the natural environment. In the environment, nanopar-ticles can adsorb NOM to form complexes (or NOM coat-ings) which may cause particles to acquire negative surfacecharge. This alters the fate and transport of nanoparti-cles in the aquatic environment.[41,42] This may also alterthe sorption of toxic hydrophobic organic compounds byNOM.[43–48]

Humic substances can significantly influence the trans-port of nanoparticles by modifying electric charge, size,and the chemical nature of the particles.[49] Recent workon the aggregation of nano-TiO2 in the presence of nat-ural organic acids, showed such influences on the stabil-ity of TiO2 suspensions.[50] The presence of oxalic andadipic acids destabilized suspensions.[50] However, experi-ments were performed at pH values far from the pHzpc andmay not realistically represent natural conditions found inthe environment. Only recently, the effects of humic sub-stances on the aggregation of nano-TiO2 were studied byusing Suwannee River Fulvic Acid (SRFA) under variousphysicochemical conditions.[51]

Diffusion coefficients and weight average diameters ofTiO2 were determined as a function of pH (2–8), ionicstrength (0.005–0.1 M), and SRFE (0.5–2.0 mg L−1). Anincrease in ionic strength usually resulted in increased ag-gregation at a given pH. Interestingly, adsorption of theSRFA onto particles, caused less aggregation of nano-TiO2,perhaps due to increased steric repulsion. This is in contrastto earlier experiments performed with oxalic acid.[50] Theinfluence of the concentration of SRFA on the aggrega-tion of nano-TiO2 is shown in Figure 5. An increase inthe concentration of SRFA increased disaggregation at allstudied pH values. At pH 8.0, which is above the pHzpc, col-loidal suspensions were less disaggregated in the presenceof 1.0 mg L−1 SRFA than its absence. At concentrationof 2.0 mg L−1 SRFA, increasing steric stabilization of theparticles likely contributed to a decrease in hydrodynamicdiameters (see Fig. 4). Results of the study[51] suggest thatthe dispersion and mobility of nano-TiO2 may occur to agreater extent than predicted from laboratory experiments.

Recently,transport properties of anatase polymorphTiO2 nanoparticles (ANTNPs) were evaluated in cleansilica, amorphous aluminum, and iron hydroxide-coatedsands.[52] In this study, the nanoparticles were also encapsu-lated by carboxymethyl cellulose (CMC) and were referredto as CMC-ANTNPs. CMC is used in several industries

Fig. 5. Variation of diffusion coefficients and weight average di-ameters of 1.0 mg L−1 of TiO2 nanoparticles as a function of theconcentration of SRFA for an ionic strength of 0.01 M and forthree different pH values: 2.0 (�), 5.5 (O) and 8.0 (�). The arrowindicates the size of the disaggregated TiO2 nanoparticles as pro-vided by NanoAmor. (Reproduced from [51] with the permissionof the American Chemical Society)

including paper and detergents, paints, ceramics, glues andadhesives and a release of such compounds may influencethe mobility of TiO2 nanoparticles in the aquatic environ-ment. Initially, influence of CMC on the surface chargeand particle size of ANTNPs was studied (Fig. 6A). Theisoelectric point (IEP) of the particles decrease with theaddition of CMC. This reduction in the IEP creates stericand double layer repulsions between particles and collectorsurfaces to restrict attachment.[52]

The influence of pH on the measured electrophoreticmobility (EPM) is also presented in Figure 5A. The EPMdecreased with increases in pH for both ANTNPs andCMC-ANTNPs. The magnitude of EPM also decreasedwith increase in the concentrations of Na+ and Ca2+ ions(Fig. 6B). Bivalent Ca2+ had a greater effect than Na+.This cationic influence may be associated with a reduc-tion of surface charge due to complexation with nega-tively charged groups (e.g. COO− and OH−) of CMC-ANTNPs.[53] Interestingly, the hydrodynamic diameter inthe presence of Ca2+ increased from 228 to 423 nm while theaddition of Na+ decreased the diameter from 228 to 147 nm(Fig. 6B).

This indicates that Ca2+ strongly suppresses the electricdouble layer and the van der Waals attractive forces be-came predominant. The addition of Ca2+ ions may alsoresult in aggregation of ANTNPs; similar to the forma-tion of large-size aggregates of fullerene nanoparticles withincreased concentration of Ca2+ ions.[54] Comparatively,the hydration shell effect of Na+ may result in the smallerhydrodynamic diameter at high salt concentrations.[55]

Overall, results suggest that high molecular weight com-pounds such as CMS influence the mobilization ofANTNPs. TiO2 nanoparticles may thus enter into natu-ral and engineered water resources more easily when CMCencapsulated.[52]

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Fig. 6. Measured electrophoretic mobility (EPM) and particle size hydrodynamic diameter (nm) of ANTNPs: (A) in the absence andpresence of CMC over pHs [ANTNPs concentration: 20 mg L−1, CMC: 0.2%, background electrolyte: 1 mM NaCl; (B) as a functionof ionic strength and cation type: pH 6.4 ± 0.4, ANTNPs: 20 mg L−1, CMC: 0.2%. (Reproduced from [52] with the permission ofthe American Chemical Society).

Toxicity

Mechanism

Because the size of nanoparticles is similar to that of typicalcellular components and proteins, nanoparticles can travelinside the human body.[56] Some studies have shown pen-

etration of nanoparticles into the skin.[57,58] However, it isimportant to know whether TiO2 particles are anatase andrutile or monodispersed or aggregated in order to under-stand their dermal toxicological impact.[59] TiO2 nanopar-ticles can cause DNA and pulmonary damages.[60] In ex-posing TiO2 nanoparticles to human endothelial cells, an

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increase in the levels of IL-8 was observed.[61] Evidence isaccumulating that nanoparticles can induce the productionof reactive oxygen species (ROS), oxidative stress (OS), andinflation in the vasculature and lungs.[5,62] During OS, theproduction of ROS overwhelms the antioxidant defense ca-pacity of the cell, causing adverse biological consequences.

It is also known that OS is the major source ofROS, which included the superoxide anion (O·−

2 ), hy-drogen peroxide (H2O2), hydroxyl radicals (·OH), andperoxynitrites.[63]

This would result in cell death due to oxidative DNAdamage and increases in the level of cellular nitricoxide.[56,64,65] Several in vitro studies have reported thatnano-TiO2 caused OS mediated toxicity in a variety of celltypes, including liver, skin fibroblast, endothelia, epithelia,Salmonella bacteria, and alveolar macrophages.[66–74] Thetoxicity of nano-TiO2 to fish cells in vitro and to algae dueto ROS has also been suggested.[75,76]

Tests were conducted to measure ROS using a fluores-cent probe by exposing Degussa P25 nano-TiO2 to brainmicroglia (BV2).[63] Figure 7 shows measured concentra-tions of O−·

2 and H2O2 as generated by the oxidative burst.Significant release of O−·

2 did not happen until 60 min-utes post-exposure (Fig. 7A). Comparatively, formation ofH2O2 was rapid (Fig. 7B). Overall, the results of this studysuggest cellular and morphological expressions of free radi-cal formation in response of brain microglia (BV2) to nano-TiO2.

The toxic effect of TiO2 nanoparticles in mouse fi-broblast cells (L929) has also been studied using homo-geneous and weakly aggregated TiO2 nanoparticles inaqueous solution.[77] As the concentration increased from3–600 µg mL−1 of TiO2, the cellular shape became morespherical and levels of ROS and lactate dehydrogenase(LDH) increased. The levels of methyl tetrazolium cyto-toxicity (MTT), glutathione (GSH), and superoxide dis-mutase (SOD) decrease; suggesting involvement of ROS inthe cytoxicity of the TiO2 nanoparticles to L929 cells.[77]

Figure 8 shows TEM images of the internalization ofTiO2 nanoparticels in L929 cells. Some particles were en-closed in lysosomes while others were engulfed. This sug-gests that incorporation of TiO2 nanoparticles into cellularmembranes[78] caused an increase in the number of lyso-somes and damaged some cytoplasmic organelles (Fig. 8).

Ecotoxicologic studies

Bacteria

Toxicity of nano-TiO2 has been tested on bacteria Vibria fis-cheri, Escherichia coli, and Bacillus subtilis.[79,80] High con-centrations of nano-TiO2 are required to inhibit the growthof bacteria. For example, 72% reduction in the growth of E.coli occurred at 5 g TiO2L−1. Comparatively, B. subtilis hap-pened to be slightly more sensitive and a similar reduction

Fig. 7. (A) Increases in O−2 were measured by the fluorescent probe

MitoSOX Red. Cells, incubated in 2 µM MitoSOX (10 min, 37◦C)showed significant (p < 0.05) increases in fluorescence (measuredat 510/580 nm) after 60 min exposure to g20 ppm P25, and flu-orescence continued to increase for 150 min postexposure. (B)Extracellular release of H2O2 was measured using OxyBURST.Cells were incubated (30 min, 37◦C) in 10 µg/mL OxyBURST inreduced-serum media, washed, and then exposed to P25 (2.5-120ppm). Significant (p < 0.05) increases of fluorescence (measuredat 508/528 nm) occurred in cells exposed to ≥ 10 ppm at 5 minand continued to increase throughout the 120 min recording pe-riod. (Reproduced from [63] with the permission of the AmericanChemical Society)

in growth occurred at 1 g TiO2L−1. Nano-TiO2 suspensionat 20 g L−1 were not acutely toxic to V. fischeri.[80]

Algae

A test of nano-TiO2 on green alga Desmodesmus subspi-catus has been conducted using 25 nm and 100 nm TiO2particles.[81] It was found that the size and crystalline formof nano-TiO2 determined the toxicity on D. subspicatus.Smaller particles were found to be more toxic to D. subspi-catus (72 hr EC50 = 44 mg L−1) than larger particles (72hr EC50 >50 mg L−1). Recently, toxicity tests of nano-TiO2(25–70 nm) and bulk-TiO2 were performed on Pseudokirch-neriella subcapitata.[77] Nano-TiO2 were more toxic thanbulk-TiO2. The EC50 values determined were 5.83 mg Ti

TiO2 nanoparticles in aquatic environment 1491

Fig. 8. TEM image of TiO2 nanoparticles internalization in L929 cells. (a) Normal cellular and mitochondrial morphology of cellscultured in DMEM (control). (b) Increase in the number of lysosomes and disappearance of cytoplasmic organelles in L929 cellscultured in media containing TiO2 nanoparticles (300 µg/mL) for 48 h. (c and d) Higher magnification of the agglomerated regionshown in b. (c) Lysosomes engulfed TiO2 nanoparticles in L929 cells by phagocytosis. Internalized TiO2 nanoparticles were presentprimarily along and inside the ysosomes. (d) Apparent discontinuities in the cell membrane and cytoplasm were observed in L929cells. (Reproduced from [77] with the permission of the American Chemical Society)

L−1 and 35.9 mg Ti L−1 for nano-TiO2 and bulk-TiO2, re-spectively. Interestingly, both nano- and bulk- TiO2 formedaggregates during incubation (Figs. 9A and D). Phase con-trast microscopy and red fluorescence confirmed the visibil-ity of algal cells (Figs. 9B, C, E and F). Significantly, largeaggregates were observed that entrapped almost all algalcells in the case of nano-TiO2 (Figs. 9 E and F). However,in using culture with bulk-TiO2, free algal cells in addi-

tion to cells trapped on small TiO2 aggregates were seen. Itseems aggregation of nano-TiO2 reduced the availability oflight to entrapped algal cells, thus inhibiting their growth.It has been shown independently that adsorption of P. sub-capitata onto surfaces of nano-TiO2 carried 2.3 times theirown weight in TiO2 particles.[82] Furthermore, kinetics andextent of adsorption depended on the pH with maximumadsorption occurring at pH 5.5.

Fig. 9. Aggregates of bulk and nano TiO2 particles in Pseudokirchneriella subcpitata culture. Aggregates of bulk TiO2 (A,B,C) andnano (D,E,F) in test medium, as visible to the naked eye (A,D), in phase contrast microscopy (B,E) and fluorescence microscopy*C,F). Arrows indicate algal cells. (Reproduced from [76] with the permission of Elsevier, Inc.)

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Table 2. Body length, number of eggs inside the worm body, and number of offspring per individual of C. elegans afterexposure to different concentrations of TiO2 nanoparticles and bulk TiO2. E. coli was added as a food to C. elegans. Eachtest was repeated for four times and n = 425. All values were expressed as mean ± standard deviation (SD) of four replicates.(Reproduced from [52] with the permission of Elsevier, Inc.)

Treatment Body length (µm) Eggs inside body Offspring per worm

Control 1126 ± 3 11.1 ± 1.2 28.4 ± 3.524.0 mg L−1nano-TiO2 1080 ± 24 9.8 ± 2.3 21.7 ± 5.847.9 mg L−1nano-TiO2 801 ± 82∗ 4.8 ± 1.9∗ 3.4 ± 3.1∗∗

95.9 mg L−1nano-TiO2 760 ± 29∗∗ 2.1 ± 0.8∗∗ 0.9 ± 1.2∗∗

167.8 mg L−1nano-TiO2 740 ± 15∗∗ 0∗∗ 0∗∗

167.8 mg L−1nano-TiO2(supernatant) 980 ± 72 8.9 ± 1.6 18.5 ± 2.424.0 mg L−1bulk TiO2 975 ± 66 9.1 ± 3.0 22.5 ± 4.247.9 mg L−1bulk TiO2 923 ± 10 7.9 ± 2.0 16.3 ± 5.595.9 mg L−1bulk TiO2 812 ± 59∗ 5.8 ± 1.5∗ 5.6 ± 3.2∗∗

167.8 mg L−1 bulk TiO2 770 ± 48∗∗ 2.2 ± 1.3∗∗ 2.3 ± 2.6∗∗

∗Significantly different from the control within each column at P < 0.05 level.∗∗Significantly different from the control within each column at P < 0.01 level.

Invertebrates

Nano-TiO2 were exposed to terrestrial arthropods (Por-cellio scaber, Isopoda, Crustacea) and feeding parameters,weight change, mortality, and the activities of catalase andglutathione-S-transferase were evaluated after 3 or 14 daysof dietary exposure.[83] It was found that the effects of nano-TiO2 depended on total consumed quantity, size, and pre-treatment of particles as well as on exposure concentrationand duration.

Daphnia magna as a test organism for aquatic inverte-brates was exposed to nano-TiO2.

[84] A low mg L−1 rangeof nanoparticles caused low toxicity, however the use ofhigh µg L−1 ranges gave some indication of chronic toxicityand behavioral changes. Another study of toxicity of nano-TiO2 was also conducted on Daphnia magna and Thamno-cephalus platyurus.[80] The tests showed 60% mortality ofD. magna at 20 g L−1 TiO2, but no toxicity forT. platyuruswas observed even at such a high TiO2 level. However il-lumination of nano-TiO2 resulted in acute toxic effects ondaphnids.[81]

Nematode

Toxicity of nano-TiO2 and bulk-TiO2 were examined forthe nematode Caenorhabditis elegans with Escherchia coli asa food source.[85] Nano-TiO2 and bulk-TiO2 has diametersof 50 nm and 285 nm, respectively. The results are presentedin Table 2. No demonstrated toxicity of bulk-TiO2 to thenematode was observed until the concentration was>95.9mg L−1. Comparatively, nano-TiO2 showed toxicity effectsat 47.9 mg L−1. Significantly, the toxicity of nano-TiO2 su-pernatant at 167.7 mg L−1 was not too different from therespective controls. Furthermore, exposure of 47.9, 95.9,and 167.8 mg L−1 nano-TiO2 gave lower numbers of off-springs per worm than those exposed to bulk-TiO2.

Rainbow trout

An in vitro study has been carried out by exposing rain-bow trout (Oncorhynchus mykiss) to 0.1, 0.5, and 1.0 mgL−1nano-TiO2 for up to 14 days.[86] Exposure caused somegill pathologies including odema and thickening of lamel-lae. Levels of metals (Na+, K+, Ca2+, and Mn2+) in tissuesdid not change, but concentration-dependent changes ofCu and Zn levels were observed, particularly in the brain.

Conclusions

Properties of nano-TiO2 differ from those of bulk-TiO2of the same composition, which results in many novel ap-plications of the nanoparticles. However, possible harmfulinteractions of nano-TiO2 with biological systems causepotential size-dependent toxic effects. The extent of ag-gregation of nano-TiO2 in the aquatic environment deter-mines the size of nano-TiO2, which indicates whether nano-TiO2 remains dispersed or produces larger-sized aggregates.Though progress has been made in understanding the in-fluence of pH, ionic composition, and ionic strength ofaqueous suspensions on aggregation, little is known aboutthe effects of the natural organic matter on the aggregationof nano-TiO2.

Furthermore, the effects of structure and properties ofhumic substances in terms of aromaticity, conformation,and polarity of aggregation of TiO2 needs examination.Available data on the toxicity of nano-TiO2 in the aquaticenvironment is limited. Future efforts should be made intowards understanding the role of properties such as size,shape, degree of agglomeration, chemical and catalytic ofparticles contribute to toxic effects on bacteria, algae, inver-tebrates, and vertebrate species. There is a possibility thatother toxicants can be associated with TiO2 nanoparticles

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and therefore likely synergistic effects must be examined foradditional toxicological concerns.

Acknowledgment

Author wishes to thank Drs. Mary Sohn and Ria Yngardfor useful comments on this paper. This paper has also beengreatly improved by the comments of the reviewer.

References

[1] Hutchison, J.E. Greener nanoscience: A proactive approach to ad-vancing applications and reducing implications of nanotechnology.ACSNano. 2008, 2(3), 395–402.

[2] Aitken, R.J.; Chaudhary, M.Q.; Boxall, A.B.A.; Hull, M. Manufac-ture and use of nanomaterials: Current status in the UK and globaltrends. Occup. Med. 2006, 56, 300–306.

[3] Brunet, L.; Lyon, D.Y.; Hotze, E.M.; Alvarez, P.J.I.; Wiesner,M.R. Comparative photoactivity and antibacterial properties ofC60 fullerenes and titanium dioxide nanoparticles. Environ. Sci.Technol. 2009, 43, 4355–4360.

[4] Lowe, T. The revolution in nanometals. Adv. Mater. Proc. 2002,160, 63–65.

[5] Emerich, D.F.; Thanos, C.G. Nanotechnology and medicine. ExpertOpin. Biol. Ther. 2003, 3, 655–663.

[6] Doumanidis, H. The manufacturing program at the National Sci-ence Foundation. Nanotechnology 2002, 13, 248–252.

[7] Mayland, A.D. Nanotechnology: A Research Strategy for Address-ing Risk. Woodrow Wilson International Center for Scholars,Washington, DC, 2006.

[8] Nel, A.; Xia, T.; Madler, L.; Li, N. Toxic potentials of materials atthe nanolevel. Science 2006, 311, 622–627.

[9] Dahl, J.A.; Maddux, B.L.S.; Hutchison, J.E. Toward greenernanosynthesis. Chem. Rev. 2007, 107, 2228–2269.

[10] Xia, T.; Kovochih, M.; Liong, M.; Madler, L.; Gilbert, B.; Shi,H.; Yeh, J.I.; Zink, J.I.; Nel, A.E. Comparison of the mechanismof toxicity of zinc oxide and cerium oxide nanoparticles based ondissolution and oxidative stress properties. ACSNano 2008, 2(10),2121–2134.

[11] Sharma, V.K.; Yngard, R.A.; Lin, Y. Silver nanoparticles: Greensynthesis and their antimicrobial activities. Adv. Coll. Int. Sci. 2009,145, 83–96.

[12] Liang, G.; Pu, Y.; Yin, L.; Liu, R.; Ye, B.; Su, Y.; Li, Y. Influ-ence of different sizes of titanium dioxide nanoparticles on hep-atic and renal functions in rats with correlation to oxidative stress.J. Toxicol. Environ. Health Pt. A Curr. Issues 2009, 72, 740–745.

[13] Soto, K.; Garza, K.M.; Murr, L.E. Cytoxic effects of aggregatednanomaterials. Acta Biomater. 2007, 3, 351–358.

[14] Soto, K.F.; Carrasco, A.; Powell, T.G.; Garza, K.M.; Murr, L.E.Comparative in vitro cytotoxicity assessment of some manufacturednanoparticulate materials characterized by transmission electronmicroscopy. J. Nanopart. Res. 2005, 7, 145–169.

[15] Soto, K.F.; Carrasco, A.; Powell, T.G.; Garza, K.M.; Murr, L.E. Bi-ological effects of nanoparticulate materials. Mater. Sci. Eng. 2006,C26, 1421–1427.

[16] Ivankovic, S.; Music, S.; Gotic, M.; Ljubesic, N. Cytotoxicity ofnanosize V2O5 particles to selected fibroblast and tumor cells. Tox-icol. in Vitro 2006, 20, 286–294.

[17] Wigginton, N.S.; Haus, K.L.; Hochella Jr. M.F. Aquatic environ-mental particles. J. Environ. Monitor. 2007, 9, 1306–1316.

[18] Deer, W.A.; Howie, R.A.; Zussman, J. An Introduction to the RockForming Minerals. Longman Group Limited, Essex, 1992.

[19] nanoRoad. Overview of Promising Nanomaterials for IndustrialApplications. http://www.nanoroad.net/download/overviewnanomaterials.pdf,” 2005.

[20] AmericanElements. Silver Nanoparticles. http://www.american-elements.com/agnp.html, 2007.

[21] Nanoscale. NanoActive Titanium Dioxide. http://www.nanoscalecorp.com/producvts and services/specialty chemicals/metal oxides/?page=tio2, 2007.

[22] Chen X.; Mao, S.S. Titanium dioxide nanomaterials: synthesis,properties, modifications, and applications. Chem. Rev. 2007, 107,2891–2959.

[23] Wigginton, N.S.; Haus, K.L.; Hochella, M.F. Aquatic environmen-tal nanoparticles. J. Enviorn. Monitor. 2007, 9, 1306–1316.

[24] Kaegi, R.; Ulrich, A.; Sinnet, B.; Vonbank, R.; Wichser, A.; Zuleeg,S.; Simmler, H.; Brunner, S.; Vonmont, H.; Burkhardt, M.; Boller,M. Synthetic TiO2 nanoparticle emission from exterior facadesinto the aquatic environment. Environ. Pollut. 2008, 156, 233–239.

[25] Mueller, N.; Nowack, B. Exposure modeling of engineered nanopar-ticles in the environment. Environ. Sci. Technol. 2008, 42, 4447–4453.

[26] Grassian, V.H. When size really matters: Size-dependent propertiesand surface chemistry of metal and metal oxide naoparticles in gasand liquid phase environments. J. Phys. Chem. C 2008, 112, 18308–18313.

[27] Gao, J.; Youn, S.; Hovsepyan, A.; Llaneza, V.L.; Wang, Y.; Bitton,G.; Bonzongo, J-C.J. Dispersion and toxicity of selected manufac-tured nanomaterials in natural river water samples: Effects of waterchemical composition. Environ. Sci. Technol. 2009, 43(9), 3322–3328.

[28] Balbus, J.M.; Maynard, A.D.; Colvin, V.L.; Castranova, V.; Daston,G.P.; Denison, R.A. Meeting Report: Hazard assessment fornanoparticles—report from an interdisciplinary workshop. Envi-ron. Health Perspect. 2007, 115, 1654–1659.

[29] Grassian, V.H.; Adamcakova-Dodd, A.; Pettibone, J.M.;O’Shaughnessy, P.T.; Thorne. P.S. Inflammatory response of miceto manufactured titanium dioxide nanoparticles: comparison ofsize effects through different exposure routes. Nanotoxicology 2007,1(3), 211–226.

[30] Grassian, V.H.; O’Shaughnessy, P.T.; Adamcakova-Dodd, A.;Pettibone, J.M.; Thorne, P.S. Inhalation exposure study of nanopar-ticulate titanium dioxide with a primary particle size of 2 to 5 nm.Environ. Health Perspect. 2007, 115, 397–402.

[31] Powers, K.W.; Brown, S.C.; Krishna, V.B.; Wasdo, S.C.; Moudgil,B.M.; Roberts, S.M. Research strategies for safety evaluationof nanomaterials. Part VI. Characterization of nanoscale par-ticles for toxicological evaluation. Toxicol. Sci. 2006, 90, 296–303.

[32] Farre, M.; Gajda-Schrantz, K.; Kantiani, L.; Barcelo, D. Ecotoxic-ity and nalysis of nanomaterials in the aquatic environment. Anal.Bioanal. Chem. 2009, 393, 81–95.

[33] Lecoanet, H.F.; Bottero, J.Y.; Wiesner, M.R. Laboratory assessmentof the mobility of nanomaterials in porous media. Environ. Sci.Technol. 2004, 38, 5164–5169.

[34] Ryan, J.N.; Elimelech, M. Colloid mobilization and transport ingroundwater. Coll. Surf. A 1996, 107, 1–56.

[35] Lecoanet, H.F.; Wiesner, M.R. Velocity effects on fullerene and ox-ide nanoparticle deposition in porous media. Environ. Sci. Technol.2004, 38, 4377–4382.

[36] Brant, J.; Lecoanet, H. Wiesner, M.R. Aggregation and deposi-tion characteristics of fullerene nanoparticles in aqueous systems. J.Nanopart. Res. 2005, 7, 545–553.

[37] Guzman, K.A.D.; Finnegan, M.P.; Banfield, J.F. Influence of sur-face potential on aggregation and transport of titania particles.Environ. Sci. Technol. 2006, 40, 7688–7693.

1494 Sharma

[38] Franch, T.A.; Burleson, D.J.; Driessen, M.D.; Penn, R.L. On thecharacterization of environmental nanoparticles. J. Environ. Sci.Health Pt. A 2004, A39(10), 2707–2753.

[39] Kosmulski, M. The significance of the difference in the point of zerocharge between rutile and anatase. Adv. Coll. Interface Sci. 2002,99, 255–264.

[40] French, R.A.; Jacobson, A.R.; Kim, B.; Isley, S.L.; Penn, R.L.;Baveye, P.C. Influence of ionic strength, pH, and cation valence onaggregation kinetics of titanium dioxide nanoparticles. Environ. Sci.Technol. 2009, 43, 1354–1359.

[41] Ghosh, S.; Mashayekhi, H.; Pan, B.; Bhowmik, P.; Xing, B. Col-loidal behavior of aluminum oxide nanoparticles as affected by pHand organic matter. Langmuir 2008, 24, 12385–12391.

[42] Hyung, H.; Fortner, J.D.; Hughes, J.B.; Kim, J.H. Natural organicmatter stabilizes carbon nanotubes in aqueous phase. Environ. Sci.Technol. 2007, 41, 179–184.

[43] Chiou, C.T. Partition and Adsorption of Organic Contaminants inEnvironmental Systems; John Wiley and Sons, New York, 2002.

[44] Yang, K.; Zhu, L.; Lou, B.; Chen, B. Correlations of nonlinear sorp-tion of organic solutes with soil/sediment physicochemical proper-ties. Chemosphere 2005, 61, 116–128.

[45] Yang, K.; Lin, D.; Xing, B. Interaction of humic acid with nanosizeinorganic oxides. Langmuir 2009, 25, 3571–3676.

[46] Wang, X.; Lu, J.; Xing, B. Sorption of organic matter by carbon nan-otubes: Influence of adsorbed organic matter. Environ. Sci. Technol.2008, 42, 3207–3212.

[47] Wang, X.; Lu, J.; Xu, M.; Xing, B. Sorption of pyrene by regularand nanoscale metal oxide particles: Influence of adsorbed organicmatter. Environ. Sci. Technol. 2008, 42, 7267–7272.

[48] Hyung, H.; Kim, J.H. Natural organic matter (NOM) adsorption tomulti-walled carbon nanotubes; Effect of NOM characteristics andwater quality parameters. Environ. Sci. Technol. 2008, 42, 4416–4421.

[49] Tipping, E.; Higgins, D.C. The effect of adsorbed humic substanceson the colloidal stability of haemitite particles. Coll. Surf. 1982, 5,85–92.

[50] Pettibone, J.M.; Cwietny, D.M.; Scherer, M.; Grassian, V.H. Ad-sorption of organic acids on TiO2 particle aggregation. Langmuir2008, 24, 6659–6667.

[51] Domingos, R.F.; Tufenki, N.; Wilkinson, K.J. Aggregation of tita-nium dioxide nanparticles: Role of fulvic acid. Environ. Sci. Tech-nol. 2009, 43, 1282–1286.

[52] Joo, S.H.; Al-abed, S.R.; Luxton, T. Influence of carboxymethylcellulose for the transport of titanium dioxide nanoparticles in cleansilica and mineral-coated sands. Environ. Sci. Technol. 2009, 43,4954–4959.

[53] Dudev, T.; Lim, C. Effect of carboxylate-binding mode on metalbinding/slectivity and function in proteins. Acc. Chem. Res. 2007,40, 85–93.

[54] Yao, K.M.; Habibian, M.M.; Omelia, C.R. Water and waste waterfiltration: Concepts and applications. Environ. Sci. Technol. 1971,5, 1105–1109.

[55] Peverill, K.I.; Sparrow, L.A.; Reuter, D.J. Soil Analyst: An Intro-duction Manual; CSIRO Publishing, Victoria, Australia, 1995; p.365.

[56] Pulskamp, K.; Diabate, S.; Krug, H.F. Carbon nanotubes show nosign of acute toxicity but induce intracellular reactive oxygen specieson dependence on contaminants. Toxicol. Lett. 2007, 168, 58–74.

[57] Tinkle, S.S.; Antonini, J.M.; Rich, B.A.; Roberts, J.R.; Salmen, R.;DePree, K.; Akkins, E.J. Skin: as a route of exposure and sensiti-zation of chronic beryllium disease. Environ. Health Perspec. 2003,111, 1202–1208.

[58] Bennat, C.; Muller-Goymann, C.C. Skin penetration and stabiliza-tion of formulations containing microfine titanium dioxide as phys-ical UV filter. Int. J. Cosmet. Sci. 2000, 22, 271–283.

[59] Tsujii, J.S.; Mayland, A.D.; Howard, P.C.; James, J.T.; Lam, C.;Warheit, D.B.; Santamariak, A.B. Research stragies for safety eval-

uation of nanomaterials, part IV: risk assessment of nanoparticles.Toxicol. Sci. 2006, 89, 42–50.

[60] Afaq, F.; Abidi, P.; Matin, R.; Rahman, Q. Cytotoxicty, pro-oxidanteffects and antitoxidant depletion in rat lung alveolar macrophageexposed to Ultrafine titanium dioxide. J. Appl. Toxicol. 1998, 18,307–312.

[61] Peters, K.; Unger, R.E.; Kirkpatrick, C.J.; Gatti, A.M.; Monari, E.Effects of nanoscale particles on endothelial cell function in vitro:Studies on viability, proliferation and inflammation. J. Mater. Sci.Mater. Med. 2004, 15, 321–325.

[62] Limbach, L.K.; Wick, P.; Manser, P.; Grass, R.N.; Bruinink, A.;Stark, W.J. Exposure of engineered nanoparticles to human lung ep-ithelial cells: Influence of chemical composition and catalyst activityon oxidative stress. Environ. Sci. Technol. 2007, 41, 4158–4163.

[63] Long, T.C.; Saleh, N.; Tilton, R.D.; Lowry, G.V.; Veronesi, B. Tita-nium dioxide (P25) produces reactive oxygen species in immortal-ized brain microglia (BV2): Implications for nanoparticle neurotox-icity. Environ. Sci. Technol. 2006, 40, 4346–4352.

[64] Montellier, C.; Tran, L.; MacNee, W.; Faux, S.; Jones, A.; Miller,B. The pro-inflammatory effects of low-toxicity surface area parti-cles, on epithelial cells in vitro: the role of low-solubility particles,nanoparticles and fine. Occup. Environ. Med. 2007, 64, 609–615.

[65] Simm, A.; Bromme, H. Reactive oxygen species (ROS) and aging:do we need them—can we measure them-should we block them?Signal Transduct. 2005, 3, 115–125.

[66] Warmer, W.G.; Yin, J.J.; Wei, R.R. Oxidative damage to nucleicacids photosensitized by titanium dioxide. Free Radical Biol. Med.1997, 23, 851–858.

[67] Gurr, J.-R.; Wang, A.S.; Chen, C.-H.; Jan, K.-Y. Ultrafine titaniumdioxide particles in the absence of photoactivation can induce ox-idative damage to human bronchial epithelial cells. Toxicology 2005,213, 66–73.

[68] Brunet, L.; Lyon, D.Y.; Hotze, E.M.; Alvarez, P.J.; Wiesner, M.R.Comparative photoactivity and antibacterial properties of C60

fullerenes and titanium dioxide nanoparticles. Environ. Sci. Tech-nol. 2009, 43, 4356–4360.

[69] Afaq, F.; Abidi, P.; Matin, R.; Rahman, Q.; Cytotoxicity, prooxidanteffects and antioxidant depletion in rat lung alveolar macrophagesexposed to ultrafine titanium dioxide. J. Appl. Toxicol. 1998, 18,307–312.

[70] Renwick, L.C.; Donaldson, K.; Clouter, A. Impairment of alveo-lar macrophage phagocytosis by ultrafine particles. Toxicol. Appl.Pharmacol. 2001, 172, 119–127.

[71] Beck-Speier, I.; Dayal, N.; Karg, E.; Maier, K.L.; Roth, C.; Ziesenis,A.; Heyder, J. Agglomeration of ultrafine particles of elementalcarbon and TiO2 induce generation of lipid mediators in alveolarmacrophages. Environ. Health Perspect. 2001, 109 Suppl 4, 613–618.

[72] Ramires, P.A.; Romito, A.; Cosentino, F.; Milella, E.; The influenceof titania/hydroxyapatite composite coatings on in vitro osteoblastsbehavior. Biomaterials 2001, 22, 1467–1474.

[73] Zhang, A.P.; Sun, Y.P. Photocatalytic killing effect of TiO2 nanopar-ticles on Ls-174-t human colon carcinoma cells. World J. Gastroen-terol 2004, 10, 3191–3193.

[74] Hussain, S.M.; Hess, K.L.; Gearhart, J.M.; Geiss, K.T.; Schlager, J.J.In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol.In Vitro 2005, 19, 975–983.

[75] Reeves, F.J.; Davies, S.J.; Dodd, N.J.F.; Jha, A.N. Hydroxyl radicals(·OH) are associated with titanium dioxide (TiO2) nanoparticles-induced cytotoxicity and oxidative DNA damage in fish cells. MutatRes. 2008, 640, 113–122.

[76] Aruoja, V.; Dubourguier, H.-C.; Kasemets, K.; Kahru, A. Toxicityof CuO, ZnO, and TiO2 to microalgae Pseudokirchneriella subcapi-tata. Sci. Total Environ. 2009, 407, 1461–1468.

[77] Jin, C.-Y.; Zhu, B.-S.; Wang, X.-F.; Lu, Q.-H. Cytotoxicity of ti-tanium dioxide nanoparticles in mouse fibroblast cells. Chem. Res.Toxicol. 2008, 21, 1871–1877.

TiO2 nanoparticles in aquatic environment 1495

[78] Wamer, W.G.; Yin, J.J.; Wei, R.R. Oxidative damage to nucleic acidsphotosensitized by titanium dioxide. Free Radical. Biol. Med. 1997,23, 851–858.

[79] Adams, L.K.; Lyon, D.Y.; Alvarez, P.J.J. Comparative eco-toxicityof nanoscale TiO2 SiO2, and ZnO water suspensions. Water Res.2006, 40, 3527–3532.

[80] Heinlaan, M.; Ivask, A.; Blinova, I.; Dubourguier, H-C.; Kahru,A. Toxicity of nanosized and bulk ZnO, CuO, and TiO2 to bacteriaVibrio fischeri and crustaceans Daphnia magna and Thamnocephalusplatyurus. Chemosphere 2008, 71, 1308–1316.

[81] Hund-Rinke, K.; Simon, M. Ecotoxic effect of photocatalytic activenanoparticles (TiO2) on algae and daphinds. Environ. Sci. Pollut.Res. Int. 2006, 13, 225–232.

[82] Huang, C.P.; Cha, D.K.; Ismat, S.S. Progress report: short-termchronic toxicity of photocatalytic nanoparticles to bacteria,algae, and zooplankton; 2005. EPA Grant number: R831721.

http://cfpub.epa.gov/ncer abstract/index.cfm/fuseaction/display.abstractDetail/abstract/7384/report/O.

[83] Drobne, D.; Jemec, A.; Tkalec, Z.P. In vivo screening to determinehazards on nanoparticles: Nanosized TiO2. Environ. Pollut. 2009,157, 1157–1164.

[84] Baun, A.; Hartmann, N.B.; Grieger, K.; Kusk, K.O. Ecotoxicity ofengineered nanoparticles to aquatic invertebrates: a brief review andrecommendations for future toxicity testing. Ecotoxicology 2008,17, 387–395.

[85] Wang, H.; Wick, R.L.; Xing, B. Toxicity of nanoparticulate andbulk ZnO, Al2O3, and TiO2 to the nematode Caenorhabditis elegans.Enviorn. Pollut. 2009, 157, 1171–1177.

[86] Federici, G.; Shaw, B.J.; Handy, R.D. Toxicity of titanium diox-ide nanoparticles to rainbow trout (Oncorhynchus mykiss): Gill in-jury, oxidative stress, and other physiological effects. Aquat. Toxicol.2007, 84, 415–430.