Ecotoxicology of Nano-TiO – An Evaluation of its Toxicity...

Int. J. Environ. Res., 6(1):33-50, Winter 2012ISSN: 1735-6865

Received 9 March 2011; Revised 12 Sep. 2011; Accepted 19 Sep. 2011

*Corresponding author E-mail: [email protected]

33

Ecotoxicology of Nano-TiO2 – An Evaluation of its Toxicity toOrganisms of Aquatic Ecosystems

Clemente, Z.1,2*, Castro, V. L.2, Jonsson, C. M.2 and Fraceto, L. F.1,3

1 Department of Biochemistry, Institute of Biology, State University of Campinas – UNICAMP,Rua Monteiro Lobato, 255, CEP 13083-862 Campinas, SP, Brazil

2 Laboratory of Ecotoxicology and Biosafety, Embrapa, Rodovia SP340 km 127.5, C.P. 69, CEP13820-000 Jaguariúna, SP, Brazil

3 Department of Environmental Engineering, São Paulo State University – UNESP, Avenida Trêsde Março, 511, CEP 18087-180 Sorocaba, SP, Brazil

ABSTRACT:The production and use of synthetic nanoparticles is growing rapidly, and therefore the presenceof these materials in the environment seems inevitable. Titanium dioxide (TiO2) presents various possibleuses in industry, cosmetics, and even in the treatment of contaminated environments. Studies about thepotential ecotoxicological risks of TiO2 nanoparticles (nano-TiO2) have been published but their results arestill inconclusive. It should be noted that the properties of the diverse nano-TiO2 must be considered in orderto establish experimental models to study their toxicity to environmentally relevant species. Moreover, thelack of descriptions and characterization of nanoparticles, as well as differences in the experimental conditionsemployed, have been a compromising factor in the comparison of results obtained in various studies. Therefore,the purpose of this paper is to make a simple review of the principal properties of TiO2, especially innanoparticulate form, which should be considered in aquatic toxicology studies, and a compilation of theworks that have been published on the subject.

Key words: Nano-TiO2, Nanotechnology, Ecotoxicology, Water, Aquatic organisms

INTRODUCTIONNanotechnology is a rapidly expanding area of

research which already has a wide variety ofcommercially available products. The material mostcommonly utilized in nanoproducts is silver, followedby carbon, titanium, silicon, zinc and gold (Meyer etal., 2009, Project on Emerging Nanotechnologies, 2009).An initial estimate indicates that nanotechnology maylead to a revolution in the development and fabricationof products that could contribute with up to one trilliondollars to the global economy by 2015 (Roco, 2001).Nanomaterials have dimensions of less than 100nanometers (nm), while nano-objects have dimensionssmaller than 100nm and nanoparticles (NPs) have threedimensions with less than 100 nm (Stone et al., 2010).However, the literature often describes NPs as particlesthat possess at least one dimension in the order of 1 to100 nanometers (nm). The Royal Society of Chemistrysuggests that 100 nm is the cut-off point above whichparticles will not enter cells through receptor-mediatedprocesses (RSCRAE, 2005), and some experimentalevidence has emerged that corroborates this dimension

(Chithrani and Chan, 2007, Clift et al., 2008). Anotherimportant cut-off dimension is particles smaller than40 nm, which can enter the nucleus, while particlessmaller than 35 nm can, potentially, cross protectivebarriers such as the hematoencephalic barrier(Oberdorster et al., 2004). However, these valuesshould serve as guidelines, since the real size to beconsidered depends on other factors of the materialand on details of its surface.

Titanium dioxide (TiO2) has been usedcommercially since 1900, particularly in coatings andpigments. In 2002, the production capacity of thisoxide was estimated at 4.6 million tons (Winkler, 2003).A review published by the United StatesEnvironmental Protection Agency (USEPA) estimatedthe annual production of TiO2 nanoparticles (nano-TiO2) to be 2000 metric tons in around 2005, with 65%of this production used in products such as cosmeticsand sunscreen lotions (USEPA, 2009). The growinguse of NPs generates effluents or wastewaters, raisingconcerns about the environmental risks and impacts

34

Clemente, Z. et al.

of nanotechnology. Due to the wide utilization andpromising uses that have emerged from nano-TiO2, thismaterial has been the target of several ecotoxicologystudies. Based on a compilation of publishes worksthat evaluate the toxicity of nano-TiO2 to aquaticorganisms, the article reviews the main properties ofTiO2, especially in nanoparticulate form, which shouldbe considered in aquatic toxicology studies.

In nature, TiO2 occurs only in the form of oxide oroxides mixed with other elements. Mineral deposits areusually of volcanic origin, but are also found in beachsand (Winkler, 2003). TiO2 can be found in threecrystalline forms: anatase (tetragonal), rutile (tetragonal)and brookite (orthorhombic), and its main reserves arelocated in Canada, the US, Scandinavia, South Africa,the Mediterranean Sea, and Australia (Titaniumart,2010). Titanium dioxide, also known as titanium oxide(IV) or titania (molecular weight 79.88), is insoluble inwater, chloric acid, nitric acid and ethanol, but is solublein concentrated and heated sulfuric, hydrogen fluorideand alkaline media (NRC, 1999).

TiO2 is obtained mainly from ore containingilmenite (FeTiO2), natural rutile (TiO2) and leucoxene-like ilmenite. TiO2 particles are referred to as primary,aggregates or agglomerates. Primary particles areindividual crystals bound by crystal planes.Aggregates are sintered primary particles connectedby their crystal faces. Agglomerates are multipleprimary particles and aggregates that are joinedtogether by van der Waal forces (IARC, 2010). Primaryparticles typically have a diameter of 0.2 to 0.3 µm,although larger aggregates are also formed (furtherdetails about bulk TiO2 are given in Diebold, 2003).Several TiO2 NPs are produced today (Xiaobo, 2009),with variations in particle size, surface area, purity (dueto doping, coating or quality control), surfacecharacteristics, crystalline shape, chemical reactivityand other properties. One of the main differencesbetween bulk TiO2 and nano-TiO2 is the larger surfacearea of a given mass or volume of NPs compared to anequivalent mass or volume of bulk TiO2 particles (Shaoand Schlossman, 1999).. Approximately 35-40% ofatoms are located on the surface of a 10 nm NPcompared with less than 20% on particles larger than30 nm. This higher surface area reinforces severalproperties, such as photocatalytic activity andultraviolet absorption at given wavelengths (Shao andSchlossman, 1999). Bulk TiO2 absorbs ultravioletradiation (<400nm). Because of its high refractive index,it is also very effective in dispersing radiation. Bothdispersion and absorption are important in theattenuation of ultraviolet radiation (UV), making it aneffective ingredient in sunscreen lotions (USEPA,2009). Small primary particles are less able to dispersevisible light and are more transparent, while larger size

particles are more opaque. Hence, sunscreenformulations containing nano-TiO2 have becomepopular due to their greater transparency on the skincompared to the white appearance of formulationscontaining bulk TiO2.

The theoretical calculations of Palmer et al. (1990)and experimental data of Sakamoto et al. (1995) showedthat the UVB attenuation of submicrometric TiO2particles is predominantly due to their absorption, whileUVA attenuation is essentially due to their dispersion.The findings of Shao and Schlossman (1999) contributeto the idea that smaller particle sizes, and hence largerspecific surface areas, are better for UVB attenuation.In contrast, the intensity of UVA dispersion is greaterthe larger the particle size (Shao and Schlossman, 1999).TiO2 is a semiconductor, i.e., a crystalline solid whoseelectrical conductivity is intermediate between that ofconductors and insulators. Thus, an importantapplication of this material is in the electronics industryand in processes of heterogeneous photocatalysis.

The principle of heterogeneous photocatalysisinvolves the activation of a semiconductor by solar orartificial radiation. A semiconductor is characterizedby two energy regions: the region of lower energy isthe valence band (EV), where the electrons cannotmove freely, and the higher region is the conductionband (EC), where the electrons move freely throughthe crystal, producing electrical conductivity similarto that of metals. These two regions are divided by a“band-gap” zone. Fig. 1 shows a schematicrepresentation of a semiconductor particle. Theabsorption of photons with energy higher than theband-gap energy (EG) causes the promotion of anelectron from the EV to the EC, with the concomitantgeneration of a gap (h+) in the EV. In the absence ofsuitable scavengers species, the stored energy isdissipated within milliseconds by recombination, withthe formation of an unpaired electron. If a suitablescavenger or a surface defect is available to containthe electron or gap, recombination is prevented and redoxreactions occur subsequently. EV gaps are potentoxidants (potential of +1.0 to +3.5 V, depending on thesemiconductor and pH) that are able to generate radicalspecies (HO•, O2•, HO2•, etc.) from water moleculesadsorbed on the semiconductor surface, which cansubsequently oxidize other molecules (Nogueira andJardim, 1998, Gaya and Abdullah, 2008, Malato et al.,2009). There are indications that the reaction occursonly in the adsorbed phase of the semiconductingparticle, hence, organic molecules that can effectivelyadhere to the surface of the photocatalyst are moresusceptible to direct oxidation (Gaya and Abdullah, 2008).

The minimum EG required for a photon to causethe photogeneration of charged species in TiO2

Int. J. Environ. Res., 6(1):33-50, Winter 2012

35

(anatase form) is 3.2 eV, which corresponds to awavelength of 388 nm. In fact, the photoactivation ofTiO2 occurs in the range of 300-388nm (Nogueira andJardim, 1998, Gaya and Abdullah, 2008). Thus, thestrong resistance of TiO2 to decomposition andphotocorrosion, its low cost, and the possibility ofusing solar UV radiation, makes it particularlyinteresting for processes of heterogeneousphotocatalysis (Malato et al., 2009).

Many studies have demonstrated the potential useof heterogeneous photocatalysis with TiO2 for thedegradation of organic and inorganic compounds(Chatterjee and Dasgupta, 2005, Fujishima and Zhang,2006). For the most part, photodegradation leads to thetotal mineralization of pollutants, generating CO2, H2Oand inorganic acids (Malato et al., 2009). This propertyis applicable in the production of self-cleaning surfaces,cleaning products, in the remediation of contaminatedsoil and water, or even the deodorization of environmentsand the destruction of gas-phase volatile compounds.The hydroxyl radicals generated during TiO2 irradiationare also able to react with most biological molecules,resulting in bactericidal and virucidal activity (Nogueiraand Jardim, 1998, Li et al., 2008).

Studies suggest that anatase and rutile havedifferent photocatalytic properties, with anatasepossessing the better combination of photoactivityand photostability (Gaya and Abdullah, 2008, USEPA,2009). The rutile form is inactive for thephotodegradation of organic compounds, although thereason for this is not completely clear (Nogueira andJardim, 1998, Malato et al., 2009). However, the low

Fig. 1. Schematic representation of a TiO2 particle, where EV and EC are the Valence Band and ConductionBand, respectively (adapted from Nogueira and Jardim, 1998)

adsorption capacity of O2 on its surface is pointed outas one of the possible factors.

Among the different titanium oxide products, TiO2P25 fabricated by Evonik Degussa Corp. (Germany) isthe one most commonly used because of its reasonablywell defined nature (typically a mixture of 70:30anatase:rutile, nonporous, surface area of about 50 m2/g, and average particle size of 30 nm) and its highphotoactivity when compared to that of other sources(Nogueira and Jardim, 1998, Malato et al., 2009).

Surface treatment of nano-TiO2 can alter its lightabsorption and photocatalytic activity. In applicationssuch as paints, coatings and cosmetics, which requirechemical stability, the photocatalytic properties of TiO2are generally suppressed by coatings it with silica andaluminum layers (Diebold, 2003, Li et al., 2008). Dopingof nanostructured TiO2 materials has also often beenemployed to modify its band-gap energy and increase itsphotocatalytic activity.TiO2 is generally used insuspension (also called slurry), but can also be usedimmobilized in an inert matrix coating surfaces (Gelover etal., 2006, Gaya and Abdullah, 2008, Malato et al., 2009).

Immobilized TiO2 has been reported to have lowcatalytic activity when compared to systems insuspension (Gaya and Abdullah, 2008, Malato et al.,2009). The mineralization rate generally increases withthe concentration of the catalyst up to a limit of highconcentration. Wei et al. (1994) used P25 for thedisinfection of E. coli in water and reported that thedisinfection rate depended mainly on two variables:the intensity of incident light and the TiO2 dose.

36

Ecotoxicology of Nano-TIO2

In general, for any photocatalytic application,the optimal concentration should be determined inorder to avoid an excess of catalyst and to ensure thetotal absorption of photons, i.e., to ensure the entireexposed surface of the particles is illuminated. Whenthe concentration of TiO2 is too high, the turbidityprevents radiation from penetrating and reaching allthe particles (Herrmann, 1999). In photocatalysisstudies, the optimal of TiO2 have been a temperatureof 20 to 80oC, a concentration of 200-500 mg/L, oxygenconcentration of pO2 ≥ 0.21 atm and pH preventingpHzpc (Malato et al., 2009).

NPs tend to aggregate in the environment and cantherefore be eliminated or captured by sedimentation.NP aggregates are generally less mobile and can interactwith filtering organisms and with organisms that feedon sediment, or even with suspended organic matter. Itis therefore important to understand the behavior ofTiO2 NPs in aquatic environments in order to understandtheir toxicology. The pH, ionic concentration and natureof the electrolytes in aqueous suspensions have beenreported as important parameters in the aggregation ofnano-TiO2 (Sharma, 2009).

The pH of aqueous solutions significantly affectsTiO2, including the particle charge, the size of aggregatesand the position of the EC and EV. The pH at which thesurface of an oxide has no electrical charge is defined asthe zero point charge (pHzpc). The pHzpc of nano-TiO2varies from 4.5 to 7, depending on the particle’s size andcrystal shape, with smaller particles presenting lowerpHzpc (Kosmulski, 2002 cited by Sharma, 2009). Finneganet al. (2007) reports pHzpc values of ~5.9 for rutile and of~6.3 for anatase. A pHzpc of 6.3 has been reported forDegussa P25 (Kosmulski, 2009).

The surface of titanium will remain positivelycharged in an acid medium and negatively charged inan alkaline medium (Gaya and Abdullah, 2008). Thelack of surface charge renders an electrostatic potentialnull, because it does not produce the repulsiveinteraction needed to separate the particles in the liquid.Therefore, TiO2 particles tend to aggregate close tothe pHzpc.

Particle aggregation interferes in the ability of thesuspension to transmit or absorb radiation. However,this variation in particle size may be an advantage whenthe objective is to separate TiO2 from water (bysedimentation and/or filtration) at the end of aphotocatalytic treatment (Malato et al., 2009).

Like other NPs, nano-TiO2 can bind to organicmatter, thus modifying its properties and behavior. Theadsorption of acid fulvic and humic acid on nano-TiO2has proved to be pH-dependent and favors the

dispersion and suspension of these particles in aquaticenvironments (Domingos et al., 2008, Yang et al., 2009).On the other hand, the adsorption of oxalic acidappears to destabilize nano-TiO2 suspensions,increasing the sedimentation rate at pH 2, although nochange in the sedimentation rate has been observedat pH 6.5 (Pettibone, 2008).

The adsorption of organic matter on nano-TiO2may also alter the adsorption of toxic compounds(Sharma, 2009). Nano-TiO2 has been reported to showadsorption behavior towards metals such as Cu(II),Cr(III), Mn(II), Ni(II), Zn(II), Cd(II), Mo(VI) (Kaur andGupta, 2009). When an aqueous suspension of bacteriaand other microorganisms is in the presence of TiO2 inthe dark, a slight reduction in the concentration ofcolonies can be observed due to the possibleagglomeration of TiO2 with the bacterial cells andsubsequent sedimentation (Malato et al., 2009).

STUDIES OF THE AQUATIC ECOTOXICOLOGYOF TIO2

NPs differ from bulk particles in terms of theirheterogeneous size distribution, surface charge,composition, degree of dispersion, etc. Therefore, in atoxicology study, it is important to determine not onlytheir exposure concentration but also other measures(Hasselov et al., 2008). At the NanoImpactNetWorkshop held in 2008, a list was proposed of the sixprincipal characteristics of nanomaterials to bediscriminated in environmental studies: size,dissolution/solubility, surface area, surface charge andsurface chemical composition. Information such as sizedistr ibution, crystal structure, morphology,agglomeration/dispersion, etc. may also be important(Stone et al., 2010). Nonetheless, the authors recognizethat the characterization of nanomaterials may be time-consuming and costly, as well as complex, andtherefore its application should depend on theobjectives of the study (Stone et al., 2010). It was alsoagreed that the properties should be characterized intest systems and not in the “bottles” that are supplied,and that certain properties such as agglomeration anddissolution should be listed as “rates” rather than“states” in view of the dynamic nature ofnanoparticulate systems.

Unfortunately, methods to measure all theproperties are not available. For example, there is stillno method available to measure the surface area in anaqueous dispersion of NPs. Moreover, there is still apaucity of information about the extent to which thelimitations of the different methods may influence thecorrect interpretation of results. The bias of a techniquecan be reduced by using multiple techniques, althoughthis is difficult due to time and cost constraints (Stone

37


et al., 2010). Hasselöv et al.’s paper (2008) presentsinformation about the main methods available for thecharacterization of NPs.

The fact that TiO2 is highly insoluble, non-reactivewith other materials, thermally stable, and non-flammable enabled it to be declared innocuous to theorganism (WHO, 1969). However, studies havedemonstrated an apparently species specificity in thegeneration of lung tumors in rats that inhaled TiO2 forlong periods (Hext et al., 2005). In addition, othersignificant data in the literature confirm the occurrenceof lung inflammation, oxidative stress and involvementof other organs after respiratory and oral exposure tonano-TiO2 (Ferin et al., 1992, Wang et al., 2007, Warheitet al., 2007a). Recently, the International Agency forResearch on Cancer (IARC) classified TiO2 as“possibly carcinogenic for humans” (IARC, 2010).

The various possible sources of contamination ofwater bodies by nano-TiO2 make it essential to assessits effects on ecosystems, i.e., its ecological, publichealth and economic consequences. There is still apaucity of studies about the presence of nano-TiO2 inthe environment. Natural TiO2 NPs have been found inriver water (Wigginton et al., 2007). In Switzerland, dueto the climatic conditions, researchers reported nano-TiO2 particles peeling off painted façades and beingcarried into surface waters, Ti concentrations of about16 µg/L were found in urban runoff (Kaegi et al., 2008).

Nanoecotoxicology studies are relativelyrecent, the first publication involving an assay withfishes dated 2004 (Orberdorster, 2004). Tables 1 to 3summarize published works about the effects of TiO2NPs on aquatic organisms.

With regard to the bioavailability of nano-TiO2 toaquatic organisms, the literature is still inconclusive.In a recent paper, Johnston et al. (2010) did not observesignificant absorption of nano-TiO2 in Oncorhynchusmykiss exposed for 10 days to concentrations of up to5 mg/L. Federici et al. (2007) also did not findaccumulation of nano-TiO2 in O. mykiss exposed for14 days to concentrations of up to 1 mg/L. On theother hand, some studies report that the nano-TiO2present in water may accumulate in Cyprinus carpio,Danio rerio and Daphnia magna, even atconcentrations of 0.1 and 1 mg/L, although low factorsof bioconcentration were determined (Zhang et al., 2006,Zhu et al., 2010a, b). Zhu et al. (2010a) report theoccurrence of trophic transfer of nano-TiO2 in D. reriofed with contaminated daphnids, but discard thepossibility of biomagnification. Other studies haveshown that the presence of nano-TiO2 may elevate theabsorption of other contaminants in fishes, such asAs and Cd (Sun et al., 2007, 2009, Zhang et al., 2007).

The results of toxicity tests have usually beenexpressed as lethal (LC50), effective or inhibitory (EC50)concentrations that cause, respectively, mortality,abnormality of inhibition to 50% of the exposedorganisms. A wide variability has been found in theresults reported in the literature with regard to toxicitytests. This variability may be due to the differentcharacteristics of nano-TiO2 and treatments applied,as well as to experimental designs. Thus, exhaustivediscussion has focused on the need for the propercharacterization of NPs under study, and for thestandardization of nanoecotoxicological evaluationmethods. The lack of information in some works makesit difficult to compare results (Warheit et al., 2008).Discussions have also focused on the lack of analyticaltechniques for the characterization of NPs in the mediautilized for ecotoxicological assays.

Lovern and Kapler (2006) reported an LC50 of 5.5ppm in D. magna exposed for 48 h to filtered nano-TiO2, but did not observe mortality or behavioralabnormalities after exposure for the same period toconcentrations of up to 500 ppm of the same nano-TiO2, although the suspension was sonicated.Although several authors considered acute exposureto nano-TiO2 of low toxicity to Daphnia (Warheit etal., 2007b, Griffith et al., 2008, Heinlaan et al., 2008, Leeet al., 2009, Strigul et al., 2009, Wiench et al., 2009, Kimet al., 2010, Rosenkranz, 2010), prolonged exposurehas presented varied results. The exposure of D.magna to Degussa P25 (sonicated) for 21 days showeda LC50 of 2.62mg/L and alteration of the reproductionand growth rates (EC50 0.46 mg/L) (Zhu et al., 2010b),while exposure for the same period to different typesof BASF nano-TiO2 (sonicated) did not cause mortalitybut reduced the reproductive capacity (EC50 26.6 mg/L) (Wiench et al., 2009). Kim et al. (2010) did not findreproductive impairment but reported a 70% mortalityrate in D. magna exposed for 21 days to 5 mg/L ofSigma Aldrich nano-TiO2.

Some studies appear to suggest that nano-TiO2has low acute toxicity for fishes, and LC50 is indicatedas 124,5 mg/L for D. rerio (Xiong et al., 2011) and >100mg/L for O. mykiss (Warheit et al., 2007b). Similarly,the exposure of D. rerio eggs to nano-TiO2 for 96 hoursat concentrations of up to 500 mg/L did not causealterations in the survival and hatching rates, ormalformations (Zhu et al., 2008). The exposure ofembryos of Pimephales promelas to concentrationsof up to 1mg/L for 7 days also caused no significantmortality or observable malformations (Jovanovic etal., 2011). On the other hand, some studies have shownthat the prolonged exposure of fish to concentrationsof 1 to 200 mg/L did not cause mortality, but observeddose-dependent elevation of the respiratory rate and

38

swimming behavior, as well as increased production ofmucus (Federici et al., 2007, Hao et al., 2009).

Evidence of adverse effects of a given contaminantat sublethal concentrations is extremely important inenvironmental risk assessment, since it may generate acascade effect with consequences at the level ofindividuals, communities and the ecosystem. Thus, theuse of biomarkers in risk assessments offers theadvantage of allowing for the detection of potentiallytoxic exposure well before real adverse effects occur(Nascimento et al., 2008, Prospéri and Nascimento, 2008).

Studies have shown that the toxicity of somenanomaterials such as TiO2 may be implicated in thegeneration of reactive oxygen species (ROS) (Kahruand Dubourguier, 2009, Pelka et al., 2009, Sharma etal., 2009). ROS can react with the majority ofbiomolecules and damage lipids, proteins and nucleicacids (Valavanidis et al., 2006).

Exposure in aqueous media appears to be moresevere than via the diet for O. mykiss (Handy et al.,2008). The prolonged exposure of fish to nano-TiO2induced biochemical and histopathological alterationsin their gills, liver and intestines (Federici et al., 2007,Hao et al., 2009, Johnston et al., 2010, Palaniappan andPramod, 2010). Exposure to nano-TiO2 can triggeroxidative stress in D. magna, fishes and mollusks(Federici et al., 2007, Hao et al., 2009, Canesi et al.,2010a, Kim et al., 2010, Xiong et al., 2011). Lysosomalinstability has also been reported in polychaetes andmollusks exposed to nano-TiO2 (Canesi et al., 2010a,Galloway et al., 2010). The intravenous administrationof high doses of nano-TiO2 in fish has shown that itaccumulated in the kidneys, with slow depuration, butno significant alterations were observed in the functionof this organ (Scown et al., 2009). An experiment withD. magna showed that even after a period of 72 hoursin clean water, the depuration of adsorbed TiO2 wasnot complete (Zhu et al., 2010b).

With regard to genotoxicity in aquatic organisms,nano-TiO2 presents controversial results. Nano-TiO2has presented genotoxicity in some studies (Griffith etal., 2009, Galloway et al., 2010, Jovanovic et al., 2011)but not in others (Lee et al., 2009). Griffith et al. (2009)reported that exposure to nano-TiO2 altered theexpression of 171 genes in D. rerio involved mainly inribosome structure and activities, but not in theregulation of oxidative stress. Jovanovic et al. (2011)also observed upregulation of genes involved ininflammatory response (especially in phagocyticprocesses), and suppression of neutrophil function infish that received an intraperitoneal dose of nano-TiO2.The immune system also appears to be an importanttarget of TiO2 NPs in bivalves (Canesi et al., 2010b).

In bioassays with aquatic organisms, the circadiancycle is usually established using fluorescent lamps.These lamps emit basically visible light, while in naturalconditions these organisms are exposed to solarradiation (infrared, visible and ultraviolet light). Thereis ample evidence of the formation of reactive oxygenspecies when TiO2 is exposed to UV radiation (Brezováet al., 2005). Several studies have reported thephototoxic effects of TiO2 normal or NPs), and itsconsequent use in the disinfection of water (Wei et al.,1994, Carp et al., 2004, Adams et al., 2006). Thephotocatalytic properties of nano-TiO2 can augmentits toxic effects in aquatic organisms underenvironmental conditions, but few studies so far havetaken this into consideration. In vitro studies haveshown that co-exposure to nano-TiO2 and ultravioletradiation increases cyto- and genotoxicity in fish cells(Reeves et al., 2008, Vevers and Jha, 2008). The pre-and co-illumination of nano-TiO2 has also been shownto elevate its toxicity in daphnids (Hund and Rinke,2006, Marcone et al., 2010).

There are still uncertainties about thecharacterization of exposure to nanoparticles in thetesting systems of all ecotoxicity assays except thosethat involve the oral administration of nanoparticles.These uncertainties include how the substance isdosed and maintained in the test medium, themeasurement and characterization of NPs in the testsystem, the understanding of the abiotic factors thatinfluence the behavior of NPs in the test system, and aconsensus about the dosimetry (Crane et al., 2008).

Today there are several guidelines for conductingecotoxicological assays (OECD, USEPA, DINStandards, etc.). However, their use fornanoecotoxicological assays is still under question(Stone et al., 2010). The use of these methodologiesmust be evaluated for each type of nanoparticle. Testingwith nano-TiO2 presents various particularities, suchas its photocatalytic properties and absorption of UVradiation, its aggregation and sedimentation behaviorin water and its interaction with organic matter.Performing assays to determine lethal and effectiveconcentrations in the proposed ranges of concentrationis particularly difficult. The OECD, for example,suggests finding the CL50 up to the concentration of100mg/L, however, nano-TiO2 forms a whitishsuspension when dissolved in water, and inconcentrations equal to or higher than 10mg/L, itprecipitates rapidly if no dispersion method is used.Wiench et al. (2009) found that TiO2 does not dispersewell at 10-100 mg/L and that sedimentation occurswithin 24-48 hours. For uncoated TiO2 (BASF, >99%,70% anatase, 30% rutile, 20-30nm, 48.6m2/g), the

Clemente, Z. et al.

39

Tabl

e 1. S

umm

ary

of p

aper

s pub

lishe

d ab

out t

he ef

fect

s of n

ano-

TiO

2 use

d in

toxi

colo

gy st

udie

s on

mic

rocr

usta

cean

s (C

ontin

ues)

Test

spec

ies

Pro

duct

test

ed

Tre

atm

ent o

f the

pro

duct

Ph

ysic

oche

mic

al

char

acte

rizat

ion

B

ioas

say

Res

ults

D. m

agna

(K

im e

t al.,

201

0)

Sigm

a A

ldric

h na

no-T

iO2

(40

nm,

30%

rutil

e, 7

0% a

nata

se)

10%

solu

tion

in w

ater

with

pH

2

(with

out s

onic

atio

n) ?

sto

ck

solu

tion

(1m

g/L

) in

mod

erat

ely

hard

syn

thet

ic w

ater

(M

HW

).

N4

and

DL

S su

bmic

ron

parti

cle

anal

yzer

. A

cute

ass

ay 4

8h. W

ithou

t fe

edin

g du

ring

the

test

. U

SEPA

199

3.

Chr

onic

ass

ay, s

emi-

stat

ic, 2

1 da

ys. R

enew

al o

f m

ediu

m a

nd d

aily

feed

ing.

C

once

ntra

tion

s te

sted

: 0, 1

, 2, 5

, 10

mg/

L.

Eva

luat

ions

wer

e m

ade

of S

OD

, GP

X, C

AT

and

GST

ac

tivity

in g

roup

s exp

osed

for

5 da

ys to

0, 0

.5, 1

, 2.5

, 5,

and

10

mg/

L o

f Ti

O2.

GPX

and

GST

wer

e al

so

test

ed a

fter

frac

tion

atio

n of

the

nano

part

icle

s (<

200,

<4

00, a

nd <

800

nm)

Acu

te a

ssay

: mor

talit

y di

d no

t rea

ch 5

0% e

ven

at 1

0mg/

l, so

the

LC 5

0 co

uld

not

be d

eter

min

ed.

Chro

nic

assa

y: h

ighe

st m

orta

lity

at 5

and

10m

g/l (

70 a

nd 8

0%,

resp

ectiv

ely)

. No

repr

oduc

tive

impa

irm

ent o

bser

ved.

Inc

reas

e in

CA

T at

10

mg/

l, no

diff

eren

ce in

SO

D, G

Px h

ighe

st at

5m

g/l,

GST

incr

ease

d at

5

and

10 m

g/l.

TiO

2 was

foun

d in

the

inte

stin

es o

f da

phni

ds a

nd g

lued

to

thei

r an

tenn

ae a

nd e

xter

nal s

urfa

ce.

D. m

agna

(R

osen

kran

z, 2

010)

Deg

ussa

P25

nan

o-Ti

O2

100m

g/L

solu

tion

was

pre

pare

d in

cu

lture

med

ium

for

daph

nids

?

soni

catio

n (3

0 m

in).

The

rem

aini

ng s

olut

ions

wer

e m

ade

from

ser

ial d

ilutio

ns o

f 1:1

0.

INA

A

cute

ass

ay 4

8h. N

o fo

od d

urin

g th

e te

st. 1

00, 1

0, 1

an

d 0.

1 m

g/L

. C

hron

ic a

ssay

21

days

. Med

ium

cha

nged

dai

ly. D

aily

fe

edin

g. C

once

ntra

tion

s: 0

.001

, 0.1

and

1 m

g/L

Acu

te a

ssay

48h

: 10%

mor

talit

y at

100

mg/

L. H

igh

mol

t fre

quen

cy,

dose

-dep

ende

nt.

Chro

nic

assa

y: h

igh

mol

t fre

quen

cy o

nly

on th

e fir

st d

ay o

f exp

osur

e, a

t 1m

g/L

.

D. m

agna

(Z

hu e

t al.,

201

0b)

Deg

ussa

P25

nan

o-Ti

O2 (

21nm

, 50

m2 /g

, 20%

rut

ile, 8

0% a

nata

se)

Size

of

agg

rega

tes

in c

ultu

re m

ediu

m:

1h -

580

.5 n

m;

12

h –

2349

.0 n

m

24

h –

3528

.6 n

m

Stoc

k so

lutio

n (1

g/l)

in u

ltrap

ure

wat

er ?

son

icat

ion

(10

min

, 50

W/L

, 40k

Hz)

? n

ew so

nica

tion

(1

0 m

in, 5

0W/L

, 40k

Hz)

prio

r to

dilu

tion

in c

ultu

re m

ediu

m fo

r da

phni

ds.

SEM

, DL

S

ICP-

OE

S (c

once

ntra

tion

of

Ti i

n th

e sol

utio

n an

d in

da

phni

ds).

Acu

te a

ssay

72h

sem

i-sta

tic. O

ECD

202

. Med

ium

re

new

ed d

aily

. No

food

dur

ing

the

test

. C

once

ntra

tion

s te

sted

: 0.1

, 0.5

, 1, 5

, 10,

50

and

100

mg/

L.

Chr

onic

ass

ay 2

1 da

ys s

emi-s

tatic

. OE

CD

211

. Dai

ly

rene

wal

of

med

ium

and

dai

ly fe

edin

g. C

once

ntra

tions

te

sted

: 0.

1, 0

.5, 1

and

5 m

g/L

. B

ioac

cum

ulat

ion

and

depu

ratio

n te

st 2

4h o

f ac

cum

ulat

ion

(sam

ples

wer

e co

llect

ed a

t 0, 2

, 6, 1

2 an

d 24

h) a

nd 7

2h o

f dep

urat

ion

(sam

ples

wer

e co

llect

ed a

t 6, 1

2, 2

4, 4

8 an

d 72

h). C

once

ntra

tions

te

sted

: 0.

1 an

d 1

mg/

L w

ith a

nd w

ithou

t dai

ly

feed

ing.

Acu

te a

ssay

In

48h

:

NO

EC <

50m

g/L

,

E

C50>

100

mg/

L,

LC 5

0 > 1

00m

g/L.

In

72

h:

NO

EC<0

.1 m

g/L

;

EC

50 =

1.6

2 m

g/L

;

L

C50 =

2.02

mg/

L.

Chro

nic

assa

y

At 0

.1 m

g/L

repr

oduc

tion

decl

ined

. At 0

.5mg

/l re

prod

uctio

n an

d gr

owth

w

ere

inhi

bite

d. M

orta

lity

was

reco

rded

in g

roup

s 1

and

5 m

g/L

afte

r 8

days

of e

xpos

ure.

EC50

= 0

.46m

g/L,

LC

50 =

2.6

2mg/

L.

Th

e fe

edin

g ra

te d

ecre

ased

as

the

expo

sure

con

cent

ratio

n in

crea

sed

(exp

osur

e of

5h)

.

B

ioac

cum

ulat

ion

test

G

roup

0.1

mg/

L:

Conc

entr

atio

n pl

atea

u in

12

h, B

CF=

5.6

6x 1

04 , ti

me

elap

sed

to

accu

mul

ate

50%

of t

he s

atur

atio

n le

vel =

3.8

7h, t

ime

to r

each

50%

de

pura

tion

= 2

6.76

h.

G

roup

1m

g/L

: Pl

atea

u in

24h

; BC

F =

1.18

x105 , t

ime

elap

sed

to a

ccum

ulat

e 50

% o

f the

sa

tura

tion

leve

l = 3

.72h

, tim

e to

rea

ch 5

0% d

epur

atio

n =

74.5

2h.

D

epur

atio

n w

as n

ot c

ompl

ete,

20%

of

the

satu

ratio

n co

ncen

tratio

n re

mai

ned

in th

e da

phni

ds a

t the

end

of t

he e

xper

imen

t.

Fe

edin

g du

ring

exp

osur

e to

TiO

2 in

crea

sed

the

accu

mul

atio

n tim

e an

d re

duce

d th

e de

pura

tion t

ime.


40

D. m

agna

and

C

hiro

nom

us r

ipar

ius

(larv

ae)

(Lee

et a

l., 2

009)

Sigm

a A

ldri

ch n

ano-

TiO

2 7n

m

(300

.81m

2 /g) a

nd 2

0 nm

(6

6.60

4m2 /

g)

Solu

tion

(1m

g/L)

in c

ultu

re m

ediu

m

? s

onic

atio

n (1

5 m

in).

TE

M, B

ET

Acu

te a

ssay

96h

. OEC

D 1

984,

199

8. C

once

ntra

tion

teste

d: 1

mg/

l. N

o ge

noto

xici

ty (c

omet

ass

ay),

alte

ratio

n in

gro

wth

, mor

talit

y or

re

prod

uctio

n w

ere

obse

rved

in a

ny g

roup

.

D. m

agna

(S

trig

ul e

t al.,

200

9)

nano

-TiO

2 pre

pare

d by

hyd

roly

sis o

f th

e tit

aniu

m su

lfat

e so

luti

on (

6 nm

, ag

glom

erat

es 0

.5 -2

mm

)

Stoc

k so

luti

on ?

son

icat

ion

(30m

in).

D

LS

Acu

te a

ssay

24

and

48h.

OEC

D 2

02. C

once

ntra

tions

te

sted:

2.5

; 8; 2

5; 8

0; 2

50 m

g/L.

T

iO2

pres

ente

d lo

w to

xici

ty a

nd L

C 50

coul

d no

t be

cal

cula

ted.

Ani

mal

s ex

pose

d to

80

and

250m

g/L

for

24h

wer

e sl

ower

.

Dap

hnia

mag

na

(Wie

nch

et a

l., 2

009)

Bu

lk T

iO2

BA

SF

nano

-TiO

2:

- non

-coa

ted

(>99

%; 7

0/30

an

atas

e/ru

tile;

20-

30nm

; 48.

6m2 /g

) - T

-LIT

E S

F (8

0%, 5

0nm

; 100

m2 /g

; ru

tile

) - T

-LIT

E S

F-S

- T

-LIT

E S

F-M

AX

It

was

fou

nd th

at 1

0-10

0 m

g/L

did

not

disp

erse

wel

l and

sed

imen

tatio

n oc

curr

ed in

24-

48h.

For

non

-coa

ted

TiO

2, af

ter 1

6h in

bid

istil

led

wat

er, t

he

conc

entra

tion

in th

e su

pern

atan

t wen

t fro

m 1

00 to

83m

g/L

and

to 3

3 m

g/L

in

surfa

ce w

ater

. For

coa

ted

TiO

2, ag

glom

erat

ion

and

sedi

men

tatio

n w

ere

slow

.

Stoc

k so

luti

on (

100m

g/L)

in

dem

iner

aliz

ed w

ater

? s

onic

atio

n (5

min

) or

mag

netic

agi

tatio

n (1

0 m

in)

or b

oth

met

hods

? d

ispe

rsio

n in

M4

or S

W m

ediu

m (

natu

ral

surfa

ce w

ater

) ?

son

icatio

n an

d fil

trat

ion

(2um

) ? U

V ir

radi

atio

n (3

0 m

in 2

0W/m

2 ).

TEM

, ultr

acen

trifu

gatio

n.

Acu

te a

ssay

48h

. OEC

D 2

02.

Chr

onic

ass

ay 2

1 da

ys. O

EC

D 2

11. O

nly

with

T-L

ite

SF-

S –s

emi-

stat

ic a

ssay

(m

ediu

m c

hang

ed 3

tim

es p

er

wee

k). D

aily

feed

ing.

Con

cent

ratio

ns te

sted

: 0.0

1 a

100

mg/

L.

Acu

te a

ssay

E

C50

>100

mg/

L in

at t

he tr

eatm

ents

EC

10 n

on-c

oate

d na

no-T

iO2 s

onic

ated

in M

4 =

85.1

mg/

L. I

n SW

= 3.

7mg/

L.

Bul

k T

iO2

soni

cate

d in

M4=

91m

g/L

; in

SW =

13.

8mg/

L.

Chr

onic

ass

ay

The

re w

as n

o m

orta

lity,

but

rep

rodu

ctiv

e ef

fect

s w

ere

obse

rved

. N

OEC

=3m

g/L

LO

EC=1

0mg/

L E

C50

=26.

6mg/

L

EC

10 =

5.0

2 m

g/L.

D. p

ulex

and

C

erio

daph

nids

dub

ia

(Gri

ffith

et

al.,

2008

)

Deg

ussa

P25

nan

o-Ti

O2 (

20%

rutil

e,

80%

ana

tase

, 45.

41m

2 /g;

20.

5±6.

7 nm

; ZP

-25

,1; p

olyd

isper

se 0

.197

, lar

gest

pa

rtic

le d

iam

eter

obs

erve

d in

su

spen

sion

was

687

.5nm

)

Stoc

k so

luti

on in

ultr

apur

e w

ater

(1

mg/

ml)

? s

onic

atio

n (6

W, 2

2.5

kHz,

6 o

ne-h

alf

seco

nd p

ulse

s).

BET

; Cou

lter L

S 13

320

; po

lydi

sper

sity

; Zet

a re

ader

M

k 21

-II;

scan

ning

m

icro

grap

hs.

Acu

te a

ssay

48h

stat

ic. A

mer

ican

Soc

iety

for

test

ing

and

mat

eria

ls g

uide

lines

. No

food

giv

en d

urin

g th

e te

st.

LC

50 >

10m

g/L

for

both

test

ed o

rgan

ism

s.

D. m

agna

and

Th

amno

ceph

alus

pl

atyu

rus

(H

einl

aan

et a

l.,

2008

)

Sigm

a A

ldri

ch n

ano-

TiO

2 (2

5-70

nm).

Ri

edel

-de

Hae

n bu

lk T

iO2

St

ock

solu

tion

in u

ltrap

ure

wat

er

(40g

/L)

? s

onica

tion

(30

min

) ?

sto

rage

at 4

o C ?

vor

tex

?

expo

sure

dos

age.

INA

D

. mag

na: A

cute

ass

ay 4

8h, i

n th

e da

rk. S

tand

ard

Ope

ratio

nal P

roce

dure

s of

Dap

htox

kit F

TM m

agna

(1

996)

. T

. pla

tyur

us (l

arva

e): A

cute

ass

ay 2

4h,

in th

e da

rk.

Tha

mno

toxk

it FT

M (1

995)

. Con

cent

ratio

ns te

sted

: 0.

01 to

200

00 m

g/L

D. m

agna

: LC

50 o

f na

no-T

iO2 ~

200

00m

g/L.

(B

ulk

TiO

2 an

d ot

her

valu

es

of to

xici

ty w

ere

not t

este

d).

T. p

laty

urus

: LC

50, L

C20

and

NO

EC

of n

ano-

and

bul

k Ti

O2 >

20.

000

mg/

L.

Tabl

e 1. S

umm

ary

of p

aper

s pub

lishe

d ab

out t

he ef

fect

s of n

ano-

TiO

2 use

d in

toxi

colo

gy st

udie

s on

mic

rocr

usta

cean

s (C

ontin

ues)


41

BC

F =

bioc

once

ntra

tion

fact

orB

ET=

Bru

naue

r, Em

met

t, Te

ller m

etho

d fo

rsu

rfac

e ar

ea c

alcu

latio

nC

AT =

cat

alas

e ac

tivity

DLS

= d

ynam

ic li

ght s

catte

ring

INA

= in

form

atio

n no

t ava

ilabl

e

D. m

agna

(L

over

n et

al.,

200

7)

Nan

o-Ti

O2 3

0nm

(in

sus

pens

ion)

TH

F w

as u

sed

to e

nsur

e di

sper

sion.

The

TH

F w

as

elim

inat

ed b

y ev

apor

atio

n an

d fil

trat

ion

and

conf

irmed

by

spec

troph

otom

etry

.

TEM

. Cha

ract

eriz

atio

n ac

cord

ing

to L

over

n an

d K

lape

r (20

06).

Acu

te a

ssay

60

min

. USE

PA 2

3. C

once

ntra

tion

test

ed: 2

ppm

(LO

EC c

alcu

late

d in

a p

revi

ous

expe

rim

ent).

TiO

2 di

d no

t sig

nifi

cant

ly a

lter

the

heat

rat

e, ju

mp,

mov

emen

t of

appe

ndic

es, a

nd c

urva

ture

of t

he a

bdom

inal

cla

w.

D. m

agna

(W

arhe

it et

al.,

20

07b)

DuP

ont

Has

kell

TiO

2:

fine

TiO

2 (38

0nm

in w

ater

, 5.8

m2 /

g,

100%

rutil

e, 9

9%Ti

O2 a

nd 1

%

alum

inum

) af

C (1

40 ±

44n

m i

n w

ater

; 38.

5m2 /g

; 79

% r

utile

21%

ana

tase

; 90%

TiO

2 ;

1% a

mor

phou

s sili

ca; 7

% a

lum

inum

).

INA

D

LS,

BET

, X-ra

y flu

ores

cenc

e, X

-ray

di

ffrac

tion.

Acu

te a

ssay

48h

, sta

tic. O

ECD

202

. Con

cent

ratio

ns

test

ed: 0

.1, 1

, 10

and

100

mg/

L.

LC 5

0 48

h >1

00m

g/L

for b

oth

type

s of T

iO2.

The

re w

as 1

0% o

f im

mo

bilit

y at

con

cent

ratio

ns o

f 10

and

100m

g/L

at t

he e

nd o

f 48h

for

both

com

poun

ds te

sted.

D. m

agna

(A

dam

s et

al.,

200

6)

Sigm

a A

ldri

ch n

ano-

TiO

2 65

nm

, 950

nm

and

44

µm.

Smal

ler

part

icle

s (65

nm

) app

eare

d la

rger

(on

aver

age

320n

m)

and

larg

er

ones

(950

nm a

nd 4

4 um

) app

eare

d sm

alle

r (3

20nm

and

1um

), re

spec

tivel

y, w

hen

in s

uspe

nsio

n.

Solu

tion

in u

ltrap

ure

wat

er

(10g

/L)

? a

gita

tion

? e

xpos

ure

dosa

ge

DL

S op

tical

mic

rosc

opy.

Pr

olon

ged

assa

y 8

days

. Con

cent

ratio

ns t

este

d: 1

, 10

and

20 p

pm.

20 p

pm o

f nan

o-T

iO2 w

as le

thal

for

40%

of t

he o

rgan

ism

s.

D. m

agna

(H

und

Rin

ke a

nd

Sim

on, 2

006)

Prod

uct 1

: 25n

m,

mai

nly

anat

ase.

Pr

oduc

t 2: 1

00nm

, 100

% a

nata

se.

The

TiO

2 sus

pens

ion

was

agi

tate

d an

d pr

e-ill

umin

ated

in S

UN

TES

T

CPS.

Par

ticle

s w

ere

was

hed

follo

win

g th

e m

anuf

actu

rer’

s in

stru

ctio

ns ?

mot

her s

olut

ion

?

soni

catio

n ?

con

tinuo

us a

gita

tion

and

irra

diat

ion

in a

sola

r lig

ht

simul

atio

n sy

stem

(300

-800

nm

25

0W, 3

0 m

in)

? s

ampl

es w

ere

trans

ferr

ed a

nd in

cuba

ted

for 7

2h

with

vis

ible

ligh

t.

INA

A

cute

ass

ay 4

8h. I

SSO

634

1, O

ECD

202

and

DIN

38

412-

30. C

once

ntra

tions

test

ed: 1

, 1.5

, 2, 2

.5, 3

m

g/L.

The

re w

as n

o co

ncen

trat

ion-

effe

ct c

urve

, so

the

EC50

cou

ld n

ot b

e de

term

ined

for a

ny g

roup

. Pre

-illu

min

atio

n in

crea

sed

the

toxi

city

of t

he

two

nano

-TiO

2 pr

oduc

ts. E

.g.:

at 1

and

2.5

mg/

L of

pro

duct

1,

imm

obi

lizat

ion

wen

t fro

m 0

to 2

0% a

nd fr

om 2

8 to

73%

, res

pect

ivel

y,

whe

n th

ere

was

pre

-illu

min

atio

n.

D. m

agna

(

Love

rn a

nd K

aple

r, 20

06)

INA

. Mea

n di

amet

er o

f fil

tere

d T

iO2:

30

nm;

in s

onic

ated

sol

utio

n: 1

00 to

50

0 nm

Solu

tions

wer

e pr

epar

ed in

thre

e w

ays:

1) D

ilutio

n in

dis

tille

d w

ater

?

soni

catio

n fo

r 30

min

.

2) 2

0mg

wer

e pl

aced

in

200

ml

THF

? p

ulve

rizat

ion

with

ni

troge

n ?

ove

r-ni

ght o

n m

ovin

g pl

ate

? fi

ltrat

ion

? d

iluti

on in

de

ioni

zed

wat

er ?

eva

pora

tion

of

the

TH

F ?

filtr

atio

n.

3) S

ame

as 2

, but

with

out T

HF.

TEM

spe

ctro

phot

omet

ry.

Acu

te a

ssay

48h

. USE

PA 2

024.

No

food

giv

en

duri

ng th

e tes

t. G

roup

s: 1

) con

trol

, 2) T

HF

grou

p, 3

) fil

tere

d Ti

O2

(0.2

, 1, 2

, 5, 6

, 8, a

nd 1

0 pp

m),

and

4)

soni

cate

d an

d no

n-fil

tere

d Ti

O2 (

50, 2

00, 2

50, 3

00,

400,

and

500

ppm

).

Filte

red

TiO

2: th

ere

was

no

mor

talit

y at

0.2

ppm

, but

1%

mor

talit

y at

1p

pm. L

C50

=5.5

ppm

; L

OEC

= 2

ppm

; N

OE

C=

1 pp

m.

So

nica

ted

TiO

2: n

o gr

oup

suff

ered

mor

talit

y >

9%. N

OEC

, LO

EC a

nd

LC 5

0 no

t app

licab

le.

Whe

n th

ere

was

no

mor

talit

y, n

o im

mob

ility

or s

wim

min

g ab

norm

aliti

es w

ere

obse

rved

in a

ny g

roup

.

EC

10 =

eff

ectiv

e co

ncen

tratio

n fo

r 10%

of e

xpos

ed o

rgan

ism

sEC

50 =

eff

ectiv

e co

ncen

tratio

n fo

r 50%

of e

xpos

ed o

rgan

ism

sG

PX –

glu

tath

ione

per

oxid

ase

activ

ityG

ST =

glu

tath

ione

S-tr

ansf

eras

e ac

tivity

ICP-

OES

= in

duct

ivel

y co

uple

d pl

asm

a op

tical

em

issi

onsp

ectr

osco

pyLC

50 =

leth

al c

once

ntra

tion

for 5

0% o

f exp

osed

org

anis

ms

LOEC

= lo

wes

t obs

erve

d ef

fect

con

cent

ratio

nN

OEC

= n

o ob

serv

ed e

ffec

t con

cent

ratio

nZP

= z

eta

pote

ntia

l

SEM

= s

cann

ing

elec

tron

mic

rosc

opy

SOD

= s

uper

oxid

e di

smut

ase

activ

ityTE

M=

trans

mis

sion

ele

ctro

n m

icro

scop

yTH

F =

tetra

hydr

ofur

an

Tabl

e 1. S

umm

ary

of p

aper

s pub

lishe

d ab

out t

he ef

fect

s of n

ano-

TiO

2 use

d in

toxi

colo

gy st

udie

s on

mic

rocr

usta

cean

sInt. J. Environ. Res., 6(1):33-50, Winter 2012

Tabl

e 2. S

umm

ary

of p

aper

s pub

lishe

d ab

out t

he ef

fect

s of n

ano-

TiO

2 use

d in

toxi

colo

gy st

udie

s on

fishe

s (C

ontin

ues)

Tes

t spe

cies

Prod

uct t

ested

Tr

eatm

ent o

f the

prod

uct

Phys

icoc

hem

ical

char

acte

rizat

ion

Bioa

ssay

Res

ults

D. re

rio a

dults

(X

iong e

t al.,

2011

)

nano

-TiO

2 fr

om N

anjin

g Un

ivers

ity of

Tec

hnol

ogy

(ana

tase,

puri

ty 99

%, d

iamete

r 20

-70n

m, h

ydro

dyna

mic

diame

ter 2

51 –

630

nm, Z

P -13

,1mV

) bu

lk T

iO2

from

Tian

jin

Guan

gche

ng C

hemi

cal R

eage

nt

Co. (

anat

ase,

purit

y 99%

, dia

mete

r 128

-949

nm,

hydr

odyn

amic

diam

eter

272-

597,

ZP

-27,

8mV

)

test

susp

ensio

n in a

erat

ed si

ngle-

disti

lled

wate

r ? so

nicat

ion (

1,5

L,10

0W, 4

0kHz

for

20 m

in).

TEM

, DLS

A

cute

assa

y 96

h, se

mi-s

tatic

(sol

utio

n ch

ange

d eve

ry

24h)

. No f

ood

given

dur

ing t

he te

st. C

once

ntrat

ions

test

ed:

0, 10

, 50,

100

, 150

, 200

and

300

mg/

L. F

rom

biom

arke

rs

anal

ysis,

fish

wer

e ex

pose

d to 5

0mg/

L un

der l

ight

or d

ark

cond

ition

s.

nano

-TiO

2 LC

50 =

124

,5 m

g/L

SOD

activ

ity de

crea

sed i

n liv

er tis

sues

and

incre

ased

in g

ut tis

sues

, in b

oth

grou

ps (u

nder

light

or d

ark c

ondi

tions

). CA

T ac

tivity

in li

ver t

issue

was

ob

serve

d to

be re

duce

d in

both

grou

ps. T

here

was

eleva

ted p

rote

in ca

rbon

yl le

vels

. Lip

id pe

roxid

es w

ere a

lso f

ound

in th

e gill

s and

gut

tis

sues

. GSH

con

tent

incre

ased

in g

ut tis

sue,

and

(und

er d

ark c

ondi

tions

) de

crea

sed

in liv

er. M

DA co

ncen

tratio

ns in

crea

sed

in gi

lls an

d gu

t tiss

ues.

Mor

pholo

gica

l cha

nges

in g

ill ce

lls

( cell

mem

bran

e dam

age,

irreg

ular c

ell o

utline

s, py

knot

ic nu

clei

and

a tre

nd o

f com

plet

e disr

uptio

n of g

ill ce

lls).

bulk

TiO 2

LC

50 >

300

mg/L

no

chan

ges

in SO

D an

d CAT

activ

ities

and

in M

DA

conte

nt. T

here

was

an

incre

ase i

n GSH

in gu

t tis

sue.

O.

myk

iss

(John

ston

et al

., 20

10)

Nano

-TiO

2 (3

4.2±1

,73n

m, Z

P -

9), b

ulk T

iO2 an

d ion

ic tit

anium

(ti

taniu

m m

etal

stan

dard

so

lutio

n, Fi

sher

Scie

ntific

).

Stoc

k solu

tion (

250µ

g/L)

in

ultra

pure

wate

r ? so

nica

tion

(30m

in) ?

expo

sure

dos

age.

TEM

, ICP

-MS,

DLS

, pa

rticl

e siz

er, C

ARS

, mu

ltiph

oton

mic

rosco

py.

Prol

onge

d assa

y 10

day

s, se

mi-s

tatic

(cha

nge o

f 50%

of

the w

ater

ever

y 2 d

ays)

. Con

centr

ation

s tes

ted:

500

(nano

-T

iO2)

and 5

000

µg/L

(na

no-

and b

ulk

TiO 2

and

ionic

Ti).

T

est ex

posu

re via

diet

21 d

ays.

Con

centr

atio

ns te

sted

: 0.0

1 an

d 0.1

% n

ano-

TiO

2 in

food.

No s

ignifi

cant

abso

rptio

n of

Ti w

as de

tecte

d in

any

grou

p. T

he T

i co

ncen

tratio

n in

the g

ills i

ncrea

sed

in th

e gro

up ex

pose

d to

ionic

Ti.

High

lev

els o

f Ti w

ere fo

und

in th

e sto

mac

h of

fish

fed w

ith m

ediu

m an

d hi

gh

dose

s of T

iO2.

TiO

2 ag

greg

ates

were

foun

d on

the

surfa

ce o

f the

gill

ep

ithel

ium af

ter 2

4 and

96h

of e

xpos

ure a

nd in

side

lamell

ae a

fter 1

4 da

ys

of e

xpos

ure.

D. re

rio a

dult

(P

alani

appa

n et

al.,

2010

)

Sigm

a Al

dric

h nan

o-Ti

O 2

(pur

ity 9

9.7%

, ana

tase,

20nm

, 20

0±20

m2 /g).

Parti

cle si

ze:

14.1

±1.52

nm.

Nice

Che

mica

ls bu

lk T

iO2

(99.7

% p

urity

, ana

tase)

.

Stoc

k solu

tion (

10 p

pm)

in ul

trapu

re w

ater ?

soni

catio

n (6

h) ?

stor

age

at -2

0o C ?

son

icat

ion (3

0 min

) ?

expo

sure

dos

age.

TEM

. Pr

olon

ged a

ssay

14 d

ays.

Con

centr

atio

ns te

sted

: 10p

pm o

f na

no-T

iO2

or 10

0ppm

of b

ulk T

iO2.

Mor

talit

y was

not o

bser

ved

durin

g th

e ex

perim

ent.

The

bioch

emica

l co

nstitu

ents

of t

he g

ills s

how

ed al

tera

tions

. The

se alt

erati

ons w

ere g

reate

r in

the g

roup

exp

osed

to na

no-T

iO2 t

han t

he on

e ex

pose

d to

bulk

TiO 2

. Ex

ampl

e: al

terat

ions

in th

e am

ide I

band

s.

D. re

rio

(Zhu

et al

., 201

0a)

Degu

ssa

P25

nano

-TiO

2 (21

nm).

Stoc

k solu

tion (

1g/L

in u

ltrap

ure

wate

r ? so

nica

tion

(10

min,

50

W/L

, 40k

Hz).

SEM

, DLS

. T

roph

ic tr

ansf

er te

st. D

aphn

ids w

ere ex

pose

d to

0.1

or

1mg/

L of

TiO

2 for

24h,

afte

r whic

h th

ey w

ere

colle

cted

and

wash

ed in

cult

ure

medi

um a

nd su

pplie

d to

D. re

rio a

s fo

od. T

he t

est in

volv

ed 14

day

s of a

bsor

ptio

n fol

lowe

d by

7

days

of de

purat

ion (

feed

ing w

ith no

n-co

ntam

inat

ed

daph

nids

). Th

e Ti

O2 c

once

ntrat

ion

in th

e da

phnid

s was

de

term

ined

as fo

llows

: 4.5

2 ± 0.

36 m

g/g (

in th

e gro

up

expo

sed

to 0

.1mg/

L) a

nd 6

1.09

± 3.

24 m

g/g (

in th

e gro

up

expo

sed

to 1

mg/L

). T

he fi

sh w

ere

samp

led o

n da

ys 0

, 1, 3

, 5,

7, 1

0, 14

, 15,

17, 1

9 an

d 21

. Pr

olon

ged e

xpos

ure

test.

14 d

ays,

follo

wed

by 7

day

s of

depu

ratio

n. S

emi-s

tatic

(wate

r ch

ange

d da

ily).

C

once

ntrati

ons

teste

d: 0.1

and

1mg/

L.

No m

orta

lity o

r abn

orm

alitie

s wer

e obs

erve

d. T

roph

ic tr

ansfe

r of

TiO2

oc

curre

d. Th

ere

was

no ap

pare

nt bi

omag

nific

atio

n. Tr

ophic

tran

sfer

test

Co

ncen

trati

on o

f Ti i

n the

fish

gro

up fe

d wi

th da

phnid

s 0.1

mg/l

= 1

06.5

7 ±

14.8

9 mg

/ kg a

nd g

roup

fed w

ith d

aphn

ids 1

mg/l

= 5

22.0

2 ±

12.9

4 mg

/ kg

. BC

F< 1.

Pr

olon

ged

expo

sure

test

Fish

acc

umul

ated

TiO

2, rea

chin

g a p

latea

u of a

bout

1.5

mg/kg

on

day

3 (g

roup

0.1

mg/L

) an

d of

100

mg/k

g on

day

10 (

grou

p 1m

g/L)

. BC

F= 25

.38

and 1

81.38

(at

equi

libriu

m fo

r gro

ups

0.1 a

nd 1m

g/L

resp

ectiv

ely).

Du

ring

the d

epur

atio

n pha

se, th

e con

cent

ratio

n of

TiO 2

in th

e en

tire b

ody

was f

ound

to de

clin

e.

Clemente, Z. et al.

42

D. r

erio

fem

ale

adul

ts

(Grif

fith

et a

l.,

2009

)

Deg

ussa

P25

nan

o-T

iO2

(45.

41m

2 /g; Z

P -2

5.1m

V).

A

ggre

gate

s in

pow

der

20.5

±6.7

nm; i

n su

spen

sion

22

0.8

to 6

87.5

nm.

Stoc

k so

lutio

n in

ultr

apur

e w

ater

?

soni

catio

n (6

s, 6W

, 22k

Hz)

?

expo

sure

dos

age.

BET

, SEM

, sca

nnin

g m

icro

grap

hs.

Acu

te a

ssay

48h

sta

tic. C

once

ntra

tion

test

ed: 1

000µ

g/L

. Si

gnif

ican

t di

ffer

ence

in t

he e

xpre

ssio

n of

171

gen

es (

mic

roar

ray)

- 6

0 up

-reg

ulat

ed a

nd 1

11 d

own-

regu

late

d (5

3 of

thes

e ge

nes

wer

e af

fect

ed

by e

xpos

ure

to n

ano-

copp

er a

nd n

ano-

silv

er).

The

affe

cted

gen

es w

ere

invo

lved

in

ribos

ome

struc

ture

and

act

ivit

y. N

o al

tera

tion

was

obs

erve

d in

gen

es r

elat

ed t

o re

gula

tion

of o

xida

tive

str

ess.

No

hist

opat

holo

gica

l di

ffer

ence

s wer

e ob

serv

ed in

the

gills

com

pare

d w

ith

the

cont

rol g

roup

.

Cypr

inus

car

pio

juve

nile

s (H

ao e

t al.,

200

9)

Hon

gshe

ng M

ater

ial n

ano-

TiO

2 (5

0nm

, 30±

10m

2 /g,

rutil

e 98

%).

So

lutio

n ?

son

icat

ion

(30m

in,

100W

, 40k

Hz)

. IN

A

Prol

onge

d as

say

8 da

ys s

emi-s

tatic

(so

lutio

n ch

ange

d da

ily).

No

food

giv

en d

urin

g th

e te

st.

Ani

mal

s w

ere

colle

cted

on

days

1, 2

, 4 a

nd 6

for

bio

chem

ical

ana

lyse

s. Fo

r hi

stop

atho

logy

, th

e an

imal

s w

ere

expo

sed

for

20

days

. Con

cent

rati

ons

test

ed: 1

0, 5

0, 1

00 a

nd 2

00 m

g/L.

No

mor

talit

y oc

curr

ed, b

ut a

fter

1h

of e

xpos

ure

the

resp

irat

ory

rate

and

sw

imm

ing

rate

s in

crea

sed,

as

wel

l as

the

pro

duct

ion

of m

ucus

, in

a

conc

entra

tion-

depe

nden

t w

ay. T

he b

iom

arke

rs o

f ox

idat

ive

stre

ss v

arie

d w

ith t

he c

once

ntra

tion

and

expo

sure

tim

e. A

t 10

0 an

d 20

0mg/

L th

ere

was

an

incr

ease

in

LPO

and

dec

reas

e in

SO

D,

CAT

and

PO

D a

ctiv

ity.

The

liver

was

mor

e se

nsiti

ve th

an t

he g

ills

and

brai

n. H

istop

atho

logi

cal

alte

ratio

ns w

ere

obse

rved

mai

nly

at th

e hi

ghes

t con

cent

ratio

ns. T

he li

ver

show

ed v

acuo

lizat

ion

of c

ytop

lasm

and

aut

osom

es,

incl

udin

g ne

crot

ic

cell

bodi

es a

nd n

ucle

ar f

ragm

ents

that

loo

ked

like

apop

toti

c bo

dies

and

so

me

foci

of

lipid

osis.

The

gill

s sh

owed

thi

cken

ing,

ede

ma,

fusi

on a

nd

hype

rplas

ia o

f th

e la

mel

lae

and

fila

men

ts.

Onc

orhy

nchu

s m

ykiss

juve

nile

s

(Sco

wn

et a

l., 2

009)

Sig

ma

Ald

rich

nano

-TiO

2 (3

2.4n

m, 4

6.3m

2 /g, p

urity

>

99.9

%, a

nata

se a

nd ru

tile)

. P

artic

le s

ize:

34.

2nm

, 18.

6m2 /g

(i

n po

wde

r).

400-

1100

nm

(in

ring

er a

nd

wat

er).

Z

P: 0

at -0

.6m

V.

Solu

tion

(100

mg/

L) i

n rin

ger

?

soni

catio

n (3

0 m

in).

BE

T

In

trav

enou

s ad

min

istra

tion

(1.3

mg/

kg).

Fi

sh a

nd b

lood

sa

mpl

es c

olle

cted

6h

and

90 d

ays p

ost-

inje

ctio

n.

10 to

19%

of i

njec

ted

Ti a

ccum

ulat

ed in

the

kidn

eys

(up

to 2

3µg/

g). T

he

conc

entra

tion

in th

e ki

dney

s di

d no

t cha

nge

sign

ifica

ntly

from

6h

to 2

1 da

ys p

ost-

inje

ctio

n, b

ut a

fter

90

days

the

con

cent

rati

on i

n th

e ki

dney

s w

as s

igni

fica

ntly

low

er. T

he T

i lev

el in

the

liver

was

app

roxi

mat

ely

15-

fold

low

er. P

relim

inar

y st

udie

s sh

owed

that

Ti d

id n

ot a

ccum

ulat

e in

the

brai

n, g

ills

or s

plee

n. N

o si

gnif

ican

t di

ffer

ence

was

fou

nd i

n bl

ood

TBA

RS

at a

ny t

ime

com

pare

d w

ith t

he c

ontr

ol.

The

hist

opat

holo

gica

l an

alys

is s

how

ed n

o al

tera

tion

in th

e ki

dney

s, bu

t the

TEM

sho

wed

sm

all

aggr

egat

es a

ppar

ently

enc

apsu

late

d ar

ound

the

tubu

les.

Cre

atin

ine

leve

ls

fluct

uate

d in

bot

h th

e co

ntro

ls an

d th

e in

ject

ed a

nim

als,

but

no

effe

ct

was

fou

nd in

the

plas

ma

prot

ein

conc

entr

atio

n.

C. c

arpi

o

(Sun

et a

l., 2

009)

D

egus

sa P

25 n

ano-

TiO

2 (5

0m2 /

g, 2

5nm

)

A

rsen

ite (A

s II

I) p

repa

red

from

A

s 2O

3.

Stoc

k so

lutio

n of

nan

oTiO

2 (1g

/l)

? so

nica

tion

(10m

in, 5

0W/l,

40

kHz)

? e

xpos

ure

dosa

ge.

INA

C

hron

ic

assa

y.

Gro

ups:

1)

co

ntro

l, 2)

on

ly

As

III

(200

µg/L

± 1

0.2)

; 3)

As

III

+ T

iO2

(10m

g/L

±1.

3).

Ani

mal

s w

ere

plac

ed i

n th

e aq

uari

ums

2h

afte

r th

e ad

diti

on o

f As a

nd T

iO2.

Sem

i-sta

tic

test

(wat

er c

hang

ed

daily

). A

nim

als

wer

e co

llect

ed o

n da

ys 2

, 5,

10,

15,

20

and

25.

Food

was

giv

en o

nce

a da

y du

ring

the

test

. Sp

ecia

tion

was

eva

luat

ed o

f A

s in

wat

er, i

n th

e pr

esen

ce

of T

iO2,

with

and

with

out s

unlig

ht.

The

conc

entra

tion

of A

s in

the

car

ps i

ncre

ased

fro

m 4

2% (

20 d

ays)

to

185%

(s

econ

d da

y)

in t

he

pres

ence

of

nan

o-Ti

O2.

The

orde

r of

ac

cum

ulat

ion

of A

s an

d T

iO2

in th

e di

ffer

ent

tissu

es w

as:

visc

era

> g

ills

> sk

in a

nd s

cale

s >

mus

cle.

In

the

abs

ence

of

sunl

ight

, on

ly a

sm

all

amou

nt o

f A

s II

I m

oved

to

As

V (

load

ed,

and

ther

efor

e w

ith

less

ca

paci

ty t

o pa

ss t

hrou

gh b

iolo

gica

l m

embr

anes

). W

ith s

unlig

ht, a

bout

75

% o

f th

e A

s II

I mov

ed to

AsV

in 1

h.

Dan

io re

rio

adul

ts

and

juve

nile

s (G

riffit

h et

al.,

20

08)

Deg

ussa

P25

nan

o-T

iO2 (

20%

ru

tile,

80%

ana

tase

, 45.

41m

2 /g;

20

.5±6

.7 n

m; Z

P -2

5.1;

po

lydi

sper

sion

0.19

7, la

rges

t pa

rticl

e di

amet

er o

bser

ved

in

susp

ensi

on =

687

.5nm

).

Stoc

k so

lutio

n (1

mg/

ml)

in

ultr

apur

e w

ater

? s

onic

atio

n (6

W,

22.5

kHz,

6 h

alf-s

econ

d pu

lses)

.

BE

T, C

oulte

r LS

13

320;

po

lydi

sper

sity

; Zet

a re

ader

Mk

21-I

I; sc

anni

ng

mic

rogr

aphs

.

Acu

te a

ssay

48h

sta

tic.

LC

50>

10m

g/L

of n

anop

artic

les

Tabl

e 2. S

umm

ary

of p

aper

s pub

lishe

d ab

out t

he ef

fect

s of n

ano-

TiO

2 use

d in

toxi

colo

gy st

udie

s on

fishe

s (C

ontin

ues)


43

O. m

ykis

s juv

enile

s (F

eder

ici e

t al.,

20

07)

Deg

ussa

P25

nan

o-T

iO2

(21n

m,

50±1

5m2 /g

, 75%

rutil

e, 2

5%

anat

ase,

pur

ity 9

9%).

Pa

rtic

le si

zes w

ere

clos

e to

th

ose

spec

ified

by

the

man

ufac

ture

r (24

.2±2

.8nm

).

The

con

cent

ratio

n of

TiO

2 (s

pect

rom

etry

) in

the

tank

re

ache

d 95

-98%

of t

he ta

rget

va

lue

10 m

in a

fter d

osin

g. T

he

conc

entr

atio

n in

wat

er w

as

mea

sure

d be

fore

cha

ngin

g th

e so

lutio

n, to

con

firm

that

the

conc

entr

atio

n re

mai

ned

unch

ange

d in

12h

.

Stoc

k so

lutio

n (1

0g/L

) in

ultr

apur

e w

ater

? so

nica

tion

(6h,

35k

Hz)

?

stor

age

? s

onic

atio

n (3

0 m

in)

?

expo

sure

dos

age.

TEM

, spe

ctra

l sca

ns.

Prol

onge

d as

say

14 d

ays

sem

i-st

atic

(80%

of t

he w

ater

ch

ange

d ev

ery

12h)

. Con

cent

ratio

ns te

sted

: 0.1

; 0.5

and

1

mg/

L. F

ood

was

with

held

24h

pri

or to

and

duri

ng th

e te

st

(exc

ept o

n da

y 10

). F

ish

wer

e sa

mpl

ed o

n da

ys 7

and

14.

The

re w

as n

o m

orta

lity.

The

fish

did

not

acc

umul

ate

Ti.

Cha

nges

in

beha

vior

and

muc

us s

ecre

tion

wer

e ob

serv

ed a

t th

e hi

ghes

t con

cent

ratio

n.

The

gil

ls sh

owed

incr

ease

d oc

curr

ence

of e

dem

a in

seco

ndar

y la

mel

lae,

m

orph

olog

ical

cha

nges

in m

ucoc

yte,

hyp

erpl

asia

of p

rim

ary

lam

ella

e,

and

aneu

rysm

. Vac

uoliz

atio

n an

d er

osio

n of

vill

ositi

es in

the

inte

stin

es

was

obs

erve

d, a

s w

ell a

s lo

ss o

f sin

usoi

dal s

pace

, som

e fo

ci o

f lip

idos

is,

occa

siona

l nec

roti

c cel

ls a

nd a

popt

otic

bod

ies

in th

e liv

er. N

o ch

ange

s w

ere

obse

rved

in th

e br

ain.

The

re w

as n

o cl

ear e

ffect

of t

he tr

eatm

ent o

r of

tim

e on

the

Ti l

evel

s in

the

gills

, liv

er o

r m

uscl

e. N

o he

mat

olog

ical

ch

ange

was

foun

d. T

here

was

alte

ratio

n of

the

leve

ls of

tiss

ue Z

n an

d C

u. A

con

cent

ratio

n-de

pend

ent

redu

ctio

n w

as fo

und

in th

e N

a-K

ATP

ase

activ

ity in

the

gills

, int

esti

nes

and

brai

n at

the

end

of th

e ex

peri

men

t (s

igni

fica

nt d

iffer

ence

s onl

y am

ong

som

e gr

oups

). In

gen

eral

, the

re w

as

an i

ncre

ase

in T

BA

RS

at th

e en

d of

the

expe

rimen

t in

gills

, int

estin

es a

nd

brai

n, b

ut n

ot in

liv

er. C

once

ntra

tion-

depe

nden

t glu

tath

ione

dep

letio

n oc

curr

ed o

nly

in l

iver

on

day

14.

C. c

arpi

o

(Zha

ng e

t al.,

200

7)

Deg

ussa

P25

nan

o-T

iO2

(50m

2 /g; 2

1nm

).

Stoc

k so

lutio

n in

ultr

apur

e w

ater

. L

aser

par

ticle

ana

lyze

r,

zeta

pot

entia

l ana

lyze

r, IC

P-O

ES,

ato

mic

fl

uore

scen

ce s

pect

rosc

opy.

Chro

nic

assa

y. A

dsor

ptio

n of

Cd

on T

iO2 an

d na

tura

l se

dim

ent

part

icle

s (SP

) w

ere

eval

uate

d. C

d w

as a

dded

to

the

wat

er (9

7.3

± 6

.9µg

/L) f

irst,

follo

wed

by

TiO

2 (1

0mg/

L) o

r SP

(10m

g/L

). T

he a

nim

als

wer

e pl

aced

in

the

wat

er 2

hou

rs la

ter.

Foo

d w

as g

iven

tw

ice

a da

y du

ring

the

test

. Fis

h w

ere

trans

ferr

ed to

new

sol

utio

ns

ever

y da

y. T

he a

nim

als

wer

e sa

mpl

ed o

n da

ys 2

, 5, 1

0,

15, 2

0 an

d 25

.

TiO

2 sh

owed

hig

her c

apac

ity to

ads

orb

Cd

than

SP

. SP

did

not h

ave

a si

gnifi

cant

infl

uenc

e on

Cd

in fi

sh. T

he p

rese

nce

of T

iO2 e

leva

ted

the

accu

mul

atio

n of

Cd.

Aft

er 2

5 da

ys o

f ex

posu

re, t

he c

once

ntra

tion

of C

d in

crea

sed

by 1

46%

, and

was

22µ

g/g.

The

re w

as a

pos

itive

cor

rela

tion

be

twee

n th

e co

ncen

trat

ion

of T

iO2 a

nd C

d. T

iO2

and C

d ac

cum

ulat

ed

mai

nly

in th

e vi

scer

a an

d gi

lls.

C. c

arpi

o

(Sun

et a

l., 2

007)

D

egus

sa P

25 n

ano-

TiO

2 (5

0m2 /

g, 2

5nm

, agg

rega

tes

of

50- 4

00nm

in w

ater

.)

A

rsen

ate

(As

V) (

prep

ared

from

N

a 3A

sO4•

12H

2O).

Stoc

k so

lutio

n of

nan

o-T

iO2

(1g/

l) ?

soni

cati

on (

10m

in, 5

0W/l,

40

kHz)

? e

xpos

ure

dosa

ge.

TEM

Ch

roni

c as

say,

sem

i-sta

tic

(wat

er c

hang

ed d

aily

). G

roup

s:

1) c

ontro

l, 2)

onl

y A

s V (

200u

g/l ±

10.

2); 3

) As

V +

TiO

2 (1

0mg/

l ±1.

3).

Ani

mal

s w

ere

plac

ed i

n th

e aq

uariu

ms 2

h af

ter t

he a

dditi

on o

f As

and

TiO

2. A

nim

als

wer

e co

llect

ed o

n da

ys 2

, 5, 1

0, 1

5, 2

0 an

d 25

. Foo

d gi

ven

once

a d

ay d

urin

g th

e te

st.

O .

myk

iss

juve

nile

s

(War

heit

et a

l.,

2007

b)

DuP

ont H

aske

ll;

Fine

TiO

2 (38

0nm

in w

ater

, 5.

8m2 /g

, 100

% r

utile

, 99%

TiO

2 an

d 1%

alu

min

um).

af

C (

140±

44 n

m in

wat

er;

38.5

m2 /

g; 7

9% r

utile

, 21%

an

atas

e, 9

0%T

iO2;

1%

am

orph

ous

silic

a; 7

%

alum

inum

).

INA

D

LS,

BET

, X-r

ay

fluo

resc

ence

, X-r

ay

diffr

acti

on.

Acu

te a

ssay

96h

stat

ic. O

EC

D 2

03. C

once

ntra

tions

te

sted

: 0.1

, 1, 1

0 an

d 10

0 m

g/L

. L

C50

96h>

100m

g/L

for b

oth

type

s of

TiO

2. T

here

was

10%

of i

mm

obili

ty

at t

he c

once

ntra

tions

of 1

0 an

d 10

0mg/

L a

t end

of 9

6h in

bot

h gr

oups

ex

pose

d to

fine

TiO

2.

B

CF

= bi

ocon

cent

ratio

n fa

ctor

BET

= B

runa

uer,

Emm

ett,

Telle

r met

hod

for s

urfa

ce ar

ea ca

lcul

atio

nC

AR

S =

cohe

rent

ant

i-Sto

kes

Ram

an s

catte

ring

CAT

= c

atal

ase

activ

ityD

LS =

dyn

amic

ligh

t sca

tterin

gIC

P-O

ES =

ind

uctiv

ely

coup

led

plas

ma

optic

al e

mis

sion

spe

ctro

scop

y

ICP-

MS

= i n

duct

ivel

y co

uple

d pl

asm

a m

ass

spec

trosc

opy

LC50

= le

thal

con

cent

ratio

n fo

r 50%

of e

xpos

ed o

rgan

ism

sLP

O =

lip

id p

erox

idat

ion

NO

EC =

no

obse

rved

eff

ect c

once

ntra

tion

POD

= p

erox

idas

eZP

= z

eta

pote

ntia

l

SEM

= s

cann

ing

elec

tron

mic

rosc

opy

SOD

= s

uper

oxid

e di

smut

ase

activ

ityTB

AR

S =

thio

barb

ituric

aci

d re

activ

e su

bsta

nce

assa

yTE

M =

tran

smis

sion

ele

ctro

n m

icro

scop

yTH

F =

tetra

hydr

ofur

anIN

A =

info

rmat

ion

not a

vaila

ble

Tabl

e 2. S

umm

ary

of p

aper

s pub

lishe

d ab

out t

he ef

fect

s of n

ano-

TiO

2 use

d in

toxi

colo

gy st

udie

s on

fishe

s


44

Tabl

e 3. S

umm

ary

of p

aper

s pub

lishe

d ab

out t

he ef

fect

s of n

ano-

TiO

2 us

ed in

toxi

colo

gy st

udie

s on

othe

r aqu

atic

org

anism

s

Test

spec

ies

Prod

uct t

ested

Tr

eatm

ent o

f the

prod

uct

Phys

icoc

hem

ical c

hara

cteri

zatio

n B

ioassa

y Re

sults

poly

chae

te Ar

enic

ola

marin

a

(Gal

loway

et a

l.,

2010

)

Sigm

a-A

ldric

h nan

o-Ti

O2 ca

t.

no. 6

3466

2-1 (

23.2

nm,

equi

valen

t sph

erica

l dia

meter

32.4

nm, 4

6.3

m2 /g,

99.9

%,

mixt

ure o

f ana

tase

and r

utile

; K

82.3

ppm

, Zn 9

.7 p

pm, N

a 6.0

pp

m, F

e 3.1

ppm

, Li 0

.4 pp

m).

bulk

TiO

2

Stoc

k so

lutio

n in

ultra

pure

wate

r ?

soni

catio

n (3

0 min

) ? m

ixed w

ith

natu

ral t

reate

d sed

imen

t (co

llect

ed

at th

e sa

me si

te w

here

the

anim

als

wer

e col

lecte

d).

TEM

, X-r

ay di

ffrac

tion,

ICP-

OES

Prol

onge

d ass

ay 1

0 day

s. O

ECD/

ASTM

1990

. Exp

osur

e in s

eaw

ater.

Sem

i-sta

tic

test

(wate

r cha

nged

eve

ry 4

8h).

Feed

ing

durin

g the

test

. Con

cent

ratio

ns te

sted:

1 to

3 g/

kg o

f sed

imen

t.

The o

rgan

ic co

nten

t of t

he s

edim

ent w

as 0.

33±0

.4%. N

o beh

avior

al

altera

tions

wer

e det

ecte

d. A

chan

ge w

as ob

serv

ed in

the

feedin

g ra

te of

the

grou

p exp

osed

to 2

g/kg

of n

ano-

TiO

2 but

not

in th

e gro

up e

xpos

ed to

1g/kg

. No

effec

t of

expo

sure

time

was

foun

d. A

t 2 a

nd 3

g/kg o

f nan

o-

TiO 2

, an

impa

ct w

as d

etect

ed in

the

lipos

ome s

tabi

lity (n

eutra

l red

reten

tion)

and

an i

ncrea

se in

gen

etic i

mpa

irmen

t (co

met a

ssay

). Bu

lk T

iO2

did n

ot al

ter t

he ra

te of

gene

tic da

mage

com

pared

to th

e co

ntrol

.

Micr

osco

py r

evea

led T

iO2

aggr

egat

es of

>200

nm su

rroun

ding

inte

stina

l

micro

villo

sities

, but

no a

bsor

ption

by

the in

test

inal e

pith

elium

, alth

ough

TiO 2

rem

ained

in th

e lu

men.

BCF

= 0.

156±

0.0

75 (g

roup

1g/

kg) a

nd

0.196

±0.0

38 (g

roup

3g/

kg).

mollu

sk M

ytilu

s

gallo

prov

incia

lis

(Can

esi e

t al.,

2010

)

Degu

ssa

P25

nano

-TiO

2 (pu

rity

>99.

5%)

Stoc

k su

spen

sion (

100µ

g/m

l) in

artif

icial

seaw

ater

? so

nica

tion

(15m

in, 1

00W

, in a

cold

bath

) ? st

orag

e ? s

onica

tion

? ex

posu

re d

osag

e.

TEM

, BET

, DLS

. A

cute

assa

y 24

h. N

o fee

ding

durin

g the

test.

Con

cent

ratio

ns te

sted

: 0.05

; 0.2

; 1; 5

mg/

l.

No m

ortal

ity w

as fo

und

in an

y exp

osur

e co

ndit

ion. T

here

was

desta

biliz

ation

of th

e ly

soso

mal m

embr

ane i

n hem

ocyt

es at

1 an

d 5m

g/L

and

in th

e dig

estiv

e gla

nds

at 0.

2, 1

and

5 mg/

L; as

wel

l as a

ccum

ulati

on

of lip

ofus

cin a

nd ly

soso

mal

neutr

al li

pids i

n th

e dige

stive

glan

ds at

1 an

d 5

mg/L

, and

an in

creas

e in

CAT

at 1

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46

The literature reports nano-TiO2 aggregates ofabout 500 nm in water, but this number varies greatlyas a function of products and treatments used. Mostaquatic tests have been performed starting from thesonication of a stock solution, while few have involvedonly agitation or filtration of the solution (Tables 1, 2and 3). Adams et al. (2006) employed only agitation ofSigma Aldrich nano-TiO2 in water and observed that65nm sized particles formed aggregates of 320 nm, whilelarger particles of 950 nm and 44 µm formed aggregatesof 320 nm and 1 µm, respectively. Zhu et al. (2010b)report that in a culture medium for daphnids, even withsonication, P25 formed aggregates that increased overtime: 580 nm (1h), 2349 nm (12h) and 3528.6 nm (24h).The aggregation state of NPs inevitably changes withdilution, but there is a growing discussion about theuse of dispersants or sonication processes to increasethe dispersion of NPs in suspension in aquatictoxicology studies, in view of their environmentalapplicability (Baveye and Laba, 2008, Crane et al.,2008). One argument is that the study of non-dispersivematerials would be of greater relevance to what actuallytakes place in the environment. Moreover, sonicationmay cause structural changes in nanomaterials, in fact,when performed in natural waters or in the presence ofany electron donor, it may result in the generation ofreactive oxygen species. The sonication time requiredchanges according to the total concentration of thenanomaterial, and once sonication or agitation hasstopped, the material does not remain dispersed forvery long. On the other hand, the existence of naturaldispersants in the environment, such as organic matter,would validate such studies (Crane et al., 2008).However, one should not assume that aggregatematerials will necessarily not be bioavailable. They maysimply change the mode of respiratory exposure onthe water column to exposure via diet through sediment(Handy et al., 2008). Benthic organisms may be moreexposed to NPs aggregates than to the material in theliquid phase. Similarly, the high concentration of ionsin hard or marine waters will tend to cause aggregationof NPs, modifying the mode of exposure or organismsin these ecosystems (Handy et al., 2008).

A large part of acute exposure studies have beenperformed by withholding food from animals on the

day prior to and during the bioassay. In the case ofprolonged exposure, daily feeding has generally beenmaintained, with a few exceptions (Federici et al., 2007,Hao et al., 2009). However, it should be noted that thisis also a point to be evaluated carefully andstandardized, in view of the capacity of organic matterto adsorb TiO2.

The diversity of manufactured TiO2 NPs, thequality of the medium, the aquatic species tested, andthe objectives of each research, require that exposureconditions be evaluated separately.

CONCLUSIONEvaluating the potential biological impact of

nanomaterials has become increasingly important inrecent years. This is particularly relevant because therapid pace of nanotechnology development has notbeen accompanied by a complete investigation of itssafety or by the development of suitablemethodologies for this investigation.

Concern about the environmental consequencesof nanotechnology has been growing and has reachedpublic opinion. Nano-TiO2 is a nanoproduct withapplications in a variety of areas, and is also promisingfor the remediation of contaminated environments.However, its potentially harmful effects should beinvestigated in depth to ensure its sustainable use.Because water bodies are the final destination ofcontaminants, the evaluation of the effects of nano-TiO2 on aquatic organisms is extremely necessary.Several groups have started research in this area,however, their results are still not conclusive and theneed remains to continue researching. In fact, the resultsvary considerably, probably due to differences in theexperimental models and products tested. Therefore,we agree with the recommendation thatnanoecotoxicology studies focus on thecharacterization of NPs and that the best exposureconditions for the different NPs be analyzed(considering their particular properties), in the attemptto standardize bioassays and facilitate the comparisonof results. In addition, the standardization ofnanoecotoxicological methodologies is useful for theconstruction of protocols to underpin and guide publicpolicies.

ACKNOWLEDGMENTSThe authors thank CAPES and Rede Nanobiotec

for awarding a doctoral grant to Zaira Clemente, aswell as the Brazilian research funding agencies FAPESP,CNPq and FUNDUNESP for their financial support ofthis work.

Clemente, Z. et al.

concentration in supernatant after 16 hours went from100 to 83 mg/L in bidistilled water and to 33 mg/L insurface water, while agglomeration and sedimentationof coated TiO2 were slow. Some studies have involvedsemi-static aquatic bioassays, changing the exposuremedium every 24-48 hours (Tables 1, 2 and 3), whileothers have performed static assays involving mainlyacute exposure.

47

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