The popularity of TiO2 nanoparticles (nano-TiO2) lies in their wide range of nanotechnological applications, together with low toxicity. Meanwhile, recent studies have shown that the photocatalytic properties of this material can result in alterations in their behavior in the environment, causing effects that have not yet been fully elucidated. The objective of this study was to evaluate the toxicity of two formulations of nano-TiO2 under different illumination conditions, using an experimental model coherent with the principle of the three Rs of alternative animal experimentation (reduction, refinement, and replacement). Embryos of the fish Danio rerio were exposed for 96h to different concentrations of nano-TiO2 in the form of anatase (TA) or an anatase/rutile mixture (TM), under either visible light or a combination of visible and ultraviolet light (UV). The acute toxicity and sublethal parameters evaluated included survival rates, malformation, hatching, equilibrium, and overall length of the larvae, together with biochemical biomarkers (specific activities of catalase (CAT), glutathione S-transferase (GST), and acid phosphatase (AP)). Both TA and TM caused accelerated hatching of the larvae. Under UV irradiation, there was greater mortality of the larvae of the groups exposed to TM, compared to those exposed to TA. Exposure to TM under UV irradiation altered the equilibrium of the larvae. Alterations in the activities of CAT and GST were indicative of oxidative stress, although no clear dose-response relationship was observed. The effects of nano-TiO2 appeared to depend on both the type of formulation and the illumination condition. The findings contribute to elucidation of the factors involved in the toxicity of these nanoparticles, as well as to the establishment of protocols for risk assessments of nanotechnology. © 2013 Elsevier B.V.147 129139Aebi, H., (1984) Catalase In Vitro, 105, pp. 121-126. , Academic PressAllouni, Z.E., Hol, P.J., Cauqi, M.A., Gjerdet, N.R., Cimpan, M.R., Role of physicochemical characteristics in the uptake of TiO2 nanoparticles (2012) Toxicol. In Vitro, 26 (3), pp. 469-479Aoyama, H., Silva, T.M.A., Miranda, M.A., Ferreira, C.V., Proteínas tirosina fosfatases: propriedades e funções biológicas (2003) Quím Nova., 6, pp. 896-900. , PortugueseBan, S., Ohi, N., Leong, S.C.Y., Takahashi, K.T., Riser, C.W., Taguchi, S., Effect of solar ultraviolet radiation on survival of krill larvae and copepods in the Antarctic Ocean (2007) Polar Biol., 30 (10), pp. 1295-1302Banerjee, S., Gopal, J., Muraleedharan, P., Tyagi, A.K., Raj, B., Physics and chemistry of photocatalytic titanium dioxide: visualization of bactericidal activity using atomic force microscopy (2006) Curr. Sci., 90 (10), pp. 1378-1383Bar-Ilan, O., Louis, K.M., Yang, S.P., Pedersen, J.A., Hamers, R.J., Peterson, R.E., Heideman, W., Titanium dioxide nanoparticles produce phototoxicity in the developing zebrafish (2012) Nanotoxicology, 6 (6), pp. 670-679Bar-Ilan, O., Chuang, C.C., Schwahn, D.J., TiO2 nanoparticle exposure and illumination during zebrafish development: mortality at parts per billion concentrations (2013) Environ. Sci. Technol., 47 (9), pp. 4726-4733Blaise, C., Gagne, F., Ferard, J.F., Eullaffoy, P., Ecotoxicity of selected nano-materials to aquatic organisms (2008) Environ. Toxicol., 23, pp. 591-598Blokhina, O., Virolainen, E., Fagerstedt, K.V., Antioxidants, oxidative stress and oxygen deprivation stress: a review (2003) Ann. Bot., 91, pp. 179-194Boyle, D., Al-Bairuty, G.A., Henry, T.B., Handy, R.D., Critical comparison of intravenous injection of TiO2 nanoparticles with waterborne and dietary exposures concludes minimal environmentally-relevant toxicity in juvenile rainbow trout Oncorhynchus mykiss (2013) Environ. Pollut., 182, pp. 70-79Braunbeck, T., Lammer, E., (2006) Fish Embryo Toxicity Assays (UBA contract number 203 85 422), , Umwelt Bundes Amt, University of Heidelberg, Heidelberg, GermanyCampos, B., Rivetti, C., Rosenkranz, P., Navas, J.M., Barata, C., Effects of nanoparticles of TiO2 on food depletion and life-history responses of Daphnia magna (2013) Aquat. Toxicol., pp. 174-183Canesi, L., Fabbri, R., Gallo, G., Vallotto, D., Marcomini, A., Pojana, G., Biomarkers in Mytilus galloprovincialis exposed to suspensions of selected nanoparticles (Nano carbon black, C60 fullerene, Nano-TiO2, Nano-SiO2) (2010) Aquat. Toxicol., 100 (2), pp. 168-177Charron, R.A., Fenwick, J.C., Lean, D.R., Moon, T.W., Ultraviolet-B radiation effects on antioxidant status and survival in the zebrafish, Brachydanio rerio (2000) Photochem. Photobiol., 72 (3), pp. 327-333Chen, J., Dong, X., Xin, Y., Zhao, M., Effects of titanium dioxide nano-particles on growth and some histological parameters of zebrafish (Danio rerio) after a long-term exposure (2011) Aquat. Toxicol., 101, pp. 493-499Chen, T.H., Lin, C.Y., Tseng, M.C., Behavioral effects of titanium dioxide nanoparticles on larval zebrafish (Danio rerio) (2011) Mar. Pollut. Bull., 63, pp. 303-308(1999) CIE Collection in Photobiology and Photochemistry, , http://www.cie.co.at/index.php/Publications/index.php?i_ca_id=410, CIE (Commission Internationale de l'Eclaraige), Available from: (cited 07.12.11)Clemente, Z., Castro, V.L., Feitosa, L.O., Lima, R., Jonsson, C.M., Maia, A.H., Fraceto, L.F., Fish exposure to nano-TiO2 under different experimental conditions: methodological aspects for nanoecotoxicology investigations (2013) Sci. Total Environ., 463-464, pp. 647-656(2009) How do anatase an rutile differ in their photocatalytic activity, , http://www.coatingsys.com/yahoo_site_admin/assets/docs/How_do_anatase_and_rutile_differ_in_their_photocatalytic_activity.120225304.pdf, Available from, Coatingsys, (cited 26.02.13)Cong, S., Xu, Y., Explaining the high photocatalytic activity of a mixed phase TiO2: a combined effect of O2 and crystallinity (2012) J. Phys. Chem. C, 115 (43), pp. 21161-21168Cui, Y., Gong, X., Duan, Y., Hepatocyte apoptosis and its molecular mechanisms in mice caused by titanium dioxide nanoparticles (2010) J. Hazard. Mater., 183 (1-3), pp. 874-880Dong, Q., Svoboda, K., Tiersch, T.R., Monroe, W.T., Photobiological effects of UVA and UVB light in zebrafish embryos: evidence for a competent photorepair system (2007) J. Photochem. Photobiol. B, 88 (2-3), pp. 137-146Elliott, D.G., The many functions of fish integument (2011) Encyclopedia of Fish Physiology: From Genome to Environment, pp. 471-475. , Academic Press, San Diego, A.P. Farrell (Ed.)Federici, G., Shaw, B.J., Handy, R.D., Toxicity of titanium dioxide nanoparticles to rainbow trout (Onchorhynchus mykiss): gill injury, oxidative stress, and other physiological effects (2007) Aquat. Toxicol., 84 (4), pp. 415-430Fent, K., Weisbrod, C.J., Wirth-Heller, A., Pieles, U., Assessment of uptake and toxicity of fluorescent silica nanoparticles in zebrafish (Danio rerio) early life stages (2010) Aquat. Toxicol., 100, pp. 218-228Fouqueray, M., Dufils, B., Vollat, B., Effects of aged TiO2 nanomaterial from sunscreen on Daphnia magna exposed by dietary route (2012) Environ. Pollut., 163, pp. 55-61Franco, R., Sánchez-Olea, R., Reyes-Reyes, E.M., Panayiotidis, M.I., Environmental toxicity, oxidative stress and apoptosis: Ménage à Trois (2009) Mutat. Res., 674, pp. 3-22French, R.A., Jacobson, A.R., Kim, B., Isley, S.L., Penn, R.L., Baveye, P.C., Influence of ionic strength, pH, and cation valence on aggregation kinetics of titanium dioxide nanoparticles (2009) Environ. Sci. Technol., 43, pp. 1354-1359Gaya, U.I., Abdullah, A.H., Heterogeneous photocatalytic degradation of organic contaminants over titanium dioxide: a review of fundamentals, progress and problems (2008) J. Photochem. Photobiol. C: Photochem. Rev., 9, pp. 1-12Gottschalk, F., Sonderer, T., Scholz, R.W., Nowack, B., Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, Fullerenes) for different regions (2009) Environ. Sci. Technol, 43 (24), pp. 9216-9222Grassian, V.H., Adamcakova-Dodd, A., Pettibone, J.M., O'Shaughnessy, P.T., Thorne, P.S., Inflammatory response of mice to manufactured titanium dioxide nanoparticles: comparison of size effects through different exposure routes (2007) Nanotoxicology, 1 (3), pp. 211-226Griffith, R.J., Luo, J., Gao, J., Bonzongo, J.C., Barber, D.S., Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms (2008) Environ. Toxicol. Chem., 27 (9), pp. 1972-1978Hao, L., Wang, Z., Xing, B., Effect of sub-acute exposure to TiO2 nanoparticles on oxidative stress and histopathological changes in juvenile carp (Cyprinus carpio) (2009) J. Environ. Sci., 21 (10), pp. 1459-1466Howe, K., Clark, M.D., Torroja, C.F., The zebrafish reference genome sequence and its relationship to the human genome (2013) Nature, 496, pp. 498-503Kaweewat, K., Hofer, R., Effect of UV-B radiation on goblet cells in the skin of different fish species (1997) J. Photochem. Photobiol. B: Biol., 41 (3), pp. 222-226Keen, J.H., Habig, W.H., Jakoby, W.B., Mechanism for several activities of the glutathione S-transferase (1976) J. Biol. Chem., 251, pp. 6183-6188Kim, K.T., Klaine, S.J., Cho, J., Kim, S.H., Kim, S.D., Oxidative stress responses of Daphnia magna exposed to TiO2 nanoparticles according to size fraction (2010) Sci. Total Environ., 408, pp. 2268-2272Kiser, M.A., Westerhoff, P., Bennt, T., Wang, Y., Pérez-Rivera, J., Hristovsk, K., Titanium nanomaterial removal and release from wastewater treatment plants (2009) Environ. Sci. Technol., 43, pp. 6757-6763Knobel, M., Basser, F.J.M., Rico, A.R., Kramer, N.I., Hermens, J.M.L., Hafner, C., Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoint, compound properties, and exposure concentration analysis (2012) Environ. Sci. Technol., 46, pp. 9690-9700Lammer, E., (2009) The Faculty of Bio Sciences>Institut für Zoologie. Refinement of the fish embryo toxicity test (FET) with zebrafish (Danio rerio): Is it a real replacement of the acute fish toxicity test?, , http://archiv.ub.uni-heidelberg.de/volltextserver/9552/, (Dissertation)Lardinois, O.M., Mestdagh, M.M., Rouxhet, P.G., Reversible inhibition and irreversible inactivation of catalase in presence of hydrogen peroxide (1996) BBA - Protein Struct. M, 1295 (2), pp. 222-238Lawrence, C., The husbandry of zebrafish (Danio rerio): a review (2007) Aquaculture, 269, pp. 1-20Leung, T.S., Bulkley, R.V., Effects of petroleum hydrocarbons on length of incubation and hatching success in Japanese medaka (1976) Bull. Environ. Contam. Toxicol., 23, pp. 236-243Lin, S., Zhao, Y., Nel, A.E., Lin, S., Zebrafish: an in vivo model for nano EHS studies (2013) Small, 9 (9-10), pp. 1608-1618Ma, H., Brennan, A., Diammond, S.A., Photocatalytic reactive oxygen species production and phototoxicity of titanium dioxide nanoparticles are dependent on the solar ultraviolet radiation spectrum (2012) Environ. Toxicol. Chem., 31 (9), pp. 2099-2107Ma, H., Brennan, A., Diamond, S.A., Phototoxicity of TiO2 nanoparticles under solar radiation to two aquatic species: Daphnia magna and Japanese medaka (2012) Environ. Toxicol. Chem., 31 (7), pp. 1621-1629Malato, S., Fernández-Ibáñez, P., Maldonado, M.I., Blanco, J., Gernjak, W., Decontamination and disinfection of water by solar photocatalysis: recent overview and trends (2009) Catal. Today, 147, pp. 1-59Marcone, G.P.S., Oliveira, A.C., Almeida, G., Umbuzeiro, G.A., Jardim, W.F., Ecotoxicity of TiO2 to Daphnia similis under irradiation (2012) J. Hazard. Mater., pp. 436-442Massarsky, A., Dupuis, L., Taylor, J., Eisa-Beygi, S., Strek, L., Trudeau, V.L., Moon, T.W., Assessment of nanosilver toxicity during zebrafish (Danio rerio) development (2013) Chemosphere, 92, pp. 59-66Nogueira, R.F.P., Jardim, W.F., A fotocatálise heterogênea e sua aplicação ambiental (1998) Quim. Nova, 21 (1), pp. 69-72(1992) Fish, acute toxicity test, , OECD Test Guideline 203Pagnout, C., Jomini, S., Dadhwal, M., Caillet, C., Thomas, F., Bauda, P., Role of electrostatic interactions in the toxicity of titanium dioxide nanoparticles toward Escherichia coli (2012) Colloid Surf. B, 92, pp. 315-321Palaniappan, P.L.R.M., Pramod, K.S., FTIR study of the effect of nTiO2 on the biochemical constituents of gill tissues of zebrafish (Danio rerio) (2010) Food Chem. Toxicol., 48 (8-9), pp. 2337-2343Paterson, G., Ataria, J.M., Hoque, M.E., Burns, D.C., Metcalfe, C.D., The toxicity of titanium dioxide nanopowder to early life stages of the Japanese medaka (Oryzias latipes) (2011) Chemosphere, 82, pp. 1002-1009Prazeres, J.N., Ferreira, C.V., Aoyama, H., Acid phosphatase activities during the germination of glycine max seeds (2004) Plant Physiol. Biochem., 42, pp. 15-20Rawson, D.M., Zhang, T., Kalicharan, D., Jongebloed, W.L., Field emission scanning electron microscopy and transmission electron microscopy studies of the chorion, plasma membrane and syncytial layers of the gastrula-stage embryo of the zebrafish Brachydanio rerio: a consideration of the structural and functional relationships with respect to cryoprotectant penetration (2000) Aquac. Res., 31, pp. 325-336Robichaud, C.O., Uyar, A.E., Darby, M.R., Zucker, L.G., Wiesner, M.R., Estimates of upper bounds and trends in nano-TiO2 production as a basis for exposure assessment (2009) Environ. Sci. Technol., 43, pp. 4227-4233Scown, T.M., Aerle, R., Johnston, B.D., Cumberland, S., Lead, J.R., Owen, R., Tyler, C.R., High doses of intravenously administered titanium dioxide nanoparticles accumulate in the kidney of rainbow trout but with no observable impairment of renal function (2009) Toxicol. Sci., 109 (2), pp. 372-380Stewart, H.S., Hopfield, R.F., Atmospheric effects (1965) Applied Optics and Optical Engineering (vol. 1), pp. 127-152. , Academic Press, New York, NY, USA, R. Kingslake (Ed.)Sun, Q., Xu, Y., Evaluating intrinsic photocatalytic activities of anatase and autile TiO2 for organic degradation in water (2010) J. Phys. Chem. C, 114 (44), pp. 18911-18918Tong, T., Binh, C.T.T., Kelly, J.J., Gaillard, J.F., Gray, K.A., Cytotoxicity of commercial nano-TiO2 to Escherichia coli assessed by high-throughput screening: effects of environmental factors (2013) Water Res., 47, pp. 2352-2362(2010) EPA/600/R-10/089. State of the Science Literature Review: Nano Titanium Dioxide - Environmental Matters. Scientific, Technical, Research, Engineering and Modeling Support (STREAMS) Final Report, , U.S Environmental Protection Agency (U.S.EPA), Office of Research and Development, WashingtonVutukuru, S.S., Raparthi, S., Basani, K., Acute exposure to nano titanium dioxide cause biochemical and physiological alterations in the Zebrafish (Danio rerio) - a case study (2013) Int. J. ChemTech Res., 5 (2), pp. 646-653Wang, J., Chen, C., Liu, Y., Potential neurological lesion after nasal instillation of TiO2 nanoparticles in the anatase and rutile crystal phases (2008) Toxicol. Lett., 183 (1-3), pp. 72-80Xiong, D., Fang, T., Yu, L., Sima, X., Zhu, W., Effects of nano-scale TiO2, ZnO and their bulk counterparts on zebrafish: acute toxicity, oxidative stress and oxidative damage (2011) Sci. Total Environ., 409, pp. 1444-1452Xiong, S., George, S., Ji, Z., Size of TiO2 nanoparticles influences their phototoxicity: an in vitro investigation (2013) Arch. Toxicol., 87, pp. 99-109Yeo, M.K., Kang, M., The effect of nano-scale Zn-doped TiO2 and pure TiO2 particles on Hydra magnipapillata (2010) Mol Cell Toxicol., 6, pp. 9-17Zhang, J., Wages, M., Cox, S.B., (2012) Effect of titanium dioxide nanomaterials and ultraviolet light coexposure on African clawed frogs (Xenopus laevis), 31 (1), pp. 176-183Zhu, X., Zhu, L., Duan, Z., Qi, R., Li, Y., Lang, Y., Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to zebrafish (Danio rerio) early developmental stage (2008) J. Environ. Sci. Health A, 43, pp. 278-284Zhu, X., Chang, Y., Chen, Y., Toxicity and bioaccumulation of TiO2 nanoparticle aggregates in Daphnia magna (2010) Chemosphere, 78, pp. 209-215Zhu, X., Zhou, J., Cai, Z., The toxicity and oxidative stress of TiO2 nanoparticles in marine abalone (Haliotis diversicolor supertexta) (2011) Mar. Pollut. Bull., 63 (5-12), pp. 334-338