Aquatic Toxicology 168 (2015) 90–97 Contents lists available at ScienceDirect Aquatic Toxicology j o ur na l ho me pag e: www.elsevier.com/locate/aquatox On the mechanism of nanoparticulate CeO 2 toxicity to freshwater algae Brad M. Angel a,∗ , Pascal Vallotton b , Simon C. Apte a a Centre for Environmental Contaminants Research, CSIRO Land and Water Flagship, Locked Bag 2007, Kirrawee, NSW 2232, Australia b Digital Productivity Flagship, CSIRO, North Ryde, NSW 1670, Australia a r t i c l e i n f o Article history: Received 26 July 2015 Received in revised form 22 September 2015 Accepted 27 September 2015 Available online 1 October 2015 Keywords: Cerium dioxide ROS Membrane sorption CytoViva Dissolved organic carbon a b s t r a c t The factors affecting the chronic (72-h) toxicity of three nanoparticulate (10–34 nm) and one micron- sized form of CeO 2 to the green alga, Pseudokirchneriella subcapitata were investigated. To characterise transformations in solution, hydrodynamic diameters (HDD) were measured by dynamic light scatter, zeta potential values by electrophoretic mobility, and dissolution by equilibrium dialysis. The protective effects of humic and fulvic dissolved organic carbon (DOC) on toxicity were also assessed. To investi- gate the mechanisms of algal toxicity, the CytoViva hyperspectral imaging system was used to visualise algal–CeO 2 interactions in the presence and absence of DOC, and the role of reactive oxygen species (ROS) was investigated by ‘switching off’ ROS production using UV-filtered lighting conditions. The nanoparticulate CeO 2 immediately aggregated in solution to HDDs measured in the range 113–193 nm, whereas the HDD and zeta potential values were significantly lower in the presence of DOC. Negligible CeO 2 dissolution over the time course of the bioassay ruled out potential toxicity from dissolved cerium. The nanoparticulate CeO 2 concentration that caused 50% inhibition of algal growth rate (IC50) was in the range 7.6–28 mg/L compared with 59 mg/L for micron-sized ceria, indicating that smaller particles were more toxic. The presence of DOC mitigated toxicity, with IC50s increasing to greater than 100 mg/L. Significant ROS were generated in the nanoparticulate CeO 2 bioassays under normal light conditions. However, ‘switching off’ ROS under UV-filtered light conditions resulted in a similar IC50, indicating that ROS generation was not the toxic mechanism. The CytoViva imaging showed negligible sorption of nanoparticulate CeO 2 to algal cells in the presence of DOC, and strong sorption in its absence, suggesting that this was the toxic mechanism. The results suggest that DOC in natural waters will coat CeO 2 particles and mitigate toxicity to algal cells. Crown Copyright © 2015 Published by Elsevier B.V. All rights reserved. 1. Introduction Nanotechnology has developed rapidly in the last decade, as nanomaterials are incorporated into an ever-increasing number of products. Nanoparticulate cerium dioxide (CeO 2 ) has been used increasingly in applications such as ceramics, photosensitive glass, fuel catalysts, sunscreens and paints, raising a number of concerns regarding their exposure, fate, effects and mechanisms of toxicity to aquatic organisms (Rogers et al., 2010; Röhder et al., 2014; Van Hoecke et al., 2009). Important issues for regulators are (i) whether the small size of nanoparticles will result in greater toxicity to aquatic organ- isms in natural waters than the corresponding bulk forms that are ∗ Corresponding author. Fax: +61 2 9710 6800. E-mail address: Brad.Angel@csiro.au (B.M. Angel). often already regulated; and (ii) the role of environmental trans- formations in modifying the toxicity of the nanomaterials. The mechanisms of toxicity of various metal and metal oxide nanopar- ticles have included exposure to dissolved metals via dissolution, oxidative damage caused by reactive oxygen species generated by photocatalysis, and for algae, shading effects, nutrient limitation and membrane damage (Angel et al., 2013; He et al., 2012; Campos et al., 2013; Fang et al., 2010; Franklin et al., 2007; Krishnamoorthy et al., 2014; Rogers et al., 2010). For example, similar rates of disso- lution explained why nanoparticulate and bulk ZnO had the same toxicity to the freshwater algae, Psuedokirchneriella subcapitata, (Franklin et al., 2007), and why nanoparticulate silver was more toxic than bulk silver to P. subcapitata and the freshwater daphnid, Ceriodaphnia dubia (Angel et al., 2013). For insoluble particles such as TiO 2 and CeO 2 , dissolution does not explain toxicity; hence, other mechanisms must be involved (Rogers et al., 2010; Schultz et al., 2014; Van Hoecke et al., 2009). http://dx.doi.org/10.1016/j.aquatox.2015.09.015 0166-445X/Crown Copyright © 2015 Published by Elsevier B.V. All rights reserved.