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In the current study, the toxicity mechanism of nanosized CuO (nCuO) to the freshwater ciliated protozoa Tetrahymena thermophila was studied. Changes in fatty acid profile, lipid peroxidation metabolites and reactive oxygen species (ROS) were measured. Bulk CuO and CuSO(4) served as controls for size and solubility and 3,5-dichorophenol (3,5-DCP) as a control for a chemical known to directly affect the membrane composition. Exposure to all copper compounds induced the generation of ROS, whereas nCuO was most potent. The latter effect was not solely explained by solubilized Cu-ions and was apparently particle-related. 24 h exposure of protozoa to 80 mg/L of nCuO (EC50) significantly decreased the proportion of two major unsaturated fatty acids (UFA) (C18:3 cis-6,9,12, C18:2 cis-9,12), while it increased the relative amount of two saturated fatty acids (SFA) (C18:0, C16:0). Analogous effect was not observed when protozoa were exposed to equitoxic suspensions of bulk CuO, Cu-ions or 3,5-DCP. As changes in the UFA:SFA upon exposure of protozoa to nCuO were not detected at 2 h exposure and no simultaneous dose- or time-dependent lipid peroxidation occurred, it is likely that one of the adaptation mechanisms of protozoa to nCuO was lowering membrane fluidity by the inhibition of de novo synthesis of fatty acid desaturases. This is the first study of the effects of nanoparticles on the membrane fatty acid composition.