Toxicity effects induced by nanosuspensions of CuO (<50 nm; Sigma-Aldrich) on macrophytic algae cells of Nitellopsis obtusa (96-h median lethal concentration [LC50]), microphytic algae Chlorella (30-min median inhibitory concentration [IC50]), shrimp Thamnocephalus platyurus (24-h LC50), and rotifer Brachionus calyciflorus (24-h LC50) were investigated. No substantial differences between the effects of nonsonicated and sonicated nCuO suspensions were observed. The particle size distribution analysis accomplished by the laser diffraction technique at suspension concentration from 3 to 100 mg/L revealed rapid (within 5 min) reagglomeration of the particles after the sonication. The observed adverse effects on N. obtusa cells may be attributed to nanoparticles per se, but not to ionic Cu, because neither chemical analysis nor biological testing (algae survival in the supernatants of suspensions) confirmed the presence of cupric ions in toxic amounts. Contrary to ionic Cu form, nCuO delayed the initial phase of N. obtusa cell membrane depolarization. Lethality tests with rewash demonstrated that the least used 5-min exposure in 100 mg/L nCuO sonicated suspension induced 70% mortality in charophyte cells after 8 d, whereas the rewash after a short exposure to a noticeably toxic concentration of Cu(2+) prevented cell mortality. The obtained data suggested the possible influence of a thick charophyte cell wall on the dynamics of nanotoxicity effects.

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Cu accumulation in the internodal cell of charophyte Nitellopsis obtusa or its compartments was investigated after 3-h-exposure to lethal effective concentrations (8-day LC) of CuO nanoparticle (nCuO) suspension or CuSO solution, i.e. 100 mg/L nCuO or 3.18 mg Cu/L as CuSO. In both cases, the major part of Cu accumulated in the cell walls. The presence of CuO NPs in the cell wall and within the cell was visualized by scanning electron microscope images as well as confirmed by energy dispersive X-ray spectrum data. Although a threefold higher intracellular concentration of Cu was found after treatment with nCuO suspension, 3.18 mg Cu/L as CuSO induced fast and substantial depolarization of cell membrane potential contrary to that of 100 mg/L nCuO. A delayed effect of nCuO on the survival of the cells was also observed. This suggests that internally accumulated Cu was far less active and further supports the hypothesis of delayed toxicity of internalized nCuO NPs to charophyte cells.

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