Co-exposure to titanium dioxide nanoparticles does not affect cadmium toxicity in radish seeds (Raphanus sativus)

Recent developments on environmental fate models indicate that as nano waste, engineered nanomaterials (ENMs) could reach terrestrial ecosystems thus potentially affecting environmental and human health. Plants can be therefore exposed to ENMs but controversial data in terms of fate and toxicity are currently available. Furthermore, there is a current lack of information on complex interactions/transformations to which ENMs undergo in the natural environment as for instance interacting with existing toxic compounds. The aim of the present study was to assess the behavior and biological effects of titanium dioxide nanoparticles (n-TiO₂) (Aeroxide P25, Degussa Evonik) and its interaction with cadmium (CdCl₂) in plants using radish seeds (Raphanus sativus L. Parvus) as model species. Radish seeds were exposed to n-TiO₂ (1-1000mg/L) and CdCl₂ (1-250mg/L) alone and in combination using a seed germination and seedling growth toxicity test OECD 208. Percentage of seed germination, germination index (GI) and root elongation were calculated. Cell morphology and oxidative stress parameters as glutathione-S-transferase (GST) and catalase activities (CAT) were measured in radish seeds after 5 days of exposure. Z-Average, PdI and Z-potential of n-TiO₂ in Milli-Q water as exposure medium were also determined. DLS analysis showed small aggregates of n-TiO₂, negative Z-potential and stable PdI in seed's exposure media. Germination percentage, GI and root length resulted affected by n-TiO₂ exposure compared to controls. In particular, n-TiO₂ at 1mg/L and 100mg/L did not affect radish seeds germination (100%) while at concentration of 10mg/L, 200mg/L, 500mg/L, and 1000mg/L a slight but not significant decrease of germination % was observed. Similarly root length and GI resulted significantly higher in seeds exposed to 10mg/L and 200mg/L compared to 1mg/L, 100mg/L, 500mg/L, 1000mg/L and control (p < 0.05). On the opposite, CdCl₂ significantly abolished germination % and GI compared to control seeds and a concentration dependent decrease on root elongation was observed against controls (p < 0.05). As well, significant decrease of germination %, GI and root elongation was observed in seeds co-exposed to n-TiO₂ and CdCl₂ at the highest concentrations (1000mg/L n-TiO₂ and 250mg/L CdCl₂) compared to co-exposed seeds at low concentration (1mg/L n-TiO₂ and 1mg/L CdCl₂) and controls (p < 0.05). Root elongation significantly increase compared to control at the lowest co-exposure concentration (p < 0.05). Similarly at intermediate concentrations of 10 and 100mg/L in co-exposure conditions, n-TiO₂ did not affect CdCl₂ toxicity. Concerning antioxidant enzymes, a significant increase of CAT activity in seeds exposed to single high n-TiO₂ concentration (1000mg/L) was observed while n-TiO₂ (1mg/L), CdCl₂ (1 and 250mg/L) and co-exposure resulted significantly decreased compared to controls (p < 0.05). Regarding GST activity, a slight increase in seeds exposed to 1000mg/L n-TiO₂ but no significantly was observed, however both n-TiO₂ and CdCl₂ alone (1 and 250mg/L, respectively) or in combinations caused a significant decrease in GST activity (p < 0.05). Therefore, overall data support the hypothesis that the presence of n-TiO₂ do not affect the toxicity of CdCl₂ at least at the highest concentration (100 and 250mg/L) in radish seeds. Morphological alterations in nuclei, vacuoles and shape of radish root cells were observed upon single Cd exposure and not abolished in the presence of n-TiO₂. Nevertheless, although n-TiO₂ seems not to reduce Cd toxicity at high concentration (up to 250mg/L), interactions cannot be excluded based on obtained results.