Recent developments on environmental fate models indicate that as nano waste, engineered nanomaterials/nanoparticles (ENM/Ps) could reach terrestrial ecosystems thus potentially affecting environmental and human health. Plants can be therefore exposed to ENM/Ps 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 ENM/Ps undergo in the natural environment as for instance with existing toxic compounds. The main aim of current study is to evaluate potential toxicological risks due to the exposure of plants to ENM/Ps in their natural environment, and investigating different routes of exposure (i.e. water and soil). The aim of the first study reported in chapter 1 was to asses 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 parvus) as model species. Radish seeds were exposed to different concentrations of n-TiO2 (range 1-1000 mg/L) and CdCl2 ( range 1-250 mg/L) alone and in combination using a seed germination and seedling growth toxicity test OECD 208. Percentages 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. Exposure of 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 250 mg/L CdCl2) compared to co-exposed seeds at low concentration (1mg/L n-TiO2 and 1 mg/L CdCl2) and controls (p<0.05). Root elongation significantly increase compared to controls at the lowest co-exposure concentration (p<0.05). Similarly at intermediate concentrations of 10 and 100 mg/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 (1000 mg/L) was observed while n-TiO2 (1 mg/ L), CdCl2 (1 and 250 mg/L) and co-exposure resulted significantly decreased compared to controls (p<0.05). Regarding GST activity, a slight increase in seeds exposed to 1000 mg/L n-TiO2 but no significantly was observed, however both n-TiO2 and CdCl2 alone (1 and 250 mg/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 250 mg/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 250 mg/L), interactions cannot be excluded based on obtained results. The aim of the second study reported in chapter 2 was to assess if the presence of n-TiO2 might affect elutriate toxicity to radish seeds (R. sativus parvus) seeds as a model species. Radish seeds were exposed to 11 soils (elutriates) alone and in combination with 1 mg/L of n-TiO2 collected from an industrial site located in Taranto area (South East of Italy). Exposure of seeds was performed according to OECD (208) guideline. Then, root elongation, percentages of seed germination and germination index% (GI) were analyzed. In addition, levels of several trace elements were also determined in soils in order to assess their level of contamination and effects on root elongation, seed germination and GI% further discussed. Main results revealed that the presence of n-TiO2 seems not affecting root length, GI % and germination% of seeds compared to seeds exposed to elutriates alone with the exception of only 2 sites. Moreover, the absence of any clear relationship between effects of elutriate on radish seed germination and trace elements levels was observed. Only slight but not significant changes based on levels of trace elements present in soil were observed in growth parameters. In particular levels of Co, Ni, Zn, Cu, Ti and Sn seem to affect radish seeds germination more than others. Regarding co-exposed seeds, the presence of n-TiO2 caused 100% of germination of radish seeds. Furthermore, in comparison to exposed seeds to elutriates alone, root length and GI % resulted more stimulated. Only slight effects on GI% and root length were observed which might be linked to interaction of these elements with n-TiO2. Likewise, it seemed that Co, Se, Sb and As in presence of n-TiO2 are responsible for changes on growth parameters. According on the overall results, soil elutriates alone could not be able to show real toxicity of a contaminated soil on seeds germination and future study should be performed in order to assess their suitability in real exposure scenarios. Therefore, based on observed data further investigations are required in order to assess real environmental scenarios where such particles could be present in soils together with existing contaminants such trace elements. The purpose of third study reported in chapter 3 chapter was to assess the impact of n-TiO2 alone and in combination with CdCl2 on germination and growth of radish seeds (R. sativus) exposed in vitro (experiment 1) and in vivo (directly into soils) (experiment 2). In experiment 1(in vitro) radish seeds were exposed to n-TiO2 (1 and 1000 mg/L and CdCl2) and CdCl2 (1 and 250 mg/L) alone and in combination (n-TiO2 1, 10, 100, 1000 mg/L and CdCl2 1, 10, 100, 250 mg/L) using a seed germination and seedling growth toxicity test OECD 208. In experiment 2 (in vivo), radish exposed only to water and then seedling transferred to soils contaminated with n-TiO2 (1 and 1000 mg/L and CdCl2) and CdCl2 (1 and 250 mg/L) alone and in combination (n-TiO2 1, 10, 100, 1000 mg/L and CdCl2 1, 10, 100, 250 mg/L), still following OECD 208 test conditions. Root length, shoot length and numbers of secondary leaves of all plants from the two experiments (1 and 2) were recorded at day 10 and day 21. Growth parameters of radish at both day 10 and day 21 showed that plants from seeds exposed during germination (experiment 1) resulted more affected by single and co-exposure to n-TiO2 and CdCl2 than those exposed directly in soil (experiment 2). Furthermore, presence of CdCl2 at 250 mg/L alone and in combination with 1000 mg/L of n-TiO2 seemed affect the root and shoot length in both experiments 1and 2 at day 10 and day 21. Growth parameter analysis of single and co-exposed groups in experiment 1 at day 10, showed a decrease in root length in all tested plants with exception of those exposed to n-TiO2 (1mg/L), co-exposed to n-TiO2 and CdCl2 (1mg/L and 1mg/L), (10 mg/L and 10 mg/L) and (100 mg/L and 10 mg/L) which showed slight increase compared to control. In experiment 2 at day10 only exposed plants to 1000 mg/kg of n-TiO2 revealed significant increase of root length while other all single and co-exposure groups showed a decrease of root length respect to control. Shoot length in exposed plants to all single and co-exposure groups in both experiments 1 and 2 at day 10 showed a decrease compared to control except plants exposed to n-TiO2 and CdCl2 (100 mg/kg and 10 mg/kg) in experiment 2 which showed an increase. Obtained results on day 21 showed a decrease of root length respect to control on tested plants to all single and co-exposure groups in both experiments 1 and 2 with exception of exposed radish to 1 mg/L of CdCl2 in experiment 1. Shoot length of all tested single and co-exposure groups in experiments 1 and 2 showed a decrease compared to control except radish exposed to 1000 mg/kg of n-TiO2 which revealed an increase in experiment 2. Regarding secondary leaves, in both experiments 1and 2 at day 10 no leaves were shown. On the opposite, (2 leaves) were present at day 21 in most plants exposed to single and in combination, while those exposed to CdCl2 (250 mg/kg), n-TiO2 and CdCl2 (10 and 100 mg/L) and (1000 mg/kg and 250 mg/kg) in experiment 1 showed no leaves. Likewise, exposure n-TiO2 (1000 mg/kg) and co-exposure of n-TiO2 and CdCl2 (10 mg/kg and 100) showed only one secondary leaf in experiment 2 at day 21.
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