Oxidative stress and genotoxicity of an organic and an inorganic nanomaterial to Eisenia andrei: SDS/DDAB nano-vesicles and titanium silicon oxide.

In the past few years the number of studies on the toxic effects of nanomaterials (NMs) in the environment increased significantly. Nonetheless, the data is still scarce, since there is a large number of NMs and new ones are being developed each day. Soils are extremely important for life, and are easily exposed to the released NMs, thus enhanced efforts are needed to study the impacts on soil biota. The objective of the present work was to determine if different concentrations of two NMs, one inorganic (TiSiO4) and other organic (nano-vesicles of sodium sodecyl sulfate/ didodecyl dimethylammonium bromide - SDS/DDAB), are genotoxic to soil invertebrates. Additionally, it was intended to understand whether, in the event of occurring, genotoxicity was caused by the incapability of the cells to deal with the oxidative stress caused by these NMs. With that purpose, Eisenia andrei were exposed for 30 days to the artificial OECD soil contaminated with different concentrations of the NMs being tested. After the exposure, coelomocytes were extracted from earthworms and DNA damage was measured by the comet assay. The activity of antioxidant enzymes (e.g. glutathione peroxidase, glutathione reductase and glutathione-S-Transferase) and lipid peroxidation were also assessed. The results showed that both NMs were genotoxic, particularly TiSiO4 for which significant DNA damages were recorded for concentrations above 444mg of TiSiO4-NM/kg of soildw. Since no statistically significant differences were found in the tested antioxidant enzymes and in lipid peroxidation, the mechanism of genotoxicity of these NMs seemed to be unrelated with oxidative stress.

Ecotoxicity of titanium silicon oxide (TiSiO4) nanomaterial for terrestrial plants and soil invertebrate species.

The huge evolution of nanotechnology and the commercialization of nanomaterials (NMs) positively contributed for innovation in several industrial sectors. Facing this rapid development and the emergence of NMs in the market, the release of this nanometric sized materials in the environment and the possible impact on different ecosystem components attracted the attention of researchers in the last few years. In our study we aimed to assess the impact of titanium silicon oxide nanomaterial (nano-TiSiO4) on soil biota to estimate a risk limit for this material. In the present research a battery of standardized ecotoxicological assays aimed at evaluating a wide range of endpoints (avoidance and reproduction of earthworms and collembolans, emergence/growth of four selected terrestrial plants) were carried out, using OECD artificial soil as test substrate spiked with aqueous suspension of different concentrations of nano-TiSiO4. The results showed a maximum avoidance percentage of 40% for earthworms (Esenia andrei) at the highest concentration tested (1000mgkg(-1) soildw of nano-TiSiO4). No significant effect on the reproductive function of both invertebrate species was recorded. Nevertheless, significant phytotoxic data was registered at least for the growth of dicotyledonous plant species (Lactuca sativa and Lycopersicon lycopersicum) with EC20 values ranging between 236 and 414 mg kg(-1) soildw of nano-TiSiO4 for L. sativa dry mass and fresh mass, respectively. Further, the characterization of nano-TiSiO4 in suspensions used to spike the soil, performed by Dynamic Light Scattering, showed the formation of aggregates with important average size diameter, thus demonstrating that the toxic effects observed were likely not size dependent. A deterministic PNEC (predicted no effect concentration) for this NM of 10.02mg kg(-1) soildw of nano-TiSiO4, is suggested, while no more ecotoxicological information exists.

Ecotoxicity to Freshwater Organisms and Cytotoxicity of Nanomaterials: Are We Generating Sufficient Data for Their Risk Assessment?

The aim of the present study was to investigate the eco-cytotoxicity of several forms of nanomaterials (NM), such as nano-CuO, nano-TiO2, nano-SiO2 and nano-ZnO, on different aquatic species (Raphidocelis subcapitata, Daphnia magna and Lemna minor) following standard protocols and on human cell lines (Caco-2, SV-80, HepG2 and HaCaT). Predicted no-effect concentrations (PNEC) or hazard concentrations for 5% of the species (HC5) were also estimated based on the compilation of data available in the literature. Most of the NM agglomerated strongly in the selected culture media. For the ecotoxicity assays, nano-CuO and nano-ZnO even in particle agglomeration state were the most toxic NM to the freshwater organisms compared to nano-TiO2 and nano-SiO2. Nano-ZnO was the most toxic NM to R. subcapitata and D. magna, while nano-CuO was found to be very toxic to L. minor. Nano-CuO was very toxic to Caco-2 and HepG2 cells, particularly at the highest tested concentrations, while the other NM showed no toxicity to the different cell lines. The HC5 and PNEC values are still highly protective, due to data limitations. However, the present study provides consistent evidence of the potential risks of both nano-CuO and nano-ZnO against aquatic organisms and also their effects on public health.

Dataset of the preparation and characterization of an artificial sludge for ecotoxicological purposes.

This work describes the protocol developed for preparing an artificial digested sludge to be used for the ecotoxicological evaluation and risk assessment of chemical substances that are previewed to attain the soil through sewage sludge applications for fertilization (e.g. pharmaceutical compounds). Such evaluations have been made with standard protocols for ecotoxicological tests with terrestrial species in which the chemical substances are directly spiked to the test soil. This procedure lacks ecological relevance in terms of the role the sludge organic matter plays on the bioavailability and fate of chemical substances. Here we describe the protocol and the composition for obtaining the artificial sludge, prepared with commercial pet food and eggs white to mimic the composition of domestic sewage sludge in terms of proteins, carbon hydrates, fat and fibers content. Further, the conditions ascertained for the anaerobic digestion of the organic mixture are described, and the final properties of the sludge are presented, after repeating the procedure twice.

Contribution for the derivation of a soil screening value (SSV) for uranium, using a natural reference soil.

In order to regulate the management of contaminated land, many countries have been deriving soil screening values (SSV). However, the ecotoxicological data available for uranium is still insufficient and incapable to generate SSVs for European soils. In this sense, and so as to make up for this shortcoming, a battery of ecotoxicological assays focusing on soil functions and organisms, and a wide range of endpoints was carried out, using a natural soil artificially spiked with uranium. In terrestrial ecotoxicology, it is widely recognized that soils have different properties that can influence the bioavailability and the toxicity of chemicals. In this context, SSVs derived for artificial soils or for other types of natural soils, may lead to unfeasible environmental risk assessment. Hence, the use of natural regional representative soils is of great importance in the derivation of SSVs. A Portuguese natural reference soil PTRS1, from a granitic region, was thereby applied as test substrate. This study allowed the determination of NOEC, LOEC, EC20 and EC50 values for uranium. Dehydrogenase and urease enzymes displayed the lowest values (34.9 and <134.5 mg U Kg, respectively). Eisenia andrei and Enchytraeus crypticus revealed to be more sensitive to uranium than Folsomia candida. EC50 values of 631.00, 518.65 and 851.64 mg U Kg were recorded for the three species, respectively. Concerning plants, only Lactuca sativa was affected by U at concentrations up to 1000 mg U kg(1). The outcomes of the study may in part be constrained by physical and chemical characteristics of soils, hence contributing to the discrepancy between the toxicity data generated in this study and that available in the literature. Following the assessment factor method, a predicted no effect concentration (PNEC) value of 15.5 mg kg-1dw was obtained for U. This PNEC value is proposed as a SSV for soils similar to the PTRS1.

Can physiological endpoints improve the sensitivity of assays with plants in the risk assessment of contaminated soils?

Site-specific risk assessment of contaminated areas indicates prior areas for intervention, and provides helpful information for risk managers. This study was conducted in the Ervedosa mine area (Bragança, Portugal), where both underground and open pit exploration of tin and arsenic minerals were performed for about one century (1857-1969). We aimed at obtaining ecotoxicological information with terrestrial and aquatic plant species to integrate in the risk assessment of this mine area. Further we also intended to evaluate if the assessment of other parameters, in standard assays with terrestrial plants, can improve the identification of phytotoxic soils. For this purpose, soil samples were collected on 16 sampling sites distributed along four transects, defined within the mine area, and in one reference site. General soil physical and chemical parameters, total and extractable metal contents were analyzed. Assays were performed for soil elutriates and for the whole soil matrix following standard guidelines for growth inhibition assay with Lemna minor and emergence and seedling growth assay with Zea mays. At the end of the Z. mays assay, relative water content, membrane permeability, leaf area, content of photosynthetic pigments (chlorophylls and carotenoids), malondialdehyde levels, proline content, and chlorophyll fluorescence (Fv/Fm and ΦPSII) parameters were evaluated. In general, the soils near the exploration area revealed high levels of Al, Mn, Fe and Cu. Almost all the soils from transepts C, D and F presented total concentrations of arsenic well above soils screening benchmark values available. Elutriates of several soils from sampling sites near the exploration and ore treatment areas were toxic to L. minor, suggesting that the retention function of these soils was seriously compromised. In Z. mays assay, plant performance parameters (other than those recommended by standard protocols), allowed the identification of more phytotoxic soils. The results suggest that these parameters could improve the sensitivity of the standard assays.