Protein profiling as early detection biomarkers for TiO2 nanoparticle toxicity in Daphnia magna.

The mode of action for nanoparticle (NP) toxicity in aquatic organisms is not yet fully understood. In this work, a strategy other than toxicity testing was applied to Daphnia magna exposed to TiO2-NPs: the use of nuclear microscopy and the assessment of protein profile. D. magna is a keystone species broadly used as a model system in ecotoxicology. Titanium (Ti) was found in the D. magna digestive tract, mainly in the gut. The penetration of Ti into the epithelial region was greater at higher exposure levels and also observed in eggs in the brood pouch. The protein profile of individuals exposed to different concentrations showed that 2.8 and 5.6 mg/L TiO2-NP concentrations induced an over-expression of the majority of proteins, in particular proteins with molecular weight of ∼120, 85 and 15 kDa, while 11.2 mg/L TiO2-NP had an inhibitory effect on protein expression. The Matrix-assisted laser desorption ionization with tandem time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis of these proteins consistently identified them as vitellogenin (Vtg)-like proteins, associated with enzymes involved in redox balance. These results indicate that Vtg-like proteins are up-regulated in D. magna exposed to TiO2-NPs. Vitellogenesis is associated with the reproduction system, suggesting that TiO2-NP exposure can impair reproduction by affecting this process. The precise mode of action of TiO2-NPs is still unclear and the results from this study are a first attempt to identify specific proteins as potential markers of TiO2-NP toxicity in D. magna, providing useful information for future research.

Liver alterations in two freshwater fish species (Carassius auratus and Danio rerio) following exposure to different TiO2 nanoparticle concentrations.

The toxicity of titanium dioxide nanoparticles (TIO2 NPs) and oxidative stress effects were studied in two freshwater fish species (Carassius auratus and Danio rerio) exposed for 21 days to different concentrations (0.01, 0.1, 1, 10, 100/mgL) of TiO2 NPs and to a control (tap water). Additional fish were transferred to clean water for 14 days to assess the ability to recover from exposure to TiO2 NPs. Activities of the enzyme glutathione-S-transferase (GST) and lipid peroxidation (LPO) (malondialdheyde) were measured as indicators of oxidative stress. Histological and ultra-structural changes in livers from both species of fish were evaluated by light and electron microscopy. Results show a general GST activity increase according to TiO2 NPs concentrations, which is in agreement with data from LPO. After 21 days, GST activities decreased possibly caused by suppression of GST synthesis as a result of severe stress. Histological and ultra-structural analysis of livers from exposed fish show degeneration of the hepatic tissue and alterations in hepatocytes such as glycogen depletion and an increase in lipofucsin lysosome-like granules. After a depuration period a partial recovery for biochemical markers and cells was observed. The results suggest that TiO2 promotes alterations in hepatic tissues compatible with oxidative stress.

Effects of diamond nanoparticle exposure on the internal structure and reproduction of Daphnia magna.

Nanomaterials have significant technological advantages but their release into the environment also carry potential ecotoxicological risks. Carbon-based nanoparticles and particularly diamond nanoparticles have numerous industrial and medical applications. The aim of the present study was to evaluate the toxic effects of diamond nanoparticles with an average particle size of 20 nm on the survival, reproduction and tissue structure of the freshwater crustacean Daphnia magna. The chronic toxicity test results showed 100% mortality at concentrations higher than 12.5 mg l(-1) and that reproduction inhibition occurred in concentrations higher than 1.3 mg l(-1). Light microscopy showed that diamond nanoparticles adhere to the exoskeleton surface and accumulate within the gastrointestinal tract, suggesting that food absorption by the gut cells may be blocked. The results support the use of chronic approaches in environmental protection as part of an integrated environmental monitoring and assessment strategy.

Ecotoxicity tests in the environmental analysis of wastewater treatment plants: case study in Portugal.

A global evaluation of wastewaters should include ecotoxicological tests to complement the chemical characterization, with advantages especially in the case of complex wastewaters. A European project developed in Trancão River Basin (Portugal), integrated the ecotoxicological and physicochemical studies of wastewater samples from two municipal sewer networks and respective wastewater treatment plants. Wastewater samples were analysed for physicochemical parameters, ecotoxicological acute and chronic tests performed and the potential for endocrine disruption evaluated. Organic load parameters and total suspended solids showed significant correlations with Microtox and ThamnoToxKit test results. Data analysis showed that treated treatment plant effluent samples are associated with less organic contamination and less toxicity in ThamnoToxKit test. Chronic toxicity test and endocrine disruption assay of treatment plant effluent samples indicated that, in a long term, potential population effects could arise in the receiving waters. A test battery to monitor this type of wastewaters is proposed, including tests with a bacterium, an alga and a crustacean. In a screening phase the most sensitive test, Microtox, can be used. The use of an ecotoxicological approach can have added value to hazard and risk assessment of discharges to the receiving waters and can contribute to the environmental management of the treatment plant.

Ecotoxicological assessment of industrial wastewaters in Trancão River Basin (Portugal).

It is important to assess the toxicity of complex effluents, since chemical evaluation alone is insufficient to protect the environment. Direct Toxicity Assessment is valuable in the decision process regarding the final disposal of complex wastewaters as it measures the total effects of the discharge, because of its known and unknown chemicals, additionally having some degree of ecological relevance. In Portugal, ecotoxicity tests are not used on a regular basis to control wastewaters. So, an integrated ecotoxicological, physical, and chemical study of wastewaters from 17 industries, in the Trancão River Basin, was carried out viewing proposing a test battery to be used in wastewater evaluation. An approach which does not include an ecotoxicological characterization may not properly evaluate the potential risks of effluent discharges, especially when they are complex. From the study carried out the use of a battery of assays to apply in the monitoring of complex wastewaters was proposed, including Microtox test, Daphnia test, and an algal test. Moreover, the added value of the ecotoxicological assessment of industrial wastewaters was demonstrated and could support the implementation of EU Directives (e.g. IPPC, WFD) within the Portuguese situation.

Performance of a miniaturized algal bioassay in phytotoxicity screening.

A miniaturized and low-cost algal growth-inhibition assay, with Pseudokirchneriella subcapitata, based on the standard ISO 8692 and using 96-well microplates, was tested and optimized in this work, to be used as a useful tool for pollutant phytotoxicity screening. For validation, the performance of the microplate algal growth-inhibition assay was first compared with the standard flask assay for the toxicity testing of five reference toxicants (copper(II) sulfate, zinc sulfate, potassium permanganate, potassium dichromate and 3,5-Dichlorophenol) and six wastewater samples. Statistical evaluation of EC(50) results from both methods demonstrated a good agreement between microplate and flask assays either in testing chemicals (r (2) = 0.975, p < 0.0017) or environmental samples toxicity (r (2) = 0.984, p < 0.0001). In addition, the performance of this algal microplate bioassay was also evaluated in comparison with Lemna test, ISO 20079, for phytotoxicity assessment of 27 wastewater samples from industries and treatment plants. The results showed that the algal test was more sensitive for most of the samples, but a significant agreement between both tests was observed (r (2) = 0.644, p < 0.0001). In conclusion, this miniaturized test can be a good tool to include in a battery of tests for phytotoxicity screening of a wide range of chemicals and environmental samples, with the advantage of requiring low sample volumes for the test, allowing large numbers of samples to be tested, and generating low volumes of waste.

Biodegradation of natural phenolic compounds as single and mixed substrates by Fusarium flocciferum

The mycelium of Fusarium flocciferum was assayed for its ability to degrade aromatic compounds, namely, gallic, protocatechuic, vanillic, syringic, caffeic, and ferulic acids and syringic aldehyde, commonly found in agro-industrial wastes. The biodegradation assays were performed in liquid medium with the phenolic compounds as single substrates and as a synthetic mixture containing the seven aromatic compounds. The results with single substrates indicated that in 24 hrs of incubation the fungus was able to reduce the phenolic concentration from 200 mg/l to below detection limits, except for syringic acid, being the lowest degradation rates found for this acid and its aldehyde. The biodegradation experiments with the mixture of phenolic compounds showed that after 8 hrs the total phenolic concentration was reduce from 350 mg/l to below the detection limits of all the tested compounds. In all the experiments a rise in the pH and an effective detoxification of the phenolic solutions were also observed.