Sérgio Marques

Toxicity and organic content characterization of olive oil mill wastewater undergoing a sequential treatment with fungi and photo-Fenton oxidation.

Olive oil mill wastewater (OOMW) is responsible for serious environmental problems. In this study, the efficiency of two treatments involving fungi and photo-Fenton oxidation, sequentially applied to OOMW was analyzed for organic compounds degradation and toxicity mitigation. The treatment with fungi (especially Pleurotus sajor caju) of diluted OOMW samples promoted a reduction of their acute toxicity to Daphnia longispina. Although this fungi species have not induced significant color reduction it was responsible for 72,91 and 77% reductions in chemical oxygen demand (COD), total phenolic and organic compound contents. After biological treatment, photo-Fenton oxidation seemed to be an interesting solution, especially for color reduction. However, the OOMWs remained highly toxic after photo-Fenton oxidation. Considering the second sequence of treatments, namely photo-Fenton oxidation followed by biological treatment, the former revealed, once more, a great potential because it can be applied to non-diluted OOMW, with significant reductions in COD (53-76%), total phenolic content (81-92%) and organic compounds content (100%). Despite fungal species still have demonstrated a high capacity for bioaccumulation of organic compounds, resulting from photo-Fenton oxidation, the biological treatment did not cause substantial benefits in terms of COD, total phenolic content and toxicity reduction.

Histopathological changes and erythrocytic nuclear abnormalities in Iberian green frogs (Rana perezi Seoane) from a uranium mine pond

In spite of their sensitivity to anthropogenic stressors, adults of Rana perezi Seoane were found inhabiting effluent ponds from a uranium mine. Due to the presence of such organisms in this environment, it becomes of paramount importance to assess the damages induced by local contamination on these aquatic vertebrates, in order to integrate this information on a site-specific risk assessment that is being carried out in the area. To attain this purpose an ethically and statistically acceptable number of green frogs were captured in the mine pond (M) and in a pristine river (VR), a few kilometres from the mine. Bioaccumulation of metals and histopathological alterations were evaluated in the liver, kidneys, spleen, lungs and testes of the animals. Simultaneously, blood samples were collected for the evaluation of genotoxic damage on erythrocytes. Animals captured in the M pond showed significantly increased levels of Be, Al, Mn, Fe and U in the liver, as well as Pb and U in the kidney. The liver was the main target organ for the bioaccumulation of Be, Al, Fe and U. However, renal histopathologies were more severe than those of liver. The main tissue alterations recorded in animals from the mine were: a slight increase in melanomacrophagic centers (MMC) in liver, lung and kidneys; dilatation of the renal tubules lumen associated with tubular necrosis. A significantly higher number of erythrocytic abnormalities (lobed, notched and kidney shaped nuclei and micronuclei) were recorded in frogs from M than in frogs from VR, along with a significantly lower frequency of immature erythrocytes. Both observations suggested that the removal of abnormal blood cells might be compromised.

Impaired microbial activity caused by metal pollution: A field study in a deactivated uranium mining area.

European frameworks for the ecological risk assessment (ERA) of contaminated sites integrate information from three lines of evidence: chemical, ecotoxicological, and ecological. Regarding the last one, field observations at the contaminated sites are compared to reference site(s) and the differences recorded are analysed at the light of a cause-effect relationship, taking into account the site-specific contamination. Thus, included in the tier 2 of a site-specific risk assessment that is being carried out in an deactivated uranium mining area, a battery of soil enzyme activities (dehydrogenases, urease, arysulphatase, cellulase, acid phosphate) and potential nitrification were assessed in seven sampling sites (A-D-E-F-G-H-I) at different distances from the mine pit. These parameters have been considered good indicators of impacts on soil microbial communities and, subsequently, on soil functions. Soil enzyme activities were impaired in the most contaminated site (A, near the mine pit), for which a higher degree of risk was determined in the tier 1 of ERA. Three other sites within the mining area (F, G, and D) were discriminated on the basis of their low microbial activity, using uni- and multivariate approaches, and validating what had been previously found with chemical and ecotoxicological lines of evidence. We observed considerable among-site heterogeneity in terms of soil physical and chemical properties, combined with seasonal differences in enzyme activities. Still, the correlation between microbial parameters and soil general physical and chemical parameters was weak. In opposition, significant and negative correlations were found between soil enzyme activities and several metallic elements (Al, Be, Cu, U). These findings suggest a clear correlation between compromised soil function (nutrient recycling) and metal contamination. Such information reinforces the evidence of risks for some sites within the mining area and is an important contribution for the usefulness of soil enzyme activities for evaluating changes in soil health.

The effectiveness of a biological treatment with Rhizopus oryzae and of a photo-Fenton oxidation in the mitigation of toxicity of a bleached kraft pulp mill effluent.

Huge efforts have been made both in adopting more environmental-friendly bleaching processes, and in developing advanced oxidation processes and more effective biological treatments for the reduction of deleterious impacts of paper mill effluents. Even so, the success of such treatments is frequently reported in terms of chemical parameters without a proper evaluation of the effluents toxicity mitigation. This is the first study reporting an exhaustive evaluation of the toxicity of a secondary bleached kraft pulp mill effluent, after either tertiary treatment with the soft-rot fungi Rhizopus oryzae or with a photo-Fenton oxidation, using a battery of freshwater species. As it has been reported the photo-Fenton/UV treatment has proved to be the most effective in reducing the colour and the COD (chemical oxygen demand) of the effluent. Nevertheless, extremely low EC(50) values were reported for almost all species, after this tertiary treatment. The treatment with R. oryzae was less effective in terms of colour removal and COD reduction, but proved to be the most promising in reducing toxicity.

Effects of a uranium mine effluent in the early-life stages of Rana perezi Seoane

Amphibians have been reported as sensitive organisms whose survival has been impaired by several environmental factors. Nevertheless, sometimes amphibians are found inhabiting extreme environments. Thus, in order to perceive how Iberian green frogs (Rana perezi Seoane) are able to survive in the ponds of a uranium mine (Central Portugal) this study aimed to assess the ecotoxicological effects promoted by the mine effluent in the early-life stages of this species. To attain this objective, eggs (collected in a nearby reference river) and laboratory hatching larvae were exposed during 96 h to different dilutions of the effluent. All the effects on the hatch success were recorded. The highest concentration of the effluent produced a significant decrease in body length of larvae, as well as a decrease in stimulus reactions and an increase in pigmentation along with tail deformities. A recovery assay showed an increased bioaccumulation of metals, uranium included, resulting from increased effluent exposure.

Antioxidant response and metal accumulation in tissues of Iberian green frogs (Pelophylax perezi) inhabiting a deactivated uranium mine

Human mining activities tend often to generate greatly impacted areas which remain contaminated for long periods of time, giving rise to extreme habitats. Mining sites are usually characterized for the production of metal rich effluents with very low pH. In this work we analyzed physical and chemical parameters of water from a deactivated uranium mine pond (M) and a reference site (REF) as well as their metal content. Furthermore, we determined and compared metal accumulation in liver, kidney, bones, muscle and skin of Pelophylax perezi from REF with P. perezi from M. We also determined the enzymatic activities of glutathione-S-transferases (GSTs), catalase (CAT), glutathione reductase (Gred), and glutathione peroxidase (GPx; both selenium-dependent and selenium-independent) in liver, kidney, lung and heart. Additionally, lipoperoxidation (LPO) was also assessed in the same tissues via thiobarbituric acid reactive substances (TBARS) assay and lactate dehydrogenase (LDH) activity was determined in muscle. Our results revealed that the majority of metals were in higher concentrations in tissues of organisms from M. This trend was especially evident for U whose content reached a difference of 1350 fold between REF and M organisms. None of the organs tested for antioxidant defenses revealed LPO, nonetheless, with exception for liver, all organs from the M frogs presented increased total GPx activity and selenium-dependent GPx. However, this response was significant only for the lung, probably as a consequence of the significant inhibition of CAT upstream and to cope with the subsequent increase in H2O2. Lungs were the organs displaying greater responsiveness of the anti-oxidant stress system in frogs from the uranium mine area.

Evaluation of growth, biochemical and bioaccumulation parameters in Pelophylax perezi tadpoles, following an in-situ acute exposure to three different effluent ponds from a uranium mine.

Mining activities invariably produce metal contaminated effluents. Depending on factors such as pH and metal concentration the toxicity of the effluent may vary. To assess the effects of three characteristically different effluent ponds from a deactivated uranium mine, with toxicologically relevant data, an in situ exposure with Pelophylax perezi tadpoles, was conducted. Tadpoles were exposed to the three effluent ponds, ranked by increasing order of metals concentrations (REF, M1, M2). Survival, growth, metal accumulation, antioxidant enzymes (catalase, glutathione peroxidase and glutathione reductase) and lipid peroxidation (LPO) were determined in tadpoles. As well, physical and chemical variables of the effluents were measured. Death percentage in the effluents was 3.17 (REF), 9.84 (M1) and 42.86% (M2) and was not coincident with metal accumulation which was highest in tadpoles exposed to M1, while metal contents in M2 tadpoles were quite similar to those recorded in REF tadpoles. However, high mortality in M2 was attributed to the extremely low pH (≈3.77). From the three effluents M2 tadpoles had the lowest growth and the antioxidant enzymatic activity was only affected in the case glutathione peroxidase (GPx) with significantly higher activity in M1, being in accordance with the highest accumulation of metals. LPO, usually associated with metal accumulation, had the following pattern M1>REF>M2. Overall, effluent toxicity in tadpoles exposed to M2 effluent seems to be primarily an effect of pH while in M1 toxicity is mainly owed to high metal concentrations. The effluent acidity seems to reduce metal accumulation probably due to damage in the integument, affecting ion uptake. The results obtained bring a better understanding of the toxicological processes that local P. perezi population is subjected to, mainly in the early life stages. Furthermore this study highlights the influence of pH in the toxicity of metal rich effluents.

Uranium mining wastes: The use of the Fish Embryo Acute Toxicity Test (FET) test to evaluate toxicity and risk of environmental discharge

Active and abandoned uranium mining sites often create environmentally problematic situations, since they cause the contamination of all environmental matrices (air, soil and water) with stable metals and radionuclides. Due to their cytotoxic, genotoxic and teratogenic properties, the exposure to these contaminants may cause several harmful effects in living organisms. The Fish Embryo Acute Toxicity Test (FET) test was employed to evaluate the genotoxic and teratogenic potential of mine liquid effluents and sludge elutriates from a deactivated uranium mine. The aims were: a) to determine the risk of discharge of such wastes in the environment; b) the effectiveness of the chemical treatment applied to the uranium mine water, which is a standard procedure generally applied to liquid effluents from uranium mines and mills, to reduce its toxicological potential; c) the suitability of the FET test for the evaluation the toxicity of such wastes and the added value of including the evaluation of genotoxicity. Results showed that through the FET test it was possible to determine that both elutriates and effluents are genotoxic and also that the mine effluent is teratogenic at low concentrations. Additionally, liquid effluents and sludge elutriates affect other parameters namely, growth and hatching and that water pH alone played an important role in the hatching process. The inclusion of genotoxicity evaluation in the FET test was crucial to prevent the underestimation of the risks posed by some of the tested effluents/elutriates. Finally, it was possible to conclude that care should be taken when using benchmark values calculated for specific stressors to evaluate the risk posed by uranium mining wastes to freshwater ecosystems, due to their chemical complexity.

Differential gene expression in Iberian green frogs (Pelophylax perezi) inhabiting a deactivated uranium mine.

Iberian green frogs (Pelophylax perezi) were found inhabiting a deactivated uranium mine, especially an effluent pond, seriously contaminated with metals and radionuclides. These animals were previously assessed for oxidative stress parameters and did not revealed significant alterations. In order to better understand which mechanisms may be involved in the ability to withstand permanent contamination gene expression analysis was performed in the liver, through suppression subtractive hybridization (SSH). The SSH outcome in the liver revealed the up-regulation of genes coding for the ribosomal protein L7a and for several proteins typical from blood plasma: fibrinogen, hemoglobin and albumin. Besides their normal function, some of these proteins can play an important role as protective agents against oxidative stress. This work provides new insights on possible basal protection mechanisms that may act in organisms exposed chronically to contamination.

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.