First ecotoxicological characterization of paraffin microparticles: a biomarker approach in a marine suspension-feeder, Mytilus sp.

Microplastics are one of the main environmental pollutants in marine ecosystems, and their presence in seawater is a consequence of the widespread use of plastic materials in modern commodities. This wide usage of plastics includes the employment of microspheres in common personal care products, which end up being ultimately released into the aquatic compartment. Known ecotoxicological effects of microplastics favoured the search for technologically viable and environmentally safer alternatives, such as paraffin wax microparticles, whose ecotoxicological risks have not been entirely characterized. To address this gap, the present study exposed mussels (Mytilus sp.) for 96 h to three densities (5 mg/L, 20 mg/L and 80 mg/L) of four size ranges (100-300 µm, 300-500 µm, 400-850 µm, and 800-1200 µm) of paraffin wax particles. Toxicological endpoints were the activities of four enzymes involved in key cellular processes, including antioxidant defence (catalase (CAT), glutathione reductase (GRed) and glutathione peroxidase (GPx)) and phase II metabolism (glutathione S-transferases (GSTs)), as well as lipid content and fatty acid profiles of the digestive gland. Significant interactions between the presence/absence of food and paraffin particle density were found, as food sometimes worked as a confounding factor in the analysed biomarkers. Despite this uncertainty, some overall patterns emerged. In general, smaller paraffin wax particles (100-300 µm) caused little effects on the activity of the four enzymes tested, whereas larger particles (800-1200 µm) caused significant effects on almost all biomarkers. CAT activity was enhanced in animals exposed to larger paraffin particles, whilst GPx activity was depressed; GRed activity was not affected by the exposure to paraffin particles. The activity of GSTs was enhanced, but only in one tested condition. No effects were observed in terms of the total lipid content and fatty acids of exposed animals. Overall, data obtained in this work suggest that, at densities of paraffin wax particles comparable to the levels found in the environment for microplastic beads, no toxicity is expected to occur in the tested mussel species, by measuring the here-assessed toxicological endpoints.

Feeding inhibition in Corbicula fluminea (O.F. Muller, 1774) as an effect criterion to pollutant exposure: Perspectives for ecotoxicity screening and refinement of chemical control.

Bivalves are commonly used in biomonitoring programs to track pollutants. Several features, including its filter-feeding abilities, cumulatively argue in favour of the use of the Asian clam (Corbicula fluminea) as a biosentinel and an ecotoxicological model. Filtration in bivalves is very sensitive to external stimuli and its control is dictated by regulation of the opening/closure of the valves, which may be used as an avoidance defence against contaminants. Here, we investigate the filter-feeding behaviour of the Asian clam as an endpoint for assessing exposure to pollutants, driven by two complementary goals: (i) to generate relevant and sensitive toxicological information based on the ability of C. fluminea to clear an algal suspension, using the invasive species as a surrogate for native bivalves; (ii) to gain insight on the potential of exploring this integrative response in the refinement of chemical control methods for this pest. Clearance rates and proportion of algae removed were measured using a simple and reproducible protocol. Despite some variation across individuals and size classes, 50-90% of food particles were generally removed within 60-120 min by clams larger than 20 mm. Removal of algae was sensitive to an array of model contaminants with biocide potential, including fertilizers, pesticides, metals and salts: eight out of nine tested substances were detected at the µg l-1 or mg l-1 range and triggered valve closure, decreasing filter-feeding in a concentration-dependent manner. For most toxicants, a good agreement between mortality (96 h - LC50 within the range 0.4-5500 mg l-1) and feeding (2 h - IC50 within the range 0.005-2317 mg l-1) was observed, demonstrating that a 120-min assay can be used as a protective surrogate of acute toxicity. However, copper sulphate was very strongly avoided by the clams (IC50 = 5.3 µg l-1); on the contrary, dichlorvos (an organophosphate insecticide) did not cause feeding depression, either by being undetected by the clams' chemosensors and/or by interfering with the valve closure mechanism. Such an assay has a large potential as a simple screening tool for industry, environmental agencies and managers. The ability of dichlorvos to bypass the Asian clam's avoidance strategy puts it in the spotlight as a potential agent to be used alone or combined with others in eradication programs of this biofouler in closed or semi-closed industrial settings.

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.

An in situ bioassay integrating individual and biochemical responses using small fish species.

The interest in the ecological relevance of risk assessments and, thus, in in situ bioassays has been increasing in the last years. The present study developed a time- and cost-effective in situ bioassay, aiming at obtaining, in a short period of time and with a minimum of resources, a set of ecologically relevant toxicological information in a site-specific approach. Poecilia reticulata and Gambusia holbrooki were chosen as test species. Post-exposure feeding inhibition and the biomarkers acetylcholinesterase, lactate dehydrogenase and glutathione S-transferases were the endpoints tested. The battery of biomarkers as a whole was sensitive to the in situ exposure in an acid mine drainage impacted effluent, although responses varied between test species. Post-exposure feeding inhibition was the most sensitive endpoint, and its association with biomarker responses was discussed. The linkage between individual responses, such as feeding, and biomarkers suggested that, at least in this case, biomarkers can be relevant at higher levels of biological organization. Altogether, the proposed short-term in situ bioassay seems to be a promising tool, since it represents a reasonable compromise between sensitivity, time/cost-effectiveness and ecological relevance.