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Environmental disturbance on freshwater ecosystems significantly impacts all levels of the trophic web. Salinity in lentic freshwater ecosystems, as a consequence of climatic changes, is rising concern in the scientific community. These alterations affect water quality, the composition and diversity of the aquatic communities. This study aimed to assess the seston quality of two lentic ecosystems, Crestuma reservoir and lake Vela, and evaluate the effects of salinity on food performance of Daphnia magna and Daphnia longispina (isolated from the referred ecosystems). Feeding inhibition tests were used to conduct these two evaluations. Physical and chemical water classification revealed a poor or good ecological potential of the two lentic ecosystems, according to WFD approach. To evaluate seston quality, filtered and unfiltered water samples from the lentic ecosystems were used as treatments. Although only summer samples from Crestuma presented good water quality, the results from the feeding inhibition tests showed that seston from lake Vela presented more nutritional quality. Concerning the assay to evaluate the salinity effect on Daphnia spp., a significant reduction in the filtration rate was observed. The NOEC value was 0.7 g/L NaCl and 0.8 g/L NaCl for D. magna and D. longispina C and V, respectively. The LOEC value was also distinct between species, being 1.0 g/L and 0.9 g/L, respectively. This work allowed to verify that feeding inhibition tests are not sensitive enough for assess the quality of natural waters. However, these tests are economically viable and have high sensitivity to evaluate NaCl effects on Daphnia spp.

The assessment of toxic effects caused by complex mixtures of contaminants in the marine environment requires previous validation of toxicological criteria, which may include biomarker end points with distinct biological meanings. This is the case of oxidative stress/phase II detoxification (glutathione-S-transferases activity), oxidative damage (thiobarbituric acid reactive substances), and neurotransmission (cholinesterase activity), which are likely to be affected after toxic insults by common marine pollutants. The main purpose of the present study was to assess potential biological alterations in the mollusk species Pollicipes pollicipes (gooseneck barnacle) caused by human contamination and seasonality, during a period of 1 year, in three different areas of the North Atlantic shore of Portugal. Our results indicate that fluctuations of the mentioned biomarkers were strongly related to seasonality, but they may also suffer influence by the already documented patterns of chemical contamination. Organisms collected in contaminated sampling sites (urban areas and oil refinery) showed greater levels of metabolic enzymes and increased levels of lipid peroxidation. These alterations were more evident during the summer, and, in some cases, spring months, suggesting an association between the presence of chemical stressors and temperature-dependent seasonal physiological fluctuations, which contribute to the modulation of the toxic response. In general terms, P. pollicipes was shown to be a promising organism in coastal biomonitoring programs, with an adequate sensitivity toward contamination and/or seasonal fluctuations. However, it is of the utmost importance to consider seasonal fluctuations in physiological parameters that modulate the toxic response. These factors can ultimately compromise the development and interpretation of data from marine biomonitoring programs if a thorough characterization of biological responses is not previously performed.

As a part of the Ecological Risk Assessment of a deactivated uranium mining area (Cunha Baixa), the aim of this study was to assess the drivers of litter arthropod community (ecological line of evidence) inhabiting soils with different degrees of contamination. Litter arthropods were collected in the mining area using a total of 70 pitfall traps, in the spring and autumn of 2004. Unlike information previously collected in the chemical and ecotoxicological lines of evidence, we found no clear evidence of impacts of soil contamination on the edaphic arthropod assemblage. Multivariate analyses were unable to extract relevant environmental gradients related to contamination, as most of the sites shared the same taxa overall. Given the consistency of the chemical and ecotoxicological lines of evidence, we must conclude that the litter arthropod assemblage underestimated the impacts of contamination in this abandoned mining area. In part, this could be due to the uncertainty caused by confounding factors that affect the litter arthropod community in the area. Nevertheless, despite the overall lack of responsiveness of the epigeic arthropod community data, a few taxa were negatively correlated with metal concentrations (Clubionidae and Staphylinidae), while Pseudoscorpionida were associated with the toxicological profile of the sites. These evidences suggest that community-level approaches with other animal and plant assemblages are necessary to reduce uncertainty relatively to the assessment of risks in higher evaluation tiers in the Cunha Baixa mine area.

Oxytetracycline (OTC), an antibacterial agent, is extensively used in aquaculture practices all over the world, but also in human and veterinary medicines. Because of its intensive use, low rates of absorption by treated animals, inadequate disposal, and low efficiency of removal in wastewater treatment plants, the potential harmful effects on aquatic organisms are of great concern. This work aimed to assess the effects of this antibiotic in rainbow trout, following both acute and chronic exposures. Catalase (CAT), total glutathione peroxidase (GPx), glutathione reductase (GRed) activities and lipid peroxidation (TBARS levels) were quantified as oxidative stress biomarkers, in gills and liver. Genotoxic endpoints, reflecting different types of genetic damage in blood cells, were also determined, by analysis of genetic damage (determination of the genetic damage index, GDI, measured by comet assay) and erythrocytic nuclear abnormalities (ENAs). The obtained results showed a mild pattern of antioxidant response, with modifications in CAT and GPx activities in gills, and lipid peroxidation in liver. These results suggest that despite the occurrence of oxidative effects, a full scenario of oxidative stress is not likely. However, exposure to OTC resulted in the establishment of genotoxic alterations with the induction of DNA strand breaks in blood cells (increase of GDI), and of chromosome breakage and/or segregational abnormalities (increase of ENAs). Considering that the oxidative response was not totally devisable, other mechanisms may be involved in the genotoxic effects reported. published

Daphnia magna is widely used as a standard organism in ecotoxicology assays. It plays a key role in energy transfer in freshwater food webs as a primary consumer, grazing on microalgae, yeast and bacteria. Daphnids are commonly reared in the laboratory using microalgae cultures but alternative or complementary sources are important to reduce the dependency on a single food source. The role played in nature by planctomycetes as a food source for other higher trophic levels is still unknown. In this study, we aimed to evaluate the potential of Rhodopirellula rubra strain LF2 as a nutritional or a supplementary food source for D. magna and Daphnia longispina. Life-history assays were conducted with daphnids fed with R. rubra in exponential and stationary growth phases, in three concentrations. Additionally, its adequacy as a supplement to the microalga Raphidocelis subcapitata was tested. In general, both daphnids showed impairment in all the parameters evaluated, especially when fed with R. rubra. However, when daphnids were fed with the two food sources, no changes were recorded for the rate of population increase. At the tested concentrations, R. rubra was not a good alternative food source in the daphnid diet. © EDP Sciences, 2016. This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT (Foundation for Science and Technology) and the European Regional Development Fund (ERDF), in the framework of the programme PT2020.

During the past century, the amount of chemicals released into water bodies has increased, with particular emphasis being attributed to xenobiotics with endocrine disruption properties and/or pro-oxidant effects. Among these, it is possible to identify a specific chemical class, alkylphenols, which are of widespread use, and include a variety of chemicals with multiple uses. Bisphenol A is an important chemical used in industrial production of plastics, and has been extensively described as an endocrine disruptor. Paracetamol is a pharmaceutical compound used in human medicine, known for its therapeutic action but also for its evident pro-oxidant features. Additionally, previous studies have suggested that paracetamol may also exert endocrine disruption. The main goal of this study was to assess the effects of both paracetamol and bisphenol A as endocrine disruptors, and as promoters of oxidative stress and damage, on the freshwater microcrustacean Daphnia magna. The obtained results showed that bisphenol A was capable of altering population traits of exposed organisms, by impairing molting. On the contrary, paracetamol was not causative of any significant change in this parameter, despite having caused extensive oxidative stress., Bruno Nunes was hired under the programme Investigador FCT, co-funded by the Human Potential Operational Programme (National Strategic Reference Framework 2007-2013) and European Social Fund (European Union (EU)). This work was supported by the strategic programme UID/BIA/04050/2013 (CBMA: POCI-01-0145-FEDER-007569), UID/Multi/04423/2013 (CIIMAR) and UID/AMB/50017/2013 (CESAM), funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POO)., info:eu-repo/semantics/publishedVersion

This study assessed the effects of episodic contamination on a drainage canal adjacent to an area of intensive rice production (Coimbra, Portugal). Four monitoring periods were considered [i) before herbicide application (day –14), ii) at the first application day (day 0), iii) 3 or 5 and iv) 6 days after]. Each one consisted in three complementary evaluation lines: a) physico-chemical analyses, b) whole effluent toxicity (WET) assays with Pseudokirchneriella subcapitata , c) in situ bioassays to assess microalgae ( P. subcapitata ) growth, and the feeding rate and survival of Daphnia longispina and Daphnia magna . Study sites were located upstream, in a protected wetland (L1), and downstream, in the vicinity of rice fields (L2). Along with the application of agrochemicals, there was a general decrease of the water quality, especially in L2, due to nutrient and herbicide inputs. Herbicide peaks (on days 0, 5 and 6) in L2 water samples were recorded concomitantly or immediately after their application. Regarding the in situ bioassessment, the algae growth decrease from day 0 onwards in L1, whilst in L2 its inhibition was generally coherent with the decline of the water quality. Apparently, WET tests indicated that the limitation of nutrients could be affecting algae growth in L1, however, conclusions should be cautious. The feeding depression of daphnids occurred on days 0 and 5 for D. longispina and only on day 0 for D. magna , while significant reductions on survival were restricted to day 0 for both species. The impairments occurring on day 0 were linked to a potential increased toxicity driven by the ingestion of particle-bound herbicides and suspended particles. The feeding rate of daphnids provided an earlier indication of toxic impairments, though it is prompted the use of complementary endpoints and trophic levels in order to understand the cumulative effects due to various herbicide pulses.

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 seleniumindependent) 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. published

This study focused the ecotoxicological evaluation of four different pesticides (chlorpyrifos, glyphosate, vinclozolin, endosulfan), sprayed into an agricultural soil, using a standard battery of aquatic bioassays for testing of soil elutriates: Vibrio fischeri -Microtox ® ; Pseudokirchneriella subcapitata growth inhibition; Daphnia magna acute and chronic toxicity. Despite relevant pesticide residues were recovered from the soil matrix (concentrations higher than 1000 μg kg −1 ), much lower concentrations could be retrieved from elutriates (highest records for endosulfan of ca. 250 ng L −1 and 1400 ng L −1 ; dissolved and particulate concentration, respectively) and little effects were generally found in the bioassays. Lethal effects ( D. magna 48 h-EC50 of 36.8%) could be noticed following exposure to the endosulfan elutriate. Elutriates induced no toxicity on V. fischeri ; algal growth was generally inhibited at high elutriate dilutions and stimulated at the lower elutriate dilutions; and no overall impairment of D. magna life-history was noticed. Results revealed that cross-contamination during field application, input of organic matter and nutrients by elutriates in test solutions, and choice of test species and endpoints may constrain the ecotoxicological assessment. Suitability of established aquatic bioassay test batteries for these purposes, and questioning on whether direct assays with soil organisms could be more protective tools is discussed.

The great number of abandoned uranium mines within the Portuguese territory led to the production of specific legislation establishing the rehabilitation of these areas and the definition of those requiring prior intervention. Cunha Baixa uranium mine (Mangualde, Central Portugal) was one of these areas and a site-specific risk assessment is being carried out for this mine, following European frameworks. In this study the Ames test with soil elutriates, and emergence and growth assays with Lactuca sativa L. and Zea mays L. were performed to assess soil retention and habitat functions. Levels of metals bioaccumulated in plant tissues, grown above soil surface, during the assays, were determined as well. No genotoxic effects were recorded for soil eluates (1:10 and 1:2, m/v) except for site F within the mine area, which is under the effluence of runoffs from the effluent treatment pond. Nevertheless, most of all were highly toxic to Salmonella typhimurium (especially for TA98) strains, used in the assay. The results gathered suggested that during the natural process of soil percolation by rainwater, the leachates yielded are contributing with toxic but no genotoxic metals to underground water resources. Phytotoxicity assays demonstrated that soil habitat function was compromised in almost all the sampling sites selected within the mine area. These results have clearly demonstrated how evaluations based on extractable metal concentrations may underestimate the risks of contaminated soils. Lettuce was the most sensitive species probably due to the great ability of this species in accumulating high concentrations of metals in their aboveground tissues. The highest bioaccumulation factors (BAFs) values were recorded for this species namely for Co (16.5), Cu (28.5), Ba (7.3), Sr (7.2) and Ni (6.1). Copper (9.0) and nickel (3.6) were the main elements bioaccumulated by maize. The dilution of mine soils with a local reference soil has proved to mitigate its toxicity in almost all the sites analysed.

Three white-rot fungi (Pleurotus sajor caju, Trametes versicolor and Phanerochaete chrysosporium) and one soft-rot fungi (Rhizopus oryzae) species confirmed their potential for future applications in the biological treatment of effluents derived from the secondary treatment of a bleached kraft pulp mill processing Eucalyptus globulus. Among the four species P. sajor caju and R. oryzae were the most effective in the biodegradation of organic compounds present in the effluent, being responsible for the reduction of relative absorbance (25-46% at 250 nm and 72-74% at 465 nm) and of chemical oxygen demand levels (74 to 81%) after 10 days of incubation. Laccase (Lac), lignin (Lip) and manganese peroxidases (MnP) expression varied among fungal species, where Lac and LiP activities were correlated with the degradation of organic compounds in the effluent treated with P. sajor caju. The first two axes of a principal component analysis explained 88.9% of the total variation among sub-samples treated with the four fungus species, after different incubation periods. All the variables measured contributed positively to the first component except for the MnP enzyme activity which was the only variable contributing negatively to the first component. Absorbances at 465 nm, LiP and Lac enzyme activities were the variables with more weight on the second component. P. sajor caju revealed to be the only species able to perform the biological treatment without promoting an increment in the toxicity of the effluent to the Vibrio fischeri, as it was assessed by the Microtox assay. The opposite was recorded for the treatments with the other three species of fungus. EC(50-5 min) values ranging between 28 and 57% (effluent concentrations) were recorded even after 10 to 13 days of treatment with P. chrysosporium, R. oryzae or with T. versicolor.

Forest fires can result in severe economic and environmental consequences, and little is known about the ecological patterns and processes that may lead to the recovery of burnt areas. In the last decades, Portugal has been the Southern European country with the highest number of fire events and with the highest burnt area per hectare. With this work, we proposed to study the effect of a forest fire on the terrestrial ecosystem. More specifically, this work intended to evaluate the short-term recovery of several soil chemical, biochemical (microbial enzymatic activities) and biological (edaphic macro-arthropod community) variables in a burnt pine tree forest area. Soil and macro-arthropod sampling was carried out in a burnt area (transects BI, BII and BIII) and in a neighbouring unburnt area (U) 3 and 8 months after the fire, coinciding with autumn and spring. Soil was collected for the determination of physical (pH and conductivity) and chemical parameters (moisture and organic matter) and soil enzymes (cellulase, acid phosphatase and nitrogen mineralisation rate). Edaphic macro-fauna was captured using pitfall traps. Univariate and multivariate statistics revealed, overall, that burnt sites displayed lower acid phosphatase and cellulase activities and higher conductivity and pH values than the unburnt area. There was a recovery in the measured soil parameters between autumn and spring in the most interior parts of the burnt areas (BII and BIII), but the outer transect (BI, close to a road) still displayed considerable differences to the remaining burnt transects as well as to the unburnt area. A total of 47 macro-arthropod taxa were captured in both seasons, with Linyphiidae spiders (20.2%) and insect families Formicidae (13.4%) and Staphylinidae (11.9%) being the most abundant. Dominance by some taxa was overall stronger in the burnt than in the unburnt area, although dominant taxa varied between seasons. In autumn, the burnt area was dominated by ants and had also a high abundance of scavengers, carrion feeders and some ground active hunters. In spring, there was a general increase in taxa diversity, richness, and total catches; in the burnt area, there was a re-colonisation by several organisms sensitive to litter quality, such as isopods and pseudoscorpions, particularly in the outer transect (closest to the unburnt area). Differences in soil parameters between burnt and unburnt areas were most likely due to the deposition of nutrient-rich alkaline ashes. However, low cellulase activity in the outer part of the burnt area (BI) indicated compromised microbial activity in both sampling seasons. Recovery of soil functional parameters was delayed in the outer zone of the burnt area because of (i) fire intensity in that area or (ii) proximity to the road (enhancing erosion and exposure to contaminants). The pattern of arthropod re-colonisation of the burnt area followed the inverse recovery pattern (from the outer zone to the inner zone), stressing the primary role of the adjacent unburnt area as a source of potentially colonizing organisms. Direct and indirect effects of fire on soil parameters (soil alkalinisation and nutrient enrichment) and edaphic fauna had a short-term persistence in the burnt area, and signs of recovery were evident 8 months after the fire (spring). The adjacent unburnt area seemed to act as an important source of arthropod colonisers. More prolonged studies on the recovery of soil functional parameters and arthropod community structure are required to understand long-term re-colonisation patterns. Researchers and authorities should also endeavour in the implementation of measures that favour and protect survivors and new indigenous colonisers (microbes, plants and animals) after a forest fire.

The Ecological Risk Assessment of pesticides requires data regarding their toxicity to aquatic and terrestrial non-target species. Such requirements concern active ingredient(s), generally not considering the noxious potential of commercial formulations. This work intends to contribute with novel information on the effects of short-term exposures to two herbicides, with different modes of action (Spasor®, Stam Novel Flo 480®), and an insecticide (Lannate®), as well as to corresponding active ingredients (Glyphosate, Propanil and Methomyl, respectively). The microalga Pseudokirchneriella subcapitata (growth inhibition), the cladoceran Daphnia magna (immobilisation), and the earthworm Eisenia andrei (avoidance behaviour) were used as test species. Both herbicides were innocuous to all test organisms at environmentally realistic concentrations, except for Stam and Propanil (highly toxic for Pseudokirchneriella; moderately toxic to Daphnia). Lannate and Methomyl were highly toxic to Daphnia and caused Eisenia to significantly avoid the spiked soil at realistic application rates. The toxicity of formulations either overestimated (e.g. Stam/Propanil for P. subcapitata) or underestimated (e.g. Stam/Propanil for D. magna) that of the active ingredient.

The goal of this study was to develop an in situ bioassay with Eisenia andrei, deploying it in several locations of an abandoned mining area. Our objectives were two-fold: (i) we intended to validate the in situ soil bioassay procedures, while (ii) providing ecologically relevant data to complement the ongoing risk evaluation based on laboratorial assays. To promote cost- and time-effectiveness, the in situ exposure was short (48 h) and the endpoints analysed included oxidative stress biomarkers and metal content in soil and organisms. The bioassay was carried out under different experimental conditions, simulating local (natural soil) vs. control conditions (LUFA soil), and irrigation with artificial rainwater vs. irrigation with diluted acidic effluent. Variation in the data was mostly due to soil type, rather than irrigation water, and substantial spatial heterogeneity was observed. Oxidative stress biomarkers did not fully work as sensitive parameters to environmental contamination. Earthworm metal burdens suggested a potential concern in terms of bioaccumulation of some metallic elements.

As a way of getting a comprehensive assessment of soil quality it is necessary to gather information on several appropriate soil parameters as well as also to perceive their spatial variation between geographic regions. The present work aimed to characterize and compare soil samples from different geographical regions and vegetation covers in the Portuguese territory through the use of physicochemical, biological and biochemical parameters. The study also intended to ascertain if those parameters are sensitive tools that consistently respond to geographical variations. Physical and chemical (soil organic matter contents, moisture, pH and conductivity), biological (macroarthropod es and diversity) and biochemical (potential nitrification, dehydrogenase and acid phosphatase activities) soil parameters were determined for three different regions of Portugal. Several sub-areas were chosen within each region, in order to include main vegetation patches. At each sampling sub-area, 5 traps were randomly set up for the collection of macroarthropods. For each sampling location, diversity descriptors of soil macrofauna were calculated: species richness, β diversity, the Shannon diversity (H′) and Pielou evenness (J′) indices. A principal component analysis (PCA) was used to plot the different sampling locations based on physical and chemical parameters; a canonical correspondence analysis (CCA) was performed to analyse the relationships between soil fauna and environmental variables (physicochemical parameters and enzyme activities) and a partial canonical correspondence analysis (pCCA), with vegetation patches as covariables, was carried out to perceive their contribution to the variation in macroarthropod distribution. Physical and chemical parameters discriminated soils from the different geographic regions in the PCA. Nevertheless, a CCA of environmental variables (physical and chemical properties, dehydrogenase activity, acid phosphatase activity and potential nitrification) and soil macroarthropod catches only discriminated continental soils (Arouca and S. Domingos Mine) from insular ones (Porto Santo Island). The former were correlated with high dehydrogenase activity (DHA) and potential nitrification values, while soils from the Porto Santo Island were particularly correlated with high pH values and low moisture percentages. However, in spite of the extreme conditions, Porto Santo sampling sub-areas presented a great diversity of macroarthropods, especially when compared to the S. Domingos Mine. The pCCA showed that the different vegetation patches only explained 15% of the variation in edaphic species distribution. Even though physical and chemical parameters were sufficient to discriminate the different geographical regions included in this study, biochemical and biological parameters grouped continental regions, discriminating them from the insular one. According to the CCA triplot, it was evident that the sampling sub-areas from the island were negatively correlated with soil moisture, suggesting that this was probably the main factor for the suppression of the microbial parameters in the island. On the other hand, the location of the S. Domingos mine area, in a metallogenetic province — the Iberian Pyrite Belt — may have contributed to the dominance of taxa more adapted to higher background levels of heavy metals. In order to perceive geographical variation among different regions, more information is needed regarding different vegetation covers and the chemical composition of their corresponding litter, as well as about other soil chemical parameters. The data gathered in this study will help to distinguish variation in soil biological and biochemical parameters induced by abiotic conditions from the one promoted by anthropogenic disturbances.

The superficial aquatic system of the Cunha Baixa uranium mine area is comprised by the flooded mine pit (M), which receives the acidic mine effluent resultant from in situ leaching of pore ore, a pond where this effluent is neutralised (T), and a potential reference pond (Ref). As part of the first tiers of an ecological risk assessment that is being performed in this area, the aim of this work was to evaluate the potential sediment toxicity of these ponds. To perform this work, elutriates were produced from sediments collected at the three ponds in two distinct seasons (Spring and Winter). Acute and chronic toxicity of elutriates was evaluated by standard assay protocols, using Daphnia spp. as test species (D. magna-standard species and D. longispina-native species). In opposition to what could be previewed based on total metal concentrations, results showed acute toxicity only in M site (low pH, high metal levels) in spring for both species with EC50=94.7% and 96.3% (D. longispina and D. magna respectively). A stimulatory effect (in growth and fecundity) was observed in the chronic assays, for almost all of the tested concentrations of the three elutriates tested-except for the highest concentrations of the M elutriate. Some differences were observed in the responses of Daphnia, both between test species and seasons. Differences between the two sampling periods were also found for pH. Although some toxicity was observed in M, overall no toxicity was found for sediments of the aquatic system, corroborating previous results from our team (including whole-sediment tests). Differences in pH may explain the observed toxicity, acting both as a stressor and mobilizing contaminants. Stimulatory phenomena, typical when dealing with natural samples, worked as confounding factors. Several explanations should be considered for these stimulatory effects, however the role of radionuclides (not measured in this work) cannot be ignored. Supported by the results gathered in this study and in previous evaluations already performed, it is possible to state that in the present situation sediments plays a secondary role in the toxicity of the Cunha Baixa uranium mine, probably working more as a barrier than as a source of contaminants to the water column. However, future reclamation works in this area should carefully consider the remobilisation of sediments, especially from pond M. Radiochemical contamination is expected to be higher in field situations where radiation emitter isotopes are present in all the sediment compartment and overlying water, emitting radiations in all the directions. Thus whole-sediment and elutriate laboratory bioassays are not representative of long-term field exposures to radiation. Based on this supposition, these laboratory responses should be validated by field surveys of the benthic and planktonic freshwater communities of these ponds and freshwater receiving resources, at the higher tiers of the local ecological risk assessment.

Inactive or abandoned mines represent a significant source of environmental, chemical, physical, and aesthetic impact. Among concerning situations, the occurrence of abandoned or semi-abandoned mine-associated ponds (for sedimentation of solids, for effluent neutralization, or for washing the ore) is a common feature in this type of system. These ponds are a source of contamination for the groundwater resources and adjacent soils, because they lack appropriate impermeabilization. The use of this water for agriculture may also pose chronic risks to humans. In Portugal, these problems have been diagnosed and some remediation projects have been developed. The purpose of our study was to evaluate the acute and chronic toxicity of water samples collected from the aquatic system surrounding an abandoned uranium mine (Cunha Baixa, Mangualde, Central Portugal). The present study focuses on the water compartment, whose toxicity was evaluated by means of standard toxicity assays using two Daphnia species (D. longispina and D. magna). Three different ponds were used in the characterization of the aquatic system from Cunha Baixa mine: a reference pond (Ref), a mine effluent treatment pond (T), and a mine pit pond (M). Metal analyses performed in the water samples from these ponds showed values that, in some cases, were much higher than maximum recommendable values established (especially Al, Mn) by Portuguese legislation for waters for crop irrigation. Acute toxicity was only observed in the mine pit pond, with EC(50) values of 28.4% and 50.4% for D. longispina and D. magna, respectively. The significant impairment of chronic endpoints, translated in reductions in the population growth rate for both species, gives rise to concerns regarding the potential risks for aquatic zooplanktonic communities, from local receiving waters, potentially exposed to point source discharges of the treated and nontreated effluent from Cunha Baixa uranium mine.

Uranium mining activities in Cunha Baixa, Mangualde (Portugal), were extensive between 1967 and 1993, with high production of poor ore. Ore exploitation left millions of tons of tailings in the surrounding area, close to human houses. Contamination of the area (water and soil compartment) presently represents a serious hazard to humans and wildlife. The aim of this work was to evaluate the acute toxicity of water and sediments from a pond that floods a uranium mine pit, in two periods (spring and autumn). High contents of metals were found in water samples (chiefly Mn, Fe, Al, U, Sr). A battery of assays was applied to screen the acute toxicity of the different compartments using algae, crustaceans and dipterans. Results showed that the sediments were non-toxic, unlike the superficial water. Water toxicity was higher in the autumn, when the effluent was more acidic, compared to spring. In the water toxicity assays, the relative sensitivity of the test species used was Daphnia longispina>Pseudokirchneriella subcapitata>Daphnia magna. The present study is part of the chemical and ecotoxicological characterisation of the aquatic compartment performed in the Tier 1 of the Ecological Risk Assessment of the Cunha Baixa mining area.

Benzalkonium chloride (BAC) is one of the most used conservatives in pharmaceutical preparations. However, its use is limited to a small set of external use formulations, due to its high toxicity. Benzalkonium chloride effects are related to the potential exertion of deleterious effects, mediated via oxidative stress and through interaction with membrane enzymes, leading to cellular damage. To address the ecotoxicity of this specific compound rainbow trouts were chronically exposed to BAC at environmental relevant concentrations (ranging from 0.100 to 1.050mg/L), and the biological response of cholinergic neurotoxicity, modulation of the antioxidant defense, phase II metabolism, lipid peroxidation and genotoxicity was studied. The obtained results showed a dual pattern of antioxidant response, with significant alterations in catalase activity (starting at 0.180mg/L), and lipid peroxidation, for intermediate (0.180 and 0.324mg/L) concentrations. No significant alterations occurred for glutathione-S-transferases activity. An unexpected increased of the acetylcholinesterase activity was also recorded for the individuals exposed to higher concentrations of BAC (starting at 0.180mg/L). Furthermore, exposure to BAC resulted in the establishment of genotoxic alterations, observable (for the specific case of the comet assay results) for all tested BAC concentrations. However, and considering that the oxidative response was not devisable, other mechanisms may be involved in the genotoxic effects reported here. published

To assess the environmental impact of As and Hg, bioindicator organisms such as bivalves have been used. Nevertheless, few studies have assessed the impacts of As and Hg in Ruditapes decussatus and Ruditapes philippinarum, which are native and exotic species in Europe, respectively. The main goal of the present study was to assess elements' partitioning and detoxification strategies of R. decussatus and R. philippinarum. Both clams showed a higher capacity to bioconcentrate Hg (BCF 2.29–7.49), when compared to As (0.59–1.09). Furthermore, As accumulation in both species was similar in the soluble and insoluble fractions, while in both species the majority of Hg was found in the insoluble fraction. Clams exposed to As showed different detoxification strategies, since R. decussatus had higher ability to enhance antioxidant enzymes and metallothioneins in order to reduce toxicity, and R.philippinarum increased glutathione S-transferase Ω activity, that catalyzes monomethyl arsenate reduction, the rate-limiting reaction in arsenic biotransformation. When exposed to Hg, R. decussatus presented, higher synthesis of antioxidant enzymes and lower LPO, being able to better tolerate Hg than the exotic species R. philippinarum. Thus under relevant levels of As and Hg contamination our work evidenced the higher ability of R. decussatus to survive and inhabit coastal environments not heavily contaminated by Hg and As.

Erythromycin (ERY) is a macrolide antibiotic used in human and veterinary medicine, and has been detected in various aquatic compartments. Recent studies have indicated that this compound can exert biological activity on non-target organisms environmentally exposed. The present study aimed to assess the toxic effects of ERY in Oncorhynchus mykiss after acute and chronic exposures. The here adopted strategy involved exposure to three levels of ERY, the first being similar to concentrations reported to occur in the wild, thus ecologically relevant. Catalase (CAT), total glutathione peroxidase (GPx), glutathione reductase (GRed) activities and lipid peroxidation (TBARS levels) were quantified as oxidative stress biomarkers in gills and liver. Genotoxic endpoints, reflecting different types of genetic damage in blood cells, were also determined, by performing analysis of genetic damage (determination of the genetic damage index, GDI, measured by comet assay) and of erythrocytic nuclear abnormalities (ENAs). The results suggest the occurrence of a mild, but significant, oxidative stress scenario in gills. For acutely exposed organisms, significant alterations were observed in CAT and GRed activities, and also in TBARS levels, which however are modifications with uncertain biological interpretation, despite indicating involvement of an oxidative effect and response. After chronic exposure, a significant decrease of CAT activity, increase of GPx activity and TBARS levels in gills was noticed. In liver, significant decrease in TBARS levels were observed in both exposures. Comet and ENAs assays indicated significant increases on genotoxic damage of O. mykiss, after erythromycin exposures. This set of data (acute and chronic) suggests that erythromycin has the potential to induce DNA strand breaks in blood cells, and demonstrate the induction of chromosome breakage and/or segregational abnormalities. Overall results indicate that both DNA damaging effects induced by erythromycin may be related to the oxidative damage observed, shown to occur at environmentally relevant concentrations of erythromycin. published

The increasing occurrence of pharmaceutical drugs in the aquatic environment is cause of concern, due to the possibility of toxic phenomena in non-target species, including oxidative stress and neurotoxicity. The present study aimed to assess the acute effect of four widely used therapeutic agents: acetaminophen (analgesic), chlorpromazine (antipsychotic), diclofenac (anti-inflammatory) and propranolol (antihypertensive), in the cladoceran species Daphnia magna. Considering the involvement of the mentioned compounds in the impairment of cholinesterasic activity and modifications in cellular redox systems, the purpose of this study was to analyze their effects on biomarkers of neuronal regulation, such as total cholinesterases (ChEs), and enzymatic oxidative stress defense, including as catalase (CAT), glutathione-S-transferases (GSTs), and total and selenium-dependent glutathione-peroxidase (total GPx; Se-GPx) activities. Exposure to acetaminophen caused a significant inhibition of AChE and Se-GPx activities in D. magna relative to the control. Among the biomarkers of oxidative stress, only the activity of CAT was significantly altered in concentration of 0.001mg L(-1) of chlorpromazine, which was not always consistent with the literature. Diclofenac caused a significant inhibition of AChE and Se-dependent GPx, and also in total GPx activities. Propranolol was responsible for a significant decrease in the activity of the latter two enzymes, and also a slight increase of GSTs activity. The results indicated that the exposure to all the tested compounds induced alterations on the cellular redox status in the studied species. In addition, acetaminophen and diclofenac were shown to have the capability of interfering with D. magna neurotransmission, through the inhibition of ChEs. Our data enlighten the need for more research on the ecological consequences of pharmaceuticals in non-target organisms.

The release of acidic effluents, naturally enriched in metals and radionuclides, is the main legacy of uranium mines. Generally, metals dissolved by these acidic effluents can cause significant alterations in exposed organisms, with distinct toxicological outcomes. In this study, 72 individuals of the freshwater fish species Carassius auratus were exposed in situ for different periods (8, 16, 24, and 48 h) to water from a pond (treatment pond (TP)) with a chemically treated effluent and a reference pond (PRP), in the vicinity of the Cunha Baixa uranium mine (Portugal). Comparing the water of the two ponds, the PRP pond was characterized by higher pH and oxygen values and lower conductivity and hardness values. Regarding total metal concentrations, among others, magnesium (56,000 μg/L), sodium (17,400 μg/L), zinc (86 μg/L), manganese (6340 μg/L), and uranium (1380 μg/L) concentrations in the TP pond were above the values obtained for the PRP pond. The values of manganese and uranium exceeded the values of quality criteria established for surface waters for cyprinids and for irrigation purposes. After exposure to pond water, significant differences were recorded for several biomarkers: (i) between ponds for acetylcholinesterase (AChE) with higher activities for animals from the PRP and glutathione-S-transferase (GST) activities that were particularly enhanced in animals from the TP pond; (ii) between ponds and exposure periods for lactate dehydrogenase (LDH) activity, since organisms from PRP pond presented always higher values than those from the TP pond, and among these, organisms exposed for the longer period presented a further depression in LDH activity; and (iii) between exposure periods for erythrocyte micronucleus. GSTs and LDH were the most sensitive biomarkers within the timeframe of the in situ assay performed. Despite the alleged efficacy of the chemical treatment (evidenced by a significantly lower pH), some metals persisted in the treated effluent (TP pond), potentially contributing to the induction of oxidative stress or increased conjugation metabolic activity in fish. published

Lake Vela is a polymictic shallow lake exhibiting some characteristics typical of an advanced trophic state, namely, the permanently turbid water, the reduction in biodiversity, and the recurrent occurrence of Cyanobacteria blooms, which occasionally lead to large fish kills. This study was carried out in order to understand the seasonal variation of Lake Vela’s phytoplankton and cladoceran communities and their interactions under advanced eutrophic conditions. When comparing our results with general models of plankton succession observed in other temperate and tropical lakes we found some coherence between several seasonal events. Our results suggest that phytoplankton is mainly regulated by nutrients (“bottom-up” effect). However, the warm mediterranean temperatures had an important role in the phytoplankton succession, being responsible for the rapid and intense Cyanobacteria development in spring and summer. Our work also demonstrated that phytoplankton is one of the main factors responsible for the seasonal structure of the community of cladocerans, which are well related to changes in algae diversity and abundance, being the Cyanobacteria having major impact. Moreover, the occurrence of a massive Cyanobacteria bloom during the study, which induced anoxia and consequent fish kill, enhanced the structuring role that fish have on the cladoceran seasonal succession and the effects of this episode in the normal seasonal succession of plankton.

Resting eggs are a fundamental reproductive strategy among freshwater cladocerans. Under adverse environmental conditions, whole Daphnia populations can disappear from a lake and a new community will arise from ephippial eggs. Since these new populations are subjected to genetic variation, their responses to environmental stress or contaminants can be different from the “original” population. In the present study, life history responses (reproduction and growth) of Daphnia longispina to different food concentrations was studied. Two Daphnia populations were tested: (a) field clones and (b) ephippial clones. Food ( Selenastrum capricornutum ) concentration was the stressor tested (absence of food, and low to high food concentrations). The results showed that reproductive responses of D. longispina to the tested food concentrations varied among clones, independently from their origin.

In recent decades, scientific research about the effects of anthropogenic xenobiotics on non-target organisms has increased. Among the likely effects, some studies reported the evaluation of biochemical and morphological changes in specific tissues or organs of fishes, such as gills, which are key organs for the direct action of pollutants in the aquatic environment. This work intended to assess biochemical [oxidative stress/phase II conjugation isoenzymes glutathione S-transferase (GSTs)] and morphological [secondary lamellar length (SLL), secondary lamellar width (SLW), interlamellar distance (ID), basal epithelial thickness (BET) and proportion of the secondary lamellae available for gas exchange (PAGE)] changes in gills, after acute exposure to the pesticide chlorfenvinphos, the detergent sodium dodecylsulphate (SDS) and to the anticholinesterasic pharmaceuticals (neostigmine and pyridostigmine). Our results point to a significant, eventually hormetic, effect in the activity of GSTs following exposure to chlorfenvinphos that significantly increased the activity of GSTs at concentration of 0.2 mg/L. The activity of GSTs increased significantly after exposure to 100 mg/L of neostigmine. Considering the morphometric analysis of the gills, the data obtained showed that chlorfenvinphos exerted mainly minor architectural alterations in gills, with the exception of the highest tested concentration of chlorfenvinphos that produced also a slight decrease of the PAGE. The overall conclusions point to a null or negligible toxicity of the selected toxicants towards L. gibbosus, which may be reverted if exposure is withdrawn.

The presence of pharmaceutical drugs in aquatic ecosystems has been widely reported during the past years. Salicylic acid (SA) is mainly used in human medicine as an analgesic and antipyretic drug, being also active in preventing platelet aggregation. To study the ecotoxicological effects potentially elicited by SA in freshwater fish, brown trout individuals (Salmo trutta fario) were chronically exposed (28 days) to this drug, in order to evaluate the enzymatic and histological effects, in both gills and liver. A qualitative and semi-qualitative evaluation of the gills and liver was performed, and also a quantitative evaluation of various lamellar structures. Oxidative stress was quantified trough the determination of glutathione S-transferases (GSTs), glutathione reductase (GRed), total and selenium-dependent glutathione peroxidase (GPx) and Catalase (Cat) activities. Lipid peroxidative damage was also assessed by the quantification of thiobarbituric acid reactive substances (TBARS) in the liver. The here-obtained data showed the occurrence of oxidative stress, reflected by an increased activity of GPx and GRed in the liver; additionally, it was possible to observe non-specific histological changes in gills. The global significance of the entire set of results is discussed, giving emphasis to the ecological relevance of the responses.

Paracetamol is one of the most prescribed drugs globally, due to its antipyretic and analgesic properties. However, it is highly toxic at elevated doses, with involvement of an already described oxidative stress pathway. Despite this, the number of ecotoxicological studies on potential effects of paracetamol in wild organisms is still scarce. The present article presents a comprehensive series of standardized assays for the assessment of paracetamol effects in freshwater organisms. The results show that paracetamol toxicity is widely variable among species, even when these species are phylogenetically related. Furthermore, comparisons between data from the literature and our results reinforce this conclusion, providing evidence of the inadequacy of standardized toxicity testing guidelines for pharmaceutical compounds in wild organisms. Paracetamol toxicity can be modulated by unpredictable physiological conditions that might compromise extrapolations and comparisons of responsiveness among species. The ecological relevance of data obtained from classical tests for this compound is further discussed.

The simultaneous presence of distinct compounds in the aquatic environment can be causative of various toxicological interactions. This scenario challenges ecotoxicologists, since the assessment of toxicological effects caused by the simultaneous presence of multiple substances is by far more complicated. An illustrative example can be given by mentioning the anticholinesterasic compounds: by studying the level of cholinesterase impairment of an exposed organism, it is possible to ascertain the level of exposure to all anticholinesterasics (despite their chemical classes and natures) that the organism was subjected to. In this work, we describe the effects of three chemically different, albeit mechanistically, and toxicologically similar compounds (copper, chlorfenvinphos, and pyridostigmine) on cholinesterases of the fish Gambusia holbrooki. The results showed that the combinatorial effects may be of considerable extent, even for levels of exposure that are close to the ones already reported in the wild, for each isolated compound.

Acetaminophen is globally one of the most prescribed drugs due to its antipyretic and analgesic properties. However, it is highly toxic when the dosage surpasses the detoxification capability of an exposed organism, with involvement of an already described oxidative stress pathway. To address the issue of the ecotoxicity of acetaminophen, we performed acute exposures of two aquatic plant species, Lemna gibba and Lemna minor, to this compound. The selected biomarkers were number of fronds, biomass, chlorophyll content, lipid peroxidation (TBARS assay), and proline content. Our results showed marked differences between the two species. Acetaminophen caused a significant decrease in the number of fronds (EC50 = 446.6 mg/L), and the establishment of a dose-dependent peroxidative damage in L. minor, but not in L. gibba. No effects were reported in both species for the indicative parameters chlorophyll content and total biomass. However, the proline content in L. gibba was substantially reduced. The overall conclusions point to the occurrence of an oxidative stress scenario more prominent for L. minor. However, the mechanisms that allowed L. gibba to cope with acetaminophen exposure were distinct from those reported for L. minor, with the likely involvement of proline as antioxidant.

The presence of pharmaceutical residues in the aquatic environment is receiving great attention since significant levels of contamination have been found, not only in sewage treatment plant effluents, but also in open waters. In our study, the toxicity of three anticonvulsant drugs commonly found in the environment (diazepam, carbamazepine, and phenytoin) was evaluated in Lepomis gibbosus (pumpkinseed sunfish). This study focused on oxidative stress parameters, namely: glutathione reductase (GRed), glutathione S-transferases (GSTs), catalase (CAT), and lipid peroxidation (thiobarbituric acid reactive substances, TBARS) in the hepatic, digestive, and gill tissues of exposed animals. Simultaneously, we assessed the effects of these drugs in terms of behavioural parameters, such as scototaxis and activity. Exposure to diazepam caused an increase in GST activities in the gills and an inhibition of GRed in the digestive tract, relative to control, suggesting an antioxidant response. It also caused fish to spend more time swimming and less time in a refuge area (black compartment of an aquarium). Exposure to carbamazepine caused an increase in GSTs and GRed activity in the digestive tract, which is not always consistent with the literature. A significant positive correlation was found between carbamazepine concentration and time spent in motion and a negative correlation with time spent in black compartment. Exposure to phenytoin was responsible for adaptive responses in the activities of CAT and GSTs (in the liver), but it did not elicit any behavioural alterations. Although all three drugs seemed to induce oxidative stress in some organs, peroxidative damage (measured as TBARS concentrations) was not found at the selected range of concentrations. Our results enlighten the need for more research on the ecological consequences of pharmaceuticals in the aquatic environment, especially drugs that interfere with the CNS and behaviour, because the net outcome of these effects may be difficult to predict. This work was partially funded by Fundação para a Ciência e Tecnologia, by project BiOtoMetal (PTDC/AMB/70431/2006). Bruno B. Castro is hired under the programme Ciência 2008 (FCT, Portugal), co-funded by the Human Potential Operational Programme (National Strategic Reference Framework 2007–2013) and European Social Fund (EU). None of the funding sources had any contribution for the experimental design adopted. published

Acetaminophen (paracetamol) is one of the most used pharmaceutical drugs, due to its antipyretic and analgesic properties that turn it into a primary choice in varied pathologies and conditions. However, and despite its massive use, acetaminophen is not exempt of adverse effects, especially when administered in over dosage, which are related to the formation of toxic metabolites by oxidative pathways. It is thus possible to observe that toxicity caused by acetaminophen is usually mediated by reactive oxygen species and can result in multiple effects, ranging from protein denaturation to lipid peroxidation and DNA damage. The occurrence of acetaminophen has been reported in the aquatic environment, being important to address the potential exertion of toxic effects on nontarget environmentally exposed organisms. The present study intended to characterize the effects of acute acetaminophen exposure on physiological traits (antioxidant defense, oxidative damage) of two species of bivalves, namely, the edible clams Venerupis decussata and Venerupis philippinarum. Results showed a significant increase in all oxidative stress biomarkers, evidencing the bioactivation of acetaminophen into a deleterious prooxidant, triggering the onset of deleterious effects. Furthermore, strong interspecific differences were observed among responses of the two tested species, which was a major issue due to intrinsic ecological implications when one considers that both species share the same habitat.

Several researchers investigated the induction of metallothioneins (MTs) in the presence of metals, namely Cadmium (Cd). Fewer studies observed the induction of MTs due to oxidizing agents, and literature comparing the sensitivity of MTs to different stressors is even more scarce or even nonexistent. The role of MTs in metal and oxidative stress and thus their use as a stress biomarker, remains to be clearly elucidated. To better understand the role of MTs as a biomarker in Cerastoderma edule, a bivalve widely used as bioindicator, a laboratory assay was conducted aiming to assess the sensitivity of MTs to metal and oxidative stressors. For this purpose, Cd was used to induce metal stress, whereas hydrogen peroxide (H2O2), being an oxidizing compound, was used to impose oxidative stress. Results showed that induction of MTs occurred at very different levels in metal and oxidative stress. In the presence of the oxidizing agent (H2O2), MTs only increased significantly when the degree of oxidative stress was very high, and mortality rates were higher than 50 percent. On the contrary, C. edule survived to all Cd concentrations used and significant MTs increases, compared to the control, were observed in all Cd exposures. The present work also revealed that the number of ions and the metal bound to MTs varied with the exposure conditions. In the absence of disturbance, MTs bound most (60-70 percent) of the essential metals (Zn and Cu) in solution. In stressful situations, such as the exposure to Cd and H2O2, MTs did not bind to Cu and bound less to Zn. When organisms were exposed to Cd, the total number of ions bound per MT molecule did not change, compared to control. However the sort of ions bound per MT molecule differed; part of the Zn and all Cu ions where displaced by Cd ions. For organisms exposed to H2O2, each MT molecule bound less than half of the ions compared to control and Cd conditions, which indicates a partial oxidation of thiol groups in the cysteine residues through ROS scavenging. The present results suggest that MTs are excellent markers of metal stress, but not of oxidative stress.

The presence of pharmaceutical residues in the aquatic environment has recently received great attention, as potential adverse effects may arise from their presence. Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. However, other widespread anthropogenic contaminants - including pharmaceutical drugs - can exert toxic effects through ChE inhibition. Studies with aquatic species have shown that inhibition of ChE is associated with behavioral changes. Bearing this in mind, this work aimed to study the effects on individual behavior and acetylcholinesterase (AChE) activity of selected tissues of Lepomis gibbosus, after exposure to the anticholinesterasic drugs neostigmine and pyridostigmine. Results revealed that neostigmine significantly decreased the activity of AChE in the head (LOEC = 100 mg L(-1)), but not in dorsal muscle. On the other hand, pyridostigmine significantly decreased the activity of AChE in both the head (LOEC = 0.001 mg L(-1)) and dorsal muscle homogenates (LOEC = 100 mg L(-1)). The impairment of this enzymatic form was attained at ecologically relevant concentrations (in the case of pyridostigmine, for head AChE), which is an interesting finding, specially considering that these toxic effects occurred for a pharmaceutical compound. Contrarily, there were no significant differences in the behavior of L. gibbosus in any parameter, for neither drug. These results suggest that the behavioral parameters analyzed (scototaxis and lethargy) in L. gibbosus could not be regarded as suitable markers to assess the effects of drugs such as neostigmine and pyridostigmine. In contrast, the pattern of response elicited by cholinesterasic inhibition showed the usefulness of this toxicological parameter for the assessment of pharmaceuticals in the environment, namely anticholinesterasics. published

This study characterizes the cholinesterasic forms present in the bivalve Corbicula fluminea, through the analysis of differential affinity towards various substrates and inhibitors. This approach also permitted the validation of cholinesterase inhibition in the mentioned species for monitoring purposes by evaluating the inhibitory activity of a detergent (SDS) and a pesticide (chlorfenvinphos), both in vitro and in vivo. The cholinesterasic form present in the tissues of C. fluminea showed intermediate properties in terms of its relation with substrates and inhibitors. Furthermore, it was not inhibited by the detergent SDS under any of the test conditions, but was clearly inhibited by the selected organophosphate. The data show clearly that the main cholinesterasic form in C. fluminea is responsive to the organophosphate chlorfenvinphos, thus supporting the use of this biomarker for the environmental monitoring of specific contaminants such as anticholinesterasic pesticides. Despite not being commonly attained in the wild, the levels of chlorfenvinphos required to elicit a significant ChE inhibition were only one order of magnitude above actual values of aquatic contamination by this pesticide. Ana Ramos was recipient of a FCT scholarship (BII/CESAM/46539/2009). published

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. published

Inhibition of cholinesterases (ChEs) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. More recently, this biomarker has been suggested as a putative biomarker for exposure to detergents. The use of cholinesterase inhibition as effect criterion in Ecotoxicology requires the previous characterization of the specific enzymatic forms that may be present in different tissues or organs. Different ChEs isoforms may be present in the same tissue and may exhibit distinct sensitivities towards environmental contaminants. This work intended to characterize the soluble ChEs present in pumpkinseed sunfish (Lepomis gibbosus) total head and dorsal muscle homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Also, the in vitro effects of sodium dodecylsulphate (SDS – anionic detergent) and chlorfenvinphos (organophosphate pesticide) on the enzymatic activity of the mentioned species were investigated. In general terms, the predominant cholinesterasic form present in both tissues was acetylcholinesterase. Chlorfenvinphos was responsible for inhibitory effects on AChE activity, while SDS did not cause any significant effect. These results suggest that in environmental monitoring programs, L. gibbosus head and dorsal muscle AChE can be an adequate diagnostic tool for exposure to OP pesticides; this conclusion however is not applicable to detergent residues. We also discuss the usefulness of L. gibbosus as an alternative model system and valuable option for freshwater ecotoxicological monitoring programs. The present work was funded by project “BiOtoMetal” (PTDC/AMB/70431/2006) from the Portuguese Foundation for Science and Technology (FCT, Fundação para a Ciência e a Tecnologia). FCT also financed Sara C. Antunes with a Post-doctoral fellowship (SFRH/BPD/40052/2007). Bruno B. Castro is hired under the programme Ciência 2008 (FCT, Portugal), co-funded by the Human Potential Operational Programme (National Strategic Reference Framework 2007–2013) and European Social Fund (EU). published

Submitted by Patrícia Correia (patriciacorreia@ua.pt) on 2019-02-05T16:06:55Z No. of bitstreams: 1 Goncalves et al. - 2011 - Ecological risk assessment of Ervedosa mine (Portu.pdf: 82345 bytes, checksum: 2610122c20ee90a69508988c8dec1249 (MD5) Approved for entry into archive by Patrícia Correia (patriciacorreia@ua.pt) on 2019-02-05T16:18:23Z (GMT) No. of bitstreams: 1 Goncalves et al. - 2011 - Ecological risk assessment of Ervedosa mine (Portu.pdf: 82345 bytes, checksum: 2610122c20ee90a69508988c8dec1249 (MD5) Made available in DSpace on 2019-02-05T16:18:23Z (GMT). No. of bitstreams: 1 Goncalves et al. - 2011 - Ecological risk assessment of Ervedosa mine (Portu.pdf: 82345 bytes, checksum: 2610122c20ee90a69508988c8dec1249 (MD5) Previous issue date: 2011 published

Included in the 2nd tier of a site specific risk assessment that is being carried out in an abandoned uranium mine (Cunha Baixa uranium mine, Central Portugal), fatty acids biomarkers and sterols were analyzed to assess the impact of soil contamination with metals and radionuclides in the structure of the microbial community in seven sampling sites located at different distances from the mine. Surface soil samples were collected in those sampling sites in the four different seasons of the year. Principal component analysis (PCA) was performed on fatty acid biomarkers and sterols. Subsequently PCA scores obtained for both components were used to test the effect of sites and seasons, on soil samples collected in the Cunha Baixa uranium mine, through bi-factorial ANOVAs. Through PCA analysis, two distinct groups were set apart along the first two components. One group included sites at a great distance from the mine which were negatively correlated with higher contents of iC15:0 and iC17:0, both indicators of Gram-positive bacteria, as well as with ergosterol, cholestanol and cholesterol. The second group, in turn, was composed of the sampling sites most impacted by ore exploration, in situ leaching of poor ore, and spread of sludge from the effluent treatment pond. These sites were positively correlated with higher levels of iC16:0 (Gram-positive bacteria indicator), cyC17:0 (generally common in gram negative bacteria) and C18:0 and C17:0 biomarkers of non-specific bacteria. The profile of fatty acids obtained in the sampling sites revealed variable predominance of groups of bacteria which are a clear indication of differences in the soil microbial communities that are directly related to the environmental conditions prevailing in the uranium mine area. published

Submitted by Patrícia Correia (patriciacorreia@ua.pt) on 2020-03-11T16:04:27Z No. of bitstreams: 1 Nunes et al. - 2011 - The use of biomarkers to assess aquatic pollution .pdf: 82529 bytes, checksum: d424812728c3e0e3ba63d520f53fe402 (MD5) Approved for entry into archive by Patrícia Correia (patriciacorreia@ua.pt) on 2020-03-11T16:04:44Z (GMT) No. of bitstreams: 1 Nunes et al. - 2011 - The use of biomarkers to assess aquatic pollution .pdf: 82529 bytes, checksum: d424812728c3e0e3ba63d520f53fe402 (MD5) Made available in DSpace on 2020-03-11T16:04:45Z (GMT). No. of bitstreams: 1 Nunes et al. - 2011 - The use of biomarkers to assess aquatic pollution .pdf: 82529 bytes, checksum: d424812728c3e0e3ba63d520f53fe402 (MD5) Previous issue date: 2011 published

The present study describes an approach for toxicity estimates based on the in situ exposure of the local phytoplankton community immobilized in calcium-alginate beads. The assay was deployed in an acid mine drainage-impacted system, at a reference site and an impacted site. Abundances and growth rates of major taxonomic groups (green algae, flagellates, diatoms, and cyanobacteria) and of the entire community were measured. The ecological relevance of classical toxicity estimates was evaluated by performing in situ and laboratory assays with the standard species Pseudokirchneriella subcapitata and Chlorella vulgaris. Site waters were tested without and with nutrient enrichment, to discriminate toxic effects from those caused by nutrient limitations. Despite the occurrence of growth-limiting conditions for the local phytoplankton within the alginate matrix, an impact of the mine effluent on the community was detected. The growth of the community and of all taxonomic groups, except for the cyanobacteria, was less at the impacted site than at the reference site, although adding nutrients partially compensated for the effluent detrimental effects. Shifts in the community structure were also provoked by the mine effluent; decreases in the abundance of green algae were paralleled by increases in the abundance of flagellates and diatoms, but mainly of cyanobacteria. Effects on the growth of both standard species of green algae were in conformity with those on the local phytoplankton, probably because the latter was dominated by green algae. Although future improvements regarding the cell immobilization technique should be attempted, the proposed easy and inexpensive approach to assess both the growth and the structure of local phytoplankton communities will allow more environmentally realistic site-specific assessments of water quality. Environ. Toxicol. Chem. 2011;30:196–205. © 2010 SETAC

Antioxidant defences are the primary mechanisms by which organisms compensate oxidative damage caused by contact with several types of substances prone to establish oxidative cycles. Biomarkers are one of the most useful tools to assess effects consequent to environmental exposure to a large number of xenobiotics. However, the use of biomarkers requires previous standardization of enzymatic protocols in order to guarantee accuracy and relevance of obtained results. This article describes standardized conditions for the use of several enzymatic biomarkers (oxidative stress: glutathione peroxidase, lipid peroxidation, glutathione reductase, glutathione-S-transferase and catalase; respiration: lactate dehydrogenase) for the evaluation of responses of specimens of Eisenia andrei, Pelophylax perezi (formerly Rana perezi) and Apodemus sylvaticus. Furthermore, we also present the normal values of activity for the given markers, for non-exposed organisms, that can serve as comparative indexes for subsequent studies. We conclude that these species are thus a suitable target to serve as sentinels in studies of environmental contamination by chemicals, which can lead to oxidative and physiological modifications. published

Soil avoidance by earthworms has been generally considered a relevant and sensitive endpoint for assessing soil contamination by xenobiotics. However, when pesticide ecotoxicological assessment is concerned, the sensitivity of the recently standardized avoidance assay has been questioned. We hypothesized that this controversy may be due to the specific pesticide mode of action of the chemicals used rather than reveal inconsistencies in the test feasibility, i.e. provided that no pesticides interfering with neuronal pathways are tested, this bioassay should keep expected high levels of sensitivity. In this study, the avoidance behaviour of the earthworm Eisenia andrei under exposure to the carbamate insecticide methomyl [S-methyl N-(methylcarbamoyloxy)thioacetimidate] was linked to the corresponding acetylcholinesterase (AChE) inhibition. Significant AChE inhibition occurred at lower concentrations (from 0.86 mg Kg(-1) onwards) than significant avoidance of spiked soil (from 5.62 mg Kg(-1) onwards). This indicates that assessments regarding pesticides that have neurotoxic activity may be biased if behavioral endpoints are selected. Despite theoretical hypothesis that have been raised, this should be the first study providing preliminary experimental evidence on such a link between avoidance behavior and neuronal impairment levels in earthworms. Further studies are ongoing that should refine conclusions of this study. published

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.

As part of the tier 2 of a site-specific risk assessment, this study was the first reporting an intensive in situ application of the bait-lamina assay; two exposure periods (7 and 14 days) were tested during four seasons in ten different sites, within a uranium mine area and at two different depths. The most contaminated sites (by deposition of sludge from the effluent treatment pond) were discriminated after 14 days of exposure because extremely low percentages of feeding activity were recorded. Previous sub-lethal ecotoxicological assays, already had demonstrated that the habitat function of these soils is compromised. Nevertheless, seasonality has proved to have a significant influence on responses. Thus to strength conclusions about the impact of contaminants, the in situ bait-lamina assay should be performed on different annual seasons, at least for temperate regions. It was also found that some environmental parameters (e.g. soil moisture and litter) can act as confounding factors in the bait-lamina assay.

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 effluent's 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.

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.

The main objective of this study is to assess the soil diversity and temporal and spatial distribution of litter macro-arthropods, captured with pitfall traps, on different vegetation covers from Porto Santo Island (Portugal) with different soil physical and chemical characteristics. The PCA clearly separated sampling areas geographically more exposed to winds and solar radiation, from the others that were not. In this analysis, seasons seemed to have no influence on this distribution. Non-exposed areas were characterized by high soil temperatures, water and organic matter contents. Among these areas, those that were covered by Pinus halepensis and Eucalyptus ficifolia showed the highest C/N ratios in winter, which may be explained by likely inhibitory effects of phenolic compounds of the leaves of both species on the decomposition process. The highest number of organisms was recorded on the exposed areas. Variance partitioning using CCA showed that the different vegetation covers and environmental variables explained 19% and 30%, respectively, of the variance in macro-arthropods distribution. Four environmental variables (exposure, temperature, C/N ratio and exchangeable K) were positively correlated with the taxa abundance matrix. The results showed that non-exposed areas with E. ficifolia and Cupressus macrocarpa seemed to favour the establishment of a high diversity of taxa, especially in the summer, when soil conditions tend to be extreme. http://www.sciencedirect.com/science/article/B6VR7-4PSJR3H-8/1/16443e80f3b5d685d1f812cd120358ae

A chemical characterization of the soil compartment per se does not supply any information about the synergistic/antagonistic effects of mixtures of chemicals, resulting in an under- or overestimation of the risks. Therefore the existence of rapid and ecologically relevant toxicity assays becomes of paramount importance, allowing the evaluation of invertebrate's behavioural parameters with equal consequences in terms of functionality of the edaphic community. The aim of this study was to evaluate the sensitivity of avoidance assays, with the earthworm Eisenia andrei, in discriminating natural soils under different anthropogenic disturbances. Although earthworms were able to discriminate soils with similar contamination, it is nonetheless questionable whether the preference for some soils is determined by the reduced bioavailability of contaminants, the great affinity of the species for organic matter-rich soils, or the inability of chemoreceptors to detect some contaminants from complex environmental mixtures, usually present in natural contaminated soils.

This study presents the first ecotoxicological data concerning the soils of the area surrounding the Cunha Baixa uranium mine. Our main goal was to categorise soils from the area based on their toxicity profiles using a battery of cost- and time-effective bioassays (elutriate approach – Microtox® and Daphnia acute tests; whole-soil approach – Microtox® and avoidance assays with Eisenia andrei), as a part of tier 1 of an ongoing Environmental Risk Assessment. No acute toxicity was found for any of the 10 sites/soils using Microtox® or Daphnia. On the contrary, the behavioural response of E. andrei was found to be an extremely sensitive endpoint, allowing the discrimination of highly to moderately toxic soils based on their toxicity profiles (as a function of soil concentration). Soils exhibiting highest toxicity corresponded to areas subjected to runoffs or sludge deposition from the aquatic effluent, while non-toxic soils were farthest to the mine. Data obtained in avoidance assays strengthen the previous evaluation of risks based on chemical data and supported decisions about proceeding for tier 2.