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Mountains are universally regarded as key sources of high-quality water resources, fundamental for the local populations as well as for communities living downstream. In recent years, global awareness has been growing among citizens, as well as public and private institutions concerning the need to protect mountain water resources. Many mountain catchments are usually situated in regions of low population density, which are the object of special nature protection status. Water pollution in these regions is often related to road de-icing operations. The objective of this research is to recognise and characterise the environmental implications of water pollution due to road de‐icing in a mountain-protected area (Serra da Estrela, Central Portugal) by means of a geoethical perspective encompassing hydrological and ecological processes along with socioeconomic conditions. A geoethically supported decision-making on road de-icing in Serra da Estrela should bear in mind the local hydrosocial cycle, that is to say, the decision-making should be aware of the complex and dialectic interactions between water flow and quality and the socioeconomic dynamics. Costs and benefits of alternative road de-icing techniques should also be assessed, including the impacts on ecological values. This approach encompasses the ability of the territory to produce fundamental ecosystem services, including hydrological services.

The profiles of total fatty acids (TFAs) and the neutral lipid fatty acids (NLFAs) were compared for the bacterium Rhodopirellula rubra and the alga Raphidocelis subcapitata (conventional food source for Daphnia magna). D. magna NLFAs were assessed when this crustacean was fed with bacterium and alga, individually or in combination. After NLFA extraction, the profiles of the various organisms were characterized by gas chromatography. Results evidenced the relevance of the different composition of the fatty acid (FAs) fractions in the different organisms, R. rubra and R. subcapitata. In these species, the NFLA analyses revealed high amounts of long chain FAs (C19). The FA profile of D. magna was influenced by the different diets provided although the preferred diet was the alga. D. magna showed the capacity to adapt to the available food resources as it defines its FA profile according to its needs, namely for the long chain FAs (C19).

The occurrence of pharmaceuticals in aquatic ecosystems and the need to study them have increased over the years since they enter continuously the environment. Besides, these compounds are not intended for applications with environmental purposes, and therefore, little is known about their ecological effects, particularly in non-target organisms, as invertebrate species. Inside these substances, endocrine disrupting compounds (EDCs) have recently come into the limelight, due to environmental concentrations and consequently their detrimental effects on different organisms. 17α-ethinylestradiol (EE2) has been detected in the aquatic environment in various locations around the globe since it is the main synthetic hormone used as a female oral contraceptive and is also applied in veterinary medicine and animal production. The present study was intended to assess the chronic effects of EE2, in the non-target organism as Daphnia magna. Thus, to analyze the individual and subindividual impact, this aquatic organism was chronically exposed (21 days) to 0.00 (control group), 0.10, 1.00, 10.0, and 100 μg/L of EE2. Results here obtained demonstrated that D. magna exposed to the EE2 concentrations had significant effects in individual (life-history) and sub-individual (biochemical levels) parameters. Alterations as anticipation in the age at first reproduction, a decrease of the growth rate, oxidative stress, and lipid peroxidation were detected, as well as genotoxic damage. Therefore, it was possible to infer that EE2 can disrupt several metabolic pathways and physiological functions of D. magna, since EE2 demonstrated ecotoxicity, at environmentally relevant concentrations. This work reinforces the importance of examining the effects of more relevant exposures (more prolonged and with ecologically pertinent concentrations) of potential endocrine disruptors like EE2, to the freshwater organisms and ecosystem.

The daily use of the planctomycete Rhodopirellula rubra as an alternative or supplementary food source for Daphnia magna and its feasibility in the nutrition of transgenerational populations were studied. The life history parameters, fatty acids (saturated, mono- and polyunsaturated; SFAs, MUFAs and PUFAs), glycogen and protein contents of organisms during feeding assays and of the first generation were analysed. An increase in the yields of D. magna with the increase of the cell concentration of R. rubra was evident, but overall, bacteria supplied as the only food source was nutritionally insufficient as observed for all the parameters analysed. However, when R. rubra was added as supplement to the microalgae Raphidocelis subcapitata a significant improvement in the life history parameters was observed namely in the reproductive output and the somatic growth rate. The identified SFAs, MUFAs and PUFAs were the fatty acids more abundant in daphniids, and the feed regimens influenced daphniids fatty acid profiles. Additionally, the mixed diet resulted in a larger number and size of offspring in the different F1 broods as also observed with the results of F0 generation. The pink colouration present in D. magna body and eggs confirmed that bacteria were absorbed, the pigment(s) retained and passed on to the next generation. Our results showed that R. rubra can play an essential role in D. magna diet as a nutritional supplement showing potential biotechnological applications.

Knee pain is a common reason for adolescent calls or visits to a primary care clinician. The authors present a case of an 14-year-old male adolescent with progressive bilateral nociceptive somatic knee pain. The pain was worse with weight bearing, and relieved at rest. It was located over the medial joint line. The patient was treated conservatively with nonsteroidal anti-inflammatory drugs and a rehabilitation program. Magnetic Resonance Imaging (MRI) showed bone marrow oedema, and Focal Periphyseal Oedema (FOPE) diagnosis was made. After a total of 6 months of treatment, the patient was asymptomatic. The literature is limited to a few case reports and radiologic studies describing this symptomatic physeal pathology, not reporting physical examination or treatment. The goals of this case report were to elucidate physicians of this condition, a newly reported cause of knee pain in adolescent, and to elicit the importance of a patient-tailored rehabilitation program in the treatment of this finding. FOPE is a clinical entity that should not be ignored and must be treated for the patient’s comfort. It is a self-limited condition and has an excellent prognosis.

Oxytetracycline (OTC) is a tetracycline antibiotic, widely used in human and veterinary medicines, including in aquaculture. Given this use, OTC has been detected in different aquatic environments. Some recent works have demonstrated unintentional biological activity of OTC in non-target aquatic organisms. This study investigated the acute and chronic effects of OTC on the physiology of the fish species Oncorhynchus mykiss (rainbow trout), namely through the quantification of the activity of enzymes involved in different biochemical pathways, such as detoxification (phase II - glutathione S-transferases - GSTs, uridine-diphosphate-glucuronosyltransferases - UGTs), neurotransmission (acetylcholinesterase - AChE) and energy production (lactate dehydrogenase - LDH). The here-obtained data demonstrated the induction of GSTs activity in gills, and inhibition of AChE activity in eyes tissue, in chronically exposed organisms, as well as alterations in LDH activity following both exposures. Considering this set of results, we can infer that OTC exposure may have induced the glutathione pathway of detoxification in gills with the involvement of GSTs, or indirectly due to the metabolites that may have been produced. In turn, these metabolites may have interfered with the mechanism of neurotransmission, also causing physiological and biochemical disturbances in rainbow trout after OTC exposure, namely disturbances in energetic metabolism. In addition, it is important to stress that such occurrences took place at low, environmentally realistic levels of OTC, suggesting that organisms exposed in the wild may be putative targets of toxic effects by commonly used drugs such as antibiotics. published

The nutritional quality of daphnids diet can influence their growth, reproduction and survival. In aquatic ecosystems, bacteria can contribute significantly to Daphnia diet by supporting, for instances, their high needs for phosphorus. The laboratory feeding of the model organisms Daphnia spp. is algal based, but should be improved to allow their better performance. The aim of this study was to evaluate the potential of two planctomycetes, Gemmata obscuriglobus and Rhodopirellula rubra, from exponential and stationary growth phases as alternative or supplementary food source for Daphnia magna. The actinobacterium Arthrobacter sp. was used for comparison. The feeding with only bacteria showed the inefficacy of both planctomycetes and actinobacteria as the only food source. However, when used in supplement to Raphidocelis subcapitata, a decrease in the age of first reproduction, a significant increase in reproductive output, in somatic growth and in rate of population increase was found for the highest cell densities of bacteria tested. The typical pink coloration of these bacteria present in daphnids body and eggs confirmed bacterial absorption and metabolization of their pigment. Planctomycetes yielded better results than the actinobacteria Arthrobacter but G. obscuriglobus that possesses sterols did not induce a better performance comparatively to R. rubra. No relation could be established between the feeding treatments that allowed improvement of Daphnia performance and the different kind of Daphnia’ Fatty Acid Methyl Esters. The use of sonication to separate planctomycetal cells before feeding the daphnids proved to be efficient. We confirmed that R. subcapitata supplemented by bacteria allows a better growth performance of D. magna. © 2017, Springer International Publishing AG, part of Springer Nature. Acknowledgements 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 European Regional Development Fund (ERDF), in the framework of the programme PT2020. Sara Antunes is recipient of individual post-doctoral grant (SFRH/ BPD/109951/2015) by the Portuguese Foundation for Science and Technology (FCT). The authors are grateful to Damien Devos for providing us a culture of Gemmata obscuriglobus.

Pharmaceuticals are found in the aquatic compartment due to their continuous release in wastewater effluents or direct dispersal in aquaculture practices raising serious threats to human and environmental health. Erythromycin (ERY) is a macrolide antibiotic widely prescribed in human and veterinary medicine to threat a number of bacterial infections, being consequently found in the aquatic environment. The present work intends to evaluate the sub-lethal effects of ERY on juveniles of rainbow trout (Oncorhynchus mykiss) in terms of tissue damage using histochemical staining procedures. Individuals were exposed for 96 h (acute exposure: 0.001–10 mg/L) and 28 days (chronic exposure: 0.05–0.8 μg/L) to environmentally realistic concentrations of ERY. Qualitative and quantitative approaches were used to assess O. mykiss gills and liver tissue alterations after exposure to ERY. For both exposures the most common gill changes recorded were progressive (e.g. hypertrophy of mucous cells and hyperplasia of the epithelial cells). However, circulatory (e.g. aneurysms and oedemas) and regressive (e.g. epithelial lifting of lamellae and lamellar fusion) changes were also observed in the acute assay. Gill morphometric analysis revealed to be a good indicator of subtle alterations in gill architecture in agreement with the qualitative scoring system. In liver, regressive (e.g. cytoplasmic vacuolization, pyknotic nucleus and hepatocellular degeneration) and circulatory disturbances (e.g. hemorrhage and increase of sinusoidal space) were the most frequently observed alterations, but only for the acute assay. Furthermore, all histological changes observed contributed to a significant increase in the pathological index for both organs. The current data demonstrate the existence of a direct dose-effect relationship between the exposure to this specific macrolide antibiotic and the histological disorders recorded in different tissues of the exposed fish. The histopathological findings observed in this study may have been the result of several physio-metabolic dysfunctions. However, the observed tissue lesions were of minimal or moderate pathological importance, non-specific and reversible. Further investigation into the cellular mechanism of action of ERY is needed.

During the last decades, the presence of antibiotics in different aquatic compartments has raised increasing interest and concern, since these compounds are usually persistent and bioactive pseudo pollutants. Erythromycin (ERY) is a macrolide antibiotic, prescribed for human and veterinary medicines but also used in aquaculture and livestock production. Taking into account the recorded environmental levels of ERY, its toxicity to non-target organisms has become a still poorly studied issue, particularly in fish. In this sense, this study investigated the acute and chronic effects of realistic levels of ERY on Oncorhynchus mykiss (rainbow trout), namely, through the quantification of the activity of enzymes involved in different biochemical pathways, such as detoxification (phase I-7-ethoxyresorufin O-deethylase (EROD); phase II-glutathione S-transferases (GSTs), uridine-diphosphate-glucuronosyltransferases (UGTs)), neurotransmission (acetylcholinesterase (AChE)), and energy production (lactate dehydrogenase (LDH)). Both types of exposure caused significant increases in EROD activity in liver of O. mykiss; an increase in GST activity in gills after chronic exposure was also observed. UGT branchial activity was significantly depressed, following the long-term exposure. Thus, EROD, GST, and UGT enzymatic forms seem to be involved in the biotransformation of ERY. In terms of neurotransmission and preferential pathway of energy homeostasis, the exposed organisms appear not to have been affected, as there were no significant alterations in terms of AChE and LDH activities, respectively. The here-obtained data suggest that the observed alterations in terms of detoxification enzymes may have prevented the establishment of a set of toxic responses, namely, neurotoxic and metabolic disorders. published

Predictions of the International Panel for Climate Changes on sea level rise foresee that the number of coastal regions impacted with salinization will increase in a near future. The present work intended to evaluate the sensitivity to salinization of two freshwater vertebrate species (the frog Pelophylax perezi and the fish Lepomis gibbosus) and their ability to acclimate to this stressor. For this, three specific objectives were targeted: (i) to assess if NaCl may be used as a safe surrogate for risk assessment of seawater (SW) intrusion for freshwater vertebrates; (ii) to evaluate the sensitivity of two freshwater vertebrate models to increased salinity (both due to NaCl or SW); (iii) to determine the capacity of the studied species to acclimate to low levels of salinization. To assess specific objectives (i) and (ii), organisms were exposed to serial concentrations of NaCl or SW dilutions. To assess the capacity of acclimation of both species to salinization, organisms were exposed to low serial concentrations of NaCl during the embryonic development or for a period of two months, respectively, and their sensitivity to NaCl was re-evaluated after this period.Results showed that fish juveniles were more tolerant (96-h LC50 of 21.3 mS cm(-1) for NaCl and 23.6 mS cm(-1) for SW) than frog embryos (96-h LC50 of 10.7 mS cm(-1) for NaCl and 10.7 mS cm(-1) for SW) and tadpoles (96-h LC50 of 19.4 mS cm(-1) for NaCl and 8.72 mS cm(-1) for SW). The fish was able to cope with conductivities of almost one third of SW conductivity, while effect conductivities computed for the amphibian were much lower than SW conductivity (approximate to 52 mS cm(-1)). The two-fold difference between the sensitivity of the two tested species reinforces the idea that ecological risk assessment for amphibians based on fish toxicity data may underestimate the risk to the former. Acclimation to low levels of salinity caused an increase in tolerance to salinization in P. perezi tadpoles but not in fish., This research was supported by national funds (OE) through FCT/MEC and co-funded by FEDER, through COMPETE (POFC), by FSE and POPH and the research projects SALTFREE (projects: IF/00475/2013; PTDC/AAC-CLI/111706/2009), SALTFREE II (POCI-01-0145-FEDER-031022) and ReNATURE (Centro-01-0145-FEDER-000007). This research was also partially supported by Strategic Funding UID/AMB/50017/2013 through national funds provided by FCT - Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020. C. Venancio was grant holder from FCT (ref. SFRH/BD/81717/2011).

In the intertidal area, the interactions between anthropogenic contaminants and natural variations (biotic and abiotic factors) are poorly understood. Consequently, there is a great need for new assessment procedures to characterize the biological responses occurring in organisms from this extreme environment. Considering the intrinsic inter-individual variations among organisms from a single population, it is important to propose new methods that address this variability, by validating a sampling strategy in target groups of organisms, encompassing seasonal fluctuations. This strategy must however be less invasive than traditional methods, avoiding the mandatory sacrifice of the sampled organisms. By doing so, it is also possible to increase the ecological relevance of obtainable data, and contribute to minimize damage to endangered species. The main purpose of the present study was to assess the influence of seasonal variations in the responses elicited by anthropogenic compounds on a marine crustacean species, by using a biomarker-based approach. According to this purpose, the seasonal variations in key physiological responses (biomarkers) were investigated in the crustacean Pollicipes pollicipes from the Northern coast of Portugal. Biomarkers used for this purpose were the activity of the phase II biotransformation isoenzymes glutathione-S-transferases (GSTs), the activity of cholinesterases (ChEs), and the levels of lipid peroxidation (TBARS). All biomarkers were quantified in distinct tissues (such as cirri, and peduncle) and haemolymph (a non-destructive source of biological samples). The glycogen content in peduncle tissue, and the variation in haemocyte number in haemolymph were also analyzed. Samples were collected monthly, during a year, in Lavadores, located in the proximity of an estuarine area (Douro River). The results showed a seasonal pattern in all tested biomarkers. The results also showed a significant increase in GSTs activities, and in peroxidative damage, especially during warmer months. The lowest ChEs values were recorded during the rainy season. The results also showed a similar pattern among all tested tissues, validating the proposed use of the haemolymph as a source tissue for non-lethal sampling strategy for biomarker determinations. Glycogen content was apparently related to the reproductive cycle, with lower values being observed in spring and summer. Total haemocyte count (THC) increased during summer months. The results pointed to an influence of natural variations in the levels of biomarkers in P. pollicipes, highly dependent upon abiotic factors, a factor that must be considered when interpreting biological responses elicited by anthropogenic contaminants from the marine coastal environment. The validation of haemolymph as a non-lethal source tissue for the quantification of biomarkers was successfully attained, opening the possibility of less invasive and almost unlimited sampling in a small number of organisms. published

Due to their worldwide use and environmental persistence, antibiotics are frequently detected in various aquatic compartments. Their toxic properties raise environmental concerns to non-target organisms. Histopathology data is frequently applied in ecotoxicology studies to assess the effects of different classes of environmental stressors in fish, including antibiotics. Tissue alterations in gills and liver of gilthead seabream (Sparus aurata) individuals acutely (96 h) and chronically (28 days) exposed to environmentally relevant concentrations of the antibiotics erythromycin (ERY: 0.0002-200 μg/L) and oxytetracycline (OTC: 0.0004-400 μg/L), including a control non-exposed group, were evaluated. Several disorders (circulatory, regressive, progressive, and inflammatory) were observed in both organs of all exposed animals. The hereby obtained data showed a higher and significant increase in gill histopathological index of organisms acutely exposed to ERY and of those chronically exposed to OTC. In terms of categorical lesions, only a significant increase of regressive and progressive alterations occurred in gills after chronic exposure to OTC. For the liver, a significant increase in pathological index was also detected, as well as regressive changes, after chronic exposure to OTC. Furthermore, the present study indicates that most of the changes observed in gills and liver were of mild to moderate severity, which might be adaptive or protective, non-specific, and mostly reversible. Despite being observed, irreversible lesions were not significant in any of the fish organs analyzed. Although there were histological changes, gill apparatus was considered still functionally normal, as well as liver tissue, not supporting the occurrence of severe toxicity. In general, the observed histological changes were not stressor-specific, and toxicological mechanistic explanations for the alterations observed in gills and liver are presented. The obtained data showed that histopathological biomarkers can be successfully applied in ecotoxicological studies, evidencing their relevance, responsivity, and complementarity to other biochemical biomarker-based approaches. published

Erythromycin (ERY) is one of the most common antibiotics used in human and veterinary practices, leading to ubiquitous environmental distribution and possible toxicity to non-target organisms. The purpose of this study was to determine sub-lethal effects of ERY towards the marine fish Sparus aurata (gilthead seabream). S. aurata were acutely (0.3–323 μg/L, 96 h) and chronically (0.7–8.8 μg/L, 28 d) exposed to ERY. Detoxification [7-ethoxyresorufin O-deethylase (EROD), glutathione S-transferases (GSTs), uridine-diphosphate-glucuronosyltransferase (UGT)], oxidative stress [catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GRed)], lipid peroxidation [thiobarbituric acid reactive substances - (TBARS)], genotoxicity [genetic damage index (GDI) and erythrocytic nuclear abnormalities (ENAs)], neurotransmission [acetylcholinesterase (AChE)] and energy metabolism [lactate dehydrogenase (LDH)] biomarkers were evaluated. Results showed that ERY did not promote significant effects in detoxification biomarkers, but induced slight pro-oxidative effects (decrease of GPx activity in the liver after acute exposure and an increase in gills after chronic exposure; and an increase of hepatic GRed activity following chronic exposure). There was a significant decrease in TBARS after chronic exposure, which contradicts a full scenario of oxidative stress. In terms of genotoxicity, both ERY exposures caused only a significant increase of GDI. Neurotransmission and energy metabolism were not also affected by ERY. Although few toxic effects of ERY have been previously documented (involving different metabolic pathways, as tested in this work), these were mainly observed for freshwater species. These findings suggest low vulnerability of S. aurata to ERY at levels close to the ones found in the wild.

Oxytetracycline (OTC) is an antibiotic widely used in human and veterinary medicines. Since the primary toxicity occurs mainly at molecular/biochemical levels, the study of different biological responses corresponds to a sensitive and crucial approach. The aim of the present study was to assess the toxic effects of OTC in gilthead seabream (Sparus aurata) through the use of multibiomarkers and elucidate about the possible toxicological mechanisms involved. S. aurata were acutely (96 h: 0.04–400 μg/L) and chronically (28 days: 0.0004–4 μg/L) exposed to OTC. Detoxification, antioxidant defense, lipid peroxidation, genotoxicity, neurotransmission and energy metabolism biomarkers were evaluated. OTC impaired the detoxification pathways and caused peroxidative damage and genotoxicity. The relevance of the here-obtained data is high, since significant effects were recorded for levels already reported to occur in the wild, meaning that environmentally-exposed marine organisms (including those cultured at fish farms) are not completely exempt of risks posed by OTC.

The occurrence of pharmaceuticals in the aquatic environment has received increasing attention in recent years, as concerns have risen about their environmental persistence, biological activity and different effects toward nontarget organisms. Considering the magnitude of concentrations (ng L(-1) to mg L(-1)) and their often-specific modes of action, the assessment of physiological responses of exposed aquatic biota may provide significant information regarding the potential ecological consequences of exposure to these contaminants. The present study intended to assess the acute and chronic effects of four pharmaceuticals: acetaminophen, chlorpromazine, diclofenac sodium and propranolol in the cladoceran species Daphnia magna. Parameters such as immobility, total of offspring and rate of population increase were analyzed. Results of acute exposures showed a considerable variability of toxicity among pharmaceuticals, with the following ranking of toxicity: diclofenac (EC50 = 123.3 mg L(-1)) propranolol (EC50 = 5.531 mg L(-1)) acetaminophen (EC50 = 2.831 mg L(-1)) chlorpromazine (EC50 = 1.805 mg L(-1)). The chronic toxicity data showed the exertion of reproductive adverse effects. The compounds chlorpromazine and propranolol caused a significant decrease in fecundity, and the rate of population increase parameter suffered a significant decrease from 0.33 mg L(-1) to 0.128 mg L(-1) onwards, respectively. The levels of exposure to which our test organism was acutely and chronically exposed were above those already reported in the wild. Nevertheless, the extensive production, prescription and release of pharmaceuticals drugs will continue to grow in the future, and consequently their loadings to the environment can result in potential long-term ecological risks to aquatic biota.

The assessment of transgenerational effects should be incorporated in standard chronic toxicity protocols for the sake of a realistic extrapolation of contaminant effects to the population level. We propose a simple add-on to the standard 21-day chronic Daphnia magna assay, allowing the assessment of the reproductive performance of the offspring (F-1 generation) born from the first clutch of the parental (F-0) generation. The extended generational assay was performed simultaneously with the standard reproduction assay. With this design, we evaluated the lethal, reproductive, and transgenerational effects of four widespread and extensively used substances: a biocide/anti-fouling (copper sulphate), an industrial oxidizing agent (potassium dichromate), a pharmaceutical (paracetamol), and a quaternary ammonium compound (benzalkonium chloride). Benzalkonium chloride was the most toxic in terms of lethality, whereas paracetamol, copper sulphate, and potassium dichromate caused deleterious effects in the reproductive performance of exposed D. magna. Adverse effects in the fitness of the daughter (F-1) generation were observed in the case of maternal exposure to paracetamol and copper sulphate, although they were not very pronounced. These findings highlight the usefulness of our approach and reinforce the view-shared by other authors-of the need for a generalised formal assessment of the transgenerational effects of pollutants., 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 European Regional Development Fund (ERDF), in the framework of the programme PT2020. Sara Antunes is recipient of individual post-doctoral grant (SFRH/BPD/109951/2015) by the Portuguese Foundation for Science and Technology (FCT). Bruno Nunes was supported by FCT (Researcher Contract IF/01744/2013)., info:eu-repo/semantics/publishedVersion

The aim of this study was to investigate the histopathological effects of oxytetracycline (OTC) on the gill and liver tissues of rainbow trouts (Oncorhynchus mykiss) following acute (96h: 0.005-50mg/L) and chronic (28days: 0.3125-5μg/L) exposures. Results suggest the existence of a cause-and-effect relationship between the exposure to OTC and tissue damage. Most predominant disorders observed in gills were progressive (e.g. hypertrophy of mucous cells and hyperplasia of epithelial cells) in acute exposure and regressive (e.g. lamellar fusion, epithelial lifting of lamellae and some changes in tissue architecture) in chronic exposure. However, only the acute exposure was responsible for a significant increase of the total gill pathological index. PAGE index, reflecting the extent of gill tissue available for gas exchanges in fish, remained unchanged for both exposures. In liver, circulatory (e.g. hemorrhage and increase of sinusoidal space), regressive (e.g. pyknotic nucleus, vacuolization and hepatocellular degenerations) and progressive (e.g. hypertrophy of hepatocytes) changes were observed, but just after acute exposure. After chronic exposure, only inflammatory changes (e.g. leucocytes infiltration) were observed. Following both exposures, a significant increase of the total liver pathological index was recorded. Despite the increase of the histological damage in individuals exposed to OTC, lesions observed were of minimal or moderate pathological importance, non-specific and reversible. The data gathered following acute and chronic exposures also suggest the onset of adaptive mechanisms of fish, namely for longer exposure periods. Furthermore the observed histological alterations appear to be result of several physio-metabolic disorders consequence of the biochemical and molecular modes of action of OTC. published

Daphnia spp. (pulgas-de-água) - Os organismos do género Daphnia são micro-crustáceos planctónicos de água doce pertencentes à ordem Cladocera (classe Branchiopoda), que ocorrem em ecossistemas lênticos. Devem o nome de “pulga-de-água” aos movimentos natatórios irregulares, análogos aos saltos das pulgas “verdadeiras”., info:eu-repo/semantics/publishedVersion

The increasing presence of pharmaceutical drugs in nature is cause of concern due to the occurrence of oxidative stress in non-target species. Acetaminophen is widely used in human medicine as an analgesic and antipyretic drug, and it is one of the most sold non-prescription drugs. The present study aimed to assess the toxic effects of acetaminophen (APAP) in Oncorhynchus mykiss following acute and chronic exposures in realistic levels. In order to evaluate the APAP effects in the rainbow trout, gills and liver were analyzed with biochemical biomarkers, such as catalase (CAT), total and selenium-dependent glutathione peroxidase (GPx), glutathione reductase (GRed) and glutathione-S-transferases (GSTs) activity and also lipid peroxidation levels (TBARS). The results obtained in all tests indicate that a significant response of oxidative stress was established, along with the increase of APAP concentrations. The establishment of an oxidative stress scenario occurred with the involvement of all tested biomarkers, sustaining a generalized set of pro-oxidative effects elicited by APAP. Additionally, the occurrence of oxidative damage strongly suggests the impairment of the antioxidant defense mechanism of O. mykiss. It is important to note that the occurrence of oxidative deleterious effects and peroxidative damages occurred for concentrations similar to those already reported for several freshwater ecosystems. The importance of these assumptions is further discussed under the scope of ecological relevance of the assessment of effects caused by pharmaceuticals in non-target organisms.

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

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

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

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.

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.

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.

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

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.

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

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

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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

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.