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Several types of stressors come into natural water bodies, degrading their quality, and having harmful effects on aquatic biota. As a result, many attempts have been made to develop complementary techniques to those imposed by the Water Framework Directive (WFD) to improve the water quality assessment strategy in a shorter time and be more faithful to the components and contaminants of the ecosystem. Daphnia magna has been extensively used as a model organism for ecotoxicity testing, and its ecotoxicological responses to several disturbance factors have been being well characterized. From this perspective, this work aimed to evaluate the applicability of the feeding bioassays with D. magna, as well as early distress tools (biochemical biomarkers), in the assessment of water quality of natural waters of reservoirs. Samplings were performed in several sites in three Portuguese reservoirs and were conducted in the spring of 2020. Bioassays and biomarkers results showed sensitivity to different reservoirs since the sites are minimally or moderately impacted. Biological responses can be related to several environmental factors, such as surrounding areas, seston composition, and chemical analysis (WFD), among others not quantified. This set of biological responses has presented good concordance with the ecological potential of the reservoirs.

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).

Despite the key role of zooplankton communities in regulating the water quality of lentic ecosystems, they are absent from the list of biological elements of the Water Framework Directive (WFD) for the assessment of ecological status. Based on this, the present work was a case study that aimed to understand the relevance of zooplankton as a bioindicator for classifying the water quality of reservoirs. For one year and in each season, the water quality of the Torrão reservoir was assessed using the mandatory elements proposed by the WFD in the sampling year (second cycle) and the legislation currently applicable (third cycle). Additionally, zooplankton samples were collected to characterize the community dynamic. The water quality of the Torrão reservoir does not reach the WFD good ecological potential. Furthermore, with the updating of the criteria, the classification tends to get worse. Concerning the zooplankton, the occurrence of Cyclopoida and Bosmina are associated with lower water quality, as well as the collapse or low abundance of the Daphnia population, in the same periods. Low abundances of zooplankton are associated with better water quality, and the Shannon-Wiener diversity values decrease with the increase of the trophic state. High-efficiency feed filters and macrofiltrator organisms dominate the Torrão reservoir in all seasons, which is associated with low water quality. The Calanoida/Cyclopoida ratio shows a strong and negative association with the trophic state. The occurrence and abundance of species, Shannon-Wiener diversity, functional groups (high and low efficiency feed filters and macrofiltrators), and different ratios (large Cladocera/total Cladocera and Calanoida/Cyclopoida) are promising and valuable tools to determine the water quality status, and should be considered within the WFD metrics. Notwithstanding this, further research including reservoirs from different geographical areas with various trophic status and pressures must be analyzed.

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.

In the wild various organisms contribute to daphnids diet. This study, intendeds to evaluate the potential of the concentration ofFeeding assays were performed according to standard guidelines for chronic assays (21 days), and life-history parameters and several biomarkers (protein content, oxidative stress, energetic reserves and pigments) were measured. Five food regimens were conducted with 20 individual replicates (A -The five diets showed a different C, N, and carotenoids composition, with an increase in the mixed diets. The results confirmed that the mixed diets improvedResults demonstrated that different diets provided a nutritional diversified food to the daphnids that induced differences in

The Water Framework Directive (WFD) is used to evaluate the water quality of aquatic ecosystems. Phytoplankton is the only biological element considered in the reservoirs water quality assessment. In this study, we aimed to assess the use of the bacterioplankton community as an indicator of water quality, using a culture-independent assay (denaturing gradient gel electrophoresis, DGGE). Four Portuguese reservoirs (Miranda, Pocinho, Aguieira and Alqueva) were analysed in four periods (autumn 2018, spring and autumn 2019, and spring 2020). Bacterial total abundance had similar values for Miranda, Pocinho and Aguieira, and generally lower values for Alqueva. Diversity and richness values did not show a clear trend. Negative correlations were observed between some nutrients and the bacterial community. Overall, members of Actinobacteria, Bacteroidetes, Cyanobacteria, Nitrospirae and Proteobacteria were identified in all sampling sites. In Alqueva, no spatial, temporal or water body quality relationships with bacterial community were observed, which may be due to its higher size, low water velocity rate and higher residence times. However, in Miranda, Pocinho and Aguieira, a strong spatial and temporal bacterial community dynamic was observed. Furthermore, the presence of some species (e.g., Acinetobacter sp.) may reflect the poor water quality that was not detected by the WFD approach.

Intermittent rivers and streams are aquatic ecosystems that lose hydrological connectivity during drought periods. Anthropogenic pressures combined with climate change accelerate the spatial and temporal intermittency of these ecosystems, promoting alterations in ecology and ecosystem functions. This study aimed to assess the ecological status of Ribeira de Silveirinhos, located in the municipalities of Valongo and Gondomar (north of Portugal), using the metrics described in the Water Framework Directive. Thus, five sampling sites were selected along Ribeira de Silveirinhos. Sampling was done in three different periods, spring of 2019 and 2020, and autumn of 2019. At each site, physical and chemical water parameters were measured, and the benthic macroinvertebrates community was characterized. Physical and chemical parameters showed low pH values at upstream sites (where the dry phase occurs and is surrounded by Eucalyptus monoculture) and high concentrations of phosphorus at the downstream sites (subjected to several anthropogenic activities, iron waters, and agriculture). The macroinvertebrate community revealed low values of specific abundance and richness, especially during the dry period. Sensitive taxa (Ephemeroptera, Plecoptera, and Trichoptera) were negatively correlated with flow intermittency. Results showed that Ribeira de Silveirinhos is classified between “poor” and “moderate” in terms of ecological status. Intermittent streams are undervalued, so their protection is at risk. Thus, it is important to consider the specific ecological functioning of these ecosystems and to adjust the planning and management models in order to guarantee ecological quality and conservation processes.

The Water Framework Directive (WFD) is the European legislation on water policy that assesses water quality according to time-consuming metrics and specific taxonomic needs. In this sense, the objective of this study was to evaluate the sensitivity of Daphnia magna feeding rate assays to assess/discriminate the water quality of heavily modified and artificial water bodies. Monthly, for one year, the quality of the Crestuma-Lever reservoir (in two sampling sites: Crestuma and Marina) was assessed using physical, chemical, and biological (concentration of chlorophyll-a) elements proposed by the WFD. Additionally, D. magna was exposed to the collected water samples and the feeding rates were evaluated to include an ecosystem function evaluation in water quality assessment. The WFD metrics showed that, overall, the Crestuma-Lever reservoir has a rating of Good to Moderate Ecological Potential, regardless of site. Feeding rates varied with the sampling site and months, demonstrating that feeding behavior evaluation is a sensitive tool that allows discriminate potential effects indicative of a lower water quality. This finding was recorded by the decrease in the feeding rate (Crestuma: May, Sept; Marina: Nov, Jan, May), despite the WFD classification, and once the organisms are affected by the components present in the water samples.

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.

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 (C

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

Reservoirs have been subject to anthropogenic stressors, becoming increasingly degraded. The evaluation of ecological potential in reservoirs is remarkably challenging, and consistent and regular monitoring using the traditional in situ methods defined in the WFD is often time- and money-consuming. Alternatively, remote sensing offers a low-cost, high frequency, and practical complement to these methods. This paper proposes a novel approach, using a C2RCC processor to analyze Sentinel-2 imagery data to retrieve information on water quality in two reservoirs of Portugal, Aguieira and Alqueva. We evaluate the temporal and spatial evolution of Chl a and total suspended solids (TSS), between 2018 and 2020, comparing in situ and satellite data. Generally, Alqueva reservoir allowed lower relative (NRMSE = 8.9% for Chl a and NRMSE = 21.9% for TSS) and systematic (NMBE = 1.7% for Chl a and NMBE = 2.0% for TSS) errors than Aguieira, where some fine-tuning would be required. Our paper shows how satellite data can be fundamental for water-quality assessment to support the effective and sustainable management of inland waters. In addition, it proposes solutions for future research in order to improve upon the methods used and solve the challenges faced in this study.

Seaweed are ecologically important organisms playing a central structural and functional role in coastal ecosystems (providing habitat, mating and nursery grounds, food and refuge for several marine organisms). Additionally, their commercial potential has been rising since seaweed are directly or indirectly used by humans in many countries. This work aimed to evaluate the temporal and spatial variation of target species biomass and the composition of seaweed assemblages at an exposed rocky shore, in Northern Portugal. The targeted species were: Codium spp., Chondrus crispus, Mastocarpus stellatus, Osmundea pinnatifida and Chondracanthus acicularis. Two sampling sites were defined and in each one 18 haphazard placed quadrats (50 × 50 cm) were sampled: 9 in the low-shore and 9 in the mid-shore. Sampling was conducted periodically from April 2017 to January 2018, in six sampling occasions. Significant temporal variation was detected in the mean biomass values of Codium spp. and O. pinnatifida, at low-shore, where high abundance of these species in typically cold months could be related to the atypical warmer year 2017 in Portugal. C. acicularis revealed a significant spatial variation at both shore levels, which could be explained by different water channels orientations at the two sampling sites. The multivariate analysis showed that the effect of sampling occasion on the seaweed assemblage structure depended on site and revealed that some species, such as Ulva spp., apparently play an important role in seaweed assemblages of Belinho-Mar intertidal and highlights its ecological importance for these community. This study provides a baseline data for future work in similar areas, allowed the development of effective management plans as basis for seaweed exploitation guidelines.

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

Salinization of coastal freshwater ecosystems is already occurring in some regions of the world. This phenomenon raises serious concerns on the protection of coastal freshwater ecosystems, since many of them support and shelter a large number of species and are considered hotspots of biodiversity. This work intended to assess the adverse effects that salinization, caused by the intrusion of seawater (SW), may pose to freshwater organisms. In this study, three specific goals were addressed: (i) to assess if sodium chloride (NaCl) may be used as a surrogate of natural SW at early-stages of risk assessment; (ii) to identify the most sensitive freshwater species to salinity NaCI; and (iii) to determine if increased tolerance to salinity may be acquired after multigenerational exposure to low levels of salinization (induced with NaCl). A total of 12 standard monospecific bioassays were carried out by exposing organisms from different taxonomic groups (Cyanobacteria: one species, Tracheophyta: two species, Rotifera: one species, Arthropoda: two species and Mollusca: one species) to a series of concentrations of NaCI (ranging from 0.95 to 22.8 mS cm(-1) ) or dilutions of SW (ranging from 1.70 to 52.3 mS cm(-1) ). In general, NaCl exerted similar or higher toxicity than SW, both at lethal and sublethal levels, suggesting that it may be proposed as a protective surrogate of SW for first tiers of salinization risk assessment. Among all tested species, the cyanobacterium Cylindrospermopsis racihorskii, the daphnid Daphnia longispina and the rotifer Brachionus plicatilis were the most sensitive taxa to salinization (EC50, This study was funded by National Funds (OE) through FCT/MEC and co-funded by FEDER, through COMPETE (POFC), by FSE and POPH (Programme Cieˆncia 2007-8) and the research project SALTFREE (PTDC/AAC-CLI/111706/2009). C.V. is a grant holder from FCT (ref. SFRH/BD/81717/2011)., info:eu-repo/semantics/publishedVersion

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

Alpine ponds are shallow waterbodies formed in alpine zones characterized by usual pristine conditions, however subject to harsh conditions that enable the emergence of unique communities with high conservational value. Therefore, it is important to gather information about these ecosystems and understand the changes in the biota. This work aimed to use Portugal’s alpine ponds as a case of study in assessed the macroinvertebrates communities in the Mediterranean ecozone. To fulfill this goal, we aimed to understand the alterations on macroinvertebrates community and how are they modulated by environmental variables. Five alpine ponds in Serra da Estrela (Portugal) were studied for one year. Macroinvertebrate communities and physical and chemical water parameters were determined in June/16, October/16, and April/17. Water chemistry values were similar to other studied lentic waterbodies in alpine zones in Europe, with slightly acidic water and low conductivity. Although accentuated macroinvertebrates differences between ponds, no significant differences in diversity were recorded, indeed the main differences recorded were influenced by ponds habitat types and environmental variables. Canonical Correspondence Analysis showed differences in the communities between ponds, namely the high influence of several physical and chemical parameters (conductivity, phosphate content, and pH) in the macroinvertebrates community composition. Indeed, Spearman’s correlation revealed significant interaction in diversity, abundance and richness of macroinvertebrates and several physical and chemical parameters (pH, dissolved oxygen and nitrates concentration).

Serra da Estrela is Portugal's mainland highest mountain, with distinctive geological, climatic and geomorphological characteristics which play a key role on surface and groundwater resources. These mountains have the largest set of glacial ponds of Portugal and are scarcely studied. Besides, there are concerns of possible impacts of the use of salts in road de-icing on ponds' biotic communities. This research intends to characterize the hydrology of five Mediterranean alpine ponds and assess their planktonic communities. The biotic index proposed by the Water Framework Directive for lentic systems was used to check its applicability to these systems. Sampling was conducted monthly from June to November 2015 alongside with hydrological characterization. Water parameters and nutrients' content were quantified. Phytoplankton and macrozooplankton were identified. Ponds' water level is controlled by precipitation (input) and evaporation, infiltration and streamflow (output). Ponds were in good ecological quality. However, the current ecological assessment tools proposed by the WFD for lentic systems do not fit alpine ponds, showing a gap in the current methods. Salts used for road de-icing did not impact the communities and the ecological state of ponds. Phytoplankton was dominated by Cyanobacteria, Dinophyceae and Bacillariophyceae; Cyclopoida was the most abundant zooplanktonic group. For macrozooplankton, RDA shows association between cladocerans and phosphorus content. Phosphorus changes promoted shifts in zooplankton, what may be of major concern from a conservation point of view, once alpine ponds and its species are still mostly unknown. Efforts should be done to develop tools to classify aquatic ecosystems from harsh environments.

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.

Antibiotics (e.g. ciprofloxacin) have been detected in surface water and groundwater for several decades. In order to understand the potential impact of the continuous exposure of aquatic organisms to ciprofloxacin, a chronic assay was carried out with Daphnia magna. This approach allowed evaluation of the effects of ciprofloxacin on life-history and sub-individual parameters (antioxidant status and metabolic response: activities of catalase and glutathione S-transferases – GSTs; peroxidative damage; thiobarbituric acid reactive substances and genotoxic effects (genetic damage index, measured by the comet assay). Life-history parameters of D. magna showed no significant effects after ciprofloxacin exposure. Concerning oxidative stress and metabolism parameters, no significant alterations were reported for catalase and GSTs activities. However, a dual response was observed, with a significant decrease in lipid peroxidation levels at low ciprofloxacin concentrations (

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.

Among emerging contaminants, pharmaceuticals are one of the most relevant groups of substances found in aquatic environment, due to their universal use, specific physical-chemical properties, and mode of action in aquatic organisms at low concentrations. Considering the magnitude of concentrations and the considerable small amount of information about their potential ecotoxicological effects on wildlife (especially towards organisms from tropical areas), the present study intended to assess the acute and chronic effects of three pharmaceuticals (the analgesic acetaminophen, the anti-inflammatory diclofenac, and the antihypertensive propranolol) in the tropical crustacean species Ceriodaphnia silvestrii. Results of acute exposures showed a considerable variation in toxicity among pharmaceuticals: acetaminophen (EC50 = 40.3 mg L−1) < diclofenac (EC50 = 37.9 mg L−1) < propranolol (EC50 = 3.17 mg L−1), C. silvestrii being more sensitive to the drug propranolol. Data of chronic toxicity tests showed the occurrence of reproductive adverse effects, but also stimulatory phenomena. Propranolol caused a significant increase in fecundity for the concentrations up to 0.0313 mg L−1, and the rate of population increase was significantly augmented from the lower concentration tested. Considering these results and the wide distribution of C. silvestrii in tropical regions, we suggest the use of this species to be used as test organism in ecotoxicity assays performed in such areas, especially for biomonitoring and/or the determination of effects of pharmaceutical drugs. © 2018, Springer International Publishing AG, part of Springer Nature. Acknowledgments We are grateful to the Brazilian Research Council (CNPq) for the financial support provided for this research project (No. 142131/2010-5). Bruno Nunes was hired under the program Investigador FCT, co-funded by the Human Potential Operational Program (National Strategic Reference Framework 2007–2013) and European Social Fund (EU). Sara Antunes was funded by a post-doctoral fellowship (SFRH/BPD/109951/2015) by Fundação para a Ciência e Tecnologia (Government of Portugal).

Standard assays for phytotoxicity provide a reductionist view on the performance of plants under toxic stress. To address two of the most prominent issues in plant toxicity studies, our aims were (1) to assess how well physiological and biochemical parameters complement standard toxicological endpoints when testing natural soils and (2) to assess the suitability of three commonly used control soils as comparative references. We compared the performance of Zea mays and Helianthus annuus in three control soils (artificial Organisation for Economic Co-operation and Development (OECD) soil, standard LUFA 2.2 soil, and turf-perlite) against three natural soils representing a gradient of contamination (from a deactivated uranium mine). Standard endpoints (emergence and biomass) were estimated, along with pigment content, photosynthetic parameters, cellular injury, and proline content. The toxicological profile of natural soils was highly dependent on the control soil used as reference; also, plant physiological performance was influenced by the soils’ properties. We discuss the need to interpret and combine multiple lines of evidence as a way to increase the degree of confidence one classifies soils based on their ecotoxicity, and this is where the integration of physiological and biochemical parameters bring added value. When facing large variability in soil characteristics, it is best to collect and integrate as much information possible to strengthen conclusions about phytotoxicity of natural soils. Obviously, this refutes reductionist views and places the final conclusion in the hands of expert judgment.

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.

In vivo phycocyanin (PC) fluorescence allows assessing cyanobacterial abundance in an easy, fast and cost-effective way. However, the establishment of PC thresholds is necessary for its use in routine monitoring programmes and there has been no consensus regarding their definition. This work aimed: (1) to assess the potential species-specific variation in fluorometric PC content among Microcystis aeruginosa, Nostoc muscorum and Cylindrospermopsis raciborskii; (2) to propose specific PC thresholds based on World Health Organization alert levels; and (3) to evaluate the in vivo PC signal reliability to interferences from massive algal growth and mixtures of bloom-forming cyanobacteria. Strong linear relationships were recorded between PC and cell counts, biovolume and chlorophyll a. However, a significant species-specific variation in PC was observed using cell counts. Increased microalgal densities did not cause significant interference of PC signals. Also, dual mixtures of cyanobacteria revealed strong relationships between measured and expected PC content. Results suggest that fluorometric PC is a good predictor for cyanobacterial biomass but cell number is not the best parameter to define thresholds. Biovolume should be used instead. Nevertheless, species-specific thresholds must be considered, rather than a general cyanobacterial threshold.

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 edible clam Venerupis decussata is widely distributed in European aquatic systems, some of which are under strong anthropogenic pressure, which can contribute to trophic transfer of xenobiotics to humans. Accordingly, the present study focused on the tolerance, bioconcentration, and biochemical responses of V. decussata after exposure to ecologically relevant concentrations of lead. Health risks to humans after consumption of clams was also explored. An acute toxicity assay (96 h) was conducted with wild clams, using Pb exposure concentrations ranging from 0 mg L−1 to 1.80 mg L−1. Lethality, bioconcentration factor (BCF), intracellular partitioning, and a relevant set of biomarkers were used as endpoints. Clams, interstitial water, water column, and sediment samples were collected to analyze Pb concentration. The Pb concentration in wild clams was below international consumption guidelines. Under laboratory conditions, clams revealed high sensitivity to Pb (median lethal concentration of 0.65 mg L−1), with a high bioconcentration ability (bioconcentration factor > 1) during exposure. The intracellular partitioning data showed that most of the Pb had accumulated in the insoluble fraction (>80%). Several significant biochemical changes were observed, namely on catalase and glutathione-S-tranferase activities and metalothionein content. Overall, it was demonstrated that the European clam has a reduced tolerance to Pb, compared with other bivalves. However, consumption of clams from the Ria de Aveiro lagoon (Portugal) does not raise public health concerns in terms of Pb. Environ Toxicol Chem 2014;33:2786–2794. © 2014 SETAC

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.