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(1) An approach with great potential for fast and cost-effective profiling and identification of diatoms in lake ecosystems is presented herein. This approach takes advantage of Raman spectroscopy. (2) The study was based on the analysis of 790 Raman spectra from 29 species, belonging to 15 genera, 12 families, 9 orders and 4 subclasses, which were analysed using chemometric methods. The Raman data were first analysed by a partial least squares regression discriminant analysis (PLS-DA) to characterise the diatom species. Furthermore, a method was developed to streamline the integrated interpretation of PLS-DA when a high number of significant components is extracted. Subsequently, an artificial neural network (ANN) was used for taxa identification from Raman data. (3) The PLS interpretation produced a Raman profile for each species reflecting its biochemical composition. The ANN models were useful to identify various taxa with high accuracy. (4) Compared to studies in the literature, involving huge datasets one to four orders of magnitude larger than ours, high sensitivity was found for the identification of Achnanthidium exiguum (67%), Fragilaria pararumpens (67%), Amphora pediculus (71%), Achnanthidium minutissimum (80%) and Melosira varians (82%).

Centro de Investigación en Sanidad Animal (CISA), Research on immunotherapeutic agents has become a focus for the treatment of fish diseases. The ability of algae to produce secondary metabolites of potential interest as immunotherapeutics has been documented. The present research intended to assess antiviral and antibacterial activities of macro- and microalgae extracts against viral and bacterial pathogens and explore their immunomodulatory potential using zebrafish (Danio rerio) larvae as a model organism. The cytotoxicity and antiviral activity of eight methanolic and ethanolic extracts from two macroalgae (Fucus vesiculosus, Ulva rigida) and two microalgae (Nannochloropsis gaditana, Chlorella sp.) were analyzed in established fish cell lines. Six extracts were selected to evaluate antibacterial activity by disk diffusion and growth inhibition assays. The three most promising extracts were characterized in terms of fatty acid composition, incorporated at 1% into a plant-based diet, and evaluated their effect on zebrafish immune response and intestinal morphology in a short-term feeding trial. All extracts exhibited in vitro antiviral activity against viral hemorrhagic septicemia and/or infectious pancreatic necrosis viruses. Methanolic extracts from F. vesiculosus and U. rigida were richer in saturated fatty acids and exhibited in vitro antibacterial action against several bacteria. Most promising results were obtained in vivo with F. vesiculosus methanol extract, which exerted an anti-inflammatory action when incorporated alone into diets and induced pro-inflammatory cytokine expression, when combined with the other extracts. Moreover, dietary inclusion of the extracts improved intestinal morphology. In summary, the results obtained in this study support the potential of algae as natural sources of bioactive compounds for the aquaculture industry., M. Monteiro, A. S. Lavrador, R. A. Santos, and F. Rangel were supported by grants SFRH/BD/114995/2016, ZEBRALGRE_BM_2019-003, SFRH/BD/131069/2017, and SFRH/BD/138375/2018, respectively, from FCT — Foundation for Science and Technology, under the POCI program. A. Couto C. Serra and P. Enes have a scientific employment contract supported by national funds through FCT. E. da Costa and M. R. Domingues are financially supported by FCT/MCTES (Portugal) through CESAM (UIDB/50017/2020 + UIDP/50017/2020), QOPNA (FCT UID/QUI/00062/2019), LAQV/REQUIMTE (UIDB/50006/2020), and RNEM (LISBOA-01–0145-FEDER-402–022125). This research was partially supported by the Strategic Funding to UID/Multi/04423/2019 (POCI-01–0145-FEDER-007621) through national funds provided by FCT under the project PTDC/CVT-WEL/5207/2014.

14 Pág. Centro de Investigación en Sanidad Animal (CISA), The search for natural immunotherapeutic agents has become increasingly important for fish disease treatment and prophylaxis. The present research explored the effects of macro- and microalgal extracts on zebrafish juvenile growth and immune status. A plant-based diet was used as a control (diet C), and seven other diets were prepared similar to diet C, supplemented with 10 g Kg−1 of each algal extract (Fucus vesiculosus, F; Nannochloropsis gaditana, N; and Ulva rigida, U), or a combination of extracts (diets FN, FU, NU and FNU). A fishmeal-based diet was included as a positive control (diet C+). Diet C+ outperformed the remaining dietary treatments, growth-wise. None of the extracts improved diet C negative effects. However, the inclusion of U. rigida and N. gaditana extracts promoted an immunomodulatory action after 1 week of trial, by upregulating cytokine expression. This effect subsided after 30 days, suggesting tolerance may be developed over time. In contrast, effects on growth-related genes were still observed after 5 weeks of feeding F, N, U and FN diets. Algal extract dietary inclusion did not compromise intestinal integrity. These findings support the potential of algal extracts as functional feed additives during short periods (, Fincanced by the Foundation for Science and Technology (grants number: SFRH/BD/114995/2016,EBRALGRE_ BM_2019-003 UID/Multi/04423/2019 POCI-01-0145-FEDER-007621 PTDC/CVT-WEL/5207/2014).

The disruption of pathogen communication or quorum-sensing (QS) via quorum-quenching (QQ) molecules has been proposed as a promising strategy to fight bacterial infections. Bacillus spp. have recognizable biotechnology applications, namely as probiotic health-promoting agents or as a source of natural antimicrobial molecules, including QQ molecules. This study characterized the QQ potential of 200 Bacillus spp., isolated from the gut of different aquaculture fish species, to suppress fish pathogens QS. Approximately 12% of the tested Bacillus spp. fish isolates (FI). were able to interfere with synthetic QS molecules. Ten isolates were further selected as producers of extracellular QQ-molecules and their QQ capacity was evaluated against the QS of important aquaculture bacterial pathogens, namely Aeromonas spp., Vibrio spp., Photobacterium damselae, Edwardsiela tarda, and Shigella sonnei. The results revealed that A. veronii and E. tarda produce QS molecules that are detectable by the Chr. violaceum biosensor, and which were degraded when exposed to the extracellular extracts of three FI isolates. Moreover, the same isolates, identified as B. subtilis, B. vezelensis, and B. pumilus, significantly reduced the pathogenicity of E. tarda in zebrafish larvae, increasing its survival by 50%. Taken together, these results identified three Bacillus spp. capable of extracellularly quenching aquaculture pathogen communication, and thus become a promising source of bioactive molecules for use in the biocontrol of aquaculture bacterial diseases.

In late 2014, Portugal implemented a national program for the treatment of patients with chronic hepatitis C with directacting antiviral agents. This program has made Portugal one of the first European countries to implement a structured measure of treatment to eliminate this serious public health problem. The aim of this study was to assess the effectiveness of direct-acting antiviral therapy in the treatment of patients with chronic hepatitis C virus infection.A retrospective observational study was conducted at Centro Hospitalar de Lisboa Ocidental on the national online platform from December 2014 until February 2017 and included patients with hepatitis C virus infection who underwent treatment. The primary endpoint was sustained virologic response at least 12 weeks post treatment. Data was analyzed with the SPSS 17.0 program.During the study period, 820 patients completed therapy and achieved sufficient follow-up time to assess sustained virologic response with an overall response rate of 97.2% (n = 797) and a response rate of 98.0%, 99.5%, 90.9%, 95.1% and 94.2% for genotypes 1a, 1b, 2, 3 and 4, respectively. Data suggested that advanced fibrosis (F3/F4), human immunodeficiency virus co-infection and treatment failure with interferon and ribavirin were not negatively related with sustained virologic response in our population. Most patients (80.1%) completed treatment with ledipasvir/sofosbuvir ± ribavirin. The most common adverse events were fatigue and insomnia followed by headache and weight loss.Patients predominantly had genotype 1 infection which correlates with HCV distribution in Europe, but we found a major proportion in genotype 4 which can be explained by immigration from African countries. Our patients' ages ranging from 22 to 90 years, reflected a new approach with no upper age limit. Direct-acting antivirals regimens resulted in remarkably high SVR rates compared to interferon-based regimens, which were consistent with clinical trials data.Our data showed that direct-acting antiviral-based regimens are safe and have a high success rate in the treatment of patients with hepatitis C virus infection in a real-world setting.Introdução: No final de 2014 foi implementado em Portugal um programa nacional para o tratamento de doentes com infecção crónica por vírus da hepatite C com recurso a antivíricos de acção directa. Este programa fez com que Portugal fosse um dos primeiros países europeus a implementar uma medida estruturante para a eliminação da hepatite C. Este estudo tem como objectivo a avaliação da efectividade dos antivíricos de acção directa no tratamento da hepatite C crónica. Material e Métodos: Estudo retrospectivo observacional dos doentes seguidos no Centro Hospitalar de Lisboa Ocidental, entre dezembro de 2014 e fevereiro de 2017. O objectivo primário do estudo é avaliar a resposta virológica sustentada a partir das 12 semanas pós tratamento. Analisámos os dados com o programa SPSS 17.0. Resultados: Durante o período do estudo 820 doentes completaram o tratamento e o tempo necessário para avaliação da resposta virológica sustentada. A resposta virológica sustentada global foi de 97.2% (n = 797), com taxas de resposta de 97,2%, 98,5%, 90,9%, 95,1% e 94,2% para os genótipos 1a, 1b, 2, 3 e 4, respectivamente. Os dados sugerem não haver relação entre a fibrose avançada (F3 / F4), a coinfecção pelo vírus da imunodeficiência humana e a falência do tratamento com interferão e ribavirina e uma menor resposta ao tratamento. A maioria dos doentes (80,1%) concluiu o tratamento com ledipasvir/sofosbuvir ± ribavirina. Os eventos adversos mais frequentes foram a fadiga e a insónia, seguida de dor de cabeça e perda de peso. Discussão: A população em estudo apresentou maior prevalência de infecção pelo genótipo 1, à semelhança dos restantes países Europeus, contudo a prevalência do genótipo 4 foi superior, reflectindo a imigração africana. A faixa etária (22 - 90 anos) dos doentes tratados reflecte uma nova abordagem sem limite superior de idade. A taxa de RVS obtida, muito superior à obtida com regimes baseados em interferão, foi consistente com os dados dos ensaios clínicos. Conclusão: Os dados encontrados demonstram que os regimes baseados em antivirais de acção directa, em contexto de vida real, são seguros e eficazes no tratamento de doentes com infecção por vírus da hepatite C.

Freshwater quality has been changing due to the ever greater use of water resources and the contamination load resulting from human activities. Management of these systems, thus, requires constant diagnose of water quality with fast and efficient methodologies. The conventional methods adopted are, however, time-consuming, often very expensive, and require specialised expertise. Raman spectroscopy (RS) is a simple, fast and label-free technique that can be applied to environmental diagnosis using diatoms. Here, we developed a diagnostic method based on Raman spectroscopy applied to freshwater diatoms. For this, Raman spectra were recorded from diatoms of three lakes of a natural city park. The data acquired was analysed by chemometrics methods to describe the data (Partial Least Squares Regression), infer relationships in the dataset (Cluster Analysis) and produce classification models (Artificial Neural Network). The classification models developed diagnosed the lakes with excellent accuracy (89%) without requiring taxonomic information about the diatom species recorded. This study provides a proof-of-concept for the application of diatom Raman spectroscopy to diagnosing water quality, laying an important foundation for future environmental studies aiming at assessing freshwater systems, to be replicated at larger scales and to varied geographic settings.

Objectives Since the outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the pressure to minimise its impact on public health has led to the implementation of different therapeutic strategies, the efficacy of which for the treatment of coronavirus disease 2019 (COVID-19) was unknown at the time. Remdesivir (REM) was granted its first conditional marketing authorisation in the EU in June 2020. The European Medicines Agency (EMA) and local health authorities all across the EU have since strongly recommended the implementation of pharmacovigilance activities aimed at further evaluating the safety of this new drug. The objective of this study was to evaluate adverse drug reactions (ADRs) attributed to either REM or hydroxychloroquine (HCQ) in patients hospitalised for COVID-19 in Centro Hospitalar de Lisboa Ocidental, a Portuguese hospital centre based in Lisbon. We present the preliminary results reporting plausible adverse effects of either HCQ or REM. Methods An observational cohort study was carried out between 16 March and 15 August 2020. Participants were divided into two cohorts: those prescribed an HCQ regimen, and those prescribed REM. Suspected ADRs were identified using an active monitoring model and reported to the Portuguese Pharmacovigilance System through its online notification tool. The ADR cumulative incidence was compared between the two cohorts. Results The study included 149 patients, of whom 101 were treated with HCQ and the remaining 48 with REM. The baseline characteristics were similar between the two cohorts. A total of 102 ADRs were identified during the study period, with a greater incidence in the HCQ cohort compared with the REM cohort (47.5% vs 12.5%; p

Contamination by microplastics is increasing steadily worldwide, affecting all environments. Additionally, aquatic organisms are often exposed to mixtures of other contaminants, including various chemicals. Numerous studies reported adsorption of chemicals to microplastics, raising concern about their possible role as vehicles of exposure through transfer to biota. Nevertheless, until recently, the studies on the topic were mostly focused on the marine environment. In the past five years, however, plenty of publications contributed empirical data about freshwater ecosystems, raising the need for a critical appraisal of the information. Herein the scientific literature was reviewed and multivariate data analysis was done. The analysed studies employed widely different experimental designs, endpoints, test species, shapes and concentrations of various polymer types and chemicals, often not relevant for the freshwater environment. Our integrated analytical approach revealed unfathomable research gaps, given the theoretical knowledge available and lessons learned from research about the marine environment. Greater harmonization of laboratory studies investigating this topic is needed, as well as testing conditions reflecting real exposure scenarios. Furthermore, standardized testing protocols are urgently required to guide such experiments and improve the comparability of the results obtained.

This study aimed at assessing the effects of the dietary tryptophan (Trp) supplementation on growth and feed utilization, brain serotonin content, and expression of selected liver genes (involved in the liver serotonin pathway, protein synthesis degradation, and antioxidant activity) in zebrafish. A growth trial was conducted with zebrafish juveniles fed five experimental isoproteic (40%DM) and isolipidic (8%DM) fishmeal-based diets containing graded levels of Trp: a Trp-non-supplemented diet (diet Trp0, with 0.22% Trp) and four Trp-supplemented diets containing 2–16 times higher Trp content (diets Trp2, Trp4, Trp8, and Trp16 with 0.40, 0.91, 2.02, and 3.34% Trp, respectively). Diets were tested in quadruplicate, with fish being fed twice a day, 6 days a week for 6 weeks to apparent visual satiation. At the end of the trial, growth performance and feed utilization were assessed, and fish from all experimental groups were sampled for whole-body composition analysis. In addition, fish fed low (Trp0), medium (Trp4), and high (Trp16) Trp diets were also sampled for analysis of brain serotonin content and liver gene expression. Tested tryptophan levels did not influence growth performance nor feed intake. However, values of energy and nitrogen retention as well as body energy content indicate a better feed utilization with diets containing around 0.9% and 2.0% DM Trp. Brain serotonin content increased with increasing dietary tryptophan levels. In addition, regarding liver genes, dietary treatment had a modulatory effect on the expression of Htr1aa and Htr2cl1 genes (encoding for serotonin receptors), TPH1a gene (encoding for tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of serotonin from tryptophan), TOR gene (involved in protein synthesis), and Keap1 gene (involved in antioxidant responses).

Zebrafish is currently intensively reared in laboratories around the world due to its increasing importance as an experimental model in biomedical research. Recirculating aquaculture systems (RAS) have been generally used for zebrafish rearing, allowing the maintenance of high densities of fish with minimal water renewal. Biofilters play a pivotal role in RAS by converting the highly toxic ammonia excreted by fish into nitrate. Due to its much lower acute toxicity, nitrate accumulation in RAS is usually neglected, and fish can be exposed to relatively high levels of this compound for extended periods. This study evaluated the impact of chronic exposure to relevant levels of nitrate on the zootechnical performance and histology of selected organs (gills, integument, kidney, liver, and intestine) of juvenile zebrafish, with the aim to define safety levels of this nitrogenous compound for zebrafish rearing. For that, groups of 30-day-old zebrafish were exposed to − 1 nitrate-N for 28 days. No mortality was registered in fish exposed up to 200 mg L − 1 nitrate, and all individuals seemed externally healthy; however, in fish exposed to the highest nitrate concentration mortality reached 47% at the end of the trial, and many individuals showed lethargy, abnormal swimming, emaciation, lordosis, and/or superficial lesions. Although final growth was not significantly different among groups, growth parameters tend to decrease with increasing levels of nitrate, and a significant negative correlation was found between weight gain and nitrate levels, suggesting a dose-dependent negative effect of nitrate on growth. Except for the lowest nitrate concentration (100 mg L − 1 nitrate-N), the histological survey revealed significant changes induced by nitrate in all examined organs, and a dose-dependent effect of nitrate on the overall histopathological changes is suggested. In conclusion, this study shows that the chronic exposure of zebrafish juveniles to nitrate induces histopathological changes that would lead to a negative impact on the general health condition of fish. Fish growth tended to decrease and the overall histological damages tended to increase with increasing nitrate levels, particularly above 100 mg L − 1 (the lowest tested value). Thus, we recommend that this limit of 100 mg L − 1 nitrate-N should not be exceeded in RAS during rearing of juvenile zebrafish.

Electro-Fenton technology was able to decrease zebrafish embryotoxicity of fluoxetine and pirimicarb, when single or in mixture.

Diatom species are good pollution bioindicators due to their large distribution, fast response to changes in environmental parameters and different tolerance ranges. These organisms are used in ecological water assessment all over the world using autoecological indices. Such assessments commonly rely on the taxonomic identification of diatom species-specific shape and frustule ornaments, from which cell counts, species richness and diversity indices can be estimated. Taxonomic identification is, however, time-consuming and requires years of expertise. Additionally, though the diatom autoecological indices are region-specific, they are often applied indiscriminately across regions. Raman spectroscopy is a simpler, fast and label-free technique that can be applied to environmental diagnosis with diatoms. However, this approach has been poorly explored. This work reviews Raman spectroscopy studies involving the structure, location and conformation of diatom cell components and their variation under different conditions. A critical appreciation of the pros and cons of its application to environmental diagnosis is also given. This knowledge provides a strong foundation for the development of environmental protocols using Raman spectroscopy in diatoms. Our work aims at stimulating further research on the application of Raman spectroscopy as a tool to assess physiological changes and water quality under a changing climate.

A standard diet for zebrafish, based on their specific nutritional requirements, is of primary importance to improve experimental outcomes with this model organism and optimize its large-scale production. However, the main basic nutritional requirements of zebrafish are yet to be determined. This study aimed at contributing to fill this gap by evaluating the dietary protein requirement of zebrafish juvenile. Ten isoenergetic fishmeal-based diets with increasing protein levels (15%-60%) were formulated, and each diet was assigned to duplicated groups of zebrafish (53.6 mg/17.8 mm initial mean body weight/fork length), fed to apparent satiation during 8 weeks. Weight gain, protein retention, and feed efficiency significantly increased in fish fed diets with increasing protein levels up to 35%-40% and then stabilized. Based on dose-response models, the dietary protein requirement of zebrafish juvenile was estimated at 37.6% and 44.8% for maximum weight gain and maximum protein retention, respectively (with a crude protein-to-energy ratio of about 22.5 g/MJ), corresponding to a protein intake of 14 mg/g average body weight/day. Feed intake increased linearly when fish were fed diets with decreasing protein levels below the estimated requirement, suggesting that zebrafish would regulate feed intake primarily to meet protein needs. On the other hand, the efficiency of protein utilization and retention linearly decreased when fish were fed diets with increasing protein levels above the estimated requirement, indicating that the excess of dietary protein would be deaminated, contributing to increased ammonia excretion. The whole-body composition of fish was affected by the dietary protein level, with fish fed diets with higher protein levels having higher water and protein contents and lower energy content. Considering that zebrafish juveniles are often reared with diets containing excessive amounts of protein, we suggest that the estimated protein requirement should be taken into account to formulate a more suitable, cost-effective, and less pollutant diet for this species.

Lymnaea stagnalis, also referred to as great or common pond snail, is an abundant and widespread invertebrate species colonizing temperate limnic systems. Given the species importance, studies involving L. stagnalis have the potential to produce scientifically relevant information, leading to a better understanding of the damage caused by aquatic contamination, as well as the modes of action of toxicants. Lymnaea stagnalis individuals are easily maintained in laboratory conditions, with a lifespan of about two years. The snails are hermaphrodites and sexual maturity occurs about three months after egg laying. Importantly, they can produce a high number of offspring all year round and are considered well suited for use in investigations targeting the identification of developmental and reproductive impairments. The primary aims of this review were two-fold: i) to provide an updated and insightful compilation of established toxicological measures determined in both chronic and acute toxicity assays, as useful tool to the design and development of future research; and ii) to provide a state of the art related to direct toxicant exposure and its potentially negative effects on this species. Relevant and informative studies were analysed and discussed. Knowledge gaps in need to be addressed in the near future were further identified.

Crustaceans often undergo periods of starvation, due to natural food shortage or physiological constraints. During these periods, several metabolic and behavioral changes can occur. This study evaluates how the brown shrimp Crangon crangon (Linnaeus, 1758) responds to prolonged deprivation of food in two seasons of the year, and how this species mobilizes its energetic reserves. Shrimps caught in June (summer) and October (autumn) 2010 in the Minho estuary (north of Portugal) were placed in individual cages in experimental aquaria and kept in starvation until the last shrimp died or was sacrificed (six shrimp per aquarium every week). The energetic content, total lipids and total protein, and the oxygen consumption rate were compared between seasons, sacrificed and naturally dead shrimp, and considering the weeks of starvation. Summer shrimp proved to be better prepared to endure starvation than those caught in autumn: they survived 2.5 times longer, had a higher Fulton condition factor, higher e...

Both field and experimental data examined the influence of exposure to environmental contaminant tributyltin (TBT) on marine organisms. Although most attention focused on the imposex phenomenon in gastropods, adverse effects were also observed in other taxonomic groups. It has been shown that imposex induction involves modulation of retinoid signaling in gastropods. Whether TBT influences similar pathways in fish is yet to be addressed. In this study, larvae of the model teleost Danio rerio were exposed to natural retinoids, all-trans-retinoic acid, 9-cis-retinoic acid, and all-trans-retinol, as well as to the RXR synthetic pan-agonist methoprene acid (MA) and to TBT. Larvae were exposed to TBT from 5 days post fertilization (dpf) to adulthood, and reproductive capacity was assessed and correlated with mode of action. TBT significantly decreased fecundity at environmentally relevant levels at 1 μg TBT Sn/g in diet. Interestingly, in contrast to previous reports, TBT altered zebrafish sex ratio toward females, whereas MA exposure biased sex toward males. Since fecundity was significantly altered in the TBT-exposed group with up to 62% decrease, the potentially affected pathways were investigated. Significant downregulation was observed in brain mRNA levels of aromatase b (CYP19a1b) in females and peroxisome proliferator activated receptor gamma (PPARg) in both males and females, suggesting an involvement of these pathways in reproductive impairment associated with TBT.

Obesity, a risk factor for the development of type-2 diabetes, hypertension, cardiovascular disease, hepatic steatosis and some cancers, has been ranked in the top 10 health risk in the world by the World Health Organization. Despite the growing body of literature evidencing an association between the obesity epidemic and specific chemical exposure across a wide range of animal taxa, very few studies assessed the effects of chemical mixtures and environmental samples on lipid homeostasis. Additionally, the mode of action of several chemicals reported to alter lipid homeostasis is still poorly understood. Aiming to fill some of these gaps, we combined an in vivo assay with the model species zebrafish (Danio rerio) to screen lipid accumulation and evaluate expression changes of key genes involved in lipid homeostasis, alongside with an in vitro transactivation assay using human and zebrafish nuclear receptors, retinoid X receptor α and peroxisome proliferator-activated receptor γ. Zebrafish larvae were exposed from 4 th day post-fertilization until the end of the experiment (day 18), to six different treatments: experimental control, solvent control, tributyltin at 100 ng/L Sn and 200 ng/L Sn (positive control), and wastewater treatment plant influent at 1.25% and 2.5%. Exposure to tributyltin and to 2.5% influent led to a significant accumulation of lipids, with white adipose tissue deposits concentrating in the perivisceral area. The highest in vitro tested influent concentration (10%) was able to significantly transactivate the human heterodimer PPARγ/RXRα, thus suggesting the presence in the influent of HsPPARγ/RXRα agonists. Our results demonstrate, for the first time, the ability of complex environmental samples from a municipal waste water treatment plant influent to induce lipid accumulation in zebrafish larvae. This work was supported by the Norte2020 and FEDER (Coral—Sustainable Ocean Exploitation—Norte-01-0145-FEDER-000036), by the Spanish Agencia Estatal de Investigación (CTM2017-84763-C3-2-R), the Galician Council of Culture, Education and Universities (ED431C2017/36) and FEDER/ERDF, and by the project NOR-WATER (0725), financed by “Programa de Cooperação Interreg Portugal/Espanha, (POCTEP) 2014-2020. Ricardo Capela was supported by grant SFRH/BD/112483/2015 SI

Anatoxin-a is a neurotoxin produced by various bloom-forming cyanobacteria. Although it shows widespread occurrence and is highly toxic to rodents, its mechanisms of action and biotransformation, and effects in fish species are still poorly understood. The main aim of this study was, thus, to investigate sub-lethal effects of anatoxin-a on selected biochemical markers in rainbow trout fry in order to get information about the mechanisms of toxicity and biotransformation of this toxin in fish. Trout fry were administered sub-lethal doses of anatoxin-a (0.08-0.31 μg g⁻¹) intraperitoneally. Livers and muscle tissue were collected 72 h later for quantification of key enzyme activities as biochemical markers. Enzymes assessed in muscle tissues were related to cholinergic transmission (acetylcholinesterase [AChE]), energy metabolism (lactate dehydrogenase [LDH] and NADP⁺-dependent isocitrate dehydrogenase [IDH]). Enzymes assessed in the liver were involved in biotransformation (ethoxyresorufin-O-deethylase [EROD] and glutathione S-transferases [GST]). The results indicated a significant increasing trend for AChE activity with the dose of anatoxin-a, possibly representing an attempt to cope with overstimulation of muscle activity by the toxin, which competes with acetylcholine for nicotinic receptors binding. Anatoxin-a was also found to significantly induce the activities of liver EROD and GST, indicating the involvement of phase I and II biotransformation in its detoxification. Likewise, lactate dehydrogenase activity recorded in fry muscle increased significantly with the dose of anatoxin-a, suggesting an induction of the anaerobic pathway of energy production to deal with toxic stress induced by the exposure. Altogether, the results suggest that under continued exposure in the wild fish may experience motor difficulties, possibly becoming vulnerable to predators, and be at increased metabolic demand to cope with energetic requirements imposed by anatoxin-a biotransformation mechanisms.

This study investigated to what extent a single exposure to low doses of ionizing radiation can induce genotoxic damage in irradiated adult zebrafish (Danio rerio) and its non-irradiated F1 progeny. Four groups of adult zebrafish were irradiated with a single dose of X-rays at 0 (control), 100, 500 and 1000 mGy, respectively, and couples of each group were allowed to reproduce following irradiation. Blood of parental fish and whole-body offspring were analysed by the comet assay for detection of DNA damage. The level of DNA damage in irradiated parental fish increased in a radiation dose-dependent manner at day 1 post-irradiation, but returned to the control level thereafter. The level of DNA damage in the progeny was directly correlated with the parental irradiation dose. Results highlight the genotoxic risk of a single exposure to low-dose ionizing radiation in irradiated individuals and also in its non-irradiated progeny.

The alkaloid cylindrospermopsin has been recognized of increased concern due to the global expansion of its main producer, Cylindrospermopsis raciborskii. Previous studies have shown that bivalves can accumulate high levels of cylindrospermopsin. Based on the potential for human health risks, a provisional tolerable daily intake of 0.03μg/kg-body weight has been recommended. However, the human exposure assessment has been based on the cylindrospermopsin concentration in raw food items. Thus, this study aimed to assess the changes on cylindrospermopsin concentration in edible mussels with storage and processing time as well as cylindrospermopsin bioaccessibility. Mussels, (. Mytilus galloprovincialis) fed cylindrospermopsin-producing C.raciborskii, were subjected to the treatments and then analyzed by LC-MS/MS. Mussels stored frozen allowed a significantly higher recovery of cylindrospermopsin (52.5% in 48h and 57.7% in one week). The cooking treatments did not produce significant differences in cylindrospermopsin concentration in the mussel matrices (flesh), however, cylindrospermopsin was found in the cooking water, suggesting that heat processing can be used to reduce the availability of cylindrospermopsin. The invitro digestion considerably decreased the cylindrospermopsin availability in uncooked and steamed mussels, highlighting the importance in integrating the bioaccessibility of cylindrospermopsinin in the human health risk assessment. © 2015 Elsevier. Marisa Freitas thanks the Fundação para a Ciência e Tecnologia (FCT) for the Ph.D. grant SFRH/BD/85490/2012. This work was partially funded by the FCT project PesT-C/MAR/LA0015/2013.

The present study aimed to determine the effect of feeding time on growth and nitrogen excretion in juvenile sole. An 84-day growth trial was conducted, in which food was supplied to three triplicate groups of juvenile Senegalese sole (3 g wet weight) at different schedules – diurnal, nocturnal and mixed. At the end of the growth trial, ammonia and urea excretion was assessed during a 24 h cycle. Improved growth (1.3% vs. 0.9% day−1, specific growth rate), higher nitrogen retention (0.35 vs. 0.27 g N kg−1 day−1), lower ammonia excretion (209 vs. 272 mg N-NH4 kg−1 day−1) and lower total nitrogen excretion (278 vs. 352 mg N kg−1 day−1) were found in daytime-fed fish compared with night-fed fish. Fish in the mixed feeding regime showed intermediate values of ammonia and total nitrogen excretion, but did not differ from day-fed fish regarding the other parameters. Results indicate that juvenile sole at a period of their life cycle appear to use more efficiently dietary protein for somatic growth under a diurnal than under a nocturnal feeding regime. This suggest that at least during a time-window in the juvenile rearing a diurnal feeding regime might be more effective in the production of this species.

Early life stage tests with zebrafish (Danio rerio) were used to detect toxic effects of compounds from a Microcystis aeruginosa natural bloom extracton their embryolarval development. We carried out the exposure of developing stages of fish to complex cyanobacterial blooms containing hepatotoxic molecules - microcystins. Fish embryo tests performed with the bloom extract containing 3 mg·L-1 Eq microcystin-LR showed that after 24 h of exposure all fish embryos died. The same tests performed with other diluted extracts (containing 0.3, 0.1 and 0.03 mg·L-1 Eq microcystin-LR) were shown to have an influence on zebrafish development and a large number of embryos showed malformation signs (edema, bent and curving tail). After hatching the larvae were transferred to a medium without toxins to follow the larval development under the new conditions. The specific growth of the pre-exposed larvae was significantly more important than that of the control larvae. This may represent a compensatory growth used to reduce the difference in size with the control fish noted after hatching.

A partial life-cycle test with the model fish Danio rerio was performed in order to evaluate the genotoxic potential of binary mixtures of xenoandrogenic (tributyltin — TBT; triphenyltin — TPT) and an estrogenic compound (ethinylestradiol — EE2). Five days post-fertilisation larvae were diet-exposed to environmental relevant concentrations of TBT and TPT (25 ng/g–100 ng/g), and water-exposed to ethinylestradiol (3.5 ng/L) for a four-month period; binary mixtures of TBT plus EE2 and TPT plus EE2 were run in parallel. The erythrocytic nuclear abnormalities (ENA) assay in circulating erythrocytes was used to evaluate genotoxicity in the end of the four-month exposure period. A significant increase (p

Owing to the increasing importance of zebrafish as a vertebrate model in many fields of research, efforts must be made to breed and maintain this species in laboratory. Zebrafish larvae are traditionally reared on cultured live paramecia during the first 9 days of exogenous feeding, followed by a combination of paramecia and artemia nauplii until day 21, making larval rearing expensive, labour intensive and unpredictable. Thus, a trial was conducted with zebrafish larvae in order to evaluate the suitability of artificial diets as an alternative to live food during the first 21 days of exogenous feeding. Five dietary treatments were tested: (1) artemia nauplii; (2) a commercial; (3) a purified; (4) a practical diet, all delivered continuously; (5) the same practical diet delivered manually. The best overall larval performance was achieved in the group fed artemia nauplii (86% survival, 14.3 mm standard length, 46.1 mg wet weight). Compared with existing results obtained with the traditional live food schedule, our results suggest that paramecia might not be the most suitable first food for zebrafish, and that artemia nauplii could be used as the only live food. The present work demonstrates that zebrafish larvae can be reared without live food with a significant growth and a high survival, provided that an appropriate artificial diet is presented in a continuous way. Among the diets tested, the practical diet, if continuously delivered, led to the best performance assuring a mean standard length of 72% of that obtained with artemia and a similar survival rate. Moreover, the purified diet, supporting over 50% survival and an appreciable growth, could be useful in some toxicological studies in which a well-defined diet is needed.

The present work aimed at evaluating whether solubility and peptide profile of dietary protein can affect survival and growth performance of common carp during early stages of life. Water-insoluble casein and two forms of soluble casein (non-hydrolysed and hydrolysed) were used as protein sources at different proportions in the experimental diets for first-feeding carp larvae. When provided as the only protein source water-insoluble casein led to high mortality and depressed growth after 21 days. The partial or total substitution of water-insoluble casein by soluble non-hydrolysed casein effectively improved survival and enhanced growth of larvae. An optimum level of soluble non-hydrolysed protein was found at 25% of total protein. The addition of some hydrolysed casein (25% of soluble protein) further improved results. However, high dietary levels of hydrolysed casein led to negative effects on larval performance, which would be related with a surplus of di-/tripeptides or/and amino acids. Protein solubility appears to have only a temporary effect on larval performance, probably related with the ontogeny of the digestive tract, since about 2 weeks after the onset of exogenous feeding larvae became able to use efficiently water-insoluble protein. Results also suggest the need for a balance in the peptide profile to optimise the utilization of dietary protein by fish larvae.

In recent years, a great deal of interest has emerged in the development of microdiets as an economic alternative to live food, in the larval culture of marine fish species. The ability to grow Sparus aurata larvae on a prototype microparticulate diet was examined. To achieve this objective, four feeding regimes differing in the time when the microdiet was introduced (3, 7 or 12 days) and one based exclusively on an inert diet were tested, during the first 22 days of larval life. Significant differences in larval growth were found between the experimental feeding regimes and their corresponding controls (enriched rotifers during the whole experimental period); the larvae in the co-feeding regimes and with an exclusive microparticulate diet were always significantly smaller than larvae fed on rotifers alone. However, the difference was minimised by introducing the inert diet at a later date. A lower survival was found in larvae with a co-feeding regime, in comparison with the control treatments and the survival was significantly lower in larvae fed exclusively on a microparticulate diet. The fatty acid analysis revealed that the experimental microencapsulated diet and the rotifers enriched with Protein Selco® presented relatively similar fatty acid content. In spite of the slightly higher (n−3)/(n−6) and Docosahexaenoic acid (DHA)/Eicosapentaenoic acid (EPA) ratios and somewhat lower highly unsaturated fatty acid (HUFA) content found in the inert diet, the fatty acid composition of the diets cannot explain the differences found in larval performance. The results revealed that the complete replacement of live prey with the tested microparticulate diet is still not possible in S. aurata larval rearing. Nevertheless, better growth and survival results and a substantial reduction in the daily supply of live food can be achieved with a combination of microdiet and live prey.

Anatoxin-a is a potent neurotoxin produced by several genera of cyanobacteria. Deaths of wild and domestic animals due to anatoxin-a exposure have been reported following a toxic response that is driven by the inhibition of the acetylcholine receptors at neuromuscular junctions. The consequent neuron depolarization results in an overstimulation of the muscle cells. In order to unravel further molecular events implicated in the toxicity of anatoxin-a, a proteomic investigation was conducted. Applying two-dimensional gel electrophoresis (2DE) and MALDI-TOF mass spectrometry, we report early proteome changes in brain and muscle of zebrafish (Danio rerio) caused by acute exposure to anatoxin-a. In this regard, the test group of male and female zebrafish received an intraperitoneal (i.p.) injection of an anatoxin-a dose of 0.8µgg(-1) of fish body weight (bw) in phosphate buffered saline solution (PBS), while the control received an i.p. injection of PBS only. Five minutes after i.p. injection, brain and muscle tissues were collected, processed and analyzed with 2DE. Qualitative and quantitative analyzes of protein abundance allowed the detection of differences in the proteome of control and exposed fish groups, and between male and female fish (gender specific responses). The altered proteins play functions in carbohydrate metabolism and energy production, ATP synthesis, cell structure maintenance, cellular transport, protein folding, stress response, detoxification and protease inhibition. These changes provide additional insights relative to the toxicity of anatoxin-a in fish. Taking into account the short time of response considered (5min of response to the toxin), the changes in the proteome observed in this work are more likely to derive from fast occurring reactions in the cells. These could occur by protein activity regulation through degradation (proteolysis) and/or post-translational modifications, than from a differential regulation of gene expression, which may require more time for proteins to be synthesized and to produce changes at the proteomic level.

The life cycle of the red swamp crayfish, Procambarus clarkii from a shallow lagoon in the Aveiro region was studied for 14 months. Analysis of the size frequency distribution and growth provided evidence for an extended recruitment period with a peak in autumn (October/November). Females clearly outnumbered males and the average sex-ratio (males/females) was 0.36. The values for the Von Bertalanffy growth parameters were L∞ 62.01 mm, K = 0.23, and t0 = 0.11 month. = The high abundance of this species and the extended recruitment period, support the need for a proper management and exploitation of this biological resource. O ciclo de vida do lagostim-vermelho-da-Louisiana Procambarus clarkii da ria de Aveiro foi estudado ao longo de 14 meses. A analise da distribuicao das frequencias de tamanho e de crescimento mostrou um periodo de recrutamento extenso com um maximo no Outono (Outubro/Novembro). As femeas ocorreram com maior abundância que os machos, o que se traduziu numa proporcao de sexos (machos/femeas) de 0,36. Os valores calculados para os parâmetros de crescimento de Von Bertalanffy foram L∞ = 62,01 mm, K = 0,23 e t0 = 0,11 mes. A elevada abundância desta especie e o largo periodo de recrutamento apontam para a necessidade de uma adequada gestao e exploracao deste recurso biologico.

A 21 day feeding trial was carried out at 24 °C aiming to evaluate the effect of diets containing high levels of protein hydrolysates on growth, survival and body composition of common carp, Cyprinus carpio L., larvae since first feeding. Eight semipurified diets based on a fish protein hydrolysate (CPSP) with or without additional nitrogen sources, such as casein, casein hydrolysates or other hydrolysates from several origins, were tested, using a diet based on yeast as a reference. High survival rates of larvae were observed at the end of the trial for all experimental diets, ranging from 97% for the yeast diet to 71% when CPSP was used as the only nitrogen source. In terms of growth, the best results were achieved with the CPSP-casein diet (1:1) and the yeast-based diet (17.5 mm total length / 82.8 mg wet weight and 16.3 mm total length / 55.4 mg wet weight, respectively). Growth of larvae fed on diets with protein hydrolysates as the only nitrogen source was considerably lower than that of larvae fed a mixture of intact protein and protein hydrolysates.

In this study, a 12-day growth trial was conducted to compare the effect of the variation in microcystin (MC) composition in two Microcystis aeruginosa bloom samples on the growth performance and MC accumulation/transfer in the common carp (Cyprinus carpio L.) larvae. Fish were fed Artemia salina nauplii that had been preexposed to extracts from two M. aeruginosa natural blooms with different microcystins (MCs) profiles. Bloom A had MC-LR as major toxin (74.05%) while bloom B had a diversity of MC (MC-RR; MC-(H4)YR; MC-YR; MC-LR; MC-FR; MC-WR) with no dominance of MC-LR. Newly-hatched Artemia nauplii were exposed separately to the two M. aeruginosa extracts A and B (100 microg L(-1)EqMC-LR) for 2h. The MC concentration in the nauplii was 73.60+/-7.88ngEqMC-LRg(-1)FW (n=4, mean+/-SE) for bloom A and 87.04+/-10.31ngEqMC-LRg(-1)FW for bloom B. These contaminated nauplii were given at the same ration to different groups (A and B) of fish larvae. Larval weight and length from day 9 were significantly different between groups A and B, and in both cases lower than that of a control group fed non-exposed nauplii. MCs accumulation by larvae, inversely correlated with the growth performance, was also significantly different between groups A and B (37.43+/-2.61 and 54.55+/-3.01ngEqMC-LRg(-1) FW, respectively, at the end of the experimental period). These results indicate that MC profile of a bloom may have differential effects on toxin accumulation/transfer and toxicity.

The solubility of protein nitrogen at pH 8 in the organisms used as live food for fish larvae (rotifers and Artemia) was determined and the molecular weight profile of soluble protein nitrogen was analysed by HPLC-gel filtration chromatography. Soluble nitrogen accounted for 61% of total nitrogen in microalgae-fed rotifers, 47% in starved rotifers and 54% in newly hatched Artemia. A remarkable similarity in the profile of molecular weight distribution of soluble nitrogen was noticed among these three types of live food. The molecular weights of soluble nitrogen was partitioned as follows, respectively, in rotifers and Artemia: 84–88% and 89% >500 Da (corresponding mainly to proteins, poly- and oligopeptides), 8–11% and 4% from 200 to 500 Da (corresponding mainly to di- and tripeptides), 3–4% and 7%

Recently, it has been demonstrated that the biocide tributyltin (TBT) can interfere with fish sex differentiation, leading to a bias of sex toward males. On the contrary, it is well known that estrogenic compounds can induce fish feminization. Yet, the combined effects of mixtures of androgenic and estrogenic compounds on fish sex differentiation have never been investigated before, even though in the environment animals are frequently exposed to both groups of xenobiotics. Therefore, in order to investigate whether exposure to estrogenic compounds can block the masculinizing effect of TBT, 5 days post-fertilization zebrafish (Danio rerio) larvae were exposed for a four month period to TBT and to the synthetic estrogen-ethinylestradiol (EE2). The fish were fed a diet containing TBT at nominal concentrations of 25 and 100 ng TBT/g, and two groups of animals were also dosed with TBT plus EE2 at nominal water concentration of 3.5 ng/L, using a flow-through design. As expected, fish exposed to TBT showed a bias of sex toward males (62.5% males in control tanks and 86% and 82% in TBT 25 and TBT 100 ng TBT/g, respectively). Co-exposure to EE2 completely blocked the masculinizing effect of TBT, with 7% males in the TBT 25 ng/g + EE2 treatment and 0% in the EE2 alone and in the TBT 100 ng/ + EE2 exposed groups. These results clearly indicate that EE2, at environmentally relevant concentrations, can block the TBT masculinizing effects in zebrafish, which suggests that in the aquatic environment the presence of estrogens may neutralize the fish masculinizing effect of TBT. Our findings highlight the need of testing the combined effects of contaminants, as single exposure studies may not be sufficient to predict the effects of mixtures of xenobiotics with antagonistic properties.

Although chemicals are usually present in the environment in complex mixtures, ecotoxicological risk assessments often rely on data from single exposures to contaminants. The present study aimed at examining the effects of two ubiquitous endocrine disrupting chemicals (EDCs), 17α-ethynilestradiol (EE2) and tributyltin (TBT), single and combined, in zebrafish (Danio rerio) reproduction and embryonic development, and to investigate the impact on DNA repair molecular pathways. Chronic life-cycle exposure to environmental relevant concentrations of both EDCs led to significant effects on reproduction and embryonic development. TBT exposure at 50 ng/L impacted the percentage of fertilized eggs. EE2 exposure at 0.75 and 1.75 ng/L and the mixture treatments (0.75 and 1.75 ng/L EE2 plus 50 ng/L TBT) led to a sex bias towards females. Furthermore, EE2 exposure at 1.75 ng/L and the mixture of EE2 (1.75 ng/L) and TBT (50 ng/L) impaired embryonic development. The study of DNA repair pathways indicated that EE2 and TBT, single and combined, significantly modulated the expression levels of key DNA repair genes in male gonads, such as xpc nucleotide excision repair (NER) pathway gene as well as the tumor suppressor p53, its target effectors, i.e. p21, gadd45α and bax, and p53 key regulator (mdm2). The observed impact in the transcription of these genes supports the hypothesis of an effect of EE2, TBT, and their combined mixtures, on DNA repair pathways, thus suggesting that impairment of embryonic development of F2 generation could be linked to the disruption of DNA repair processes in the gonads of individuals from the parental generation. Taken together, these findings contribute to a better understanding of the impact of mixtures of the ubiquitous contaminants EE2 and TBT, strongly supporting the inclusion of embryonic development and gene expression studies in the screening of endocrine disruption in wildlife. © 2017 Elsevier Joana Soares participation was funded by Fundação para a Ciência e a Tecnologia (FCT-SFRH/BD/30211/2006). This paper was developed under the project INNOVMAR – Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035), with- in Research Line ECOSERVICES – Assessing the environmental quality, vulnerability and risks for the sustainable management of NW coast natural resources and ecosystem services, supported by North Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement.

Accumulation of microcystin-LR (MC-LR) in edible aquatic organisms, particularly in bivalves, is widely documented. In this study, the effects of food storage and processing conditions on the free MC-LR concentration in clams (Corbicula fluminea) fed MC-LR-producing Microcystisaeruginosa (1 × 105 cell/mL) for four days, and the bioaccessibility of MC-LR after in vitro proteolytic digestion were investigated. The concentration of free MC-LR in clams decreased sequentially over the time with unrefrigerated and refrigerated storage and increased with freezing storage. Overall, cooking for short periods of time resulted in a significantly higher concentration (P < 0.05) of free MC-LR in clams, specifically microwave (MW) radiation treatment for 0.5 (57.5%) and 1 min (59%) and boiling treatment for 5 (163.4%) and 15 min (213.4%). The bioaccessibility of MC-LR after proteolytic digestion was reduced to 83%, potentially because of MC-LR degradation by pancreatic enzymes. Our results suggest that risk assessment based on direct comparison between MC-LR concentrations determined in raw food products and the tolerable daily intake (TDI) value set for the MC-LR might not be representative of true human exposure.

International audience; A 21 day feeding trial was carried out at 24 °C aiming to evaluate the effect of diets containing high levels of protein hydrolysates on growth, survival and body composition of common carp, Cyprinus carpio L., larvae since first feeding. Eight semipurified diets based on a fish protein hydrolysate (CPSP) with or without additional nitrogen sources, such as casein, casein hydrolysates or other hydrolysates from several origins, were tested, using a diet based on yeast as a reference. High survival rates of larvae were observed at the end of the trial for all experimental diets, ranging from 97% for the yeast diet to 71% when CPSP was used as the only nitrogen source. In terms of growth, the best results were achieved with the CPSP-casein diet (1:1) and the yeast-based diet (17.5 mm total length / 82.8 mg wet weight and 16.3 mm total length / 55.4 mg wet weight, respectively). Growth of larvae fed on diets with protein hydrolysates as the only nitrogen source was considerably lower than that of larvae fed a mixture of intact protein and protein hydrolysates.