Microplastics and copper induce apoptosis, alter neurocircuits, and cause behavioral changes in zebrafish (Danio rerio) brain

The knowledge regarding the neurological and behavioral toxic effects associated with microplastics (MPs) and heavy metals exposure is still scarce. The present study aimed to evaluate the potential chronic (30 days) toxic effects of MPs (2 mg/L) and copper (Cu, 25 μg/L), alone or combined, in the zebrafish (Danio rerio) brain antioxidant system, cell proliferation/death, cholinergic-, serotonergic- and dopaminergic pathways and, consequently, in locomotor, anxiety, and social behaviors. Our findings showed that MPs and Cu exposure modulated the antioxidant system of zebrafish brain, with superoxide dismutase (SOD) and glutathione reductase (GR) having higher activity in the Cu25 +MPs group, but glutathione peroxidase (GPx) being inhibited in MPs, Cu25 and Cu25 +MPs. Moreover, an increase in acetylcholinesterase (AChE) activity was observed in all exposed groups. When considering neurogenesis genes, a downregulation of proliferating cell nuclear antigen (pcna) was noticed in zebrafish exposed to the mixture treatment, while for dopaminergic system-related genes (th and slc6a3) an upregulation was observed in MPs, Cu25 and Cu25 +MPs groups. An increase in apoptosis-related genes expression (casp8, casp9 and casp3) was observed in the MPs exposed group. Changes in zebrafish behavior, particularly in mean speed, total distance moved, inactivity in the aquaria, and social/shoaling behavior was also observed in the MPs and Cu exposed groups. Overall, our results highlight the multiplicity of toxic effects of MPs, alone or combined with Cu, in zebrafish brain, namely apoptosis and alterations in adult neurogenesis, neurocircuits and, consequently, behavior.Versión del editor2,29

The role of aquatic ecosystems (River Tua, Portugal) as reservoirs of multidrug-resistant Aeromonas spp.

The inappropriate use of antibiotics, one of the causes of the high incidence of antimicrobialresistant bacteria isolated from aquatic ecosystems, represents a risk for aquatic organisms and the welfare of humans. This study aimed to determine the antimicrobial resistance rates among riverine Aeromonas spp., taken as representative of the autochthonous microbiota, to evaluate the level of antibacterial resistance in the Tua River (Douro basin). The prevalence and degree of antibiotic resistance was examined using motile aeromonads as a potential indicator of antimicrobial susceptibility for the aquatic environment. Water samples were collected from the middle sector of the river, which is most impacted area by several anthropogenic pressures. Water samples were plated on an Aeromonas-selective agar, with and without antibiotics. The activity of 19 antibiotics was studied against 30 isolates of Aeromonas spp. using the standard agar dilution susceptibility test. Antibiotic resistance rates were fosfomycin (FOS) 83.33%, nalidixic acid (NA) 60%, cefotaxime (CTX) 40%, gentamicin (CN) 26.67%, tobramycin (TOB) 26.67%, cotrimoxazole (SXT) 26.67%, chloramphenicol (C) 16.67%, and tetracycline (TE) 13.33%. Some of the nalidixic acid-resistant strains were susceptible to fluoroquinolones. Multiple resistance was also observed (83.33%). The environmental ubiquity, the natural susceptibility to antimicrobials and the zoonotic potential of Aeromonas spp. make them optimal candidates for studying antimicrobial resistance (AMR) in aquatic ecosystems. Aquatic environments may provide an ideal setting for the acquisition and dissemination of antibiotic resistance because anthropogenic activities frequently impact them. The potential risk of multi- and pan-resistant bacteria transmission between animals and humans should be considered in a “One Health-One World” concept.This research was supported by the Portuguese Foundation for Science and Technology (FCT) and the Operational Competitiveness Programme (COMPETE), under the projectsUIDB/04033/2020 (CITAB-UTAD) and the UIDB/00690/2020 (CIMO-IPB).info:eu-repo/semantics/publishedVersio

Multi-scale approach using phytoplankton as a first step towards the definition of the ecological status of reservoirs

The growing need to analyse the present state of ecosystems and predict their rate of change has triggered a demand to explore species environment relationships for assessing alterations under anthropogenic influence. The Water Framework Directive (WFD) requires the definition of different types of water bodies which are of relevance when assessing their ecological status. The main aim of this study was to define of the types of Portuguese reservoirs located in the North and Centre of Portugal and to assess their ecological status using phytoplankton as water quality indicators. In this study, sampling was carried out in 34 reservoirs during four seasons (spring, summer, autumn and winter), through a period of 8 years (1996-2004).http://www.sciencedirect.com/science/article/B6W87-4ST4CF7-1/1/911b46293dcdcf046f40a1c1863a619

Antimicrobial susceptibility profiles of multidrug-resistant aeromonads isolated from Northern Portugal freshwater ecosystem

The inappropriate use of antibiotics, one of the causes of the high incidence of antimicrobial-resistant bacteria isolated from aquatic ecosystems, represents a risk for aquatic organisms and the welfare of humans. This study aimed to determine the antimicrobial resistance rates among riverine Aeromonas, taken as representative of autochthonous microbiota, to evaluate the level of antibacterial resistance in the Tua River (Douro basin). The prevalence and degree of antibiotic resistance were examined using motile aeromonads as a potential indicator of antimicrobial susceptibility for the aquatic environment. Water samples were collected from the middle sector of the river, which is impacted by several anthropogenic pressures. Water samples were plated on an Aeromonas-selective agar, with and without antibiotics. The activity of 19 antibiotics was studied against 30 isolates of Aeromonas spp. using the standard agar dilution susceptibility test. Antibiotic resistance rates were fosfomycin (FOS) 83.33%, nalidixic acid (NA) 60%, cefotaxime (CTX) 40%, gentamicin (CN) 26.67%, tobramycin (TOB) 26.67%, cotrimoxazole (SXT) 26.67%, chloramphenicol (C) 16.67%, and tetracycline (TE) 13.33%. Some of the nalidixic acid-resistant strains were susceptible to fluoroquinolones. Multiple resistance was also observed (83.33%). The environmental ubiquity, the natural susceptibility to antimicrobials and the zoonotic potential of Aeromonas spp. make them optimal candidates for studying antimicrobial resistance in aquatic ecosystems. Aquatic environments may provide an ideal setting for the acquisition and dissemination of antibiotic resistance because anthropogenic activities frequently impact them. The potential risk of multi- and pan-resistant bacteria transmission between animals and humans should be considered in a “One Health-One World” concept.info:eu-repo/semantics/publishedVersio

Artificial substrates as sampling devices for marine epibenthic fauna: A quest for standardization

In temperate regions, macroalgae are naturally abundant habitat builders for many organisms of ecological and economic importance. Hence, macroalgae are good targets for monitoring studies based on colonization processes as, through them, it is possible to sample the epifauna that uses them as habitat. Nevertheless, macroalgae collection may not be sustainable, can compromise the survival of the target macroalgae populations and destroy fragile or threatened communities.The search for an adequate procedure that can overcome the problems related to destructive quantitative sampling of the epifauna associated with macroalgae and the development of a methodology that can be used for comparative macrofauna monitoring, regardless of the location, were the motivations for this study. The evaluation of the mobile epifauna associated with Artificial Substrates (AS) with different degrees of complexity and natural subtidal macroalgae was implemented, as a means to evaluate the viability of AS as an alternative approach for epifauna monitoring.Cystoseira baccata and Halidrys siliquosa were chosen as natural macroalgae. The hypotheses tested were (1) macroalgae and AS with similar structure will support similar assemblages of mobile epifauna; (2) different complexity AS will shelter different assemblages of mobile epifauna. The results obtained after 3 and 6 months showed that AS and macroalgae, both with similar structure, supported different assemblages of mobile epifauna, differing also when the time factor is considered. Moreover, different complexity AS supported different epifaunal assemblages. Our results also show that a period of colonization of 3 months was enough to accurately discriminate locations but the 3 additional months reinforce these differences and provide more coherent results with the species colonizing natural macroalgae in both locations studied. Hence, AS could be used as a valid, replicable, standard and representative alternative tool for monitoring studies.The present study was cofinanced by NextSea: Next generation monitoring of coastal ecosystems in a scenario of global change (operation NORTE-01-0145-FEDER-000032) and by FSE: Fundo Social Europeu, Portugal through NORTE 2020. Diego CarreiraFlores was supported by a PhD Fellowship (BD/Do*Mar/1010/2016) do Programa de Ciencia, Tecnologia e Gestao Marinha (Do*Mar), Portugal, operation number NORTE-08-5369-FSE-000039. This work was also supported by the strategic programme UID/BIA/04050/2019 funded by national funds through the FCT I.P, Portugal

Antimicrobial susceptibility profiles of multidrug-resistant aeromonads isolated from Northern Portugal freshwater ecosystem.

Extensive use of water and anthropogenic activities contribute to water body pollution. Agricultural, urban, and animal waste, often characterized by numerous toxic and carcinogenic chemicals, pathogenic bacteria, and antibiotics, as well as antibiotic resistance genes (ARGs), loaded with microflora, can contaminate water and enter the food chain, posing a considerable danger to public health [1,2]. The inappropriate use of antibiotics, one of the causes of the high incidence of antimicrobial-resistant bacteria isolated from aquatic ecosystems, represents a risk for aquatic organisms and the welfare of humans. Infectious diseases, both human and animal, are closely related through the environment in the One World - One Medicine - One Health concept, in order to deal with the growing problem of antibiotic resistance. Aeromonas spp. can acquire antimicrobial resistance mechanisms, with the potential to spread via horizontal gene transfer, so they could be a good candidate as an indicator to follow antimicrobial resistance dissemination in water [3,4]. This study aimed to determine the antimicrobial resistance rates among riverine Aeromonas spp., taken as representative of the autochthonous microbiota, to evaluate the level of antibacterial resistance in the Tua River (Douro basin).N/

Ecological integrity assessment in aquatic ecosystems: A multi-scale approach towards the definition of the ecological status of Mediterranean reservoirs

Tese de Doutoramento em Ciências do AmbienteMundialmente os sistemas aquáticos têm sido afectados por diversas pressões a larga escala, nomeadamente a intensificação agrícola e o aumento de fontes tópicas e difusas, que se sobrepõem no espaço e no tempo e têm levado à ruptura da estrutura e do funcionamento destes sistemas. Face a esta alteração global, o declínio da qualidade ambiental dos rios e respectivas albufeiras, tem sido alvo de uma crescente preocupação. Neste contexto, tem surgido uma crescente procura de novas tecnologias para a monitorização de albufeiras que permitam estabelecer prioridades de conservação, protecção e restauração, que possibilitem alcançar o estado ecológico requerido ou um bom potencial ecológico para estas massas de água fortemente modificadas. Assim, o objectivo desta tese foi o desenvolvimento de novas ferramentas assentes em análises estatísticas multivariadas e metodologias de modelação, que possibilitam a determinação do estado ecológico de albufeiras mediterrâneas, no âmbito da Directiva Quadro da Água DQA). Este trabalho pretende desenvolver metodologias integradas que captem a complexidade inerente aos ecossistemas aquáticos através de uma abordagem multi escala, de modo a compreender melhor os mecanismos locais e regionais que operam nestas massas de água. Para uma melhor compreensão das alterações ocorridas na estrutura e funcionamento das albufeiras, vários componentes destes ecossistemas foram incorporados a diferentes escalas temporais e espaciais de análise, nomeadamente, parâmetros hidromorfológicos, físicos, químicos e biológicos e ainda vários factores de perturbação (i.e., alterações dos usos do solo, variações hidrométricas e fontes de poluição tópicas e difusas). A tese foi estruturada em sete capítulos cuja análise foi suportada por extensas bases de dados, nomeadamente as séries temporais de longo-termo, referentes ao período 1996-2004, compiladas pelo Laboratório de Ambiente do Grupo Electricidade de Portugal (EDP, LABELEC). A base de dados engloba um conjunto de 34 albufeiras que se estendem por cinco regiões hidrográficas distintas (Bacias do Lima, Cávado e Ave, Douro, Mondego e Tejo). Os principais objectivos desta tese foram: Estabelecer a tipologia e as condições de referência de cada Tipo, das albufeiras Portuguesas com fins hidroeléctricos definidos, através de uma abordagem multi escala baseada nos elementos abióticos e bióticos, nomeadamente nas comunidades fitoplânctonicas como indicadores da qualidade da água (Capítulo 2) Determinar a importância das variáveis ambientais na estruturação da comunidade defitoplâncton nos Tipos de albufeiras estudadas a diferentes escalas espaciais (geográfica, regional e local) e temporais (estações do ano, anos) (Capítulo 3) Determinar, como proposto pela DQA, o grau de perturbação das diversas albufeiras, ao longo do tempo, através da comparação com os locais de referência para cada um dos Tipos determinados (Capítulo 4) Desenvolver um modelo Estocástico Dinâmico (StDM), baseado numa abordagem multi escala,para prever o estado ecológico de albufeiras Mediterrâneas face a alterações de uso do solo nas respectivas bacias hidrográficas. Neste contexto, a bacia do Douro foi utilizada para testar e validar o modelo desenvolvido (Capítulo 5), assim como avaliar a sua performance na previsão de alteração da qualidade da água de uma albufeira, nomeadamente ao nível ambiental e biológico (fitoplâncton), face a expectáveis cenários espaciais de alteração de uso do solo (Capítulo 6). Os resultados das diferentes abordagens foram consistentes na definição da tipologia das albufeiras portuguesas estudadas, assim como nos gradientes de poluição observados, que de algum modo parecem reflectir a degradação associada à conversão de áreas agro florestais em áreas ndustriais ou semi urbanas. Assim, foi possível identificar dois Tipos de massas de água com características hidromorfológicas, químicas e biológicas distintas, nomeadamente com comunidades fitoplântonicas específicas: Tipo 1, albufeiras localizadas nos rios principais (Douro e Tejo) caracterizadas por elevadas concentrações de nutrientes e minerais. Este Tipo de albufeira “fio de água”, assemelha se muito mais a um sistema lótico do que a um sistema lêntico, apresentando tempos de residência baixos (dias), boa mistura e uma relativa elevada velocidade da corrente, nunca ou muito raramente ocorre estratificação. Em geral, situam se em áreas densamente povoadas, industriais ou agrícolas sujeitas a elevados inputs de matéria orgânica e descargas industriais. Tipo 2, verdadeiros reservatórios, profundos e situados a altitudes elevadas. Geralmente, encontram se localizadas nos tributários e apresentam elevados tempos de residência (semanas a meses). Consequentemente, são fortemente afectados por fenómenos de estratificação. Foi possível, determinar as comunidades fitoplânctonicas características de cada um dos Tipos de albufeiras referidos. Para ambos os tipos de sistemas regulados, foi também possível estabelecer os taxa característicos dos locais perturbados e dos locais menos perturbados ou de referência. Esta caracterização é um pré requisito essencial para o desenvolvimento de um processo de avaliação de acordo com a DQA, dado que a avaliação deve efectuar se através da comparação da composição de espécies actual com a que estaria presente em condições de referência. Os resultados relativos à importância das escalas local e regional na estrutura e composição do fitoplâncton vieram corroborar estudos anteriores, reflectindo a importância do habitat e da paisagem nos ecossistemas lênticos. Estes resultados parecem demonstrar que o fitoplâncton pode ser um bom indicador ecológico na avaliação holística dos efeitos de pressões cumulativas ou multi escala, podendo vir a ser integrado no futuro, a nível regional ou mundial, na monitorização de albufeiras. Relativamente ao modelo Estocástico-Dinânmico (StDM) desenvolvido, de uma maneira geral os resultados foram encorajadores dado que parecem demonstrar a consistência desta ferramenta em captar a dinâmica ambiental aleatória das métricas seleccionadas face a cenários espaciais expectáveis. Este tipo de abordagem holística pode eventualmente facilitar o ordenamento destas massas de água assim como a tomada de decisão segundo as exigências da DQA. Globalmente, as metodologias desenvolvidas nesta tese poderão contribuir para a implementação da DQA em Portugal, especialmente na avaliação do estado ecológico, fornecendo novas ferramentas para a avaliação da integridade ecológica de sistemas lênticos, e pretende ainda contribuir para uma maior compreensão das alterações estruturais e funcionais que ocorrem nos sistemas aquáticos portugueses devido a pressões antropogénicas.Worldwide aquatic ecosystems have been impacted by broad scale environental pressuressuch as agriculture, point and non point source pollution and land use changes overlapping in space and time, leading to the disruption of the structure and functioning of these systems. In face of this global change, declines in environmental quality of rivers respective and reservoirs are of increasing concern. This has lead to an increasing search for new technologies for the monitoring of reservoirs to establish priorities of conservation, protection and restoration to achieve the Maximun Ecological Potencial of these waterbodies in the context, the objective of this thesis was to develop new tools, supported by multivariate statistical analyses and modelling methodologies, which can be applied in assessing the ecological status of Mediterranean reservoirs, in the scope of the Water Framework Directive (WFD). This work intended to design integrated methodologies to capture the aquatic ecosystems complexity through a multi scale approach, in order to develop and understand the local and regional mechanisms that operate in reservoirs to better understand the occurring changes in the structure and functioning of this systems several ecosystem components (e.g. hydromorphological, physical, chemical and biological) and several disturbance factors (e.g. land use changes, hydrometric variations, point and non point source pollution, etc.) were incorporated, at different spatial and temporal scales of analysis the thesis was structured into seven chapters based in the analysis of extensive data bases, namely long term data bases collected in the field by the Laboratory of Environmental and Applied Chemistry (LABELEC) managed by the Portuguese Electricity Group (EDP), in five different Portuguese hydrographic regions (Lima, Cavado and Ave, Douro, Mondego and Tagus catchment), through a period of nine years (1996 to 2004) the major objectives of this thesis were: To establish the typology and the Type-specific reference condition of the portuguese Hydroelectric reservoirs based on biotic and abiotic quality elements, namely phytoplankton as Water quality indicator, through a multi scale approach. To determine the importance of environmental variables at different spatial (geographical,Regional and local) and time scales (seasons, years) on the phytoplankton community structure of the studied reservoirs Types. To determine, overtime, the degree of impairment ofthese reservoirs by contrasting it with a reference site, as proposed by the WFD. To develop a modeling multi scale approach, based on an holistic Stochastic Dynamic Methodology (StDM), in order to predict the ecological status tendencies in Mediterranean reservoirs as a response to land use changes in the respective watersheds. Therefore, the Douro’s basin was used to test and validate the StDM model, as well as evaluate the StDM performance in capturing how expected changes at land use level scenarios will alter the Reservoir water quality, namely at physicochemical and phytoplankton levels. The results of the different approaches were consistent in the typology defined for the Portuguese reservoirs, as well as in the observed pollution gradients, which in some extend reflected the degradation associated to the conversion of the agroforestry areas into industrialized and semi Urban areas. Therefore, it was possible to identify two types of dammed water bodies which were characterized by different hydromorphological features, water chemistry characteristics and by a specific phytoplankton taxa composition: Type 1 represents lowland reservoirs located in the main rivers (Douro and Tagus) characterized by higher concentrations of nutrients and water mineral content. This “riverine reservoirs”, resemble more a river than a lake, with short hydraulic retention times, good mixing and relative high water velocities, never or rarely affected by stratification phenomena. In general, is located in densely populated, industrialized or agricultural areas, receiving high inputs of organic matter and industrial discharge. Type 2 represents deeper high altitude “artificial lake reservoirs”, largely located in tributaries, with a high residence time, with longer water storage and release cycles operating under seasonal influence, strongly affected by stratification phenomena.It was possible to assign characteristic phytoplankton taxa assemblages to each type of reservoir. For Both regulated systems types, was also possible to establish he characteristic taxa of disturbed and Less or non disturbed sites. Such ascription is an essential prerequisite for the development of an Assessment procedure according to the WFD, where the assessment should be done by comparing The actual species composition to the one that would be present under reference conditions. Our findings of the importance of local and regional scale factors on phytoplankton community structure lend support to earlier studies, reflecting the importance of habitat and large scale (landscape) factors on reservoir ecosystems and demonstrate that Phytoplankton could be a good ecological indicator for multi scale and cumulative disturbance effects with a view to integrate future Worldwide monitoring in reservoirs. Regarding the developed modelling multi scale approach (StDM), the overall results were encouraging since they seem to demonstrate the tool reliability in capturing the stochastic environmental dynamics of the selected metrics facing spatial explicit scenarios. The ultimate goal of this approach was to couple monitoring assessment and the described modeling techniques to ease management and decision making regarding the requirements of the WFD. Overall, the methodologies presented in this thesis could contribute to the implementation of the WFD in Portugal, with regard specifically to the evaluation of the ecological status, providing new Tools for the ecological integrity assessment of lentic waters, and pretend to contribute to a larger Understanding of the structural and functional human induced alterations that occur in Portuguese aquatic ecosystems

Phytoplankton analysis of portuguese reservoirs: a cluster analysis with R

In this work, cluster analysis is applied to a real dataset of biological features of several Portuguese reservoirs. All the statistical analysis is done using R statistical software. Several metrics and methods were explored, as well as the combination of Euclidean metric and the hierarchical Ward method. Although it did not present the best combination in terms of internal and stability validation, it was still a good solution and presented good results in terms of interpretation of the problem at hand