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Fluoxetine Arrests Growth of the Model Diatom Phaeodactylum tricornutum by Increasing Oxidative Stress and Altering Energetic and Lipid Metabolism
Publication . Feijão, Eduardo; Cruz de Carvalho, Ricardo; Duarte, Irina A.; Matos, Ana Rita; Cabrita, Maria Teresa; Novais, Sara C.; Lemos, Marco F. L.; Caçador, Isabel; Marques, João Carlos; Reis-Santos, Patrick; Fonseca, Vanessa F.; Duarte, Bernardo
Pharmaceutical residues impose a new and emerging threat to aquatic environments and its biota. One of the most commonly prescribed pharmaceuticals is the antidepressant fluoxetine, a selective serotonin re-uptake inhibitor that has been frequently detected, in concentrations up to 40 μg L-1, in aquatic ecosystems. The present study aims to investigate the ecotoxicity of fluoxetine at environmentally relevant concentrations (0.3, 0.6, 20, 40, and 80 μg L-1) on cell energy and lipid metabolism, as well as oxidative stress biomarkers in the model diatom Phaeodactylum tricornutum. Exposure to higher concentrations of fluoxetine negatively affected cell density and photosynthesis through a decrease in the active PSII reaction centers. Stress response mechanisms, like β-carotene (β-car) production and antioxidant enzymes [superoxide dismutase (SOD) and ascorbate peroxidase (APX)] up-regulation were triggered, likely as a positive feedback mechanism toward formation of fluoxetine-induced reactive oxygen species. Lipid peroxidation products increased greatly at the highest fluoxetine concentration whereas no variation in the relative amounts of long chain polyunsaturated fatty acids (LC-PUFAs) was observed. However, monogalactosyldiacylglycerol-characteristic fatty acids such as C16:2 and C16:3 increased, suggesting an interaction between light harvesting pigments, lipid environment, and photosynthesis stabilization. Using a canonical multivariate analysis, it was possible to evaluate the efficiency of the application of bio-optical and biochemical techniques as potential fluoxetine exposure biomarkers in P. tricornutum. An overall classification efficiency to the different levels of fluoxetine exposure of 61.1 and 88.9% were obtained for bio-optical and fatty acids profiles, respectively, with different resolution degrees highlighting these parameters as potential efficient biomarkers. Additionally, the negative impact of this pharmaceutical molecule on the primary productivity is also evident alongside with an increase in respiratory oxygen consumption. From the ecological point of view, reduction in diatom biomass due to continued exposure to fluoxetine may severely impact estuarine and coastal trophic webs, by both a reduction in oxygen primary productivity and reduced availability of key fatty acids to the dependent heterotrophic upper levels.
Unlocking Kautsky’s dark box: development of an optical toxicity classification tool (OPTOX index) with marine diatoms exposed to emerging contaminants
Publication . Duarte, Bernardo; Feijão, Eduardo; Cruz de Carvalho, Ricardo; Franzitta, Marco; Carlos Marques, João; Caçador, Isabel; Cabrita, Maria Teresa; Fonseca, Vanessa F.
Chlorophyll a induction curves, or Kautsky curves, have been extensively used to study physiological stress conditions in phototrophic organisms, with the analysis of several derived parameters. Nevertheless, these variables use only about 10 % of the information comprised in the complete Kautsky curve dataset, leaving 90 % of the photochemical data within an underutilized dark box, that is not translated into photochemically relevant variables. By observing the variable fluorescence profiles from marine diatoms exposed to a myriad of emerging and classical contaminants, several fluorescence profile alterations were detected, with significant deviations from the control conditions concomitant with the degree of growth inhibition imposed by the chemical stressor. The Linear Discriminant Analysis (LDA) analysis based on the normalized variable chlorophyll a fluorescence profiles revealed a high discriminatory efficiency of the type of contaminant to which the cultures were exposed, indicating that the exposure to different chemical stressors (contaminants) results in specific fluorescence profiles that can be used as descriptors of these exposure conditions. Analysing the individual contaminant LDA analysis, a very low overlap between samples exposed to different concentrations was observed, indicating a high discriminatory power of the variable fluorescence profiles. When evaluating the blind-test classification efficiencies, provided by this contaminant-specific LDA approach, it was possible to observe a high degree of efficiency in almost all contaminants tested, and for most of the concentrations applied. With this in mind, the produced linear discriminants and proportion of traces was used to compute an optical toxicity classification tool - the OPTOX index. The index revealed a high degree of correlation with the growth inhibition observed and/or with the exogenous dose of contaminant applied. The developed OPTOX index, a unifying tool enclosing all the fluorescence data provided by the chlorophyll a induction curve, proved to be an efficient tool to apply in ecotoxicological assays using marine model diatoms with a high degree of reliability for classifying the exposure of the cells to emerging contaminants. Additionally, the data analysis pipeline, as well as the index development methodology here proposed, can be easily transposed to other autotrophic organisms subjected to different ecotoxicological test conditions calibrated and validated against known biochemical or morphological descriptors of stress, integrating this way a large amount of data that was until know completely overlooked and left within an underutilized and undervalued dark box.
Pharmaceuticals in the environment : occurrence and exposure effects in non-target fish species
Publication . Duarte, Irina; Fonseca, Vanessa; Cabral, Henrique; Fick, Jerker
A contaminação por fármacos nas zonas costeiras constitui um problema ambiental emergente, onde os fármacos neuroativos são de particular importância porque bioacumulam em espécies não-alvo, afetando o sistema nervoso central e causando efeitos ao nível das populações. Os resultados demonstram, na literatura existente, os diversos efeitos adversos da exposição de fármacos neuroativos em peixes, apesar de incidir sobre poucos compostos e maioritariamente espécies de água-doce. A bioconcentração é também insuficientemente considerada e raramente estudada em combinação com outras respostas biológicas, dificultando a conjugação entre concentração interna e efeitos observados. A estimativa da bioconcentração dos fármacos neuroativos através da sua lipofilicidade não é direta e depende de múltiplos fatores experimentais, o que dificulta a previsão de risco. Contudo, concentrações ambientais de nove fármacos neuroativos excedem ou estão próximas de concentrações que causam efeitos deletérios em peixes. Dados ambientais, com a deteção de até 28 fármacos neuroativos em águas superficiais e peixes, evidenciam a ubiquidade e diversidade destes compostos em estuários, sendo que o padrão de bioacumulação em sete espécies de peixes foi independente da lipofilicidade dos compostos, do uso do habitat ou do nível-trófico das espécies, com maior frequência e concentrações observadas no cérebro, seguido do fígado e músculo. Experiências de curta e longa exposição a fármacos neuroativos com duas espécies estuarinas/marinhas demonstraram a toxicidade de fármacos com diferentes modos-de-ação, revelando maior acumulação e toxicidade do fármaco neuroativo, fluoxetina, em comparação com outros fármacos frequentemente detetados. As respostas sub-individuais revelaram efeitos em processos essenciais (e.g. mecanismos antioxidantes, biotransformação, metabolismo energético), enquanto efeitos individuais de relevância ecológica (e.g. crescimento, comportamentos alimentares e locomotores) ocorreram após exposição crónica, ou aguda a concentrações mais elevadas. Em suma, novos conhecimentos relativos à presença, acumulação e efeitos da exposição em peixes, demonstram a necessidade de priorizar a investigação e monitorização dos fármacos neuroativos em ecossistemas costeiros.
Comparative analysis of biomarker responses to environmental contamination in estuaries: a multi-taxa approach
Publication . Duarte, Irina Almeida; Fonseca, Vanessa Filipa Simão, 1981-; Cabral, Henrique N., 1969-
Estuaries are highly productive areas of high ecological and economic importance, providing various benefits and services for mankind. The estuarine watershed is a preferential location for human settlement and associated anthropogenic activities, such as industrial and agricultural development, resource exploitation and shipping activities, which result in continuous pressures inevitably leading to environmental degradation. Biomarkers are considered early-warning signs able to provide a predictive perspective of the long-term effects of exposure to pollutants in organisms. Therefore, biomarkers are considered useful tools for environmental quality assessment that integrate biological responses and the degree of stressors, usually applied in a single taxa context. Exposure to xenobiotics compounds and their metabolites lead to enhanced oxidative stress and potentially to major molecular damages such as oxidation of proteins, DNA and peroxidation of unsaturated lipids in cell membranes. Hence, defence mechanisms play a major role in preventing damages and include several enzymes such as antioxidant enzymes superoxide dismutase enzymes (SOD) and catalase (CAT), as well as phase I ethoxyresorufin-O-deethylase (EROD) and phase II glutathione S-transferase (GST) biotransformation enzymes. Accordingly, these enzymes activities as well as both biomarkers of effects lipid peroxidation (LPO) and DNA damage (DNAd) were determined in the present study aiming at assessing overall contamination impacts on various organisms. In this context, the aim of this work is to quantify multiple biomarker responses in a multi-taxa approach, in order to assess the diversity in response patterns among species from two differently impacted estuarine systems. Two Portuguese estuaries, Tejo and Ria de Aveiro, were sampled in two months, June and September 2015, specifically in two sites in each estuary: Alcochete (ALC) and Vila Franca de Xira (VFX) in Tejo and Mira channel (AVSUL) and Murtosa (MUR) in Ria de Aveiro. Several species were selected based on their abundance and estuarine occurrence as well as on their prior use as bioindicator species. Two fish species were considered, the European sea bass Dicentrarchus labrax, a marine migrant species whose juveniles use estuarine areas as nursery areas; and the common goby Pomatoschistus microps a resident estuarine species. Four invertebrate species were also sampled, two common infaunal species, the bivalve Scrobicularia plana and the ragworm Hediste diversicolor and two epibenthic crustaceans, the brown shrimp Crangon crangon and the green shore crab Carcinus maenas. Overall, biomarker responses signaled environmental chemical exposure and some degree of deleterious effects for all species. Low variability among sites and months was observed in antioxidant enzymes responses for most species and no clear pattern was discernible amongst species. In fish species, induction of both biotransformation enzymes was observed with significant spatial variability, with lower variability of GST comparing to EROD activity, though with overall concordant higher levels in Tejo estuarine sites, especially in ALC. Less marked responses in biotransformation enzymes were observed in invertebrate species. A similar GST response pattern was observed for H. diversicolor, S. plana and C. maenas, identifying higher contamination levels in AVSUL, considered the least contaminated site in this study. Very low or even incipient spatial and temporal variability in EROD activity was observed in all invertebrates. Nevertheless, these species signaled mostly Tejo sites according to phase I enzyme responses, especially VFX, which is in agreement with previously reported environmental contamination levels. Concerning monthly variation in general all species showed higher enzyme activities and effects in September, which may reflect the effects of continuous exposure. Notwithstanding the significant activity of detoxification enzymes, deleterious effects were reported for all species, suggesting an overall contamination level above the capacity of the molecular defence mechanisms to limit exposure effects in these species. Biomarkers of effects response patterns differed amongst species, yet akin LPO variability patterns were observed among species, namely between C. crangon, C. maenas and P. microps, signalling VFX and for D. labrax, H. diversicolor and S. plana, signalling MUR. Most species also showed higher mean DNAd values in VFX, except for C. crangon and C. maenas that signalled MUR. A positive correlation between biomarkers of effects was observed for all invertebrate species (except for H. diversicolor), highlighting site-specific contamination. IBR indices also varied throughout sampled sites for all species studied, yet the major pattern observed was the opposed response pattern between a highly mobile fish species D. labrax and both infaunal invertebrate species (S. plana and H. diversicolor). Species-specific responses were evident from lack of concordance among species biomarker responses and IBR scores, most likely due to biological differences (in terms of physiology, vulnerability and overall capacity of defence mechanisms), but also to ecological differences such as differential habitat use, feeding habits, life-strategies and consequently differential contaminants exposure. Overall, Tejo sites were considered more impacted than Ria de Aveiro, yet significant responses were also found in the latter for all species studied. In conclusion, this multi-biomarker and multi-taxa approach provided important insights into the variability of species responses to contaminants exposure in estuaries. The complexity of biomarker response patterns for all species in this study evidenced species differential response and differential exposure to environmental contamination, on top of the complex environmental stimuli, such as pollutants’ mixtures and natural variability characteristic of the estuarine environment. This emphasizes the difficulties associated with effective multi-species ecological risk assessment, and application must carefully consider the potential added ecological value of a multispecific approach (similar to a multi-biomarker approach) versus more complex results interpretation and assessment of the environmental quality.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/MAR-EST/3048/2014