Elemental and spectral chemometric analyses of Octopus vulgaris beaks as reliable markers of capture location.

The high demand and economic relevance of cephalopods make them prone to food fraud, including related to harvest location. Therefore, there is a growing need to develop tools to unequivocally confirm their capture location. Cephalopod beaks are nonedible, making this material ideal for traceability studies as it can also be removed without a loss of commodity economic value. Within this context, common octopus (Octopus vulgaris) specimens were captured in five fishing areas along the Portuguese coast. Untargeted multi-elemental total X-ray fluorescence analysis of the octopus beaks revealed a high abundance of Ca, Cl, K, Na, S, and P, concomitant with the keratin and calcium phosphate nature of the material. We tested a suite of discrimination models on both elemental and spectral data, where the elements contributing most to discriminate capture location were typically associated with diet (As), human-related pressures (Zn, Se, and Mn), or geological features (P, S, Mn, and Zn). Among the six different chemometrics approaches used to classify individuals to their capture location according to their beaks' element concentration, classification trees attained a classification accuracy of 76.7%, whilst reducing the number of explanatory variables for sample classification and highlighting variable importance for group discrimination. However, using X-ray spectral features of the octopus beaks further improved classification accuracy, with the highest classification of 87.3% found with partial least-squares discriminant analysis. Ultimately, element and spectral analyses of nonedible structures such as octopus beaks can provide an important, complementary, and easily accessible means to support seafood provenance and traceability, whilst integrating anthropogenic and/or geological gradients.

Neuroactive pharmaceuticals in estuaries: Occurrence and tissue-specific bioaccumulation in multiple fish species.

Contamination of surface waters by pharmaceuticals is an emerging problem globally. This is because the increased access and use of pharmaceuticals by a growing world population lead to environmental contamination, threatening non-target species in their natural environment. Of particular concern are neuroactive pharmaceuticals, which are known to bioaccumulate in fish and impact a variety of individual processes such as fish reproduction or behaviour, which can have ecological impacts and compromise fish populations. In this work, we investigate the occurrence and bioaccumulation of 33 neuroactive pharmaceuticals in brain, muscle and liver tissues of multiple fish species collected in four different estuaries (Douro, Tejo, Sado and Mira). In total, 28 neuroactive pharmaceuticals were detected in water and 13 in fish tissues, with individual pharmaceuticals reaching maximum concentrations of 1590 ng/L and 207 ng/g ww, respectively. The neuroactive pharmaceuticals with the highest levels and highest frequency of detection in the water samples were psychostimulants, antidepressants, opioids and anxiolytics, whereas in fish tissues, antiepileptics, psychostimulants, anxiolytics and antidepressants showed highest concentrations. Bioaccumulation was ubiquitous, occurring in all seven estuarine and marine fish species. Notably, neuroactive compounds were detected in every water and fish brain samples, and in 95% of fish liver and muscle tissues. Despite variations in pharmaceutical occurrence among estuaries, bioaccumulation patterns were consistent among estuarine systems, with generally higher bioaccumulation in fish brain followed by liver and muscle. Moreover, no link between bioaccumulation and compounds' lipophilicity, species habitat use patterns or trophic levels was observed. Overall, this work highlights the occurrence of a highly diverse suite of neuroactive pharmaceuticals and their pervasiveness in waters and fish from estuarine systems with contrasting hydromorphology and urban development and emphasizes the urgent need for toxicity assessment of these compounds in natural ecosystems, linked to internalized body concentration in non-target species.

Untargeted Metabolomics Reveals Antidepressant Effects in a Marine Photosynthetic Organism: The Diatom Phaeodactylum tricornutum as a Case Study.

The increased use of antidepressants, along with their increased occurrence in aquatic environments, is of concern for marine organisms. Although these pharmaceutical compounds have been shown to negatively affect marine diatoms, their mode of action in these non-target, single-cell phototrophic organisms is yet unknown. Using a Fourier-transform ion cyclotron-resonance mass spectrometer (FT-ICR-MS) we evaluated the effects of fluoxetine in the metabolomics of the model diatom Phaeodactylum tricornutum, as well as the potential use of the identified metabolites as exposure biomarkers. Diatom growth was severely impaired after fluoxetine exposure, particularly in the highest dose tested, along with a down-regulation of photosynthetic and carbohydrate metabolisms. Notably, several mechanisms that are normally down-regulated by fluoxetine in mammal organisms were also down-regulated in diatoms (e.g., glycerolipid metabolism, phosphatidylinositol signalling pathway, vitamin metabolism, terpenoid backbone biosynthesis and serotonin remobilization metabolism). Additionally, the present work also identified a set of potential biomarkers of fluoxetine exposure that were up-regulated with increasing fluoxetine exposure concentration and are of high metabolic significance following the disclosed mode of action, reinforcing the use of metabolomics approaches in ecotoxicology.

Elemental Fingerprinting of Wild and Farmed Fish Muscle to Authenticate and Validate Production Method.

In the context of expanding fish production and complex distribution chains, traceability, provenance and food safety tools are becoming increasingly important. Here, we compare the elemental fingerprints of gilthead seabream (Sparus aurata) muscle from wild and different aquaculture productions (semi-intensive earth ponds and intensive sea cages from two locations) to confirm their origin and evaluate the concentrations of elements with regulatory thresholds (Cu, Hg, Pb and Zn). Using a chemometric approach based on multi-elemental signatures, the sample origin was determined with an overall accuracy of 90%. Furthermore, in a model built to replicate a real-case scenario where it would be necessary to trace the production method of S. aurata without reliable information about its harvesting location, 27 of the 30 samples were correctly allocated to their original production method (sea-cage aquaculture), despite being from another location. The concentrations of the regulated elements ranged as follows: Cu (0.140-1.139 mg/Kg), Hg (0-0.506 mg/Kg), Pb (0-2.703 mg/Kg) and Zn (6.502-18.807 mg/Kg), with only Pb presenting concentrations consistently above the recommended limit for human consumption. The present findings contribute to establishing elemental fingerprinting as a reliable tool to trace fish production methods and underpin seafood authentication.

Harnessing the Full Power of Chemometric-Based Analysis of Total Reflection X-ray Fluorescence Spectral Data to Boost the Identification of Seafood Provenance and Fishing Areas.

Provenance and traceability are crucial aspects of seafood safety, supporting managers and regulators, and allowing consumers to have clear information about the origin of the seafood products they consume. In the present study, we developed an innovative spectral approach based on total reflection X-ray fluorescence (TXRF) spectroscopy to identify the provenance of seafood and present a case study for five economically relevant marine species harvested in different areas of the Atlantic Portuguese coast: three bony fish-Merluccius merluccius, Scomber colias, and Sparus aurata; one elasmobranch-Raja clavata; one cephalopod-Octopus vulgaris. Applying a first-order Savitzky-Golay transformation to the TXRF spectra reduced the potential matrix physical effects on the light scattering of the X-ray beam while maintaining the spectral differences inherent to the chemical composition of the samples. Furthermore, a variable importance in projection partial least-squares discriminant analysis (VIP-PLS-DA), with k - 1 components (where k is the number of geographical origins of each seafood species), produced robust high-quality models of classification of samples according to their geographical origin, with several clusters well-evidenced in the dispersion plots of all species. Four of the five species displayed models with an overall classification above 80.0%, whereas the lowest classification accuracy for S. aurata was 74.2%. Notably, about 10% of the spectral features that significantly contribute to class differentiation are shared among all species. The results obtained suggest that TXRF spectra can be used for traceability purposes in seafood species (from bony and cartilaginous fishes to cephalopods) and that the presented chemometric approach has an added value for coupling with classic TXRF spectral peak deconvolution and elemental quantification, allowing characterization of the geographical origin of samples, providing a highly accurate and informative dataset in terms of food safety.

Fatty acid-based index development in estuarine organisms to pinpoint environmental contamination.

Estuaries have long been preferred areas of human settlement, where multiple anthropogenic activities take place, which have contributed to a significant decrease in environmental quality of these ecosystems. Accordingly, environmental monitoring and management have long relied on the development of tools that summarize and simplify complex information and provide direct interpretation of quality status. Here, the fatty acid profiles of three abundant estuarine species, namely Hediste diversicolor, Carcinus maenas and Pomatoschistus microps, were used to develop and validate a multimetric index, based on the Euclidean dissimilarities of profiles between sites, in response to contamination gradient in a large urban estuary. Spatial differences were generally related to unsaturated fatty acids (mono- and polyunsaturated, of the n-3 and n-6 series) in all species, albeit more pronounced in P. microps. Multivariate models returned high classification accuracies for the three sampled sites, varying from 73.3% in the invertebrate species to 100.0% in the fish species. Results show the applicability of the developed FA-based index, particularly due to the easy of communication, for managers and the public alike, but also highlight the need for prior validation on species suitability or sensitivity to depict environmental contamination.

Elemental Chemometrics as Tools to Depict Stalked Barnacle (Pollicipes pollicipes) Harvest Locations and Food Safety.

The stalked barnacle Pollicipes pollicipes is an abundant species on the very exposed rocky shore habitats of the Spanish and Portuguese coasts, constituting also an important economical resource, as a seafood item with high commercial value. Twenty-four elements were measured by untargeted total reflection X-ray fluorescence spectroscopy (TXRF) in the edible peduncle of stalked barnacles sampled in six sites along the Portuguese western coast, comprising a total of 90 individuals. The elemental profile of 90 individuals originated from several geographical sites (N = 15 per site), were analysed using several chemometric multivariate approaches (variable in importance partial least square discriminant analysis (VIP-PLS-DA), stepwise linear discriminant analysis (S-LDA), linear discriminant analysis (LDA), random forests (RF) and canonical analysis of principal components (CAP)), to evaluate the ability of each approach to trace the geographical origin of the animals collected. As a suspension feeder, this species introduces a high degree of background noise, leading to a comparatively lower classification of the chemometric approaches based on the complete elemental profile of the peduncle (canonical analysis of principal components and linear discriminant analysis). The application of variable selection approaches such as the VIP-PLS-DA and S-LDA significantly increased the classification accuracy (77.8% and 84.4%, respectively) of the samples according to their harvesting area, while reducing the number of elements needed for this classification, and thus the background noise. Moreover, the selected elements are similar to those selected by other random and non-random approaches, reinforcing the reliability of this selection. This untargeted analytical procedure also allowed to depict the degree of risk, in terms of human consumption of these animals, highlighting the geographical areas where these delicacies presented lower values for critical elements compared to the standard thresholds for human consumption.

Bioconcentration of neuroactive pharmaceuticals in fish: Relation to lipophilicity, experimental design and toxicity in the aquatic environment.

Uptake of contaminants is linked to their toxicity and is usually estimated through their lipophilicity (logKow). Here, we review current literature regarding bioconcentration, i.e. uptake of contaminants from the external environment only, and the effects of exposure to neuroactive pharmaceuticals in fish. We aim to determine if lipophilicity is a suitable predictor of bioconcentration of these compounds in fish, to identify major drivers of bioconcentration and explore the link between bioconcentration potential and toxicity, focusing on survival, growth, condition, behaviour and reproduction endpoints. Additionally, we compare concentrations known to elicit significant effects in fish with current environmental concentrations, identifying exposure risk in ecosystems. The majority of studies have focused on antidepressants, mainly fluoxetine, and encompasses mostly freshwater species. Few studies determined pharmaceuticals bioconcentration, and even a smaller portion combined bioconcentration with other toxicity endpoints. Results show that lipophilicity isn't a good predictor of neuroactive pharmaceuticals' bioconcentration in fish, which in turn is highly influenced by experimental parameters, including abiotic conditions, species and life-stage. The need for increased standardization of experimental settings is key towards improving accuracy of environmental risk assessments and application in future regulatory schemes. Still, increased fish lethality was linked to increased bioconcentration, yet no other correlations were observed when considering effects on growth, condition, behaviour or reproduction, likely as a result of insufficient and variable data. In the context of current environmental concentrations, several neuroactive pharmaceuticals were found to be potentially threatening, while data on occurrence is lacking for some compounds, particularly in brackish/marine systems. Specifically, nine compounds (fluoxetine, citalopram, sertraline, amitriptyline, venlafaxine, clozapine, carbamazepine, metamfetamine and oxazepam) were found at concentrations either above or critically close to minimum response concentrations, thus likely to affect fish in freshwater and brackish or marine environments, which supports further exploration in risk management strategies and monitoring programs in aquatic environments.

Environmental risk assessment and bioaccumulation of pharmaceuticals in a large urbanized estuary.

We screened for the presence of 66 different pharmaceutical residues in surface waters and in multiple invertebrate and fish species of the Tejo estuary to produce an environmental risk assessment of individual pharmaceuticals and their mixtures, as well as evaluate the bioaccumulation of pharmaceuticals in one of Europe's largest estuarine systems. Sixteen pharmaceutical residues, from seven therapeutic classes, were detected in estuarine waters, with environmental mixture concentrations ranging from 42 to 1762 ng/L. Environmental risk assessment via the determination of risk quotients, demonstrated high ecological risk for the antibiotic amoxicillin and angiotensin II receptor blockers irbesartan and losartan. Moderate risk was estimated for antidepressants, antiepileptics, anxiolytics and beta-blockers, but the risk quotient of the accumulated mixture of compounds was over 380-fold higher than the no risk threshold, driven by antibiotics and angiotensin II receptor blockers. In biota, higher risk therapeutic groups were found in higher concentrations, with nine pharmaceutical residues detected, including six antibiotics and two neuroactive compounds, and maximum tissue concentrations up to 250 µg/kg. Bioaccumulation was species- and compound-specific, with only two compounds found simultaneously in water and biota, likely a result of the complex dynamics and fate of pharmaceuticals in estuarine waters. Nonetheless, higher detection frequencies were observed in species living directly on or just above the substrate (i.e. benthic and demersal species), underpinning the importance of habitat use, as well the potential role of sediment and diet based routes for pharmaceutical uptake. Ultimately, results support urgent action on managing the impact of pharmaceuticals in coastal environments, striving for improved monitoring schemes tailored to the dynamic nature and ecological diversity of estuaries and coastal ecosystems.

Study on the efficiency of a covalent organic framework as adsorbent for the screening of pharmaceuticals in estuary waters.

Herein, we demonstrate, for the first time, that covalent organic frameworks (COFs) can be efficient adsorbents for the screening of pharmaceuticals in real water samples, obtaining highly representative data on their occurrence and avoiding the cost of carrying high volume samples and tedious and costly clean-up and preconcentration steps. Of the 23 pharmaceuticals found present in the water samples from the Tagus river estuary using state-of-the-art solid-phase extraction (SPE), 22 were also detected (adsorbed and recovered for analysis) using a COF as the adsorbent material with adsorption efficiency of over 80% for nearly all compounds. In specific cases, acidification of the water samples was identified to lead to a dramatic loss of adsorption efficiency, underlining the effect of sample pre-treatment on the results. The COF efficiently adsorbed (>80%) 19 pharmaceuticals without acid treatment of the sample, highlighting the potential of this class of materials for representative in situ passive adsorption of pharmaceuticals, making this material suitable for being used in water monitoring programs as a simple and cost-efficient sample preparation procedure. In the case of α-hydroxyalprazolam and diclofenac, the COF outperformed the SPE procedure in the recovery efficiency. Although further efforts should be made in tailoring the desorption of the pharmaceuticals from the COF by using different solvents or solvent mixtures, we propose COFs as convenient adsorbent for broad-scope screening and as an efficient adsorbent material to target specific classes of pharmaceuticals. To the best of our knowledge, this is the first study on the use of COFs for contaminant screening in real, naturally contaminated water samples.

Effects of Propranolol on Growth, Lipids and Energy Metabolism and Oxidative Stress Response of Phaeodactylum tricornutum.

Present demographic trends suggest a rise in the contributions of human pharmaceuticals into coastal ecosystems, underpinning an increasing demand to evaluate the ecotoxicological effects and implications of drug residues in marine risk assessments. Propranolol, a non-selective ß-adrenoceptor blocker, is used worldwide to treat high blood pressure conditions and other related cardiovascular conditions. Although diatoms lack ß-adrenoceptors, this microalgal group presents receptor-like kinases and proteins with a functional analogy to the animal receptors and that can be targeted by propranolol. In the present work, the authors evaluated the effect of this non-selective ß-adrenoceptor blocker in diatom cells using P. tricornutum as a model organism, to evaluate the potential effect of this compound in cell physiology (growth, lipids and energy metabolism and oxidative stress) and its potential relevance for marine ecosystems. Propranolol exposure leads to a significant reduction in diatom cell growth, more evident in the highest concentrations tested. This is likely due to the observed impairment of the main primary photochemistry processes and the enhancement of the mitochondrial respiratory activity. More specifically, propranolol decreased the energy transduction from photosystem II (PSII) to the electron transport chain, leading to an increase in oxidative stress levels. Cells exposed to propranolol also exhibited high-dissipated energy flux, indicating that this excessive energy is efficiently diverted, to some extent, from the photosystems, acting to prevent irreversible photoinhibition. As energy production is impaired at the PSII donor side, preventing energy production through the electron transport chain, diatoms appear to be consuming storage lipids as an energy backup system, to maintain essential cellular functions. This consumption will be attained by an increase in respiratory activity. Considering the primary oxygen production and consumption pathways, propranolol showed a significant reduction of the autotrophic O2 production and an increase in the heterotrophic mitochondrial respiration. Both mechanisms can have negative effects on marine trophic webs, due to a decrease in the energetic input from marine primary producers and a simultaneous oxygen production decrease for heterotrophic species. In ecotoxicological terms, bio-optical and fatty acid data appear as highly efficient tools for ecotoxicity assessment, with an overall high degree of classification when these traits are used to build a toxicological profile, instead of individually assessed.

Comfortably numb: Ecotoxicity of the non-steroidal anti-inflammatory drug ibuprofen on Phaeodactylum tricornutum.

Emerging pollutants such as pharmaceuticals are continuously released to aquatic environments posing a rising threat to marine ecosystems. Yet, monitoring routines and ecotoxicity data on biota worldwide for these substances are lacking. Non-steroidal anti-inflammatory drugs are among the most prescribed and found pharmaceuticals in aquatic environments. The toxicity effects of environmentally relevant concentrations of ibuprofen on primary productivity, oxidative stress and lipid metabolism of the diatom Phaeodactylum tricornutum were assessed. Diatom cultures were exposed to 0, 0.8, 3, 40, 100 and 300 µg L-1 ibuprofen concentrations, usually found in the vicinity of wastewater treatment plants and coastal environments. Higher concentrations (100 and 300 µg L-1) had a negative impact in P. triconutum growth, inhibiting the chloroplastic energy transduction in the electron transport chain resulting in lower energy reaching the PS I (r2 = -0.55, p < 0.05). In contrast, the mitochondrial electron transport and available energy increased (r2 = 0.68 and r2 = 0.85, p < 0.05 respectively), mostly due to enhancements in lipid and protein contents as opposed to reduction of carbohydrates. A general up-regulation of the antioxidant enzymes could contributed to alleviate oxidative stress resulting in the decrease of lipid peroxidation products (r2 = 0.77, p < 0.05). Canonical analysis of principal components was performed and successfully discriminated exposure groups, with optical data excelling in classifying samples to different ibuprofen concentrations, being potentially used as environmental indicators. Finally, the identified mild to severe effects of ibuprofen on diatoms are likely to be exacerbated by the sustained use of this drug worldwide, underpinning the urgency of evaluating the impacts of this pharmaceutical on coastal and marine trophic webs.

Effects of antidepressants in the reproduction of aquatic organisms: a meta-analysis.

Pharmaceuticals are widespread contaminants across the aquatic systems and despite the role that these compounds play in society today, little is known about their effects in aquatic organisms once they are released into the environment. This study aims to provide quantitative insight on the effects of antidepressants on the reproduction of fish and crustaceans with a multilevel meta-analysis. A systematic literature search identified 19 studies investigating effects of antidepressant exposure in fish or crustaceans' reproduction, according to specific selection criteria. Heterogeneity analysis was performed and the moderating effect of the range of exposure concentrations, exposure time, organism group and toxicant was tested. Additionally, publication bias was also addressed. The results showed that, overall, there is no significant association between antidepressant exposure and the reproduction of fish and crustaceans, however, moderator analysis revealed that the range of concentrations is a significant moderator for fish and crustacean fecundity, showing contrary results between the two groups. Antidepressant concentration had a small, yet positive effect on fish fecundity, meaning that increased concentrations resulted in increased fish fecundity, whilst a negative effect on crustaceans' fecundity was apparent with increasing concentrations. This difference could be related to data artifacts, or, more likely, evidencing a hormetic dose-response curve, with different ranges of exposure concentrations considered in studies on fish and crustaceans. Antidepressants have shown effects on reproductive outcomes in aquatic organisms, based on individual studies and narrative reviews. However, our results show that other factors can have an important role. Additionally, data available for a quantitative assessment is scarce, focusing mainly on a few freshwater species, low concentration ranges and one SSRI compound, fluoxetine. Thus, more research on the subject is needed since meta-analysis are only as statistically powerful as the number of, good quality, studies they include.

Fluoxetine Arrests Growth of the Model Diatom Phaeodactylum tricornutum by Increasing Oxidative Stress and Altering Energetic and Lipid Metabolism.

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 ß-carotene (ß-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.

Depressed, hypertense and sore: Long-term effects of fluoxetine, propranolol and diclofenac exposure in a top predator fish.

Pharmaceutical compounds are continuously released into the aquatic environment, resulting in their ubiquitous presence in many estuarine and coastal systems. As pharmaceuticals are designed to produce effects at very low concentrations and target specific evolutionary conserved pathways, there are growing concerns over their potential deleterious effects to the environment and specifically to aquatic organisms, namely in early life-stages. In this context, the long-term effects of exposure of juvenile meagre Argyrosomus regius to three different pharmaceuticals were investigated. Fish were exposed to environmental concentrations of one of three major used pharmaceuticals: the antidepressant fluoxetine (0.3 and 3 µg/L for 15 days), the anti-hypertensive propranolol and the non-steroidal anti-inflammatory agent diclofenac (0.3 and 15 µg/L for 30 days). Pharmaceuticals bioconcentration in fish muscle was examined, along with biomarkers in different tissues related with antioxidant and biotransformation responses (catalase, superoxide dismutase, ethoxyresorufin-O-deethylase and glutathione S-transferase), energetic metabolism (lactate dehydrogenase, isocitrate dehydrogenase and electron transport system activities), neurotransmission (acetylcholinesterase activity) and oxidative damage (DNA damage and lipid peroxidation levels). Overall, each pharmaceutical had different potential for bioconcentration in the muscle (FLX > PROP > DCF) and induced different biological responses: fluoxetine was the most toxic compound to juvenile meagre, affecting fish growth, triggering antioxidant defense responses, inhibiting detoxification mechanisms and increasing lipid peroxidation and DNA damage in the liver; propranolol exposure increased DNA damage and decreased aerobic metabolism in fish muscle; and diclofenac showed no potential to bioconcentrate, yet it affected fish metabolism by increasing cellular energy consumption in the muscle and consequently reducing fish net energy budget. The diverse response patterns evidence the need for future research focused on pharmaceuticals with different modes of action and their exposure effects on organismal physiological mechanisms and homeostatic status. Ultimately, the combination of sub-individual and individual responses is key for ecologically relevant assessments of pharmaceutical toxicity.

Biomarker and behavioural responses of an estuarine fish following acute exposure to fluoxetine.

Antidepressants such as fluoxetine are frequently detected in estuaries and can have profound effects on non-target organisms by interfering with the neural system and affecting essential physiological processes and behaviours. In this context, short-term effects of fluoxetine exposure were analysed in the common goby Pomatoschistus microps, an estuarine resident fish species. Two experiments were conducted with fish exposed to: i) fluoxetine concentrations within the µg/L range for 96 h (0.1, 0.5, 10 and 100 µg/L) and ii) fluoxetine concentrations within the mg/L range for 1 h (1, 5 and 10 mg/L). Acute toxicity was assessed via multiple biomarker responses, namely: activity levels of antioxidant (superoxide dismutase and catalase) and detoxification enzymes (ethoxyresorufin O-deethylase and glutathione S-transferase); and biomarkers of effects (lipid peroxidation and DNA damage) and of neurotoxicity (acetylcholinesterase inhibition). Furthermore, behavioural responses concerning activity (active time, movement delay and number of active individuals) and feeding (number of feeding individuals) were also recorded and analysed. Acute fluoxetine exposure for 96 h (in the µg/L range) reduced antioxidant CAT activity with increasing concentrations but had no significant effect on SOD activity. Biotransformation enzymes showed bell-shaped response curves, suggesting efficient fluoxetine metabolism at concentrations up to 10 µg/L. No significant damage (LPO and DNAd) was observed at both concentration ranges (µg/L and mg/L), yet 1 h exposure to higher fluoxetine concentrations (mg/L range) inhibited acetylcholinesterase activity (up to 37%). Fluoxetine (at mg/L) also decreased the number of both feeding and active individuals (by 67%), decreased fish active time (up to 93%) and increased movement delay almost 3-fold (274%). Overall, acutely exposed P. microps were able to cope with fluoxetine toxicity at the µg/L range but higher concentrations (mg/L) affected fish cholinergic system and behavioural responses.

Short-term variability of fish condition and growth in estuarine and shallow coastal areas.

Short-term variability in condition factor: relative condition factor Kn; biochemical condition: RNA:DNA and protein content; and instantaneous growth rates were determined in estuarine and coastal fish. Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps were sampled in the Tejo estuary, while Trachurus trachurus was sampled in an adjacent shallow coastal area. Variation of condition indices was more frequent at the week scale (sampling periods with fortnight intervals) than at the daily scale (consecutive days in each sampling period) in all species. Water temperature was correlated with biochemical indices, while salinity showed no effect, evidencing the influence of environmental short-term variation (temperature) on biochemical condition in natural populations. Yet, decreasing individual variability in fish condition was observed along the sampled weeks, resulting in a more homogeneous condition of populations, particularly for T. trachurus likely due to a more stable coastal environment. Biochemical indices proved to be sensitive to short-term environmental variability, despite species-specific responses.

Biomarker responses to environmental contamination in estuaries: A comparative multi-taxa approach.

Estuaries are highly productive ecosystems subjected to numerous anthropogenic pressures with consequent environmental quality degradation. In this study, multiple biomarker responses [superoxide dismutase (SOD), catalase (CAT), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities, as well as lipid peroxidation (LPO) and DNA damage (DNAd)] were determined in two fish (Dicentrarchus labrax and Pomatoschistus microps) and four macroinvertebrate species (Carcinus maenas, Crangon crangon, Hediste diversicolor and Scrobicularia plana) from the Ria de Aveiro and Tejo estuaries over distinct months. Two sites per estuarine system were selected based on anthropogenic pressures and magnitude of environmental contamination. Antioxidant enzyme activities in fish species suggested a ubiquitous response to oxidative stress, while biotransformation and effect biomarkers exhibited higher spatial and temporal variation. In invertebrate species, biotransformation enzyme activity was clearly less variable than in fish evidencing lower xenobiotic transformation capability. Overall, largest biomarker responses were found in the most contaminated sites (Tejo), yet species-specific patterns were evident. These should be factored in multi-taxa approaches, considering that the differential functional traits of species, such as habitat use, life-stage, feeding or physiology can influence exposure routes and biomarker responses. The Integrated Biomarker Response index highlighted patterns in biomarker responses which were not immediately evident when analyzing biomarkers individually. Overall, results provided insights into the complexity of species responses to contamination in naturally varying estuarine environments. Ultimately, multi-taxa and multi-biomarker approaches provide a comprehensive and complementary view of ecosystem health, encompassing diverse forms of biological integration and exposure routes, and allow the validation of results among markers and species.