Lipids as biomarkers to assess the nutritional and physiological status of two diadromous fish (Anguilla anguilla and Chelon auratus) at early life stages in a temperate macrotidal estuary.

Estuaries are considered as key habitats for the early life stages of fish. However, in the face of massive destruction of many estuarine intertidal areas, management and conservation measures are needed. Fish condition indicators may be used as a proxy of habitat quality and provide valuable information for management of coastal areas. In this study, the larvae of golden mullet (Chelon auratus) and European glass eels (Anguilla anguilla) were sampled in three sites of the Gironde Estuary. Different lipid classes and fatty acids were quantified: phospholipids (globally, phosphatidylethanolamine and phosphatidylcholine), triglycerides, omega-3 (particularly docosahexaenoic and eicosapentaenoic acids), omega-6 and C18:1. These biomarkers provide information on the nutritional status of the larvae as well as on prey availability and larvae diet between sites. One site significantly differed from the others as it seemed to offer abundant and better-quality prey. The very high levels of omega-3 contained in mullet larvae suggested that this site provided a high amount of diatoms. However, the mullet larvae that colonized this site also showed physiological stress that could be explained by exposure to pollutants through their prey. This work constitutes an essential baseline for developing biomarkers to assess the quality of habitats in a global change context.

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.

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.

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.

Biochemical impacts in adult and juvenile farmed European seabass and gilthead seabream from semi-intensive aquaculture of southern European estuarine systems.

The nutritional value and developmental variations of cultured fish were assessed for European seabass and gilthead seabream specimens reared in semi-intensive aquaculture systems in two Portuguese estuaries. Quantification of total protein and of carbohydrate and fatty acid profiles was carried out to determine differences between the composition of the same species in two development stages reared in four distinct farms. A significant influence of the rearing site on the nutritional composition of the same species was found for adult European seabass regarding saturated, monounsaturated and highly unsaturated fatty acids contents, both between estuaries and within each estuary. In gilthead seabream, saturated, monounsaturated, polyunsaturated and highly unsaturated fatty acids content were also influenced by the rearing site. Carbohydrate analysis showed a significant influence of the rearing site on free sugar and polysaccharide content in fish of both species, and there was no influence on the species' protein content. Differences in fatty acid and carbohydrate content among juvenile and adult stages were found for all the groups studied. The present study supported the existing evidence that semi-intensive rearing systems are subjected to the variability of extrinsic factors in the rearing sites, influencing the nutritional value of the same species, namely regarding lipid and carbohydrate profiles, depending on the production site. From a consumer's perspective, such differences may come as a disadvantage of the rearing method, as it is expected for a product to provide equal nutritional properties and benefits regardless its origin, especially within the same country.

Ecotoxicity of the lipid-lowering drug bezafibrate on the bioenergetics and lipid metabolism of the diatom Phaeodactylum tricornutum.

Pharmaceutical residues impose a new and emerging threat to the marine environment and its biota. In most countries, ecotoxicity tests are not required for all pharmaceutical residues classes and, even when mandatory, these tests are not performed using marine primary producers such as diatoms. These microalgae are among the most abundant class of primary producers in the marine realm and key players in the marine trophic web. Blood-lipid-lowering agents such as bezafibrate and its derivatives are among the most prescribed drugs and most frequently found human pharmaceuticals in aquatic environments. The present study aims to investigate the bezafibrate ecotoxicity and its effects on primary productivity and lipid metabolism, at environmentally relevant concentrations, using the model diatom Phaeodactylum tricornutum. Under controlled conditions, diatom cultures were exposed to bezafibrate at 0, 3, 6, 30 and 60 µg L-1, representing concentrations that can be found in the vicinity of discharges of wastewater treatment plants. High bezafibrate concentrations increased cell density and are suggested to promote a shift from autotrophic to mixotrophic metabolism, with diatoms using light energy generated redox potential to breakdown bezafibrate as carbon source. This was supported by an evident increase in cell density coupled with an impairment of the thylakoid electron transport and consequent photosynthetic activity reduction. In agreement, the concentrations of plastidial marker fatty acids showed negative correlations and Canonical Analysis of Principal coordinates of the relative abundances of fatty acid and photochemical data allowed the separation of controls and cells exposed to bezafibrate with high classification efficiency, namely for photochemical traits, suggesting their validity as suitable biomarkers of bezafibrate exposure. Further evaluations of the occurrence of a metabolic shift in diatoms due to exposure to bezafibrate is paramount, as ultimately it may reduce O2 generation and CO2 fixation in aquatic ecosystems with ensuing consequences for neighboring heterotrophic organisms.

Screening of human and veterinary pharmaceuticals in estuarine waters: A baseline assessment for the Tejo estuary.

We investigated the presence of 66 human and veterinary pharmaceuticals from seven therapeutic groups in surface waters of the Tejo estuary. Collection sites covered the entire estuary and included areas near main river inflows and wastewater treatment outfalls, traversing urban, agriculture, aquaculture, and nature reserve areas. Detection of pharmaceuticals was performed via UHPLC-TOF-MS. Pharmaceuticals were found in all sites (32 different compounds in total). Antibiotics, ß-blockers, antihypertensives and anti-inflammatories were the most frequently detected (>90%), with variation in concentrations reflecting the multifaceted nature of estuarine surroundings (accumulated site contamination between 15 and 351 ng L-1). Higher concentrations of antidepressant Sertraline (304 ng L-1), non-steroidal anti-inflammatory Diclofenac (51.8 ng L-1), lipid regulator Gemfibrozil (77.0 ng L-1), antihypertensive Ibersartan (161.9 ng L-1) or antibiotic Doxycycline (128.0 ng L-1), among others, though localized may potentially impact key estuarine functions or services. Ultimately, results provide a baseline for regulatory information and future biota evaluations.

Thermal stress and energy metabolism in two circumtropical decapod crustaceans: Responses to acute temperature events.

Extreme events associated with global warming, such as ocean heat waves, can have contrasting fitness consequences for different species, thereby modifying the structure and composition of marine communities. Here, we examined the effects of a laboratory simulated heat wave on the physiology and performance of two Indo-Pacific crustacean species: the shrimp Rhynchocinetes durbanensis and the hermit crab Calcinus laevimanus. We exposed the crustaceans to a control temperature or to a +5 °C temperature (25 °C vs 30 °C) for two consecutive weeks, and weekly analyzed protective proteins, antioxidant activity, and lipid peroxides in muscle and visceral mass. Fulton's K, total protein, %C, and C:N molar ratio of muscle tissue were also analyzed at the end of the experiment. Results showed that 1) the most responsive tissues were the muscle in the shrimp species and the visceral mass in the hermit crab species; 2) biomarker responses in both species occurred mostly after 7 days of exposure; 3) temperature stress led to an increase in biomarker levels; 4) highest biomarker fold-changes were detected in protective chaperones and antioxidants superoxide dismutase and glutathione-S-transferase; 4) integrated biomarker indices suggested poorer health status in individuals subjected to the heat wave; 5) performance changes at the organism level were only detected in R. durbanensis; and 6) mortality rates of both species remained unchanged with the heat wave. Finally, we concluded that these species are capable of physiological adjustments in response to rapid environmental changes, which ultimately confers them with enough thermal tolerance to withstand this simulated heat wave without major consequences for fitness.

Extrinsic and intrinsic factors shape the ability of using otolith chemistry to characterize estuarine environmental histories.

Reconstructing habitat use and environmental histories of fish via otolith chemistry relies on linking otolith chemical composition to the surrounding environment, as well as disentangling the consequences of ontogenetic or physiological effects that may mask environmental signals. We used multiple linear and linear mixed models to analyse the importance of environmental (temperature, salinity, water chemistry) and individual based (fish size) factors on otolith chemical composition and incorporation (Li, Mg, Mn, Sr, and Ba) of juvenile Dicentrarchus labrax, over their time within an estuarine nursery area. Multi model inference highlighted the importance of ontogeny, as well as the influence of temperature and salinity on otolith chemistry and elemental incorporation, with results indicating that intrinsic effects may potentially outweigh environmental effects. Ultimately, understanding if otolith chemistry accurately reflects fine-scale environmental variation is key to reconstruct environmental histories of juvenile fishes in estuaries and will contribute to determining the impact changing estuarine conditions may have on growth and survival.

Effect of underwater visual survey methodology on bias and precision of fish counts: a simulation approach.

Bias in underwater visual census has always been elusive. In fact, the choice of sampling method and the behavioural traits of fish are two of the most important factors affecting bias, but they are still treated separately, which leads to arbitrarily chosen sampling methods. FishCensus, a two-dimensional agent-based model with realistic fish movement, was used to simulate problematic behavioural traits in SCUBA diving visual census methods and understand how sampling methodology affects the precision and bias of counts. Using a fixed true density of 0.3 fish/m2 and a fixed visibility of 6 m, 10 counts were simulated for several combinations of parameters for transects (length, width, speed) and point counts (radius, rotation speed, time), generating trait-specific heatmaps for bias and precision. In general, point counts had higher bias and were less precise than transects. Fish attracted to divers led to the highest bias, while cryptic fish had the most accurate counts. For point counts, increasing survey time increased bias and variability, increasing radius reduced bias for most traits but increased bias in the case of fish that avoid divers. Rotation speed did not have a significant effect in general, but it increased bias for fish that avoid divers. Wider and longer transects and a faster swim speed are beneficial when sampling mobile species, but a narrower, shorter transect with a slow swim is beneficial for cryptic fish.

Reconciling differences in natural tags to infer demographic and genetic connectivity in marine fish populations.

Processes regulating population connectivity are complex, ranging from extrinsic environmental factors to intrinsic individual based features, and are a major force shaping the persistence of fish species and population responses to harvesting and environmental change. Here we developed an integrated assessment of demographic and genetic connectivity of European flounder Platichthys flesus in the northeast Atlantic (from the Norwegian to the Portuguese coast) and Baltic Sea. Specifically, we used a Bayesian infinite mixture model to infer the most likely number of natal sources of individuals based on otolith near core chemical composition. Simultaneously, we characterised genetic connectivity via microsatellite DNA markers, and evaluated how the combined use of natural tags informed individual movement and long-term population exchange rates. Individual markers provided different insights on movement, with otolith chemistry delineating Norwegian and Baltic Sea sources, whilst genetic markers showed a latitudinal pattern which distinguished southern peripheral populations along the Iberian coast. Overall, the integrated use of natural tags resulted in outcomes that were not readily anticipated by individual movement or gene flow markers alone. Our ecological and evolutionary approach provided a synergistic view on connectivity, which will be paramount to align biological and management units and safeguard species' biocomplexity.

Environmental health assessment of warming coastal ecosystems in the tropics - Application of integrative physiological indices.

According to climate science, ocean warming is one of the current and future greatest threats to coastal ecosystems. Projection scenarios for the end of this century show that tropical intertidal ecosystems are particularly at risk. In this study we optimized and tested a holistic method for bio-monitoring present and projected thermal pressure in such ecosystems, in order to assess organism vulnerability to ocean warming. Several species representative of different animal groups (fish, crustaceans and gastropods) were collected from the field and subjected to an experimental trial for 28 days, testing two temperatures: control (present seawater summer temperature) and elevated temperature (+3 °C, projected seawater temperature anomaly for 2100). Muscle samples were collected weekly to quantify several biomarkers of: i) macromolecular damage (protein unfolding and denaturation, and lipid peroxidation), ii) reactive oxygen species (ROS) scavengers (antioxidant enzymes), and iii) body condition (energy reserves and body mass). These biomarkers were combined in integrated biomarker response (IBR) indices, either in three separate stress response categories (as previously defined) or in a unique combined analysis of overall physiological performance. Both approaches suggest that temperature affected IBRs, with increasing temperatures significantly impairing the overall health of individuals. Biomarkers of lower levels of biological organization indicated deleterious effects of temperature, whereas biomarkers of higher levels suggested maintenance of performance after chronic exposure. Overall indices combining the estimates of biomarkers across levels of biological organization are essential to predict the vulnerability of species, or populations, to climate warming. Such indices may assist managers and stakeholders in the establishment of monitoring programs and environmental policies toward the conservation of fragile coastal systems.

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.

Comparing biomarker responses during thermal acclimation: A lethal vs non-lethal approach in a tropical reef clownfish.

Knowledge of thermal stress biology for most tropical fish species in reef ecosystems under climate change is still quite limited. Thus, the objective of this study was to measure the time-course changes of thermal stress biomarkers in the commercially exploited coral reef fish Amphiprion ocellaris, during a laboratory simulated event of increased temperature. Heat shock protein 70kDa (Hsp70) and total ubiquitin (Ub) were determined in the muscle (lethal method) and in the fin (non-lethal alternative method) under two temperature treatments (control - 26°C and elevated temperature - 30°C) throughout one month with weekly samplings. Results suggest that biomarker basal levels are tissue-specific and influence the degree of response under temperature exposure. Responses were highly inducible in the muscle but not in fin tissue, indicating that the latter is not reliable for monitoring purposes. Thermal stress was observed in the muscle after one week of exposure (both biomarkers increased significantly) and Ub levels then decreased, suggesting the animals were able to acclimate by maintaining high levels of Hsp70 and through an effective protein turnover. In addition, the results show that mortality rates did not differ between treatments. This indicates that A. ocellaris is capable of displaying a plastic response to elevated temperature by adjusting the protein quality control system to protect cell functions, without decreasing survival. Thus, this coral reef fish species presents a significant acclimation potential under ocean warming scenarios of +4°C. Monitoring of thermal stress through a non-lethal method, fin-clipping, although desirable proved to be inadequate for this species.

Global patterns and predictors of fish species richness in estuaries.

1. Knowledge of global patterns of biodiversity and regulating variables is indispensable to develop predictive models. 2. The present study used predictive modelling approaches to investigate hypotheses that explain the variation in fish species richness between estuaries over a worldwide spatial extent. Ultimately, such models will allow assessment of future changes in ecosystem structure and function as a result of environmental changes. 3. A comprehensive worldwide data base was compiled of the fish assemblage composition and environmental characteristics of estuaries. Generalized Linear Models were used to quantify how variation in species richness among estuaries is related to historical events, energy dynamics and ecosystem characteristics, while controlling for sampling effects. 4. At the global extent, species richness differed among marine biogeographic realms and continents and increased with mean sea surface temperature, terrestrial net primary productivity and the stability of connectivity with a marine ecosystem (open vs. temporarily open estuaries). At a smaller extent (within a marine biogeographic realm or continent), other characteristics were also important in predicting variation in species richness, with species richness increasing with estuary area and continental shelf width. 5. The results suggest that species richness in an estuary is defined by predictors that are spatially hierarchical. Over the largest spatial extents, species richness is influenced by the broader distributions and habitat use patterns of marine and freshwater species that can colonize estuaries, which are in turn governed by history contingency, energy dynamics and productivity variables. Species richness is also influenced by more regional and local parameters that can further affect the process of community colonization in an estuary including the connectivity of the estuary with the adjacent marine habitat, and, over smaller spatial extents, the size of these habitats. In summary, patterns of species richness in estuaries across large spatial extents seem to reflect from global to local processes acting on community colonization. The importance of considering spatial extent, sampling effects and of combining history and contemporary environmental characteristics when exploring biodiversity is highlighted.

Early contamination of European flounder (Platichthys flesus) by PCDD/Fs and dioxin-like PCBs in European waters.

Contamination levels and profiles of 7 polychlorinated-p-dioxins, 10 polychlorinated furans (PCDD/Fs) and 12 dioxin-like polychlorinated biphenyls (dl-PCBs) were investigated in juvenile European flounder (Platichthys flesus) captured in different nursery areas in the northeastern Atlantic coast across its geographical distribution range. The toxic equivalent concentrations (WHO-TEQfish) were also determined in order to evaluate which P. flesus population was more exposed to dioxin-like toxicity. Juveniles caught in the Sørfjord (Norway) showed the lowest WHO-TEQfish concentration (0.052 pg WHO-TEQfish g(-1)wet weight) whereas the highest value was observed in fish from the Wadden Sea (The Netherlands; 0.291 pg WHO-TEQfish g(-1)ww), mainly due to the greater contribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin, the most toxic congener. Nonetheless, when comparing the results with existent tissue residue-based toxicity benchmarks, no adverse effects resulting from PCDD/Fs and dl-PCBs are expected to occur in flounder from the studied systems.

Fishers' behaviour in response to the implementation of a Marine Protected Area.

Marine Protected Areas (MPAs) have been widely proposed as a fisheries management tool in addition to their conservation purposes. Despite this, few studies have satisfactorily assessed the dynamics of fishers' adaptations to the loss of fishing grounds. Here we used data from before, during and after the implementation of the management plan of a temperate Atlantic multiple-use MPA to examine the factors affecting the spatial and temporal distribution of different gears used by the artisanal fishing fleet. The position of vessels and gear types were obtained by visual surveys and related to spatial features of the marine park. A hotspot analysis was conducted to identify heavily utilized patches for each fishing gear and time period. The contribution of individual vessels to each significant cluster was assessed to better understand fishers' choices. Different fisheries responded differently to the implementation of protection measures, with preferred habitats of target species driving much of the fishers' choices. Within each fishery, individual fishers showed distinct strategies with some operating in a broader area whereas others kept preferred territories. Our findings are based on reliable methods that can easily be applied in coastal multipurpose MPAs to monitor and assess fisheries and fishers responses to different management rules and protection levels. This paper is the first in-depth empirical study where fishers' choices from artisanal fisheries were analysed before, during and after the implementation of a MPA, thereby allowing a clearer understanding of the dynamics of local fisheries and providing significant lessons for marine conservation and management of coastal systems.

Ecological quality assessment of small estuaries from the Portuguese coast based on benthic macroinvertebrate assemblages indices.

Benthic macroinvertebrates communities are the most consistently emphasized biotic component of aquatic ecosystems and are one of the biological indicators required for assessment by the European Water Framework Directive. In this context, several indices based on these communities have been developed in order to assess ecological quality of estuarine systems. In the present work we used AMBI, M-AMBI, BENTIX and BAT to distinguish ecological status of five small estuarine systems of the Portuguese south and southwest coasts. Although indices outputs did not differ between systems and sampling seasons, results indicated that the metrics in which these indices are based could differentiate community structures as a result of two main gradients that force these communities: the natural variability, and the anthropogenic impact.

Assessing food web dynamics and relative importance of organic matter sources for fish species in two Portuguese estuaries: a stable isotope approach.

Stable carbon and nitrogen isotopes (δ¹³C, δ¹5N) were used to analyse food web dynamics of two of the main estuaries of the Portuguese coast: Tejo and Mira. The ultimate sources of organic matter supporting production of some of the most abundant and commercially important fish species were determined; and seasonal, inter- and intra- estuarine differences in the trophic relations among producers and consumers were identified. Stable isotope analysis was performed in different producers, primary consumers (main prey items for fish) and fish species (Solea solea, Solea senegalensis, Pomatoschistus microps, Dicentrarchus labrax, Liza ramada, Diplodus vulgaris and Atherina presbyter) of two areas in each estuary, in July and October 2009. Model calculations showed that the main prey for the fish species in the Tejo estuary used mostly salt marsh-derived organic matter as nutritional sources, with no marked differences between the sampled months. Trophic levels of fish species from the same estuary differed at multiple scales: inter-species, seasonally and spatially (both between and within estuaries). Significant differences in isotopic composition of fish species were more pronounced spatially (between the two sampled areas in the estuary) than seasonally (between sampled months). Trophic relationships in both estuaries demonstrated that organic matter is transferred to higher trophic positions mainly through benthic pathways. This shows the flexibility of these species to share resources and to exploit temporary peaks in prey populations. The present results showed that extensive disturbance in intertidal habitats from both estuaries may potentially change the balance of organic matter in the base of these complex food webs.