Climate-driven shifts in decapod larvae assemblages in a temperate estuary.

The study examines the complex impact of climatic patterns, driven by the North Atlantic Oscillation (NAO), on regional climate, hydrology, and sea surface temperatures. Focused on the period from 2003 to 2012, the research specifically investigates the influence of thermal variability on decapod larval communities. Monthly zooplanktonic sampling conducted at the Mondego Estuary, Portugal, entrance over a decade revealed the prevalence of Carcinus maenas, Diogenes pugilator, and Pachigrapsus marmoratus larvae. These assemblages displayed notable interannual and seasonal fluctuations, often corresponding with changes in sea surface temperatures. Significant system shifts around 2007, instigated by the large-scale NAO, led to subsequent modifications in sea surface temperature and decapod larvae communities' dynamics. Post-2007, there was an upward trajectory in both species' abundance and richness. Phenologically during the former period, the community exhibited two abundance peaks, with the earlier peak occurring sooner, attributed to heightened temperatures instead of the unique peak exhibited before 2007. The research further elucidated the occurrences of Marine Heatwaves (MHW) in the region, delving into their temporal progression influenced by the NAO. Although water temperature emerged as a crucial factor influencing decapod larvae communities annually and seasonally, the study did not observe discernible impacts of MHW events on these communities. These communities represent essential trophic links and are crucial for the survival success of adult decapods. Given the rapid pace of climate change and increasing temperatures, it is imperative to assess whether these environmental shifts, particularly in thermal conditions, affect these meroplanktonic communities.

Seasonal characterization of mercury contamination along the Portuguese coast: human health and environmental risk assessment.

A seasonal characterization of mercury (Hg) accumulation in three different estuaries along the Portuguese coast (i.e. Ria de Aveiro, Tagus estuary and Ria Formosa) was done. For that, it was evaluated: (1) Hg concentrations in abiotic (water) and biotic matrices (flora and fauna); (2) the risk of consumption of local seafood species (e.g. bivalves) to human health; and (3) the environmental risk to Hg exposure. During 1 year, water and biological samples were collected during low tide, in each system for Hg quantification. Our findings revealed that total Hg concentrations in surface waters were higher in Ria de Aveiro and Tagus estuary than in Ria Formosa. In Ria de Aveiro, a particular attention should be given in autumn periods, where Hg levels (≈ 100 µg L-1) were considered quite high according to European quality parameters. The same was observed for the Tagus estuary during spring time. Regarding macrofauna Hg levels, no clear seasonal trend was observed. Also, total Hg concentrations in edible species (< 0.5 µg. g-1 ww) represent no risk for consumption. However, considering the environmental risk, in Ria de Aveiro, there is a moderate risk (RQ > 0.1) in autumn periods, which can be a matter of concern.

Function of estuaries and coastal areas as nursery grounds for marine fish early life stages.

The present study describes the larval and juvenile fish fauna of an estuary and its adjacent coastal area (Mondego estuary, Northwest coast of Portugal) and evaluates their function as nurseries for marine fish. For this, larvae and juveniles were sampled in both systems. The temporal and spatial patterns of the ichthyoplankton community were described for each system and related to the influence of environmental factors. Additionally, the recruitment pattern was evaluated based on the composition of juveniles. Results show a seasonal variation of larval density and community structure between and within systems, indicating a degree of segregation according to their ecological functional classification. Temperature was the most important environmental factor structuring the communities. The juvenile recruitment patterns observed show a different nursery function of the estuary and coastal area for early life stages of different species, reinforcing the need to integrate larval and juvenile stages to better understand fish life cycles and the connectivity between systems.

The functional composition of nearshore fish communities demonstrated by trait analysis: Response to environmental gradients.

There is limited understanding on nearshore fish community functioning in relation to changing environmental gradients. To address this, we investigated the trait composition of fish communities in five regions along the Portuguese coast during 4 years. Northern regions were characterized by higher Chlorophyll-α and small-sized species with short lifespans. The southernmost region was characterized by warmer waters, more intense upwelling events and typical warm-water species. Higher species richness and functional redundancy at the southernmost region suggested that in case of potential species loss, community functioning would be less affected in this region, and thus, a certain level of community resilience is maintained. The central region presented lower functional redundancy and high functional richness, which indicated the presence of rare species with rare traits, suggesting a more vulnerable community. This information on the nearshore fish community distribution may be particularly important towards managing coastal ecosystems in response to changing environmental conditions.

Meta-analysis of multidecadal biodiversity trends in Europe.

Local biodiversity trends over time are likely to be decoupled from global trends, as local processes may compensate or counteract global change. We analyze 161 long-term biological time series (15-91 years) collected across Europe, using a comprehensive dataset comprising ~6,200 marine, freshwater and terrestrial taxa. We test whether (i) local long-term biodiversity trends are consistent among biogeoregions, realms and taxonomic groups, and (ii) changes in biodiversity correlate with regional climate and local conditions. Our results reveal that local trends of abundance, richness and diversity differ among biogeoregions, realms and taxonomic groups, demonstrating that biodiversity changes at local scale are often complex and cannot be easily generalized. However, we find increases in richness and abundance with increasing temperature and naturalness as well as a clear spatial pattern in changes in community composition (i.e. temporal taxonomic turnover) in most biogeoregions of Northern and Eastern Europe.

Environmental and human health risk indicators for agricultural pesticides in estuaries.

The present study aims to contribute to a better assessment of pesticide environmental and human health (here evaluated in the context of human exposure via food items) risks for the estuarine system by comprehensively studying the spatial and temporal occurrence of the pesticides atrazine, azoxystrobin, bentazon, λ-cyhalothrin, penoxsulam and terbuthylazine in the River Mondego estuary (Portugal). Pesticide quantification was performed in surface water, sediment, macroalgae (Ulva spp., Gracilaria gracilis, Fucus vesiculosus), aquatic plants (Zostera noltii, Spartina maritime, Scirpus maritimus) and bivalves (Scrobicularia plana). Since intense precipitation could promote the runoff of pesticides from the surrounding agricultural fields, a single long-duration flood event was also studied in this estuarine system. Under normal flow conditions, quantified concentrations were determined mostly during summer in agreement with the pesticide application period. Azoxystrobin presented the highest detection frequency and atrazine (an herbicide used globally but banned in the EU) presented the second highest frequency, thus highlighting the need to include legacy pesticides in monitoring programmes. Pesticide concentrations in surface water determined in the present study suggest low risk to estuarine organisms. However, all the pesticides were bioaccumulated by S. plana, leading us to consider that pesticides may not only cause adverse effects on the aquatic organism itself, but should also be an alert for human exposure, for this is an edible species and is considered of economic interest. Concern is also expressed about edible seaweeds, since s-triazine pesticides were found in Ulva spp. and G. gracilis. Acknowledging these concerns, developing and establishing allowable pesticide safety values for edible seaweeds and bivalves is recommended, as well as monitoring bivalve pesticide levels, using the whole animal, as a human health exposure indicator for estuarine systems. During the studied flood event, it appears that no serious pesticide contamination has occurred in the River Mondego estuary.

Sharing fishers´ ethnoecological knowledge of the European pilchard (Sardina pilchardus) in the westernmost fishing community in Europe.

BACKGROUND: With the present difficulties in the conservation of sardines in the North Atlantic, it is important to investigate the local ecological knowledge (LEK) of fishermen about the biology and ecology of these fish. The ethnoecological data of European pilchard provided by local fishermen can be of importance for the management and conservation of this fishery resource. Thus, the present study recorded the ethnoecological knowledge of S. pilchardus in the traditional fishing community of Peniche, Portugal. METHODS: This study was based on 87 semi-structured interviews conducted randomly from June to September 2016 in Peniche. The interview script contained two main points: Profile of fishermen and LEK on European pilchard. The ethnoecological data of sardines were compared with the scientific literature following an emic-etic approach. Data collected also were also analysed following the union model of the different individual competences and carefully explored to guarantee the objectivity of the study. RESULTS: The profile of the fishermen was investigated and measured. Respondents provided detailed informal data on the taxonomy, habitat, behaviour, migration, development, spawning and fat accumulation season of sardines that showed agreements with the biological data already published on the species. The main uses of sardines by fishermen, as well as beliefs and food taboos have also been mentioned by the local community. CONCLUSIONS: The generated ethnoecological data can be used to improve the management of this fishery resource through an adaptive framework among the actors involved, in addition to providing data that can be tested in further ecological studies. Therefore, this local knowledge may have the capacity to contribute to more effective conservation actions for sardines in Portugal.

New climatic targets against global warming: will the maximum 2 °C temperature rise affect estuarine benthic communities?

The Paris Agreement signed by 195 countries in 2015 sets out a global action plan to avoid dangerous climate change by limiting global warming to remain below 2 °C. Under that premise, in situ experiments were run to test the effects of 2 °C temperature increase on the benthic communities in a seagrass bed and adjacent bare sediment, from a temperate European estuary. Temperature was artificially increased in situ and diversity and ecosystem functioning components measured after 10 and 30 days. Despite some warmness effects on the analysed components, significant impacts were not verified on macro and microfauna structure, bioturbation or in the fluxes of nutrients. The effect of site/habitat seemed more important than the effects of the warmness, with the seagrass habitat providing more homogenous results and being less impacted by warmness than the adjacent bare sediment. The results reinforce that most ecological responses to global changes are context dependent and that ecosystem stability depends not only on biological diversity but also on the availability of different habitats and niches, highlighting the role of coastal wetlands. In the context of the Paris Agreement it seems that estuarine benthic ecosystems will be able to cope if global warming remains below 2 °C.

Determination and validation of an aquatic Maximum Acceptable Concentration-Environmental Quality Standard (MAC-EQS) value for the agricultural fungicide azoxystrobin.

The main goal of the present study was to determine and validate an aquatic Maximum Acceptable Concentration-Environmental Quality Standard (MAC-EQS) value for the agricultural fungicide azoxystrobin (AZX). Assessment factors were applied to short-term toxicity data using the lowest EC50 and after the Species Sensitivity Distribution (SSD) method. Both ways of EQS generation were applied to a freshwater toxicity dataset for AZX based on available data, and to marine toxicity datasets for AZX and Ortiva® (a commercial formulation of AZX) obtained by the present study. A high interspecific variability in AZX sensitivity was observed in all datasets, being the copepoda Eudiaptomus graciloides (LC50,48h = 38 µg L-1) and the gastropod Gibbula umbilicalis (LC50,96h = 13 µg L-1) the most sensitive freshwater and marine species, respectively. MAC-EQS values derived using the lowest EC50 (≤0.38 µg L-1) were more protective than those derived using the SSD method (≤3.2 µg L-1). After comparing the MAC-EQS values estimated in the present study to the smallest AA-EQS available, which protect against the occurrence of prolonged exposure of AZX, the MAC-EQS values derived using the lowest EC50 were considered overprotective and a MAC-EQS of 1.8 µg L-1 was validated and recommended for AZX for the water column. This value was derived from marine toxicity data, which highlights the importance of testing marine organisms. Moreover, Ortiva affects the most sensitive marine species to a greater extent than AZX, and marine species are more sensitive than freshwater species to AZX. A risk characterization ratio higher than one allowed to conclude that AZX might pose a high risk to the aquatic environment. Also, in a wider conclusion, before new pesticides are approved, we suggest to improve the Tier 1 prospective Ecological Risk Assessment by increasing the number of short-term data, and apply the SSD approach, in order to ensure the safety of aquatic organisms.

Climate influence on juvenile European sea bass (Dicentrarchus labrax, L.) populations in an estuarine nursery: A decadal overview.

Estuarine systems support the life cycle stages of commercially important marine fish and are influenced by large and local-scale climatic patterns. In this study, performed in the Mondego estuary, Portugal, we used an 11-year database (2003-2013) for analyzing the variability in the population of a marine juvenile migrant fish, the European sea bass Dicentrarchus labrax, regarding changes in abundance, population structure, growth rates, secondary production and annual day of peak abundance. Higher densities and production occurred in 2003, but no differences in 0-group growth could be observed. In order to detect change points in both biological and climatic data, the cumulative sum (CUSUM) of the deviations from the mean for the 2003-2013 period were determined for each parameter. The relationship between large and local-scale drivers and 0-group biological attributes were evaluated using a Spearman rank correlation analysis of CUSUM of biological and environmental data, considering the correspondent yearly values and with a time-lag of 1 year. The North Atlantic Oscillation (NAO) index, sea surface temperature (SST) and their respective winter values were tested as large-scale factors, while river runoff, salinity and estuarine water temperature were considered as local climate patterns. The significant factors explaining D. labrax 0-group abundance and production were salinity and the NAO, the latter being also a significant predictor considering the 1-year lag. The NAO with 1-year lag was also positively correlated with the day of peak abundance. The early stages of European sea bass were demonstrated to have a climate-dependent life cycle, controlled by variations in both large-scale climatic patterns and local features. In southern European marine populations, the effects of the NAO seem less direct, and dependent on the magnitude of its expressions and on the time scale considered.

A multiresidue approach for the simultaneous quantification of antibiotics in macroalgae by ultra-high performance liquid chromatography-tandem mass spectrometry.

Together with fish, algae reared in aquaculture systems have gained importance in the last years, for many purposes. Besides their use as biofilters of effluents, macroalgae's rich nutritional profiles have increased their inclusion in human diets but also in animal feeds as sources of fatty acids, especially important for the fish industry. Nonetheless, algae are continuously exposed to environmental contaminants including antibiotics and possess the ability for bioaccumulation of such compounds. Therefore, the present paper describes the development and validation of an ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of antibiotics in the green macroalgae Ulva lactuca. This multi-residue method enables the determination of 38 compounds distributed between seven classes and was fully validated according to EU Decision 2002/657/EC.

Multi-matrix quantification and risk assessment of pesticides in the longest river of the Iberian peninsula.

The distribution of pesticides in dissolved aqueous phase (DAP), suspended particulate matter (SPM) and Scrobicularia plana soft tissues from the Tagus River estuary was determined to evaluate the chemicals pollution status and their hazard potential in this area. Samples were collected in 6 campaigns (December 2012-October 2013), from 3 strategical sites, and analysed via different extraction procedures followed by gas chromatography tandem mass spectrometry (GC-MS/MS) determination. The contamination profile among matrices (DAP, SPM, and soft tissue from bivalves (STB)) was marked by average concentrations of 345ng/L, 0.51mg/kg, and 0.02mg/kg, respectively, with several samples above the 2013/39/EU Directive of environmental quality standards (EQS); no differences were observed between sex. A wider range of pesticides was present in STB (n=53) than in SPM (n=36) and DAP (n=19) matrices. Sediment-water partition coefficient, bioaccumulation factor in both DAP and SPM fraction were estimated ranging between 2.5 and 4.4 and 0.008-2799, respectively. The spatial distribution of most pesticides and physicochemical parameters were consistent, indicating a pollution pattern primarily near the Trancão River mouth. Due to the presence of the target compounds, calculated risk quotients pointed out potential hazards for aquatic organisms, mainly to invertebrates. The estimated average daily intake, theoretical maximum daily intake, and hazard quotient of the studied pesticides-via bivalve ingestion-indicated no risk for human health, although it is important to note possible biomagnification processes that may happen along the estuarine food-chain.

A single-step pesticide extraction and clean-up multi-residue analytical method by selective pressurized liquid extraction followed by on-line solid phase extraction and ultra-high-performance liquid chromatography-tandem mass spectrometry for complex matrices.

Pesticides, a group of compounds linked to human activity, may, when in toxic levels, have a profound effect on water quality, and hence result in adverse consequences to aquatic life and ultimately to human health. Analytical challenges arise when successfully trying to determine these levels in environmental complex matrices. Therefore, fast, simple, sensitive and selective analytical methodologies for multi-residue determination of pesticides (atrazine, azoxystrobin, bentazon, λ-cyhalothrin, penoxsulam and terbuthylazine) in sediment, macrophytes (algae and aquatic plants) and aquatic animals were developed and validated. The established methods were matrix-dependent and were based on Selective Pressurized Liquid Extraction (SPLE) followed by on-line Solid Phase Extraction and Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (on-line SPE-UPLC-ESI-MS/MS). This cutting-edge research methodology uses a small amount of sample, is time saving and reduces the use of organic solvents in compliance with Green Chemistry principles. The analytical features were adequate for all compounds in all studied matrices. The established methodology was applied on real marine samples and no pesticide concentrations above their respective method quantification limits were measured in sediments or aquatic plants. However, terbuthylazine was found in the macroalgae Ulva spp. (108ngg(-1)dw) and all the prospected pesticides were measured above their respective method quantification limits in the bivalve Scrobicularia plana (atrazine: 48ngg(-1)dw, azoxystrobin: 64ngg(-1)dw, bentazon: 33ngg(-1)dw, λ-cyhalothrin: 2531ngg(-1)dw, penoxsulam: 50ngg(-1)dw, and terbuthylazine: 44ngg(-1)dw).

Development and application of a QuEChERS-based extraction method for the analysis of 55 pesticides in the bivalve Scrobicularia plana by GC-MS/MS.

A method for quantitative determination of 55 pesticides in a bivalve matrix was established, based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction and using gas chromatography (GC)-ion trap (IT) mass spectrometry (MS/MS). Accomplishing the European SANCO guidelines, this method was validated using 5 g of homogenized soft tissue, allowing the quantification of pesticides at ng/g of wet weight (ww). Quantification limits and recovery rates ranged from 0.33 to 10.3 µg/L and from 78 to 119 %, respectively. As an important mollusc, not only from an ecological perspective but also for food consumption, the peppery furrow shell (Scrobicularia plana) was sampled at three strategical sites (Ria Formosa Lagoon, in the south of Portugal) during 2012-2013, over six campaigns. A total of 2160 animals were pooled by place and sex. No statistical differences were found among sites or between sexes. Forty percent of the sampled pools were above quantification limits, reaching total annual average concentrations of ∑800 ng/g ww. Additionally, 83 % of the selected compounds showed concentrations above the legal limits set by the European Directive 2013/39/EU. In conclusion, the applied method was successful and proved that bivalves were contaminated by the selected pesticides. In future work, this methodology can be used to monitor body burdens and obtain data for predicting impacts in shellfish consumers. Graphical Abstract Resume of pesticides extraction and analyses process from S. plana.

Pollution by oestrogenic endocrine disruptors and ß-sitosterol in a south-western European river (Mira, Portugal).

The Mira River is a Portuguese water body widely known for its wilderness and is advertised as one of the less polluted European rivers. On this presumption, the levels of endocrine-disrupting compounds (EDCs) in Mira waters were never measured. However, because environmentalists have claimed that the Mira could be moderately polluted, a range of 17 EDCs were measured not only at the estuary but also along the river. The targeted EDCs included natural and pharmaceutical oestrogens (17ß-oestradiol, oestrone and 17α-ethynylestradiol), industrial/household pollutants (octylphenols, nonylphenols and their monoethoxylates and diethoxylates and bisphenol A), phytoestrogens (formononetin, biochanin A, daidzein, genistein) and the phytosterol sitosterol (SITO). For this propose, waters from six sampling sites were taken every 2 months, over a 1-year period (2011), and analysed by gas chromatography-mass spectrometry. Unexpectedly high levels of oestrogens and of industrial/household pollutants were measured at all sampling sites, including those located inside natural protected areas. Indeed, the annual average sum of EDCs was ≈57 ng/L for oestrogens and ≈1.3 µg/L for industrial/household chemicals. In contrast, the global average levels of phytoestrogens (≈140 ng/L) and of SITO (≈295 ng/L) were lower than those reported worldwide. The EDC concentrations were normalised for ethynylestradiol equivalents (EE2eq). In view of these, the oestrogenic load of the Mira River attained ≈47 ng/L EE2eq. In addition, phosphates were above legal limits at both spring and summer (>1 mg/L). Overall, data show EDCs at toxicant relevant levels in the Mira and stress the need to monitor rivers that are allegedly less polluted.

Environmental assessment of pesticides in the Mondego River Estuary (Portugal).

The Mondego River estuary, located on the North Atlantic Ocean Ecoregion, is a basin affected by agricultural run-off with increasing signs of eutrophication. We evaluated the amounts and distribution of 56 priority pesticides belonging to distinct categories (insecticides, herbicides and fungicides). Temporal trends were considered and a total of 42 surface water samples were collected between 2010 and 2011. More than 55% of the GC-MS/MS-quantified pesticides were above the maximum amounts established by the European Directives (98/83/EC and 2013/39/EU). Based on the concentration addition (CA) and independent action (IA) models, we used a two-tiered approach to assess the hazard of the pesticide mixture, at the maximum concentration found, reflecting a potential risk. Short-term exposure using Artemia salina indicated a significant toxic effect where the locomotion of the animals was clearly affected.

Pollution by endocrine disruptors in a southwest European temperate coastal lagoon (Ria de Aveiro, Portugal).

Ria de Aveiro is a Portuguese lagoon renowned for its ecological and economic importance. Nonetheless, in literature, few data exist about its organic pollution. Accordingly, this study chemically monitored for the first time a series of 17 endocrine disruptor compounds (EDCs). The target EDCs include natural (17ß-oestradiol, oestrone) and pharmaceutical (17α-ethynylestradiol) oestrogens, industrial/household xenoestrogenic pollutants (octylphenols, nonylphenols and their mono and diethoxylates and bisphenol A), phytoestrogens (formononetin, biochanin A, daidzein, genistein) and the phytosterol sitosterol (SITO). For the investigation of these EDCs, water samples were taken from eight sampling sites widely spread along the lagoon, at three different occasions in 2011, and analysed by gas chromatography-mass spectrometry. Results not only proved the ubiquitous distribution of all analysed EDCs but revealed that their amounts were extremely high at all sampling sites. The annual average concentrations were ≈ 46 ng/L for oestrogens, ≈ 3962 ng/L for industrial/household chemicals, ≈ 1740 ng/L for phytoestrogens and ≈ 908 ng/L for SITO. Normalising these values in ethynylestradiol equivalents (EE2eq), the oestrogenic load in this lagoon attained ≈ 50 ng/L EE2eq, which is a value well above that known to produce oestrogenic-induced disorders in aquatic fauna. Additionally, phosphate concentrations were also above the legal limits (>1 mg/L). Overall, data show EDCs at toxic relevant levels in the Ria de Aveiro and stress the need to enforce depollution measures in this habitat.

Occurrence and seasonal loads of pesticides in surface water and suspended particulate matter from a wetland of worldwide interest--the Ria Formosa Lagoon, Portugal.

Two novel methods were developed to extract and quantify 56 pesticides in surface waters, considering their content in both dissolved aqueous phase (DAP) and suspended particulate matter (SPM) fractions. These procedures were applied to coastal samples taken seasonally during 2012-2013, from three strategic sampling sites along the Ria Formosa Lagoon (south of Portugal). Briefly, 500 mL of water samples were filtrated, separating both fractions. The DAP fraction was extracted and pre-concentrated by solid-phase extraction (SPE), while the SPM was extracted using ultrasonic extraction technique (USE). Both fractions were then analyzed, and the pesticides were quantified and identified, within 35 min, by gas chromatography (GC) coupled to mass spectrometry (GC-MS and GC-MS/MS), respectively. The extraction of pesticides from the SPM fraction showed average recoveries of 102%, detection limits below 2.2 ng/L, and quantification limits ranging from 0.3 to 6.6 ng/L. Considering the real water samples, 73% of the selected pesticides were quantified in both DAP and SPM fractions (ΣDAP+SPM 2.3 µg/L) and their maximum levels were measured in autumn and winter. By category, the global loads of fungicides, herbicides, and insecticides were ≈407, ≈323, and ≈1.6 µg/L, respectively. Thirty-one percent of the quantified pesticides exceeded the European directives levels (2008/105/EC and 98/83/EC). From the total loads, the SPM fraction contribution was 32%, showing the importance of measuring pesticides in that fraction. The water physicochemical parameters revealed that the total nitrogen amounts were very high relatively to the legal required values, mainly close to the city of Faro (2.6 mg/L). In light of the above, measures are in need to meet European directives and protect both fauna and humans that use this area for leisure.

Toxicological relevance of endocrine disruptors in the Tagus River estuary (Lisbon, Portugal).

The Tagus is the longest Iberian river, notwithstanding, the levels of natural and xenoestrogenic endocrine-disrupting compounds (EDCs) were never measured in its estuary. Suspecting for their presence, we made a major survey of 17 EDCs that include: (i) natural (17ß-oestradiol and estrone) and pharmaceutical oestrogens (17α-ethynylestradiol); (ii) industrial and household pollutants (octylphenols, nonylphenols and their mono and diethoxylates, and bisphenol A); (iii) phytoestrogens (biochanin A, daidzein, formononetin, genistein); and (iv) the phytosterol (sitosterol). Water samples from the Tagus estuary were taken from nine locations every 2 months over a 1-year period and analysed by gas chromatography. Oestrogens, industrial/household pollutants were consistently higher at two sites-at Tagus River mouth and close to the Trancão tributary, both at Lisbon region. The overall oestrogenic load, in ethynylestradiol equivalents, was 13 ng/L for oestrogens, 2.3 ng/L for industrial/household pollutants and 43 ng/L for phytoestrogens; well in the range of toxicological significance. Water physicochemical parameters also indicated anthropogenic pollution, mainly at the two above-referred sampling sites.

Biochemical and physiological responses of Carcinus maenas to temperature and the fungicide azoxystrobin.

Research on the effects of thermal stress is currently pertinent as climate change is expected to cause more severe climate-driven events. Carcinus maenas, a recognised estuarine model organism, was selected to test temperature-dependence of azoxystrobin toxicity, a widely applied fungicide. Crabs' responses were assessed after a 10-d acclimation at different temperatures (5°C, 22°C, and 27°C) of which the last 72h were of exposure to an environmental concentration of azoxystrobin. SOD and GST activities, mitochondrial oxygen consumption rates and protein content, as well as the Coupling Index were determined. The hypothesis proposed that extreme temperatures (5°C and 27°C) and azoxystrobin would affect crabs' responses. Results showed statistically significant different effects of SOD and all oxygen rates measured promoted by temperature, and that neither 30.3µgL(-1) of azoxystrobin nor the combined effect were crab-responsive. Protein content at 5°C was statistically higher when compared with the control temperature (22°C). The Coupling Index revealed both a slight and a drastic decrease of this index promoted by 5°C and 27°C, respectively. Regarding azoxystrobin effects, at 22°C, this index only decreased slightly. However, at extreme temperatures it fell 47% at 5°C and slightly increased at 27°C. Results provided evidence that crabs' responses to cope with low temperatures were more effective than their responses to cope with high temperatures, which are expected in future climate projections. Moreover, crabs are capable of handling environmental concentrations of azoxystrobin. However, the Coupling Index showed that combined stress factors unbalance crabs' natural capability to handle a single stressor.