The H9c2(2-1) cell-based sulforhodamine B assay is a non-animal alternative to evaluate municipal wastewater quality over time.

The present study validates the potential of the in vitro H9c2(2-1) cell-based sulforhodamine B (SRB) assay to evaluate the temporal variability of wastewater quality. The impact of effluent disposal on water quality and the efficiency of the wastewater treatment process were also assessed. To correlate standard analytical method results with in vitro results, a total of 16 physicochemical parameters, such as nutrients, pH, chemical oxygen demand, total suspended solids and metals, were determined in both raw and treated wastewater samples. Results revealed that the H9c2(2-1) cell-based SRB assay has an enormous potential to evaluate municipal wastewater quality over time and to discriminate influent and effluent toxic characteristics, as well as for water quality monitoring and surveillance of the efficacy of treatment processes. Finally, the gathered results alerted to the impact of phosphates in a biological system, leading us to recommend the selection of this parameter as a potential environmental health indicator.

Screening-level evaluation of marine benthic dinoflagellates toxicity using mammalian cell lines.

Complementary studies at different levels of the biological organization are fundamental to fully link environmental exposure to marine benthic dinoflagellate toxins and their effects. In order to contribute to this transdisciplinary evaluation, and for the first time, the present study aims to study the effects of Gambierdiscus excentricus, Ostreopsis cf. ovata, Prorocentrum hoffmannianum and Prorocentrum lima extracts on seven functionally different mammalian cell lines: HEK 293, HepG2, HNDF, H9c2(2-1), MC3T3-E1, Raw 264.7 and SH-SY5Y. All the cell lines presented cell mass decrease in a concentration-dependence of dinoflagellate extracts, exhibiting marked differences in cell toxicity. Gambierdiscus excentricus presented the highest effect, at very low concentrations with EC50,24h (i.e., the concentration that gives half-maximal response after a 24-h exposure) between 1.3 and 13 cells mL-1, followed by O. cf. ovata (EC50,24h between 3.3 and 40 cells mL-1), and Prorocentrum species (P. lima: EC50,24h between 191 and 1027 cells mL-1 and P. hoffmannianum: EC50,24h between 152 and 783 cells mL-1). Cellular specificities were also detected and rat cardiomyoblast H9c2(2-1) cells were in general the most sensitive to dinoflagellate toxic compounds, suggesting that this cell line is an animal-free potential model for dinoflagellate toxin testing. Finally, the sensitivity of cells expressing distinct phenotypes to each dinoflagellate extract exhibited low relation to human poisoning symptoms.

Rat cardiomyocyte H9c2(2-1)-based sulforhodamine B assay as a promising in vitro method to assess the biological component of effluent toxicity.

The treatment of wastewaters is crucial to maintain the ecological status of receiving waters, and thereby guarantee the protection of aquatic life and human health. Wastewater quality evaluation is conventionally based on physicochemical parameters, but increasing attention has been paid to integrate physicochemical and biological data. Nevertheless, the regulatory use of fish in biological testing methods has been subject to various ethical and cost concerns, and in vitro cell-based assays have thus become an important topic of interest. Hence, the present study intends: (a) to evaluate the efficiency of two different sample pre-concentration techniques (lyophilisation and solid phase extraction) to assess the toxicity of municipal effluents on rat cardiomyoblast H9c2(2-1) cells, and (b) maximizing the use of the effluent sample collected, to estimate the environmental condition of the receiving environment. The gathered results demonstrate that the H9c2(2-1) sulforhodamine B-based assay is an appropriate in vitro method to assess biological effluent toxicity, and the best results were attained by lyophilising the sample as pre-treatment. Due to its response, the H9c2(2-1) cell line might be a possible alternative in vitro model for fish lethal testing to assess the toxicity of municipal effluents. The physicochemical status of the sample suggests a high potential for eutrophication, and iron exceeded the permissible level for wastewater discharge, possibly due to the addition of ferric chloride for wastewater treatment. In general, the levels of carbamazepine and sulfamethoxazole are higher than those reported for other countries, and both surpassed the aquatic protective values for long-term exposure.

Evidence of extensive plasma glucose recycling following a glucose load in seabass.

Seabass and other carnivorous fish are highly dependent on gluconeogenesis from dietary amino acids to maintain glycemia. Glucose recycling (glucose→C3-intermediate→glucose) may potentiate the effects of glucose administration in sparing amino acid gluconeogenesis. To date, very few measurements of glucose recycling have been reported in fish. Thus, to determine the extent of glucose recycling following a glycemic challenge, juvenile seabass were given an intraperitoneal glucose load (2gkg-1) enriched with [U-13C]glucose. 13C NMR analysis of plasma glucose 13C-isotopomers was used to determine the fractional contributions of glucose derived directly from the load versus that from glucose recycling at 48h after the load. Both fed and 21-day fasted fish (20 per condition) were studied. In fasted fish, 18±4% of plasma glucose was directly derived from the load while 13±2% was derived from glucose recycling. In fed fish, the load accounted for 6±1% of plasma glucose levels while glucose recycling contributed 16±4%. 13C NMR analysis of plasma lactate revealed 13C-isotopomers corresponding to the expected C3-intermediates of peripheral [U-13C]glucose catabolism indicating that circulating lactate was a key intermediate in glucose carbon recycling under these conditions. In conclusion, glucose recycling was shown to contribute a significant portion of plasma glucose levels in both fed and fasted seabass 48h after an intraperitoneal glucose challenge and circulating lactate was shown to be an intermediate of this pathway.

Effects of dietary carbohydrate on hepatic de novo lipogenesis in European seabass (Dicentrarchus labrax L.).

Farmed seabass have higher adiposity than their wild counterparts and this is often attributed to carbohydrate (CHO) feeding. Whether this reflects a reduction in fat oxidation, increased de novo lipogenesis (DNL), or both, is not known. To study the effects of high CHO diets on hepatic TG biosynthesis, hepatic TG deuterium ((2)H) enrichment was determined following 6 days in (2)H-enriched tank water for fish fed with a no-CHO control diet (CTRL), and diets with digestible starch (DS) and raw starch (RS). Hepatic fractional synthetic rates (FSRs, percent per day(-1)) were calculated for hepatic TG-glyceryl and FA moieties through (2)H NMR analysis. Glyceryl FSRs exceeded FA FSRs in all cases, indicating active cycling. DS fish did not show increased lipogenic potential compared to CTRL. RS fish had lower glyceryl FSRs compared with the other diets and negligible levels of FA FSRs despite similar hepatic TG levels to CTRL. DS-fed fish showed higher activity for enzymes that can provide NADPH for lipogenesis, relative to CTRL in the case of glucose-6-phosphate dehydrogenase (G6PDH) and relative to RS for both G6PDH and 6-phosphogluconate dehydrogenase. This approach indicated that elevated hepatic adiposity from DS feeding was not attributable to increased DNL.

Contribution of dietary starch to hepatic and systemic carbohydrate fluxes in European seabass (Dicentrarchus labrax L.).

In the present study, the effects of partial substitution of dietary protein by digestible starch on endogenous glucose production were evaluated in European seabass (Dicentrarchus labrax). The fractional contribution of dietary carbohydrates v. gluconeogenesis to blood glucose appearance and hepatic glycogen synthesis was quantified in two groups of seabass fed with a diet containing 30% digestible starch (DS) or without a carbohydrate supplement as the control (CTRL). Measurements were performed by transferring the fish to a tank containing water enriched with 5% (2)H2O over the last six feeding days, and quantifying the incorporation of (2)H into blood glucose and hepatic glycogen by (2)H NMR. For CTRL fish, gluconeogenesis accounted for the majority of circulating glucose while for the DS fish, this contribution was significantly lower (CTRL 85 (SEM 4) % v. DS 54 (SEM 2) %; P < 0.001). Hepatic glycogen synthesis via gluconeogenesis (indirect pathway) was also significantly reduced in the DS fish, in both relative (CTRL 100 (SEM 1) % v. DS 72 (SEM 1) %; P < 0.001) and absolute terms (CTRL 28 (SEM 1) v. DS 17 (sem 1) µmol/kg per h; P < 0.001). A major fraction of the dietary carbohydrates that contributed to blood glucose appearance (33 (sem 1) % of the total 47 (SEM 2) %) had undergone exchange with hepatic glucose 6-phosphate. This indicated the simultaneous activity of hepatic glucokinase and glucose 6-phosphatase. In conclusion, supplementation of digestible starch resulted in a significant reduction of gluconeogenic contributions to systemic glucose appearance and hepatic glycogen synthesis.

Expressional regulation of key hepatic enzymes of intermediary metabolism in European seabass (Dicentrarchus labrax) during food deprivation and refeeding.

We hypothesized that the analysis of mRNA level and activity of key enzymes in amino acid and carbohydrate metabolism in a feeding/fasting/refeeding setting could improve our understanding of how a carnivorous fish, like the European seabass (Dicentrarchus labrax), responds to changes in dietary intake at the hepatic level. To this end cDNA fragments encoding genes for cytosolic and mitochondrial alanine aminotransferase (cALT; mALT), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were cloned and sequenced. Measurement of mRNA levels through quantitative real-time PCR performed in livers of fasted seabass revealed a significant increase in cALT (8.5-fold induction) while promoting a drastic 45-fold down-regulation of PK in relation to the levels found in fed seabass. These observations were corroborated by enzyme activity meaning that during food deprivation an increase in the capacity of pyruvate generation happened via alanine to offset the reduction in pyruvate derived via glycolysis. After a 3-day refeeding period cALT returned to control levels while PK was not able to rebound. No alterations were detected in the expression levels of G6PDH while 6PGDH was revealed to be more sensitive specially to fasting, as confirmed by a significant 5.7-fold decrease in mRNA levels with no recovery after refeeding. Our results indicate that in early stages of refeeding, the liver prioritized the restoration of systemic normoglycemia and replenishment of hepatic glycogen. In a later stage, once regular feeding is re-established, dietary fuel may then be channeled to glycolysis and de novo lipogenesis.

Effects of food-deprivation and refeeding on the regulation and sources of blood glucose appearance in European seabass (Dicentrarchus labrax L.).

Sources of blood glucose in European seabass (initial weight 218.0±43.0g; mean±S.D., n=18) were quantified by supplementing seawater with deuterated water (5%-(2)H2O) for 72h and analyzing blood glucose (2)H-enrichments by (2)H NMR. Three different nutritional states were studied: continuously fed, 21-day of fast and 21-day fast followed by 3days of refeeding. Plasma glucose levels (mM) were 10.7±6.3 (fed), 4.8±1.2 (fasted), and 9.3±1.4 (refed) (means±S.D., n=6), showing poor glycemic control. For all conditions, (2)H-enrichment of glucose position 5 was equivalent to that of position 2 indicating that blood glucose appearance from endogenous glucose 6-phosphate (G6P) was derived by gluconeogenesis. G6P-derived glucose accounted for 65±7% and 44±10% of blood glucose appearance in fed and refed fish, respectively, with the unlabeled fraction assumed to be derived from dietary carbohydrate (35±7% and 56±10%, respectively). For 21-day fasted fish, blood glucose appearance also had significant contributions from unlabeled glucose (52±16%) despite the unavailability of dietary carbohydrates. To assess the role of hepatic enzymes in glycemic control, activity and mRNA levels of hepatic glucokinase (GK) and glucose 6-phosphatase (G6Pase) were assessed. Both G6Pase activity and expression declined with fasting indicating the absence of a classical counter-regulatory stimulation of hepatic glucose production in response to declining glucose levels. GK activities were basal during fed and fasted conditions, but were strongly stimulated by refeeding. Overall, hepatic G6Pase and GK showed limited capacity in regulating glucose levels between feeding and fasting states.

Disposition of [U-2H7]glucose into hepatic glycogen in rat and in seabass.

The stimulation of hepatic glycogenesis is a ubiquitous response to a glucose challenge and quantifying its contribution to glucose uptake informs its role in restoring euglycemia. Glycogenesis can be quantified with labeled water provided that exchange of glucose-6-phosphate hydrogen 2 (G6P-H2) and body water via glucose-6-phosphate isomerase, and exchange of positions 4, 5 and 6 hydrogens (G6P-H456) via transaldolase, are known. These exchanges were quantified in 24-h fasted rats (Rattus norvegicus; n=6) and 21-day fasted seabass (Dicentrarchus labrax; n=8) by administration of a glucose load (2000mg·kg(-1)) enriched with [U-(2)H7]glucose and by quantifying hepatic glycogen (2)H-enrichments after 2h (rats) and 48h (seabass). Direct pathway contributions of the glucose load to glycogenesis were also estimated. G6P-H2 and body water exchange was 61±1% for rat and 47±3% for seabass. Transaldolase-mediated exchange of G6P-H456 was 5±1% for rat and 10±1% for seabass. Conversion of the glucose load to hepatic glycogen was significant in seabass (249±54mg·kg(-1)) but negligible in rats (12±1mg·kg(-1)). Preload plasma glucose levels were similar for seabass and rats (3.3±0.7 and 4.4±0.1mmol·L(-1), respectively) but post-load plasma glucose was significantly higher in seabass compared to rats (14.6±1.8 versus 5.8±0.3mmol·L(-1), p<0.01). In conclusion, G6P-H2 and body water exchange is incomplete for both species and has to be accounted for in estimating hepatic glycogen synthesis and direct pathway activities with labeled water tracers. Transaldolase-mediated exchange is insignificant. Hepatic direct pathway glycogenesis plays a prominent role in seabass glucose load disposal, but a negligible role in the rat.

PCB bioaccumulation in three mullet species--a comparison study.

Polychlorinated biphenyls (PCBs) are lipophilic contaminants that tend to accumulate in organisms. PCBs were detected in Chelon labrosus, Liza aurata and Liza ramada, along different age groups. L. ramada presented the highest concentration, and it increased with age, whereas C. labrosus and L. aurata concentration remained constant. L. ramada high concentration can be attributed to its ecological niche, since this species is able to accumulate PCBs along its different age groups even in low environmental contamination conditions. PCBs 101, 118, 138, 149, 153, 170 and 180 were the congeners that more contributed to these species contamination, being PCB 138 and 153 the congeners with higher concentration. Mullets are edible in many countries, being important in fisheries and aquaculture. L. ramada is the most common mullet for capture and human consumption. All species presented concentrations below the regulation limit establish by the European Union, and therefore safe for human consumption.

Are mercury levels in fishery products appropriate to ensure low risk to high fish-consumption populations?

The main goal of the present study is to determine the sources of methylmercury (MeHg) for high fish-consumption populations with the Portuguese population as showcase, as Portugal is the EU country with the highest fish consumption per capita (2019: 59.91 kg year-1). Since limited information is available on the effective levels of mercury after culinary treatments, cooked and raw codfish (Gadus morhua), hake (Merluccius merluccius), octopus (Octopus vulgaris), horse mackerel (Trachurus trachurus) and sardine (Sardina pilchardus) were considered. The mercury concentration ranking Hake > Horse mackerel > Codfish > Octopus > Sardine was observed in all situations (cooked and raw samples) for both MeHg and total mercury (T-Hg). The gathered results reinforce the general assumption that the loss of moisture during cooking increases MeHg and T-Hg concentrations in fish, but the idea that MeHg in fish muscle tissue represents the bulk of T-Hg cannot be generalised, as our study determined a MeHg/T-Hg ratio of 0.43 for grilled sardines.

Assessing pharmaceuticals in the green seaweed Ulva lactuca through a multi-residue UHPLC-ToF-MS strategy.

Seaweeds have become an important asset in several sectors, including the food and feed industries, cosmetics, and pharmaceuticals, among others. Whether harvested or reared, interest in algae has been growing worldwide due to the resources they offer, including proteins, vitamins, minerals, carbohydrates, essential fatty acids, and dietary fiber, as well as sources of biologically active compounds. However, given their morphology and physiology, as well as their harvest and cultivation environments, algae are prone to the presence of hazards, including pharmaceuticals taken up from the water. Thus, to ensure human and animal safety as well as environmental health, monitoring is essential. Therefore, this work describes the development and validation of a sensitive screening and confirmatory analytical method based on ultra-high-performance liquid chromatography coupled with time-of-flight mass spectrometry (UHPLC-ToF-MS). This multi-residue method enables the determination of 62 pharmaceuticals distributed between 8 therapeutic classes and was fully validated according to Commission Implementing Regulation (EU) 2021/808.

Short and long-term temperature variations drive recruitment variability in marine and estuarine juvenile fishes.

Understanding the long-term effects of climatic factors on key species' recruitment is crucial to species management and conservation. Here, we analysed the recruitment variability of key species (Dicentrarchus labrax, Platichthys flesus, Solea solea, Pomatoschistus microps and Pomatoschistus minutus) in an estuary between 2003 and 2019, and related it with the prevailing local and large-scale environmental factors. Using a dynamic factor analysis (DFA), juvenile abundance data were grouped into three common trends linked to different habitat uses and life cycle characteristics, with significant effect of temperature-related variables on fish recruitment: Sea surface temperature and the Atlantic Multidecadal Oscillation. In 2010, a regime shift in the North Atlantic coincided with a shift in the common trends, particularly a decline in P. flesus and S. solea trend. This work highlights the thermophilic character of fish recruitment and the necessity to investigate key biological processes in the context of species-specific responses to climate change.

Hepatic glycogen synthesis in farmed European seabass (Dicentrarchus labrax L.) is dominated by indirect pathway fluxes.

Hepatic glycogen synthesis fluxes from direct and indirect pathways were quantified in seabass by postmortem (2)H NMR analysis of plasma water (PW) and glycogen glucosyl (2)H enrichments from (2)H-enriched seawater. Eighteen fish (28.0 ± 1.7 cm and 218.0 ± 43.0 g) were divided into three groups of 6 and studied over 24 days with transfer to 5% (2)H-seawater after day 21. Over this period, one group was fed daily with fishmeal, a second group was fasted, and a third group was fasted for 21 days followed by 3 days refeeding. Glycogen turnover and sources were determined from the ratio of glucosyl position 5 enrichment to that of plasma water (H5/PW). Glycogen levels of fed fish were significantly higher than fasted (665.4 ± 345.2 µmol.g(-1) liver versus 77.2 ± 59.5 µmol.g(-1) liver, P<0.05) while refed fish had comparable levels to fed (584.6 ± 140.4 µmol.g(-1) liver). Glycogen enrichment of fed fish was undetectable indicating negligible turnover over 3 days. For fasted fish, H5/PW was ~50% indicating that half of the glycogen had turned over via indirect pathway flux. For refed fish, H5/PW was ~100% indicating that the indirect pathway accounted for all net glycogen synthesis. Direct pathway conversion of dietary carbohydrate to glycogen was not detected in any of the groups.

A Stereological Study of the Three Types of Ganglia of Male, Female, and Undifferentiated Scrobicularia plana (Bivalvia).

Neurotransmitters modulate gonadal maturation in bivalves. However, it remains unclear whether there are differences in the nervous system structure between sexes, maturation, and ganglia. Therefore, a stereological study was conducted on the ganglia of adult peppery furrow shell (Scrobicularia plana). Equal-sized males, females, and undifferentiated (gamete absence) animals were fixed with 10% formalin and processed for light microscopy. They were serially cut into 35 µm paraffin thick sections and stained with hematoxylin-eosin. Sections with cerebral (cerebropleural), pedal, and visceral ganglia were studied. The parameters estimated were the volumes of the ganglia, the total and relative volumes of their cortex (outer layer) and medulla (neuropil), and the total number of cells (neurons, glia, and pigmented) per ganglia and compartment. The volumes and numbers were estimated, respectively, by the Cavalieri principle and by the optical fractionator. Females show a larger glia to neuron numerical ratio. Further, females have a greater ganglionic volume than undifferentiated adults, with males showing intermediate values. These facts indicate that the ganglia size is related somehow to maturation. The cell size forms the basis of the differences because total cellularity is equal among the groups. The three ganglion types differ in total volumes and the volume ratio of the cortex versus the medulla. The greater volumes of the pedal ganglia (vis-a-vis the cerebral ones) and of the visceral ganglia (in relation to all others) imply more voluminous cortexes and medullae, but more neuronal and non-neuronal cells only in the visceral. The new fundamental data can help interpret bivalve neurophysiology.

Influences of Climate Change and Variability on Estuarine Ecosystems: An Impact Study in Selected European, South American and Asian Countries.

It is well-known that climate change significantly impacts ecosystems (at the macro-level) and individual species (at the micro-level). Among the former, estuaries are the most vulnerable and affected ecosystems. However, despite the strong relations between climate change and estuaries, there is a gap in the literature regarding international studies across different regions investigating the impacts of climate change and variability on estuaries in different geographical zones. This paper addresses this need and reviews the impacts of climate change, variability and extreme weather on estuaries. It emphasises the following: (i) a set of climate parameters governing estuarine hydrology and processes; and (ii) a sample of countries in Asia (Bangladesh), Europe (Portugal) and South America (Uruguay). We reviewed the influences of the climatic drivers of the estuarine hydrology, ecological processes and specific species in estuarine communities across the selected geographical regions, along with an analysis of their long-term implications. The key results from the three estuaries are as following: (i) Hilsa fish, of which the catches contribute to 10% of the total earnings of the fishery sector (1% of GDP), are affected by climate-forced hydrological and productivity changes in the Meghna; (ii) extreme droughts and short-term severe precipitation have driven the long-term abundance and spatial distribution of both fish larvae and juveniles/adults in the Mondego; and (iii) the river inflow and fluctuations increases since the early 1970s have contributed to variations in the salinity, the stratification, the oxygen, nutrient and trophic levels and the spatial pattern for the life stages of planktonic species, fish biomass and captures in the Rio de la Plata. The results suggested that immediate action is needed to reduce the vulnerability of estuaries to climate stressors, mainly the changing river flows, storms and sea-level rise. As a contribution to addressing current problems, we described a set of adaptation strategies to foster climate resilience and adaptive capacity (e.g., early-warning systems, dam management to prevent overflows and adaptive fisheries management). The implications of this paper are two-fold. Firstly, it showcases a variety of problems that estuaries face from changing climate conditions. Secondly, the paper outlines the need for suitable adaptive management strategies to safeguard the integrity of such vital ecosystems.

Analysis of glucose metabolism in farmed European sea bass (Dicentrarchus labrax L.) using deuterated water.

Glucose metabolism in free-swimming fasted and fed seabass was studied using deuterated water ((2)H(2)O). After transfer to seawater enriched with 4.9% (2)H(2)O for 6-h or for 72-h, positional and mole percent enrichment (MPE) of plasma glucose and water were quantified by (2)H NMR and ESI-MS/MS. Plasma water (2)H-enrichment reached that of seawater within 6h. In both fasted and fed fish, plasma glucose MPE increased asymptotically attaining ~55% of plasma water enrichment by 72 h. The distribution of (2)H-enrichment between the different glucose positions was relatively uniform. The gluconeogenic contribution to glucose that was synthesized during (2)H(2)O administration was estimated from the ratio of position 5 and 2 glucose enrichments. For both fed and fasted fish, gluconeogenesis accounted for 98±1% of the glucose that was produced during the 72-h (2)H(2)O administration period. For fasted fish, gluconeogenic contributions measured after 6h were identical to 72-h values (94±3%). For fed fish, the apparent gluconeogenic contribution at 6-h was significantly lower compared to 72-h (79±5% versus 98±1%, p<0.05). This may reflect a brief augmentation of gluconeogenic flux by glycogenolysis after feeding and/or selective enrichment of plasma glucose position 2 via futile glucose-glucose-6-phosphate cycling.

Lifelong mercury bioaccumulation in Atlantic horse mackerel (Trachurus trachurus) and the potential risks to human consumption.

Atlantic horse mackerel is one of the most commercially important species in Europe. It can reach a longevity of 30 years, with potential implications in lifespan mercury contamination. This study conducted along the Portuguese coast aimed at evaluating the total Hg content and tissue distribution, to determine the annual mercury bioaccumulation patterns and the associated risk for consumption. The T-Hg accumulation patterns observed followed the order: muscle (0.34) > liver (0.28) > heart (0.19) > gills (0.11) > brain (0.041 mg kg-1). Significant differences between tissues reflect the role of the different tissues in storage and redistribution. Significant relationships observed between age and T-Hg for all tissues highlight the continuous nature of the bioaccumulation process. European food safety guidelines signalled significant risk of consumption in about 30% of the samples. Still, there was an overall low risk from the consumption of this species, which can be further minimized through consumer options to avoid health issues.

Effects of climate variability on an estuarine green crab Carcinus maenas population.

The increase in frequency and intensity of extreme climate events over the last few decades has been leading to profound changes in estuarine and marine ecosystems worldwide, with strong implications for the species inhabiting these ecosystems as well as for the services provided by them. In this study, we analysed the effects of climate variability on the temporal and spatial variations in population dynamics of the green crab Carcinus maenas in the Mondego estuary (Portugal), between 2003 and 2018. In this 15-year period, a greater recruitment of C. maenas was observed during drought periods, periods which was matched by an increase in secondary production. Ontogenic stage segregation was also observed, with juveniles being found mainly in the further upriver areas of the estuary. The estuarine population was mainly composed of the green morphotype, with the orange and red morphotypes present in more downstream areas of the estuary. Redundancy analysis (RDA) showed high spatial and temporal variability of C. maenas in the estuary which was related with environmental changes over the 15-year period. A correlation between C. maenas biological features and several local-scale (salinity and river runoff) and large-scale (North Atlantic Oscillation index and Eastern Atlantic pattern) environmental variables was identified through cumulative sums analysis (CUSUM), indicating a strong environmental control on C. maenas population dynamics. This paper shows the importance of relatively long-term datasets to unravel the effects of extreme weather events due to climate change on key epibenthic estuarine species, and also how they might cope with a changing marine environment.

Determination of intestinal absorption of the paralytic shellfish toxin GTX-5 using the Caco-2 human cell model.

Contributing to the human health risk assessment, the present study aims to evaluate the ability of paralytic shellfish toxins (PSTs) to cross the human intestinal epithelium by using the Caco-2 permeability assay. A crude extract prepared from the PST producer dinoflagellate Gymnodinium catenatum strain, GCAT1_L2_16, and the PST analogue gonyautoxin-5 (GTX-5) prepared from a certified reference material (CRM) were tested. In the conditions of the assay, none of the compounds altered Caco-2 viability, or the integrity of cell monolayers. The GTX-5 apparent permeability coefficients are 0.9×10-7 and 0.6×10-7 cm s-1 for the crude extract and CRM, respectively, thus, <10-6 cm s-1, which indicates that humans absorb this PST analogue poorly. The present study also reveals that, during a 90-min exposure, GTX-5 is not metabolised to a high extent by Caco-2 or retained in the Caco-2 cytoplasm. Since it is known that GTX-5 can be found in the spleen, liver or kidney of the victims, as well as in the urine samples of patients who consumed contaminated seafood, further research is needed to clarify the transport mechanisms involved, permeation time and dose-dependence, and the possible role of intestinal microflora.