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.

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.

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.

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.

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.

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.

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.

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.

Spatial and seasonal distribution of 17 endocrine disruptor compounds in an urban estuary (Mondego River, Portugal): evaluation of the estrogenic load of the area.

The Mondego River estuary demonstrates signs of pollution, but the levels of endocrine disrupting compounds (EDCs), such as the natural (17ß-estradiol and estrone) and pharmaceutical (17α-ethynylestradiol) estrogens, xenoestrogenic industrial pollutants (4-octylphenol, 4-nonylphenol, and their mono- and diethoxylates and bisphenol A), phytoestrogens (formononetin, biochanin A, daidzein, and genistein), and sitosterol were either poorly or never measured in this area. Thus, to conclude about the influx of EDCs in this estuary, water samples were taken every 2 months, during 1 year (2010) in low tide, at eight sites distributed along the estuary. Water samples (1 L) were preconcentrated in the Oasis HLB cartridges and cleaned in silica cartridges before their analysis by GC-MS. In summer, potentially hazardous amounts of estrogens (≈ 26 ng L(-1)), alkylphenols (≈ 11.5 µg L(-1)), alkylphenolethoxylates (≈ 13 µg L(-1)), and phytoestrogens (≈ 5.6 µg L(-1)) were measured. These data suggest that changes in the hydrodynamics of the estuary coupled with the increase of water temperatures interfere with the amount of EDCs in the water. Complementary physicochemical parameters also point to high levels of anthropogenic pollution in this area. Globally, the estrogenic load, expressed in ethynylestradiol equivalents, attained 71.8 ng L(-1) demonstrating that, all together, the measured EDCs pose important health risks for both biota and humans.

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.

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.

Determination of seventeen endocrine disruptor compounds and their spatial and seasonal distribution in Ria Formosa Lagoon (Portugal).

In spite of its outstanding ecological and touristic importance the Ria Formosa Lagoon shows signs of anthropogenic pollution. Nonetheless, until the present survey no studies had ever documented the measurement of natural and pharmaceutical estrogens (17ß-estradiol, estrone, and 17α-ethynylestradiol), xenoestrogenic industrial pollutants (4-octylphenol, 4-nonylphenol, and their mono and diethoxylates and bisphenol A), phytoestrogens (formononetin, biochanin A, daidzein, genistein), and sitosterol in this area. The 17 compounds measured herein are known as endocrine disrupters (EDCs) and act over the endocrine system even in few amounts (ng L(-1)-µg L(-1)). Thus to conclude about the influx of EDCs in the lagoon, water samples were taken every 2 months, during 1 year (2010), in low tide at nine sites distributed along the coastline. Water samples (1 L) were preconcentrated in the Oasis HLB cartridges and cleaned in silica cartridges before their analysis by GC-MS. Data showed the ubiquitous presence of potentially hazardous amounts of estrogens (particularly of ethynylestradiol, up to 24.3 ng L(-1)), nonylphenol (up to 547 ng L(-1)), and sitosterol (up to 12,300 ng L(-1)), mainly in summer, suggesting that the increase of the local number of inhabitants (tourists), the rise of the water temperature (up to 26 °C), and the blooming of local flora may interfere with the water quality parameters. This makes the lagoon a potential model to study. Taking into account the data, it was concluded that there are conditions for the occurrence of endocrine disruption in aquatic animals, even in areas included in the natural park of the Formosa. Besides, both the high amounts of un-ionized ammonia (up to 0.3 mg L(-1)) and phosphates (up to 1.6 mg L(-1)) my pose risks for local fauna and humans.

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.