A Proof-of-Concept for a Hypolipidemic Brown Trout Model.

The impacts of hypolipidemic pharmaceuticals on fish lipid metabolism remain unexplored. However, data points to similar effects and mechanisms of action between fish and humans. Therefore, fish may be a strong model for screening hypolipidemic drug candidates and water pollution by lipid-modulating agents. This study aimed to test a new hypolipidemic model assay with juvenile brown trout using atorvastatin (ATV)-a hypolipidemic chemical. We selected 17α-ethinylestradiol (EE2), known to cause hyperlipidemia in fish, to ensure model functionality. Fish received intramuscular injections of 4 µL/g for two weeks under the following experimental conditions: control-C (0.7% NaCl), solvent control-SC (0.7% NaCl, 0.9% ethanol, 0.1% dimethyl sulfoxide), ATV (0.3 µg/g), EE2 (2 µg/g), and a mixture of both compounds-MIX (0.3 µg/g ATV and 2 µg/g EE2). Endpoints included blood lipid biochemistry, hepatic lipid droplet quantification, and liver mRNA expression of lipid-related target genes (related to lipogenesis, lipid transport, and ß-oxidation pathways). ATV lowered blood total cholesterol, high-density lipoproteins (HDL), and low-density lipoproteins (LDL) levels, whilst triglycerides and very-low-density lipoproteins (VLDL) were highest under EE2. Hepatic lipid droplet deposition significantly increased in the ATV, EE2, and MIX groups. ATV and MIX caused a significant downregulation of the peroxisome proliferator-activated receptor γ (pparγ) and acetyl Co-A oxidase 3 (acox3). EE2 upregulated acyl-CoA long-chain synthetase 1 (acsl1) and downregulated both fatty acid binding protein 1 (fabp1) and acetyl Co-A oxidase 1-3I (acox1-3I). ATV caused hypolipidemic effects in juvenile brown trout and could even counteract EE2-stimulated hyperlipidemia, reinforcing the potential of fish hypo- and hyperlipidemic models.

Fish as models to study liver and blood lipid-related effects of fibrates and statins and screen new hypolipidemic drugs.

Fibrates and statins lead worldwide prescriptions of lipid-lowering drugs, whose consumption is increasing considerably due to the growing incidence of dyslipidemias, particularly in high-income areas. Consequently, these chemicals are frequently found in aquatic environments, usually closer to highly urbanized and populated areas, reaching the water systems primarily through waste-water treatment plant (WWTP) effluents. Despite that, the knowledge regarding the effects caused by fibrates and statins in fish, namely in liver lipid metabolism and blood-related parameters, is still very limited. There is yet no standardized fish model for testing the effects of those drugs. However, experimental evidence suggests that the mechanisms of action (MoA) of fibrates and statins are fairly similar to those observed in humans, which makes these aquatic organisms viable alternatives for toxicological and mechanistic studies. This graphical review serves as a state point regarding the potential use of fish as a model for the study of hypolipidemic compounds, addressing (I) the current state of aquatic pollution caused by statins and fibrates, (II) the experimental designs used in the literature to assess effects on fish, (III) the liver metabolism and blood effects caused by exposure to fibrates and statins, as well as (IV) the MoA of both drugs. It further focuses on the current and future benefits of establishing a standardized fish model(s) for testing hypolipidemic drugs.

Chemical Survey and Risk Assessment of 56 Pesticides in the Sado River Estuary (Iberian Peninsula, Portugal).

The Sado basin (~8000 km2) is an area where intensive agriculture occurs. However, this region still has few data about the water levels of priority pesticides such as fungicides, herbicides, and insecticides. Therefore, water samples were collected every two months at nine sites along the Sado River Estuary and analyzed by GC-MS/MS to determine the influx of pesticides in that ecosystem. More than 87% of the pesticides were quantified, and 42% and 72% were above the maximum totals established by the European Directives 98/83/EC and 2013/39/EU, respectively. Fungicides (91%), herbicides (87%), and insecticides (85%) attained average annual amounts of ≈3.2 µg/L, ≈1.0 µg/L, and ≈12.8 µg/L, respectively. A mathematical approach was used to evaluate the hazard of the pesticide mixture at the maximum concentrations found in this area. The assessment identified invertebrates as the most at-risk trophic level and identified two chemicals (chlorpyriphos and cyfluthrin) as the primary culprits. This assumption was supported by acute in vivo assays using Daphnia magna. These observations, and the high concentrations of phosphates, indicate that the status of the Sado waters poses environmental and potential human health risks.

Chemical monitoring and risk assessment of 56 pesticides in the Ave River and adjoining Atlantic coastline (Iberian Peninsula, Portugal).

The Ave basin (North Atlantic Ocean Ecoregion) is highly disturbed by anthropogenic activities. The amounts and distribution patterns of 56 priority pesticides (fungicides, herbicides, and insecticides) dissolved in the aqueous phase (DAP) and suspended particle matter (SPM) were evaluated seasonally along the river, estuary and coastline. Over 83 % of the GC-MS/MS-identified pesticides were quantified in DAP and 91 % in SPM. Pesticides' average annual amounts were ≅316 ng/L and 1.3 µg/g (fungicides), ≅272 ng/L and 2.2 µg/g (herbicides) and ≅1344 ng/L and 6.6 µg/g (insecticides). Invertebrates were the most vulnerable trophic level in the theoretical assessment of pesticide mixture hazards. This hypothesis was strengthened by acute in vivo assays using Artemia salina and Daphnia magna. These data, and the fact that 38 % of pesticides showed concentrations above the maximum established by the European legislation, plus the high concentrations of nitrates, nitrites, ammonia and phosphates, request further depollution actions in this region.

Morvan's Syndrome: The Importance of Knowing Different Risk-Associated Phenotypes and Antibodies in Identifying the Correct Underlying Tumor.

Paraneoplastic neurologic syndromes (PNS) are neurologic disorders that can affect any part of the nervous system, occur in association with cancer, and have an immune-mediated mechanism that produces direct damage to the neural tissue. Neurological symptoms frequently precede, in months to years, the symptoms directly attributed to the primary tumor, requiring a high clinical suspicion for adequate investigation. We report the case of a man in his early 80s admitted for an altered level of consciousness, alternating between periods with stupor and drowsiness, short-term waking states and psychomotor agitation, respiratory failure and dysautonomia, resembling a Morvan's syndrome. Anti-leucine-rich glioma-inactivated 1 and anti-contactin-associated protein-like 2 antibodies were both positive and, after exclusion of infectious and autoimmune systemic causes, the possibility of PNS was raised. Screening for the primary tumor was pursued, and an 18F-fluorodeoxyglucose (18F-FDG)/PET showed only an intensely hypermetabolic, apparent parietal thickening of the lower rectum. Due to the frequent association of Morvan's syndrome to thymoma, a review of the CT of the thorax images was requested and a mediastinal image with features of thymoma was identified. PNS treatment and prognosis depend on finding and treating the underlying tumor, with benefits in both resolution of neurological symptoms and in the prognosis of the underlying tumor itself. Therefore, clinicians should be aware of this frequent but underdiagnosed and underreported condition, in order to improve the chances of better outcomes.

Synthetic Progestins in Waste and Surface Waters: Concentrations, Impacts and Ecological Risk.

Synthetic progestins (PGs) are a large family of hormones used in continuously growing amounts in human and animal contraception and medicinal therapies. Because wastewater treatment plants (WWTPs) are unable to eradicate PGs after excretion, they are discharged into aquatic systems, where they can also be regenerated from conjugated PG metabolites. This review summarises the concentrations of 12 PGs in waters from 2015 to 2021. The selected PGs were considered of particular interest due to their wide use, activity, and hormonal derivation (from testosterone, progesterone, and spirolactone). We concluded that PGs had been analysed in WWTPs influents and effluents and, to a lesser extent, in other matrices, including surface waters, where their concentrations range from ng/L to a few µg/L. Because of their high affinity for cell hormone receptors, PGs are endocrine disruptor compounds that may alter the reproductive fitness and development of biota. This review focused on their biological effects in fish, which are the most used aquatic model organisms to qualify the impacts of PGs, highlighting the risks that environmental concentrations pose to their health, fecundity, and fertility. It is concluded that PGs research should be expanded because of the still limited data on their environmental concentrations and effects.

Temporal-spatial survey of PAHs and PCBs in the Atlantic Iberian northwest coastline, and evaluation of their sources and risks for both humans and aquatic organisms.

Herein, sixteen priority sixteen priority polycyclic aromatic hydrocarbons (PAHs) and seven polychlorinated biphenyls (PCBs), considered seawater quality indicators, were quantified in four beaches of the Iberian (Portuguese) northwest coastline, including one ornithological reserve. These seashores are close to an important industrial region and a harbour. Water samples were extracted for PAHs and PCBs in their aqueous phase (DAP) and suspended particulate matter (SPM) and analysed by GC-MS/MS. Annual average concentrations reached ∼8 ng/L (DAP) and ∼100 ng/g dw (SPM) for PAHs and ∼3 ng/L (DAP) and ∼19 ng/g dw (SPM) for PCBs. PAHs and PCBs had fluctuations suggestive of seasonality. The possible origins of PAHs point to pyrogenic (forest fires) and petrogenic activities linked to maritime transports. The source(s) of PCBs appear to be related with leaks from waste sorting centres located in the vicinity of the sampling sites, together with the burning of contaminated materials. Toxic equivalents (TEQs) suggested a low carcinogenic potential for PAHs in seawater samples (∼0.3 ng/L and ∼4 ng/g dw, for DAP and SPM). Evaluation of risk coefficients for Σ16 PAHs revealed "Low-risk" of both matrices in all sampling areas. PCBs thyroid toxicity equivalents (TEQs-TH), suggested a low impact on biota (∼7.0E-05 ng/L and ∼5.2E-04 ng/g dw, for DAP and SPM). However, in vivo acute assays with Artemia salina exposed to environmental concentrations of PAHs, PCBs, and mixtures, confirmed the theoretical approach, showing that this coastline is not "Risk-free". This fact calls for further toxicological approaches to fully understand the risks posed by these compounds locally.

Concentrations, sources and risks of PAHs in dissolved and suspended material particulate fractions from the Northwest Atlantic Coast of the Iberian Peninsula.

Anthropogenic activities can introduce polycyclic aromatic hydrocarbons (PAHs) in coastal waters. Here, 16 priority PAHs were quantified by gas chromatography-mass spectroscopy (GC-MS) in seawater samples from three Iberian Atlantic habitats. Results showed global concentrations of ≅9 ng/L in the aqueous phase (DAP) and ≅94 ng/g, dw in suspended particulate matter (SPM). The identified sources were both petrogenic and pyrogenic. Nonetheless, the application of several quantitative approaches, including Principal Component Analysis, point to distinct PAHs inputs amongst the sampling areas and demonstrate that sources in S1 (fire forests/sea harbor) are different from those at S2 and S3 (oil refinery/sea harbor). The calculated carcinogenicity potential in both DAP and SPM was low for humans (high percentage of Group 3 PAHs). However, both toxic equivalent factors (TEQs) and environmental risks coefficients (RQs) reveal that the evaluated areas are not risk-free, as confirmed by the practical Artemia salina acute-test assay.

Histological characterization of the maturation stages of the ovarian follicles of the goldfish Carassius auratus (Linnaeus, 1758).

The goldfish is a model organism showing great potential for research, particularly in comparative endocrinology concerning the neuroendocrine signalling and regulation of vertebrate reproduction. Furthermore, this teleost is increasingly stressed as a relevant alternative to more common fish model organisms, namely zebrafish. However, quality descriptions and illustrations of the complete goldfish gonadal histology are surprisingly scarce, but needed, to support research using this fish. Therefore, the main aim of this work is to describe in detail and adequately illustrate the goldfish oogenesis, from oogonia to late maturation, by applying routine stains (haematoxylin-eosin) and special procedures (periodic acid-Schiff and Goldner's trichrome). We hypothesized that the combined strategies would enable not only to observe the most general features but also to perceive some poorly described details of oocytes better. We describe the details of the following maturation stages: oogonia proliferation, chromatin-nucleolus, primary growth (one nucleolus step, multiple nucleoli step, perinucleolar step, cortical alveoli step) and secondary growth (early secondary growth step, late secondary growth step). Additionally, we report aspects of early and late follicular atresia. The study allowed comparisons with other species and showed that the Goldner's trichrome has the best discriminative power and should be the preferred stain, despite more time-consuming.

Magnetic nanosorbents with siliceous hybrid shells of alginic acid and carrageenan for removal of ciprofloxacin.

Water contamination with antibiotics is a serious environmental threat. Ciprofloxacin (CIP) is one of the most frequently detected antibiotics in water. Herein, silica-based magnetic nanosorbents prepared using three seaweed polysaccharides, alginic acid, κ- and λ-carrageenan, were developed and evaluated in the uptake of ciprofloxacin. The sorbents were firstly characterized in detail to assess their morphology and composition. A systematic investigation was conducted to study the adsorption performance towards CIP, by varying the initial pH, contact time and initial CIP concentration. The maximum adsorption capacity was 464, 423 and 1350 mg/g for particles prepared from alginic acid, κ- and λ-carrageenan respectively. These high values indicate that these materials are among the most effective sorbents reported so far for the removal of CIP from water. The kinetic data were consistent with the pseudo-second-order model. The CIP adsorption on λ-carrageenan particles followed a cooperative process with sigmoidal isotherm that was described by the Dubinin-Radushkevich model. The high charge density of λ-carrageenan and the propensity of CIP molecules to self-aggregate may explain the cooperative nature of CIP adsorption. The sorbents were easily regenerated in mild conditions and could be reused in CIP removal up to 4 times without a significant loss of adsorptive properties.

PAHs in water and surface sediments from Douro River estuary and Porto Atlantic coast (Portugal)-impacts on human health.

This study investigated the presence of 16 priority polycyclic aromatic compounds (PAHs) in waters from the Douro River estuary and nearby Atlantic seacoast, which both bath the Porto metropolis. In the area, there is an oil refinery, an important harbour, an intense maritime traffic, small marinas and highly inhabited cities. For the analysis of PAHs, water samples were taken from four sampling sites, at six different times of the year (2011), and extracted by solid-phase extraction (dissolved fraction) and by ultrasound technique (suspended fraction), before their quantification by gas chromatography-mass spectrometry. Results not only proved the ubiquitous distribution of all analysed PAHs in the present habitat, but also that their global amounts (∑16PAHs) were extremely high at all sampling sites. Their average concentrations attained ≈ 55 ng/L and ≈ 52 µg/g dry weight (dw), respectively, in water and surface sediments. Accordingly, the surveyed area was classified as highly polluted by these organics and so, in view of the concentrations, mutagenic/carcinogenic responses in both humans and aquatic animals are possible to occur. The percentages of carcinogenic PAHs for humans (group 1) dissolved in water and in surface sediments were ca. 5 and 6%, respectively. These results are the first reported in the area and can be used as a baseline for future control of the PAHs levels locally while serving the building of global scenarios of PAHs pollution in Europe. Graphical abstract Percentage of PAHs, from different categories acordingly to WHO (2016), in both surface sediments and surface waters from Douro River estuary and Porto Atlantic seacoast; group 1 - carcinogenic, group 2A - probably carcinogenic, group 2B - possibly carcinogenic, and group 3 - not classifiable as carcinogenic to humans.

Determination of 54 pesticides in waters of the Iberian Douro River estuary and risk assessment of environmentally relevant mixtures using theoretical approaches and Artemia salina and Daphnia magna bioassays.

As a case study, the estuary of the international Douro River (Iberian Peninsula) was sampled over a year (2010) at six sampling sites to determine the presence of 56 pesticides of different categories (insecticides, herbicides, and fungicides). 96% of measured pesticides were detected in 79% of the quantified samples. Individual average pesticide concentrations ranged from 39 to 1 265ng/L, indicating a ubiquitous presence of the selected compounds; moreover, twelve pesticides were above the 2013/39/EU Directive limits. Due to its highly impacted profile, a theoretical hazard assessment was done considering the average and maximum environmental mixtures of all measured pesticides to identify the most sensitive trophic level. For both environmental mixtures, the theoretical approach suggested that invertebrates were the most sensitive group. Therefore, short-time exposure assays using both invertebrates Artemia salina and Daphnia magna, were done using the referred mixtures. Data demonstrated significant toxic effects ─ high mortality rate and abnormal swimming behaviour ─ of the exposed animals. Both approaches (theoretical and experimental) support the analytical results, alerting for an intervention on this estuarine environment and of other comparable.

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.

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.

A mollusk VDR/PXR/CAR-like (NR1J) nuclear receptor provides insight into ancient detoxification mechanisms.

The origin and diversification of the metazoan endocrine systems represents a fundamental research issue in biology. Nuclear receptors are critical components of these systems. A particular group named VDR/PXR/CAR (NR1I/J) is central in the mediation of detoxification responses. While orthologues have been thoroughly characterized in vertebrates, a sparse representation is currently available for invertebrates. Here, we provide the first isolation and characterization of a lophotrochozoan protostome VDR/PXR/CAR nuclear receptor (NR1J), in the estuarine bivalve the peppery furrow shell (Scrobicularia plana). Using a reporter gene assay, we evaluated the xenobiotic receptor plasticity comparing the human PXR with the S. plana NR1Jß. Our results show that the molluscan receptor responds to a natural toxin (okadaic acid) in a similar fashion to that reported for other invertebrates. In contrast, the pesticide esfenvalerate displayed a unique response, since it down regulated transactivation at higher concentrations, while for triclosan no response was observed. Additionally, we uncovered lineage specific gene duplications and gene loss in the gene group encoding NRs in protostomes with likely impacts on the complexity of detoxification mechanisms across different phyla. Our findings pave the way for the development of multi-specific sensor tools to screen xenobiotic compounds acting via the NR1I/J group.

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.

Reproductive hormones affect follicular cells and ooplasm of Stage I and II oocytes in zebrafish.

The basic pathway of oocyte development and its regulation is evolutionarily conserved among vertebrates; however, little is known about the role of hormones at the first stages (Stages I and II) of follicle development in fish. In the present study, zebrafish follicles at Stages I and II were exposed in vitro to the reproductive hormones 17ß-oestradiol (E2), 11-ketotestosterone (11KT), 17,20ß-dihydroxy-4-pregnen-3-one (DHP) and to the secondary messenger dibutyryl cyclic adenosine monophosphate (db-cAMP) at a concentration of 1µM for a 48-h period. Morphological alterations of the ooplasm were assessed by transmission electron microscopy and of the granulosa cell layer by quantitative stereology. Expression of mRNA was analysed for cell-cycle genes (cyclin B and E) and resident proteins of the endoplasmic reticulum (calnexin and 78-kDa glucose-regulated protein (grp78/bip)). E2 and db-cAMP stimulated the presence of endoplasmic reticulum in the ooplasm and calnexin mRNA increased in the db-cAMP treatment, but also in response to 11KT and DHP. 11KT, DHP and db-cAMP inhibited the progression of the cell cycle in the granulosa-theca cell layer, indicated by a reduction of the nucleus volume-weighted size of granulosa cells and of increased cyclin E mRNA expression. Reproductive hormones had different effects on the ooplasm and the granulosa-theca cell layer of zebrafish follicles, predominantly at Stage II.

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.