Search

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 β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The concentrations of seventeen endocrine disruptor compounds (EDCs) that included oestrogens, phytoestrogens, sitosterol, and banned industrial pollutants were investigated at ten sites of the Douro River estuary. Surface waters were collected during 2019. After evaluating the physicochemical data (ammonia, nitrates, nitrites and phosphates), the waters were filtrated and submitted to solid-phase extraction (SPE) to extract and pre-concentrate (4000-fold) the EDCs. The extracts were derivatized with BSTFA + 1% TMS and analysed by gas chromatography-mass spectrometry (GC-MS). All EDCs showed a high detection rate (97%, on average), exhibiting ubiquity in this estuary. The finding of biologically relevant amounts of oestrogens (up to 8.5 ng/L for oestradiol, E2), phytoestrogens (up to 827 ng/L for biochanin A, BIO-A) and industrial pollutants (up to 2.7 µg/L for nonylphenol di-ethoxylated, NP2EO) strongly support ongoing risks of endocrine disruption for the local aquatic wildlife. Globally, there was an E2-equivalents (E2-EQs) concentration of 25 ng/L and a hazard index (HI) of 26, which further indicates considerable potential for adverse effects on local biota. Moreover, the physicochemical data suggest direct sewage discharges. Beyond possible toxicological effects on fauna, the detected contaminants may pose risks to humans via direct contact (bathing at local fluvial beaches) or by ingestion (local fish). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The main aim of this study was to histochemically estimate the relative copper content in the liver during the breeding cycle of female Ohrid trout, a wild population endemic to the Ohrid Lake. The histology of the oogenetic cycle in this species was documented for the first time. Histochemistry with semi-quantification revealed positive correlations between the gonadosomatic indices and copper load. Uptake of the metal increases in the liver as the ovaries increase in size, with the highest hepatic copper load occurring postspawning. The plasmatic levels of 17β-oestradiol were also quantified, but no correlations to copper load were found, so there is no direct estrogenic control on the uptake and release of copper from the bloodstream to either organ. Therefore, when interpreting field studies or assays on hepatic copper pollution and toxicity, breeding-related seasonal changes in Ohrid trout must be taken into account. [ABSTRACT FROM AUTHOR]

Estrone, 17β-estradiol, 17α-ethynylestradiol, alkylphenols and alkylphenol ethoxylates, are endocrine disruptor compounds. In Portugal, the report of male fish feminization in Minho, Ave and Mondego River estuaries made pertinent their evaluation in surface water samples. Data, got by specific enzyme-linked immunosorbent assays, showed that estrogens concentrations were lower in Minho than in Ave or Mondego estuaries: [Estrone] > [17β-estradiol] > [17α-ethynylestradiol] (p < 0.05). Converting estrogen concentrations in 17α-ethynylestradiol equivalents, the contribution of estrogens was 1.3 ng/L, 3.5 ng/L, and 2.4 ng/L, respectively, for Minho, Ave, and Mondego estuaries stressing out a high risk for local aquatic species. The concentrations of alkylphenols and alkylphenol ethoxylates were lower at both Minho and Mondego estuaries ≈600 ng/L and 2700 ng/L, respectively than at the Ave, ≈1070 ng/L and 4855 ng/L (p < 0.05). In conclusion, this study reports the first systematic data about the presence of the targeted endocrine disruptor compounds in the Minho River estuary and, in parallel, provides new information about their concentrations in two other habitats previously monitored. This study also shows that in Ave and Mondego estuaries the concentrations of industrial estrogens were still excessive, whereas the load of the pure estrogens has decreased significantly facing previous surveys. [ABSTRACT FROM AUTHOR]

Copyright of Contributions. Section of Natural, Mathematical & Biotechnical Sciences is the property of Macedonian Academy of Sciences & Arts (MANU) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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 17b-oestradiol (E2), 11-ketotestosterone (11KT), 17,20b-dihydroxy-4-pregnen-3-one (DHP) and to the secondary messenger dibutyryl cyclic adenosine monophosphate (db-cAMP) at a concentration of 1 mM 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. [ABSTRACT FROM AUTHOR]

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 (17β-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. [ABSTRACT FROM AUTHOR]

This study describes the simultaneous quantification of 56 pesticides in surface coastal water, supported by the development and validation of a gas chromatography (GC)–ion trap (IT) mass spectrometry (MS) method. Samples (500 mL) were pre-concentrated 2500 times by solid phase extraction (OASISTMHLB). The compounds were identified and quantified, within 35 minutes, by GC tandem mass spectrometry (GC-MS/MS) and GC-MS, respectively. The methodology proved to be highly specific for all target pesticides, with an average linearity of 0.99. Detection limits and recovery rates ranged from 0.4 to 1.3 ng L−1and 71% to 120%, respectively. The performance of the method was checked using water samples collected from nine sampling sites along the Ria Formosa Lagoon Natural Park (south of Portugal, n = 54) in each season (2010). The total annual concentrations of all pesticides in each category (fungicides, herbicides and insecticides) were 1.4, 0.6 and 9.0 µg L−1, respectively. Moreover, 89% of the pesticides tested for were detected, 84% could be quantified and 25% had concentrations above the European recommended levels (2013/39/EU). The highest total loads of pesticides were found in the spring, which is in agreement with their seasonal application. Physicochemical parameters such as, nitrites, nitrates, ammonia and phosphates, also indicate poor water quality, supporting the fact that the Ria Formosa lagoon actually needs an effective monitoring programme for effective preservation of its natural reserve status. [ABSTRACT FROM PUBLISHER]

To achieve more information about growth and development of oocytes in teleost fish or concerning toxicity testing, it is necessary to develop adequate in vitro oocyte culture conditions. Herein, initial stages of zebrafish oocytes (I, primary; II, cortical; III, vitellogenic) were analyzed under serum-free medium conditions as gonadal fragments or as separated oocyte-follicle complexes. Two vital dye staining methods (MTT, trypan blue) were applied to assess mitochondrial activity and membrane integrity of the oocytes during 4 days, and compared to morphological alterations studied by transmission electron microscopy. Vital dye staining indicated reduced viability at day 4 for all stages in both in vitro culture methods. Additionally, the viability decreased significantly in gonadal fragments at day 2 for stages III (MTT, TB) and II (TB only). Signs of degradation at the ultrastructural level (vacuoles, disintegration of endoplasmic reticulum and detachment of follicular cell layers) appeared in gonadal fragments at day 4 for stages II and III, and in separated oocyte-follicle complexes both at day 4 for stages I-III, and at day 2 for stage III. In conclusion, zebrafish oocytes at stages I and II seemed viable for 2 days as separated oocyte-follicle complexes considering their mitochondrial activity, membrane integrity and ultrastructural morphology. Cultured as gonadal fragments, the majority of analyses indicated similar results for stages I and II oocytes. In contrast, stage III oocytes seemed viable for not longer than 24 h. Results should be taken into consideration for the experimental design of in vitro assays using teleost fish oocytes. [ABSTRACT FROM AUTHOR]

A gas chromatography with mass spectrometric detection (GC-MS) method was developed and optimized for the determination of 17 endocrine disrupting compounds (EDCs) in coastal water samples. The evaluated EDCs were from different origins and included estrogens, bisphenol A, alkylphenolethoxylates, alkylphenols, phytoestrogens and sitosterol (SITO). The EDCs were extracted from samples using Oasis HLB (Hydrophilic--Lipophilic Balance) cartridges and derivatized with N,Obis( trimethylsilyl)trifluoroacetamide (BSTFA) added with 1% trimethylchlorosilane (TMCS). The validation parameters revealed that this method was highly specific for all assayed compounds (>99%) and the linearity of the calibration curves showed a correlation higher than 0.99. The detection limits were at low ng/L level and the recovery rates were higher than 70%. The performance of the method was checked using coastal water samples taken every 2 months during 2009-2010 from the Douro River estuary and the Porto coastline (Portugal). These data revealed that approximately 98.0% of the analyzed compounds showed levels above their limits of detection (LODs). The measured estrogens (2-20 ng/L) and industrial pollutants (up to 1.1 μg/L) were in biologic hazardous concentrations. Besides, a clear seasonal pattern of fluctuation was established for phytoestrogens and SITO. The physicochemical data, namely the amounts of nitrates, nitrites and phosphorous, confirmed the low water quality of this area. [ABSTRACT FROM AUTHOR]

The Ave River flows through the most urbanized and industrialized Porto district areas. Despite ovotestis was recently reported in male fish from the estuary, no data exist on the water levels of natural and pharmaceutical estrogens (17β-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, and genistein), and sitosterol. Absence of analyses applies to the river, estuary, and nearby coastline. Those compounds mimic the action of endogenous estradiol, being well-recognized endocrine disrupters (EDCs). To conclude about suspected influxes of estrogenic EDCs into the river and coastline, water samples were taken at eight sites every two months, during one year (2010), at low tide. Data showed ubiquitous presence of potentially hazardous amounts of estrogens (particularly ethynylestradiol, up to 10 ng L−1), nonylphenol (up to 250 ng L−1), and sitosterol (up to 6 μg L−1), which helps explaining the ovotestis emergence in local fish. Also, because physicochemical parameters used in water quality evaluation, such as pH, dissolved oxygen, nitrates, and nitrites levels, were within legal limits – suggesting good quality – our study supports that assessment of human and environmental risks by targeting surface waters requires integrating EDCs monitoring in routine analyses. [ABSTRACT FROM AUTHOR]

The Sado waters show signs of anthropogenic pollution. However, hardly any information exists on the levels of endocrine disrupters (EDCs) such as estrogens (17β-estradiol, estrone and 17α-ethynylestradiol) and there is no information at all on xenoestrogenic industrial pollutants (4-octylphenol, 4-nonylphenol and their mono and diethoxylates and bisphenol A). Moreover, data on plant derived phytoestrogens (formononetin, biochanin A, daidzein, genistein) and sitosterol are scarce or inexistent. To assess on the suspected influx of EDCs in this aquatic system, water samples were taken every two months, during 2010, at nine sites distributed along the Sado Estuary and River (Alcácer do Sal) being afterward analyzed by GC-MS. Data showed the ubiquitous presence of potentially hazardous amounts of estrogens (mainly of estrone and estradiol, reaching over 10 ng L−1), nonylphenol diethoxylate (up to 900 ng L−1) and nonylphenol (up to 230 ng L−1). It was concluded that there are conditions for the occurrence of endocrine disruption in local aquatic animals, even in areas included inside the Natural Reserve. Moreover, a clear seasonal pattern of fluctuation was found for phytoestrogens and sitosterol. Besides, there were high amounts of unionized ammonia (up to 0.6 mg L−1) and phosphates (up to 3.2 mg L−1) suggesting that the current status poses risks for local fauna and humans. [ABSTRACT FROM PUBLISHER]

An analytical method based on solid-phase extraction followed by gas chromatography mass spectrometry (GC-MS) was developed and validated for the quantification of 39 pesticides with distinct physico-chemical characteristics (including some degradates), in estuarine water samples. The method includes pre-concentration of samples (2500-fold factor), in OASIS HLB cartridges, by solid phase extraction. The analysed pesticides included organochlorines, organophosphorous, triazines, pyrethroids and other miscellaneous compounds, that are usually analysed separately. Method detection limits were between 3.6 and 61.2 ng L−1. The obtained sensitivity and accuracy, associated with the inherent confirmatory potential of GC-MS, validate the method as a toll in environmental monitoring. Analyses of water samples (n = 84) taken from the Douro River estuary, from March to May 2009, showed the presence of all assayed pesticides, among which 71.6% where above the limits of quantification. Besides, the feasibility of the method for chemical monitoring was highlighted by establishing a first pattern of spatial-temporal fluctuation for all classes of pesticides in the estuary, showing a significant increase of their concentrations in April and mainly at the margin bordering the city of Porto. Moreover, the finding of environmental hazardous amounts of hexachlorbenzene, endrin, endosulfan (all forms), DDTs, chlorfenvinfos Z and simazine pose a clear risk for local aquatic wildlife, and eventually to humans, by direct exposure or by consumption of contaminated fish. [ABSTRACT FROM AUTHOR]

The Leça River and two beaches close to the north and south margins of the estuary, nowadays an important seaport harbor, show signs of contamination. However, the chemical nature of that contamination is uncertain. Therefore, this study checked for the presence of 11 endocrine disrupting compounds (EDCs) of animal (17β-estradiol and estrone), pharmaceutical (17α-ethynylestradiol), and industrial (alkylphenols, alkylphenol ethoxylates, and bisphenol A) origins in water samples collected from six points at the river gradient and at two sites in the coastline (Atlantic Ocean). Along with the EDCs, evaluated by gas chromatography – mass spectroscopy (GC-MS) during spring, summer, and autumn, physico-chemical parameters were also assessed and particular attention was given to dissolved oxygen (DO) levels. Data showed the presence of high amounts of estrogens (up to 10 ng L−1 for estrone) and industrial compounds (up to 2 µg L−1 for the nonylphenol ethoxylates) at both river and seacoast. Along with this, clear signs of hypoxia were found in the river (DO < 3 mg L−1 at several sampling sites). Taking into account these issues, it was concluded that there are local conditions for the occurrence of endocrine disruption in aquatic animals. [ABSTRACT FROM PUBLISHER]

Previous studies in the Douro River estuary, based on occasional sampling, showed the presence of several estrogenic disrupting chemicals (EDCs). In sequence, we hypothesized that such type of pollution was more likely an enduring issue than an occasional phenomenon, and that it may even affect recreational beaches in each side of the estuarine mouth. Thus to conclude about the continuous influx of EDCs, water samples were taken twice a day, once per a week, from March to May of 2009, at four sites within the estuary and at two sites in the coastline. After solid-phase extraction, the extracts were prepared for GC-MS analysis of 11 reference EDCs. These embraced natural and pharmaceutical estrogens (17β-estradiol, estrone and 17α-ethynylestradiol) and xenoestrogenic industrial pollutants (4-octylphenol, 4-nonylphenol, and their mono and diethoxylates and bisphenol A). Data showed the ubiquitous presence of potentially hazardous amounts of natural estrogens (particularly of estradiol, ca 5.5 ng L−1) and persistent organic pollutants such as nonylphenol mono (up to 550 ng L−1) and diethoxylate (up to 2000 ng L−1). It was concluded that the targeted area is continuously polluted by the assayed EDCs, and as a consequence, conditions exist for endocrine disturbance in the biota by chronic exposure to EDCs. [ABSTRACT FROM PUBLISHER]