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Seven seawater polychlorinated biphenyls (PCBs) were measured in water (DAP), suspended particulate matter (SPM), and blue mussels (Mytilus edulis) collected from four beaches in northwest Portugal. PCBs were extracted using solid-phase-extraction, ultrasound-extraction and QuEChERS before GC-MS analysis. The two-year annual average concentrations of PCBs in DAP, SPM and the four-year analysis in mussels were ∼ 4.4 ng/L, ∼15.9 μg/kg, and ∼ 56.0 μg/kg. The results suggest higher concentrations of PCBs in summer for SPM and in spring for mussels, mainly those collected close to the Ave River estuary. The origins of PCBs remain uncertain. Risk assessment shows that PCBs in water are unlikely to harm local biota due to their low thyroid hormone toxicity equivalents (TEQ-TH; ∼1.4E-04 ng/L and ∼ 4.1E-04 μg/kg) and on WHO toxicity equivalents (TEQ-WHO; ∼2.1E-05 ng/L and ∼ 4.9E-05 μg/kg). However, the lifetime carcinogenic risk (LCR) for humans consuming local bivalves is concerning, as it exceeds 1.0E-06., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 S 1 (fire forests/sea harbor) are different from those at S 2 and S 3 (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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 (∑ 16 PAHs) 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.

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 NR1Jβ. 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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 (17β-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.

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.

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 (17β-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.

The Lima River is a Spanish–Portuguese water body. Notwithstanding the fact that the river incorporates protected natural areas, levels of endocrine-disrupting compounds (EDCs) within its waters have never been measured; such EDCs include the following: natural and pharmaceutical oestrogens (17β-estradiol, E1, and 17α-ethynylestradiol), industrial and household pollutants (4-octylphenol, 4-nonylphenol, and their monoethoxylates and diethoxylates, and bisphenol A), phytoestrogens (formononetin, biochanin A, daidzein, genistein), and phytosterols (namely, sitosterol). To obtain an understanding of levels of EDCs, water samples were taken from eight sampling sites along the river every 2 months during a 1-year period (2011). The water samples were preconcentrated (Oasis HLB cartridges), cleaned (silica cartridges), and analysed using gas chromatography. Results showed that levels of oestrogens and industrial and household pollutants were higher in summer than in other seasons. Although oestrogens were more abundant (approximately 40 ng/L) on the southern margin of the river, levels of other pollutants were higher (approximately 124 ng/L) in the north. Phytoestrogens and sitosterol showed clear seasonal fluctuations with higher amounts of formononetin (approximately 389 ng/L), biochanin A (approximately 160 ng/L), and sitosterol (≥5 µg/L) measured in summer. The overall oestrogenic load, expressed in ethynylestradiol equivalents, was 18 ng/L for oestrogens, 0.5 ng/L for industrial and household pollutants, and 13 ng/L for phytoestrogens. Water physicochemical parameters indicate anthropogenic pollution because Σnitrites,nitrates (>1 mg/L) and phosphates (approximately 0.4 mg/L) were high. The study showed that the waters of the Lima River are subject to impacts and that levels of EDCs pose risks to the river’s biota.

The Mondego River estuary demonstrates signs of pollution, but the levels of endocrine disrupting compounds (EDCs), such as the natural (17β-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.

A new and fully validated QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction and gas chromatography-tandem mass spectrometry methodology was developed and subsequently implemented for the quantification of 16 polycyclic aromatic hydrocarbons (PAHs) in wild (from Matosinhos Beach, Portugal) and commercial (from Ria de Arousa, Spain) mussels. The method proved to be robust, precise, and accurate, with recoveries ranging from 89.2 to 111.8 %. Total sums of 16 PAHs were 52.91 and 37.58 ng/g of wet weight for wild and commercial specimens, respectively. The three- to four-ring PAHs were the most abundant, and a mixture of petrogenic and pyrolytic sources were suspected to occur in both origin areas. Although the contamination levels were below the European regulated limits, specifically for commercial mussels (this despite wild specimens are also consumed), care should be taken in terms of human health, since we are still not aware of the low-dose versus long-term effects, even more in high-risk population groups.

A newly analytical method based on QuEChERS extraction followed by gas chromatography with mass spectrometry (GC-MS) analysis was developed and validated for the quantification of 18 PCBs in wild (from Matosinhos Beach, Portugal) and cultivated (from Ria de Arousa, Spain) mussel samples, pooled by sex. Wild animals showed higher PCB levels than cultivated mussels, with males from both origins, presenting an upper contamination profile comparing with females. This fact seems to be correlated with few biometric parameters, but other interdependencies, not addressed herein, such as distinct lipid contents between sexes, as a consequence of the gametogenic stage, may also explain this data. Overall, data reiterate the importance of investigating the presence of PCBs in marine biological samples, which can act both as bioindicators of environmental contamination, either as food quality controls for human health.

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 (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, 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.

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,O-bis(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.

Qualitative and quantitative approaches were tested to assess zebrafish liver effects after sub-acute exposures of certain pharmaceuticals. Carbamazepine, fenofibric acid, propranolol, sulfamethoxazole and trimethoprim were tested individually and in mixtures, including low environmental levels. Overall, data showed sex specific reactions in liver, with the major alterations being observed in males. Males treated with propranolol, fenofibric acid and with mixtures, showed an increase of vitellogenin immunostaining, compared with the control. Males also evidenced a tendency for an increased hepatic mass, after individual and mixture exposures. The volume-weighted nuclear volume of hepatocytes was high in males after exposures to either mixture, which together with the greater cytoplasmic eosinophilia and changes in cytochrome P450 1A immunoreactivity, point to an increase in metabolic/detoxification activity. These investigations revealed distinct impacts depending on the exposure type, and strengthened the importance of studying non-steroidal compounds in mixtures, including environmental levels and both sexes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Concerns associated with pharmaceuticals in aquatic systems demand the establishment of links between xenobiotics and their respective concentrations and impacts on aquatic organisms. Herein, effects of non-steroidal pharmaceuticals in the gonadal maturation of zebrafish (Danio rerio) were evaluated by histopathological and stereological analyses after 21 days of exposure. Carbamazepine, fenofibric acid, propranolol, sulfamethoxazole and trimethoprim were selected, considering their detection in the Douro estuary (Portugal). Exposures were performed with single compounds and mixtures, the exposure concentrations including environmental levels. Overall, quantitative analyses showed a decreasing trend for late maturation stages in male and female gametogenesis with parallel increases in immature gametes. In females, and at the highest concentration mixture, a significant switch between the volume densities of late/mature oocytes versus primary oocytes was observed. On the verge of statistical significance, oocyte atresia was higher in both mixtures (5.75 ± 4.02% for MXA and 5.65 ± 5.27% for MXB) versus control (2.21 ± 1.88%), in accordance with the histological identification of large atretic areas in some fish. Unlike females, males showed significant effects with single exposures. Spermatozoa in controls totalled 53.25 ± 7.13% of the testis volume, decreasing with carbamazepine (47.19 ± 5.30%), fenofibric acid (46.36 ± 4.30%), propranolol (37.22 ± 2.38%) and sulfametoxazole (39.37 ± 5.15%). An increase in spermatocyte percentage was noted with propranolol (40.13 ± 7.36%) and sulfametoxazole (40.84 ± 1.66%) versus control (30.93 ± 6.53%). The changes in maturation dynamics did not impact the gonadosomatic index. The results show that pharmaceuticals from various therapeutic classes can disrupt the maturation dynamics of fish ovaries and testes. Further studies are justified to tackle the underlying mechanisms and to gauge the full extent of effects/risks., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Fish embryos are a particularly vulnerable stage of development, so they represent optimal targets for screening toxicological effects of waterborne xenobiotics. Herein, the toxicity potential of two mixtures of pharmaceuticals was evaluated using a zebrafish embryo test. One of the mixtures corresponds to an environmentally realistic scenario and both have carbamazepine, fenofibric acid, propranolol, trimethoprim and sulfamethoxazole. The results evidenced morphological alterations, such as spinal deformities and yolk-sac oedemas. Moreover, heart rates decreased after both mixture exposures, e.g., at 48hpf, highest mixture versus blank control (47.8±4.9 and 55.8±3.7 beats/30s, respectively). The tail lengths also diminished significantly from 3208±145μm in blank control to 3130±126μm in highest mixture. The toxicological effects were concentration dependent. Mortality, hatching rate and the number of spontaneous movements were not affected. However, the low levels of pharmaceuticals did interfere with the normal development of zebrafish, which indicates risks for wild organisms., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The amount and distribution of six pharmaceutical compounds belonging to distinct therapeutic classes were investigated along the navigation channel of the Douro River estuary. Distinct spatial and temporal trends were considered and a total of 87 water samples were pre-concentrated by solid-phase extraction (SPE) and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with an ion trap (IT) analyzer and electrospray ionization (ESI). The maximum concentrations found were 178ng/L for carbamazepine, 3.65ng/L for diazepam, 70.3ng/L for fenofibric acid, 3.18ng/L for propranolol, 15.7ng/L for trimethoprim and 53.3ng/L for sulfamethoxazole. Carbamazepine was the most ubiquitous compound with 100% positive detection frequency followed by propranolol (38%), trimethoprim (34%) and sulfamethoxazole (33%). The pharmaceutical compounds were quantified at higher levels in the lower stretch of the estuary, especially near the wastewater treatment plant (WWTP). The data proves that pollution of the Douro River estuary by pharmaceuticals is consistent and is occurring in a fairly constant manner in time, covering a wide area and displaying hot-spots. Individually, the concentration levels are not likely to cause acute effects, based on reference experimental data. However, the fact that complex mixtures exist gives cause for concern as regards potentially relevant toxicological risks. The study points out the need for continuous monitoring of contamination levels not only in the Douro River estuary but also in other major estuaries. Finally, the scenario supports the need for experimental studies on toxicological impacts on aquatic organisms at environmentally relevant concentrations., (Copyright © 2010 Elsevier B.V. All rights reserved.)

This paper describes the development and validation of a simple analytical method using solid-phase extraction followed by a high-performance liquid chromatography with diode array detection (HPLC-DAD) analysis. Target compounds included six pharmaceuticals (carbamazepine, diazepam, fluoxetine, propranolol, sulfamethoxazole, and trimethoprim) and the active metabolite of fenofibrate (fenofibric acid). Briefly, this method consisted of the preconcentration of water samples (2 L) on 500 mg Oasis HLB cartridges and HPLC analysis using a RP-(18) analytical column in a gradient mode with a flow rate of 1 mL/min. The validation parameters revealed that this method was highly specific for all assayed compounds (> 99%), and the linearity of the calibration curves always showed a correlation higher than 0.99. The detection limits were in the ng/L level, and recovery rates were > 70% for most of the target compounds. Analysis of samples from polluted areas of the Douro River estuary indicated that propranolol and carbamazepine are present in concentrations ranging from 22.0 to 54.0 ng/L and 21.3 to 32.7 ng/L, respectively. Thus, it is concluded that this method can be successfully applied for screening pharmaceuticals in polluted estuarine areas.

The important Portuguese Sado River estuary has never been investigated for the presence of potentially endocrine-disrupting chemicals (EDCs), such as natural estrogens (estradiol, estrone), pharmaceutical estrogens (17alpha-ethynylestradiol), phytoestrogens (daidzein, genistein and biochanin A), or industrial chemicals (4-octylphenol, 4-nonylphenol, and bisphenol A). Thus, the main objective of this study was to evaluate their presence at 13 sampling points distributed between both the industrial and the natural reserve areas of the estuary, zones 1 and 2, respectively. For that, water samples collected in summer and winter were processed by solid phase extraction and analyzed by high-performance liquid chromatography with photodiode array detection and gas chromatography-mass spectroscopy. Results showed that estrone, ethynylestradiol, all the aforementioned phytoestrogens as well as bisphenol A and 4-octylphenol were found in zone 1. In zone 2, neither estrogens nor 4-OP were found. However, in the same zone, daidzein (500 ng/L) and genistein (320 ng/L) attained their highest levels in summer, whereas biochanin A peaked in winter (170 ng/L). Furthermore, bisphenol A was also found in some areas of zone 2, but showed similar concentrations in both surveys (about 220 ng/L). This study demonstrated that the Sado River estuary had low EDCs levels, suggesting that the Sado's high hydrodynamic activity may be involved in the dilution of local pollution. It was suggested that at the current levels of concentrations, all assayed EDCs are unlikely to individually cause endocrine disruption in local animals. However, under a continuous exposure scenario, an additive and/or synergistic action of the estrogenic chemicals load can not be excluded, and so, continuous monitoring is advisable.

An analytical method based on solid-phase extraction followed by liquid chromatography tandem mass spectrometry with an ion trap analyser was developed and validated for the quantification of a series of pharmaceutical compounds with distinct physical-chemical characteristics in estuarine water samples. Method detection limits were between 0.03 and 16.4 ng/L. The sensitivity and the accuracy obtained associated with the inherent confirmatory potential of ion trap tandem mass spectrometry (IT-MS/MS) validates its success as an environmental analysis tool. Two MS/MS transitions were used to confirm compound identity. Almost all pharmaceuticals were detected at ng/L level in at least one sampling site of the Douro River estuary, Portugal.

Recent studies in the Mondego River estuary demonstrated signs of pollution in the area, but the nature of the contamination remains unexplored. Because there seems to be any studies in that zone checking for the simultaneous presence of xenoestrogenic endocrine disrupting compounds, either of animal (estradiol and estrone), vegetal (daidzein, genistein and biochanin A), pharmaceutical (17alpha-ethynylestradiol) or industrial (bisphenol A, 4-octylphenol, 4-nonylphenol) origins, the main objective of this study was to investigate their presence, in every year season and at eight points along the estuarine gradient. For this propose, water samples (2 L) were collected in high and low tides, preconcentrated in the Oasis HLB cartridges and cleaned in silica cartridges before their analysis by HPLC-DAD and GC-MS. The current data showed the absence of contamination by animal or pharmaceutical estrogens and by alkylphenols, but demonstrated the presence of high levels of phytoestrogens (up to 1.1 mug/L) and of bisphenol A (up to 880.0 ng/L). Because these two chemicals existed in high environmental concentrations in the most upstream sampling station, and taking in account that these pollutants may additively contribute to endocrine disruption, namely on fish, it was concluded that at least this particularly area of the Mondego estuary deserves continuous monitoring programmes.

Recent studies in the Douro River estuary show signs of pollution in the area and of fish endocrine disruption. However, the chemical nature of the local contamination has not been fully investigated nor have studies checking for the simultaneous presence of endocrine-disrupting chemicals (EDCs), either of animal (estrone, E1; estradiol, E2), pharmaceutical (17alpha-ethynylestradiol, EE2), vegetal (daidzein, DAID; genistein, GEN; biochanin A, BIO-A), or industrial (bisphenol A, BPA; 4-octylphenol, 4-OP; 4-nonylphenol, 4-NP) origins. Thus, the main objective of this study was to examine the presence of these EDCs in estuarine water samples collected, in every season of the year, at nine sampling stations along the estuarine gradient. All samples were processed by two-step solid-phase extraction (Oasis HLB followed by silica) prior to high-performance liquid chromatography with diode-array detection (HPLC-DAD) and gas chromatography-mass spectrometry (GC-MS) analyses. The current data showed that E1 and EE2, all phytoestrogens, and BPA were identified and measured in this estuary. In contrast, 4-OP was only detected by GC-MS and E2 and 4-NP were not found. Additionally, E1 (up to 112.9 ng/L) and EE2 (up to 101.9 ng/L) were both measured in biologically hazardous amounts in winter. In the year sampled, the phytoestrogens suggested a possible seasonal pattern of fluctuation. Both DAID (up to 888.4 ng/L) and GEN (183.6 ng/L) were maximal in early summer, whereas BIO-A (up to 191.4 ng/L) reached its highest concentrations in winter. BPA (up to 10.7 microg/L) also attained highest levels in winter. In December 2005, it is hypothesized that E1, EE2, and BPA concentrations were atypically high due to current drought conditions. Almost all assayed EDCs existed in all seasons and, therefore, might have contributed to endocrine disruption of aquatic animals, previously documented by the high rate of ovotestis in fish caught in this estuary.