Catarina Cruzeiro

The toxicity potential of pharmaceuticals found in the Douro River estuary (Portugal): Evaluation of impacts on fish liver, by histopathology, stereology, vitellogenin and CYP1A immunohistochemistry, after sub-acute exposures of the zebrafish model

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.

Testing the effects of ethinylestradiol and of an environmentally relevant mixture of xenoestrogens as found in the Douro River (Portugal) on the maturation of fish gonads—A stereological study using the zebrafish (Danio rerio) as model

In natural environments fish populations are exposed to many potential xenoestrogens, whereby understanding the impacts of mixtures continue to be of great interest. The main objective of this study was, therefore, to understand whether and how an environmentally relevant mixture of xenoestrogens found in the Douro River estuary can disrupt the normal gametogenesis in fish. For this purpose, adult zebrafish of both sexes were exposed for 21 days to an environmental mixture (MIX) of 11 xenoestrogens from diverse sources. A 100 ng/L ethinylestradiol (EE2) positive control was added. A quantitative (stereological) analysis with systematic sampling was made in the gonads, and using light microscopy both the relative and the absolute volumes of the gametogenic stages were estimated. Data point that the EE2 stimulus induced changes in structural compartments; with decreasing trends for the advanced maturation stages both in males and females. There was also a trend for a greater amount of interstitial tissue in males. Along with an interstitial fibrosis increase detected, the presence of a proteinaceous fluid was observed in both sexes and experimental groups (EE2 and MIX). Other histopathologic alterations were observed in the EE2 female group, such as the presence of foci of granulomatous inflammation and follicular mineralization in the germinal parenchyma and luminal areas. The most interesting finding of this study was that the exposure to the MIX caused a decrease of the relative volume of spermatozoa in zebrafish. This kind of estrogenic effect has not earlier been structurally quantified in such a fine detail with unbiased stereology in fish gonads. Despite the ultimate consequences of such disruptions being unknown, it could be logically argued that reduction or slowing-down of the appearance of the most mature cohorts and/or eventual interstitial fibrosis and other pathologic changes can adversely affect breeding. The findings add further explanatory bases for understanding the negative impacts of xenoestrogens.

The toxicity potential of pharmaceuticals found in the Douro River estuary (Portugal): Assessing impacts on gonadal maturation with a histopathological and stereological study of zebrafish ovary and testis after sub-acute exposures

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.

Quantification of 17 endocrine disruptor compounds and their spatial and seasonal distribution in the Iberian Ave River and its coastline

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.

Development and validation of a GC-MS method for determination of 39 common pesticides in estuarine water – targeting hazardous amounts in the Douro River estuary

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.

Determination of Polycyclic Aromatic Hydrocarbons in Coastal Sediments from the Porto Region (Portugal) by Microwave-Assisted Extraction, Followed by SPME and GC-MS

A simple low-cost, analytical method based on microwave-assisted extraction of sediments, followed by solid phase micro-extraction and gas chromatography mass spectrometry, was developed and validated for the quantification of sixteen polycyclic aromatic hydrocarbons (PAHs) in marine and estuarine sediment samples. The PAHs were those included in the United States Environmental Protection Agency (US EPA) priority list. Method detection limits were between 0.07 and 0.76 µg/kg dry weight (dw), which makes the current method suitable for environmental analysis. Sediments screened for PAHs from the Douro River estuary and the Porto seacoast exhibit total concentrations that ranged from 58.98 to 156.45 µg/kg dw, and from 51.98 to 54.79 µg/kg dw, respectively. The presence of almost all human carcinogenic PAHs in the analyzed areas indicate that these sediments can be considered polluted, suggesting that future monitoring programs together with an effective coastal management program must be implemented to guarantee the safe usage of the current areas for fishing and bathing.

Endocrine disruptors in the Leça River and nearby Porto Coast (NW Portugal): presence of estrogenic compounds and hypoxic conditions

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.

Occurrence of endocrine disruptor compounds in the estuary of the Iberian Douro River and nearby Porto Coast (NW Portugal)

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.

Development and validation of a GC–MS method for the evaluation of 17 endocrine disruptor compounds, including phytoestrogens and sitosterol, in coastal waters – their spatial and seasonal levels in Porto costal region (Portugal)

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.

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.