Maria João Rocha

Spatiotemporal distribution of pharmaceuticals in the Douro River estuary (Portugal)

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.

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.

Pharmaceutical trace analysis in aqueous environmental matrices by liquid chromatography–ion trap tandem mass spectrometry

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.

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.

Development and Validation of a HPLC‐DAD Method for Determination of Several Endocrine Disrupting Compounds in Estuarine Water

Abstract This paper presents the development and validation of an analytical procedure, which allows the simultaneous quantification of nine endocrine disrupters (EDCs) in polluted surface waters. The compounds selected for this study were the natural hormones (17β‐estradiol and estrone), the synthetic hormone (17α‐ethynylestradiol), the bisphenol A, the alkylphenols (4‐octylphenol and 4‐nonylphenol), and the phytoestrogens (daidzein, genistein, and biochanin A). Briefly, this method consisted of the preconcentration of water samples (2 L) in 500 mg Oasis HLB cartridges, followed by a cleanup step in 1 g silica cartridges, and analysis of all EDCs by High Performance Liquid Chromatography with Diode Array Detection (HPLC‐DAD). The chromatographic separation occurred in a RP‐18 analytical column in a gradient mode of CH3CN:H2O (pH 2) at a flow rate of 1 mL/min. The validation parameters revealed that this method was highly specific for all assayed EDCs (>99%), the linearity of the calibration curves, obtained either by external patronization or by fortified matrix calibration techniques, always showed a correlation higher than 0.99 and their intermediate precision and repeatability, evaluated in terms of intra and inter assays, were optimal for all EDCs (RSD<3%). The precision and accuracy, estimated as the recovery rates that occurred during the preconcentration and the cleanup processes showed, for all EDCs, excellent results (RSD<3% and recoveries up to 116%), and preconcentration factors (enrichment) up to 10000. The limits of detection (LOD) and quantification (LOQ) were mathematically estimated from the calibration curves. As expected, these values were different for each of the nine assayed EDCs and were higher when calculated from the addition standard calibration curves. Here, 4‐octylphenol was the compound with the lowest LOD and LOQ (3.8 ng/L and 12.0 ng/L) and 17α‐ethynylestradiol the one with the highest levels for both cited parameters (18.0 ng/L and 54.5 ng/L). Finally, the performance of the method was checked with water samples from a highly polluted area in the Douro river estuary (Portugal). The field results revealed high levels of estrogens (up to 176 ng/L), bisphenol A (up to 5.1 µg/L), alkylphenols (up to 449 ng/L), and phytoestrogens (up to 74 ng/L), in accordance with the high local pollution load, and suggested that this method can be easily and successfully used in water monitoring surveys of, at least, highly polluted estuarine areas.

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.