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

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

Liver and Plasma Fatty Acid Characterization in Cultured Brown Trout at Distinct Reproductive Stages.

Fatty acids are energy sources, and their profiles are used as biomarkers of metabolic status and physiological changes in fish. Within this context, the main aim of this study was to identify the fatty acids that best discriminate the reproductive status of male and female farmed brown trout. The fatty acid composition in liver and plasma samples from the adults of both sexes was monitored along four distinct reproductive stages, namely the spawning capable (December), regressing (March), regenerating (July), and developing (November) stages. Irrespective of the sex and stage, the most representative fatty acids were palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1 n-9), arachidonic acid (20:4 n-6), eicosapentaenoic acid (EPA, 20:5 n-3), and docosahexaenoic acid (DHA, 22:6 n-3). There were no significant sex differences in fatty acid classes in the liver and plasma. Despite this, there were several changes in individual fatty acid levels between the sexes. In the liver, both males and females showed high monounsaturated fatty acid and low polyunsaturated fatty acid (PUFA) levels during the regressing and regenerating stages. At spawning capable and developing stages, a reverse profile was noted. The plasma profiles were mainly influenced by changes in saturated fatty acids and PUFAs in males and by PUFA in females. Based on the most representative fatty acids, four patterns were established for female plasma samples, one for each reproductive stage. This scenario suggests that female plasma samples are promising for the discrimination of gonadal reproductive status, and this potential can be further explored in aquaculture and environmental monitoring studies.

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

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

Silencing of PPARαBb mRNA in brown trout primary hepatocytes: effects on molecular and morphological targets under the influence of an estrogen and a PPARα agonist.

The crosstalk between peroxisome proliferator-activated receptor α (PPARα) and estrogenic pathways are shared from fish to humans. Salmonid fish had an additional genome duplication, and two PPARα isoforms (PPARαBa and PPARαBb) were previously identified. Since a negative regulation between estrogen signaling and PPARα was described, a post-transcriptional gene silencing for PPARαBb was designed in primary brown trout hepatocytes. The aims of the study were to: (i) decipher the effects of PPARαBb knock-down on peroxisome morphology and on mRNA expression of potential target genes, and (ii) to assess the cross-interferences caused by an estrogenic compound (17α-ethinylestradiol - EE2) and a PPARα agonist (Wy-14,643 - Wy) using the established knock-down model. A knock-down efficiency of 70% was achieved for PPARαBb and its silencing significantly reduced the volume density of peroxisomes, but did not alter mRNA levels of the studied genes. Exposure to Wy did not change peroxisome morphology or mRNA expression, but under silencing conditions Wy rescued the volume density of peroxisomes to control levels, and increased acyl-coenzyme A oxidase 1-3l (Acox1-3l) mRNA. Exposure to EE2 caused a reduction of peroxisome volume density, but under silencing conditions this effect was abolished and ApoA1 mRNA level was diminished. The morphological alterations of peroxisomes by WY and EE2 demonstrated that obtained results are PPARαBb dependent, and suggest the regulation of unknown downstream targets of PPARαBb. In summary, PPARαBb is involved in the control of peroxisome size and/or number, which opens future opportunities to explore its regulation and molecular targets.

Sex-steroids and hypolipidemic chemicals impacts on brown trout lipid and peroxisome signaling - Molecular, biochemical and morphological insights.

Lipid metabolism involves complex pathways, which are regulated in a similar way across vertebrates. Hormonal and hypolipidemic deregulations cause lipid imbalance from fish to humans, but the underlying mechanisms are far from understood. This study explores the potential of using juvenile brown trout to evaluate the in vivo interferences caused by estrogenic (17α-ethinylestradiol - EE2), androgenic (testosterone - T), and hypolipidemic (clofibrate - CLF) compounds in lipidic and/or peroxisomal pathways. Studied endpoints were from blood/plasma biochemistry, plasma fatty acid profile, ultrastructure of hepatocytes and abundance of their peroxisomes to mRNA expression in the liver. Both T and CLF caused minimal effects when compared to EE2. Estrogenized fish had significantly higher hepatosomatic indexes, increased triglycerides and very-low density lipoproteins (VLDL) in plasma, compared with solvent control. Morphologically, EE2 fish showed increased lipid droplets in hepatocytes, and EE2 and T reduced volume density of peroxisomes in relation to the hepatic parenchyma. Polyunsaturated fatty acids (PUFA) in plasma, namely n-3 PUFA, increased with EE2. EE2 animals had increased mRNA levels of vitellogenin A (VtgA), estrogen receptor alpha (ERα), peroxisome proliferator-activated receptor alpha (PPARα), PPARαBa and acyl-CoA long chain synthetase 1 (Acsl1), while ERß-1, acyl-CoA oxidase 1-3I (Acox1-3I), Acox3, PPARγ, catalase (Cat), urate oxidase (Uox), fatty acid binding protein 1 (Fabp1) and apolipoprotein AI (ApoAI) were down-regulated. In summary, in vivo EE2 exposure altered lipid metabolism and peroxisome dynamics in brown trout, namely by changing the mRNA levels of several genes. Our model can be used to study possible organism-level impacts, viz. in gonadogenesis.

Genome specific PPARαB duplicates in salmonids and insights into estrogenic regulation in brown trout.

Peroxisome proliferator-activated receptors (PPARs) are key regulators of many processes in vertebrates, such as carbohydrate and lipid metabolism. PPARα, a member of the PPAR nuclear receptor gene subfamily (NR1C1), is involved in fatty acid metabolism, namely in peroxisomal ß-oxidation. Two gene paralogues, pparαA and pparαB, were described in several teleost species with their origin dating back to the teleost-specific genome duplication (3R). Given the additional salmonid-specific genome duplication (4R), four genes could be theoretically anticipated for this gene subfamily. In this work, we examined the pparα gene repertoire in brown trout, Salmo trutta f. fario. Data disclosed two pparα-like sequences in brown trout. Phylogenetic analyses further revealed that the isolated genes are most likely genome pparαB duplicates, pparαBa and pparαBb, while pparαA is apparently absent in salmonids. Both genes showed a ubiquitous mRNA expression across a panel of 11 different organs. In vitro exposed primary brown trout hepatocytes strongly suggest that pparα gene paralogues are differently regulated by ethinylestradiol (EE2). PparαBb mRNA expression significantly decreased with dosage, reaching significance after exposure to 50µM EE2, while pparαBa mRNA increased, significant at 1µM EE2. The present data enhances the understanding of pparα function and evolution in teleost, and reinforces the evidence of a potential crosstalk between estrogenic and pparα signaling pathways.

Cross-interference of two model peroxisome proliferators in peroxisomal and estrogenic pathways in brown trout hepatocytes.

Peroxisome proliferators cause species-specific effects, which seem to be primarily transduced by peroxisome proliferator-activated receptor alpha (PPARα). Interestingly, PPARα has a close interrelationship with estrogenic signaling, and this latter has already been promptly activated in brown trout primary hepatocytes. Thus, and further exploring this model, we assess here the reactivity of two PPARα agonists in direct peroxisomal routes and, in parallel the cross-interferences in estrogen receptor (ER) mediated paths. To achieve these goals, three independent in vitro studies were performed using single exposures to clofibrate - CLF (50, 500 and 1000µM), Wy-14,643 - Wy (50 and 150µM), GW6471 - GW (1 and 10µM), and mixtures, including PPARα agonist or antagonist plus an ER agonist or antagonist. Endpoints included gene expression analysis of peroxisome/lipidic related genes (encoding apolipoprotein AI - ApoAI, fatty acid binding protein 1 - Fabp1, catalase - Cat, 17 beta-hydroxysteroid dehydrogenase 4 - 17ß-HSD4, peroxin 11 alpha - Pex11α, PPARαBb, PPARαBa and urate oxidase - Uox) and those encoding estrogenic targets (ERα, ERß-1 and vitellogenin A - VtgA). A quantitative morphological approach by using a pre-validated catalase immunofluorescence technique allowed checking possible changes in peroxisomes. Our results show a low responsiveness of trout hepatocytes to model PPARα agonists in direct target receptor pathways. Additionally, we unveiled interferences in estrogenic signaling caused by Wy, leading to an up-regulation VtgA and ERα at 150µM; these effects seem counteracted with a co-exposure to an ER antagonist. The present data stress the potential of this in vitro model for further exploring the physiological/toxicological implications related with this nuclear receptor cross-regulation.

Peroxisome proliferator-activated receptor gamma (PPARγ) in brown trout: Interference of estrogenic and androgenic inputs in primary hepatocytes.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a pivotal regulator of lipid and glucose metabolism in vertebrates. Here, we isolated and characterized for the first time the PPARγ gene from brown trout (Salmo trutta f. fario). Hormones have been reported to interfere with the regulatory function of PPARγ in various organisms, albeit with little focus on fish. Thus, primary hepatocytes isolated from juveniles of brown trout were exposed to 1, 10 and 50µM of ethinylestradiol (EE2) or testosterone (T). A significant (3 fold) decrease was obtained in response to 50µM of EE2 and to 10 and 50µM of T (13 and 14 folds), while a 3 fold increase was observed at 1µM of EE2. Therefore, trout PPARγ seems a target for natural/synthetic compounds with estrogenic or androgenic properties and so, we advocate considering PPARγ as another alert sensor gene when assessing the effects of sex-steroid endocrine disruptors.

Acyl-coenzyme A oxidases 1 and 3 in brown trout (Salmo trutta f. fario): Can peroxisomal fatty acid ß-oxidation be regulated by estrogen signaling?

Acyl-coenzyme A oxidases 1 (Acox1) and 3 (Acox3) are key enzymes in the regulation of lipid homeostasis. Endogenous and exogenous factors can disrupt their normal expression/activity. This study presents for the first time the isolation and characterization of Acox1 and Acox3 in brown trout (Salmo trutta f. fario). Additionally, as previous data point to the existence of a cross-talk between two nuclear receptors, namely peroxisome proliferator-activated receptors and estrogen receptors, it was here evaluated after in vitro exposures of trout hepatocytes the interference caused by ethynylestradiol in the mRNA levels of an inducible (by peroxisome proliferators) and a non-inducible oxidase. The isolated Acox1 and Acox3 show high levels of sequence conservation compared to those of other teleosts. Additionally, it was found that Acox1 has two alternative splicing isoforms, corresponding to 3I and 3II isoforms of exon 3 splicing variants. Both isoforms display tissue specificity, with Acox1-3II presenting a more ubiquitous expression in comparison with Acox1-3I. Acox3 was expressed in almost all brown trout tissues. According to real-time PCR data, the highest estrogenic stimulus was able to cause a down-regulation of Acox1 and an up-regulation of Acox3. So, for Acox1 we found a negative association between an estrogenic input and a directly activated PPARα target gene. In conclusion, changes in hormonal estrogenic stimulus may impact the mobilization of hepatic lipids to the gonads, with ultimate consequences in reproduction. Further studies using in vivo assays will be fundamental to clarify these issues.

Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors.

Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol-EE2) at 1, 10 and 50µM; (2) an ER antagonist (ICI 182,780) at 10 and 50µM; and (3) mixtures of both (Mix I-10µM EE2 and 50µM ICI; Mix II-1µM EE2 and 10µM ICI and Mix III-1µM EE2 and 50µM ICI). The mRNA levels of the estrogenic targets (ERα, ERß-1 and vitellogenin A-VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase-Uox, 17ß-hydroxysteroid dehydrogenase 4-17ß-HSD4, peroxin 11α-Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50µM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα mRNA increased after all EE2 treatments, while ERß-1 had an inverse pattern. The EE2 action was reversed by ICI 182,780 in a concentration-dependent manner, for VtgA, ERα and Uox. Overall, our data show the great value of primary brown trout hepatocytes to study the effects of estrogenic/anti-estrogenic inputs in peroxisome kinetics and in ER and PPARα signaling, backing the still open hypothesis of crosstalk interactions between these pathways and calling for more mechanistic experiments.

Estimation of volume densities of hepatocytic peroxisomes in a model fish: catalase conventional immunofluorescence versus cytochemistry for electron microscopy.

Accurately accessing changes in the intracellular volumes (or numbers) of peroxisomes within a cell can be a lengthy task, because unbiased estimations can be made only by studies conducted under transmission electron microscopy. Yet, such information is often required, namely for correlations with functional data. The optimization and applicability of a fast and new technical proceeding based on catalase immunofluorescence was implemented herein by using primary hepatocytes from brown trout (Salmo trutta f. fario), exposed during 96 h to two distinct treatments (0.1% ethanol and 50 µM of 17α-ethynylestradiol). The time and cost efficiency, together with the results obtained by stereological analyses, specifically directed to the volume densities of peroxisomes, and additionally of the nucleus in relation to the hepatocyte, were compared with the well-established 3,3'-diaminobenzidine cytochemistry for electron microscopy. With the immuno technique it was possible to correctly distinguish punctate peroxisomal profiles, allowing the selection of the marked organelles for quantification. By both methodologies, a significant reduction in the volume density of the peroxisome within the hepatocyte was obtained after an estrogenic input. The most interesting point here was that the volume density ratios were quite correlated between both techniques. Overall, the immunofluorescence protocol for catalase was evidently faster, cheaper and provided reliable quantitative data that discriminated in the same way the compared groups. After this validation study, we recommend the use of catalase immunofluorescence as the first option for rapid screening of changes of the amount of hepatocytic peroxisomes, using their volume density as an indicator.

A step forward using QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) based extraction and gas chromatography-tandem mass spectrometry-levels of priority polycyclic aromatic hydrocarbons in wild and commercial mussels.

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.

Contamination levels of polychlorinated biphenyls in wild versus cultivated samples of female and male mussels (Mytilus sp.) from the Northwest Coast of Iberian Peninsula--new application for QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methodology.

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.

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.

The toxicity potential of pharmaceuticals found in the Douro River estuary (Portugal)--experimental assessment using a zebrafish embryo test.

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.

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.

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.

A column-switching method for quantification of the enantiomers of omeprazole in native matrices of waste and estuarine water samples.

This work reports the use of a two-dimensional liquid chromatography (2D-LC) system for quantification of the enantiomers of omeprazole in distinct native aqueous matrices. An octyl restricted-access media bovine serum albumin column (RAM-BSA C(8)) was used in the first dimension, while a polysaccharide-based chiral column was used in the second dimension with either ultraviolet (UV-vis) or ion-trap tandem mass spectrometry (IT-MS/MS) detection. An in-line configuration was employed to assess the exclusion capacity of the RAM-BSA columns to humic substances. The excluded macromolecules had a molecular mass in the order of 18 kDa. Good selectivity, extraction efficiency, accuracy, and precision were achieved employing a very small amount (500 microL or 1.00 mL) of native water sample per injection, with detection limits of 5.00 microg L(-1), using UV-vis, and 0.0250 microg L(-1), using IT-MS/MS. The total analysis time was only 35 min, with no time spent on sample preparation. The methods were successfully applied to analyze a series of waste and estuarine water samples. The enantiomers were detected in an estuarine water sample collected from the Douro River estuary (Portugal) and in an influent sample from the wastewater treatment plant (WWTP) of São Carlos (Brazil). As far as we are concerned, this is the first report of the occurrence of (+)-omeprazole and (-)-omeprazole in native aqueous matrices.

Development and optimization of a HPLC-DAD method for the determination of diverse pharmaceuticals in estuarine surface waters.

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.

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.