Occurrence of venlafaxine residues and its metabolites in marine mussels at trace levels: development of analytical method and a monitoring program.

Coastal areas are subject to growing pressures and impacts because of the increase in human activities. Lipophilic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs) or polychlorinated biphenyls (PCBs), have been monitored for decades within monitoring programs. However, until now, little information on the detection of so-called "emerging contaminants" such as hydrophilic organic compounds in the marine environment and no data on its metabolites or transformation products in marine organisms is available. In this report, a sensitive analytical methodology for identification and confirmation of venlafaxine (VEN) residues and five of its main metabolites in the marine mussels Mytilus galloprovincialis was validated. The sample preparation procedure was based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) approach. An analytical method was developed to quantify these compounds at trace levels by liquid chromatography coupled to high-resolution mass spectrometry. The method was then applied to marine mussels collected from the Mediterranean Sea in southeastern France. Residues of the antidepressant VEN were occasionally detected at ng/g dw level. In addition, the approach allowed us to identify several transformation products in the analyzed samples. N-desmethylvenlafaxine (NDV) was the most frequently detected metabolite followed by N,O-di-desmethylvenlafaxine (NODDV).

Estrogen-like and dioxin-like organic contaminants in reclaimed wastewater: transfer to irrigated soil and groundwater.

In semi-arid regions, treated wastewater reuse for irrigation is a common practice since wastewater is considered as a non negligible water resource in these areas. However, treated wastewater contains traces of organic compounds which may contaminate the receiving environment i.e. soil and groundwater. Some of these organic compounds have the ability to bind to estrogen receptor (ER) or dioxin receptor (AhR, aryl hydrocarbon receptor). The fate of these compounds in effluent reused for irrigation, irrigated soils and groundwater is not well addressed yet. In the present study, estrogenic and dioxin-like activities were studied in three media: i) effluents reused for irrigation, ii) soils samples collected from the reclaimed water irrigated plot during six month irrigation, and iii) groundwater sampled before and after irrigation periods. Effluents reused for irrigation exhibited ER and AhR activities at 38.5 +/- 9.9 ng estradiol-equivalent/L (ng E2-EQ/L) and 113.3 +/- 27.7 ng dioxin-equivalent/L (ng TCDD-EQ/L), respectively. Soils showed ER activity (0.05 ng E2-EQ/g) only after 4 months of irrigation. AhR activities detected in all soil samples have not changed during irrigation. In groundwater, ER activities were detected in two piezometers indicating transfer of some estrogenic compounds.

Elucidating venlafaxine metabolism in the Mediterranean mussel (Mytilus galloprovincialis) through combined targeted and non-targeted approaches.

Exposure of aquatic organisms to antidepressants is currently well documented, while little information is available on how wild organisms cope with exposure to these pharmaceutical products. Studies on antidepressant metabolism in exposed organisms could generate information on their detoxification pathways and pharmacokinetics. The goal of this study was to enhance knowledge on the metabolism of venlafaxine (VEN)-an antidepressant frequently found in aquatic ecosystems-in Mytilus galloprovincialis, a bivalve that is present worldwide. An original tissue extraction technique based on the cationic properties of VEN was developed for further analysis of VEN and its metabolites using targeted and non-targeted approaches. This extraction method was assessed in terms of recovery and matrix effects for VEN metabolites. Commercial analytical standards were applied to characterize metabolites found in mussels exposed to 10 µg/L VEN for 3 and 7 days. Targeted and non-targeted approaches using liquid chromatography (LC) combined with high-resolution mass spectrometry (HRMS) were implemented to screen for expected metabolites based on the literature on aquatic species, and for metabolites not previously documented. Four venlafaxine metabolites were identified, namely N-desmethylvenlafaxine and O-desmethylvenlafaxine, which were clearly identified using analytical standards, and two other metabolites revealed by non-target analysis. According to the signal intensity, hydroxy-venlafaxine (OH-VEN) was the predominant metabolite detected in mussels exposed for 3 and 7 days.

Impact of urban wastewater discharges on the sediments of a small Mediterranean river and associated coastal environment: assessment of estrogenic and dioxin-like activities.

The Mediterranean region includes many small coastal rivers about which little is known concerning organic contaminant loads in their sediment. This study was designed to assess organic contamination in one of these small coastal rivers (Lez River) and associated coastal sediments. Levels of alkylphenols (APs), polycylic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs) were measured in sediments of the Lez River and two coastal lagoons impacted by wastewater discharges. In parallel, sediment surrounding a recently built submarine outfall that discharges treated wastewater, from an area encompassing some 450,000 inhabitants, into the sea was monitored a year after the beginning of emission via the outfall. Finally, these sediments were characterized by screening estrogenic, PAH-like and dioxin-like activities using in vitro bioassays. Both chemical analyses and bioassays revealed that wastewater inputs were a source of organic contamination of sediments from the Lez and lagoons, which still persisted 2 years after the discharges were stopped. APs could explain a small proportion of the overall estrogenic activities (up to 31%), suggesting that other estrogenic compounds were also present in the sediments. PAHs explained a great share (83% on average) of the EROD induction potency of the extracts. This survey should be the first step in the long-term monitoring of these sites.

Anthropic impacts on Sub-Saharan urban water resources through their pharmaceutical contamination (Yaoundé, Center Region, Cameroon).

Sub-Saharan urban centers have to tackle high population growth, lack of sanitation infrastructures and the need for good quality water resources. To characterize the impacts of anthropization on the water resources of the capital of Cameroon (Yaoundé), a multi-disciplinary approach was used in ten sub-watersheds (peri-urban and urban) of the Méfou watershed. Pharmaceutical residues were used as tracers of surface and groundwater contamination caused by the release of domestic wastewater from pit latrines and landfills. A water use survey was conducted in the vicinity of the sampling sites to better assess water use, treatment and management. Available land use and hydro-geomorphological data completed characterization of the sub-watersheds. The combined data showed that natural features (elevation, slope, and hydrography) and human activities (land use) favor rainfall-runoff events and hence surface water contamination. Pharmaceutical monitoring revealed contamination of both surface and groundwater especially in the urban sub-watersheds. Analgesics/anti-inflammatory drugs and anti-epileptic carbamazepine were the most frequently found compounds (in up to 91% of water samples) with concentrations of acetaminophen reaching 5660 ng/L. In urban sub-watersheds, 50% of the groundwater sites used for drinking water were contaminated by diclofenac (476-518 ng/L), carbamazepine (263-335 ng/L), ibuprofen (141-276 ng/L), sulfamethoxazole (<2-1285 ng/L) and acetaminophen (110-111 ng/L), emphasizing the need for a deeper understanding of the interactions between surface and groundwater. The use of groundwater as drinking water by 68% of the total population surveyed raises concerns about population exposure and potential health risks. This case study highlights the need for strategies to limit contamination of the water resource given the predicted future expansion of Sub-Saharan urban centers.

Fate of endocrine disrupters in waste stabilization pond systems.

The performance in the removal of estrogenicity from wastewater was studied in three wastewater treatment plants (WWTPs). Different treatment processes were evaluated: stabilization ponds and trickling filter. Sampling was performed from the input to the output of the treatment systems. The total estrogenic activity was determined with MCF-7-derived cell lines which express the endogenous estrogen receptor alpha. The two wastewater stabilization ponds with long retention time had high removal of estrogenicity (90% to 95%). Trickling filters despite being effective at removing organic load were less effective in removing estrogenicity (42%), and post tertiary ponds enhanced estrogenicity removal.

POCIS passive samplers as a monitoring tool for pharmaceutical residues and their transformation products in marine environment.

In the last years, several scientific studies have shown that carbamazepine (CBZ) is one of the most frequently detected pharmaceutical in aquatic environment. However, little data is available on its detection and its transformation products (TPs) in marine water. The use of polar organic chemical integrative sampling (POCIS) passive samplers as a semi-quantitative and qualitative tool for screening of pharmaceuticals and TPs in seawater has been studied. Furthermore, the uptake rates of the target compounds were also determined under laboratory experiments to characterize the levels accumulated in devices. The results confirmed the presence of residues of anticonvulsant CBZ as well as some of its main metabolites, over a 1-year monitoring campaign carried out in French coast on the Mediterranean Sea. The work reports for the first time the presence of two TPs (10,11-dihydro-10,11-trans-dihydroxycarbamazepine (TRANS) and 10-hydroxy-10,11-dihydrocarbamazepine (10OH)) in marine water. The results contribute in assessing the environmental and human health risk of pharmaceuticals on coastal areas.

Estrogenic activity of cosmetic components in reporter cell lines: parabens, UV screens, and musks.

In this work, the estrogenic effects of three classes of substances included in cosmetic formulations-parabens, ultraviolet (UV) screens, and musk fragrances-were studied. Their estrogenic activity was measured with the use of three reporter cell lines: HELN, HELN ERalpha, and HELN ERbeta. These three cell lines allowed for the measurement of estrogenic activity toward estrogen receptors alpha and beta (ERalpha and ERbeta, while taking nonspecific interactions into account. Eight of the 15 substances tested showed specific estrogenic activity with the following degree of potency on ERalpha butylparaben > propylparaben > homosalate = octyl-dimethyl-PABA = 4-methyl-benzylidenecamphor = octyl-methoxycinnamate > ethylparaben = galaxolide. Among these active substances, parabens activated ERalpha and ERbeta similarly, UV screens activated ERalpha moderately and had almost no effect on ERbeta, and fragrances did not activate ERbeta. Methylparaben, ethylparaben, musk moskene, celestolide, and cashmeran did not activate estrogenic responses up to 10(-5) M. Musk ketone and benzophenone-3 were not considered estrogenic at 10(-5) M.

In vivo exposure of marine mussels to carbamazepine and 10-hydroxy-10,11-dihydro-carbamazepine: Bioconcentration and metabolization.

Aquatic organisms are exposed to pharmaceuticals present in natural waters, but few data are available on the accumulation of these substances in such organisms. The present study evaluated the in vivo bioconcentration of two anticonvulsants--carbamazepine (CBZ) and 10-hydroxy-10,11-dihydro-carbamazepine (10 OH)--in marine mussels (Mytilus galloprovincialis) exposed to nominal 10 µg L(-1) concentrations for one week. The bioconcentration factors (BCFs) were 3.9 and 4.5 L kg(-1) dry weight (dw) for CBZ and 10 OH, respectively. CBZ accumulation reached an average tissue concentration of 29.3 ± 4.8 ng g(-1) dw, and 10 OH accumulated up to 40.9 ± 4.6 ng g(-1) dw in tissues within one week, showing first-order kinetics. BCF obtained with linear QSAR models correctly estimated the CBZ bioconcentration and overestimated the 10 OH bioconcentration to some extent. The detection of two metabolites (carbamazepine-10,11-epoxide and acridine) among the five sought suggested an active metabolism for CBZ. In contrast, none of the 10 OH metabolites were detected in mussels exposed to 10 OH. CBZ showed higher accumulation in the digestive gland, where some relevant metabolites were detected, than in other studied tissues. The implication of those findings on field biomonitoring is discussed.

Kinetics of abiotic hydrolysis of isoxaflutole: influence of pH and temperature in aqueous mineral buffered solutions.

A kinetic study of the chemical hydrolysis of isoxaflutole [5-cyclopropyl-4-(2-methanesulfonyl-4-trifluoromethylbenzoyl)is oxazol e (IFT)], a new herbicide recently developed by Rhône-Poulenc Agro, in buffered, sterile aqueous solutions was carried out in the dark at 295, 308, and 323 K and at nine pH values between 1.8 and 10.1. Samples were analyzed by HPLC-UV. The decrease in IFT concentration was accompanied by an increase in the concentration of its diketonitrile derivative (DKN). Obeying pseudo-first-order kinetics, isoxaflutole hydrolysis increased with increasing pH and temperature: for 295 K and pH 9.3 the rate of degradation was 100-fold faster than at pH 3.8. Using the Arrhenius equation, the rate constants K(obsd), activation energies E(a), and entropies DeltaS() were calculated, and plotting log(K(obsd)) against pH showed that the effect of pH varied with temperature. According to DeltaS() values the mechanism of the reaction was found to be different with respect to pH range. The benzoic acid derivative, known as a degradation product of DKN in plants, was not detected in the present study.

Fate of a phenylpyrazole in vegetation and soil under tropical field conditions.

The fate of fipronil, a phenylpyrazole insecticide, and its metabolites under tropical conditions was studied in soil and in vegetation after treatment for locust control. Two different plots were treated with a formulation of fipronil at doses of 5 and 10 g of active ingredient ha(-)(1), respectively. Vegetation and soil at depths of 0-5 and 5-20 cm were sampled for up to 2 months after treatment. After extraction and purification on fipronil immunoaffinity cartridges, residues were analyzed by gas chromatography using electron capture and mass detectors. In soil, a rapid initial decrease of fipronil was observed with a rapid formation of the sulfone and the photodegradate; the amide and the sulfide were not detected. In vegetation, a rapid initial decrease of fipronil was also observed with a rapid formation of mostly the sulfone; the photodegradate and the sulfide were also detected but at much lower concentrations. The metabolites resulting from the degradation of fipronil were similar in both soil and vegetation, but their relative concentrations were different.

Reporter cell lines to study the estrogenic effects of xenoestrogens.

In order to characterize the estrogenic activity of chemicals, we established complementary in vitro recombinant receptor-reporter gene assays in stably transfected MCF-7 and HeLa cells. MCF-7 cells which express the endogenous estrogen receptor alpha (ER alpha) were stably transfected with only an estrogen-regulated luciferase gene. These cells enable the detection of compounds which bind to ER alpha or interfere with the induction of ER alpha mediated gene expression. Furthermore, HeLa cells, which do not express endogenous ERs, were transfected with an ER alpha or an ER beta construct together with an estrogen-regulated luciferase gene, or a chimeric GAL4-ER alpha receptor and the corresponding luciferase reporter gene. Finally, we tested these four cellular models as tools to check the estrogenic activities of several potential xenoestrogens and to detect estrogenic activity in wastewater sewage treatment effluents. In all of the models, nonylphenol mixture (NPm), 4n-nonylphenol (4nNP), 2,4'-DDE, 4,4'-DDE and wastewater sewage treatment effluent were active, while PCB mixture (Aroclor 1254), PCB 77, atrazine and lindane (gamma hexachlorocyclohexane) were inactive. Dioxin partially activates the estrogen receptor in MCF-7 cells while in HeLa-derived cell lines, it decreased the estrogenic-induced expression of luciferase.

Kinetics of chemical degradation of isoxaflutole: influence of the nature of aqueous buffers (alkanoic acid/sodium salt vs phosphate).

A kinetic study of the chemical degradation of isoxaflutole (5-cyclopropyl-1,2-oxazol-4-yl alpha alpha alpha-trifluoro-2-mesyl-p-tolyl ketone) into its diketonitrile derivative (DKN), which is its active herbicide principle, in organic buffers at different pH values was carried out using a HPLC/UV detection method. The values of the pseudo-first-order rate constants Kobs for the reaction were calculated and compared with those previously obtained in inorganic buffers. In both cases, Kobs was found to be dependent on pH and temperature, but at pH 5.2 the degradation of isoxaflutole in CH3COOH/CH3COONa buffers was considerably faster than in KH2PO4/Na2HPO4 buffers, indicating that the compound was sensitive to the nature of the reagents used to prepare buffered solutions. The influence of phosphate and acetate concentrations and the influence of the R-substituent in RCOOH/RCOONa buffers were investigated. For the HA/A- buffers studied, the values of Kobs were linearly dependent on HA and A- concentrations, which meant that the degradation of isoxaflutole was subject to general catalysis. The values of Kobs were also found to be dependent on the number and the position of the CH3 groups of the R-substituent. The known degradation product of DKN (a benzoic acid derivative) was not detected throughout this study.

Detection of emerging contaminants (UV filters, UV stabilizers and musks) in marine mussels from Portuguese coast by QuEChERS extraction and GC-MS/MS.

The UV filters and musk fragrances have come into focus because these compounds are contained and increasingly used not only in sunscreen products but also in many products of daily use, such as cosmetics, skin creams, plastics or varnish. In view of this, the main objective of the present work was to develop and validate a method for the determination of three UV filters, two UV stabilizers and four musks in mussel samples (Mytilus galloprovincialis). The procedure combined a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction method with an analysis by gas chromatography-tandem mass spectrometry (GC-MS/MS). The methodology allowed the determination of target analytes at trace concentration levels (ng/g), with mean recoveries ranging from 91 to 112%. A monitoring study was conducted in four beaches in the Portuguese coast which are impacted by recreational activities and outflow of treated waste water effluents in rivers. The results are used to assess the occurrence of UV filters in comparison with UV stabilizers and musk fragrances which indicate other activities than bathing.

Fast and easy extraction combined with high resolution-mass spectrometry for residue analysis of two anticonvulsants and their transformation products in marine mussels.

Environmental field studies have shown that carbamazepine (Cbz) is one of the most frequently detected human pharmaceuticals in different aquatic compartments. However, little data is available on the detection of this substance and its transformation products in aquatic organisms. This study was thus mainly carried out to optimize and validate a simple and sensitive analytical methodology for the detection, characterization and quantification of Cbz and oxcarbazepine (Ox), two anticonvulsants, and six of their main transformation products in marine mussels (Mytilus galloprovincialis). A modified QuEChERS extraction method followed by analysis with liquid chromatography coupled to high resolution mass spectrometry (HRMS) was used. The analyses were performed using two-stage fragmentation to reveal the different fragmentation pathways that are highly useful for the identification of isomeric compounds, a common problem when several transformation products are analyzed. The developed analytical method allowed determination of the target analytes in the lower ng/g concentration levels. The mean recovery ranged from 67 to 110%. The relative standard deviation was under 11% in the intra-day and 18% in the inter-day analyses, respectively. Finally, the method was applied to marine mussel samples collected from Mediterranean Sea cultures in southeastern France. Residues of the psychiatric drug Cbz were occasionally found at levels up to 3.5ng/g dw. Lastly, in this study, other non-target compounds, such as caffeine, metoprolol, cotinine and ketoprofen, were identified in the real samples analyzed.

Polycyclic aromatic hydrocarbon metabolites and 7-ethoxyresorufin O-deethylase activity in caged European eels.

This study investigated the contribution of two biomarkers, bile polycyclic aromatic hydrocarbon (PAH) metabolites and 7-ethoxyresorufin O-deethylase (EROD), activity in the assessment of PAH contaminated sites. European eels (Anguilla anguilla) were caged in a freshwater stream upstream and downstream from local industrial effluent outlets. Bile PAH metabolites were recorded as fluorescent aromatic compounds by synchronous fluorescence spectroscopy and as a marker for total PAH metabolism: 1-hydroxypyrene (1-OH Pyr) was isolated by high-pressure liquid chromatography and quantified. After 14 and 28 days of caging, EROD activity, bile fluorescence (synchronous fluorometric measurement), and 1-OH Pyr concentrations in bile were higher at the downstream site than at the upstream site. This increase was similar after 2 and 4 weeks of caging. During a reversibility study, EROD activity, bile fluorescence, and 1-OH Pyr concentrations decreased, and this trend was similar for the three markers. These results suggest that PAHs could be the main factor responsible for EROD induction in eels caged at the downstream site.

Laboratory and field-caging studies on hepatic enzymatic activities in European eel and rainbow trout.

Caging field experiments were conducted on young European eel (Anguilla anguilla) and on rainbow trout (Onchorynchus mykiss) for the determination of a biomonitoring protocol of freshwater streams. Hepatic monooxygenase and conjugation enzyme activities were measured as biomarkers of exposure to some organic pollutants. Laboratory studies were performed to compare the responsiveness of these two species concerning their enzymatic activities. The induction of monooxygenase activity, measured as the deethylation of 7-ethoxyresorufin (EROD activity) was examined by beta-naphtoflavone (beta-NF) treatment. Dose-response study revealed that EROD activity increased significantly from 1 mg beta-NF/kg bw and was on a plateau at 50 mg beta-NF/kg bw for both species. No significant effect on glutathione S-transferase (GST) activity was observed. During a time-course study, EROD activity increased, starting from the second day for both species. In European eel, this enzymatic activity decreased significantly from the seventh day, whereas no significant decrease was observed for rainbow trout after 14 days. During a 2-week field study, EROD and GST activities were measured in fish held in cages upstream and downstream of a polluted area. For both species, the induction of EROD activity was observed in the polluted site after 14 days of caging; GST activity was not significantly affected. Monooxygenase activity appeared to be a sensitive tool for biomonitoring freshwater streams for both species.

Estrogenic activity in water and sediments of a French river: contribution of alkylphenols.

Alkylphenols, known to possess estrogenic activity, have been found in the aquatic environment. In this study, we focused on the contribution of alkylphenols to total estrogenic activity in sediment and water extracts of French rivers. Four sites representing rural, agricultural, urban, and industrial watersheds were studied. The concentrations of alkylphenols in water and sediment were quantified by GC/MS. Estrogen-responsive reporter cell lines (MELN) have been used for investigating estrogenic activity at these sites. These observed activities were compared with activities mediated by known concentrations of alkylphenols. In water, the concentration of alkylphenols, from 0.06 to 0.550 microg x L(-1) and from < 0.001 microg x L(-1) to 0.077 microg x L(-1) for nonylphenols and 4t-octylphenol, respectively, were too low to contribute to the observed estrogenic activity. In sediment of the industrial, rural, and urban sites, the observed estrogenic activities could be explained in great part by the alkylphenol concentrations from 0.26 to 2.87 microg x g(-1) and from 0.005 microg x g(-1) to 0.49 microg x g(-1) for nonylphenols and 4t-octylphenol, respectively. In the agricultural site, the alkylphenols (0.022 microg x g(-1) of nonylphenols) poorly contribute to the observed estrogenic activity. Other compounds, such as natural and synthetic hormones, present in water and sediments could act additively in the overall activity.