Toxicokinetic and mass balance of morpholine in rats.

Morpholine (MOR) has a broad spectrum of use and represents high risk of human exposure. Ingested MOR can undergo endogenous N-nitrosation in the presence of nitrosating agents forming N-nitrosomorpholine (NMOR), classified as possible human carcinogen by the International Agency for Research on Cancer.In this study, we evaluated the MOR toxicokinetics in six groups of male Sprague-Dawley rats orally exposed to 14C-radiolabelled MOR and NaNO2. The major urinary metabolite of MOR, N-nitrosohydroxyethylglycine (NHEG), was measured through HPLC as an index of endogenous N-nitrosation. Mass balance and toxicokinetic profile of MOR were determined by measuring radioactivity in blood/plasma and excreta.MOR reached maximum blood concentration 30 minutes after administration. Elimination rate was rapid (70% in 8h). Most of the radioactivity was excreted in the urine (80.9 ± 0.5%) and unchanged 14C-MOR was the main compound excreted in the urine (84% of the dose recovered). 5.8% of MOR is not absorbed and/or was not recovered.Endogenous nitrosation of MOR was demonstrated by the detection of NHEG. The maximum conversion rate found was 13.3 ± 1.2% and seems to be impacted by the MOR/NaNO2 ratio.These results help refining our knowledge of the endogenous production of NMOR, a possible human carcinogen.

A Critical Review on Chemical Speciation of Chlorine-Produced Oxidants in Seawater. Part 3: Chromatographic- and Mass Spectrometric-Based Methodologies.

Chlorination of seawater forms a range of secondary oxidative species - collectively called "chlorine-produced oxidants" (CPOs) - having different biocidal, environmental and ecotoxicological properties. The chemical speciation of these compounds is an important step in attempts to assess the effectiveness of chlorination and the potential impacts of its releases. However, comprehensive determination of CPOs represents a significant analytical challenge for many reasons, including the following: CPO species are numerous, highly reactive, with short-lifetimes, difficult to isolate and generally present at low concentrations in a complex salt matrix. Literature review reveals the development of a wide variety of analytical approaches for analysis of CPOs, either collectively via group parameters or individually. A first category of these approaches was the subject of article II (also including sampling and sample preparation) of a trilogy devoted to the chemical speciation of CPOs in seawater. In this third article - which closes the trilogy - emphasis is placed on chromatographic- and mass spectrometric-based approaches. It reviews more than 80 methods, reported from 1981 to date, and thoroughly discusses their principles and performances. Methodologies involving chemical derivatization of CPOs prior to their analysis by gas or liquid chromatography coupled to mass spectrometry provide the best sensitivities, achieving sub-ppb detection limits for species for which suitable derivatization reagents are available. Online mass spectrometry approaches are attracting increasing interest for their ability to analyze multiple CPO species in real time without extensive sample preparation steps, reaching detection limits of about ppb for less polar oxidants. At the current state of metrological development, neither the methodologies based on chromatography nor those based on online mass spectrometry allow complete speciation of CPOs. Future trends and major challenges related to these approaches are discussed.

A Critical Review on Chemical Speciation of Chlorine-Produced Oxidants (CPOs) in Seawater. Part 1: Chlorine Chemistry in Seawater and Its Consequences in Terms of Biocidal Effectiveness and Environmental Impact.

Seawater chlorination has three main industrial uses: disinfection of water and installations, control of biofouling, and preventing the transport of aquatic invasive species. Once in contact with seawater, chlorine reacts rapidly with water constituents (e.g. bromide ions, ammonia, and nitrogen-containing compounds) to form a range of oxidative species (e.g. bromine and N-haloamines), termed "chlorine-produced oxidants" (CPOs) or "total residual oxidants" (TRO). The chemical nature of CPOs and their concentration are a function of two categories of parameters related to treatment modality (e.g. chlorine dose) and water quality (e.g. temperature, pH, ammonia concentration, and organic constituents). The chlorination process may result in continuous or intermittent releases of CPOs in seawater. The reactivity and potential ecotoxicity of CPO species largely depend on their physical and chemical properties. Therefore, evaluation of the biocidal effectiveness of chlorination and its potential impacts requires not only determining the sum of CPOs (via a bulk parameter), but also their chemical speciation. The aim of this article - which is the first of a trilogy dedicated to the chemical speciation of CPOs in seawater - is to provide an overview of current knowledge about chlorine chemistry in seawater and to discuss the biocidal efficacy and the environmental fate of resulting CPOs. The 2nd and 3rd articles delineate a comprehensive and critical review of analytical methods and approaches for the determination of CPOs in seawater.

A Critical Review on Chemical Speciation of Chlorine-Produced Oxidants (CPOs) in Seawater. Part 2: Sampling, Sample Preparation and Non-Chromatographic and Mass Spectrometric-Based Methods.

Chlorination of seawater forms a range of secondary oxidative species, collectively termed "chlorine-produced oxidants" (CPOs). These compounds do not have the same biocidal efficacy, the same fate and behavior in the marine environment, the same potential formation of chlorination by-products (CBPs), nor the same effects on marine organisms. Their chemical speciation is an important step toward an accurate assessment of the effectiveness of chlorination and the potential impacts of its releases, among others. The aim of this paper - which is the second of a trilogy dedicated to the chemical speciation of CPOs in seawater - is to cover all aspects related to CPOs analysis in seawater, from sampling to instrumental determination. First, it discusses the procedures involved in synthesis, storage, and standardization of analytical standards. Second, it deals with sampling and sample preparation, addressing all relevant issues related to these two key steps. Third, it provides a comprehensive and up-to-date overview of the colorimetric, titrimetric, and electrochemical methods used for CPOs determination and thoroughly discusses their advantages and limitations. Finally, this review ends with some recommendations for progress in the field of CPO analysis with the three aforementioned approaches. Chromatographic and mass spectrometric-based methods will be covered in the third and final article (Part III).

Challenges and opportunities for on-line monitoring of chlorine-produced oxidants in seawater using portable membrane-introduction Fourier transform-ion cyclotron resonance mass spectrometry.

The present study reports the first evaluation of a MIMS device equipped with a high-resolution Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS) for comprehensive speciation of chlorine-produced oxidants (CPO) in seawater. A total of 40 model compounds were studied: 4 inorganic haloamines (mono-, di-, and trichloramine and monobromamine), 22 organic N-haloamines, 12 N-haloamino acids, and 2 free oxidants (HOCl/ClO- and HOBr/BrO-). The main key factors influencing the analytes' introduction and their detection were optimized. Under optimized conditions, the rise and fall times of the MIMS signal ranged from 8 to 79 min and from 7 to 73 min, respectively, depending on the compound. Free oxidants and N-haloamino acids, which are ionic or too polar at seawater pH, hardly crossed the membrane, and MIMS analysis was thus unsuitable. Nevertheless, better enrichment and therefore better sensitivity were achieved with organic N-haloamines than with inorganic haloamines. The observed detection limits ranged from tens of µM to sub-µM levels. Oxidant decomposition occurred inside the MIMS device, at a higher rate for N-bromamines than for chlorinated analogues.Graphical abstract.

SPIX: A new software package to reveal chemical reactions at trace amounts in very complex mixtures from high-resolution mass spectra dataset.

RATIONALE: High-resolution mass spectrometry based non-targeted screening has a huge potential for applications in environmental sciences, engineering and regulation. However, it produces large datasets for which full appropriate processing is a real challenge; the development of processing software is the last building-block to enable large-scale use of this approach. METHODS: A new software application, SPIX, has been developed to extract relevant information from high-resolution mass spectral datasets. Dealing with intrinsic sample variability and reducing operator subjectivity, it opens up opportunities and promising prospects in many areas of analytical chemistry. SPIX is freely available at: http://spix.webpopix.org. RESULTS: Two features of the software are presented in the field of environmental analysis. An example illustrates how SPIX reveals photodegradation reactions in wastewater by fitting kinetic models to significant changes in ion abundance over time. A second example shows the ability of SPIX to detect photoproducts at trace amounts in river water, through comparison of datasets from samples taken before and after irradiation. CONCLUSIONS: SPIX has shown its ability to reveal relevant modifications between two series of large datasets, allowing, for instance, the study of the consequences of a given event on a complex substrate. Most of all - and it is to our knowledge the only software currently available allowing this - it can reveal and monitor any kind of reaction in all types of mixture.

Determination of monochloramine dissipation kinetics in various surface water qualities under relevant environmental conditions - Consequences regarding environmental risk assessment.

A total 190 experiments were performed to study the dissipation kinetics of monochloramine (NH2Cl, CAS no 10599-90-3) in surface water samples from six rivers (Loire, Rhône, Meuse, Garonne, Seine and Moselle) and an artificial reservoir (Mirgenbach), all located in France. Experiments were conducted in an open reactor, under relevant controlled environmental conditions. The impact of various parameters such as initial NH2Cl concentration, temperature, pH, presence of sediments, sampling site and collection period was investigated. It was found that NH2Cl dissipated rapidly without any lag phase, and that decay follows an apparent first-order kinetics (r2 > 0.99). Presence of sediment greatly accelerated decay. Half-lives were generally <1 h in river water in presence of natural sediment, but of several hours without sediment. The impact of pH was low for the normal river water pH range. However, increase in temperature significantly accelerated decay. The combination of high initial NH2Cl concentrations and elevated temperatures generally gives half-lives similar to those obtained at lower temperatures and lower concentrations. Short half-lives (0.06 to 1.50 h) were found in all the surface waters examined, regardless of geographic location of sampling site or collection period, indicating no temporal or site-specific effects on NH2Cl dissipation. Decay was slightly faster at lower initial concentrations, which supports extrapolation of half-lives measured in this study to a wide range of environmental concentrations. It can thus be assumed that NH2Cl degradation in river and reservoir waters is mainly determined by presence of sediments and temperature.

Tracking Monochloramine Decomposition in MIMS Analysis.

Membrane-introduction mass spectrometry (MIMS) has been presented as one of the promising approaches for online and real-time analysis of monochloramine (NH2Cl) in diverse matrices such as air, human breath, and aqueous matrices. Selective pervaporation of NH2Cl through the introduction membrane overcomes the need for sample preparation steps. However, both the selectivity and sensitivity of MIMS can be affected by isobaric interferences, as reported by several researchers. High-resolution mass spectrometry helps to overcome those interferences. Recent miniaturization of Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR MS) technology coupled to the membrane-introduction system provides a potent tool for in field analysis of monochloramine in environmental matrices. Monochloramine analysis by MIMS based FT-ICR MS system demonstrated decomposition into ammonia. To further clarify the origin of this decomposition, headspace analyses after bypassing the membrane were undertaken and showed that monochloramine decomposition was not exclusively related to interactions within the membrane. Adsorption inside the MIMS device, followed by surface-catalyzed decomposition, was suggested as a plausible additional mechanism of monochloramine decomposition to ammonia.

Development and validation of a multiclass method for the determination of organohalogen disinfectant by-products in water samples using solid phase extraction and gas chromatography-tandem mass spectrometry.

An analytical method employing solid phase extraction (SPE) and gas chromatography-tandem mass spectrometry (GC-MS/MS) has been developed for quantitative determination of twenty-six organohalogen disinfectant by-products (OXBPs) in water samples. Target analytes include four trihalomethanes (THMs), four iodohalomethanes (I-HMs), one haloacetaldehyde (HAL), six haloketones (HKs), four halonitromethanes (HNMs), and seven haloacetonitriles (HANs). The sample preparation procedure includes pretreatment with ascorbic acid to quench residual oxidants, followed by analyte enrichment using solid-phase extraction. Five SPE sorbents were tested. The best results for the majority of target analytes were obtained using a styrene-divinylbenzene copolymer phase and methyl tert-butyl ether (MTBE) as the elution agent. GC-MS analysis was performed using electron ionization (EI) and selected reaction monitoring (SRM) for analyte detection. The performance of the method was assessed according to the French standard NF T90-210. The method showed LOQs ranging from 3 to 3000 ng L-1. The applicability of the method has been demonstrated by analyzing both river water and tap water samples. The OXBPs detected most often in the tap water were dibromochloromethane (in 100% of the samples, 4.3-4.7 µg L-1), bromodichloromethane (100%, 1.3-1.7 µg L-1), tribromomethane (100%, 0.8-4.4 µg L-1), trichloromethane (100%, 0.6-0.7 µg L-1), dibromoacetonitrile (75%, 0.5-0.9 µg L-1), with dichloroacetonitrile and 1,1,1-trichloro-2-propanone detected at concentrations around the LOQ levels. In the treated river water samples, the only OXBPs measured at levels above the limit of quantification were trichloromethane and 1,1,1-trichloro-2-propanone. To the best of our knowledge, this is the first SPE-GC-MS/MS method enabling the analysis of an extensive list of OXBPs.

A sensitive and specific solid-phase extraction-gas chromatography-tandem mass spectrometry method for the determination of 11 haloacetic acids in aqueous samples.

A method for the analysis of 11 haloacetic acids in water samples has been developed. It involves enrichment of the target analytes from water samples by solid-phase extraction, derivatization to methyl esters, and gas chromatography coupled with tandem mass spectrometry determination. Gas chromatography conditions were optimized for a good separation of all haloacetic acids in a short runtime. Data were acquired in the multiple reaction monitoring mode. Six solid-phase extraction sorbents among the most widely used in environmental analysis were tested. Bakerbond SDB was retained because it has been shown to provide the best results for a large class of targeted haloacetic acids. The performances of the developed method have been assessed according to the French Standard NF T 90-210. The calibration curves for all the studied haloacetic acids had consistent slopes with r2 values > 0.99. Quantification limits between 0.01 and 0.50 µg l-1 were achieved. Satisfactory repeatability (relative standard deviation ≤ 14.3%) and intermediate precision (relative standard deviation ≤ 15.7%) were obtained. Applied to the analysis of 15 untreated water samples collected from three rivers, the method allowed the detection of five haloacetic acids including monochloroacetic acid (in 100% of the samples, <0.5-1.85 µg l-1), dichloroacetic acid (87%, <0.05-0.22 µg l-1), trichloroacetic acid (93%, <0.05-0.52 µg l-1), dibromoacetic acid (53%, <0.01-0.40 µg l-1), tribromoacetic acid (20%, <0.05-0.14 µg l-1), and bromodichloroacetic acid (6%, < 0.05 µg l-1).

Determination of adsorbable organic halogens in surface water samples by combustion-microcoulometry versus combustion-ion chromatography titration.

Adsorbable Organic Halogen (AOX) is an analytical parameter of considerable interest since it allows to evaluate the amount of organohalogen disinfection by-products (OXBPs) present in a water sample. Halogen speciation of AOX into adsorbable organic chlorine, bromine and iodine, respectively AOCl, AOBr and AOI, is extremely important since it has been shown that iodinated and brominated organic by-products tend to be more toxic than their chlorinated analogues. Chemical speciation of AOX can be performed by combustion-ion chromatography (C-IC). In the present work, the effectiveness of the nitrate wash according to ISO 9562 standard method protocol to eliminate halide ions interferences was firstly examined. False positive AOX values were observed when chloride concentration exceeded 100 ppm. The improvements made to the washing protocol have eliminated chloride interference for concentrations up to 1000 ppm. A C-IC method for chemical speciation of AOX into AOCl, AOBr, and AOI has been developed and validated. The most important analytical parameters were investigated. The following optimal conditions were established: an aqueous solution containing 2.4 mM sodium bicarbonate/2.0 mM sodium carbonate, and 2% acetone (v/v) as mobile phase, 2 mL of aqueous sodium thiosulfate (500 ppm) as absorption solution, 0.2 mL min-1 as water inlet flow rate for hydropyrolysis, and 10 min as post-combustion time. The method was validated according to NF T90-210 standard method. Calibration curves fitted through a quadratic equation show coefficients of determination (r2) greater than 0.9998, and RSD less than 5%. The LOQs were 0.9, 4.3, and 5.7 µg L-1 Cl for AOCl, AOBr, and AOI, respectively. The accuracy, in terms of relative error, was within a ±â€¯10% interval. The applicability of the validated method was demonstrated by the analysis of twenty four water samples from three rivers in France. The measurements reveals AOX amounts above 10 µg L-1 Cl in all untreated samples, suggesting the presence of organohalogen compounds in the sampled rivers. On weight concentration basis, AOCl accounted for 77-100% of AOX in the treated water samples. A good agreement between the conventional AOX method and the developed C-IC method was found.

Photodegradation of fluorene in aqueous solution: Identification and biological activity testing of degradation products.

Degradation of fluorene under UV-vis irradiation in water was investigated and structural elucidation of the main photoproducts was achieved using gas chromatography coupled with mass spectrometry. Twenty-six photoproducts were structurally identified, mainly on the basis of electron ionization mass spectra interpretation. The main generated transformation products are hydroxy derivatives. Some secondary photoproducts including fluorenone, hydroxy fluorenone, 2-biphenyl carboxylic acid, biphenylene, methanol fluorene congeners and hydroxy fluorene dimers were also observed. A photodegradation pathway was suggested on the basis of the chemical structures of photoproducts. Fluorene as well as its main photoproducts for which chemical standards were commercially available were tested for their ability to elicit cytotoxic, estrogenic and dioxin-like activity by using in vitro cell-based bioassays. None of the tested compounds was cytotoxic at concentrations up to 100 µM. However, 2-hydroxyfluorene and 3-hydroxyfluorene exerted significant estrogenic and dioxin-like activity on a concentration range of 3-30 µM, while fluorene and 9-hydroxyfluorene were weakly or not active, respectively, in our assays. This supports the view that photodegradation processes can generate by-products of higher toxicological concern than the parent compound and strengthens the need to further identify transformation products in the aquatic environment.

Selective and trace determination of monochloramine in river water by chemical derivatization and liquid chromatography/tandem mass spectrometry analysis.

Monochloramine (MCA) may enter the aquatic environment through three main sources: wastewater treatment plant effluents, industrial effluents and thermal power plant wastes. Up to date, there are no available data about the concentration levels of this chemical in river water due to lack of appropriate analytical methods. Therefore, sensitive and selective analytical methods for monochloramine analysis in river water are required to evaluate its environmental fate and its effects on aquatic ecosystems. Thus, in this study we describe a highly specific and sensitive method for monochloramine determination in river water. This method combines chemical derivatization of monochloramine into indophenol followed by liquid chromatography coupled to electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) analysis. Two precursor-to-product ion transitions were monitored (200→127 and 200→154) in positive ionisation mode, fulfilling the criteria of selectivity, in accordance with the European Legislation requirements (decision 2002/657/EC). Ion structures and fragmentation mechanisms have been proposed to explain the selected transitions. Linearity range, accuracy and precision of the method have been assessed according to the French method validation standard NF T90-210. Detecting the derivatized monochloramine (indophenol) in Multiple Reaction Monitoring (MRM) mode provided a limit of quantification of 40 ng L(-1) equivalent monochloramine. Applied to Loire river water (France), the developed method occasionally detected monochloramine at concentrations less than 300 ng L(-1), which could be explained by punctual discharges of water containing active chlorine upstream of the sampling point. Indeed, it is widely reported in the literature that the addition of chlorine to water containing ammonia (e.g., wastewater effluents and river water) may result in the instantaneous formation of monochloramine. The proposed method is a powerful tool that can be used in environmental research (e.g., assessment of environmental fate and generating of ecotoxicological data) as well as in research studies concerning the evaluation of water disinfection efficiency; but it is not currently appropriate for routine use in industrial applications given the complexity of the procedure, the instability of indophenol and the use of certain toxic reagents.

Photolysis of estrone generates estrogenic photoproducts with higher activity than the parent compound.

In the present study, we aimed to evaluate the effect of UV-visible irradiation on the estrogenicity of an estrone aqueous solution by using chemical analysis associated with an in vitro bioassay and in silico analysis. An estrone aqueous solution was irradiated with an UV-visible high-pressure mercury lamp. By using the MELN in vitro cellular bioassay, based on the induction of a luciferase reporter gene upon the activation of the estrogen receptor by chemicals, we showed that the estrogenic potency of the solution increased after irradiation. High-performance liquid chromatography fractionation of the photolyzed solution followed by in vitro testing of fractions allowed the quantitation of the estrogenic potency of each fraction. Nine photoproducts were detected and characterized by liquid chromatography-mass spectrometry coupling. The observed estrogenic activity is mediated by mono- and multi-hydroxylated photoproducts; it is influenced by the position of hydroxyl groups on the steroidal skeleton. In addition, a structure-activity analysis of the hydroxylated photoproducts confirmed their ability to act as estrogen receptor ligands.

Effect-directed analysis of endocrine-disrupting compounds in multi-contaminated sediment: identification of novel ligands of estrogen and pregnane X receptors.

Effect-directed analysis (EDA)-based strategies have been increasingly used in order to identify the causative link between adverse (eco-)toxic effects and chemical contaminants. In this study, we report the development and use of an EDA approach to identify endocrine-disrupting chemicals (EDCs) in a multi-contaminated river sediment. The battery of in vitro reporter cell-based bioassays, measuring estrogenic, (anti)androgenic, dioxin-like, and pregnane X receptor (PXR)-like activities, revealed multi-contamination profiles. To isolate active compounds of a wide polarity range, we established a multi-step fractionation procedure combining: (1) a primary fractionation step using normal phase-based solid-phase extraction (SPE), validated with a mixture of 12 non-polar to polar standard EDCs; (2) a secondary fractionation using reversed-phase-based high-performance liquid chromatography (RP-HPLC) calibrated with 33 standard EDCs; and (3) a purification step using a recombinant estrogen receptor (ER) affinity column. In vitro SPE and HPLC profiles revealed that ER and PXR activities were mainly due to polar to mid-polar compounds, while dioxin-like and anti-androgenic activities were in the less polar fractions. The overall procedure allowed final isolation and identification of new environmental PXR (e.g., di-iso-octylphthalate) and ER (e.g., 2,4-di-tert-butylphenol and 2,6-di-tert-butyl-α-methoxy-p-cresol) ligands by using gas chromatography coupled with mass spectrometry with full-scan mode acquisition in mid-polar fractions. In vitro biological activity of these chemicals was further confirmed using commercial standards, with di-iso-octylphthalate identified for the first time as a potent hPXR environmental agonist.

Investigation of the dissociation pathways of metolachlor, acetochlor and alachlor under electron ionization - application to the identification of ozonation products.

With the future aim of using gas chromatography coupled with mass spectrometry to characterize the transformation products of ozonated herbicides: metolachlor, acetochlor and alachlor, an interpretation of their electron ionization mass spectra is presented. Fragmentation mechanisms are proposed on the basis of isotopic labelling and multiple-stage mass spectrometry experiments carried out on an ion trap mass spectrometer. We also give examples in order to demonstrate how the elucidation of such fragmentation mechanisms for herbicides may simplify the characterization of their ozonation products.

Elucidation of the decomposition pathways of protonated and deprotonated estrone ions: application to the identification of photolysis products.

With the future aim of elucidating the unknown structures of estrogen degradation products, we characterized the dissociation pathways of protonated estrone (E1) under collisional activation in liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments employing a quadrupole time-of-flight mass spectrometer. Positive ion and negative ion modes give information on the protonated and deprotonated molecules and their product ions. The mass spectra of estrone methyl ether (CH(3)-E1) and estrone-d(4) (E1-d(4)) were compared with that of E1 in order (i) to elucidate the dissociation mechanisms of protonated and deprotonated molecules and (ii) to propose likely structures for each product ions. The positive ion acquisition mode yielded more fragmentation. The mass spectra of E1 were compared with those of estradiol (E2), estriol (E3) and 17-ethynylestradiol (EE2). This comparison allowed the identification of marker ions for each ring of the estrogenic structure. Accurate mass measurements have been carried out for all the identified ions. The resulting ions revealed to be useful for the characterization of structural modifications induced by photolysis on each ring of the estrone molecule. These results are very promising for the determination of new metabolites in the environment.

Bioanalytical characterisation of multiple endocrine- and dioxin-like activities in sediments from reference and impacted small rivers.

A comprehensive evaluation of organic contamination was performed in sediments sampled in two reference and three impacted small streams where endocrine disruptive (ED) effects in fish have been evidenced. The approach combined quantitative chemical analyses of more than 50 ED chemicals (EDCs) and a battery of in vitro bioassays allowing the quantification of receptor-mediated activities, namely estrogen (ER), androgen (AR), dioxin (AhR) and pregnane X (PXR) receptors. At the most impacted sites, chemical analyses showed the presence of natural estrogens, organochlorine pesticides, parabens, polycyclic aromatic hydrocarbons (16 PAHs), bisphenol A and alkylphenols, while synthetic steroids, myco-estrogens and phyto-estrogens were not detected. Determination of toxic-equivalent amounts showed that 28-96% of estrogenic activities in bioassays (0.2-6.3 ng/g 17beta-estradiol equivalents) were explained by 17beta-estradiol and estrone. PAHs were major contributors (20-60%) to the total dioxin-like activities. Interestingly, high PXR and (anti)AR activities were detected; however, the targeted analysed compounds could not explain the measured biological activities. This study highlighted the presence of multiple organic EDCs in French river sediments subjected to mixed diffuse pollution, and argues for the need to further identify AR and PXR active compounds in the aquatic environment.

Evaluation of an hPXR reporter gene assay for the detection of aquatic emerging pollutants: screening of chemicals and application to water samples.

Many environmental endocrine-disrupting compounds act as ligands for nuclear receptors. Among these receptors, the human pregnane X receptor (hPXR) is well described as a xenobiotic sensor to various classes of chemicals, including pharmaceuticals, pesticides, and steroids. To assess the potential use of PXR as a sensor for aquatic emerging pollutants, we employed an in vitro reporter gene assay (HG5LN-hPXR cells) to screen a panel of environmental chemicals and to assess PXR-active chemicals in (waste) water samples. Of the 57 compounds tested, 37 were active in the bioassay and 10 were identified as new PXR agonists: triazin pesticides (promethryn, terbuthryn, terbutylazine), pharmaceuticals (fenofibrate, bezafibrate, clonazepam, medazepam) and non co-planar polychlorobiphenyls (PCBs; PCB101, 138, 180). Furthermore, we detected potent PXR activity in two types of water samples: passive polar organic compounds integrative sampler (POCIS) extracts from a river moderately impacted by agricultural and urban inputs and three effluents from sewage treatment works (STW). Fractionation of POCIS samples showed the highest PXR activity in the less polar fraction, while in the effluents, PXR activity was mainly associated with the dissolved water phase. Chemical analyses quantified several PXR-active substances (i.e., alkylphenols, hormones, pharmaceuticals, pesticides, PCBs, bisphenol A) in POCIS fractions and effluent extracts. However, mass-balance calculations showed that the analyzed compounds explained only 0.03% and 1.4% of biological activity measured in POCIS and STW samples, respectively. In effluents, bisphenol A and 4-tert-octylphenol were identified as main contributors of instrumentally derived PXR activities. Finally, the PXR bioassay provided complementary information as compared to estrogenic, androgenic, and dioxin-like activity measured in these samples. This study shows the usefulness of HG5LN-hPXR cells to detect PXR-active compounds in water samples, and further investigation will be necessary to identify the detected active compounds.

Extraction and purification procedures for simultaneous quantification of phenolic xenoestrogens and steroid estrogens in river sediment by gas chromatography/ion trap mass spectrometry.

A sensitive and simple method based on ultrasonication extraction with a hexane/acetone (2:1, v/v) mixture, followed by clean up of the extract by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC/MS) detection, has been developed and validated for the analysis of 20 estrogenic endocrine-disrupting chemicals (EEDCs) including phenolic xenoestrogens, synthetic and natural estrogens in river sediment. After extraction and purification, analytes are derivatised with a BSTFA/TMCS/pyridine (49:1:50, v/v/v) mixture and quantified by GC/MS. The GC/MS method involves switching between electron ionisation (EI) and chemical ionisation (CI); it also switches between selected ion storage and tandem mass spectrometry detection. The applicability of the method has been demonstrated by analysing extracts of French river sediments for which bioanalytical tests (in vitro) had already shown that they were impacted by estrogenic endocrine disrupters. The biological contribution of all the products detected in each sediment extract was compared to the estrogenic activity measured by bioassays.