Emmanuelle Vulliet

Development of a multi-residue analysis of diclofenac and some transformation products in bivalves using QuEChERS extraction and liquid chromatography-tandem mass spectrometry. Application to samples from mesocosm studies.

Pharmaceuticals are ubiquitously present in the aquatic environment, mainly due to insufficient removal in wastewater treatment plants. Although these compounds are often found at trace levels in waters, long-term exposure can have negative impacts on biotic communities due to their inherent biological activity. The non-steroidal anti-inflammatory drug diclofenac (DCF) is one of the most frequently detected human pharmaceuticals in water and has recently been included in the watch list of the European Union. However little data are available on the detection of this substance and its transformation products in aquatic organisms. In this context, an analytical methodology has been developed to quantify traces of DCF along with its biotic and abiotic transformation products in a wild species of bivalve, the zebra mussel Dreissena polymorpha. A modified QuEChERS extraction was implemented on a small quantity of soft bivalve tissue (100mg). This was followed by liquid chromatography coupled to tandem-mass spectrometry (LC-MS/MS) with electrospray ionization in positive mode (ESI+). Whole analytical method was validated on spiked real samples, with regard to linearity (from 1 to 50 or 100ng/g depending on the target compounds, R(2)>0.99), intra-day precision (relative standard deviation (RSD)<18%), inter-day precision (RSD <25%), (recoveries 78-117%), and limits of detection and of quantification (both inferior or equal to 1ng/g). The optimized method was successfully applied to organisms collected from mesocosm experiments. Bioconcentration factors comprised between 4 and 13 were observed for DCF in the zebra mussels. To the best of our knowledge, the product 2-indolone was for the first time detected in bivalves, with levels up to 6ng/g.

Two-year survey of specific hospital wastewater treatment and its impact on pharmaceutical discharges

It is well known that pharmaceuticals are not completely removed by conventional activated sludge wastewater treatment plants. Hospital effluents are of major concern, as they present high concentrations of pharmaceutically active compounds. Despite this, these specific effluents are usually co-treated with domestic wastewaters. Separate treatment has been recommended. However, there is a lack of information concerning the efficiency of separate hospital wastewater treatment by activated sludge, especially on the removal of pharmaceuticals. In this context, this article presents the results of a 2-year monitoring of conventional parameters, surfactants, gadolinium, and 13 pharmaceuticals on the specific study site SIPIBEL. This site allows the characterization of urban and hospital wastewaters and their separate treatment using the same process. Flow proportional sampling, solid-phase extraction, and liquid chromatography coupled with tandem mass spectrometry were used in order to obtain accurate data and limits of quantification consistent with ultra-trace detection. Thanks to these consolidated data, an in-depth characterization of urban and hospital wastewaters was realized, as well as a comparison of treatment efficiency between both effluents. Higher concentrations of organic carbon, AOX, phosphates, gadolinium, paracetamol, ketoprofen, and antibiotics were observed in hospital wastewaters compared to urban wastewaters. Globally higher removals were observed in the hospital wastewater treatment plant, and some parameters were shown to be of high importance regarding removal efficiencies: hydraulic retention time, redox conditions, and ambient temperature. Eleven pharmaceuticals were still quantified at relevant concentrations in hospital and urban wastewaters after treatment (e.g., up to 1xa0μg/L for sulfamethoxazole). However, as the urban flow was about 37 times higher than the hospital flow, the hospital contribution appeared relatively low compared to domestic discharges. Thanks to the SIPIBEL site, data obtained from this 2-year program are useful to evaluate the relevance of separate hospital wastewater treatment.

Utilisation of an enzyme-linked immunosorbent assay (ELISA) for determination of alkylphenols in various environmental matrices. Comparison with LC–MS/MS method

Among the wide range of substances discharged continuously in the environment, alkylphenols became a major focus of environmental research in the last decades, as it was found that they possess endocrine disrupting properties. Knowledge about the occurrence and levels of alkylphenols in environment is critical for the risk assessment of these compounds on both ecosystem and human health. However, the analysis of traces of alkylphenols in environmental matrices is a very difficult task, and the suitable methods involve generally an extraction followed by an extensive sample clean-up before detection, steps often time-consuming and costly. In order to reduce the analysis time, obtain a high throughput of analysis and thus improve work efficiency, the objective of the present study is to investigate the use of immunochemical technique (ELISA) for the determination of nonylphenol and octylphenol in soils and various kinds of water. To our knowledge, this is the first time that the determination of alkylphenols in soil using immunoassay technique is described. A methodology is developed, based on the combination of a single preparation step and the use of a simply ELISA kit. The performances of the method are compared with LC-MS/MS, considered as reference. The developed procedure offers the sensitivity and selectivity necessary for the detection of the target alkylphenols in the ng/g or ng/L range, and is successfully applied to the analysis of several samples. Results indicate that alkylphenols are quantified with concentrations in the same order than LC-MS/MS, meaning that ELISA may be useful not only in screening the samples and get a positive/negative response, but also it allows a good approximation of the concentrations.

Determination of carbamazepine and 12 degradation products in various compartments of an outdoor aquatic mesocosm by reliable analytical methods based on liquid chromatography-tandem mass spectrometry

The aims of this work are to develop suitable analytical methods to determine the widely used anticonvulsant carbamazepine and 12 of its degradation/transformation products in water, sediment, fish (Gasterosteus aculeatus) and mollusc (Dreissena polymorpha). Protocols based on solid phase extraction for water, pressurized-liquid extraction for sediments and QuEChERS (quick easy cheap efficient rugged and safe) extraction for both organisms followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are developed, validated and finally applied to samples collected during a 6-month experiment in outdoor mesocosms. Very low detection limits are reached, allowing environmentally realistic doses (namely, 0.05, 0.5 and 5xa0μg/L nominal concentrations) to be employed. The results indicate several metabolites and/or transformation products in each compartment investigated, with concentrations sometimes being greater than that of the parent carbamazepine. Biotic degradation of carbamazepine is demonstrated in water, leading to 10,11-dihydrocarbamazepine and 10,11-epoxycarbamazepine. In sediment, the degradation results in the formation of acridine, and 2- and 3-hydroxycarbamazepine. Finally, in both organisms, a moderate bioaccumulation is observed together with a metabolization leading to 10,11-epoxycarbamazepine in fish and 2-hydroxycarbamazepine in mollusc. Acridone is also present in fish. This study provides new and interesting data, helping to elucidate how chronic exposure to carbamazepine at relevant concentrations may affect impact freshwater ecosystems.

Non-targeted investigation of benthic invertebrates (Chironomus riparius) exposed to wastewater treatment plant effluents using nanoliquid chromatography coupled to high-resolution mass spectrometry

Nanoliquid chromatography (nanoLC) was coupled to high-resolution mass spectrometry (HRMS) to perform a non-targeted investigation on benthic invertebrates, Chironomus riparius exposed to wastewater treatment plant (WWTP) effluents. Insect larvae represent a complex and low-weight matrix that required the use of a miniaturized Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method of extraction followed by nanoLC-HRMS to perform the analysis. The optimization of this coupling in terms of separation conditions including trapping step, detection conditions and data treatment provided reproducible fingerprints on insect larvae exposed to WWTP effluents with both in situ and ex-situ approaches. Statistical treatments such as principal component analysis highlighted the impact of WWTP effluents on the metabolome of insect larvae and showed the influence of exposure conditions. The identification of discriminating signals (m/z, tR) matched with several potential endogenous biomarkers. These are mainly fatty acids, indicating a change in lipid metabolism that can be correlated with exposure to WWTP effluents. Several xenobiotics have also been detected, including ibuprofen and propranolol, whose identities have been confirmed by analytical standards. This work demonstrates the effectiveness and sensitivity of nanoLC-HRMS based environmental non-targeted approaches in ecotoxicological studies and provides the first profiling data for a very small aquatic invertebrate.

Development of a method for the simultaneous determination of multi-class pesticides in earthworms by liquid chromatography coupled to tandem electrospray mass spectrometry.

AbstractAgricultural intensification, and in particular the use of pesticides, leads over the years to a loss of biodiversity and a decline of ecosystem services in cultivated zones and agricultural landscapes. Among the animal communities involved in the functioning of agro-ecosystems, earthworms are ubiquitous and recognized as indicators of land uses and cultural practices. However, little data is available on the levels of pesticides in such organisms in natura, which would allow estimating their actual exposure and the potentially resulting impacts. Thus, the objective of this study was to develop a sensitive analytical methodology to detect and quantify 27 currently used pesticides in earthworms (Allolobophora chlorotica). A modified QuEChERS extraction was implemented on individual earthworms. This step was followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The whole analytical method was validated on spiked earthworm blank samples, with regard to linearity (from 1 to 100 method limit of quantification, r2u2009>u20090.95), intra-day precision (relative standard deviation (RSD) <u200915%), inter-day precision (RSD <u200920%), recoveries (mainly in the range 70–110%), and limits of detection and of quantification (inferior to 5xa0ng/g for most of the pesticides). The developed method was successfully applied to determine the concentrations of pesticides in nine individuals collected in natura. Up to five of the selected pesticides have been detected in one individual.n Graphical abstract

Phenotypic defects in newborn Gammarus fossarum (Amphipoda) following embryonic exposure to fenoxycarb

During morphogenesis numerous morphogenetic factors ensure the production of a target phenotype. By disrupting these processes, a toxic exposure during this period could cause an increase of phenotypic defects. In the present study, embryos of the freshwater amphipod Gammarus fossarum were exposed throughout the embryogenesis to increasing concentrations of fenoxycarb (0, 0.5µgL-1, 5µgL-1 and 50µgL-1), a growth regulator insecticide analog of the insect juvenile hormone. In addition, to identify morphogenesis sensitive period, embryos were exposed during either early or late embryonic development to 5µgL-1 of fenoxycarb. In newborn individuals from exposed embryos, three phenotypes were investigated: i) eye pigmentation, ii) length of the antenna and gnathopod of both left and right sides and iii) midgut tissue state. Developmental homeostasis was assessed by measuring fluctuating asymmetry and inter-individual variance of both the antenna and gnathopod. Exposure to 5µgL-1 and 50µgL-1 fenoxycarb throughout the embryonic development induced a delayed hatching and altered appendages size. Moreover, exposure to 5µgL-1 throughout the embryogenesis and during the gastrulation phase impaired eye pigmentation, while exposure to 50µgL-1 resulted in increased tissue damages of the midgut. No significant increase of fluctuating asymmetry was observed in exposed individuals, neither for the antenna nor for the gnathopod. These results demonstrate that fenoxycarb can alter embryonic development of G. fossarum without disrupting developmental homeostasis.

A rapid and easy method based on hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC-MS/MS/MS) to quantify iodinated X-ray contrast in wastewaters

This work proposes the first method based on hydrophilic interaction liquid chromatography coupled to multiple reaction monitoring with triple stage fragmentation (HILIC-MRM3) to quantify polar organic micropollutants in complex sewage waters. A fast HILIC-MRM3 analytical method, without sample preparation except a dilution step, was developed and validated to quantify seven iodinated contrast media (ICMs) in sewage waters, namely iohexol, iomeprol, ioversol, iopamidol, diatrizoic acid, iopromide and iopentol. Several chromatographic columns and mobile phase conditions were investigated and a good separation of the ICMs was obtained with a mixed-mode column (Acclaim Mixed-mode WAX) used in HILIC conditions. The validation was performed using a synthetic matrix: the limits of quantification (LOQ) were inferior to 1u202fµg/L and the linearity of each compound was comprised within the [0.5-50] µg/L range. The applicability of the HILIC-MRM3 method was assessed by the analysis of several raw waters. The results highlighted the presence of ICMs in most samples, at concentrations up to several mg/L in hospital sewage waters.

Determination of a new index of sexual maturity (ISM) in zebra mussel using flow cytometry: interest in ecotoxicology

The global dynamic spread of chemical contamination through the aquatic environment calls for the development of biomarkers of interest. Reproduction is a key element to be considered because it is related to the sustainability of species. Spermatogenesis is a complex process that leads to the formation of mature germ cells, whose steps and impairments need to be finely described in ecotoxicological analyses. The physiological process has been commonly described by histological analyses of gonads in different taxa. In the present paper, we describe the development of a novel technique to characterize spermatogenesis based on the analysis of the DNA content of germ cells by flow cytometry, using a DNA-intercalating agent. This new biomarker, referred to as an index of sexual maturity, proved relevant to describe the seasonal reproductive cycle of the zebra mussel, Dreissena polymorpha (Pallas, 1771), used as a sentinel species in the biomonitoring of continental waters and sensitive to highlight the reprotoxicity of carbamazepine (an anti-epileptic pharmaceutical) tested under ecosystemic conditions (mesocosms).

Occurrence of multi-class surfactants in urban wastewater: contribution of a healthcare facility to the pollution transported into the sewerage system

Healthcare facility discharges, by their nature, are often considered as non-domestic effluent, which can provide significant pollution comparatively to other domestic sources. In this context, a total of 12 monthly sampling campaigns were collected from a healthcare facility as well as the output of a sewerage system of Sitexa0Pilote de Bellecombe (SIPIBEL) observatory. This study focuses more specifically on 12 surfactants and biocides: four anionics, four cationic, two non-ionic, one zwitterionic, and one dispersive agent, among the most commonly used commercial surfactants. Particular attention was also provided to routine wastewater quality parameters. Both effluents were heavily contaminated by most anionic surfactants; they displayed median concentrations up to 1 to 2xa0mg/L for linear alkylbenzene sulfonates and between 10 and 100xa0μg/L for other sodium sulfate congeners (lauryl and laureth). Overall, for the majority of surfactants, the healthcare facility contribution to the total flux reaching the wastewater treatment plant ranges between 5 and 9%.