Development of innovative analytical tools based on nanoliquid chromatography coupled to mass spectrometry for the assessment of bioaccumulation and biotransformation of emerging pollutants in freshwater invertebrates

L'écosystème aquatique est le résultat d'un équilibre entre l'environnement naturel et les organismes qui s'y développent. Cet équilibre peut être modifié par l'introduction de substances chimiques dues aux activités humaines. Aujourd'hui, l'impact de cette micropollution est encore mal connu, particulièrement pour les organismes constituant les premiers maillons des chaînes trophiques qui requièrent des efforts analytiques importants en raison de leur faible taille. L'objectif de ces travaux est centré sur le développement, la validation et l'application d'outils analytiques permettant d'étudier la bioaccumulation et la biotransformation de polluants émergents chez des invertébrés benthiques. Cette étude s'est focalisée sur 3 organismes aquatiques d'eau douce : C. riparius, G. fossarum et P. antipodarum. Une méthode analytique pour la quantification de 35 polluants émergents chez les 3 espèces sélectionnées a ainsi été développée. Elle permet d'accéder aux premières données de bioaccumulation des substances d'intérêts à l'échelle d'un individu grâce à la mise en oeuvre d'une stratégie analytique entièrement miniaturisée, incluant une extraction MicroQuEChERS suivie d'une analyse par nanochromatographie liquide couplée à la spectrométrie de masse en tandem. Afin de mieux comprendre l'impact d'une telle pollution sur les espèces sélectionnées et d'obtenir une vue globale des capacités de biotransformation de celles-ci, une approche métabolomique a été mise en place. Enfin, un nouveau mode de quantification par MRM3 a été utilisé pour dépasser la complexité de telles matrices, et fournir une évaluation fiable des cinétiques de bioaccumulation de potentiels traceurs de pollution anthropiqueThe aquatic ecosystem is the result of a balance between the natural environment and the organisms that inhabit it. This balance can be modified by the input of excessive amount of substances generated from human activities. Nowadays, the impact of this pollution is still little known, especially regarding the risk of bioaccumulation in the first trophic levels. This lack of data could be partially explained by the lack of suitable analytical method for small organisms. In this context, the aim of this study is to establish the development, the validation and the application of analytical tools for the assessment of bioaccumulation and biotransformation of emerging pollutants in freshwater invertebrates. Three benthic invertebrates are chosen for this project: C. riparius, G. fossarum and P. antipodarum. Analytical method has been developed for the quantification of 35 emerging pollutants in the selected species. This method provides the first bioaccumulation data of targeted substances in individual scale through the implementation of miniaturized analytical strategy, including an extraction step based on MicroQuEChERS followed by nanoliquid chromatography coupled to tandem mass spectrometry analysis. To better understand the impact of such pollution and to obtain a global view of the biotransformation capacities of the selected organisms, metabolomics approach has been put in place. Finally, a new quantification mode based on MRM3 was used to overcome biotic matrix complexity and assess the bioaccumulation kinetics of potential tracers of anthropic pollutio

Mass spectrometry characterization of anticoagulant rodenticides and hydroxyl metabolites

International audienceRationaleAnticoagulant rodenticides (ARs) are used worldwide for rodent population control to protect human health and biodiversity, and to prevent agricultural and economic losses. Rodents may develop a metabolic resistance to ARs. In order to help understand such metabolic resistance, mass spectrometry was used to position the hydroxylated group of hydroxyl metabolites of second‐generation ARs (SGARs).MethodsMost AR pesticides are derived from the 4‐hydroxycoumarin/thiocoumarin family. We used low‐resolution and high‐resolution mass spectrometry to understand the fragmentation pathways of the ARs and their respective metabolites, and to better define the structure of their tandem mass spectrometry product ions.ResultsSeven specific product ions were evidenced for five ARs, with their respective chemical structures. Those ions were obtained as well from the mass spectra of the hydroxyl metabolites of four SGARs, difenacoum (DFM), brodifacoum (BFM), difethialone (DFTL) and flocoumafen (FLO), with different positions of the hydroxyl group.ConclusionsThe differences in chemical structure between DFM on the one hand and BFM, FLO and DFTL on the other could explain the differences in bioavailability between these two groups of molecules. The defined product ions will be used to investigate the part played by the metabolic issue in the field resistance of SGARs

Mass spectrometry characterization of anticoagulant rodenticides and hydroxyl metabolites

International audienceRationaleAnticoagulant rodenticides (ARs) are used worldwide for rodent population control to protect human health and biodiversity, and to prevent agricultural and economic losses. Rodents may develop a metabolic resistance to ARs. In order to help understand such metabolic resistance, mass spectrometry was used to position the hydroxylated group of hydroxyl metabolites of second‐generation ARs (SGARs).MethodsMost AR pesticides are derived from the 4‐hydroxycoumarin/thiocoumarin family. We used low‐resolution and high‐resolution mass spectrometry to understand the fragmentation pathways of the ARs and their respective metabolites, and to better define the structure of their tandem mass spectrometry product ions.ResultsSeven specific product ions were evidenced for five ARs, with their respective chemical structures. Those ions were obtained as well from the mass spectra of the hydroxyl metabolites of four SGARs, difenacoum (DFM), brodifacoum (BFM), difethialone (DFTL) and flocoumafen (FLO), with different positions of the hydroxyl group.ConclusionsThe differences in chemical structure between DFM on the one hand and BFM, FLO and DFTL on the other could explain the differences in bioavailability between these two groups of molecules. The defined product ions will be used to investigate the part played by the metabolic issue in the field resistance of SGARs

Multi-residue analysis of emerging pollutants in benthic invertebrates by modified micro-QuEChERS extraction and nanoLC-MS/MS analysis: a new strategy to evaluate bioaccumulation.

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Development of a multi-residue method of emerging pollutants in benthic invertebrates using modified micro-QuEChERS extraction followed by nanoLC-MS/MS analysis: a new strategy to evaluate bioaccumulation

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Quantification of of emerging micropollutants in an amphipod crustacean by nanoliquid chromatography coupled to mass spectrometry using multiple reaction monitoring cubed mode

International audienceAn innovative analytical method has been developed to quantify the bioaccumulation in an amphipod crustacean (Gammarus fossarum) of three micropollutants regarded as anthropic-pollution markers: carbamazepine, oxazepam, and testosterone. A liquid-liquid extraction assisted by salts, known as QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) was miniaturised and optimised, so it could be adapted to the low mass samples (approximatively 5 mg dry weight). For this same reason and in order to obtain good sensitivity, ultra-trace analyses were carried out by means of nanoliquid chromatography. A pre-concentration system by on-column trapping was optimised to increase the injection volume. In order to improve both sensitivity and selectivity, the multiple reaction monitoring cubed mode analyses (MRM3) were carried out, validated and compared to the classic MRM. To the best of our knowledge, this is the first time that MRM3 is coupled to nanoliquid chromatography for the analysis and detection of organic micropollutants <300 Da. The optimised extraction method exhibited recoveries superior to 80%. The limits of quantification of the target compounds were 0.3, 0.7 and 4.7 ng/g (wet weight) for oxazepam, carbamazepine and testosterone, respectively and the limits of detection were 0.1, 0.3 and 2.2 ng/g (wet weight), respectively. The intra- and inter-day precisions were inferior to 7.7% and 10.9%, respectively, for the three levels of concentration tested. The analytical strategy developed allowed to obtain limits of quantification lower than 1 ng/g (wet weight) and to establish the kinetic bioconcentration of contaminants within G. fossarum

Quantification of emerging micropollutants in an amphipod crustacean by nanoliquid chromatography coupled with mass spectrometry in MRM3 mode

International audienceOver the last decades, human activities (industries, agriculture, transports, consumption, urbanization…) and their impacts on the environment have not ceased to increase. Many molecules related to these activities are released to the environment and their occurrences within aquatic ecosystem become an important issue. In particular, the dose-response relationships of emerging pollutants on organisms at lower trophic levels remain little-known. In order to conduct biomonitoring studies, it is necessary to acquire knowledge about the kinetics of bioconcentration and depuration of these molecules within species, taking into account the inter-individual variability. In this context, an innovative analytical method was developed to quantify, in an amphipod crustacean (Gammarus fossarum), three emerging micropollutants known as anthropic pollution markers: carbamazepine, oxazepam and testosterone which are respectively an anticonvulsant, an anxiolytic and an endogenous hormone. A salting out liquid-liquid extraction, known as QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) was miniaturized and optimized. The miniaturization of the method made it possible to adapt to the low mass sample (approximatively 5 mg dry weight) and to perform the analysis at the scale of a single organism. Beside an efficient sample preparation, ultra-trace analysis of compounds in such matrices of small size requires a good sensitivity. Therefore, an original coupling was developed. It consisted of a separation by nanoliquid chromatography (nanoLC) coupled to tandem mass spectrometry using multiple reaction monitoring cubed mode (MRM3). For the nanoLC separation, a preconcentration system by on-column trapping was optimized to increase the injection volume. In order to keep both sensitivity and selectivity, detection is usually performed by mass spectrometry in multiple reaction monitoring mode (MRM). Nevertheless, proteomic studies showed that sensitivity and selectivity could be improved using MRM3 mode. This technic was developed in this project and compared to the classic MRM. In the best of our knowledge, this was the first time that MRM3was used for small molecule ( The analytical strategy developed allowed to obtain limits of quantification lower than 1 ng.g-1 (wet weight) and to establish the kinetics of bioconcentration of contaminants within G. fossarum