Metabolomics to study the sublethal effects of diazepam and irbesartan on glass eels (Anguilla anguilla)

Since glass eels are continuously exposed to contamination throughout their migratory journey in estuaries, to a certain extent the fall in the population of this endangered species might be attributed to this exposure, which is especially acute in estuaries under high urban pressure. In this work, metabolomics was used to address the main objective of this study, to evaluate the effects of two pharmaceuticals previously identified as potential concerning chemicals for fish (diazepam and irbesartan) on glass eels. An exposure experiment to diazepam, irbesartan and their mixture was carried out over 7 days followed by 7 days of depuration phase. After exposure, glass eels were individually sacrificed using a lethal bath of anesthesia, and then an unbiased sample extraction method was used to extract separately the polar metabolome and the lipidome. The polar metabolome was submitted to targeted and non-targeted analysis, whereas for the lipidome only the non-targeted analysis was carried out. A combined strategy using partial least squares discriminant analysis and univariate and multivariate statistical analysis (ANOVA, ASCA, t-test, and fold-change analysis) was used to identify the metabolites altered in the exposed groups with respect to the control group. The results of the polar metabolome analysis revealed that glass eels exposed to the diazepam-irbesartan mixture were the most impacted ones, with altered levels for 11 metabolites, some of them belonging to the energetic metabolism, which was confirmed to be sensitive to these contaminants. Additionally, the dysregulation of the levels of twelve lipids, most of them with energetic and structural functions, was also found after exposure to the mixture, which might be related to oxidative stress, inflammation, or alteration of the energetic metabolism.Authors acknowledge financial support from the Agencia Estatal de Investigación (AEI) of Spain and the European Regional Development Fund through CTM2017–84763-C3–1-R and CTM2020–117686RB-C31 projects and the Basque Government through the financial support as a consolidated group of the Basque Research System (IT1446–22). Naroa Lopez-Herguedas is grateful to the Spanish Ministry of Economy, Industry and Competitivity for her predoctoral scholarship FPI 2018. Iker Alvarez-Mora is grateful to the University of the Basque Country and the Université de Pau et des Pays de L' Adour for his cotutelle predoctoral scholarship

Mercury interaction with S-containing molecules: implications for methylation and demethylation processes in a sulfate reducing bacteria

Mercury methylation by anaerobic microorganisms, including sulfate-reducing bacteria (SRB), is a key process in the production of neurotoxic methylmercury (MeHg). The chemical speciation of mercury (Hg) strongly influences its bioavailability as well as its potential for methylation and demethylation, with sulfur-containing ligands playing a critical role in these processes. In this study, we used isotopically enriched mercury species (199Hg(II), Me202Hg) to investigate how molecular speciation of mercury affects both methylation and demethylation processes by the sulfate-reducer Pseudodesulfovibrio hydrargyri BerOc1. Experimental assays were carried out: (i) without external addition of S-ligands, (ii) with the addition of increasing concentrations of exogenous cysteine (Cys) (0.01, 0.1, and 0.5 mM), or (iii) with the addition of exogenous sulfide (0.1 mM). We showed that the highest methylation rate (Kmeth) was obtained without the external addition of S-ligands, whereas the addition of Cys or sulfide decreased Hg methylation regardless of Cys concentration. By quantitatively determining Hg(II) speciation in extracellular fractions, we demonstrated that Hg(II) was mostly present in the form of Hg(Cys)2, when Cys was added. However, metabolically sulfide production from Cys degradation shifted the chemical speciation of Hg(II) from Hg(Cys)2 to a more insoluble fraction (HgS(S)). In the assay without externally added ligands (Cys or sulfide), speciation models were generated by taking in account the metabolically produced thiols. These models established the predominance of Hg(II) complexes with a mixed ligation involving biosynthesized thiols, OH−, and Cl− ions. Our results suggest that these complexes with lower thermodynamic stabilities enhance the MeHg formation rate compared to the more stable Hg(Cys)2 or HgS(s) species. Unlike Hg(II) methylation, the addition of S-ligands did not affect the rates of demethylation (Kdemeth) of MeHg, even though it caused a shift in the chemical speciation of MeHg (from MeHgCl to MeHgCys and MeHgSH). These findings contribute to our understanding of the potential role of specific S-ligands and chemical speciation in governing the environmental fate and toxicity of mercury

SETApp: A machine learning and image analysis based application to automate the sea urchin embryo test

[EN] Since countless xenobiotic compounds are being found in the environment, ecotoxicology faces an astounding challenge in identifying toxicants. The combination of high-throughput in vivo/in vitro bioassays with high-resolution chemical analysis is an effective way to elucidate the cause-effect relationship. However, these combined strategies imply an enormous workload that can hinder their implementation in routine analysis. The purpose of this study was to develop a new high throughput screening method that could be used as a predictive expert system that automatically quantifies the size increase and malformation of the larvae and, thus, eases the application of the sea urchin embryo test in complex toxicant identification pipelines such as effect-directed analysis. For this task, a training set of 242 images was used to calibrate the size-increase and malformation level of the larvae. Two classification models based on partial least squares discriminant analysis (PLS-DA) were built and compared. Moreover, Hierarchical PLS-DA shows a high proficiency in classifying the larvae, achieving a prediction accuracy of 84 % in validation. The scripts built along the work were compiled in a user-friendly standalone app (SETApp) freely accessible at https://github.com/UPV-EHU-IBeA/SETApp. The SETApp was tested in a real case scenario to fulfill the tedious requirements of a WWTP effect-directed analysis.Authors gratefully acknowledge financial support from the Agencia Estatal de Investigación (AEI) of Spain and the European Regional Development Fund through project CTM2017–84763-C3–1-R and the Basque Government through the financial support as a consolidated group of the Basque Research System (IT1213–19). Iker Alvarez is grateful to the University of the Basque Country and the Université de Pau et des Pays de L′ Adour for his cotutelle predoctoral scholarship

Utilisation de traceurs isotopiques stables pour la spéciation du mercure et des butylétains: application à l'étude de la biométhylation du mercure dans les environnements aquatiques

The first part of this work involves the development of new analytical methods for the speciation analysis of mercury and butyltin compounds in environmental matrices. The optimization of these new methods for speciation analysis is based on the isotope dilution technique and has been developed specially for the simultaneous determination of mercury and butyltin compounds taking into account the simultaneous sample preparation and the simultaneous detection. The analytical development has been carried out for the different environmental matrix found in the aquatic ecosystems: water samples, biological tissues and sediments.In the second part of this work, the developed analytical techniques have been used to investigate biogeochemical cycles of mercury and butyltin into aquatic environments. Experimental methods, using isotopically enriched species as tracers, have been developed and have allowed the evaluation of mercury transformation mechanisms (methylation/demethylation) in the different compartments of the aquatics systems (in the sediments and the water column). Environmental factors and biological parameters have been also investigated in order to determine the possible link between the mercury transformation processes and the biological activities (bacterio- and phyto-plankton activities).La première partie de ce travail porte sur le développement de nouvelles méthodes pour l'analyse de spéciation du mercure et des butylétains dans les matrices environnementales. La mise au point de ces nouvelles techniques analytiques de spéciation est basée sur la quantification par dilution isotopique. Les techniques développées sont axées principalement sur la détermination simultanée des composés de mercure et des butylétains ce qui comprend la préparation d'échantillon simultanée ainsi que l'analyse simultanée. Le développement porte sur les différentes matrices composant les environnements aquatiques : l'eau, les tissus biologiques et les sédiments.Dans la deuxième partie de ce travail, les méthodes analytiques développées ont été appliquées à des études environnementales sur les cycles biogéochimiques du mercure et des butylétains dans les environnements aquatiques. Des méthodes expérimentales utilisant des espèces enrichies isotopiquement comme traceurs ont été développées et mises en œuvre. Elles permettent d'évaluer les taux de transformations du mercure (méthylation/déméthylation) dans les différents compartiments des environnements aquatiques que sont les sédiments et la colonne d'eau. Les facteurs environnementaux et notamment les paramètres biologiques sont aussi contrôlés afin de déterminer le lien entre les transformations du mercure et les activités des organismes vivants tels que les bactéries et le phytoplancton

Imaging Differential Mercury Species Bioaccumulation in Glass Eels Using Isotopic Tracers and Laser Ablation Inductively Coupled Plasma Mass Spectrometry

Dramatic increases in global mercury pollution require a deeper understanding of specific toxicity mechanisms for mercury compounds in organisms. Despite numerous studies addressing mercury toxicity, the detailed mechanisms underlying its transport and accumulation in fish remain unclear. The aim of this study was to unravel differential uptake pathways for mercury compounds, metabolisation, and sequestration mechanisms in glass eels using techniques able to localize at the tissue and organ levels. A multi isotope image mapping procedure was developed to simultaneously study the uptake and distribution of both mercury compounds MeHg and Hg(II) within the organs of the whole organism. The use of isotopically labelled Hg species (methylmercury Me201Hg and inorganic mercury 199Hg(II)) and image based on isotope ratio instead of elemental signals allowed to visualize spatially and with time the differential Hg species uptake, transport, and sequestration routes. The results showed a preferential uptake of the MeHg counterpart and a dynamic transport of MeHg within different organs. The gills were the main target organs for MeHg uptake, whereas the skeletal muscle was the final MeHg storage tissue. Hg(II) was found to mainly transit by the gills and the olfactory bulbs with a very low transfer and storage in the other organs and a rapid depuration. No significant internal demethylation and methylation was observed during this experimentation.</jats:p

Multi‐residue analysis of 44 pharmaceutical compounds in environmental water samples by solid‐phase extraction coupled to liquid chromatography‐tandem mass spectrometry

International audienceA solid‐phase extraction combined with a liquid chromatography‐tandem mass spectrometry analysis has been developed and validated for the simultaneous determination of 44 pharmaceuticals belonging to different therapeutic classes (i.e., antibiotics, anti‐inflammatories, cardiovascular agents, hormones, neuroleptics, and anxiolytics) in water samples. The sample preparation was optimized by studying target compounds retrieval after the following processes: i) water filtration, ii) solid phase extraction using Waters Oasis HLB cartridges at various pH, and iii) several evaporation techniques. The method was then validated by the analysis of spiked estuarine waters and wastewaters before and after treatment. Analytical performances were evaluated in terms of linearity, accuracy, precision, detection, and quantification limits. Recoveries of the pharmaceuticals were acceptable, instrumental detection limits varied between 0.001 and 25 pg injected and method quantification limits ranged from 0.01 to 30.3 ng/L. The precision of the method, calculated as relative standard deviation, ranged from 0.3 to 49.4%. This procedure has been successfully applied to the determination of the target analytes in estuarine waters and wastewaters. Eight of these 44 pharmaceuticals were detected in estuarine water, while 26 of them were detected in wastewater effluent. As expected, the highest values of occurrence and concentration were found in wastewater influent

Imaging Differential Mercury Species Bioaccumulation in Glass Eels Using Isotopic Tracers and Laser Ablation Inductively Coupled Plasma Mass Spectrometry

Dramatic increases in global mercury pollution require a deeper understanding of specific toxicity mechanisms for mercury compounds in organisms. Despite numerous studies addressing mercury toxicity, the detailed mechanisms underlying its transport and accumulation in fish remain unclear. The aim of this study was to unravel differential uptake pathways for mercury compounds, metabolisation, and sequestration mechanisms in glass eels using techniques able to localize at the tissue and organ levels. A multi isotope image mapping procedure was developed to simultaneously study the uptake and distribution of both mercury compounds MeHg and Hg(II) within the organs of the whole organism. The use of isotopically labelled Hg species (methylmercury Me201Hg and inorganic mercury 199Hg(II)) and image based on isotope ratio instead of elemental signals allowed to visualize spatially and with time the differential Hg species uptake, transport, and sequestration routes. The results showed a preferential uptake of the MeHg counterpart and a dynamic transport of MeHg within different organs. The gills were the main target organs for MeHg uptake, whereas the skeletal muscle was the final MeHg storage tissue. Hg(II) was found to mainly transit by the gills and the olfactory bulbs with a very low transfer and storage in the other organs and a rapid depuration. No significant internal demethylation and methylation was observed during this experimentation

Prioritization based on risk assessment to study the bioconcentration and biotransformation of pharmaceuticals in glass eels (Anguilla anguilla) from the Adour estuary (Basque Country, France)

International audienceThe presence of contaminants of emerging concern in the aquatic environment directly impacts water-living organisms and can alter their living functions. These compounds are often metabolized and excreted, but they can also be accumulated and spread through the food chain. The metabolized contaminants can also lead to the formation of new compounds with unknown toxicity and bioaccumulation potential. In this work, we have studied the occurrence, bioconcentration, and biotransformation of CECs in glass eels (Anguilla anguilla) using UHPLC-HRMS. To select the target CECs, we first carried out an environmental risk assessment of the WWTP effluent that releases directly into the Adour estuary (Bayonne, Basque Country, France). The risk quotients of every detected contaminant were calculated and three ecotoxicologically relevant contaminants were chosen to perform the exposure experiment: propranolol, diazepam, and irbesartan. An experiment of 14 days consisting of 7 days of exposure and 7 days of depuration was carried out to measure the bioconcentration of the chosen compounds. The quantitative results of the concentrations in glass eel showed that diazepam and irbesartan reached BCF ≈10 on day 7, but both compounds were eliminated after 7 days of depuration. On the other hand, propranolol's concentration remains constant all along with the experiment, and its presence can be detected even in the non-exposed control group, which might suggest environmental contamination. Two additional suspect screening strategies were used to identify metabolization products of the target compounds and other xenobiotics already present in wild glass eels. Only one metabolite was identified, nordiazepam, a well-known diazepam metabolite, probably due to the low metabolic rate of glass eels at this stage. The xenobiotic screening confirmed the presence of more xenobiotics in wild glass eels, prominent among them, the pharmaceuticals exemestane, primidone, iloprost, and norethandrolone. ☆ This paper has been recommended for acceptance by. Eddy Y. Zeng. ☆☆ Contaminants of Emerging Concern in Glass Eel (Anguilla anguilla): Occurrence, Bioconcentration and Biotransformation

Imaging differential mercury and selenium species bioaccumulation by glass eels using isotopic tracers and laser ablation

International audienc