Metabolic profiling identification of metabolites formed in Mediterranean mussels (Mytilus galloprovincialis) after diclofenac exposure
Graphical abstract
Introduction
Pharmaceutically active compounds (PhACs) in aquatic environments have become major contaminants of interest over the last decade (Boxall et al., 2012, Fent et al., 2006). Their presence is due mostly to their limited removal rate in wastewater treatment plant effluents, which are considered as the main source of PhACs (Santos et al., 2010). A very small number of them have just been added to the first European Union Water Framework Directive watch list (Directive 2008/105/EC, European Commission, 2015). Diclofenac (DCF) was included on the list with the aim of gathering the first monitoring data.
Marine waters, like surface waters, are concerned by PhAC inputs because of the high population growth rates in coastal areas and the development of sea outfall wastewater treatment plants (Fenet et al., 2014). The extent of coastal PhAC contamination has been studied much less than in continental waters, under the pretext that dilution rates are high in such environments (Maruya et al., 2012). However, lagoons—which are transition zones between continental and marine waters—do not benefit from this dilution and are known to be nursery grounds for early marine fish life stages and a habitat for aquatic shellfish species such as mussels. Relatively few studies have focused on the potential impact of pharmaceuticals and their bioconcentration in organisms inhabiting coastal environments (Huerta et al., 2012). Recent studies have nevertheless been published on the occurrence and bioconcentration of drug residues in seawater and marine organisms, thus raising questions on associated environmental risks (Alvarez et al., 2014, Arpin-Pont et al., 2016, Moreno-González et al., 2016). Among PhACs found in seawater, DCF concentrations have been reported from few ng/L to approximately 1 μg/L (Gaw et al., 2014).
PhAC exposure may lead to bioconcentration in non-target organisms, particularly mussels because of their limited mobility and filter feeding behavior. Once bioconcentrated, pharmaceuticals can be metabolized by organisms. Recent studies demonstrated that mussels exposed to DCF had a bioconcentration factor (BCF) ranging from 4 to 13, depending on the exposure concentration (Daniele et al., 2016b, Ericson et al., 2010). These results implied a low bioconcentration in mussel tissues, thus raising the question of possible DCF metabolization.
To our knowledge, only a few studies have investigated DCF metabolism in aquatic organisms. Two of them were conducted in bile of rainbow trout (Oncorhynchus mykiss) (Kallio et al., 2010, Lahti et al., 2011). The first study (Kallio et al., 2010) was conducted after intraperitoneal exposure to 0.25 mg DCF/100 g fish biomass and the bile samples were collected at 2 days postinjection. The second study (Lahti et al., 2011) was carried out after 10 days of exposure to different DCF concentrations, i.e. an environmental (1–2 μg/L) and a higher (25–50 μg/L) concentration. The results of both studies highlighted the presence of DCF and some of its main metabolites, i.e. 4′-hydroxy-diclofenac and 5-hydroxy-diclofenac, as well as their sulfate and glucuronide conjugates. In both studies, the acyl glucuronide of 3′-hydroxy-diclofenac was also detected. In a third study, DCF metabolite formation was studied in three-spined sticklebacks (Gasterosteus aculeatus). The fish were exposed for 6 months to various environmental DCF concentrations (0.05; 0.45 and 4.1 μg/L). Only 4′-hydroxy-diclofenac was detected in fish exposed to the highest concentration (Daniele et al., 2016a). Finally, a last study was conducted on zebra mussels, which to our knowledge is the only study that has been performed on bivalves (Daniele et al., 2016b). Mussels were exposed to three different DCF concentrations (0.05; 0.5 and 5 μg/L) for two different durations, i.e. 3 and 6 months. Only 2-indolone, a DCF transformation product, was detected in mussel tissues. Note that these four studies were conducted on the basis of a targeted analysis, i.e. a search for already known transformation products, thus limiting the possibility of finding unacknowledged metabolites in organisms such as mussels for which information on PhAC metabolism is limited.
The use of a non-targeted approach, based on the generation of profiles of chemicals detected in organisms exposed and unexposed to a xenobiotic could be a good strategy for studying PhAC metabolism in such organisms. A semi-quantitative mass spectrometry approach based on the comparison of signal intensities detected in both groups of organisms could highlight differential signals corresponding to the administered xenobiotic and its metabolites. This kind of comprehensive approach for characterizing PhAC metabolites has been convincingly applied for drug metabolite detection in rat biological fluids (Plumb et al., 2003). However, it is not yet well applied in ecotoxicology even though it is proven powerful in toxicology (Werner et al., 2008). For example, (Southam et al., 2011) demonstrated by such an approach that the main route of fenitrothion (organophosphorus pesticide) degradation in roaches (Rutilus rutilus) was O-demethylation based on a metabolomic study performed using Fourier Transform mass spectrometry. This kind of approach could thus be suitable for studying PhACs metabolism in non-target organisms for which data is scarce.
In this context, while very little information is available on the DCF biotransformation in non-target organisms, the goal of the present study was to investigate the potential of such a non-targeted approach for identifying DCF metabolites produced in mussels after exposure. To increase the probability of metabolite detection, mussels were exposed to two DCF concentrations that were higher than those usually found in marine waters. Analyses were performed with liquid chromatography (LC) combined with high resolution mass spectrometry (HRMS). This approach allowed us to putatively identify 13 DCF metabolites in mussel tissues, two of which have yet to be assigned. This is the first time that as many DCF metabolites were reported in an aquatic organism. After metabolite identification, those for which an analytical standard was available were quantified in both tissues and seawater for the two exposure concentrations.
Section snippets
Chemicals
DCF (≥ 98%) and diclofenac-d4 (DCF-d4, ≥ 98%) were purchased from Sigma-Aldrich (Steinheim, Germany). 4′-hydroxy-diclofenac (4′OH-DCF, ≥ 97%) and 5-hydroxy-diclofenac (5OH-DCF, 98% ± 2%) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Stock standard solutions of individual compounds were prepared at 1000 mg/L concentration in methanol for DCF, DCF-d4, 4′OH-DCF and 5OH-DCF. Subsequent stock standard dilutions were prepared with methanol. All standard solutions were stored at − 20 °C.
Analytical performances
Linearity was found to be satisfactory, with R2 values better than 0.99 for all target analytes in tissue and better than 0.97 in seawater samples. The average repeatabilities for DCF-d4, DCF, 4′OH-DCF, and 5OH-DCF were around 11% and never exceeded 21% (Table 2). Absolute recovery ranges were higher than 66% for DCF-d4, DCF, 4′OH-DCF, and 5OH-DCF (Table 2).
DCF quantification
The DCF concentrations were measured in water to control the exposure of mussels in aquaria. The results are presented in Table 3. DCF was
Discussion
The observed BCF (11.3 and 16.5 for DCF exposure at 100 and 600 μg/L, respectively) were in the same range as those already obtained for mussels exposed to DCF. In a first study, the BCF reported for Baltic Sea mussels exposed to 1 mg/L of DCF was of 10 (Ericson et al., 2010). A second study conducted in zebra mussels reported a BCF ranging from 4 to 13 following exposure to DCF environmental concentrations (0.05, 0.5 and 5 μg/L) for 3 and 6 months (Daniele et al., 2016b). Based on the obtained
Conclusion
The use of a non-targeted approach was powerfully applied for screening DCF transformation products in Mediterranean mussels. It allowed us to detect the parent compound and 13 DCF metabolites, 3 of which were phase I metabolites, and 10 were phase II metabolites. Non-targeted analysis, carried out without any a priori, enabled us to detect metabolites that we might have overlooked in a targeted analysis because of their general lack of mention in the literature. We conclude that in
Acknowledgments
Funding support was obtained from the the French National Research Program for Environmental and Occupational Health of Agence Nationale de Sécurité Sanitaire de l'alimentation, de l'environnement et du travail (AMeCE 2015/1/091) and the Agence Nationale de la Recherche (IMAP ANR-16-CE34-0006-01). This research benefited from the support of the Chair Veolia Environnement - HydroSciences: Risk analysis relating to emerging contaminants in water bodies. The doctoral fellowship of Bénilde
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2023, TalantaCitation Excerpt :Bivalve mollusks as marine mussels (Mytilus galloprovincialis) are organisms tolerant to a certain degree of pollution and resistant to environmental changes; they are also ubiquitous and sedentary filter feeders that may accumulate pollutants present in the surrounding environment [14]. Because of that and their commercial interest in human consumption, mussels have been widely used as sentinels for chemical pollution biomonitoring studies, as well as in metabolomics experiments of exposure to organic microcontaminants under controlled conditions [15–18]. In addition to the environmental interest, the distribution and selling of mussels exposed to antibiotics may be a risk for consumers such as allergy and toxicity.