Metabolomic Profiling and Toxicokinetics Modeling to Assess the Effects of the Pharmaceutical Diclofenac in the Aquatic Invertebrate Hyalella azteca

Abstract

The exposure of ecologically critical invertebrate species to biologically active pharmaceuticals poses a serious risk to the aquatic ecosystem. Yet, the fate and toxic effects of pharmaceuticals on these nontarget aquatic invertebrates and the underlying mechanisms are poorly studied. Herein, we investigated the toxicokinetic (TK) processes (i.e., uptake, biotransformation, and elimination) of the pharmaceutical diclofenac and its biotransformation in the freshwater invertebrate Hyalella azteca. We further employed mass spectrometry-based metabolomics to assess the toxic effects of diclofenac on the metabolic functions of H. azteca exposed to environmentally relevant concentrations (10 and 100 μg/L). The TK results showed a quick uptake of diclofenac by H. azteca (maximum internal concentration of 1.9 μmol/kg) and rapid formation of the conjugate diclofenac taurine (maximum internal concentration of 80.6 μmol/kg), indicating over 40 times higher accumulation of diclofenac taurine than that of diclofenac in H. azteca. Depuration kinetics demonstrated that the elimination of diclofenac taurine was 64 times slower than diclofenac in H. azteca. Metabolomics results suggested that diclofenac inhibited prostaglandin synthesis and affected the carnitine shuttle pathway at environmentally relevant concentrations. These findings shed light on the significance of the TK process of diclofenac, especially the formation of diclofenac taurine, as well as the sublethal effects of diclofenac on the bulk metabolome of H. azteca. Combining the TK processes and metabolomics provides complementary insights and thus a better mechanistic understanding of the effects of diclofenac in aquatic invertebrates.

Wastewater effluent affects behaviour and metabolomic endpoints in damselfly larvae

“…Perturbations in other carnitine metabolites have been observed in other species, e.g. unionid mussel Lampsilis fasciola exposed to EE2 26 or amphipod Hyalella azteca exposed to diclofenac 80 . Carnitines consist of fatty acids and are responsible for their transport into mitochondria for subsequent oxidation for energy production.…”

Environmental Metabolomics Promises and Achievements in the Field of Aquatic Ecotoxicology: Viewed through the Pharmaceutical Lens

“…The 100 µg/L threshold was chosen as the upper environmental concentration, likely to occur in areas of the world where wastewater is untreated, whereas the most common concentrations in fresh or marine waters tend to be less than 1 µg/L [18,19]. Finally, 16 articles published between 2014 and 2022 were selected [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. Substantial information was extracted from each of them and is summarized in Table S1, such as the studied PhAC and its therapeutic class, the exposure conditions (time and concentrations), the studied organism and its sex, the sample type (e.g., organ, whole organism, and biofluide), and the analytical method used (e.g., mass spectrometry combined with liquid or gas chromatography (LC/GC-MS), and protonic nuclear magnetic resonance spectroscopy ( 1 H NMR)), and significantly modulated metabolites and their associated metabolic pathways.…”

“…In addition to providing an overview of the molecular effects triggered by contaminant exposure, environmental metabolomics has a high potential to decipher the contaminant MeOA via the identification of toxicity pathways and/or signatures [36]. Regarding examples dealing with PhACs, Fu et al, highlighted the MeOA of diclofenac in the Hyalella azteca crustacean exposed for 10 days to 10 and 100 µg/L concentrations [24]. Significant changes in metabolite abundance-more pronounced with increasing diclofenac concentrations-were measured, e.g., prostaglandin E1 (down-modulation), arachidonic acid (up-modulation), and three other prostaglandin metabolites.…”

Metabolomics study on southern hard clams (Mercenaria campechiensis) response to diclofenac exposure

“…Nonsteroidal anti-inflammatory drug (NSAID) is one of the major classes of PPCPs that have been used globally (5), and have been constantly released to the aquatic environment due to universal consumption, low human metabolic capability, improper disposal, and incomplete removal from the wastewater treatment plants (6,7). Diclofenac is one prescribed NSAID that has been widely detected in various aquatic ecosystems ranging from the ng/L to g/L range in surface seawater (8,9) and from ng/g to g/g in aquatic organisms (10,11). Diclofenac has been added to the first European Union Water Framework Directive Watch List in 2015 (12) and for continuous monitoring (13).…”

“…Diclofenac is biologically active at low doses to the targeted organism such as humans with a specific mechanism of action, therefore, its nontarget organisms in the aquatic environment are concerned by various studies. The adverse effect of diclofenac has been studied in several nontarget organisms such as marine mussels (Mytilus galloprovincialis) (14)(15)(16), zebrafish (Danio rerio) (17), rainbow trout (Oncorhynchus mykiss) (18,19), freshwater invertebrate (Hyalella azteca) (10), common carp (Cyprinus carpio) (20), brown trout (Salmo trutta f. fario) (21,22), and three-spined stickleback (Gasterosteus aculeatus) (23).…”