1H NMR-based metabolomics reveals interactive effects between the carrier solvent methanol and a pharmaceutical mixture in an amphibian developmental bioassay with Limnodynastes peronii

Abstract

Organic carrier solvents are used in aquatic toxicity testing to improve chemical solubility and facilitate the exploration of dose-response relationships. Both water- and solvent-control groups are normally included in these scenarios to ensure that the solvent itself has no effect on the test organism, but this fails to consider possible interactive effects between carrier solvents and contaminants of interest. We explored this topic by exposing Limnodynastes peronii tadpoles to a mixture of common water-soluble pharmaceuticals (diclofenac, metformin and valproic acid) in the presence and absence of the carrier solvent methanol, according to standard developmental bioassay methodology. Nuclear Magnetic Resonance (NMR) spectroscopy was applied as a platform for untargeted metabolomics, to compare broad sub-lethal hepatotoxicity in solvent- and solvent-free exposure scenarios. Considerable interactive effects were identified between the pharmaceutical mixture and a typical dose of methanol (0.003%). Specifically, pronounced differences were observed between the solvent- and solvent-free exposure groups for leucine, acetate, glutamine, citrate, glycogen, tyrosine, arginine, purine nucleotides and an unidentified metabolite at 6.53 ppm. Various other metabolites exhibited similar disparity related to the use of carrier solvent, but the interactions were non-significant. These results raise important questions about the use of carrier solvents for chemical exposures in aquatic ecotoxicology, and particularly for studies interested in sub-lethal mechanistic information and/or biomarker discovery.

Metabolite profiles of striped marsh frog (Limnodynastes peronii) larvae exposed to the anti-androgenic fungicides vinclozolin and propiconazole are consistent with altered steroidogenesis and oxidative stress

“…We would be remiss if we failed to discuss the use of carrier solvents in this and other studies, since it is becoming evident that organic solvents may have an effect on sub-lethal endpoints including aspects of the metabolome (David et al, 2012). In some cases it seems that carrier solvents may even interact with test chemicals (Aliferis and Jabaji, 2011;Melvin et al, 2017;Melvin et al, 2018). There are unfortunately currently limited options for addressing this problem, and the use of solvents is important for pushing solubility limits to facilitate testing dose-response relationships in ecotoxicology (Maes et al, 2012).…”

A multifaceted assessment of the effects of polyethylene microplastics on juvenile gilthead seabreams (Sparus aurata)

“…Samples were placed in Eppendorf tubes and immediately flashfrozen in liquid nitrogen and stored at -80 °C until processing. Samples were then lyophilized and coarsely ground with a ceramic pestle prior to extraction of polar metabolites using a modified methanol:chloroform extraction (Bligh and Dyer, 1959), as previously described (Melvin et al, 2017(Melvin et al, , 2018. Following steps toward the investigation of metabolite profiles were similar to the ones described in Belivermis et al (2020) using the same type of equipment, software, spectra treatments and analyses (NMR being a 800MHz Bruker® Avance III HDX spectrometer equipped with a Triple -TCI-Resonance 5mm Cryoprobe and controlled with IconNMR™ software).…”

(Xeno)metabolomics for the evaluation of aquatic organism’s exposure to field contaminated water

“…Cappello et.al. [27], for gill tissue, used ultraturrax homogenization with MeOH, followed by the previously exposed bi-phasic separation (with a final proportion of MeOH:H2O:CHCl3 of 2:1:4 for Melvin [28] For the rest of studies, the authors have preferred bead beating extraction for the whole body, incubation [7] or simply vortex agitation [29].…”