The ecotoxicological effects of metformin and its metabolite, guanylurea, on Japanese medaka (Oryzias latipes)

Abstract

Human pharmaceutical waste threatens many natural processes of non-target aquatic organisms, through the introduction of such contaminants into the environment. Pharmaceuticals are a large and diverse groups of medicinal compounds that are used for the diagnosis, cure, mitigation, treatment, and/or prevention of diseases in humans and animals. In recent years, the occurrence and fate of pharmaceuticals in the aquatic environment have become an emerging issue in aquatic toxicology, with one of the most prevalent contaminants being the type-2 diabetic drug, metformin, and its metabolite guanylurea. Metformin and guanylurea have been measured in surface waters in the ng- μg·L-1 range. This study aimed to investigate the toxicological effects of metformin and guanylurea on Japanese medaka (Oryzias medaka). A first experimental step was to determine the potential for uptake, bioaccumulation, and depuration of metformin in early-life stage (ELS) medaka using 14C-metformin. Results from this study showed that both embryo and larval medaka can take up, bioaccumulate, and successfully depurate metformin from the aquatic environment. Furthermore, egg chorion hardening (~6 hpf) prevented the uptake of metformin from by developing embryos. Additionally, we show that waterborne exposure to metformin and guanylurea significantly impacted the growth metrics in ELS fish, showing that guanylurea caused growth retardation at exposure concentrations an order of magnitude lower that those required by the parent compound to exert similar effects. We sought to explain these growth effects by investigating potential alterations in the metabolome and gene expression of these exposed fish. Several metabolite abundances were altered by the exposure of medaka to 3.2 μg·L-1 metformin and 1.0 ng·L-1 guanylurea, which included metabolites associated with cellular energetics, fatty acid synthesis and metabolism, and polyamine synthesis. Metabolomic results were applied to select genes of interest for RT-qPCR analyses, and the expressions of critical genes involved in lipid metabolism were shown to be significantly affected. Furthermore, a full 165 day life-cycle study assessing the effects of metformin and guanylurea alone, and in combination, was performed to determine potential population-level impacts of the compounds on Japanese medaka. While exposure to metformin and guanylurea alone, and in combination, did not significantly stunt the growth of medaka, exposure to the compounds did alter the production of important sex steroid hormones in both male and female medaka. However experimental treatments did not affect the production of the egg yolk precursor protein, vitellogenin. Interestingly, no significant effect on reproductive output was seen. Collectively, these results underscore the need for additional studies examining the effects of metformin and guanylurea on aquatic ecosystems. To our knowledge, this research is the first to investigate the effects of environmentally relevant concentrations of these compounds in this manner.