Chronic Embryo‐Larval Exposure of Fathead Minnows to the Pharmaceutical Drug Metformin: Survival, Growth, and Microbiome Responses

Abstract

Metformin is a glucose-lowering drug commonly found in municipal wastewater effluents (MWWEs). The present study investigated the chronic effects of metformin in early-life stages of the fathead minnow (Pimephales promelas). Endpoints assessed were growth, survival, and deformities. The larval gut microbiome was also examined using 16 S ribosomal RNA gene amplicon sequencing to determine microbial community composition and alpha and beta diversity. Eggs and larvae were exposed to metformin measured concentrations (mean [standard deviation]) of 0.020 (0.017) μg/L (for controls) and 3.44 (0.23), 33.6 (1.6), and 269 (11) μg/L in a daily static-renewal setup, with 20 embryos per beaker. The low and middle metformin exposure concentrations represent river and MWWE concentrations of metformin. To detect small changes in growth, we used 18 replicate beakers for controls and 9 replicates for each metformin treatment. Over the 21-d exposure (5 d as embryos and 16 d posthatch [dph]), metformin did not affect survival or growth of larval fish. Hatch success, time to hatch, deformities in hatched fry, and survival were similar across all treatments. Growth (wet wt, length, and condition factor) assessed at 9 and 16 dph was also unaffected by metformin. Assessment of the microbiome showed that the larvae microbiome was dominant in Proteobacteria and Firmicutes, with small increases in Proteobacteria and decreases in Firmicutes with increasing exposure to metformin. No treatment effects were found for microbiome diversity measures. Control fish euthanized with the anesthetic tricaine methane sulfonate had decreased alpha diversity compared to those sampled by spinal severance. This experiment demonstrates that metformin at environmentally relevant concentrations (3.44 and 33.6 μg/L) and at 10 times MWWE concentrations (269 µg/L) does not adversely affect larval growth or gut microbiome in this ubiquitous freshwater fish species.

Comparative Effects of Embryonic Metformin Exposure on Wild and Laboratory-Spawned Fathead Minnow (Pimephales promelas) Populations

“…Metformin is one of the most prevalent pharmaceuticals found in surface waters globally, and there is a growing body of evidence to suggest that current environmental exposure scenarios may adversely impact the reproduction and development of small-bodied fish. However, the nature and magnitude of effects appears to differ between species, timing of exposure, and study duration. ,,,,,− , For example, the growth of 28-DPF Japanese medaka (Oryzias latipes) was found to be significantly reduced by developmental exposure to environmentally relevant concentrations of metformin. , Similar effects were not observed in 21-DPF FHM exposed to comparable concentrations of metformin as embryos; however, other researchers have found that the growth and reproduction of FHM are affected by environmentally relevant concentrations of metformin at later life stages. − Exposure to nM concentrations of metformin also led to an increase in the occurrence of various morphological abnormalities in ELS zebrafish, along with behavioral alterations and changes in the proportion of unhatched eggs remaining at 5-DPF . The deformities observed in that study mirrored those observed in the present study, including skeletal abnormalities, cardiac edema, and yolk sac edema, with cardiac edema as the most frequently observed adverse outcome …”

“…Despite the widespread environmental occurrence of metformin in aquatic systems, the effects of developmental metformin exposure on nontarget aquatic biota have not yet been fully elucidated. Laboratory studies have found that the nature and magnitude of effects of metformin exposure differ between species, timing of exposure, and study duration. ,− Moreover, important variations in sensitivity may also exist between laboratory and wild fish populations of the same species and life stage. These differences can be attributed to a number of factors that vary between natural and artificial habitats, including the nature and intensity of environmental stress, abiotic and biotic characteristics of the environment that interact with animals, whether the potential for avoidance exists, the influence of artificial and natural selection on traits within each population, and the fate and partitioning behaviors of aquatic contaminants that affect bioavailability (and thus, exposure and risk). − Consequently, it is not uncommon for laboratory-derived water quality criteria and/or effect concentrations to under or overpredict toxicity for wild fish populations .…”

“…These differences can be attributed to a number of factors that vary between natural and artificial habitats, including the nature and intensity of environmental stress, abiotic and biotic characteristics of the environment that interact with animals, whether the potential for avoidance exists, the influence of artificial and natural selection on traits within each population, and the fate and partitioning behaviors of aquatic contaminants that affect bioavailability (and thus, exposure and risk). − Consequently, it is not uncommon for laboratory-derived water quality criteria and/or effect concentrations to under or overpredict toxicity for wild fish populations . growing body of laboratory-derived evidence suggests that current environmental concentrations of metformin may be sufficient to alter the reproduction and/or development of small-bodied prey fish. ,,,,,,− Given the potential importance of metformin as a ubiquitous pseudopersistent environmental contaminant, − ,,, the variability in laboratory study outcomes to date, ,− ,,,, and the need for highly representative toxicity data to inform regulatory decisions, , the present study utilizes a multiple-lines-of-evidence approach to investigate the developmental effects of metformin on a widely distributed small-bodied model fish species, the fathead minnow (Pimephales promelas).…”

“…Fathead minnow (FHM) embryos from a laboratory-spawned and a wild-spawned fish population were exposed to environmentally relevant concentrations of metformin from 0-days post-fertilization (DPF) through hatch (5-DPF) under controlled environmental conditions. Exposure solutions used for the lab-spawned cohort exposure were prepared using metformin-spiked reconstituted freshwater, consistent with previously conducted laboratory exposures. ,,− Exposure solutions used in the wild-spawned component of the study were subsampled from in-lake limnocorrals previously dosed with metformin to achieve environmentally relevant target concentrations (nominally, 0, 5, 50 μg/L metformin), thereby allowing us to account for environmental fate and transport processes that may influence outcomes. To our knowledge, this is the first study to directly compare the toxicological effects of metformin on both laboratory- and wild-spawned ELS FHM.…”

Pharmaceuticals in the Environment: Just One Stressor Among Others or Indicators for the Global Human Influence on Ecosystems?

“…Translating these suborganismal effects into ecological consequences that can then give regulators (and the broader community) confidence in setting environmental limits for this group of compounds is likely to be the next big challenge, because there are many different mechanisms through which they may exert an effect. Even when no effects on fish larvae and microbiome composition are noted for commonly used pharmaceuticals such as metformin (Parrott et al, 2022), the fact that some of them are so prevalent in the environment warrants ongoing evaluation in other nontarget species, in environmental compartments, and at different scales, especially as new evaluation methodology becomes available.…”