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

Metformin is a glucose-lowering drug commonly found municipal wastewater effluents (MWWEs). The current 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 16S rRNA 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 set-up, with 20 embryos per beaker. The low and mid 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 days as embryos, and 16 days post-hatch, 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 weight, length, and condition factor) assessed at 9 and 16 dph was also unaffected by metformin. Assessment of the microbiome showed 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. The anaesthetic MS222 affected alpha diversity of control larvae compared to spinal severance. This experiment demonstrates that metformin at environmentally-relevant concentrations (3.44 and 33.6 μg/L), and at 10xMWWE concentrations (269 µg/L), does not adversely affect larval growth or gut microbiome in this ubiquitous freshwater fish species. This article is protected by copyright. All rights reserved.