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 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. Environ Toxicol Chem 2022;41:635-647. © 2021 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

The gut content microbiome of wild-caught rainbow darter is altered during laboratory acclimation.

An increasing number of laboratory studies are showing that environmental stressors and diet affect the fish gut microbiome. However, the application of these results to wild populations is uncertain as little is known about how the gut microbiome shifts when fish are transitioned from the field to the laboratory. To assess this, intestinal contents (i.e. digesta) of wild-caught rainbow darter (Etheostoma caeruleum) were sampled in the field and in the lab after 14- and 42-days acclimation. In addition, from days 15-42 some fish were exposed to waterborne triclosan, an antimicrobial found in aquatic ecosystems, or to dilutions of municipal wastewater effluents, to determine how these stressors affect the bacterial communities of gut contents. 16S rRNA gene amplicon sequencing was used to determine microbial community composition, alpha, and beta diversity present in the fish gut contents. In total, there was 8,074,658 reads and 11,853 amplicon sequence variants (ASVs) identified. The gut contents of wild fish were dominant in both Proteobacteria (35%) and Firmicutes (27%), while lab fish were dominant in Firmicutes (37-47%) and had lower alpha diversity. Wild fish had greater ASVs per sample (423-1304) compared to lab fish (19-685). Similarly, the beta-diversity of these bacterial communities differed between field and lab control fish; control fish were distinct from the 10% wastewater effluent and 100 ng/L TCS treatment groups. Results indicate that the gut microbiome of wild fish changes with the transition to laboratory environments; hence, prolonged acclimation to new settings may be required to achieve a stable gut content microbiome in wild-caught fish. Research is required to understand the length of time required to reach a stable fish gut microbiome.

Rainbow darter (Etheostoma caeruleum) from a river impacted by municipal wastewater effluents have altered gut content microbiomes.

Municipal wastewater treatment plant (WWTP) effluent contains pharmaceuticals and personal care products known to affect fish health and reproduction. The microbiome is a community of bacteria integral in maintaining host health and is influenced by species, diet, and environment. This study investigated changes in the diversity and composition of the gut content microbiome of rainbow darter (Etheostoma caeruleum) at ten sites on the Grand River, Ontario, Canada. Gut contents were collected in fall 2018 from these fish at sites upstream and downstream of two municipal wastewater treatment plants (WWTPs; Waterloo and Kitchener). 16S rRNA genes were sequenced to determine the composition and diversity (alpha and beta) of microbial taxa present. Gut content bacterial alpha diversity increased downstream of both WWTP outfalls; dominance of bacterial amplicon sequence variants decreased compared to upstream fish. Fish collected at different sites had distinct bacterial communities, with upstream samples dominant in Proteobacteria and Firmicutes, and downstream samples increasingly abundant in Proteobacteria and Cyanobacteria. In mammals, increased abundance of Proteobacteria is indicative of microbial dysbiosis and has been linked to altered health outcomes, but this is not yet known for fish. This research indicates that the fish gut content microbiome was altered downstream of WWTP effluent outfalls and could lead to negative health outcomes.

Gestational triphenyl phosphate exposure in C57Bl/6 mice perturbs expression of insulin-like growth factor signaling genes in maternal and fetal liver.

Triphenyl phosphate (TPhP) is an organophosphorus flame retardant and plasticizer that has been added to numerous consumer products in recent years. TPhP is not overtly toxic, however recent studies have suggested that it may have metabolic disrupting effects following developmental exposure. The present study aimed to investigate the developmental and potential metabolic effects of TPhP in a murine model. C57Bl/6 dams were exposed on gestational days (GD) 8, 10, 12, and 14 to 0, 5, 25, or 50 mg/kg TPhP via intraperitoneal injection. Dams were euthanized on GD19, maternal organs excised and weighed, fetal measurements taken, and maternal and fetal livers retained for analysis. A significant increase in placenta size of TPhP exposed mice was found. Maternal and fetal liver gene expression of insulin-like growth factor (Igf) 1 and 2, as well as downstream genes involved in Igf signaling were measured. Additionally, Igf1 protein levels were measured in both maternal and fetal liver. A significant decrease in transcript levels of Igf1 and Irs2 was detected in maternal livers, whereas a significant increase in transcript levels of all genes measured was detected in fetal liver. A significant decrease in Igf1 protein levels was detected in maternal liver, however the increase in Igf1 protein levels in fetal livers was not found to be statistically significant. These results support previous findings that TPhP does not cause overt structural developmental toxicity. These data also support the hypothesis that TPhP could disrupt maternal and fetal metabolism, justifying the need for follow-up studies to investigate further.