Elsevier

Aquatic Toxicology

Volume 205, December 2018, Pages 58-65
Aquatic Toxicology

Effects of environmentally relevant metformin exposure on Japanese medaka (Oryzias latipes)

https://doi.org/10.1016/j.aquatox.2018.10.003Get rights and content

Highlights

Early life stage (ELS) fish can uptake/depurate waterborne metformin.

Metformin alters metabolomes, gene expression, and growth of ELS medaka.

Lifelong metformin exposure affects steroid hormones in adult fish.

Environmentally relevant concentrations of metformin may affect fish development.

Abstract

Metformin is one of the most prevalent pharmaceuticals in both surface and waste waters, yet little is known about the bioavailability and/or effects of developmental exposure on early life stage (ELS) fish. Here, we demonstrate that embryo-larval stages of medaka are capable of taking up metformin from the aquatic environment, provided exposure occurs prior to chorion hardening (∼6-hpf). Once transferred to clean water, ELS medaka are able to completely depurate metformin in <24-hours. Furthermore, ELS medaka exposed to a range of relevant concentrations of waterborne metformin (from 6 hpf through 28-days post hatch) had significantly reduced growth metrics, altered metabolomes, and changes in the expression of genes associated with cell growth. The range of concentrations investigated were 1.0, 3.2, 10, 32, and 100 μg·L−1. To examine effects of chronic, low level metformin exposure across the full medaka life-cycle, we exposed newly fertilized embryos to 3.2 μg L−1 waterborne metformin for 165-days. The weight and length of adult fish were examined, as were effects on the production of some steroid hormones, specifically a significant increase (control females: 0.161 ± 0.023 pg/mg; metformin treated females: 3.42 ± 0.543) in the production of 11-ketotestosterone was observed in adult female medaka. Collectively, these results suggest that current environmental exposure scenarios may be sufficient to cause effects on developing fish.

Introduction

The presence of pharmaceuticals in aquatic ecosystems continues to increase, with a number of pharmaceutical compounds now frequently detected in the ng·L−1 to μg·L−1 range in surface waters (Corcoran et al., 2010; Fatta-Kassinos et al., 2011; Magi et al., 2016; Tanwar et al., 2014). Those prescribed in high volumes may be continuously discharged from wastewater treatment plants (WWTPs), leading to chronic exposure scenarios for aquatic biota. Given the highly conserved nature of many receptors targeted by pharmaceuticals across taxa, the potential for adverse effects in aquatic biota exists. This is particularly true for sensitive populations like early life stage (ELS) fish. Although the concentrations measured in surface waters are considerably lower than the effective doses for target organisms (e.g. humans, cattle, canines), the effect concentrations for non-target organisms, such as fish, have not yet been fully elucidated.

Metformin is currently thought to be the highest drug by weight released into the aquatic environment (Blair et al., 2013; Kolpin et al., 2002; Oosterhauis et al., 2013; Scheurer et al., 2012; Trautwein et al., 2014). Metformin is not biotransformed by humans, and approximately 70% of the therapeutic dose is excreted unchanged in urine and feces (Gong et al., 2012; Pentikäinen et al., 1979). Despite a removal rate exceeding 90% in wastewater treatment plants (WWTPs; Scheurer et al., 2012), measurable amounts of metformin are present in effluent and surface waters at concentrations ranging from 1 to 47 μg · L−1 in effluent, and 0.06 to 3 μg · L-1 in surface waters (Oosterhuis et al., 2013; Blair et al., 2013; Ghoshdastidar et al., 2015; Oosterhuis et al., 2013; Scheurer et al., 2012). The prevalence of metformin is attributed to its widespread use, as it is prescribed for the treatment of a number of common human health disorders. These include type-2 diabetes, polycystic ovary syndrome, and cancers where insulin resistance is a factor (Tang et al., 2012).

The effects of chronic exposure to low concentrations of metformin on non-target aquatic species are largely unknown, including the potential for uptake and bioaccumulation in fish. In the present study, we describe the uptake, bioaccumulation, and depuration of metformin by embryo-larval stages of Japanese medaka (Oryzias latipes), and investigate the effects of developmental metformin exposure on 28-day post hatch (dph) medaka. We examined growth (length/weight) of larvae, and performed screening level metabolomics to gain insight to the mechanisms driving toxicity in metformin exposed ELS fish. Differences in the relative abundances of metabolites between the control and metformin-exposed fish were used to generate hypotheses regarding genes that may be differentially expressed, which we analyzed via qPCR. Additionally, we performed a full life-cycle study (165-days) in which embryonic medaka were exposed to 3.2-μg · L−1 of metformin through adulthood, to determine if life-long exposure to low levels of metformin leads to differences in growth and/or the production of the reproductive steroids estradiol, and 11-ketotestosterone (11-KT) in adult medaka.

Section snippets

Animal care

Adult Japanese medaka (FLFII strain) were obtained from the National Institute for Basic Biology (Okazaki, Japan), and housed at the University of Ontario Institute of Technology (Oshawa, ON, Canada). Animals were housed in 3, 70-L tanks, containing 40 adult fish at a ratio of 2 males: 3 females. All embryos used in the present study were obtained by gently stripping eggs from each gravid female with a gloved finger. Debris was removed from embryos by gently rolling them on clean paper towel.

Metformin uptake and depuration

Because of metformin’s log Kow (−2.64; Hernández et al., 2015; Table S1), it was thought that the compound would not be readily taken up by aquatic organisms (US EPA, 2012). Here, we show that low concentrations of waterborne metformin can be taken up in both embryonic and larval stages of medaka. Waterborne metformin uptake and accumulation was influenced by the hardening of the chorion at approximately 6 hpf. Embryos exposed to 10-μg · L−1 14C-metformin prior to hardening had significantly

Conclusion

To our knowledge, the findings of the present study provide some of the first evidence of developmental-metformin toxicity in early-life stage fish, at concentrations presently measured in the environment. Furthermore, our data provides further support for the proposed AMPK-dependent MOA. The results presented here, in conjunction with the findings of other studies (although scarce), indicate that there is potential for population level impacts in fish, should environmental concentrations

Acknowledgements

The authors would like to thank Dr. Aaron Roberts for his guidance and feedback, which improved this manuscript. The authors also wish to thank our sources of funding (Canada Research Chairs, Tier 1 #950-221924, NSERC #360557-2011) for the monetary support for this research. We would like to thank the National Institute of Natural Sciences, National BioResource Project (NBRP) and the National Institute of Basic Biology for the FLFII MT109 Japanese medaka strain used for this research. Thank you

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