Is the Environmental Risk of Metformin Underestimated?

1 This is the postprint (accepted manuscript) version of the article published by ACS in Environmental Science and Technology on May 2023. Available on-line at https://doi.org/10.1021/acs.est.3c02468 Is the environmental risk of metformin underestimated? Unax Lertxundi https://orcid.org/0000-0002-9575-1602 • Bioaraba Health Research Institute; Osakidetza Basque Health Service, Araba Mental Health Network, Araba Psychiatric Hospital, Pharmacy Service, VitoriaGasteiz, Spain. c/Alava 43, 01006 Vitoria-Gasteiz, Alava, Spain. Telephone: +34 945 00 65 33 Fax: +34 945 00 65 87 Saioa Domingo-Echaburu https://orcid.org/0000-0002-0642-9081 • Osakidetza Basque Health Service, Debagoiena Integrated Health Organisation, Pharmacy Service, Nafarroa Hiribidea 16, 20500 Arrasate, Gipuzkoa, Spain. Susana Barros. https://orcid.org/0000-0003-2427-6696 • CIIMAR/CIMAR_LA ─Interdisciplinary Centre of Marine and Environmental Research, Endocrine Disruptors and Emerging Contaminants Group, University of Porto, Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal. • CITAB - Centre for the Research and Technology of Agro-Environmental and Biological Sciences, University of Trás-os-Montes and Alto Douro (UTAD), Quinta de Prados, Pavilhão 2, 5000-801 Vila Real, Portugal Miguel Machado Santos. https://orcid.org/0000-0001-7347-0546 • CIIMAR/CIMAR_LA─Interdisciplinary Centre of Marine and Environmental Research, Endocrine Disruptors and Emerging Contaminants Group, University of Porto, Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal. • FCUP - Department of Biology, Faculty of Sciences, University of Porto (U. Porto), Rua do Campo Alegre s/n, 4169-007 Porto, Portugal. Teresa Neuparth. https://orcid.org/0000-0003-3314-0184 • CIIMAR/CIMAR_LA —Interdisciplinary Centre of Marine and Environmental Research, Endocrine Disruptors and Emerging Contaminants Group,University of Porto, Av. General Norton de Matos S/N, 4450-208 Matosinhos, Portugal Jose Benito Quintana. https://orcid.org/0000-0002-2566-8133 • Department of Analytical Chemistry, Nutrition and Food Sciences, IAQBUS - Institute of Research on Chemical and Biological Analysis, Universidade de Santiago de Compostela, Constantino Candeira S/N, 15782 Santiago de Compostela, Spain. 2 Rosario Rodil. https://orcid.org/0000-0002-7100-723X • Department of Analytical Chemistry, Nutrition and Food Sciences, IAQBUS - Institute of Research on Chemical and Biological Analysis, Universidade de Santiago de Compostela, Constantino Candeira S/N, 15782 Santiago de Compostela, Spain. Rosa Montes. https://orcid.org/0000-0002-4154-3541 • Department of Analytical Chemistry, Nutrition and Food Sciences, IAQBUS - Institute of Research on Chemical and Biological Analysis, Universidade de Santiago de Compostela, Constantino Candeira S/N, 15782 Santiago de Compostela, Spain. Gorka Orive https://orcid.org/0000-0002-0773-300X • NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country UPV/EHU, Paseo de la Universidad 7, Vitoria-Gasteiz 01006, Spain. • Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). Vitoria-Gasteiz, Spain. • Bioaraba, NanoBioCel Research Group, 01006 Vitoria-Gasteiz, Spain. • University Institute for Regenerative Medicine and Oral Implantology - UIRMI (UPV/EHU-Fundación Eduardo Anitua), Vitoria, Spain. • Singapore Eye Research Institute, The Academia, 20 College Road, Discovery Tower, Singapore. Corresponding author: Unax Lertxundi Telephone: +34 945 00 65 33 Fax: +34 945 00 65 87 [email protected] KEYWORDS: water framework directive; pharmaceuticals in the environment; predicted no-effect concentration (PNEC), surface water 3 A highly prescribed drug – a ubiquitous water pollutant The worldwide prevalence of diabetes mellitus was estimated to be 10.5% in 2021 (536.6 million people), and is expected to rise up to 12.2% in 2045 (Sun et al., 2022). For many decades, metformin has been the backbone of oral glucose-therapy for type 2 diabetes mellitus (Bailey CJ et al., 2017). It is estimated that over 150 million people each year take this drug, by far the most prescribed glucose-lowering oral medicine worldwide (Drzewoski et al., 2021). More recently, metformin has gained attention because of its capacity to partially prevent long-COVID, showing a 42% relative reduction in a placebo-controlled randomized trial (Bramante et al., 2023). Last but not least, this biguanide has also been utilized for treatment of antipsychotic induced weight gain, acne vulgaris and polycystic ovarian syndrome, among others (Bailey et al., 2017). The mechanism of action is not fully elucidated, although its therapeutic effects are believed to derive from an insulin sensitizing action. Hence, due to such widespread use, the high prescribed doses (0.5-2 g/person/day), and the fact that it is excreted by the kidney primarily unchanged, its routine detection in the aquatic environment comes as no surprise. As many other pharmaceuticals, metformin enters the aquatic environment via wastewater treatment plants (WWTPs), where removal rates are highly variable, from 22 to 99% (He et al., 2022). This leads to concentrations in the µg/L range in final effluents and even the receiving watersheds. Unsurprisingly, the Global Monitoring of Pharmaceuticals project has shown that metformin is the second most frequently detected pharmaceutical in world’s rivers, with a mean concentration of 2.8 µg/L and a maximum of 51 µg/L in Lahore, Pakistan (Wilkinson et al., 2022). In our recent 1-year study in the NW of Portugal and Spain (48 river and 15 coastal water samples) metformin median values were 0.28 and 0.07 µg/L (detection frequency: 98% and 67%), for inland and coastal surface water, respectively (Montes et al., 2023), proving that not only freshwater is contaminated. The fourth meta-study of He et al. cites a concentration up to 1.2 µg/L in drinking water (He et al., 2022). An underestimated risk 4 Metformin ecotoxicological risks may have been underestimated in the literature. As an example, the valuable tool NORMAN ecotoxicology database, that is often used by many researchers to derive risk quotients, still displays a predicted no-effect concentration (PNEC) several orders of magnitude higher than the concentrations reported in several recent fish ecotoxicity studies. In fact, PNEC values from 100 to 1030 µg/L are frequently used (European commission, 2022, NORMAN, 2022; Caldwell et al., 2019, He et al., 2022). However, several works performed by distinct institutions using different fish species, point mostly to negative effects at ng/L to low µg/L range. Thus, even if some contrasting findings were reported by different teams for fathead minnows (Pimephales promelas) (Parrot et al., 2022; Blackwell et al., 2022; Nielsen et al., 2022;), with the latest study by Nielsen et al., (2022) showing impact in embryonic development at 5 µg/L, particularly in wild-spawned fathead minnows, most studies support effects in the range of environmental concentrations. The reported effects include disruption of embryonic development in zebrafish and fathead minnows at concentrations as low as 0.39-5 µg/L (Elizalde et al., 2021; Nielsen et al., 2022; Barros et al., 2023); vitellogenin (VTG) induction and disruption of reproductive associated signaling pathways in fathead minnows, zebrafish and Japanese rice fish (Oryzias latipes) (Niemuth et al., 2015; Niemuth and Klaper 2015; Crago et al., 2015; Lee et al., 2019, Barros et al., 2023) in concentrations ranging for 0.39 to 40 µg/L; and changes in metabolism of life-cycle exposed zebrafish and in processes related to cell cycle, DNA repair and steroid hormone biosynthesis, concomitantly to alterations in apical endpoints such as growth, hepatosomatic index, and fertilization rate (Barros et al., 2022, 2023) in concentrations ranging from 0.39 to 14. 4 µg/L. The EU Water Framework Directive watch list needs to be revised Considering its wide use and the concentrations found in the environment, metformin and its main transformation product, guanylurea, have been recently proposed to be included in the 4th version of the European surface water watch list under the Water Framework Directive (WFD) (European Commission, 2022). However, an extremely high limit of quantification (LOQ) has been set (156 µg/L) as the requirement for laboratories. 5 Indeed, this LOQ is higher than what is usually found in the environment, even higher than the highest concentration found in the Global Monitoring of Pharmaceuticals project (Wilkinson et al., 2022). Besides, as highlighted, toxic effects in non-target aquatic organisms have been shown at much lower concentrations, while there is still more research needed with regards to the toxicity of its transformation product. In other words, the current fishnet mesh is too large for metformin (Figure 1). Figure 1: The EU WFD 4th watch list has set the fishnet mesh too large. As a result, it will not be able to provide any useful information on metformin levels and risks. In summary, we call for an urgent revision of the LOQ set for metformin in the 4th watch list of the WFD. Reasons for such a call include: i) methods for the determination of metformin (and its transformation product) at the ng/L range are available. ii) the drug may exert toxic effects in non-target aquatic organisms at low concentrations. iii) the consumption of metformin is expected to increase assuming the current epidemic of 6 diabetes mellitus. It is time for action. Otherwise, EU Member States would uselessly spend time and resources measuring metformin at unrealistic concentrations. REFERENCES • Bailey CJ. Metformin: historical overview. 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