Comparative toxicity of [C₈mim]Br and [C₈py]Br in early developmental stages of zebrafish (Danio rerio) with focus on oxidative stress, apoptosis, and neurotoxicity

Highlights

• Comparative toxicity of [C₈mim]Br and [C₈py]Br to zebrafish larvae was performed.

• [C₈mim]Br has much higher accumulation and neurodevelopmental toxicity than [C₈py]Br.

• [C₈mim]Br and [C₈py]Br triggered oxidative stress and apoptosis in distinct ways.

• Imidazolium and pyridinium group play a vital role in determining the toxicity of ILs.

Abstract

The increasing production and usage of ionic liquids (ILs) have raised global ecotoxicological concerns regarding their release into the environment. While the effects of side chains on the IL-induced toxicity in various aquatic organisms have been well-recognized, the role of cationic cores in determining their ecotoxicity remains to be elucidated. Herein, the comparative bioavailability and toxicity of two ILs with different cationic cores but the same anion and side chain in zebrafish embryos were determined. 1-octyl-3-methylimidazolium bromide ([C8mim]Br) has higher accumulation in zebrafish, and triggered developmental toxicity by inducing oxidative stress and apoptosis. Meanwhile, 1-octyl-1-methylpyridium bromide ([C8py]Br) enhanced SOD activity and upregulated anti-apoptotic bcl-2 gene expression, contributing to its much lower neurodevelopmental toxicity. Our study demonstrates the vital role of cationic core in determining the developmental toxicity of ILs and highlights the need for further investigations into the toxicity of imidazolium and pyridinium based ILs in aquatic ecosystems.

Introduction

Due to their unique physiochemical properties, ionic liquids (ILs) are widely used in various fields such as bio-catalysis, organic synthesis, electrochemistry, and pharmaceutical applications, and have long been considered as ideal green solvents that can replace conventional organic solvents (Firmansyah et al., 2020, Greer et al., 2020, Singh and Savoy, 2020). However, some recent studies have shown that the toxic effects of some special kinds of ILs were comparable with, or even higher than that of traditional organic solvents (Perez et al., 2017, Tsarpali and Dailianis, 2015). In addition, ILs are poorly degradable in the environment, thus are often regarded as potential persistent organic pollutants (POPs) (Modelli et al., 2008, Romero et al., 2008).

ILs are highly soluble in water and can induce toxicity to aquatic organisms. For example, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([C₄pyr]TFSI) and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C₄mim]TFSI) at μg/L levels induced morphological changes in zebrafish larvae, leading to impaired feeding and swimming (Pandey et al., 2017). Acute toxicity in response to IL exposures was also found in other aquatic organisms like Raphidocelis subcapitata, Paramisgurnus dabryanus and Hypophthalmichthys molitrix, with adverse outcomes characterized as growth inhibition, inappropriate biochemical responses, and negative effects on physiology (Ma et al., 2018, Nan et al., 2017, Samorì et al., 2015). Cation is the major toxic component of an IL, and length of alkyl chain is a critical factor determining the toxicity of the cation (Wei et al., 2021). While ILs with short alkyl chains exhibit relatively low toxicity to aquatic phytoplankton, invertebrates, and vertebrates, the IL-induced toxicity increased with the lengthening of the alkyl chains till ~C₁₀ (Latała et al., 2009, Piotrowska et al., 2018, Ruokonen et al., 2016, Samorì et al., 2015, Wei et al., 2021). Besides the alkyl chain, the cationic core is another main component of cation in IL, whose contribution to the IL-induced toxicity in zebrafish remains to be elucidated.

Imidazolium- and pyridinium-based ILs are widely used and account for > 60% of the global market for industrial and pharmaceutical sales and uses (Heckenbach et al., 2016). Even though imidazolium- and pyridinium-based ILs have been classified into categories of low to moderate toxicity to zebrafish because of their relatively high 96 h LC₅₀ values (Li et al., 2020, Mehrkesh and Karunanithi, 2016, Zhang et al., 2017a), the question whether and how could they induce adverse outcomes at non-lethal concentrations remains to be answered. Up until now, acute toxicity, oxidative stress response, and genotoxicity have been the focus of toxicological investigations of ILs in aquatic organisms, such as 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl), 1-butyl-3-methylimidazolium bromide ([C₄mim]Br), 1-methyl-3-hexylimidazolium bromide ([C₆mim]Br) and 1-butyl-3-methylimidazolium tetrafluoroborate ([C₄mim]BF₄) (Dong et al., 2016, Zhang et al., 2018, Zhang et al., 2017b). However, the toxic mechanisms underlying IL exposures are rarely reported in aquatic organisms and additional research is needed to elucidate these toxicity pathways.

In the present study, zebrafish at 2 hpf were exposed to 2.5, 5, 10 and 20 mg/L of 1-octyl-3-methylimidazolium bromide ([C₈mim]Br) or 1-octyl-1-methylpyridium bromide ([C₈py]Br) for 96 h to determine their distinct developmental toxicity to zebrafish. Based upon cell-based assays and computational methods, ILs are suspected to be inhibitors of acetylcholinesterase (AChE) (Das and Roy, 2014, Stock et al., 2004, Yan et al., 2021). We therefore hypothesized that ILs may exert neurotoxicity to early staged zebrafish. From individual and molecular levels, the potential neurotoxicity of [C₈mim]Br and [C₈py]Br were determined quantitatively. To better understand toxic mechanisms of both ILs, the oxidative stress response and apoptosis-related endpoints were also measured. This study supplements toxicological data of ILs in aquatic organisms and characterizes potential risks of IL exposure in aquatic environments.