Impact of chronic exposure to the ionic liquid ([C8mim][PF6]) on intestinal physical barrier, immunological barrier and gut microbiota in common carp (Cyprinus carpio L.)

Highlights

• ([C8mim][PF6]) exposure increased intestinal permeability and disrupted intestinal physical barrier in common carp.

• [C8mim]PF6 exposure destroyed the intestinal immune function and trigger inflammatory response in common carp.

• ([C8mim][PF6]) exposure reduced gut microbial diversity and altered microbial community structure in common carp.

Abstract

Ionic liquids (ILs) are commonly known as "green" solvents and have been widely used in various fields. However, the ecotoxicity of ILs in aquatic environment has received considerable attention from scientific researchers. This study investigated the toxic effects of different concentrations of 1-octyl-3-methylimidazolium hexafluorophosphate ([C8mim][PF6]) (0, 1.35, 2.70 and 5.40 mg/L) on intestinal physical barrier, immunological barrier, and intestinal microbiome in common carp on days 30 and 60. The results showed that ([C8mim][PF6]) exposure could reduce the intestinal villus height, decrease the mRNA expression of tight junction genes (occludin, claudin-2 and zonula occludens-1), and increase the levels of D-lactic and diamine oxidase, and reduce acid phosphatase and lysozyme activities, complement 3 and 4 contents, and anti-inflammatory cytokine TGF-β protein level, while increase pro-inflammatory cytokines TNF-α and IL-1β protein levels in common carp. Moreover, ([C8mim][PF6]) exposure was also found to significantly reduce gut microbial diversity and alter microbial community structure in common carp. Collectively, our study highlighted that exposure to ([C8mim][PF6]) could disrupt intestinal physical barrier, impair immunological barrier and alter intestinal microbiome in common carp, suggesting that ILs exert a negative effect on fish intestinal health status and may pose serious health risks in fish. The results of this study may be helpful to illuminate the toxicity mechanisms of the ILs on fish.

Introduction

Ionic liquids (ILs) are used as an alternative to traditional organic solvents, which possess unique physical and chemical features such as low vapor pressure, high thermal and chemical stability, good electrical conductivity and non-flammability (Shao et al., 2019). Nowadays, ILs have been recognized as “green solvents” and have broad application prospects in manufacturing, processing and cleaning technologies (Li et al., 2013). However, recent toxicity studies have indicated that ILs can exert toxic effects similar to other traditional solvents (Amde et al., 2015; Bubalo et al., 2015; Garcia et al., 2015). Previous researches have also demonstrated that ILs are persistent in the environment due to their high chemical stability, poor biodegradability and low solubility in water (Deng et al., 2017; Pham et al., 2016). Other studies have assessed the toxicity of ILs to aquatic organisms at different trophic levels, and highlighted the potential hazards of IL exposure in aquatic environment (Thi et al., 2010; Bubalo et al., 2014). However, the toxicological effects of ILs on the aquatic organisms have not been studied extensively and thoroughly. Therefore, it is essential to determine the toxic levels and ecological risk of IL accumulation in water environment.

As an important part of the aquatic food chain, fish can help to maintain the balance of an aquatic ecosystem. Also, fish are commonly used as the toxicological model organisms to assess the toxicity of pollutants in aquatic ecosystems (Ma et al., 2018). At present, several studies have paid attention to the toxic effects of ILs on aquatic environment using fish model, including Danio rerio, Paramisgurnus dabryanus and Hypophthalmichthys molitrix (Zhang et al., 2017; Ma et al., 2019; Nan et al., 2016; Younes et al., 2018; Thamke and Kodam, 2016). These studies mainly focused on the toxicological effects of ILs on fish liver, skin, gills and embryos. Few studies have aimed to understand the toxicological impacts of ILs on the intestine, which is the principal site of exposure to toxic substances because of its large surface area and physical properties (Sun et al., 2018; Vismaya, 2014). Thus, the intestine is proposed to be a target organ of ILs.

Normal intestinal structure and function are of basic importance for fish health (Suo et al., 2017). Typically, the intestinal health status of fish is dependent on their physical barrier, immunological barrier and intestinal microbiota (Pan et al., 2017; Chang et al., 2019). Firstly, intestinal physical barrier can maintain selective permeability by ingesting fluids, ions and nutrients, and preventing toxic substances to enter the circulatory system (Asmonaite et al., 2018). Secondly, intestinal immunological barrier is consisted of humoral immune factors such as complements, cytokines, lysozyme (LZM) and acid phosphatase (ACP) (Pan et al., 2017), which can protect the intestine against pathogen invasion (Rombout et al., 2011). Thirdly, intestinal microbiome in fish is crucial to the host by regulating immune activity, feed conversion, metabolic function and pathogen resistance (Chang et al., 2019; Wang et al., 2019). An imbalance in gut microbiota can affect the immunity of fish and lead to the development of diseases (Li et al., 2018; Gomez and Balcazar, 2008). Hence, the maintenance of a balanced microbiota is of great importance to the health of fish. Numerous factors, such as environmental determinants, geographical habitat, fish species, developmental stage and longevity have been shown to regulate the composition of gut microbiota (Li et al., 2018; Zhang et al., 2016), which in turn can effect the health status of fish.

According to previous literature, imidazolium-based ILs are one of the broadly used and investigated groups (Deng et al., 2017). Among them, 1-butyl-3-methylimidazolium hexafluorophosphate ([C4mim][PF6]), 1-hexyl-3-methylimidazolium hexafluorophosphate ([C6mim][PF6]), and 1-octyl-3-methylimidazolium hexafluorophosphate ([C8mim][PF6]) are widely used in electrochemical, chemical synthesis and biotechnological processes. And these ILs were hard biodegraded, and were very stable in the water and soils (Deng et al., 2017). According to72 h-LC₅₀ values (269.25 mg/L ([C8mim][PF6]), 328.69 mg/L ([C6mim][PF6]), and mg/L 397.54 mg/L ([C4mim][PF6])) in our pre-experimental, ([C8mim][PF6]) was the most toxic one. Thus, in order to investigate the toxicity impact of chronic exposure to low doses of ILs on fish, [C8mim][PF6] was chosen as the tested compound. Moreover, the Cyprinus carpio L., or commonly known as common carp, was employed as a model organism to determine the impact of ([C8mim][PF6]) on gut health because it is a widely farmed fish species in China, and also has been adopted as a biological indicator to investigate the effects of environmental pollution (Yesilbudak and Erdem, 2014). In this study, the common carp were exposed to 0, 1.35, 2.70 and 5.40 mg/L of ([C8mim][PF6]) for 60 days. The objectives of the present research were to evaluate whether [C8mim]PF6 has the potential effects on intestinal physical barrier, immunological barrier and gut microbiota of common carp. The changes of physical barrier function were studied by evaluating intestinal morphology, and the levels of D-lactic acid (D-Lac), diamine oxidase (DAO) and tight junction proteins genes at days 30 and 60. Additionally, the contents of complement C3, C4, tumour necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and transforming growth factor-β (TGF-β) as well as the activities of LZM and ACP were studied to investigate the effects of ([C8mim][PF6]) on intestine immunological barrier at days 30 and 60. Furthermore, high-throughput sequencing was performed to evaluate the diversity and composition of intestinal microbiota after ([C8mim][PF6]) exposure for 60 days. The results would provide more data to unravel the toxic effects of ILs on fish and to evaluate their safety in aquatic environment. In addition, our results may be helpful to illuminate the toxicity mechanisms of the ILs on fish.