Combined toxicity of nanoplastics and sodium fluoride to zebrafish liver: Impact on gut-liver axis homeostasis and lipid metabolism.

Introduction

With industrial development, the problem of compounded contamination of water bodies with nanoplastics (NPs) and inorganic fluorides (NaF), which endanger aquatic ecosystems due to their persistence and biotoxicity, is becoming increasingly serious (Habumugisha et al., 2024; Yadav et al., 2025). Field monitoring data from three water bodies in the western Songnen Plain confirm the widespread coexistence of NaF and microplastics: Fluoride levels exceeded the Class III surface water standard (1.0 mg/L) at all sites, averaging 2.47 mg/L in Chagan Lake and 1.28 mg/L in Xianghai Reservoir, with an 87.5 % exceedance rate in Longfeng Wetland. Microplastics were consistently detected, averaging 3.61±2.23 items/L in Chagan Lake and 0.29±0.11 items/L in Xianghai Reservoir, with higher abundances in areas of greater human activity (Xu et al., 2021; Yin et al., 2021). Studies have shown that human activities such as fertilizer factories, brick-making factories, and the use of fluorinated pesticides can lead to an increase in fluoride levels in surface water (Camargo et al., 2003). Three plausible real-world scenarios lead to the co-occurrence of high-concentration NaF and micro/nanoplastics: (1) Natural lakes contaminated with both pollutants via human activities; (2) Industrial discharge zones (e.g., aluminum, fertilizer, brick industries) where fluoride is discharged together with plastic pollution runoff (Wang et al., 2019); (3) Wastewater treatment plant effluents, a verified common transmission pathway for both contaminants (Naziri et al.zx, 2023; Huang et al., 2023). Environmentally relevant concentrations show that freshwater micro/nanoplastic levels range from 0.41 µg/L to 10 mg/L in polluted areas (Yang et al., 2025; Blettler et al., 2017; Xia et al., 2025), while sodium fluoride concentrations often exceed 3–10 mg/L in endemic regions (Kim et al., 2015; Chaithra et al., 2020). These fluorides not only damage liver tissue structure, leading to pathological changes such as hepatocyte vacuoles and rupture of hepatocyte membranes (Hu et al., 2023), but also produce hepatotoxicity by inducing oxidative stress and apoptosis (Chen et al., 2019; Deng et al., 2016). In the meanwhile, the global annual production of about 300 million tons of plastics makes the plastic degradation products in the aquatic environment widely distributed (Geyer et al., 2017), especially the particle size of less than 100 nm nanoplastics due to its special surface effect and small size characteristics, more likely to be absorbed by aquatic organisms to produce toxicity (Jambeck et al., 2015). Regarding the adsorption interaction between NPs and NaF, this will be the focus of in-depth follow-up research. It is worth noting that nanoplastics (70 nm) have been found to accumulate in the liver of zebrafish and cause lipid metabolism disorders (Shen et al., 2019). In addition, nanoplastics (40 nm) affect the intestinal permeability of zebrafish and cause intestinal damage (Teng et al., 2022). However, there is still a lack of clarity regarding the combined toxic effects of fluoride and nanoplastics combined exposure and their mechanisms of action.

Zebrafish have a highly conserved gut-liver axis regulatory mechanism with more than 70 % similarity to mammals (Moradian et al., 2024). Zebrafish was selected as a biological model to carry out relevant studies in this study. As a complex bidirectional regulatory system, the gut-liver axis plays an important role in nutrient absorption, immune defense, and toxin clearance through the anatomical structure of portal connection and functional metabolic interactions (Tilg et al., 2022). When the intestinal barrier is disrupted, abnormal changes in intestinal flora and their metabolites can directly affect liver function through portal circulation (Bajaj et al., 2022). The potential effects of fluoride and nanoplastics on gut and liver health focus on gut microbes and hepatic lipid metabolism. NaF exposure has been shown to cause intestinal epithelial damage in mice (Huang et al., 2024), whereas chronic exposure to microplastics also caused intestinal barrier disruption and abnormal hepatic lipid metabolism in zebrafish (Luan et al., 2024). These findings indicate that NPs and sodium NaF are harmful to intestinal and liver health, but whether they have combined exposure toxicity remains uncertain.

Zebrafish were employed to examine the impacts of co-exposure to NaF and NPs (40 nm) on the liver and intestine toxicity and its potential mechanism. They were randomly divided into a control group, the NPs group (0.1 mg/L), the NaF group (15 mg/L), and the NaF + NPs group (15 mg/L NaF and 0.1 mg/L NPs). The selected concentrations of NaF (15 mg/L, corresponding to ?6.8 mg/L free F?) and NPs (0.1 mg/L) are based on prior toxicological studies, which have been shown to induce significant yet sub-lethal biological effects in aquatic organisms including zebrafish, placing this study in an established investigative context for exploring biologically active concentration effects (Bhowmik et al., 2020; Ding et al., 2022). To systematically explain the toxicity mechanism of NPs and NaF on the enterohepatic axis, this study innovatively integrated network toxicology analysis. This study provides an efficient and precise research strategy for the study of the toxicity mechanism of environmental pollutants. By constructing a multidimensional network of compounds-targets-pathways, network toxicology can analyze the molecular mechanisms of toxicity from a systems biology perspective (Huang et al., 2024). Histopathological damage of the intestine and liver was observed after 30d of exposure. Intestinal health was assessed and the relationship between intestinal barrier disruption and intestinal microbial changes and hepatic lipid metabolism was analyzed. This study provides new ideas to elucidate the toxicity, potential mechanisms and broader risks of NaF, NPs and their combined exposure. It should be noted that this study used a single exposure concentration for each pollutant; while the co-exposure group showed the most pronounced toxic effects, a formal assessment of interaction type (synergy/additivity) requires future studies with a full dose-response matrix.