Combined effect and mechanism of microplastic with different particle sizes and levofloxacin on developing Rana nigromaculata: Insights from thyroid axis regulation and immune system

Microplastics (MPs) usually appear in the aquatic environment as complex pollutants with other environmental pollutants, such as levofloxacin (LVFX). After 45-day exposure to LVFX and MPs with different particle sizes at environmental levels, we measured the weight, snout-to-vent length (SVL), and development stages of Rana nigromaculata. Furthermore, we analyzed proteins and genes related to immune system and thyroid axis regulation, intestinal histological, and bioaccumulation of LVFX and MPs in the intestine and brain to further explore the toxic mechanism of co-exposure. We found MPs exacerbated the effect of LVFX on growth and development, and the order of inhibitory effects is as follows: LVFX-MP3>LVFX-MP1>LVFX-MP2. 0.1 and 1 μm MP could penetrate the blood-brain barrier, interact with LVFX in the brain, and affect growth and development by regulating thyroid axis. Besides, LVFX with MPs caused severer interference on thyroid axis compared with LVFX alone. However, 10 μm MP was prone to accumulating in the intestine, causing severe histopathological changes, interfering with the intestinal immune system and influencing growth and development through immune enzyme activity. Thus, we concluded that MPs could regulate the thyroid axis by interfering with the intestinal immune system.