Nanoplastics (NPs), as an emerging contaminant, have usually been found charged in the environment, posing threats to aquatic animals. However, the underlying mechanisms governing the gut toxicity of differentially charged NPs to benthic insects are not well understood. In this study, the gut toxicity in larvae of Chironomus kiinensis exposed to negatively charged NPs (PS-COOH, 50 nm) and positively charged NPs (PS-NH2, 50 nm) at 0.1 and 1 g/kg was investigated through fluorescence imaging, histopathology, biochemical approaches, and 16S rRNA sequencing. The results showed that PS-NH2 caused more adverse effect on the larval growth performance and induced more severe oxidative stress, epithelial damage, and inflammatory responses in the gut than PS-COOH. The stronger impact caused by PS-NH2 was because the gut accumulated PS-NH2 more readily than PS-COOH for its negatively charged cell membrane. In addition, PS-NH2 were less agglomerated compared with PS-COOH, leading to an increased interaction with gut cell membranes and microbiota. Furthermore, alpha diversity and relative abundance of the keystone microbiota related to gut barrier and nutrient absorption were markedly lower exposed to PS-NH2 than PS-COOH, indirectly exacerbating stronger gut and growth damage. This study provides novel insights into the effect mechanisms underlying differentially charged NPs on benthic insects.

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