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• Polystyrene nanoplastics (PSNPs) significantly affected the growth and feed intake. • DEHP-PSNPs co-exposure significantly increased PSNPs retention in the intestines. • DEHP-PSNPs co-exposure caused significant oxidative stress and histological damages. • DEHP-PSNPs co-exposure changed the intestinal microbiota composition. Organic pollutants such as di-(2-ethylhexyl) phthalate (DEHP) interact with nanoplastics (NPs) and change their bioavailability and toxicity to aquatic organisms. This study aims to assess the ecotoxicological impacts of NPs in the presence and absence of DEHP on juvenile largemouth bass (LMB) Micropterus salmoides. Therefore, LMB was fed with diets containing various concentrations (0, 2, 10, and 40 mg/g) of polystyrene nanoplastics (PSNPs) by the weight of diets. After a 21-day of PSNPs dietary exposure, LMB was treated with DEHP at 450 μg/L through waterborne exposure for three days. Our results showed that PSNPs were accumulated in the intestinal tissues, which significantly decreased the feeding and growth rates in LMB. The histopathological analysis showed the intestine and liver of LMB were subjected to various degrees of structural damage caused by PSNPs, and DEHP-PSNP co-exposure enhanced those histopathological damages in both tissues. Additionally, the co-exposure induced oxidative stress in terms of increased activities of glutathione S-transferase, catalase, and superoxide dismutase enzymes in the liver, intestine, spleen, and serum. Furthermore, the co-exposure significantly changed the intestinal microbial composition, i.e., the decrease in the abundance of probiotics (Bacteroidetes and Proteobacteria) and the increase in pathogenic bacteria (Firmicutes) posed a great threat to fish metabolism and health. Therefore, this study highlights that the presence of DEHP enhances the toxicity of NPs on LMB in freshwater and suggests the regulated use of plastic and its additives for improving the health status of aquaculture fish for food safety in humans. [Display omitted] [ABSTRACT FROM AUTHOR]