Silver nanoparticles trigger pronounced immunotoxicity via disrupting gut microbiota in estuarine fish: a study highlighting particle size and the dynamic transformation.

Silver nanoparticles (Ag0-NPs) are among the most widely used nanomaterials, yet current risk assessments predominantly focus on particle size while overlooking the role of dynamic speciation. Here, using the estuarine fish Scatophagus argus, we demonstrated that primary particle size (20, 60, and 100 nm) and the bidirectional Ag0-NPs/dissolved Ag + interconversion critically governed in vivo toxicity. Our results revealed that Ag exposure induced the most pronounced pro-inflammatory immunotoxicity, characterized by selective enrichment of gut Gram-negative bacteria, lipopolysaccharide (LPS) translocation across a compromised intestinal barrier, and subsequent activation of the TLR/MyD88/NF-κB (Toll-like receptor/myeloid differentiation primary response 88/nuclear factor kappa-B) signaling pathway, an effect most pronounced with 20-nm Ag0-NPs and Ag+ exposure. Counterintuitively, 20-nm Ag0-NPs at an environmentally relevant concentration (10 μg/L) caused significantly higher mortality than equimolar Ag+ exposure (15.75 μg/L AgNO3). This disparity arose because administered Ag+ (∼10%) was rapidly sulfidated into low-bioavailability Ag2S-NPs, constituting an endogenous detoxification mechanism, whereas 20-nm Ag0-NPs sustained elevated intracellular and luminal Ag + levels through continuous dissolution. Prolonged Ag0-NPs exposure exacerbated gut microbiota dysbiosis and systemic inflammation. Crucially, fecal microbiota transplantation from exposed donors into germ-free medaka recapitulated elevated serum LPS and enhanced inflammatory response, confirming a causal link between nanoparticle exposure, gut microbiota perturbation, and host immune dysfunction. These findings reveal that the synergistic interplay between particle size and in vivo silver transformation dictates nanotoxicity, underscoring that secondary particle formation and transformation-dependent toxicity are underestimated in conventional risk paradigms.