Development of an adverse outcome pathway for nanoplastic toxicity in Daphnia pulex using proteomics

Nanoplastics are a growing environmental and public health concern. However, the toxic mechanisms of nanoplastics are poorly understood. Here, we evaluated the effects of spherical polystyrene nanoplastics on reproduction of Daphnia pulex and analyzed the proteome of whole animals followed by molecular and biochemical analyses for the development of an adverse outcome pathway (AOP) for these contaminants of emerging concern. Animals were exposed to polystyrene nanoplastics (0, 0.1, 0.5, 1 and 2 mg/L) via water for 21 days. Nanoplastics negatively impacted cumulative offspring production. A total of 327 differentially expressed proteins (DEPs) were identified in response to nanoplastics which were further validated from gene expression and enzyme activity data. Based on these results, we propose an AOP for nanoplastics, including radical oxygen species production and oxidative stress as the molecular initiating event (MIE); followed by changes in specific signaling pathways (Jak-STAT, mTOR and FoxO) and in the metabolism of glutathione, protein, lipids, and molting proteins; with an end result of growth inhibition and decrease reproductive output. This study serves as a foundation for the development of a mechanistic understanding of nanoplastic toxicity in crustaceans and perhaps other aquatic organisms.