ABSTRACT: Understanding the potential uptake and biodistribution of engineered nanoparticles (ENPs) in soil-grown plants is imperative for realistic toxicity and risk assessment considering the oral intake of edibles by humans. Herein, growing N-fixing symbiont (Bradyrhizobium japonicum) inoculated soybean (Glycine max (L.) Merr.) for a full lifecycle of 120 days, we assessed the potential influence of particle size (25, 50, and 250 nm) and concentration (0, 50, 100, 200, and 500 mg/kg soil) of Copper oxide nanoparticles (CuONPs) on: (1) root system architecture, (2) soil physicochemical attributes at the soil-root interface, and (3) Cu transport and accumulation in root, stem, leaf, and seed in soybean, and compared them with the soluble Cu2+ ions and water-only controls. Finally, we performed a comparative assessment of total seed Cu levels in soybean with other valuable food sources for Cu intake and discussed potential human health implications. Results showed particle size- and concentration-dependent influence of CuONPs on Cu uptake and distribution in root, stem, leaf, and seed. Alterations in root architecture (root biomass, length, volume, and area) were dependent on the Cu compound types, Cu concentrations, and their interactions. Concentration-response relationships for all three sizes of CuONPs and Cu2+ ions were found to be linear. Furthermore, CuONPs and Cu2+ ions had inhibitory effects on root growth and development. Overall, soybean responses to the smallest size of CuONPs-25 nm-were greater for all parameters tested compared to the two larger-sized CuONPs (50 nm, 250 nm) or Cu2+ ions. Results suggest that minor changes in soil-root physicochemical attributes may not be a major driver for Cu uptake in soybean. Cu bioaccumulation followed the order: root > leaf > stem > seed. Despite reduction in root architecture and seed yield, the smallest size CuONPs-25 nm led to increased total seed Cu uptake compared to the larger-sized CuONPs or Cu2+ ions. Our findings also suggest that soil amendment with CuONPs, and more so with the smallest size of CuONPs-25 nm-could significantly improve seed nutritional Cu value in soybean as reflected by the % Daily Values (DV) and are rated "Good" to "Very Good" according to the "World's Healthiest Foods" rating. However, until the potential toxicity and risk from CuONP-fortified soybean seed ingestion is characterized in humans, we caution recommending such seeds for daily human consumption when addressing food Cu-deficiency and associated diseases, globally.
