Safe-by-Design Iron Oxide Nanofertilizers: Ecotoxicological Screening and Concentration- Dependent Physiological Responses in Zea mays L

The development of engineered nano formulations is a pivotal strategy to improve the bioavailability of poorly mobile micronutrients like iron (Fe), minimizing agrochemical runoff and environmental footprint. However, establishing clear ecotoxicological thresholds for these novel materials remains crucial for safe agricultural application. Here, we report the synthesis, comprehensive physicochemical characterization, and cellular toxicity screening of chitosan-coated iron oxide nanoparticles (CS:Fe ₃ O ₄ NPs). The synthesized composite was structurally and thermally characterized using Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM/EDX), and differential thermal analysis (DTA/DSC). FTIR confirmed the successful formation of the composite via characteristic N-H and Fe−O stretching vibrations, while SEM/EDX revealed spherical morphology (average diameter of 114 nm) consisting of Fe, C, and O. The physiological, biochemical, and toxicological effects were evaluated in Zea mays L. leaf discs exposed to a concentration range of 0 (0), 1 (10), 20 (200), 40 (400), 60 (600), 80 (800) and 100 (1000) µg mg ^-1 (µg L⁻¹). Moderate application doses induced a hormetic effect, significantly enhancing photosynthetic pigment biosynthesis and electron transport efficiency in photosystem II (PSII). Conversely, high concentrations triggered severe oxidative stress, characterized by significant accumulations of hydrogen peroxide (H ₂ O ₂ ) and malondialdehyde (MDA), indicating lipid peroxidation of cellular membranes. This toxicity resulted in a severe depletion of non-enzymatic defense compounds including total phenolics, flavonoids, and tannins accompanied by a reduction in guaiacol peroxidase (GPOX) and ascorbate peroxidase (APX) activities. To cope with excess iron, the plant activated a catalase (CAT) mediated antioxidant defense pathway. In conclusion, CS:Fe ₃ O ₄ NPs induce concentration-dependent modulations in PSII functionality and plant metabolism. These findings successfully establish a safe environmental and agronomic application ceiling (< 60 (600) µg mg ^-1 (µg L⁻¹)), providing critical baseline parameters for the sustainable management of iron nanofertilizers in precision agriculture.