Plant-mediated ‘green’ synthesis of nanoparticles (NPs) is widely reported, but the exact functional role of the retained phytochemical capping layer versus the core metal remains contested. Furthermore, the impact of thermal calcination—a common post-synthesis purification step—on the bio-functional and ecological profile of these NPs is poorly understood. We synthesized four distinct biogenic NPs—Ag and Fe using Salvinia molesta extract, and Cu and Zn using Mimosa pigra extract. While the Cu, Zn, and Fe NPs were evaluated in both non-calcined (as-synthesized) and thermally calcined states, the Ag NPs were evaluated exclusively in their highly active, non-calcined state. We evaluated their physicochemical properties, in vitro antioxidant capacity (with Ag NPs showing 42.08 mg TE/g), and antibacterial efficacy against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Additionally, the ecotoxicological impact was evaluated via a one-month soil microbial respiration assay for the calcined metal oxides (Cu, Zn, Fe) and the non-calcined Ag NPs. Characterization confirmed that calcination successfully formed highly crystalline metal oxides but stripped the Cu, Zn, and Fe NPs of their organic phytochemical corona. Consequently, the non-calcined NPs exhibited significant antioxidant activity, which was substantially abolished in the metal oxides following calcination. Antibacterial assays revealed a strict metal-dependency; Fe, Cu, and Zn NPs showed no significant antibacterial action even at high screening concentrations, regardless of calcination. In contrast, the non-calcined Ag NPs exhibited potent antimicrobial efficacy, with a minimum inhibitory concentration (MIC) of 7.8 ppm. Crucially, 4-week soil respiration assays (at doses up to 1000 ppm) demonstrated that neither the calcined metal-oxides (Fe, Cu, Zn) nor the highly reactive non-calcined Ag NPs exerted long-term toxic effects on the soil microbiome. Our findings demonstrate that the ‘green’ bioactivity (antioxidant potential) of biogenic NPs is primarily mediated by the uncalcined phytochemical corona, whereas cytotoxicity (antibacterial action) is governed by the core metal identity. The absence of significant suppression of CO₂ respiration suggests minimal acute metabolic disruption in soil microbiomes, providing preliminary evidence for short-term microbial tolerance. These findings warrant further investigation into the potential agricultural applications of these biogenic nanomaterials.
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