Medicago sativa L. Plant Response against Possible Eustressors (Fe, Ag, Cu)-TiO(2): Evaluation of Physiological Parameters, Total Phenol Content, and Flavonoid Quantification

The present study analyzed Medicago sativa L. crops irrigated by TiO(2) in the anatase phase and TiO(2) doped with Ag, Fe, and Cu ions at 0.1%w synthesized using the sol–gel method (SG) and the sol–gel method coupled with microwave (Mw-SG). The materials were added to the irrigation water at different concentrations (50, 100, and 500 ppm). Stress induction by nanomaterials was observed by measuring stem morphology, chlorophyll index, total phenols and flavonoids, and antioxidant activity through the DPPH (2,2-diphenyl-1-picrylhydrazy) radical inhibition assay. The nanomaterial treatments caused statistically significant reductions in parameters such as stem length, leaf size, and chlorophyll index and increases in total phenol content and DPPH inhibition percentage. However, the observed effects did not show clear evidence regarding the type of nanomaterial used, its synthesis methodology, or a concentration-dependent response. By generally grouping the results obtained to the type of dopant used and the synthesis method, the relationship between them was determined employing a two-way ANOVA. It was observed that the dopant factors, synthesis, and interaction were relevant for most treatments. Additionally, the addition of microwaves in the synthesis method resulted in the largest number of treatments with a significant increase in the total content of phenols and the % inhibition compared to the traditional sol–gel synthesis. In contrast, parameters such as stem size and chlorophyll index were affected under different treatments from both synthesis methods.