Medicago sativa responses to PAH-contaminated soils amended with leaf biomass: growth and stress biomarker assessment

Polycyclic aromatic hydrocarbons (PAHs) are persistent soil contaminants that induce plant stress. This study assessed how leaf-biomass amendments affect growth and stress-biomarker profiles in Medicago sativa exposed to PAH-contaminated soils. Soils were spiked with 100 mg kg⁻¹ pyrene and phenanthrene and amended with 10%, 30%, or 70% (w/w) leaf biomass, sand + 30% amendment, contaminated-only, and non-contaminated controls. After 30 days of soil incubation, residual PAHs were quantified. Subsequently, alfalfa was grown for 4 weeks, and plant vigor, photosynthetic pigments, oxidative stress biomarkers, antioxidant enzyme activities, and nutrient uptake were assessed. PAH dissipation was clearly amendment dependent. The 10% amendment was most effective (18.9 ± 5.85 mg kg⁻¹; 81.10% reduction), followed by 30% (33.01 ± 4.92; 66.99%), 70% (39.40 ± 8.05; 60.60%), and sand + 30% (58.10 ± 4.56; 41.90%). The unamended contaminated soil retained its full load (~ 100 mg kg⁻¹) as it was not subjected to incubation, whereas the background control declined from 2.82 to 0.50 mg kg⁻¹ (82.27% reduction) by natural attenuation. Plant vigor mirrored these trends, highest in controls and lowest in contaminated soil. Stress symptoms included chlorophyll-a depletion, lipid peroxidation, and accumulation of reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂) and superoxide anion (O₂•⁻). Antioxidant defenses—comprising catalase (CAT), which decomposes hydrogen peroxide into water and oxygen; ascorbate peroxidase (APX), which detoxifies hydrogen peroxide via the ascorbate–glutathione cycle; and phenolic compounds, which contribute to non-enzymatic ROS scavenging—were upregulated but insufficient to fully counter oxidative damage. Leaf biomass amendments—particularly at 10%—significantly enhanced PAH dissipation, reduced oxidative stress, and maintained physiological function in M. sativa. These findings support the potential use of leaf residues as low-cost organic inputs for sustainable phytomanagement and highlight M. sativa as a sensitive bioindicator for assessing soil quality in PAH-affected systems.