Heavy Metals Bioaccumulation in Riparian Vegetation: A Multi-Species Biomonitoring Framework Using Total X-Ray Reflection Fluorescence Spectroscopy

Heavy metal contamination in Mediterranean riparian ecosystems presents serious risks to biodiversity and human health, requiring innovative biomonitoring methods for pol¬luted watersheds. This study developed a comprehensive biomonitoring framework for an industrially impacted Mediterranean river basin, with approximately 100,000 tons of contaminated waste in landfills from decades of metallurgical processing. We investigated heavy metal bioaccumulation patterns in five taxonomically diverse native riparian species (Equisetum arvense, Laurus nobilis, Rubus ulmifolius, Sambucus nigra, and Salix alba) across contaminated and reference sites using Total Reflection X-ray Fluorescence (TXRF) spectroscopy. TXRF was validated against Inductively Coupled Plasma Atomic Optical Spectroscopy (ICP-AOS) using standard reference materials, demonstrating superior sen-sitivity for trace element detection in plant matrices. Heavy metal concentrations varied significantly among species and sites in riparian vegetation, with Fe (23.14 ± 5.21 mg/ kg), Mn (2.74 ± 0.89 mg/kg), and Cr (3.33 ± 1.12 mg/kg) showing the highest accumula¬tion. Laurus nobilis appeared as a multi-metal hyperaccumulator with the highest Pollu¬tion Load Index (PLI = 2.67) and exceptional accumulation of Cr (14.74 mg/kg) and Fe (63.64 mg/kg). Statistical analysis (Kruskal-Wallis test, p < 0.05) confirmed that Mn and Cr represented primary anthropogenic pollutants, with contaminated sites showing 2.20- fold and 1.92-fold increases, respectively. Principal Component Analysis revealed distinct co-accumulation networks, with Fe-Cr-Ni-Cu forming synchronized uptake mechanisms, while Mn operated independently. Biogeochemical ratio analyses (Fe/Zn, Cu/Mn) pro¬vided sensitive contamination indicators, detecting physiological stress beyond simple concentration measurements. Results support species-specific phytoremediation strategies aligned with UN Sustainable Development Goals, with Laurus suitable for multi-metal ex¬traction and Equisetum for Mn-specific remediation, advancing sustainable environmental management in industrially impacted Mediterranean watersheds.