Synergistic effect of pyrene and heavy metals (Zn, Pb, and Cd) on phytoremediation potential of Medicago sativa L. (alfalfa) in multi-contaminated soil

The environment in India is contaminated with polycyclic aromatic hydrocarbons (PAHs) due to the occurrence of large anthropogenic activities, i.e., fuel combustion, mineral roasting, and biomass burning. Hence, 13 toxic PAHs were detected: phenanthrene, anthracene, fluoranthene, pyrene, and benz(a) anthracene, ben-zo; (b) fluoranthene, benzo(k) fluoranthene, benzo(a) pyrene, benzo(ghi)perylene, dibenz (ah) anthracene, indeno1,2,3-(cd) pyrene, coronene and coronene in the environment (i.e., ambient particulate matter, road dust, sludge, and sewage) of the most industrialized area. Pollutants such as heavy metals and polycyclic aromatic hydrocarbons co-contaminate the soil and pose a significant hazard to the ecosystem because these pollutants are harmful to both humans and the environment. Phytoremediation is an economical plant-based natural approach for soil clean-up that has no negative impact on ecosystems. The aim of this study was to investigate the effects of pyrene (500 mg kg^-1), Zn (150 mg kg^-1), Pb (150 mg kg^-1), and Cd (150 mg kg^-1) alone and in combination on the phytoextraction efficiency of Medicago sativa growing in contaminated soil. Plant biomass, biochemical activities, translocation factors, accumulation of heavy metals, and pyrene removal were determined. After 60 days of planting, compared with those of the control plants, the growth parameters, biomass, and chlorophyll content of the M. sativa plants were significantly lower, and the reactive oxygen species activity, such as proline and polyphenol content and metallothionein protein content, was markedly greater in the pyrene and heavy metal-polluted soils. Furthermore, the combined toxicity of pyrene and all three metals on M. sativa growth and biochemical parameters was significantly greater than that of pyrene, Zn, Pb, or Cd alone, indicating the synergistic effect of pyrene and heavy metals on cytotoxicity. Pyrene stress increased Cd accumulation in M. sativa. After pyrene exposure alone or in combination with Zn-pyrene, a greater pyrene removal rate (85.5-81.44%) was observed than that in Pb-pyrene, Cd-pyrene, and Zn-Pb-Cd-pyrene polluted soils (62.78-71.27%), indicating that zinc can enhance the removal of pyrene from contaminated soil. The resulting hypotheses demonstrated that Medicago sativa can be used as a promising phytoremediation agent for co-contaminated soil.