Abstract
The escalating release of multi-walled carbon nanotubes (MWCNTs) into the environment has raised concerns due to their potential ecotoxicological impacts. However, their combined phytotoxicity with heavy metals such as copper (Cu) is still unclear. This study investigated the individual and combined toxic effects of MWCNTs (MWCNT, MWCNT-OH, and MWCNT-COOH) and Cu(2+) on ryegrass (Lolium multiflorum), uniquely considering different addition orders. The results show that Cu severely inhibited the growth of ryegrass while MWCNTs exhibited a hormesis effect on ryegrass. When MWCNT and Cu were combined, the malondialdehyde (MDA) content in ryegrass showed a 32.39% increase at 20 mg/L MWCNT exposure, suggesting reduced oxidative stress. However, at the higher concentration of 1000 mg/L, it led to a significant 75.22% reduction in ryegrass biomass. MWCNT-COOH had the most pronounced effect, reducing the total chlorophyll content by 39.76% compared to unmodified MWCNT and by 10.67% compared to MWCNT-OH (500 mg/L). Additionally, pre-induced MWCNTs might alleviate the Cu in the plant by 23.08-35.38% through adsorption in the nutrient solution. Small molecule organic acids and amino acids primarily mediated the response to environmental stress in ryegrass. This research provides crucial insights into understanding the complex interactions of MWCNT and Cu(2+) and their combined effects on plant ecosystems.