Growth, physiological parameters and DNA methylation in Spirodela polyrhiza (L.) Schleid exposed to PET micro-nanoplastic contaminated waters

The effects of polyethylene terephthalate micro-nanoplastics (PET-MNPs) were tested on the model freshwater species Spirodela polyrhiza (L.) Schleid., with focus on possible particle-induced epigenetic effects (i.e. alteration of DNA methylation status). MNPs (size ∼ 200-300 nm) were produced as water dispersions from PET bottles through repeated cycles of homogenization and used to prepare N-medium at two environmentally relevant concentrations (∼0.05 g L^-1 and ∼0.1 g L^-1 of MNPs). After 10 days of exposure, a reduction in fresh and dry weight was observed in treated plants, even if the average specific growth rate for both frond number and area was not altered. Impaired growth was coupled with a MNP-induced decrease of chlorophyll fluorescence parameters (i.e. ΨET_o and Pi_abs, indicators of photochemical efficiency) and starch concentration, as well as with alterations in plant ionomic profile and oxidative status. The methylation-sensitive amplification polymorphism (MSAP) technique was used to assess possible changes in DNA methylation levels induced by plastic particles. The analysis showed unusual hypermethylation in 5'-CCGG sites that could be implicated in DNA protection from dangerous agents (i.e. reactive oxygen species) or in the formation of new epialleles. This work represents the first evidence of MNP-induced epigenetic modifications in the plant world.