Testing laboratory protocols for micro- and nano-PET particles preparation by bottom-up chemical and top-down physical methods and implications for environmental studies

Micro- and nanoplastics are plastic particles with sizes below 1 or 5 mm for microplastics and 1 µm for nanoplastics. These particles can enter the environment through various pathways and subsequently affect all components of ecosystems. However, research on nanoplastics remains particularly limited, primarily due to challenges in isolating these particles from complex environmental matrices and producing them in controlled laboratory settings, especially in terms of size, shape, and purity. This study addresses the urgent need for standardized nanoplastic test materials by comparing various bottom-up and top-down laboratory methods for the preparation of polyethylene terephthalate (PET) micro- and nanoplastics, with a focus on their potential applications in environmental research. Chemical methods based on dissolution and recoagulation (using solvents such as 1,1,1,3,3,3-hexafluoro-2-propanol, trifluoroacetic acid, and chlorobenzene) and physical methods (including cryomilling and laser ablation) were evaluated. The resulting PET particles were characterized in terms of their size, polydispersity, morphology, and surface structure. For chemical methods, the presence of residual solvents was also analyzed. Chemical methods exhibited significant limitations, such as low reproducibility, polydisperse particles (PDI up to 11.4), strong aggregation tendencies, and the presence of potentially toxic solvent residues which make them unsuitable for standardized toxicity testing. From this point of view, physical methods represent solvent-free approaches. Cryomilling, however, proved ineffective due to surface melting of the particles during the process. Laser ablation produced highly monodisperse particles with regular spherical shapes, a median size of 340 nm, a size range of 193 to 9.5 µm, and a very low polydispersity index (PDI = 1.003 ± 0.002). These particles fall within the nanoplastic domain and showed no detectable contamination, making them especially suitable for environmental toxicity studies. Moreover, the method demonstrated consistent repeatability across replicates and is highly promising for producing standardized test materials for environmental fate, transport, and toxicity research.