An improved method to generate secondary nanoplastics and oligomers: application in ecotoxicology†

Recent studies have highlighted the ecotoxicological effects of conventional primary nanoplastics (NPLs); however, the impacts of secondary NPLs and oligomers (Olig), especially those derived from biodegradable plastics, formed through fragmentation and natural degradation processes (e.g., photooxidation) remain underexplored. This gap is partly due to challenges in producing sufficient quantities for toxicity testing. An improved method to generate non-photooxidized (NP) and photooxidized (P) secondary NPLs and Olig from polybutylene adipate co-terephthalate (PBAT), a biodegradable plastic commonly used in agriculture mulching, which involves the mechanical breakdown of PBAT-microbeads with or without prior photooxidation is presented. PBAT was irradiated at ∼9.34 kW m⁻² (approximately 120 times the solar irradiance) for 96 h, irradiation that corresponds to ∼16 months of average sunlight in the Iberian Peninsula (7.7 kWh m⁻² per day). The toxicological effects on Chlamydomonas reinhardtii, a model green microalga of primary producers in freshwater ecosystems, were also assessed. The protocol yielded 0.199 mg of secondary NP-PBAT-NPLs and 10.275 mg of NP-PBAT-Olig per gram of PBAT-microbeads. PBAT-NPLs presented irregular spherical morphologies and hydrodynamic sizes ranging from 56.71 to 69.86 nm. HPLC and MALDI-TOF analysis identified linear and cyclic Olig, ranging from dimers to 19 repeated-units Olig. PBAT-NPLs and PBAT-Olig exhibited negative surface charges, suggesting colloidal stability in water. While PBAT-NPLs and PBAT-Olig did not inhibit algal growth in the short term, they induced reactive oxygen species overproduction at the environmentally relevant concentration of 0.01 mg L⁻¹ and caused membrane depolarization, impaired photosynthesis and lipid peroxidation at 10 mg L⁻¹. Non-photooxidized PBAT-NPLs exhibited the highest toxicity, followed by photooxidized PBAT-NPLs and both non-photooxidized and photooxidized PBAT-Olig. This study provides an efficient method for producing reference secondary NPLs and Olig and underscores the potential risks of PBAT towards primary producers in freshwater ecosystems.