Comminution method and oxidative aging modulate the impact of tyre wear microplastics on the reproduction of the nematode C. elegans

Environmental context Each year, tonnes of tyre wear particles enter the terrestrial environment and undergo natural aging. This study investigates the effect of artificial weathering, particle concentration and preparation method on the reproduction of Caenorhabditis elegans, a soil- and water-dwelling nematode, using the standardised toxicology assay ISO 10872. Rationale Tyre abrasion is one of the largest sources of microplastic (MP) in aquatic and terrestrial environments. Despite this fact, research on tyre wear particles (TWP) and its effects on soil ecosystems is scarce, especially regarding natural weathering processes. This leaves a large knowledge gap on the interactions of TWP with soil biota. Methodology Cryo-milled tyre tread (CMTT) and diamond-ground tyre tread (DGTT) were artificially weathered through dry ozone treatment and heat exposure. Particles were analysed by scanning electron microscopy, particle size distribution and shape characterisation. TWP leachate composition was examined using liquid chromatography–mass spectrometry. The impact on the reproduction of the nematode Caenorhabditis elegans, a widely used toxicological model, was tested for leachate concentration, exposure duration and TWP aging status, using offspring per adult as the endpoint. Results The comminution method significantly influenced TWP particle size distribution, with diamond grinding yielding smaller particles and a more structured surface morphology than cryo-milling. Aging with ozone (180 min) and heat (20 min at 100°C) reduced DGTT particle sizes by 27–58%, but not in CMTT. Additionally, aging increased carboxylic functional groups and led to a brittle structure in both TWP types. Leachate composition varied with comminution method and aging. Benzothiazole, N-Cyclo-N-phenylurea and aniline were more abundant in pristine and aged CMTT and aged DGTT. Diphenylguanidine had the highest concentration in all leachates. Aged TWP leachates, regardless of comminution, had significant toxic effects on C. elegans. Leachate from pristine CMTT was more toxic than from pristine DGTT. Nematode offspring correlated negatively with ozone exposure duration in aged DGTT. Tests with aged CMTT leachate showed even short-term exposure reduced offspring numbers. Discussion The results underscore the importance of oxidative and mechanical weathering in TWP toxicity and challenge the use of pristine particles in toxicological assays for risk assessment in the natural environment.