Cytotoxic activity of settled dust in primary schools: a multi-matrix, multi-season screening approach for children’s environmental health

Children spend up to nine hours daily in school environments, yet the potential health impact of accumulated indoor dust remains largely uncharacterised. This study provides the first multi-matrix, multi-season assessment of passive dust samples from Portuguese primary schools, using the MTT assay to evaluate the biological activity of complex indoor pollutant mixtures. 207 passive samples were collected across 11 primary schools in the Lisbon Metropolitan Area, using four sampling matrices, settled dust filters, electrostatic dust cloths (EDC), electrostatic dust cloths on T-shirts (EDCT), and floor mops, across six indoor locations, warm and cold seasons, and in urban and rural contexts. Cytotoxic activity was assessed in A549 (human alveolar epithelial) and SK (swine kidney) cells, potential azole resistance was detected by growth on azole-supplemented culture media. Cytotoxic activity was detected in 64.7% (A549) and 24.6% (SK) of samples. Sampling matrix was the strongest predictor of cytotoxic variability for both cell lines (A549: R2 = 0.199; SK: R2 = 0.081; p < 0.001), with floor mops exhibiting the highest cytotoxic rates (A549: 94.4%; SK: 83.3%). Season was a significant secondary modulator exclusively for A549 cells, with cold-season positivity of 82.8% versus 50.0% (p < 0.001). Geographic context, school identity, and indoor location showed no significant effects, suggesting cytotoxic variability reflects building-level operational factors, cleaning practices and ventilation, rather than environmental setting. Spearman analysis revealed cell-line and compound-specific associations between fungal burden, potential azole resistance detected by growth on azole-supplemented culture media, and cytotoxic responses, with reduced susceptibility to itraconazole, voriconazole, and posaconazole detected in school dust isolates. These findings demonstrate that microbial contamination in school settled dust may result in potential health effects involving clinically relevant antifungal resistance species. Floor mop sampling is identified as a high-sensitivity sentinel approach for the combined biological and chemical toxic burden accumulated at floor level. Passive multi-matrix cytotoxicity assessment should be integrated into school indoor air quality surveillance to protect this vulnerable population.