Activation of Toll-like receptor 2 reveals microbial contamination beyond endotoxins on micro- and nanoplastics

Current literature on health hazards associated with micro- and nanoplastics (MNPs) is largely influenced by studies that insufficiently account for potential microbial contamination of their test materials. This may lead to misinterpretation of outcomes, as the test materials may be incorrectly considered pristine MNPs. The present study screened eight MNP test materials for microbial contaminants using Toll-like receptor (TLR) reporter cells for TLR2 and TLR4 and the commonly used Limulus amebocyte lysate (LAL) assay. Our results show that MNPs testing negative for endotoxins, based on the absence of TLR4 activation and negative LAL results, may still contain microbial ligands that selectively activate TLR2. Moreover, five of the eight MNP test materials contained microbial ligands capable of activating TLR2 and/or TLR4. Compared to the LAL assay, TLR4-based screening effectively detected endotoxin contamination. Overall, we found that the TLR reporter cell assay provides broader coverage than the LAL assay in detecting microbial ligands, which appear to be highly prevalent in MNP test materials.