Tire wear particles (TWP), also known as tire dust, are formed when friction occurs between tires and the road surface. As the particles continue to come into contact with the road surface, they can reach increasingly smaller sizes, in the micro and nanoscale, making them more susceptible to transportation by wind and/or water. Physical damage to organisms as well as parallel toxicity exerted by compounds on the TWP composition that are released over time have already been reported. However, to induce quantifiable effects, the concentrations used are amplified and therefore fall outside the sphere of environmental relevance, while studies evaluating the ageing of the particles and the parallel release of compounds from their composition over time have yet to be reported. The study aimed to evaluate the toxicity of a TWP suspension at environmentally relevant concentrations ranging between 0.5 and 512 mg TWP/L and at three ageing stages: immediately after preparation (0dA), 7 days after preparation (7dA), and 30 days after preparation (30dA). The zebrafish model species Danio rerio was chosen to perform these assays due to its utility as a homolog for potential effects that TWP may cause on humans, and due to the multiplicity of sublethal endpoints that D. rerio allows for analysis. The 96h assays followed the OECD guideline 236 (OECD, 2013) using both embryos and post-hatching larvae to determine potential different routes of uptake. Mortality and malformations were registered every 24h, in addition to the determination of the heartbeat rate (HBR), total and interocular length (TL and IL, respectively), swim bladder area (SBA), and behaviour (total swim distance and time, thigmotaxis).

No significant mortality and malformations were registered in all assays of embryos and larvae (>10%). In embryos, HBR presented a pattern of tachycardia between concentrations of 0.5 and 8 mg TWP/L, whilst a pattern of bradycardia at higher concentrations (<32 mg TWP/L), for all ageing stages. Also in embryos, TL decreased significantly (p<0.05) at the highest concentration (512 mg TWP/L) compared to the control for all ageing stages, whilst IL was significantly (p<0.05) higher at concentrations <8 mg TWP/L compared with the control for 0dA. The SBA was significantly lower at 128 and 512 mg TWP/L compared to the control in organisms exposed to 30dA (p<0.05). In the post-hatching larvae, it is to highlight the significant increment in IL at concentrations <8 mg TWP/L for 0dA and 7dA suspensions. Larvae SBA decreased significantly (p<0.05) at all tested concentrations (except 0.5 mg TWP/L) compared to the control of 0dA. About behavioural assessment carried out at the end of each exposure, it is worth noting that activity time or distance travelled were zero or almost zero during periods of exposure to light at concentrations between 32 and 512 mg TWP/L for all stages of ageing of the suspensions, compared to the control organisms. The results obtained highlight that, in addition to the lack of ecotoxicological studies on the effects of TWP, experimental designs must be carefully planned to increase the level of realism of the experiments and to increase knowledge and understanding of the impact that this type of particulate may have on ecosystems and, ultimately, human health.