The expansion of the automotive sector and the increased reliance on rubber-based components have led to the growing environmental presence of tire wear particles (TWPs), a major category of non-exhaust traffic emissions (Wik & Dave, 2009;Parker-Jurd et al., 2021). TWPs are generated predominantly through abrasion between tires and road surfaces as well as brake wear (Kole et al., 2017). They span a wide particle-size range and contain mixtures of synthetic and natural rubber together with numerous additives (Wagner et al., 2018). Relatively high density of TWPs and their resistance to weathering result in accumulation and persistence across numerous environmental compartments, where they function not only as particulate pollutants but also as vectors for numerous chemicals (Sommer et al., 2018). TWPs have been documented in diverse aquatic systems, including rivers, lakes, reservoirs, sewage networks, and marine environments, largely due to hydrological transport processes such as road runoff, stormwater flow, wastewater discharge, and atmospheric deposition (Halsband et al., 2020;Werbowski et al., 2021). Global emissions of TWPs are estimated at roughly six million tons annually (Baensch-Baltruschat et al., 2020), and they constitute a substantial fraction of microplastics detected in aquatic ecosystems (Wik & Dave, 2009;Siegfried et al., 2017). With increasing tire production and vehicle use worldwide, the environmental load and long-term impacts of TWPs are expected to intensify.

Leachates originating from tire degradation can further exacerbate ecological risks by releasing complex mixtures of chemical constituents into aquatic environments (Wik and Dave, 2009;Capolupo et al., 2020;Khan et al., 2024). These mixtures include metals, organic compounds, plasticizers, vulcanization additives, antioxidants, and antiozonants, with zinc commonly reported at elevated levels due to its central role in vulcanization (Councell et al., 2004;Camponelli et al., 2009;Halle et al., 2021;Lehtiniemi et al., 2021;Shin et al., 2022;Yang et al., 2022). Additional toxicants, such as polycyclic aromatic hydrocarbons (PAHs) and the widely used antioxidant N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) and its metabolites, have been detected in water, sediments, and biota, highlighting their environmental persistence and biological relevance (Tian et al., 2021). Aquatic contamination can be induced from direct leaching of discarded tire stockpiles, runoff from roads and landfills, and the mobilization of micro- and nanoscale TWPs (Halsband et al., 2020;Werbowski et al., 2021). Numerous studies have demonstrated that both field-collected and laboratory-generated TWP powders, along with their leachates, exert adverse effects on aquatic organisms (Turner and Rice, 2010;Hahladakis et al., 2018;Gunaalan et al., 2020;LaPlaca and van den Hurk, 2020;Capolupo et al., 2021;Halle et al., 2021;Shin et al., 2022;Zhang et al., 2025). Moreover, the combined physical and chemical stressors associated with TWPs may produce synergistic toxicity that exceeds the effects of individual components (Hermabessiere et al., 2017;Wagner et al., 2018). Despite decades of recognition of these pollutants, substantial knowledge gaps remain regarding their bioavailability and ecotoxicological impacts across taxa, underscoring the need for more comprehensive assessments.

Tire abrasion generates particles of various shapes and sizes. Yet, advancements in tire production and wear simulation have not been matched by progress in treatment technologies or globally standardized protocols for managing tire leachates. Consequently, comparable toxicity data for aquatic organisms remain scarce, underscoring the urgent need for standardized, reproducible studies to support scientific consensus and regulatory development. To evaluate the acute and chronic harmful effects of tire leachate, we used the monogonont rotifer Brachionus manjavacas as a toxicity test model. This species is widely distributed, serves as an essential food source for aquatic organisms, and is commonly used in toxicity testing (Snell and Hicks, 2011;Johnston et al., 2018;Kim et al., 2024). Using this model, we assessed 24-h acute toxicity, intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as the activities of key antioxidant defense enzymes. For chronic endpoints, we examined survival, lifespan, and reproductive output over a 10-day exposure to a sublethal concentration of tire leachate.

The monogonont rotifer B. manjavacas was maintained in continuous culture using artificial seawater within a static–renewal system at Incheon National University (Incheon, Republic of Korea). Water quality parameters, including dissolved oxygen, pH, salinity, and conductivity, were routinely monitored with an Orion Star portable pH meter (A221, Thermo Fisher Scientific Inc., MA, USA). Standardized culture conditions were maintained at 20 °C, 18 practical salinity unit (PSU), and pH 7.9–8.2, with dissolved oxygen ranging from 6.5 to 7.3 mg L^-1 under a 16:8 h light–dark cycle. Cultures were fed daily with the microalga Tetraselmis suecica at a density of 6 × 10⁴ cells mL^-1.

TWPs were produced using a tire wear simulator at the Korea Institute of Machinery and Materials (Daejeon, Republic of Korea). Leachates used in the present experiments originated from the same TWP material characterized in a previous study (Shin et al., 2022). Concentrations of metals and PAHs were quantified at the Korea Institute of Ocean Science and Technology (Geoje, Republic of Korea) using an Agilent 8800 Triple Quadrupole ICP-MS (iCAP RQ, Thermo Fisher Scientific) and an Agilent 1200 Series HPLC system coupled to a tandem mass spectrometer (API 3200; AB SCIEX, Concord, Canada). Detailed analytical results were previously reported in our previous study (Shin et al., 2022).

Acute toxicity was assessed in a 24-h static exposure test using neonate B. manjavacas (< 2 h old) exposed to tire leachate concentrations ranging from 0.1 to 1.0 g L^-1 without renewal of test media. Survival was determined after 24 h using a stereomicroscope (Olympus BX51, Tokyo, Japan), and no significant mortality was observed in the control group. Approximately 8,000 rotifers were prepared for biochemical measurements and subdivided into triplicate samples. Intracellular ROS levels were quantified using H₂DCF-DA (Molecular Probes, Eugene, OR, USA). Lipid peroxidation was evaluated via MDA, a representative thiobarbituric acid reactive substance (TBARS). Fluorescence signals were measured using a Thermo Varioskan Flash spectrophotometer (Thermo Fisher Scientific, Tewksbury, MA, USA) and normalized to protein content determined by the Pierce^TM BCA assay (Thermo Fisher Scientific Inc.). Enzymatic activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) were quantified using commercial kits (Sigma Aldrich Chemie, Switzerland) and expressed as units per milligram of protein.

Chronic endpoints were evaluated in the B. manjavacas exposed to 0.1 g L^-1 tire leachate for ten days. The detailed protocols and experimental conditions follow methodologies described in our previous studies (Kim et al., 2024, 2025). Survival, lifespan, and fecundity (number of neonates) of B. manjavacas were analyzed every 12 h using a stereomicroscope (Olympus BX51).

Overall, statistical analyses were performed using SPSS ver. 20.0 (SPSS Inc., Chicago, IL, USA). Data are presented as mean ± standard deviation. Differences among treatments were evaluated using one-way ANOVA followed by Tukey and Dunnett post-hoc tests, with significance defined at P < 0.05.

In the tire leachate, Zn (48 μg L^-1) was present at the highest concentration, followed by Cu (1.4 μg L^-1) and Ni (1.3 μg L^-1) (Shin et al., 2022). A similar highest concentration of Zn than other metals was also observed in previous studies due to the essential role of Zn as an activator in tire production (Adachi and Tainosho, 2004;Camponelli et al., 2009;Capolupo et al., 2020;Lehtiniemi et al., 2021). In the case of PAHs, retene showed the highest concentration (0.28 μg mL^-1), with pyrene being the next most abundant (0.16 μg mL^-1) (Shin et al., 2022). The total PAH concentration measured in the tire leachate was 0.70 μg mL^-1 (Shin et al., 2022). PAHs are crucial constituents of tires (Halsband et al., 2020). Therefore, the complex mixture of metals, PAHs, and numerous unquantified compounds in tire leachate may exert cocktail toxic effects on marine rotifers.

The survival rate of B. manjavacas was significantly reduced by tire leachate exposure over 24 h (Fig. 1), with a calculated 24-h LC50 of 0.56 g L^-1 as the threshold for survival and mortality. Compared to the rotifer Brachionus plicatilis, B. manjavacas showed higher sensitivity (Shin et al., 2022), which may reflect differences in habitat, genetic makeup, physiology, life history traits, and detoxification capacity between the species.

Exposure to 0.2 and 0.3 g L^-1 of tire leachate significantly increased intracellular ROS and MDA levels (P < 0.05) (Figs. 2A and 2B). Imbalances between ROS production and its removal can cause oxidative fluctuation and subsequent damage in aquatic organisms (Winston and Di Giulio, 1991). Excessive ROS can induce lipid peroxidation (Niki et al., 2005;Regoli and Giuliani, 2014), as evidenced by elevated MDA levels in B. manjavacas, suggesting that tire leachate disrupts the pro-oxidant/antioxidant balance and reduces the rotifers’ capacity to mitigate oxidative stress.

Enzymatic activities of SOD and CAT were significantly elevated in response to 0.2 and 0.3 g L^-1 of tire leachate (P < 0.05) (Figs. 2C and 2D), whereas GPx and GR activities increased significantly only at 0.3 g L^-1 of tire leachate (P < 0.05) (Figs. 2E and 2F). Aquatic invertebrates maintain oxidative homeostasis through antioxidant defenses (Valavanidis et al., 2006). SOD catalyzes the dismutation of the superoxide radical into H₂O₂ which subsequently converted by CAT into H₂O and O₂ (Halliwell and Gutteridge, 1999), while GPx and GR regenerate glutathione (GSH) to further eliminate ROS (Lesser, 2006). These elevated antioxidant responses likely contributed to the sustained survival of the marine rotifer at relatively low tire leachate concentrations (0.1–0.3 g L^-1), consistent with previous studies on this species (Kim et al., 2024, 2025).

Despite the known toxicity of tire leachate, few studies have examined its sublethal long-term effects on aquatic invertebrates. Survival rate of B. manjavacas decreased progressively over a 10-day exposure to 0.1 g L^-1 of tire leachate (Fig. 3A), suggesting cumulative damage that disrupts homeostasis and general metabolism. Life span was also significantly reduced by 0.1 g L^-1 of tire leachate in B. manjavacas (P < 0.05) (Fig. 3B), supporting the long-term detrimental effects of sublethal exposure to tire leachate. In addition, the number of newborn rotifers at day 10 was significantly lower after exposure to 0.1 g L^-1 of tire leachate (Fig. 3C), likely suggesting the combined effects of reduced survival and lifespan. Similarly, the reproduction of the rotifer B. plicatilis was significantly reduced upon chronic exposure to tire leachate, resulting in decreased population growth (Shin et al., 2022). These chronic impacts on reproduction and longevity may ultimately compromise population maintenance and ecological stability in B. manjavacas.