Evidence of impact of tire wear particles on a roadside plant community at environmentally relevant concentrations

Evidence of impact of tire wear particles on a roadside plant community at environmentally relevant concentrations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evidence of impact of tire wear particles on a roadside plant community at environmentally relevant concentrations Shin Woong Kim, Yooeun Chae, Hyun-Sook Jeong, Chang-Beom Park This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8597992/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 21 You are reading this latest preprint version Abstract Tire wear particles (TWPs) are recognized as a significant contributor of microplastic pollution, and recent research has shown their effects on plants using various types of crops. However, given the fact that abrasion of tire tread intensifies near roadways, roadside plant community should receive more attention. In this study, we established mono- and mixed-plant communities including four different species (, , , and ), and the effects of TWPs on each community were investigated across a range of concentrations (1 to 10,000 mg kg). We found the significant changes in the structure of mixed-plant community, which represented opposite growth patterns between two species ( and ), while there were no effects on each species in the mono-culture condition. Our results revealed that takes advantage in the mixed-culture condition, in terms of biomass, while experienced the negative effects on shoot mass. We measured electrical capacitances to understand plant-plant interactions in root system, and it aligned with the growth patterns ( and ) of TWP treatments. Soil pH varied depending on the cultivation of each species and TWP treatments. Consequently, we assumed that TWPs alter the soil process and plant-plant interactions. Roadside ecosystem provides ecological values, and the impacts of TWPs on roadside plant community might be crucial to sustain a wild range of environmental functions. Biodiversity Microplastics Mixed culture Soil Wild plant Figures Figure 1 Figure 2 Figure 3 1. Introduction Tire wear particles (TWPs) are generated from the friction between tire tread and road surface, and they have received extensive attention as one of microplastic types (diameter < 5 mm) in recent years (Ding et al., 2023 ; Wang et al., 2024 ). Emissions of TWPs mainly occurs in urban road traffic, and they are accumulated on roadside through dust or aerosol dispersion (Sommer et al., 2018 ). Roadside soils are main enrichment locations for TWPs, and the highest concentrations are found within five meters of the road (Siever et al., 2020 ). The high level of TWPs pollution in the roadside soils has been reported from 2,000 to 26,400 mg kg -1 , and TWPs in a motorway of Germany range from 155 to 15,898 mg kg -1 (Müller et al., 2022 ; Rødland et al., 2023 ). There are a few studies regarding the potential effects of TWPs on soil ecosystem (Ding et al., 2023 ; Wang et al., 2024 ). The impacts of TWPs on plants have been investigated using various types of crops, including Vigna radiata (mung bean), Ipomoea aquatica (water spinach), and Allium porrum (leek) (Kim et al., 2022 ; Leifheit et al., 2022 ; Zeb et al., 2024a ; 2024b ). V. radiata (mung bean) shows negative responses in growth and photosynthetic capacity at TWP concentrations of 1 and 10 g kg -1 (Kim et al., 2022 ), but by contrast, another study has reported that there was no significant impact at concentration up to 15 g kg -1 (Zeb et al., 2024a ). Aged TWPs enhance the growth of I. aquatica (water spinach) while pristine TWPs increase the plant stress at concentration of 1 and 10 g kg -1 (Zeb et al., 2024b ), and the growth of A. porrum (leek) significantly decrease at 10 to 60 g kg -1 (Leifheit et al., 2022 ). The use of crop species for toxicity test is important since a large portion of TWPs can migrate into agricultural soils through sewage sludge or rainwater erosion (Wang et al., 2024 ). However, given the fact that abrasion of tire tread intensifies near roadside with heavy traffic, the roadside plant community should receive more attention in research field. Since the landscapes are heavily disrtubed by agriculture or urban development, roadside ecosystem plays an important role for remaining fragments of native vegetation, making them crucial for the conservation of plant and animal biodiversity including rare and threatened species (Milton et al., 2015 ). Compared with agricultural mono-culture, the roadside plant communities have a complex structure (Castro, 2010 ; Ding and Eldridge, 2022 ), and they form heterogeneous habitats for soil animals, providing a range of ecological value such as pollination (Milton et al., 2015 ). The roadside environment receives high concentrations of chemicals due to road drainage and dust (De Silva et al., 2020 ; Rentch, 2005). However, compared to the current knowledge in crop species, the effects of TWPs on roadside plant community are poorly understood. In this study, we established mono- and mixed-plant communities with invasive, allelopathic, or facilitative characteristics, which naturally co-occur in roadside soils. We assessed the effect of TWPs on the growth and community structure, in mono- and mixed-cultures of four plant species ( Aegopodium podagraria , Lespedeza cuneata , Artemisia princeps , and Agastache rugosa ). We hypothesized that TWPs would alter not only growth but also community structure and that TWPs may exacerbate the effects on the mixed-culture condition. 2. Materials and methods 2.1. TWPs and soil preparation Waste tires were collected from random vehicles and trucks, and mechanically ground using granulator, rotary mill, and extruder at tire recycling company (Greenara, Haman-gun, Gyeongsangnam-do, South Korea). Further size reduction was achieved using a cryogenic-grinder (6875 Freezer/Mill®, SPEX SamplePrep, USA), and the tire particles were passed through 500-µm sieve. The prepared TWPs were observed under an optical microscope (SMZ1000, Nikon, Japan), and average size was measured using ImageJ version 1.54p (U.S. National Institute of Health, Bethesda, MA, USA). TWPs showed irregular shape with micro-sizes, and the average size was 492 ± 271 µm ( n = 200). We collected sandy loam soil (water holding capacity, 38.5%, pH, 5.9) from Jinju, South Korea, where wild plant species naturally grow. The soil was air-dried at room temperature, and then sieved through 4 mm mesh size, and mixed with TWPs at the concentrations of 0 (control, t0), 1 (t1), 10 (t2), 100 (t3), 1,000 (t4), and 10,000 (t5) mg kg -1 , which encompass environmentally relevant concentrations (155 to 26,400 mg kg -1 ) (Müller et al., 2022 ; Rødland et al., 2023 ). We prepared TWP-soils separately for each experimental unit. Each portion of TWPs was mixed into 250 g of soil, and homogenized by hand shaking for 3 min, before being placed into each individual pot (width, 13 x 13 cm, height, 12 cm), to provide an equal distribution of TWPs throughout the soil. Each pot was watered (60% of water holding capacity), and maintained for one week before planting. 2.2. Plant species We selected four plant species including A. podagraria (Apiaceae, goutweed), L. cuneata (Fabaceae, sericea lespedeza), A. princeps (Asteraceae, mugwort), and A. rugosa (Lamiaceae, Korean mint). All s pecies are frequently co-occurring grassland species that naturally grow in roadside environment. A. podagraria is widely distributed in the open habitats such as roadsides or gardens (D’Hertefeldt et al., 2014 )d cuneata has been suggested as an indicator species of roadside vegetation (Rentch et al., 2005 ). A. princeps and A. rugosa are the representative species of roadside environment in South Korea (Cho et al., 2013 ). Each seed was obtained from a commercial supplier in the region (Aramseed, Seoul, South Korea). Since the germination rates of all species were nearly zero in sterile conditions (10% sodium hypochlorite and sterile sand), we skipped the surface-sterilization process. Each seed was germinated for 14 days in trays containing potting soil (Hungnong, South Korea, cocopeat 47.2%, peatmoss 35%, zeolite 7%, vermiculite 10%, dolomite 0.6%). Individual seedlings of similar development stage were used for the plant test: average shoot lengths ( n = 10) of each seedling were 7.5 ± 1.9 cm ( A. podagraria ), 4.7 ± 1.1 cm ( L. cuneata ), 1.8 ± 0.4 ( A. princeps ), and 1.4 ± 0.2 cm ( A. rugosa ). 2.3. Experimental design In December 2024, we established the pot experiment in a controlled room with a day-light period of 16 h, and a temperature regime at 22°C with a relative humidity of 60%. We prepared 156 experimental units (pots) including mono-culture (96), mixed-culture with four species (36), and no plant group (24). Each group contained four (mono and no plant) and six (mixed) replicates per each TWP treatment (t0 to t5) (Fig. S1 ). Four holes per a pot were dug in a grid, keeping a distance of 3 cm. Seedlings were regularly distributed for the mixed-culture (upper position, A. podagraria and A. princeps ; lower position, L. cuneata and A. rugosa ; left to right in order), and each planting hole received one plant individual. Four individuals of the same species were also planted for mono-culture condition, to serve as an intraspecific interaction control. Pots were well watered every day (60% of soil water holding capacity) during the 7 weeks of growth. Before one day of the harvest, the electrical capacitances between plant-plant species in each individual pot was measured. The electrical capacitance is a physical quantity that characterizes the ability of capacitors to hold electrical charge, and it is part of bioimpedance technology (Gu et al., 2021 ). The nondestructive method is applied to the stem and soil through the electrodes (Gu et al., 2021 ; Chloupek, 1972 ), but in this study, we measured the electrical capacitance between plant-plant species. The electrical capacitance was measured after four hours of watering (60% of soil water holding capacity) using a capacitance meter (New Mate 2012R, Kyoritsu, Japan) with a frequency of 1 kHz. The plant electrodes were fixed with a clamp about 2 cm above ground of each plant in a pot. The electrical capacitance was recorded in nano Farad (nF) when it became stable after attaching the two clamps. The harvest was done in a randomized manner. A total 528 of shoots (384 from mono-culture pots; 144 from mixed-culture pots) was sorted by plant species, and shoot mass, shoot height, and number of leaves were measured immediately. Roots were carefully removed from the soil and gently washed. Each root mass was measured after drying at 40°C oven for 24 h. We measured soil pH of 156 experimental units using the soil-water suspension (1:5). 2.4. Statistical analysis Data visualization and statistical analysis were computed by R Version 4.4.2. Significant differences of the measured parameters (mass, height, and number of leaves) in each treatment were assessed by ANOVA with Tukey and Bonferroni’s multiple range tests at p < 0.05. We calculated the relative interactive index (RII) for each species to understand how each plant-plant interaction influenced shoot and root mass (Armas et al., 2004 ). RII represents the competitive effect of intra- and inter-specific interactions compared with when the species is grown alone: RII = (B mix – B mono )/(B mix + B mono ), where B mix is the biomass of a species growing in the mixed-culture condition and B mono is the biomass of a species that is grown in each mono-culture condition. Principal component analysis (PCA) was performed using the function “prcomp” and “fviz_pca” from the package “factoextra” and “factorMineR” (Kassambara and Mundt, 2017 ). To analyze changes in plant community composition, we log-transformed shoot and root mass per species, and each ellipse in the PCA graph grouped the different treatments (t0 to t5) with a confidence level of 0.95. 3. Results 3.1. Effects of TWPs on mono- and mixed-plant communities No significant changes in shoot mass, shoot height, number of leaves, and root mass were observed in the mono-culture conditions of each species (Fig. S2). However, in the mixed-culture condition, we found the significant differences of TWP treatments compared to control group (Fig. 1 ). The shoot mass and height of A. princeps significantly decreased at the highest concentration (t5, 10,000 mg kg -1 ) (Fig. 1 C), but by contrast, the shoot mass of A. rugosa significantly increased at the same concentration (Fig. 1 D). In addition, the root mass of A. rugosa was pronounced at t4 concentration (1,000 mg kg -1 ), compared to the control group (Fig. 1 D). Although total mass of shoot and root in each mixed-culture pot (each community level) showed no differences between each treatment (Fig. S3), RII (for shoot and root biomass) of both A. princeps and A. rugosa were influenced in the mixed-culture conditions (Fig. S4). The PCA results revealed the distinct differences of shoot and root mass in TWP treatments. The shoot growth of A. princeps showed a negative correlation with A. rugosa (PC1, 37.6%), and the ellipses of control and TWP treatment (t5) were significantly separated with a confidence level of 95% (Fig. 2 A). The root mass analysis indicated that the A. rugosa exhibited the negative correlation with other plant species on PC1 (41.2%) (Fig. 2 B). 3.2. Soil pH and plant-plant interactions The soil pH in no plant condition were measured as 6.01 ± 0.10, 6.02 ± 0.05, 6.16 ± 0.04, 6.26 ± 0.06, 6.25 ± 0.07, and 6.32 ± 0.06 in each TWP treatment. The increase of soil pH depending on TWP treatments were also observed in three mono-culture conditions ( A. podagraria , L. cuneata , and A. rugosa ), while A. princeps mono-culture showed no differences between each treatment (Fig. S5). The soil pH in the mixed-culture conditions reached to 6.49 ± 0.05, but there was no significant changes in all TWP treatments. The electrical capacitances in control group were shown in Fig. S6. The highest electrical capacitance was observed between A. podagraria of mono-culture, and the lowest one was L. cuneata mono-culture. In TWP treatment (t5), each electrical capacitance of plant-plant interactions was 0.38 ± 0.05 ( A. podagraria - A. podagraria ), 0.25 ± 0.04 ( A. podagraria - L. cuneata ), 0.32 ± 0.04 ( A. podagraria - A. princeps ), 0.41 ± 0.09 ( A. podagraria - A. rugosa ), 0.20 ± 0.01 ( L. cuneata - L. cuneata ), 0.22 ± 0.02 ( L. cuneata - A. princeps ), 0.24 ± 0.02 ( L. cuneata - A. rugosa ), 0.30 ± 0.03 ( A. princeps - A. princeps ), 0.30 ± 0.02 ( A. princeps - A. rugosa ), and 0.32 ± 0.04 ( A. rugosa - A. rugosa ) nF (Fig. S5B). 4. Discussion The underlying mechanism of TWP effects on plant remains unclear. There are several potential effect mechanisms of particulate contaminants on plants, in part as a function of their types, such as microplastics. Alterations of soil physical structure by microplastic addition could directly influence plant root activities, and a large amount of inert carbon in microplastics might slowly contribute the wide C:N ratio, potentially leading to nutrient immobilization (Rillig and Lehmann, 2019). In this study, we found no effects on the mono-culture treatments (Fig. S2), and total biomass of each treated pot showed no changes (Fig. S3). However, the growth patterns of individual plants in the mixed-culture were altered by TWP addition (Fig. 1 C and D), and it might be related to the shift of community structures. We assumed that there is an unrevealed mechanism in plant-plant interactions. TWPs can alter soil pH since they contain calcium carbonate as a filler (Zhu et al., 2024 ). In this study, soil pH varied depending on the cultivation of each species ( A. podagraria , 5.42 ± 0.18, L. cuneata , 5.88 ± 0.03, A. princeps , 5.79 ± 0.07, A. rugosa , 5.48 ± 0.05), and each mono-culture showed the increasing trends with the increase of TWP concentration (Fig. S5). However, in both A. princeps mono-culture and mixed-culture conditions, no significant change was observed across the TWP treatments, and A. princeps may play an important role to capture the potential shifts in soil pH by TWP addition. A. princeps is known to produce multiple biochemical compounds (Ryu et al., 2013 ), and they are crucial for maintiaing the plant growth and adapting to sress condition by regulating soil processes such as rhizosphere pH (Hättenschwiler and Vitousek, 2000 ). However, higher plant diversity can raise soil pH with the accumulation of cations such as calcium and magnesium (Furey and Tilman, 2021 ), and the control soil pH in the mixed-culture condition was already elevated, potentially exceeding the measurable capacity of TWPs to induce a further increase (Fig. S5). Consequently, we assumed that A. princeps failed to change the pH shift favorable to its growth, and TWPs alter the environment in a way that benefits to other competitors ( A. podagraria and A. rugosa ). The plant-plant interactions in a community, such as competition, facilitation, and allelopathy, are widespread and easily observed in mixtures of plants (Thorpe et al., 2011 ). A. podagraria is known as an invasive plant that creates symbiotic relationships to native plant species (Jakubczyk et al., 2020 )d princeps has compact and massive root system, leaving no space for pre-existing vegetation (Verloove and Andeweg, 2020 ). Although each plant species has different strategies of competition or facilitation, our results revealed that A. podagraria takes advantage in the mixed-culture condition, in terms of biomass, while A. princeps experienced the negative effects on shoot mass and number of leaves, as compared to the mono-culture condition (Fig. 3 A and C). L. cuneata and A. rugosa showed no change in shoot mass and height, but the leave numbers of L. cuneata was reduced (Fig. 3 B and D). We measured the electrical capacitance to understand the plant-plant interactions through the electrical properties in root systems (Gu et al., 2021 ), and the relevant differences were observed in each species interaction (Fig. S6). From the view of electrical interactions between A. podagraria and each other species in the control condition, the electrical capacitance of L. cuneata was lower than those of other species, while A. princeps and A. rugosa exhibited stronger interactions with A. podagraria . These three species have an extensive root system (Verloove and Andeweg, 2020 ), and the underground competition or facilitation seems trigger the increase of electrical capacitance between each root. After TWP exposure, the electrical capacitance of A. princeps associated with A. podagraria diminished, whereas A. rugosa experienced a slight increase. These results align with the growth patterns of these species in the TWP treatment (Fig. 1 ). 5. Conclusion Roadside ecosystem serves multiple purposes such as enhancing the aesthetics of landscape, stimulating nutrient cycling, improving air quality, and creating wildlife habitats, but the natural and anthropogenic disturbances (e.g., construction or erosion) could influence these functions (Waheed and Naeem, 2024 ). When considering that roadside ecosystem acts as an ecological conduit allowing native plant and animal species to move among landscapes such as agricultural land or urban garden (Brooker, 2006 ; Dietzel et al., 2023 ), their ecological stability might be crucial to sustain a wild range of environmental functions. In this study, we found that TWPs affect roadside plant community at environmentally relevant concentration, and the potential competition or commensalism may be altered in each plant species and shift the community structure. In other aspects, the roadside plant community has a high resistance against TWP pollution by regulating their composition and structure. However, the plant-plant interactions play a key role throughout ecosystem, not only plant community themselves (Brooker, 2006 ), and soil can be altered over time through small changes that accumulate in a part of communities. The effects of TWPs on plant communities might be another key factor in roadside environment, where the actual high level of pollution occurs, and this potential variable should be considered to understand the effects on relatively unconcerned ecosystem. This study may provide the starting point to achieving these goals, and future studies should be pursued through large-scale and multisite experiments. Declarations Acknowledgements We appreciate all members of Division of Gyeongnam Bio-Environmental Research for all service they provide. Author contributions S.W.K. conceptualised the study. S.W.K., Y.C., and H.-S.J. performed the experiment and processed the samples. S.W.K. and Y.C. analysed the data. S.W.K. led the writing with support of Y.C. and C.-B.P. and input from all authors. All authors reviewed the manuscript. Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-16070203). This research was supported by the Korea Institute of Toxicology, Republic of Korea (2710086916). Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests No, I declare that the authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper References Armas, C., Ordiales, R., Pugnaire, F. I. (2004). Measuring plant interac -tions: A new comparative index. Ecology , 85 , 2682–2686. Bennett, A. F. (2023). Linkages in the landscape: the role of corridors and connectivity in wildlife conservation. Coserving Foret Ecosystems Series No. 1, IUCN Forest Conservation Programme, Gland, Switzerland and Cambridge, UK. Brooker, R. W. (2006). Plant-plant interactions and environmental change. New Phytologist , 171 , 271–284. Castro, A., Wise, D. H. (2010). Influence of fallen coarse woody debris on the diversity and community structure of forest-floor spiders (Arachnida: Araneae). Forest Ecology and Management , 260 , 2088–2101. Chloupek, O. (1972). The relationship between electric capacitance and some other parameters of plant roots. Biologia Planttarum , 14 , 227–230. Cho, K.-J., Kang, K.-K., Han, M.-S., Na, Y.-E., Kim, M., Choe, L.-J., Kim, M.-H. (2013). Occurrence and distribution of C 4 plants under diverse agricultural field types in Korea. Korean Journal of Agricultural and Forest Meteorology , 15 , 85–101. D’Hertefeldt, T., Eneström, J. M., Pettersson, L. B. (2014). Geographic and habitat origin influence biomass production and storage translocation in the clonal plant Aegopodium podagraria. PLOS One , 9 , e85407. De Silva, S., Ball, A. S., Indrapala, D. V., Reichman, S. M. (2020). Review of the interactions between vehicular emitted potentially toxic elements, roadside soils, and associated biota. Chemosphere , 263 , 128135. Dietzel, S., Rojas-Botero, S., Kollmann, J., Fischer, C. (2023). Enhanced urban roadside vegetation increases pollinator abundance whereas landscape characteristics drive pollination. Ecological Indicators , 147 , 109980. Ding, J., Eldridge, D. J. (2022). Roadside verges support greater ecosystem functions than adjacent agricultural land in a grassy woodland. Journal of Environmental Management , 308 , 114625. Ding, J., Lv, M., Zhu, D., Leifheit, E. F., Chen, Q. L., Wang, Y. Q., Chen, L. X., Rillig, M. C., Zhu, Y. G. (2023). Tire wear particles: an emerging threat to soil health. Critical Reviews in Environmental Science & Technology , 53 , 239–257. Furey, G. N., Tilman, D. (2021). Plant biodiversity and the regeneration of soil fertility. Proceedings of the National Academy of Sciences , 118 , e2111321118. Gu, H., Liu, L., Butnor, J. R., Sun, H., Zhang, X., Li, C., Liu, X. (2021). Electrical capacitance estimates crop root traits best under dry conditions–a case study in cotton ( Gossypium hirsutum L.). Plant Soil , 467 , 549–567. Hättenschwiler, S., Vitousek, P. M. (2000). The role of polyphenols in terrestrial ecosystem nutrient cycling. Trends in Ecology & Evolution , 15 , 238–243. Jakubczyk, K., Janda, K., Stykurski, D., Łukomska, A. (2020). Goutweed ( Aegopodium podagraria L.) – botanical characteristics and prohealthy properties. Postepy Higieny i Medycyny Doswiadczalnej , 74 , 28–35. Kassambara, A., Mundt, F. (2017). factoextra: Extract and visualize the results of multivariate data analyses. R package version 1.0.5. https://CRAN.R-project.org/package=factoextra . Kim, L., Lee, T.-Y., Kim, H., An, Y.-J. (2022). Toxicity assessment of tire particles released from personal mobilities (bicycles, cars, and electric scooters) on soil organisms. Journal of Hazardous Materials , 437 , 129362. Leifheit, E. F., Kissener, H. L., Faltin, E., Ryo M., Rillig, M. C. (2022). Tire abrasion particles negatively affect plant growth even at low concentrations and alter soil biogeochemical cycling. Soil Ecology Letters, 4, 409–415. Milton, S. J., Dean, W., Sielecki, L. E., Ree, R. (2015). The function and management of roadside vegetation. Handbook of Road Ecology, 373–381. Müller, A., Kocher, B., Altmann, K., Braun, U. (2022). Determination of tire wear markers in soil samples and their distribution in a roadside soil. Chemosphere, 294, 133653. Rentch, J.S., Fortney, R. H., Stephenson, S. L., Adams, H. S., Grafton, W. N., Anderson, J. T. (2005). Vegetation–site relationships of roadside plant communities in West Virginia, USA. Journal of Applied Ecology , 42 , 129–138. Rillig, M. C., Lehmann, A., De Souza Machado, A. A., Yang, G. (2019). Microplastic effects on plants. New Phytologist , 223 , 1066–1070. Rødland, E. S., Heier, L. S., Lind, O. C., Meland, S. (2023). High levels of tire wear particles in soils along low traffic roads. Science of Total Environment , 903 , 166470 Ryu, R., Jung, U. J., Kim, H.-J., Lee, W., Bae, J.-S., Park, Y. B., Choi, M.-S. (2013). Anticoagulant and antiplatelet activities of Artemisia princeps pampanini and its bioactive components. Preventive Nutrition and Food Science , 18 , 181–187. Siever, R., Kawecki, D., Nowack, B. (2020). Dynamic probabilistic materials flow analysis of rubber release from tires into the environment. Environmental Pollution , 258 , 1313573. Sommer, F., Dietze, V., Baum, A., Sauer, J., Gilge, S., Maschowski, C., Giere, R. (2018). Tire abrasion as a major source of microplastics in the environment. Aerosol Air Quality Research , 18 , 2014–2028. Thorpe, A. S., Aschegoug, E. T., Atwater, D.Z., Callaway, R. M. (2011). Interactions among plants and evolution. Journal of Ecology , 99 , 729–740. Verloove, F., Andeweg, R. (2020). Artemisia princeps L. (Asteraceae), an overlooked invasive Far Eastern weed in Western Europe. Gorteria , 42 , 1–18. Waheed, M., Naeem, Z. (2024). The role of roadside tree planting in environmental conservation. Radinka Journal of Science and Systematic Lierature Review , 2 , 235–239. Wang, Y., Li, X., Yang, H., Wu, Y., Pu, Q., He, W., Li, X. (2024). A review of tire wear particles: Occurrence, adverse effects, and control strategies. Ecotoxicology and Environmental Safety , 283, 116782. Zeb, A., Liu, W., Ali, N., Shi, R., Lian, Y., Wang, Q., Wang, J., Li, J., Zeng, Z., Liu, J., Yu, M., Liu, J. (2024a). Integrating metabolomics and high-throughput sequencing to investigate the effects of tire wear particles on mung bean plants and soil microbial communities. Environmental Pollution , 340 , 122872. Zeb, A., Liu, W., Ali, N., Shi, R., Zhao, Y., Wang, J., Wang, Q., Khan, S., Baig, A. M., Liu, J., Khan, A. A., Ge, Y., Li, X., Yin, C. (2024b). Impact of pristine and aged tire wear particles on Ipomoea aquatica and rhizospheric microbial communities: Insights from a long term exposure study. Environmental Science & Technology , 58 , 21143–21154. Zhu, Y., Kim, S. W., Li, H., Rillig, M. C. (2024). Delivery rate alters the effects of tire wear particles on soil micobial activities. Environmental Sciences Europe , 36 , 9. Additional Declarations No competing interests reported. Supplementary Files GA.png Graphical abstract TWPRoadsideplantSM.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 07 Apr, 2026 Reviews received at journal 03 Apr, 2026 Reviews received at journal 23 Feb, 2026 Reviews received at journal 11 Feb, 2026 Reviews received at journal 08 Feb, 2026 Reviews received at journal 08 Feb, 2026 Reviews received at journal 07 Feb, 2026 Reviews received at journal 07 Feb, 2026 Reviews received at journal 03 Feb, 2026 Reviewers agreed at journal 01 Feb, 2026 Reviewers agreed at journal 30 Jan, 2026 Reviewers agreed at journal 30 Jan, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers invited by journal 27 Jan, 2026 Editor assigned by journal 18 Jan, 2026 Submission checks completed at journal 14 Jan, 2026 First submitted to journal 14 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8597992","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581828735,"identity":"4a713322-99ed-4791-84b5-a7c77745aefd","order_by":0,"name":"Shin Woong Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYDACCcYGKIuN4QBDRQIPTMKASC1nEnhgevBogbPYGBgY2xIYCGqRn93c9pinwiZP3oEt8dDNeWky9uw9BswVFQzG5g3YtRjcOdhuzHMmrdjwANuBw7nbcnh4eI4lMJ45w2AmcwCHFonENunctsOJGxvYG4BaKnh4JJIPMDa2MdhIYNfBID8DRcsckJbEBsbGf7i1MNyAapnPAHJYQw7UlgYGM1xaDEBa/pxJS9zAzJZwOOdYGg/PmWMJBxuOSRjjdlj6M8kZFTaJ89vbjD/n1CTbs7f3GD5sqLExnIHLYXDrDiNxDiDHF04g30BYzSgYBaNgFIxQAABhnlcc7nvcpQAAAABJRU5ErkJggg==","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":true,"prefix":"","firstName":"Shin","middleName":"Woong","lastName":"Kim","suffix":""},{"id":581828736,"identity":"c01b2f88-5402-4e5c-8385-f69cb397473d","order_by":1,"name":"Yooeun Chae","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Yooeun","middleName":"","lastName":"Chae","suffix":""},{"id":581828737,"identity":"69e2707b-4294-4a0e-adfd-479086e634f5","order_by":2,"name":"Hyun-Sook Jeong","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Hyun-Sook","middleName":"","lastName":"Jeong","suffix":""},{"id":581828738,"identity":"bcfba161-1cab-4008-9ef8-5b1577a3aeb3","order_by":3,"name":"Chang-Beom Park","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Chang-Beom","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2026-01-14 06:08:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8597992/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8597992/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101442733,"identity":"9d28c6e9-a519-4a13-9aa1-d21fe12ef62e","added_by":"auto","created_at":"2026-01-29 17:28:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":177074,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of TWPs on plant species (A, \u003cem\u003eAegopodium podagraria\u003c/em\u003e, B, \u003cem\u003eLespedeza cuneata\u003c/em\u003e, C, \u003cem\u003eArtemisia princeps\u003c/em\u003e, D, \u003cem\u003eAgastache rugosa\u003c/em\u003e) in the mixed-culture condition: Shoot mass, shoot height, number of leaves, and root mass of each treatment (t0, 0 mg kg\u003csup\u003e-1\u003c/sup\u003e, t1, 1 mg kg\u003csup\u003e-1\u003c/sup\u003e, t2, 10 mg kg\u003csup\u003e-1\u003c/sup\u003e, t3, 100 mg kg\u003csup\u003e-1\u003c/sup\u003e, t4, 1,000 mg kg\u003csup\u003e-1\u003c/sup\u003e, t5, 10,000 mg kg\u003csup\u003e-1\u003c/sup\u003e). Replicates of each treatment (\u003cem\u003en\u003c/em\u003e = 6) are represented by dots. The central line indicates the median value (50th percentile), and each box contains 25th to 75th percentiles of dataset. The asterisks indicate significant differences compared to each control group (\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8597992/v1/6d75245acd624b158548b42c.png"},{"id":101442730,"identity":"d6abb322-2887-47df-85a9-beb0ef59bcda","added_by":"auto","created_at":"2026-01-29 17:28:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":114509,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) of the plant community structure based on (A) shoot and (B) root mass growing under each treatment (t0, 0 mg kg\u003csup\u003e-1\u003c/sup\u003e, t1, 1 mg kg\u003csup\u003e-1\u003c/sup\u003e, t2, 10 mg kg\u003csup\u003e-1\u003c/sup\u003e, t3, 100 mg kg\u003csup\u003e-1\u003c/sup\u003e, t4, 1,000 mg kg\u003csup\u003e-1\u003c/sup\u003e, t5, 10,000 mg kg\u003csup\u003e-1\u003c/sup\u003e) of the mixed-culture condition. Replicates of each treatment (\u003cem\u003en\u003c/em\u003e = 6) are represented by dots. Arrows indicate each plant species data, and each ellipse in the PCA graph shows each different treatment (t0 to t5) with a confidence level of 95%.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8597992/v1/496af7ec913c3de1cecfceee.png"},{"id":101442734,"identity":"4ee2ea41-1ade-4dea-bbae-a1c8ca5ffb47","added_by":"auto","created_at":"2026-01-29 17:28:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":156376,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of each plant parameter between mono- and mixed-culture conditions (control): (A) \u003cem\u003eAegopodium podagraria\u003c/em\u003e, (B) \u003cem\u003eLespedeza cuneata\u003c/em\u003e, (C) \u003cem\u003eArtemisia princeps\u003c/em\u003e, and (D) \u003cem\u003eAgastache rugosa\u003c/em\u003e). The correlation and density analysis are shown based on the shoot mass and height of all treatments: (E) \u003cem\u003eAegopodium podagraria\u003c/em\u003e, (F) \u003cem\u003eLespedeza cuneata\u003c/em\u003e, (G) \u003cem\u003eArtemisia princeps\u003c/em\u003e, (H) \u003cem\u003eAgastache rugosa\u003c/em\u003e. Spearman correlation coefficients and significance are indicated by R and p, respectively.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8597992/v1/5085586c5f9037ec3bd5474e.png"},{"id":101751836,"identity":"281fdd19-dcf3-4391-b14a-60d5a986a6ef","added_by":"auto","created_at":"2026-02-03 10:23:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":997368,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8597992/v1/7da265bf-7727-4a4b-b05e-cdf83aafa2a4.pdf"},{"id":101442732,"identity":"d7548c9f-bb01-4bbd-a8ac-3cd57182e5fa","added_by":"auto","created_at":"2026-01-29 17:28:08","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":144599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-8597992/v1/752cec981fef58624d56f5e7.png"},{"id":101442735,"identity":"9f63d475-7c49-44d3-b154-f229251ceac8","added_by":"auto","created_at":"2026-01-29 17:28:09","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1238797,"visible":true,"origin":"","legend":"","description":"","filename":"TWPRoadsideplantSM.docx","url":"https://assets-eu.researchsquare.com/files/rs-8597992/v1/a9ff3b8a098d3926e256be96.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evidence of impact of tire wear particles on a roadside plant community at environmentally relevant concentrations","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTire wear particles (TWPs) are generated from the friction between tire tread and road surface, and they have received extensive attention as one of microplastic types (diameter\u0026thinsp;\u0026lt;\u0026thinsp;5 mm) in recent years (Ding et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Emissions of TWPs mainly occurs in urban road traffic, and they are accumulated on roadside through dust or aerosol dispersion (Sommer et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Roadside soils are main enrichment locations for TWPs, and the highest concentrations are found within five meters of the road (Siever et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The high level of TWPs pollution in the roadside soils has been reported from 2,000 to 26,400 mg kg\u003csup\u003e-1\u003c/sup\u003e, and TWPs in a motorway of Germany range from 155 to 15,898 mg kg\u003csup\u003e-1\u003c/sup\u003e (M\u0026uuml;ller et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; R\u0026oslash;dland et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are a few studies regarding the potential effects of TWPs on soil ecosystem (Ding et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The impacts of TWPs on plants have been investigated using various types of crops, including \u003cem\u003eVigna radiata\u003c/em\u003e (mung bean), \u003cem\u003eIpomoea aquatica\u003c/em\u003e (water spinach), and \u003cem\u003eAllium porrum\u003c/em\u003e (leek) (Kim et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Leifheit et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zeb et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e; \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). \u003cem\u003eV. radiata\u003c/em\u003e (mung bean) shows negative responses in growth and photosynthetic capacity at TWP concentrations of 1 and 10 g kg\u003csup\u003e-1\u003c/sup\u003e (Kim et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but by contrast, another study has reported that there was no significant impact at concentration up to 15 g kg\u003csup\u003e-1\u003c/sup\u003e (Zeb et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). Aged TWPs enhance the growth of \u003cem\u003eI. aquatica\u003c/em\u003e (water spinach) while pristine TWPs increase the plant stress at concentration of 1 and 10 g kg\u003csup\u003e-1\u003c/sup\u003e (Zeb et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e), and the growth of \u003cem\u003eA. porrum\u003c/em\u003e (leek) significantly decrease at 10 to 60 g kg\u003csup\u003e-1\u003c/sup\u003e (Leifheit et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The use of crop species for toxicity test is important since a large portion of TWPs can migrate into agricultural soils through sewage sludge or rainwater erosion (Wang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, given the fact that abrasion of tire tread intensifies near roadside with heavy traffic, the roadside plant community should receive more attention in research field.\u003c/p\u003e \u003cp\u003eSince the landscapes are heavily disrtubed by agriculture or urban development, roadside ecosystem plays an important role for remaining fragments of native vegetation, making them crucial for the conservation of plant and animal biodiversity including rare and threatened species (Milton et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Compared with agricultural mono-culture, the roadside plant communities have a complex structure (Castro, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ding and Eldridge, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and they form heterogeneous habitats for soil animals, providing a range of ecological value such as pollination (Milton et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The roadside environment receives high concentrations of chemicals due to road drainage and dust (De Silva et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rentch, 2005). However, compared to the current knowledge in crop species, the effects of TWPs on roadside plant community are poorly understood.\u003c/p\u003e \u003cp\u003eIn this study, we established mono- and mixed-plant communities with invasive, allelopathic, or facilitative characteristics, which naturally co-occur in roadside soils. We assessed the effect of TWPs on the growth and community structure, in mono- and mixed-cultures of four plant species (\u003cem\u003eAegopodium podagraria\u003c/em\u003e, \u003cem\u003eLespedeza cuneata\u003c/em\u003e, \u003cem\u003eArtemisia princeps\u003c/em\u003e, and \u003cem\u003eAgastache rugosa\u003c/em\u003e). We hypothesized that TWPs would alter not only growth but also community structure and that TWPs may exacerbate the effects on the mixed-culture condition.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. TWPs and soil preparation\u003c/h2\u003e \u003cp\u003eWaste tires were collected from random vehicles and trucks, and mechanically ground using granulator, rotary mill, and extruder at tire recycling company (Greenara, Haman-gun, Gyeongsangnam-do, South Korea). Further size reduction was achieved using a cryogenic-grinder (6875 Freezer/Mill\u0026reg;, SPEX SamplePrep, USA), and the tire particles were passed through 500-\u0026micro;m sieve. The prepared TWPs were observed under an optical microscope (SMZ1000, Nikon, Japan), and average size was measured using ImageJ version 1.54p (U.S. National Institute of Health, Bethesda, MA, USA). TWPs showed irregular shape with micro-sizes, and the average size was 492\u0026thinsp;\u0026plusmn;\u0026thinsp;271 \u0026micro;m (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;200).\u003c/p\u003e \u003cp\u003eWe collected sandy loam soil (water holding capacity, 38.5%, pH, 5.9) from Jinju, South Korea, where wild plant species naturally grow. The soil was air-dried at room temperature, and then sieved through 4 mm mesh size, and mixed with TWPs at the concentrations of 0 (control, t0), 1 (t1), 10 (t2), 100 (t3), 1,000 (t4), and 10,000 (t5) mg kg\u003csup\u003e-1\u003c/sup\u003e, which encompass environmentally relevant concentrations (155 to 26,400 mg kg\u003csup\u003e-1\u003c/sup\u003e) (M\u0026uuml;ller et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; R\u0026oslash;dland et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We prepared TWP-soils separately for each experimental unit. Each portion of TWPs was mixed into 250 g of soil, and homogenized by hand shaking for 3 min, before being placed into each individual pot (width, 13 x 13 cm, height, 12 cm), to provide an equal distribution of TWPs throughout the soil. Each pot was watered (60% of water holding capacity), and maintained for one week before planting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Plant species\u003c/h2\u003e \u003cp\u003eWe selected four plant species including \u003cem\u003eA. podagraria\u003c/em\u003e (Apiaceae, goutweed), \u003cem\u003eL. cuneata\u003c/em\u003e (Fabaceae, sericea lespedeza), \u003cem\u003eA. princeps\u003c/em\u003e (Asteraceae, mugwort), and \u003cem\u003eA. rugosa\u003c/em\u003e (Lamiaceae, Korean mint). All \u003cem\u003es\u003c/em\u003epecies are frequently co-occurring grassland species that naturally grow in roadside environment. \u003cem\u003eA. podagraria\u003c/em\u003e is widely distributed in the open habitats such as roadsides or gardens (D\u0026rsquo;Hertefeldt et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)d \u003cem\u003ecuneata\u003c/em\u003e has been suggested as an indicator species of roadside vegetation (Rentch et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). \u003cem\u003eA. princeps\u003c/em\u003e and \u003cem\u003eA. rugosa\u003c/em\u003e are the representative species of roadside environment in South Korea (Cho et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Each seed was obtained from a commercial supplier in the region (Aramseed, Seoul, South Korea). Since the germination rates of all species were nearly zero in sterile conditions (10% sodium hypochlorite and sterile sand), we skipped the surface-sterilization process. Each seed was germinated for 14 days in trays containing potting soil (Hungnong, South Korea, cocopeat 47.2%, peatmoss 35%, zeolite 7%, vermiculite 10%, dolomite 0.6%). Individual seedlings of similar development stage were used for the plant test: average shoot lengths (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) of each seedling were 7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 cm (\u003cem\u003eA. podagraria\u003c/em\u003e), 4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 cm (\u003cem\u003eL. cuneata\u003c/em\u003e), 1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 (\u003cem\u003eA. princeps\u003c/em\u003e), and 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 cm (\u003cem\u003eA. rugosa\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Experimental design\u003c/h2\u003e \u003cp\u003eIn December 2024, we established the pot experiment in a controlled room with a day-light period of 16 h, and a temperature regime at 22\u0026deg;C with a relative humidity of 60%. We prepared 156 experimental units (pots) including mono-culture (96), mixed-culture with four species (36), and no plant group (24). Each group contained four (mono and no plant) and six (mixed) replicates per each TWP treatment (t0 to t5) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Four holes per a pot were dug in a grid, keeping a distance of 3 cm. Seedlings were regularly distributed for the mixed-culture (upper position, \u003cem\u003eA. podagraria\u003c/em\u003e and \u003cem\u003eA. princeps\u003c/em\u003e; lower position, \u003cem\u003eL. cuneata\u003c/em\u003e and \u003cem\u003eA. rugosa\u003c/em\u003e; left to right in order), and each planting hole received one plant individual. Four individuals of the same species were also planted for mono-culture condition, to serve as an intraspecific interaction control. Pots were well watered every day (60% of soil water holding capacity) during the 7 weeks of growth. Before one day of the harvest, the electrical capacitances between plant-plant species in each individual pot was measured. The electrical capacitance is a physical quantity that characterizes the ability of capacitors to hold electrical charge, and it is part of bioimpedance technology (Gu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The nondestructive method is applied to the stem and soil through the electrodes (Gu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chloupek, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1972\u003c/span\u003e), but in this study, we measured the electrical capacitance between plant-plant species. The electrical capacitance was measured after four hours of watering (60% of soil water holding capacity) using a capacitance meter (New Mate 2012R, Kyoritsu, Japan) with a frequency of 1 kHz. The plant electrodes were fixed with a clamp about 2 cm above ground of each plant in a pot. The electrical capacitance was recorded in nano Farad (nF) when it became stable after attaching the two clamps. The harvest was done in a randomized manner. A total 528 of shoots (384 from mono-culture pots; 144 from mixed-culture pots) was sorted by plant species, and shoot mass, shoot height, and number of leaves were measured immediately. Roots were carefully removed from the soil and gently washed. Each root mass was measured after drying at 40\u0026deg;C oven for 24 h. We measured soil pH of 156 experimental units using the soil-water suspension (1:5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eData visualization and statistical analysis were computed by R Version 4.4.2. Significant differences of the measured parameters (mass, height, and number of leaves) in each treatment were assessed by ANOVA with Tukey and Bonferroni\u0026rsquo;s multiple range tests at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. We calculated the relative interactive index (RII) for each species to understand how each plant-plant interaction influenced shoot and root mass (Armas et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). RII represents the competitive effect of intra- and inter-specific interactions compared with when the species is grown alone: RII = (B\u003csub\u003emix\u003c/sub\u003e \u0026ndash; B\u003csub\u003emono\u003c/sub\u003e)/(B\u003csub\u003emix\u003c/sub\u003e + B\u003csub\u003emono\u003c/sub\u003e), where B\u003csub\u003emix\u003c/sub\u003e is the biomass of a species growing in the mixed-culture condition and B\u003csub\u003emono\u003c/sub\u003e is the biomass of a species that is grown in each mono-culture condition. Principal component analysis (PCA) was performed using the function \u0026ldquo;prcomp\u0026rdquo; and \u0026ldquo;fviz_pca\u0026rdquo; from the package \u0026ldquo;factoextra\u0026rdquo; and \u0026ldquo;factorMineR\u0026rdquo; (Kassambara and Mundt, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). To analyze changes in plant community composition, we log-transformed shoot and root mass per species, and each ellipse in the PCA graph grouped the different treatments (t0 to t5) with a confidence level of 0.95.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Effects of TWPs on mono- and mixed-plant communities\u003c/h2\u003e \u003cp\u003eNo significant changes in shoot mass, shoot height, number of leaves, and root mass were observed in the mono-culture conditions of each species (Fig. S2). However, in the mixed-culture condition, we found the significant differences of TWP treatments compared to control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The shoot mass and height of \u003cem\u003eA. princeps\u003c/em\u003e significantly decreased at the highest concentration (t5, 10,000 mg kg\u003csup\u003e-1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), but by contrast, the shoot mass of \u003cem\u003eA. rugosa\u003c/em\u003e significantly increased at the same concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition, the root mass of \u003cem\u003eA. rugosa\u003c/em\u003e was pronounced at t4 concentration (1,000 mg kg\u003csup\u003e-1\u003c/sup\u003e), compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough total mass of shoot and root in each mixed-culture pot (each community level) showed no differences between each treatment (Fig. S3), RII (for shoot and root biomass) of both \u003cem\u003eA. princeps\u003c/em\u003e and \u003cem\u003eA. rugosa\u003c/em\u003e were influenced in the mixed-culture conditions (Fig. S4). The PCA results revealed the distinct differences of shoot and root mass in TWP treatments. The shoot growth of \u003cem\u003eA. princeps\u003c/em\u003e showed a negative correlation with \u003cem\u003eA. rugosa\u003c/em\u003e (PC1, 37.6%), and the ellipses of control and TWP treatment (t5) were significantly separated with a confidence level of 95% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The root mass analysis indicated that the \u003cem\u003eA. rugosa\u003c/em\u003e exhibited the negative correlation with other plant species on PC1 (41.2%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Soil pH and plant-plant interactions\u003c/h2\u003e \u003cp\u003eThe soil pH in no plant condition were measured as 6.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, 6.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, 6.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, 6.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, 6.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, and 6.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 in each TWP treatment. The increase of soil pH depending on TWP treatments were also observed in three mono-culture conditions (\u003cem\u003eA. podagraria\u003c/em\u003e, \u003cem\u003eL. cuneata\u003c/em\u003e, and \u003cem\u003eA. rugosa\u003c/em\u003e), while \u003cem\u003eA. princeps\u003c/em\u003e mono-culture showed no differences between each treatment (Fig. S5). The soil pH in the mixed-culture conditions reached to 6.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, but there was no significant changes in all TWP treatments. The electrical capacitances in control group were shown in Fig. S6. The highest electrical capacitance was observed between \u003cem\u003eA. podagraria\u003c/em\u003e of mono-culture, and the lowest one was \u003cem\u003eL. cuneata\u003c/em\u003e mono-culture. In TWP treatment (t5), each electrical capacitance of plant-plant interactions was 0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 (\u003cem\u003eA. podagraria\u003c/em\u003e - \u003cem\u003eA. podagraria\u003c/em\u003e), 0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 (\u003cem\u003eA. podagraria\u003c/em\u003e - \u003cem\u003eL. cuneata\u003c/em\u003e), 0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 (\u003cem\u003eA. podagraria\u003c/em\u003e - \u003cem\u003eA. princeps\u003c/em\u003e), 0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 (\u003cem\u003eA. podagraria\u003c/em\u003e - \u003cem\u003eA. rugosa\u003c/em\u003e), 0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (\u003cem\u003eL. cuneata\u003c/em\u003e - \u003cem\u003eL. cuneata\u003c/em\u003e), 0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 (\u003cem\u003eL. cuneata\u003c/em\u003e - \u003cem\u003eA. princeps\u003c/em\u003e), 0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 (\u003cem\u003eL. cuneata\u003c/em\u003e - \u003cem\u003eA. rugosa\u003c/em\u003e), 0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 (\u003cem\u003eA. princeps\u003c/em\u003e - \u003cem\u003eA. princeps\u003c/em\u003e), 0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 (\u003cem\u003eA. princeps\u003c/em\u003e - \u003cem\u003eA. rugosa\u003c/em\u003e), and 0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 (\u003cem\u003eA. rugosa\u003c/em\u003e - \u003cem\u003eA. rugosa\u003c/em\u003e) nF (Fig. S5B).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe underlying mechanism of TWP effects on plant remains unclear. There are several potential effect mechanisms of particulate contaminants on plants, in part as a function of their types, such as microplastics. Alterations of soil physical structure by microplastic addition could directly influence plant root activities, and a large amount of inert carbon in microplastics might slowly contribute the wide C:N ratio, potentially leading to nutrient immobilization (Rillig and Lehmann, 2019). In this study, we found no effects on the mono-culture treatments (Fig. S2), and total biomass of each treated pot showed no changes (Fig. S3). However, the growth patterns of individual plants in the mixed-culture were altered by TWP addition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D), and it might be related to the shift of community structures. We assumed that there is an unrevealed mechanism in plant-plant interactions.\u003c/p\u003e \u003cp\u003eTWPs can alter soil pH since they contain calcium carbonate as a filler (Zhu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, soil pH varied depending on the cultivation of each species (\u003cem\u003eA. podagraria\u003c/em\u003e, 5.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18, \u003cem\u003eL. cuneata\u003c/em\u003e, 5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, \u003cem\u003eA. princeps\u003c/em\u003e, 5.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, \u003cem\u003eA. rugosa\u003c/em\u003e, 5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05), and each mono-culture showed the increasing trends with the increase of TWP concentration (Fig. S5). However, in both \u003cem\u003eA. princeps\u003c/em\u003e mono-culture and mixed-culture conditions, no significant change was observed across the TWP treatments, and \u003cem\u003eA. princeps\u003c/em\u003e may play an important role to capture the potential shifts in soil pH by TWP addition. \u003cem\u003eA. princeps\u003c/em\u003e is known to produce multiple biochemical compounds (Ryu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and they are crucial for maintiaing the plant growth and adapting to sress condition by regulating soil processes such as rhizosphere pH (H\u0026auml;ttenschwiler and Vitousek, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). However, higher plant diversity can raise soil pH with the accumulation of cations such as calcium and magnesium (Furey and Tilman, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and the control soil pH in the mixed-culture condition was already elevated, potentially exceeding the measurable capacity of TWPs to induce a further increase (Fig. S5). Consequently, we assumed that \u003cem\u003eA. princeps\u003c/em\u003e failed to change the pH shift favorable to its growth, and TWPs alter the environment in a way that benefits to other competitors (\u003cem\u003eA. podagraria\u003c/em\u003e and \u003cem\u003eA. rugosa\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThe plant-plant interactions in a community, such as competition, facilitation, and allelopathy, are widespread and easily observed in mixtures of plants (Thorpe et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). \u003cem\u003eA. podagraria\u003c/em\u003e is known as an invasive plant that creates symbiotic relationships to native plant species (Jakubczyk et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)d \u003cem\u003eprinceps\u003c/em\u003e has compact and massive root system, leaving no space for pre-existing vegetation (Verloove and Andeweg, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although each plant species has different strategies of competition or facilitation, our results revealed that \u003cem\u003eA. podagraria\u003c/em\u003e takes advantage in the mixed-culture condition, in terms of biomass, while \u003cem\u003eA. princeps\u003c/em\u003e experienced the negative effects on shoot mass and number of leaves, as compared to the mono-culture condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and C). \u003cem\u003eL. cuneata\u003c/em\u003e and \u003cem\u003eA. rugosa\u003c/em\u003e showed no change in shoot mass and height, but the leave numbers of \u003cem\u003eL. cuneata\u003c/em\u003e was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and D). We measured the electrical capacitance to understand the plant-plant interactions through the electrical properties in root systems (Gu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and the relevant differences were observed in each species interaction (Fig. S6). From the view of electrical interactions between \u003cem\u003eA. podagraria\u003c/em\u003e and each other species in the control condition, the electrical capacitance of \u003cem\u003eL. cuneata\u003c/em\u003e was lower than those of other species, while \u003cem\u003eA. princeps\u003c/em\u003e and \u003cem\u003eA. rugosa\u003c/em\u003e exhibited stronger interactions with \u003cem\u003eA. podagraria\u003c/em\u003e. These three species have an extensive root system (Verloove and Andeweg, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and the underground competition or facilitation seems trigger the increase of electrical capacitance between each root. After TWP exposure, the electrical capacitance of \u003cem\u003eA. princeps\u003c/em\u003e associated with \u003cem\u003eA. podagraria\u003c/em\u003e diminished, whereas \u003cem\u003eA. rugosa\u003c/em\u003e experienced a slight increase. These results align with the growth patterns of these species in the TWP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eRoadside ecosystem serves multiple purposes such as enhancing the aesthetics of landscape, stimulating nutrient cycling, improving air quality, and creating wildlife habitats, but the natural and anthropogenic disturbances (e.g., construction or erosion) could influence these functions (Waheed and Naeem, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). When considering that roadside ecosystem acts as an ecological conduit allowing native plant and animal species to move among landscapes such as agricultural land or urban garden (Brooker, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Dietzel et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), their ecological stability might be crucial to sustain a wild range of environmental functions. In this study, we found that TWPs affect roadside plant community at environmentally relevant concentration, and the potential competition or commensalism may be altered in each plant species and shift the community structure. In other aspects, the roadside plant community has a high resistance against TWP pollution by regulating their composition and structure. However, the plant-plant interactions play a key role throughout ecosystem, not only plant community themselves (Brooker, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and soil can be altered over time through small changes that accumulate in a part of communities. The effects of TWPs on plant communities might be another key factor in roadside environment, where the actual high level of pollution occurs, and this potential variable should be considered to understand the effects on relatively unconcerned ecosystem. This study may provide the starting point to achieving these goals, and future studies should be pursued through large-scale and multisite experiments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate all members of Division of Gyeongnam Bio-Environmental Research for all service they provide.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.W.K. conceptualised the study. S.W.K., Y.C., and H.-S.J. performed the experiment and processed the samples. S.W.K. and Y.C. analysed the data. S.W.K. led the writing with support of Y.C. and C.-B.P. and input from all authors. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-16070203). This research was supported by the Korea Institute of Toxicology, Republic of Korea (2710086916).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo, I declare that the authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArmas, C., Ordiales, R., Pugnaire, F. I. (2004). Measuring plant interac -tions: A new comparative index. \u003cem\u003eEcology\u003c/em\u003e, \u003cem\u003e85\u003c/em\u003e, 2682\u0026ndash;2686.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBennett, A. F. (2023). Linkages in the landscape: the role of corridors and connectivity in wildlife conservation. Coserving Foret Ecosystems Series No. 1, IUCN Forest Conservation Programme, Gland, Switzerland and Cambridge, UK.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrooker, R. W. (2006). Plant-plant interactions and environmental change. \u003cem\u003eNew Phytologist\u003c/em\u003e, \u003cem\u003e171\u003c/em\u003e, 271\u0026ndash;284.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCastro, A., Wise, D. H. (2010). Influence of fallen coarse woody debris on the diversity and community structure of forest-floor spiders (Arachnida: Araneae). \u003cem\u003eForest Ecology and Management\u003c/em\u003e, \u003cem\u003e260\u003c/em\u003e, 2088\u0026ndash;2101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChloupek, O. (1972). The relationship between electric capacitance and some other parameters of plant roots. \u003cem\u003eBiologia Planttarum\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e, 227\u0026ndash;230.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCho, K.-J., Kang, K.-K., Han, M.-S., Na, Y.-E., Kim, M., Choe, L.-J., Kim, M.-H. (2013). Occurrence and distribution of C\u003csub\u003e4\u003c/sub\u003e plants under diverse agricultural field types in Korea. \u003cem\u003eKorean Journal of Agricultural and Forest Meteorology\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e, 85\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026rsquo;Hertefeldt, T., Enestr\u0026ouml;m, J. M., Pettersson, L. B. (2014). Geographic and habitat origin influence biomass production and storage translocation in the clonal plant Aegopodium podagraria. \u003cem\u003ePLOS One\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, e85407.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Silva, S., Ball, A. S., Indrapala, D. V., Reichman, S. M. (2020). Review of the interactions between vehicular emitted potentially toxic elements, roadside soils, and associated biota. \u003cem\u003eChemosphere\u003c/em\u003e, \u003cem\u003e263\u003c/em\u003e, 128135.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDietzel, S., Rojas-Botero, S., Kollmann, J., Fischer, C. (2023). Enhanced urban roadside vegetation increases pollinator abundance whereas landscape characteristics drive pollination. \u003cem\u003eEcological Indicators\u003c/em\u003e, \u003cem\u003e147\u003c/em\u003e, 109980.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing, J., Eldridge, D. J. (2022). Roadside verges support greater ecosystem functions than adjacent agricultural land in a grassy woodland. \u003cem\u003eJournal of Environmental Management\u003c/em\u003e, \u003cem\u003e308\u003c/em\u003e, 114625.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing, J., Lv, M., Zhu, D., Leifheit, E. F., Chen, Q. L., Wang, Y. Q., Chen, L. X., Rillig, M. C., Zhu, Y. G. (2023). Tire wear particles: an emerging threat to soil health. Critical Reviews in \u003cem\u003eEnvironmental Science \u0026amp; Technology\u003c/em\u003e, \u003cem\u003e53\u003c/em\u003e, 239\u0026ndash;257.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFurey, G. N., Tilman, D. (2021). Plant biodiversity and the regeneration of soil fertility. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e, \u003cem\u003e118\u003c/em\u003e, e2111321118.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGu, H., Liu, L., Butnor, J. R., Sun, H., Zhang, X., Li, C., Liu, X. (2021). Electrical capacitance estimates crop root traits best under dry conditions\u0026ndash;a case study in cotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e L.). \u003cem\u003ePlant Soil\u003c/em\u003e, \u003cem\u003e467\u003c/em\u003e, 549\u0026ndash;567.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH\u0026auml;ttenschwiler, S., Vitousek, P. M. (2000). The role of polyphenols in terrestrial ecosystem nutrient cycling. \u003cem\u003eTrends in Ecology \u0026amp; Evolution\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e, 238\u0026ndash;243.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJakubczyk, K., Janda, K., Stykurski, D., Łukomska, A. (2020). Goutweed (\u003cem\u003eAegopodium podagraria\u003c/em\u003e L.) \u0026ndash; botanical characteristics and prohealthy properties. \u003cem\u003ePostepy Higieny i Medycyny Doswiadczalnej\u003c/em\u003e, \u003cem\u003e74\u003c/em\u003e, 28\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKassambara, A., Mundt, F. (2017). factoextra: Extract and visualize the results of multivariate data analyses. R package version 1.0.5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://CRAN.R-project.org/package=factoextra\u003c/span\u003e\u003cspan address=\"https://CRAN.R-project.org/package=factoextra\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, L., Lee, T.-Y., Kim, H., An, Y.-J. (2022). Toxicity assessment of tire particles released from personal mobilities (bicycles, cars, and electric scooters) on soil organisms. \u003cem\u003eJournal of Hazardous Materials\u003c/em\u003e, \u003cem\u003e437\u003c/em\u003e, 129362.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeifheit, E. F., Kissener, H. L., Faltin, E., Ryo M., Rillig, M. C. (2022). Tire abrasion particles negatively affect plant growth even at low concentrations and alter soil biogeochemical cycling. Soil Ecology Letters, 4, 409\u0026ndash;415.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMilton, S. J., Dean, W., Sielecki, L. E., Ree, R. (2015). The function and management of roadside vegetation. Handbook of Road Ecology, 373\u0026ndash;381.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026uuml;ller, A., Kocher, B., Altmann, K., Braun, U. (2022). Determination of tire wear markers in soil samples and their distribution in a roadside soil. Chemosphere, 294, 133653.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRentch, J.S., Fortney, R. H., Stephenson, S. L., Adams, H. S., Grafton, W. N., Anderson, J. T. (2005). Vegetation\u0026ndash;site relationships of roadside plant communities in West Virginia, USA. \u003cem\u003eJournal of Applied Ecology\u003c/em\u003e, \u003cem\u003e42\u003c/em\u003e, 129\u0026ndash;138.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRillig, M. C., Lehmann, A., De Souza Machado, A. A., Yang, G. (2019). Microplastic effects on plants. \u003cem\u003eNew Phytologist\u003c/em\u003e, \u003cem\u003e223\u003c/em\u003e, 1066\u0026ndash;1070.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR\u0026oslash;dland, E. S., Heier, L. S., Lind, O. C., Meland, S. (2023). High levels of tire wear particles in soils along low traffic roads. \u003cem\u003eScience of Total Environment\u003c/em\u003e, \u003cem\u003e903\u003c/em\u003e, 166470\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyu, R., Jung, U. J., Kim, H.-J., Lee, W., Bae, J.-S., Park, Y. B., Choi, M.-S. (2013). Anticoagulant and antiplatelet activities of Artemisia princeps pampanini and its bioactive components. \u003cem\u003ePreventive Nutrition and Food Science\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e, 181\u0026ndash;187.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiever, R., Kawecki, D., Nowack, B. (2020). Dynamic probabilistic materials flow analysis of rubber release from tires into the environment. \u003cem\u003eEnvironmental Pollution\u003c/em\u003e, \u003cem\u003e258\u003c/em\u003e, 1313573.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSommer, F., Dietze, V., Baum, A., Sauer, J., Gilge, S., Maschowski, C., Giere, R. (2018). Tire abrasion as a major source of microplastics in the environment. \u003cem\u003eAerosol Air Quality Research\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e, 2014\u0026ndash;2028.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThorpe, A. S., Aschegoug, E. T., Atwater, D.Z., Callaway, R. M. (2011). Interactions among plants and evolution. \u003cem\u003eJournal of Ecology\u003c/em\u003e, \u003cem\u003e99\u003c/em\u003e, 729\u0026ndash;740.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerloove, F., Andeweg, R. (2020). \u003cem\u003eArtemisia princeps\u003c/em\u003e L. (Asteraceae), an overlooked invasive Far Eastern weed in Western Europe. \u003cem\u003eGorteria\u003c/em\u003e, \u003cem\u003e42\u003c/em\u003e, 1\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaheed, M., Naeem, Z. (2024). The role of roadside tree planting in environmental conservation. \u003cem\u003eRadinka Journal of Science and Systematic Lierature Review\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e, 235\u0026ndash;239.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Y., Li, X., Yang, H., Wu, Y., Pu, Q., He, W., Li, X. (2024). A review of tire wear particles: Occurrence, adverse effects, and control strategies. \u003cem\u003eEcotoxicology and Environmental Safety\u003c/em\u003e, 283, 116782.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeb, A., Liu, W., Ali, N., Shi, R., Lian, Y., Wang, Q., Wang, J., Li, J., Zeng, Z., Liu, J., Yu, M., Liu, J. (2024a). Integrating metabolomics and high-throughput sequencing to investigate the effects of tire wear particles on mung bean plants and soil microbial communities. \u003cem\u003eEnvironmental Pollution\u003c/em\u003e, \u003cem\u003e340\u003c/em\u003e, 122872.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeb, A., Liu, W., Ali, N., Shi, R., Zhao, Y., Wang, J., Wang, Q., Khan, S., Baig, A. M., Liu, J., Khan, A. A., Ge, Y., Li, X., Yin, C. (2024b). Impact of pristine and aged tire wear particles on Ipomoea aquatica and rhizospheric microbial communities: Insights from a long term exposure study. \u003cem\u003eEnvironmental Science \u0026amp; Technology\u003c/em\u003e, \u003cem\u003e58\u003c/em\u003e, 21143\u0026ndash;21154.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, Y., Kim, S. W., Li, H., Rillig, M. C. (2024). Delivery rate alters the effects of tire wear particles on soil micobial activities. \u003cem\u003eEnvironmental Sciences Europe\u003c/em\u003e, \u003cem\u003e36\u003c/em\u003e, 9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-sciences-europe","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"eseu","sideBox":"Learn more about [Environmental Sciences Europe](http://enveurope.springeropen.com)","snPcode":"12302","submissionUrl":"https://submission.nature.com/new-submission/12302/3","title":"Environmental Sciences Europe","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biodiversity, Microplastics, Mixed culture, Soil, Wild plant","lastPublishedDoi":"10.21203/rs.3.rs-8597992/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8597992/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Tire wear particles (TWPs) are recognized as a significant contributor of microplastic pollution, and recent research has shown their effects on plants using various types of crops. However, given the fact that abrasion of tire tread intensifies near roadways, roadside plant community should receive more attention. In this study, we established mono- and mixed-plant communities including four different species (, , , and ), and the effects of TWPs on each community were investigated across a range of concentrations (1 to 10,000 mg kg). We found the significant changes in the structure of mixed-plant community, which represented opposite growth patterns between two species ( and ), while there were no effects on each species in the mono-culture condition. Our results revealed that takes advantage in the mixed-culture condition, in terms of biomass, while experienced the negative effects on shoot mass. We measured electrical capacitances to understand plant-plant interactions in root system, and it aligned with the growth patterns ( and ) of TWP treatments. Soil pH varied depending on the cultivation of each species and TWP treatments. Consequently, we assumed that TWPs alter the soil process and plant-plant interactions. Roadside ecosystem provides ecological values, and the impacts of TWPs on roadside plant community might be crucial to sustain a wild range of environmental functions.","manuscriptTitle":"Evidence of impact of tire wear particles on a roadside plant community at environmentally relevant concentrations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 17:28:04","doi":"10.21203/rs.3.rs-8597992/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-07T17:05:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-03T18:40:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-23T14:35:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-11T23:09:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-08T12:29:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-08T10:38:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-07T11:42:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-07T06:07:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-04T03:51:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198031752248165709068968071942559032185","date":"2026-02-01T16:29:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197465964725671232723247183561297719484","date":"2026-01-30T06:25:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189063812477355544378059085642068082915","date":"2026-01-30T05:09:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"325266993130226795867986581852836683091","date":"2026-01-30T03:52:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39261740211732023341193154366348776656","date":"2026-01-29T16:42:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262218978570316205211564493872371229368","date":"2026-01-29T16:26:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100210545921654926217855944220387138957","date":"2026-01-28T04:41:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8488285581452270089938928517634483887","date":"2026-01-28T00:07:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-27T16:02:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-18T10:01:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-14T07:29:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Sciences Europe","date":"2026-01-14T05:58:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-sciences-europe","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"eseu","sideBox":"Learn more about [Environmental Sciences Europe](http://enveurope.springeropen.com)","snPcode":"12302","submissionUrl":"https://submission.nature.com/new-submission/12302/3","title":"Environmental Sciences Europe","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2d7937f3-a5ee-4786-bae5-ac9920a3b0a6","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T07:39:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 17:28:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8597992","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8597992","identity":"rs-8597992","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}