Delivery rate alters the effects of tire wear particles on soil microbial activities

Delivery rate alters the effects of tire wear particles on soil microbial activities | 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 Delivery rate alters the effects of tire wear particles on soil microbial activities Yanjie Zhu, Shin Woong Kim, Huiying Li, Matthias C. Rillig This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3972392/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Tire wear particles (TWPs) produced by the abrasion between tires and road surfaces have been recognized as an emerging threat to soil health globally in recent years. They can be transported from the road surface to adjacent soil at different delivery rates, with precipitation a main driver underpinning this movement. However, studies typically assume an abrupt exposure of TWPs in their experimental design. In this study, we investigated the impacts of abrupt and gradual delivery of TWPs on soil physicochemical properties and microbial activities. We used two different delivery rates of TWPs (abrupt and gradual) and devised two experimental phases, namely the TWPs-delivery period (phase 1) and the end-of-delivery period (phase 2). Results We found that the gradual TWPs delivery treatments negatively influenced the activity of carbon cycle-related enzymes (β-glucosidase and β-D-1,4-cellobiosidase). Furthermore, the abrupt treatment highly increased the effects on nitrogen cycle-related enzyme activity (β-1,4-N-acetyl-glucosaminidase). In phase 2 (end-of-delivery period), each enzyme activity was changed to a similar level as the control group, but these changes were influenced by the prior delivery rates. Conclusion Abruptly and gradually delivered TWPs induce different responses to soil microbial activities. Our findings imply that the delivery rate of TWPs could be another key factor changing the effects of TWPs, further enhancing our understanding of the ecological impacts of TWPs. Graphical abstract Abrupt exposure Gradual exposure Microplastics Enzyme activities Soil aggregates Soil pH Soil respiration Figures Figure 1 Figure 2 Figure 3 1. Introduction Although the first tire was produced in the 19th century, tire wear particles (TWPs) pollution in road dust was not highlighted until 1966 [ 1 ]. TWPs are usually regarded as microplastics (< 5 mm) due to their size, insolubility, polymer structure, and solid state [ 2 , 3 ], and have emerged as a main contributor of microplastic pollution [ 4 – 7 ]. The global average emission of TWPs has reached 0.81 kg per year per capita [ 8 ], and recent research has reported the potential effects of TWPs on soil ecosystem. TWPs can induce adverse effects on survival and reproduction of soil fauna [ 9 , 10 , 4 , 11 ], plant growth [ 12 ], and microbial activities [ 12 , 13 ], and these effects have been underpinned with multiple pathways [ 3 ]. For instance, TWPs can physically damage the intestine of soil fauna [ 11 ], and influence soil microbial activities through changing soil properties and nutrient cycles [ 3 ]. Tires consist of rubber, filler, softener, vulcanization agents and various additives [ 7 ], including toxic chemicals such as polycyclic aromatic hydrocarbons (PAHs), benzothiazoles and heavy metals (e.g., Cd, Pb, and Zn), and these additives can be released into the environment during the abrasion of tires, contributing to the adverse effects [ 14 , 15 ]. Once TWPs are generated on the road surface, they can be delivered into the soil environment either through road runoff or airborne transport [ 6 ]. Rainfall is considered as a main factor influencing the transport of TWPs [ 16 ], and the intensity of precipitation is directly linked with TWPs delivery rates [ 6 ]. For instance, during a strong rainfall event, TWPs can be abruptly washed from the air or road surfaces to roadside soils [ 17 , 6 ], while no precipitation or weak/steady rainfall leads to a more gradual delivery of TWPs. This is important since there is still a knowledge gap regarding different delivery rates of TWPs due to the lack of empirical data [ 18 ]. The rate of changes in environmental (e.g., drought) or anthropogenic (e.g., chemical pollutants) factors have been well established in global change research [ 19 ], and different effects under different rates of changes have been observed frequently [ 19 , 20 ]. Soil is a main sink of contaminants emitted into the environment, and 67% of TWPs are delivered to the soil environment eventually [ 21 ]. In this study, we investigated whether the delivery rate influences the impacts of TWPs on soil microbial activities and physicochemical properties. We established the different delivery rates of TWPs (abrupt and gradual), and designed two phases that capture the TWPs-delivery period (phase 1) and the end-of-delivery period (phase 2). We hypothesized that (1) TWPs pollution affects soil microbial activities and physicochemical properties, (2) delivery rate alters the effects of TWPs, and (3) these effects will be changed at the end-of-delivery period, depending on the prior delivery rates. 2. Materials and methods 2.1. Test Soil and TWPs Test soil (loamy sandy; Albic Luvisol) was collected from a grassland site of the Institute of Biology of Freie Universität Berlin (52°28' N, 13°17' E) [ 22 ]. Soil bulk density and water holding capacity (WHC) were 1.2 g cm − 3 and 39.5%, and pH and EC were 5.8 and 58.8 µS cm − 1 . The soil was air-dried for one week at room temperature and passed through a 2 mm-sieve for further use. TWPs (< 500 µm) were obtained from the company KURZ-Karkassenhandel (Landau, Germany) and sieved through a 250 µm mesh. A light microscope (MDG41, Leica, Wetzlar, Germany) equipped with a camera (Flexacam C1, Leica, Wetzlar, Germany) was used to measure the average size of particles (88.4 µm, 4.5–276.8 µm) (Fig. S1 ), and this particle size is within a range of TWPs produced during driving (4–350 µm) [ 15 ]. 2.2. Experiment set-up The experiment was established in a greenhouse (22 ± 2/18 ± 2°C, day/night; daylight period, 16:8; relative humidity, 40%). We placed 200 g of test soil in 210 mL round plastic containers (diameter, 80 mm; height, 48 mm; RPC superfos a/s, Denmark; soil layer thickness, 30 mm) covered with nylon mesh (pore size, 38 µm), and pre-incubated with 60% of WHC for a week to achieve a steady state of soil microbial metabolism (n = 12 for each three treatment). After the pre-incubation, we divided the experiment into two different phases, the TWPs-delivery period (phase 1; duration of 9 weeks) and end-of-delivery period (phase 2; duration of 4 weeks). During phase 1, we treated soils with TWPs at three different delivery rates including control (no TWPs addition), gradual delivery, and abrupt delivery. The abrupt and gradual delivery treatments had the same overall dose by the addition of TWPs according to a “week-dose” principle (Fig. S2). Briefly, 0.2 g of TWPs (0.1%, total dry soil mass) was added weekly in the gradual delivery treatment, and 1.8 g of TWPs (0.9%, total dry soil mass) was added at the fifth week for the abrupt treatment. These concentrations were based on the measurements (0.2 to 2%, dry weight) of TWPs in roadside soils according to previous studies [ 6 ]. At the beginning of phase 1, TWPs were suspended in deionized water with a volume matching 100% of the soil WHC, and we evenly poured this onto the soil surface for the gradual delivery treatment. The control and abrupt delivery treatments were treated with only deionized water (no TWPs addition). The water contents in each container were checked and replenished with deionized water (35 to 40 mL) every week to keep uniform moisture (100% of WHC). This water maintenance was accompanied with TWPs addition for the gradual delivery treatment, and the same TWPs addition method was applied for the abrupt delivery treatment in the fifth week (Fig. S3). We applied TWPs to the soil surface (soil depth, 30 mm) without subsequent mixing to prevent artificial disturbance, and saturated the soil with 100% of WHC to simulate rainy season conditions and to facilitate the particle penetration into the soil. At the end of phase 1, 6 units of each treatment were harvested, and the rest of experimental units (n = 6) were incubated for 4 more weeks with no further TWPs addition (phase 2) (Fig. S3). After harvest, each soil was carefully homogenized using a spatula, and fresh soil samples were stored at 4°C for the subsequent measurements. The rest of the soil samples were air dried and then stored at room temperature. 2.3. Measurement of soil properties Soil respiration rate was indicated by the CO 2 concentration produced per hour (µM M –1 h –1 ) [ 23 ]. Thirty gram of fresh soil was placed in a 50 mL falcon tube equipped with a rubber stopper on the lid. The tubes were flushed with CO 2 -free air for 5 min to eliminate background CO 2, and then incubated for 4 h at 20°C. We sampled 1 mL of air in the headspace of the tube using a syringe and injected this sample into an infrared gas analyzer (LI-6400XT, LI-COR Inc., Bad Homburg, Germany). The activities of C-related enzymes β-glucosidase (EC3.2.1.21) and β-D-1,4-cellobiosidase (EC3.2.1.91), N-related enzyme β-1,4-N-acetyl-glucosaminidase (EC3.2.1.52), and P-related enzyme phosphatase (EC3.1.3.2) were measured with artificial p -nitrophenyl ( p NP) linked substrates, and quantified by a microplate reader [ 24 ]. Briefly, 10 mL of 50 mM acetate buffer (pH, 5.0 − 5.5) was added into 50 mL-test tubes containing 5.0 g of each soil sample. The tubes were vortexed for 5 secs, and each soil slurry (150 µL) into each well (6 wells per sample) in the 96-well plate. Then, 150 µL of each substrate was added: p NP-β-D-glucopyranoside (Sigma no. N7006), p NP-β-D-cellobioside (Sigma no. N5759), p NP-N-acetyl-β-D-glucosaminide (Sigma no. N9376), p NP- phosphate disodium salt hexahydrate (Sigma no. 71,768). After 2 h (β-glucosidase and phosphatase) and 4 h (β-D-1,4-cellobiosidase and β-1,4-N-acetyl-glucosaminidase) incubations at 20°C under dark conditions, each plate was centrifuged at 3,000 rpm for 5 min, and 100 µL of supernatant was transferred into a new plate and mixed with 200 µL of 0.1 M NaOH. Absorbances were determined at 410 nm using a microplate reader (Benchmark Plus, BioRad Laboratories GmbH, Hercules, CA, USA), and each enzyme activity was calculated as µmol p-nitrophenol g soil dry mass -1 h -1 . Water stable aggregates (WSA) content was measured following the wet sieving method of Kemper and Rosenau [ 25 ] with a slight modification. Four gram of air-dried soil was put in a small sieve with a mesh size of 0.25 mm, re-wetted by capillarity with deionized water and inserted into a sieving machine (Agrisearch Equipment, Royal Eijkelkamp B.V., Giesbeek, Netherlands) to be wet-sieved for 3 min. The soil left on the sieve (fraction 1: stable soil aggregates and coarse matter) was dried at 60°C and weighed, and then crushed manually in the wet sieve to obtain the coarse matter (fraction 2). Calculations of the percentage of WSA were according to: WSA (%) = (fraction 1 – fraction 2)/(4.0 –fraction 2) × 100%. [ 26 ]. To measure soil pH, 25 mL of distilled water was added to 5.0 g of air-dried soil in a falcon tube. The tube was shaken in 250 rpm for 30 min and then centrifuged in 3000 rpm for 5 min. The supernatant was filtered, and the pH was determined by a pH-meter (Hanna Instruments GmbH, Smithfield, USA). 2.4. Data analysis We performed all the statistical analyses and plotting in R (v.4.1.2). Unpaired t-tests were performed by the t.test() function with a confidence interval of 95% to compare the difference between each treatment and phase. The variance was estimated separately for each group and the Welch modification to the degrees of freedom was used. Boxplots were drawn using the ggplot2 package. The changes of enzyme activities between phase 1 and phase 2 were calculated by the percentage (%) of the changes in phase 2 to compared to phase 1. 3. Results We evaluated the activities of four soil enzymes at the end of two different phases (phase 1 and 2), and found that their activities were dependent on TWPs delivery rates (abrupt and gradual) (Fig. 1 ). In phase 1, the activities of β-glucosidase and β-D-1,4-cellobiosidase were significantly reduced in the gradual treatment compared to the control (Fig. 1 (a) and (b)). The activity of β-1,4-N-acetyl-glucosaminidase increased in the abrupt treatment (Fig. 1 (c)), but there was no significant change in phosphatase activity (Fig. 1 (d)). We found no significant effect on pH, soil respiration, and WSA (Fig. 2 ). After phase 1, each soil was incubated for 4 weeks with no additional TWPs addition (phase 2). The activities of none of the enzymes were significantly different in both delivery treatments, compared to control (Fig. 1 ). There was no significant effect on WSA, soil respiration, and pH (Fig. 2 ). Through a comparison between phase 1 and 2, we found that the activities of β-glucosidase (gradual) and β-D-1,4-cellobiosidase (abrupt and gradual) notably increased over time to a similar level as those observed in the control group (Fig. 1 (a) and (b), lower panels). The β-1,4-N-acetyl-glucosaminidase activity significantly decreased in the abrupt treatment (Fig. 1 (c), lower panel), and phosphatase activity increased in the control and the abrupt treatment (Fig. 1 (d), lower panel). In addition, soil pH significantly increased over time in both of the TWPs treatments (gradual and abrupt) (Fig. 2 (a), lower panel). 4. Discussion The effects of TWPs on extracellular enzyme activities have been explored in previous studies [ 27 , 28 ], and our results showed general agreement with these studies, such as the increase of β-1,4-N-acetyl-glucosaminidase activity [ 28 ], the decrease of β-glucosidase activity [ 28 ], and no significant change in phosphatase activity [ 27 ]. However, the impacts of TWPs delivery rates have not been reported before. Our findings indicated that the gradual delivery treatment induced a significant decrease in the activity of carbon cycle-related enzymes, while there was no effect in the abrupt treatment. TWPs can be presumably classified as an inert (persistent) carbon source, similar to microplastics and charcoal [ 29 ], and the fraction of persistent carbon is barley utilized by microbes through the production of hydrolytic enzymes [ 30 , 31 ]. Many classes of enzymes and environmental factors (e.g., C/N ratio and pH) are involved in the cellulose hydrolysis process [ 32 , 33 ], and our hypothesis regarding the carbon hydrolyzing enzymes may be partly supported by the results of soil parameters in our study. For instance, the enzyme secretion by microbes relies on the availability of soil nitrogen, and high N availability would increase microbial demand for carbon, inducing the production of glucosidases [ 34 ]. In this study, the increase of β-1,4-N-acetyl-glucosaminidase activity (Fig. 1 (c)), which may be abruptly caused by the release of chemical additives (PAHs and nitro-compounds as vulcanization agent) from TWPs [ 14 , 35 – 37 ], was observed in the abrupt treatment, and this change might neutralize the negative effects on β-glucosidase activity in the abrupt treatment (Fig. 1 (a)). TWPs negatively affected the carbon cycle-related enzymes during phase 1 (TWPs-delivery period), and these effects were significantly reduced in phase 2 (end-of-delivery period). This consequence may be linked to previous reports that carbon cycle-related functional genes can be abundant after TWPs exposure [ 38 ]. The soil microbial community has the capacity to establish a new equilibrium with the altered environment, which is defined as the adaptation/adjustment capacity [ 39 ], and this capacity seems to be shown in both delivery treatments. However, we assumed that abruptly and gradually delivered TWPs have elicited different adaptation/adjustment of carbon cycle-related microorganisms to TWPs pollution, since the changes (phase 1 to phase 2) of both β-glucosidase and β-D-1,4-cellobiosidase activities were more remarkable in the gradual treatment (19.82% and 59.29%) than those in the abrupt treatment (0.31% and 32.34%) (Table 1 ). In the results of β-1,4-N-acetyl-glucosaminidase, we found that the enzyme activity dramatically decreased in the abrupt treatment in phase 2 compared to in phase 1. Although the activities of most soil extracellular enzymes generally decrease over time in normal conditions [ 40 , 41 ], this reduction was remarkable in the abrupt treatment (29.80%) compared to other treatments (control and gradual, 13.16% and 5.59%) (Table 1 ). According to a previous study, abrupt TWPs pollution can stimulate anaerobic nitrogen metabolism [ 38 ], which may potentially reduce the activities of nitrogen cycle-related enzymes in a long run, and this may cause the decrease in β-1,4-N-acetyl-glucosaminidase activity over time in the abrupt delivery treatment. In general, soil pH is positively related to β-1,4-N-acetyl-glucosaminidase activity [ 42 ], but our results in the abrupt treatment showed negative correlations between pH and β-1,4-N-acetyl-glucosaminidase activity (Fig. 1 (c) and 2(a), lower panel). This might be caused by the leaching of chemicals from TWPs [ 43 ] or the interactions among multiple extracellular enzymes [ 32 ], and we assumed that these changes were influenced by the prior delivery rates. Table 1 The changes of enzyme activities from phase 1 to phase 2 in different treatments. Each value indicates the percent (%) change of each enzyme activity in phase 2 compared to the activity in phase 1. Treatment β-glucosidase β-D-1,4-cellobiosidase β-1,4-N-acetyl-glucosaminidase Phosphatase Control -0.47 ± 12.41% 2.96 ± 18.27% -13.16 ± 18.75% 7.89 ± 6.95%* Abrupt 0.31 ± 20.81% 32.34 ± 28.59%* -29.80 ± 9.16%* 19.57 ± 22.35%* Gradual 19.82 ± 8.91%* 59.29 ± 54.28%* -5.59 ± 9.51% 4.926 ± 16.12% means ± standard deviations. * Significant differences between phase 1 and phase 2 ( p < 0.05). 5. Conclusions Given the mode of arrival of TWPs in soil, it is important to carefully consider their transport from the road surface into the environment in order to understand their effects. Unfortunately, this aspect has been overlooked in previous experiments. Our results revealed that TWPs pollution influences soil microbial activities, and that these effects are altered by different delivery rate (abrupt and gradual). The adaptation/adjustment of soil microorganisms after the TWPs-delivery period was affected depending on the prior delivery rate. These findings indicated that the delivery rate of TWPs may represent another key factor altering the effect of TWPs. It is thus imperative to consider exposure scenarios when interpreting the impacts of TWPs in soil systems (Fig. 3 ), and this study enhances our understanding of the ecological effects of TWPs. Further research is warranted to investigate the responses at the microbial community level, with the use of different test soils and a variety of delivery rates, to gain a comprehensive understanding of how TWPs delivery rates influence soil ecosystems. The shifts in TWPs effects under different delivery rates are important for deciphering the true extent of TWPs effects in the field, and likely are also relevant for other types of microplastics. Declarations Supporting information The Supporting Information is available free of charge. Supplementary figure of photographic processing of tire wear particle sizes to determine average diameter and size range (Fig. S1), scheme of the gradual and abrupt TWPs treatments based on the “day-dose” principle (Fig. S2), experimental design (Fig. S3). Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article and the supplementary information files. Competing interests The authors declare that they have no competing interests. Funding M.C.R. acknowledges support from PAPILLONS (Plastics in Agricultural Production: Impacts, Lifecycles and Long-term sustainability, No.101000210) of the European Union’s Horizon 2020 research and innovation program. YZ, HL acknowledge the China Scholarship Council for a scholarship (YZ, CSC No. 202106320023; HL, CSC No. 202108080156). Author Contributions YZ: conceptualization, design of the study, experiment setup, analysis of data, and writing; SWK: conceptualization, design of the study, writing, review and editing; HL and HC: experiment setup. M.C.R.: design of the study, review and editing. All authors contributed to the article and approved the submitted version. Acknowledgements Not applicable. References Thompson, R. N., Nau, C. A., & Lawrence, C. H. (1966). Identification of vehicle tire rubber in roadway dust. American Industrial Hygiene Association Journal, 27 (6), 488-495, doi:10.1080/00028896609342461. Hartmann, N. B., Huffer, T., Thompson, R. C., Hassellov, M., Verschoor, A., Daugaard, A. E., et al. (2019). Are we speaking the same language? Recommendations for a definition and categorization framework for plastic debris. Environmental Science & Technology, 53 (3), 1039-1047, doi:10.1021/acs.est.8b05297. Ding, J., Lv, M., Zhu, D., Leifheit, E. F., Chen, Q. L., Wang, Y. Q., et al. (2022). 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Soil Biology and Biochemistry, 91 , 1-13, doi:https://doi.org/10.1016/j.soilbio.2015.08.016. Uwituze, Y., Nyiraneza, J., Fraser, T. D., Dessureaut-Rompré, J., Ziadi, N., & Lafond, J. (2022). Carbon, nitrogen, phosphorus, and extracellular soil enzyme responses to different land use. [Original Research]. Frontiers in Soil Science, 2 , doi:10.3389/fsoil.2022.814554. Xu, Z., Zhang, T., Wang, S., & Wang, Z. (2020). Soil pH and C/N ratio determines spatial variations in soil microbial communities and enzymatic activities of the agricultural ecosystems in Northeast China: Jilin Province case. Applied Soil Ecology, 155 , 103629, doi:https://doi.org/10.1016/j.apsoil.2020.103629. Asmar, F., Eiland, F., & Nielsen, N. E. (1994). Effect of extracellular-enzyme activities on solubilization rate of soil organic nitrogen. Biology and Fertility of Soils, 17 (1), 32-38, doi:10.1007/BF00418669. Tan, B., Yang, F., Lan, L. Y., You, C. M., Zhang, J., Xu, Z. F., et al. (2019). Naphthalene exerts substantial nontarget effects on soil nitrogen mineralization processes in a subalpine forest soil: A microcosm study. Plos One, 14 (5), e0217178, doi:10.1371/journal.pone.0217178. de Menezes, A., Clipson, N., & Doyle, E. (2012). Comparative metatranscriptomics reveals widespread community responses during phenanthrene degradation in soil. Environmental Microbiology, 14 (9), 2577-2588, doi:10.1111/j.1462-2920.2012.02781.x. Ding, G. C., Heuer, H., He, Z. L., Xie, J. P., Zhou, J. Z., & Smalla, K. (2012). More functional genes and convergent overall functional patterns detected by geochip in phenanthrene-spiked soils. Fems Microbiology Ecology, 82 (1), 148-156, doi:10.1111/j.1574-6941.2012.01413.x. Liu, Y., Zhou, H., Yan, M., Liu, Y., Ni, X. M., Song, J. B., et al. (2022). Toxicity of tire wear particles and the leachates to microorganisms in marine sediments. Environmental Pollution, 309 , doi:10.1016/j.envpol.2022.119744. Biagianti-Risbourg, S., Paris-Palacios, S., Mouneyrac, C., & Amiard-Triquet, C. (2013). Pollution acclimation, adaptation, resistance, and tolerance in ecotoxicology. In J.-F. Férard, & C. Blaise (Eds.), Encyclopedia of Aquatic Ecotoxicology (pp. 883-892). Dordrecht: Springer Netherlands. Zhao, T. T., Lozano, Y. M., & Rillig, M. C. (2021). Microplastics increase soil pH and decrease microbial activities as a function of microplastic shape, polymer type, and exposure time. Frontiers in Environmental Science, 9 , 675803, doi:10.3389/fenvs.2021.675803. Liu, C. H., Ma, J. Y., Qu, T. T., Xue, Z. J., Li, X. Y., Chen, Q., et al. (2023). Extracellular enzyme activity and stoichiometry reveal nutrient dynamics during microbially-mediated plant residue transformation. Forests, 14 (1), doi:10.3390/f14010034. Deng, S. P., & Tabatabai, M. A. (1996). Effect of tillage and residue management on enzyme activities in soils. Biology and Fertility of Soils, 22 (3), 208-213, doi:10.1007/BF00382514. Capolupo, M., Sørensen, L., Jayasena, K. D. R., Booth, A. M., & Fabbri, E. (2020). Chemical composition and ecotoxicity of plastic and car tire rubber leachates to aquatic organisms. Water Research, 169 , 115270, doi:https://doi.org/10.1016/j.watres.2019.115270. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.png TWPDeliverySIESE.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Mar, 2024 Reviews received at journal 12 Mar, 2024 Reviewers agreed at journal 21 Feb, 2024 Reviewers agreed at journal 21 Feb, 2024 Reviewers invited by journal 21 Feb, 2024 Editor assigned by journal 21 Feb, 2024 Submission checks completed at journal 21 Feb, 2024 First submitted to journal 20 Feb, 2024 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. 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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-3972392","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274309126,"identity":"25b87486-534d-4c2a-886e-c456158630e1","order_by":0,"name":"Yanjie Zhu","email":"","orcid":"","institution":"Freie Universität Berlin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanjie","middleName":"","lastName":"Zhu","suffix":""},{"id":274309127,"identity":"4c413246-2e26-4b06-bf5a-2fe9642aaf9e","order_by":1,"name":"Shin Woong Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYFACNoYDDBVA+gAQA+kEBgbmBiCbmYCWMyAtQEVnwFoYCWthYGxD08KAT4v8jLTEQzfn1cnxHT9/gOFgG0Mev/TBxsMFDNZyuLQY3Eg7cDh3G5ux5JlkBpCWYsm+xIbDMxjSjXFqkU5vAGrhSdxwIJmB+WPb/8QNZxgbDvMwHE5swOWw2SAtcyTqN5x/DLYlcT9USz0uLQy3QQ5rMEgwuAFxWOIGHoiWBJwOu/8s4XDOsQTDmTceGxw4cI6hWAJsi0G6IU6H9Rwz/pxTUyfPdz7x4YMDZcAQ62E+/Jmnwloely0o4ACS7URpGAWjYBSMglGAAwAAIKdgX0vzY84AAAAASUVORK5CYII=","orcid":"","institution":"Freie Universität Berlin","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shin","middleName":"Woong","lastName":"Kim","suffix":""},{"id":274309128,"identity":"378674b8-5b8a-41d8-874c-b49be32d820f","order_by":2,"name":"Huiying Li","email":"","orcid":"","institution":"Freie Universität Berlin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huiying","middleName":"","lastName":"Li","suffix":""},{"id":274309129,"identity":"67069432-28a8-4ef8-a4a5-92175527dcf1","order_by":3,"name":"Matthias C. Rillig","email":"","orcid":"","institution":"Freie Universität Berlin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Matthias","middleName":"C.","lastName":"Rillig","suffix":""}],"badges":[],"createdAt":"2024-02-20 09:45:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3972392/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3972392/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51538704,"identity":"f087c071-5ad0-4082-a1fc-bbfbbb954284","added_by":"auto","created_at":"2024-02-23 10:31:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":588641,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of TWPs delivery rates (abrupt and gradual) on the activities of (a) β-glucosidase, (b) β-D-1,4-cellobiosidase, (c) β-1,4-N-acetyl-glucosaminidase and (d) phosphatase. Boxplot indicates the median (thick black line), first quartile (bottom line), third quartile (top line), upper and lower limits (whiskers) for each group. The upper panels show the data comparison of different treatments (control, abrupt, and gradual) in each phase, and the lower panels show the rearranged data of upper panels for the comparison of different phases under each treatment. Asterisks (*) indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05) compared to the control (upper panels) or phase 1 (lower panels).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3972392/v1/d0e95918521fefc19d344491.png"},{"id":51538705,"identity":"b9f1265a-bfb8-49b5-af4e-8277cc87e3a9","added_by":"auto","created_at":"2024-02-23 10:31:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":332966,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of TWPs delivery rate (abrupt and gradual) on (a) soil pH, (b) the percentage of soil water stable aggregates (WSA), and (c) soil respiration rate. Boxplot indicates the median (thick black line), first quartile (bottom line), third quartile (top line), upper and lower limits (whiskers) for each group. The upper panels show the data comparison of different treatments (control, abrupt, and gradual) in each phase, and the lower panels show the rearranged data of upper panels for the comparison of different phases under each treatment. The asterisks (*) indicate significant differences (p\u0026lt; 0.05) compared to the control (upper panels) or phase 1 (lower panels).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3972392/v1/e862441e34f3e92aafe19656.png"},{"id":51538708,"identity":"5c3a86c0-154f-4e57-9e19-c16e2442cc89","added_by":"auto","created_at":"2024-02-23 10:31:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":161377,"visible":true,"origin":"","legend":"\u003cp\u003eConceptual interpretation of the effects of TWPs at different delivery rates.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3972392/v1/978af39daf7bc75f775638be.png"},{"id":51539059,"identity":"1defeedb-0163-431e-9226-5cb0a11e9680","added_by":"auto","created_at":"2024-02-23 10:39:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1009483,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3972392/v1/1f778d85-6f39-48e0-9f24-10fe1eaa3f0a.pdf"},{"id":51538707,"identity":"c8795b7f-c8de-4198-a1a3-ac98db46b690","added_by":"auto","created_at":"2024-02-23 10:31:20","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":120169,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-3972392/v1/f3629364d5039298ed151895.png"},{"id":51538709,"identity":"16299cf3-cdc6-4e54-b475-edecb0f8d812","added_by":"auto","created_at":"2024-02-23 10:31:20","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4073354,"visible":true,"origin":"","legend":"","description":"","filename":"TWPDeliverySIESE.docx","url":"https://assets-eu.researchsquare.com/files/rs-3972392/v1/1302d4e7ff98b9b7b0abfb12.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Delivery rate alters the effects of tire wear particles on soil microbial activities","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAlthough the first tire was produced in the 19th century, tire wear particles (TWPs) pollution in road dust was not highlighted until 1966 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. TWPs are usually regarded as microplastics (\u0026lt;\u0026thinsp;5 mm) due to their size, insolubility, polymer structure, and solid state [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and have emerged as a main contributor of microplastic pollution [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The global average emission of TWPs has reached 0.81 kg per year per capita [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and recent research has reported the potential effects of TWPs on soil ecosystem. TWPs can induce adverse effects on survival and reproduction of soil fauna [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], plant growth [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and microbial activities [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and these effects have been underpinned with multiple pathways [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. For instance, TWPs can physically damage the intestine of soil fauna [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and influence soil microbial activities through changing soil properties and nutrient cycles [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Tires consist of rubber, filler, softener, vulcanization agents and various additives [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], including toxic chemicals such as polycyclic aromatic hydrocarbons (PAHs), benzothiazoles and heavy metals (e.g., Cd, Pb, and Zn), and these additives can be released into the environment during the abrasion of tires, contributing to the adverse effects [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOnce TWPs are generated on the road surface, they can be delivered into the soil environment either through road runoff or airborne transport [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Rainfall is considered as a main factor influencing the transport of TWPs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and the intensity of precipitation is directly linked with TWPs delivery rates [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For instance, during a strong rainfall event, TWPs can be abruptly washed from the air or road surfaces to roadside soils [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], while no precipitation or weak/steady rainfall leads to a more gradual delivery of TWPs. This is important since there is still a knowledge gap regarding different delivery rates of TWPs due to the lack of empirical data [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The rate of changes in environmental (e.g., drought) or anthropogenic (e.g., chemical pollutants) factors have been well established in global change research [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and different effects under different rates of changes have been observed frequently [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSoil is a main sink of contaminants emitted into the environment, and 67% of TWPs are delivered to the soil environment eventually [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this study, we investigated whether the delivery rate influences the impacts of TWPs on soil microbial activities and physicochemical properties. We established the different delivery rates of TWPs (abrupt and gradual), and designed two phases that capture the TWPs-delivery period (phase 1) and the end-of-delivery period (phase 2). We hypothesized that (1) TWPs pollution affects soil microbial activities and physicochemical properties, (2) delivery rate alters the effects of TWPs, and (3) these effects will be changed at the end-of-delivery period, depending on the prior delivery rates.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Test Soil and TWPs\u003c/h2\u003e \u003cp\u003eTest soil (loamy sandy; Albic Luvisol) was collected from a grassland site of the Institute of Biology of Freie Universit\u0026auml;t Berlin (52\u0026deg;28' N, 13\u0026deg;17' E) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Soil bulk density and water holding capacity (WHC) were 1.2 g cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e and 39.5%, and pH and EC were 5.8 and 58.8 \u0026micro;S cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The soil was air-dried for one week at room temperature and passed through a 2 mm-sieve for further use. TWPs (\u0026lt;\u0026thinsp;500 \u0026micro;m) were obtained from the company KURZ-Karkassenhandel (Landau, Germany) and sieved through a 250 \u0026micro;m mesh. A light microscope (MDG41, Leica, Wetzlar, Germany) equipped with a camera (Flexacam C1, Leica, Wetzlar, Germany) was used to measure the average size of particles (88.4 \u0026micro;m, 4.5\u0026ndash;276.8 \u0026micro;m) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and this particle size is within a range of TWPs produced during driving (4\u0026ndash;350 \u0026micro;m) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experiment set-up\u003c/h2\u003e \u003cp\u003eThe experiment was established in a greenhouse (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2/18\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, day/night; daylight period, 16:8; relative humidity, 40%). We placed 200 g of test soil in 210 mL round plastic containers (diameter, 80 mm; height, 48 mm; RPC superfos a/s, Denmark; soil layer thickness, 30 mm) covered with nylon mesh (pore size, 38 \u0026micro;m), and pre-incubated with 60% of WHC for a week to achieve a steady state of soil microbial metabolism (n\u0026thinsp;=\u0026thinsp;12 for each three treatment). After the pre-incubation, we divided the experiment into two different phases, the TWPs-delivery period (phase 1; duration of 9 weeks) and end-of-delivery period (phase 2; duration of 4 weeks). During phase 1, we treated soils with TWPs at three different delivery rates including control (no TWPs addition), gradual delivery, and abrupt delivery. The abrupt and gradual delivery treatments had the same overall dose by the addition of TWPs according to a \u0026ldquo;week-dose\u0026rdquo; principle (Fig. S2). Briefly, 0.2 g of TWPs (0.1%, total dry soil mass) was added weekly in the gradual delivery treatment, and 1.8 g of TWPs (0.9%, total dry soil mass) was added at the fifth week for the abrupt treatment. These concentrations were based on the measurements (0.2 to 2%, dry weight) of TWPs in roadside soils according to previous studies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. At the beginning of phase 1, TWPs were suspended in deionized water with a volume matching 100% of the soil WHC, and we evenly poured this onto the soil surface for the gradual delivery treatment. The control and abrupt delivery treatments were treated with only deionized water (no TWPs addition). The water contents in each container were checked and replenished with deionized water (35 to 40 mL) every week to keep uniform moisture (100% of WHC). This water maintenance was accompanied with TWPs addition for the gradual delivery treatment, and the same TWPs addition method was applied for the abrupt delivery treatment in the fifth week (Fig. S3). We applied TWPs to the soil surface (soil depth, 30 mm) without subsequent mixing to prevent artificial disturbance, and saturated the soil with 100% of WHC to simulate rainy season conditions and to facilitate the particle penetration into the soil. At the end of phase 1, 6 units of each treatment were harvested, and the rest of experimental units (n\u0026thinsp;=\u0026thinsp;6) were incubated for 4 more weeks with no further TWPs addition (phase 2) (Fig. S3). After harvest, each soil was carefully homogenized using a spatula, and fresh soil samples were stored at 4\u0026deg;C for the subsequent measurements. The rest of the soil samples were air dried and then stored at room temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Measurement of soil properties\u003c/h2\u003e \u003cp\u003eSoil respiration rate was indicated by the CO\u003csub\u003e2\u003c/sub\u003e concentration produced per hour (\u0026micro;M M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e h\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Thirty gram of fresh soil was placed in a 50 mL falcon tube equipped with a rubber stopper on the lid. The tubes were flushed with CO\u003csub\u003e2\u003c/sub\u003e-free air for 5 min to eliminate background CO\u003csub\u003e2,\u003c/sub\u003e and then incubated for 4 h at 20\u0026deg;C. We sampled 1 mL of air in the headspace of the tube using a syringe and injected this sample into an infrared gas analyzer (LI-6400XT, LI-COR Inc., Bad Homburg, Germany). The activities of C-related enzymes β-glucosidase (EC3.2.1.21) and β-D-1,4-cellobiosidase (EC3.2.1.91), N-related enzyme β-1,4-N-acetyl-glucosaminidase (EC3.2.1.52), and P-related enzyme phosphatase (EC3.1.3.2) were measured with artificial \u003cem\u003ep\u003c/em\u003e-nitrophenyl (\u003cem\u003ep\u003c/em\u003eNP) linked substrates, and quantified by a microplate reader [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, 10 mL of 50 mM acetate buffer (pH, 5.0\u0026thinsp;\u0026minus;\u0026thinsp;5.5) was added into 50 mL-test tubes containing 5.0 g of each soil sample. The tubes were vortexed for 5 secs, and each soil slurry (150 \u0026micro;L) into each well (6 wells per sample) in the 96-well plate. Then, 150 \u0026micro;L of each substrate was added: \u003cem\u003ep\u003c/em\u003eNP-β-D-glucopyranoside (Sigma no. N7006), \u003cem\u003ep\u003c/em\u003eNP-β-D-cellobioside (Sigma no. N5759), \u003cem\u003ep\u003c/em\u003eNP-N-acetyl-β-D-glucosaminide (Sigma no. N9376), \u003cem\u003ep\u003c/em\u003eNP- phosphate disodium salt hexahydrate (Sigma no. 71,768). After 2 h (β-glucosidase and phosphatase) and 4 h (β-D-1,4-cellobiosidase and β-1,4-N-acetyl-glucosaminidase) incubations at 20\u0026deg;C under dark conditions, each plate was centrifuged at 3,000 rpm for 5 min, and 100 \u0026micro;L of supernatant was transferred into a new plate and mixed with 200 \u0026micro;L of 0.1 M NaOH. Absorbances were determined at 410 nm using a microplate reader (Benchmark Plus, BioRad Laboratories GmbH, Hercules, CA, USA), and each enzyme activity was calculated as \u0026micro;mol p-nitrophenol g soil dry mass\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e. Water stable aggregates (WSA) content was measured following the wet sieving method of Kemper and Rosenau [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] with a slight modification. Four gram of air-dried soil was put in a small sieve with a mesh size of 0.25 mm, re-wetted by capillarity with deionized water and inserted into a sieving machine (Agrisearch Equipment, Royal Eijkelkamp B.V., Giesbeek, Netherlands) to be wet-sieved for 3 min. The soil left on the sieve (fraction 1: stable soil aggregates and coarse matter) was dried at 60\u0026deg;C and weighed, and then crushed manually in the wet sieve to obtain the coarse matter (fraction 2). Calculations of the percentage of WSA were according to: WSA (%) = (fraction 1 \u0026ndash; fraction 2)/(4.0 \u0026ndash;fraction 2) \u0026times; 100%. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To measure soil pH, 25 mL of distilled water was added to 5.0 g of air-dried soil in a falcon tube. The tube was shaken in 250 rpm for 30 min and then centrifuged in 3000 rpm for 5 min. The supernatant was filtered, and the pH was determined by a pH-meter (Hanna Instruments GmbH, Smithfield, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Data analysis\u003c/h2\u003e \u003cp\u003eWe performed all the statistical analyses and plotting in R (v.4.1.2). Unpaired t-tests were performed by the t.test() function with a confidence interval of 95% to compare the difference between each treatment and phase. The variance was estimated separately for each group and the Welch modification to the degrees of freedom was used. Boxplots were drawn using the ggplot2 package. The changes of enzyme activities between phase 1 and phase 2 were calculated by the percentage (%) of the changes in phase 2 to compared to phase 1.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eWe evaluated the activities of four soil enzymes at the end of two different phases (phase 1 and 2), and found that their activities were dependent on TWPs delivery rates (abrupt and gradual) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In phase 1, the activities of β-glucosidase and β-D-1,4-cellobiosidase were significantly reduced in the gradual treatment compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a) and (b)). The activity of β-1,4-N-acetyl-glucosaminidase increased in the abrupt treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c)), but there was no significant change in phosphatase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(d)). We found no significant effect on pH, soil respiration, and WSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter phase 1, each soil was incubated for 4 weeks with no additional TWPs addition (phase 2). The activities of none of the enzymes were significantly different in both delivery treatments, compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There was no significant effect on WSA, soil respiration, and pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Through a comparison between phase 1 and 2, we found that the activities of β-glucosidase (gradual) and β-D-1,4-cellobiosidase (abrupt and gradual) notably increased over time to a similar level as those observed in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a) and (b), lower panels). The β-1,4-N-acetyl-glucosaminidase activity significantly decreased in the abrupt treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c), lower panel), and phosphatase activity increased in the control and the abrupt treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(d), lower panel). In addition, soil pH significantly increased over time in both of the TWPs treatments (gradual and abrupt) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a), lower panel).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe effects of TWPs on extracellular enzyme activities have been explored in previous studies [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and our results showed general agreement with these studies, such as the increase of β-1,4-N-acetyl-glucosaminidase activity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], the decrease of β-glucosidase activity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and no significant change in phosphatase activity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, the impacts of TWPs delivery rates have not been reported before. Our findings indicated that the gradual delivery treatment induced a significant decrease in the activity of carbon cycle-related enzymes, while there was no effect in the abrupt treatment. TWPs can be presumably classified as an inert (persistent) carbon source, similar to microplastics and charcoal [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and the fraction of persistent carbon is barley utilized by microbes through the production of hydrolytic enzymes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Many classes of enzymes and environmental factors (e.g., C/N ratio and pH) are involved in the cellulose hydrolysis process [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and our hypothesis regarding the carbon hydrolyzing enzymes may be partly supported by the results of soil parameters in our study. For instance, the enzyme secretion by microbes relies on the availability of soil nitrogen, and high N availability would increase microbial demand for carbon, inducing the production of glucosidases [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study, the increase of β-1,4-N-acetyl-glucosaminidase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c)), which may be abruptly caused by the release of chemical additives (PAHs and nitro-compounds as vulcanization agent) from TWPs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], was observed in the abrupt treatment, and this change might neutralize the negative effects on β-glucosidase activity in the abrupt treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a)).\u003c/p\u003e \u003cp\u003eTWPs negatively affected the carbon cycle-related enzymes during phase 1 (TWPs-delivery period), and these effects were significantly reduced in phase 2 (end-of-delivery period). This consequence may be linked to previous reports that carbon cycle-related functional genes can be abundant after TWPs exposure [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The soil microbial community has the capacity to establish a new equilibrium with the altered environment, which is defined as the adaptation/adjustment capacity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and this capacity seems to be shown in both delivery treatments. However, we assumed that abruptly and gradually delivered TWPs have elicited different adaptation/adjustment of carbon cycle-related microorganisms to TWPs pollution, since the changes (phase 1 to phase 2) of both β-glucosidase and β-D-1,4-cellobiosidase activities were more remarkable in the gradual treatment (19.82% and 59.29%) than those in the abrupt treatment (0.31% and 32.34%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the results of β-1,4-N-acetyl-glucosaminidase, we found that the enzyme activity dramatically decreased in the abrupt treatment in phase 2 compared to in phase 1. Although the activities of most soil extracellular enzymes generally decrease over time in normal conditions [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], this reduction was remarkable in the abrupt treatment (29.80%) compared to other treatments (control and gradual, 13.16% and 5.59%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). According to a previous study, abrupt TWPs pollution can stimulate anaerobic nitrogen metabolism [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], which may potentially reduce the activities of nitrogen cycle-related enzymes in a long run, and this may cause the decrease in β-1,4-N-acetyl-glucosaminidase activity over time in the abrupt delivery treatment. In general, soil pH is positively related to β-1,4-N-acetyl-glucosaminidase activity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], but our results in the abrupt treatment showed negative correlations between pH and β-1,4-N-acetyl-glucosaminidase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c) and 2(a), lower panel). This might be caused by the leaching of chemicals from TWPs [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] or the interactions among multiple extracellular enzymes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and we assumed that these changes were influenced by the prior delivery rates.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe changes of enzyme activities from phase 1 to phase 2 in different treatments. Each value indicates the percent (%) change of each enzyme activity in phase 2 compared to the activity in phase 1.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ-glucosidase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ-D-1,4-cellobiosidase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ-1,4-N-acetyl-glucosaminidase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePhosphatase\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;12.41%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.96\u0026thinsp;\u0026plusmn;\u0026thinsp;18.27%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-13.16\u0026thinsp;\u0026plusmn;\u0026thinsp;18.75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e7.89\u0026thinsp;\u0026plusmn;\u0026thinsp;6.95%*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbrupt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;20.81%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e32.34\u0026thinsp;\u0026plusmn;\u0026thinsp;28.59%*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-29.80\u0026thinsp;\u0026plusmn;\u0026thinsp;9.16%*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e19.57\u0026thinsp;\u0026plusmn;\u0026thinsp;22.35%*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGradual\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19.82\u0026thinsp;\u0026plusmn;\u0026thinsp;8.91%*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e59.29\u0026thinsp;\u0026plusmn;\u0026thinsp;54.28%*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-5.59\u0026thinsp;\u0026plusmn;\u0026thinsp;9.51%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.926\u0026thinsp;\u0026plusmn;\u0026thinsp;16.12%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003emeans\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003e*\u003c/b\u003e Significant differences between phase 1 and phase 2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eGiven the mode of arrival of TWPs in soil, it is important to carefully consider their transport from the road surface into the environment in order to understand their effects. Unfortunately, this aspect has been overlooked in previous experiments. Our results revealed that TWPs pollution influences soil microbial activities, and that these effects are altered by different delivery rate (abrupt and gradual). The adaptation/adjustment of soil microorganisms after the TWPs-delivery period was affected depending on the prior delivery rate. These findings indicated that the delivery rate of TWPs may represent another key factor altering the effect of TWPs. It is thus imperative to consider exposure scenarios when interpreting the impacts of TWPs in soil systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), and this study enhances our understanding of the ecological effects of TWPs. Further research is warranted to investigate the responses at the microbial community level, with the use of different test soils and a variety of delivery rates, to gain a comprehensive understanding of how TWPs delivery rates influence soil ecosystems. The shifts in TWPs effects under different delivery rates are important for deciphering the true extent of TWPs effects in the field, and likely are also relevant for other types of microplastics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eSupporting information\u0026nbsp;\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe Supporting Information is available free of charge.\u003c/p\u003e\n\u003cp\u003eSupplementary figure of photographic processing of tire wear particle sizes to determine average diameter and size range (Fig. S1), scheme of the gradual and abrupt TWPs treatments based on the \u0026ldquo;day-dose\u0026rdquo; principle (Fig. S2), experimental design (Fig. S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and the supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.C.R. acknowledges support from PAPILLONS (Plastics in Agricultural Production: Impacts, Lifecycles and Long-term sustainability, No.101000210) of the European Union\u0026rsquo;s Horizon 2020 research and innovation program. YZ, HL acknowledge the China Scholarship Council for a scholarship (YZ, CSC No. 202106320023; HL, CSC No. 202108080156).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYZ: conceptualization, design of the study, experiment setup, analysis of data, and writing; SWK: conceptualization, design of the study, writing, review and editing; HL and HC: experiment setup. M.C.R.: design of the study, review and editing. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eThompson, R. N., Nau, C. A., \u0026amp; Lawrence, C. H. (1966). Identification of vehicle tire rubber in roadway dust. \u003cem\u003eAmerican Industrial Hygiene Association Journal, 27\u003c/em\u003e(6), 488-495, doi:10.1080/00028896609342461.\u003c/li\u003e\n\u003cli\u003eHartmann, N. B., Huffer, T., Thompson, R. C., Hassellov, M., Verschoor, A., Daugaard, A. E., et al. (2019). Are we speaking the same language? Recommendations for a definition and categorization framework for plastic debris. \u003cem\u003eEnvironmental Science \u0026amp; Technology, 53\u003c/em\u003e(3), 1039-1047, doi:10.1021/acs.est.8b05297.\u003c/li\u003e\n\u003cli\u003eDing, J., Lv, M., Zhu, D., Leifheit, E. F., Chen, Q. L., Wang, Y. Q., et al. (2022). Tire wear particles: An emerging threat to soil health. \u003cem\u003eCritical Reviews in Environmental Science and Technology, 53\u003c/em\u003e, 239-257, doi:10.1080/10643389.2022.2047581.\u003c/li\u003e\n\u003cli\u003eSelonen, S., Dolar, A., Kokalj, A. J., Sackey, L. N. A., Skalar, T., Fernandes, V. C., et al. (2021). 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Chemical composition and ecotoxicity of plastic and car tire rubber leachates to aquatic organisms. \u003cem\u003eWater Research, 169\u003c/em\u003e, 115270, doi:https://doi.org/10.1016/j.watres.2019.115270.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Abrupt exposure, Gradual exposure, Microplastics, Enzyme activities, Soil aggregates, Soil pH, Soil respiration","lastPublishedDoi":"10.21203/rs.3.rs-3972392/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3972392/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTire wear particles (TWPs) produced by the abrasion between tires and road surfaces have been recognized as an emerging threat to soil health globally in recent years. They can be transported from the road surface to adjacent soil at different delivery rates, with precipitation a main driver underpinning this movement. However, studies typically assume an abrupt exposure of TWPs in their experimental design. In this study, we investigated the impacts of abrupt and gradual delivery of TWPs on soil physicochemical properties and microbial activities. We used two different delivery rates of TWPs (abrupt and gradual) and devised two experimental phases, namely the TWPs-delivery period (phase 1) and the end-of-delivery period (phase 2).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that the gradual TWPs delivery treatments negatively influenced the activity of carbon cycle-related enzymes (β-glucosidase and β-D-1,4-cellobiosidase). Furthermore, the abrupt treatment highly increased the effects on nitrogen cycle-related enzyme activity (β-1,4-N-acetyl-glucosaminidase). In phase 2 (end-of-delivery period), each enzyme activity was changed to a similar level as the control group, but these changes were influenced by the prior delivery rates.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eAbruptly and gradually delivered TWPs induce different responses to soil microbial activities. Our findings imply that the delivery rate of TWPs could be another key factor changing the effects of TWPs, further enhancing our understanding of the ecological impacts of TWPs.\u003c/p\u003e\u003ch2\u003eGraphical abstract\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Delivery rate alters the effects of tire wear particles on soil microbial activities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-23 10:31:15","doi":"10.21203/rs.3.rs-3972392/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-24T20:48:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-12T06:27:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161381a2-57c6-48bb-83b1-74422e904892","date":"2024-02-21T22:33:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a324a9f9-41bf-43e8-b868-6fac518972a2","date":"2024-02-21T22:14:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-21T20:10:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-21T11:31:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-21T11:31:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Sciences Europe","date":"2024-02-20T09:30:47+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":"ee41d693-83e7-44e9-bcb9-080475e96771","owner":[],"postedDate":"February 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-04-25T02:57:02+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-23 10:31:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3972392","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3972392","identity":"rs-3972392","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}