The effect of 1-ethyl-3-methylimidazolium chloride on oxidative stress and the functioning of the photosynthetic apparatus in maize seedlings – the modulatory role of exogenous ascorbic acid

The effect of 1-ethyl-3-methylimidazolium chloride on oxidative stress and the functioning of the photosynthetic apparatus in maize seedlings – the modulatory role of exogenous ascorbic acid | 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 The effect of 1-ethyl-3-methylimidazolium chloride on oxidative stress and the functioning of the photosynthetic apparatus in maize seedlings – the modulatory role of exogenous ascorbic acid Barbara Pawłowska, Aleksandra Lechowska, Radomír Ščurek, Robert Biczak This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8938488/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ionic liquids (ILs) are widely used chemical compounds that may pose potential risks to the environment. In the present study, the effects of 1-ethyl-3-methylimidazolium chloride (EMIMCl) on growth, photosynthetic performance, and oxidative stress in maize ( Zea mays L.) seedlings were evaluated, and the role of exogenous L-ascorbic acid (AsA) in modulating plant responses to this stress was investigated. Plants were cultivated in soil contaminated with EMIMCl at concentrations ranging from 1 to 1000 mg·kg -1 of soil dry weight and treated with AsA at concentrations of 0.5–2 mM. EMIMCl significantly inhibited plant growth, reduced photosynthetic pigment content, and impaired chlorophyll fluorescence parameters, accompanied by increased hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) levels, indicating the induction of oxidative stress. Moderate doses of AsA partially alleviated EMIMCl-induced toxicity, whereas higher AsA concentrations under severe EMIMCl contamination intensified stress symptoms. These findings demonstrate a dose-dependent and biphasic role of AsA in maize responses to EMIMCl-induced stress. phytotoxicity oxidative stress chlorophyll fluorescence maize 1-ethyl-3-methylimidazolium chloride Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The rapid advancement of chemical technologies over recent decades has resulted in a substantial increase in both the production and application of ionic liquids (ILs). Ionic liquids constitute a vast and structurally diverse class of compounds. Owing to their distinctive physicochemical properties, these substances have been widely implemented across numerous industrial sectors. In particular, imidazolium-based ionic liquids, including 1-ethyl-3-methylimidazolium chloride (EMIMCl), have found applications in chemical synthesis, lignocellulosic biomass processing, and electrochemical technologies (Bae et al. 2024; Maculewicz et al. 2024; Rowan et al. 2025; Egorova and Ananikov 2018). Market forecasts indicate that the global ionic liquids market is expected to reach USD 7,266.2 million by 2030 (Industry ARC 2025). The continuous expansion of ILs production and their growing range of applications across various industries raise concerns regarding their potential environmental impact. An increasing body of literature suggests that, despite their advantageous technological properties, many ionic liquids exhibit significant biological activity, which may pose risks to living organisms following their release into the environment. Scientific investigations have confirmed that ionic liquids have already entered environmental compartments and have been detected, among others, in river waters. Due to their water solubility and limited susceptibility to biodegradation, imidazolium-based ILs may accumulate in soils and surface waters, thereby increasing the exposure risk for terrestrial organisms, including crop plants. Numerous studies report that ionic liquids can exert toxic effects on bacteria, fungi, algae, crustaceans, fish, and plants (Maculewicz et al. 2022; Cvjetko Bubalo et al. 2014a; Matzke et al. 2007; Cvjetko Bubalo et al. 2014b). Previous research has demonstrated that ILs, including EMIMCl, may inhibit seed germination, impair plant growth and development, and disrupt metabolic processes in various plant species, including agriculturally important crops. Moreover, ionic liquids may display strong binding affinity to human blood proteins, which could contribute to their bioaccumulation in the human body (Maculewicz et al. 2022; Zheng et al. 2021; Hrubec et al. 2021; Neuwald et al. 2021). Despite numerous studies conducted to date, the precise mechanisms underlying the phytotoxic effects of ionic liquids have not yet been fully elucidated. Growing evidence suggests that disruption of biological membrane integrity and the induction of oxidative stress play central roles in their mode of action. Imidazolium cations are capable of interacting with membrane phospholipids as well as thiol-containing proteins, which may result in destabilization of cellular structures and impaired functioning of mitochondria and chloroplasts. These disturbances can lead to excessive generation of reactive oxygen species (ROS), triggering lipid peroxidation and damage to the photosynthetic apparatus (Pham et al. 2010; Cvjetko Bubalo et al. 2017). Plants have evolved complex defense strategies to maintain redox homeostasis under environmental stress conditions. A key component of these protective systems is the antioxidant network, which includes both enzymatic antioxidants and low-molecular-weight reducing compounds. Ascorbic acid (AsA) plays a particularly important role within this system by directly scavenging ROS and regenerating other antioxidants through the ascorbate–glutathione cycle. In addition, AsA functions as a cofactor for certain oxidases and participates in the biosynthesis of several phytohormones, including abscisic acid (ABA) and ethylene. It also contributes significantly to maintaining intracellular and extracellular redox balance in plants. Through these functions, AsA enables plants to respond rapidly and effectively to environmental fluctuations, thereby enhancing their tolerance to various abiotic stresses (Ding et al. 2020; Xiao et al. 2021; Wu et al. 2024; Zhang et al. 2019). Numerous studies have demonstrated that exogenous application of AsA may exert beneficial effects on plants exposed to drought, high salinity, or heavy metal stress (Zhang et al. 2019; Ali et al. 2018; Alves et al. 2022). It should be noted, however, that the effects of AsA are not invariably protective. Under conditions of severely disrupted redox balance or when applied at excessive concentrations, ascorbic acid may exhibit pro-oxidant properties. In particular, through participation in redox reactions—especially in the presence of transition metal ions—AsA can promote the overproduction of reactive oxygen species (ROS) (Smirnoff 2018). This phenomenon indicates that the effectiveness of exogenous antioxidants is strongly dependent on their concentration and on the intensity of environmental stress. Despite the increasing number of studies addressing the toxicity of ionic liquids, comprehensive analyses integrating their simultaneous effects on plant growth, photosynthetic performance, and oxidative stress—along with the potential modulation of these responses by exogenous antioxidants—remain limited. In particular, there is a scarcity of data concerning the impact of EMIMCl on economically important crop species such as maize and the potential implications for agroecosystems. Maize is the third most important cereal crop worldwide, following rice and wheat. Compared with these two cereals, however, maize has a broader range of applications. In addition to its role in human nutrition, it serves as a major component of livestock feed and is widely used in industrial and bioenergy production systems (Erenstein et al. 2022; Zhang et al. 2019). The objective of the present study was to evaluate the effects of 1-ethyl-3-methylimidazolium chloride (EMIMCl) on early growth and development, photosynthetic efficiency, and oxidative stress parameters in maize seedlings. Furthermore, the potential role of exogenously applied ascorbic acid in modulating plant responses to this chemical stressor was investigated. Particular emphasis was placed on dose–response relationships and on the possibility of biphasic effects of AsA under progressively increasing EMIMCl-induced stress. To the best of our knowledge, this is the first study to examine the influence of ascorbic acid on the phytotoxicity of ionic liquids. Materials and methods Chemicals 1-Ethyl-3-methylimidazolium chloride (EMIMCl; ≥95% purity) was obtained from Sigma-Aldrich Chemical Co. L-ascorbic acid (analytical grade) was purchased from Chempur. All other chemicals and reagents used for the respective analyses were of at least analytical reagent grade (≥ AR grade). Experimental design Phytotoxicity assays were conducted under controlled greenhouse conditions. The study was performed in accordance with the guidelines outlined in OECD/OCDE (2006). Ten uniform maize ( Zea mays L.) seeds of the cultivar ‘Rywal’ were sown in plastic pots containing 250 g of control soil (without ionic liquids) or soil amended with EMIMCl at concentrations of 1, 50, 100, 500, and 1000 mg·kg⁻¹ of soil dry weight (DW). Maize seeds were obtained from the Plant Breeding and Production Station in Nieznanice, belonging to Małopolska Hodowla Roślin – HBP Sp. z o.o. The soil used in the experiment was classified as sandy loam, containing approximately 11% particles < 0.02 mm, 8.5 g·kg⁻¹ organic carbon, and a pH (KCl) of 6.0. EMIMCl was applied to the soil in the form of aqueous solutions and thoroughly homogenized prior to sowing. Immediately after seed sowing, the soil was irrigated either with distilled water (control treatment) or with L-ascorbic acid solutions at concentrations of 0.5, 1, and 2 mM. Throughout the experiment, growth conditions were maintained at constant levels: soil moisture was kept at 70% of water-holding capacity, temperature at 20 ± 2°C, and light intensity at 170 µmol·m⁻²·s⁻¹ under a 16 h light/8 h dark photoperiod. All measurements were performed in four independent replicates (Fig. 1 ). After 14 days of growth, chlorophyll fluorescence was measured and plant morphology was visually assessed and documented using digital photography. Subsequently, the following parameters were determined: shoot and root length, fresh biomass yield, dry matter content, photosynthetic pigment concentration, and levels of H₂O₂, malondialdehyde (MDA), and ascorbic acid (AsA). Figure 1 . Experimental scheme for assessing EMIMCl phytotoxicity in maize under controlled conditions Determination of basic phytotoxicity parameters Inhibition of shoot and root growth was evaluated according to the procedure described by Wang et al. (2009). Fresh biomass yield was also determined for each treatment. Dry wieght content was assessed following the method of Kowalska (2004). Approximately 1 g of leaf tissue was dried at 105°C until a constant weight was achieved. Chlorophyll fluorescence Chlorophyll fluorescence parameters were measured using an OS1p chlorophyll fluorometer (Opti-Sciences, Inc., Hudson, USA). The following parameters were recorded: initial (minimal) fluorescence (F₀), variable fluorescence (F v ), maximal fluorescence (Fₘ), the maximum quantum efficiency of photosystem II (F v /Fₘ), and the more sensitive ratio F v /F₀. Determination of photosynthetic pigments The concentrations of photosynthetic pigments, including chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids, were determined according to the method proposed by Oren et al. (1993). Briefly, 200 mg of maize leaf tissue was homogenized in 80% acetone (4°C) and subsequently centrifuged. Pigment concentrations were quantified spectrophotometrically by measuring absorbance at 470, 647, and 664 nm, followed by calculation using the appropriate equations. Determination of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and ascorbate (AsA) Fresh plant material (0.5 g) was homogenized in 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged. The resulting supernatant was used for the determination of MDA and H₂O₂ concentrations. MDA assay. The reaction mixture consisted of the supernatant, 0.5% thiobarbituric acid (TBA) prepared in 20% TCA, and phosphate buffer (pH 7.6). Samples were incubated in a water bath at 95°C for 30 minutes and then rapidly cooled in an ice bath. After cooling, absorbance was measured at 532 and 600 nm, following the method described by Hodges et al. (1999). H₂O₂ assay. For hydrogen peroxide determination, the supernatant was mixed with 1 M potassium iodide (KI) and phosphate buffer (pH 7.6). The reaction mixture was incubated in the dark for 1 hour, after which absorbance was recorded at 390 nm according to Singh et al. (2007). AsA assay . To determine ascorbate content, 0.5 g of fresh maize leaf tissue was homogenized in 10% TCA and centrifuged. An aliquot of 0.2 cm³ of the supernatant was combined with 2% TCA, 1 M H₃PO₄, 0.8% bipyridyl, and 0.15% FeCl₃. The prepared reaction mixture was incubated at 37°C for 60 minutes in darkness. Absorbance was subsequently measured at 525 nm, and AsA concentration was calculated using a standard calibration curve, as described by Law et al. (1983). Statistical analysis The data were analyzed using two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, employing STATISTICA 13.3 software. Differences were considered statistically significant at p < 0.05. All measurements were performed in at least four independent replicates (n = 4). Results are presented as mean values ± standard deviation (SD). Principal component analysis (PCA) was performed using standardized data (z-score normalization). The analysis was based on the correlation matrix, and the first two principal components were used for graphical interpretation. PCA calculations and visualizations were carried out in the R statistical environment (R Foundation for Statistical Computing, Vienna, Austria). Results Phytotoxicity The effect of different concentrations of 1-ethyl-3-methylimidazolium chloride (EMIMCl) and L-ascorbic acid (AsA) on maize emergence is presented in Table 1 . The presence of EMIMCl in the soil reduced seedling emergence only at the highest tested concentration (1000 mg·kg⁻¹ of soil DW). In contrast, soil supplementation with AsA at all applied concentrations mitigated the adverse effect of the ionic liquid on seed germination capacity. Even at the highest EMIMCl level, exogenous AsA application restored emergence to values that did not differ significantly from the control treatment. Table 1 Shoot and root length of maize seedlings and percentage emergence of plants grown in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are presented as means of 10 replicates ± standard deviation. Values within columns followed by the same letter do not differ significantly. Concentration ILs [mg·kg − 1 of soil DW] Concentration AsA [mM] Root length [cm] Shoot length [cm] Number of plants that germinated 0 (control) 0 14.1 ± 1.5 abc 24.8 ± 2.7 bcde 9 ± 2 ab 0.5 15.0 ± 0.9 ab 29.2 ± 2.5 a 9 ± 1 ab 1 14.6 ± 1.1 abc 26.3 ± 2.3 ab 9 ± 0 ab 2 14.2 ± 1.2 abc 24.0 ± 1.8 bcdef 10 ± 1 ab 1 0 14.6 ± 1.3 abc 24.3 ± 2.3 bcde 10 ± 0 a 0.5 15.7 ± 1.2 a 25.7 ± 1.5 bcd 10 ± 1 ab 1 15.1 ± 1.3 ab 25.2 ± 2.7 bcde 10 ± 0 a 2 14.6 ± 1.0 abc 23.6 ± 2.2 bcdefg 9 ± 0 ab 50 0 13.5 ± 1.1 bcd 22.2 ± 0.8 efg 8 ± 1 bc 0.5 14.6 ± 1.3 abc 26.1 ± 2.4 abc 9 ± 2 ab 1 13.8 ± 1.1 bcd 24.5 ± 2.7 bcde 10 ± 1 ab 2 13.4 ± 1.1 bcd 22.8 ± 1.8 cdefg 10 ± 1 ab 100 0 11.9 ± 1.3 d 20.6 ± 1.8 fgh 10 ± 1 ab 0.5 12.9 ± 1.4 cd 22.4 ± 2.6 defg 9 ± 1 ab 1 14.0 ± 1.1 abc 25.1 ± 2.0 bcde 10 ± 0 a 2 13.5 ± 0.9 bcd 23.4 ± 2.5 bcdefg 9 ± 1 ab 500 0 7.4 ± 0.7 ef 17.8 ± 2.2 h 9 ± 1 ab 0.5 7.3 ± 1.1 efg 17.8 ± 2.2 h 9 ± 1 ab 1 9.1 ± 1.2 e 24.5 ± 1.7 bcde 8 ± 1 bc 2 7.8 ± 1.3 ef 20.3 ± 2.2 gh 9 ± 0 ab 1000 0 6.4 ± 1.1 fg 11.1 ± 1.3 i 6 ± 0 c 0.5 6.6 ± 1.0 fg 10.7 ± 1.7 i 8 ± 1 bc 1 7.4 ± 0.9 ef 12.1 ± 1.2 i 9 ± 1 ab 2 5.4 ± 1.1 g 10.6 ± 1.5 i 8 ± 1 abc Table 1 . Shoot and root length of maize seedlings and percentage emergence of plants grown in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are presented as means of 10 replicates ± standard deviation. Values within columns followed by the same letter do not differ significantly. Application of EMIMCl resulted in a clear, concentration-dependent inhibition of maize seedling growth, affecting both shoots and roots. The presence of AsA in the soil promoted faster shoot and root development compared with plants grown without AsA supplementation. However, AsA did not fully prevent the negative effects of the ionic liquid. Moreover, the addition of 2 mM AsA to soil containing 1000 mg·kg⁻¹ of soil DW EMIMCl slightly intensified growth inhibition of both shoots and roots (Table 1 ). Proper germination and seedling establishment strongly influenced subsequent plant morphology and root system development (Suppl. Figures. 1 and 2). The photographic documentation shows that EMIMCl inhibited plant and root growth in a dose-dependent manner. Furthermore, exposure to 500 and 1000 mg·kg⁻¹ of soil DW EMIMCl induced chlorotic symptoms in maize leaves. When AsA was applied to soils containing the highest EMIMCl concentrations, chlorosis was more pronounced compared with plants exposed to EMIMCl alone. These findings were reflected in fresh biomass production. The presence of EMIMCl at 1–50 mg·kg⁻¹ of soil DW did not significantly affect maize fresh weight. Higher concentrations led to progressively stronger biomass reduction. In control soil, supplementation with 0.5 and 1 mM AsA visibly increased fresh biomass yield. Similarly, in EMIMCl-contaminated soil, AsA application improved fresh weight compared with plants grown in the presence of EMIMCl alone. Importantly, following AsA addition, a significant reduction in fresh biomass was observed only from 500 mg·kg⁻¹ of soil DW EMIMCl onwards. The extent of AsA-mediated mitigation of EMIMCl toxicity depended on the applied AsA concentration. Under the highest EMIMCl level (1000 mg·kg⁻¹ of soil DW), a beneficial effect on fresh biomass was observed exclusively at 1 mM AsA (Table 2 ). Table 2 Fresh biomass yield and dry weight content of maize seedlings cultivated in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are expressed as means of four replicates ± standard deviation. Values within columns marked with the same letter are not significantly different. Concentration ILs [mg·kg − 1 of soil DW] Concentration AsA [mM] Yield [g·pot − 1 ] Dry weight [g·g −1 FW] 0 (control) 0 2.837 ± 0.281 bcd 0.0993 ± 0.0028 g 0.5 3.330 ± 0.269 ab 0.0965 ± 0.0031 g 1 3.184 ± 0.208 abc 0.1018 ± 0.0014 fg 2 2.862 ± 0.322 bcd 0.1027 ± 0.0005 fg 1 0 3.111 ± 0.133 abc 0.1007 ± 0.0026 fg 0.5 3.498 ± 0.079 a 0.0990 ± 0.0030 g 1 3.171 ± 0.231 abc 0.1008 ± 0.0020 fg 2 3.137 ± 0.335 abc 0.1022 ± 0.0041 fg 50 0 2.622 ± 0.107 cde 0.0986 ± 0.0010 g 0.5 3.125 ± 0.133 abc 0.0988 ± 0.0016 g 1 2.762 ± 0.185 bcd 0.0980 ± 0.0026 g 2 2.649 ± 0.388 cd 0.0987 ± 0.0021 g 100 0 2.058 ± 0.054 efg 0.0979 ± 0.0008 g 0.5 2.339 ± 0.176 def 0.0999 ± 0.0017 g 1 2.807 ± 0.006 bcd 0.0995 ± 0.0005 g 2 2.707 ± 0.053 bcd 0.0983 ± 0.0029 g 500 0 1.474 ± 0.004 hi 0.1157 ± 0.0050 cde 0.5 1.438 ± 0.063 hi 0.1171 ± 0.0020 bcd 1 1.997 ± 0.045 fgh 0.1082 ± 0.0014 ef 2 1.592 ± 0.147 ghi 0.1134 ± 0.0015 de 1000 0 1.078 ± 0.076 i 0.1254 ± 0.0025 a 0.5 1.127 ± 0.013 i 0.1214 ± 0.0012 abc 1 1.608 ± 0.111 ghi 0.1223 ± 0.0026 abc 2 1.041 ± 0.143 i 0.1242 ± 0.0040 ab Table 2 . Fresh biomass yield and dry weight content of maize seedlings cultivated in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are expressed as means of four replicates ± standard deviation. Values within columns marked with the same letter are not significantly different. The presence of the ionic liquid also affected dry weight accumulation in maize seedlings. Exposure to 500 and 1000 mg·kg⁻¹ of soil DW EMIMCl significantly increased dry weight content. The addition of AsA did not significantly alter dry weight levels compared with plants exposed to EMIMCl without antioxidant supplementation (Table 2 ). Photosynthetic pigment content and chlorophyll fluorescence The concentrations of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids), as well as the ratios of chlorophyll a to chlorophyll b (chl a/chl b) and total chlorophyll to carotenoids ((chl a + chl b)/car), are presented in Fig. 2 . Figure 2 . Photosynthetic pigment content in maize seedlings grown in soil contaminated with different concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Values represent means of four replicates ± standard deviation. Asterisks indicate significant differences compared with the respective control at the same AsA concentration ( p < 0.05). Exposure of maize seedlings to EMIMCl resulted in a significant reduction in all analyzed photosynthetic pigments. The magnitude of this decline increased with rising concentrations of the ionic liquid. In soil not contaminated with EMIMCl, AsA supplementation led to a slight decrease in pigment content. However, when 0.5 mM AsA was applied to soil containing 1–100 mg·kg⁻¹ of soil DW EMIMCl, pigment levels were higher than in plants grown without antioxidant supplementation. At higher EMIMCl concentrations (500 and 1000 mg·kg⁻¹ of soil DW), beneficial effects on pigment accumulation were observed following the application of 0.5 and 1 mM AsA. In contrast, 2 mM AsA consistently reduced pigment content relative to plants grown without AsA. When high EMIMCl levels were combined with 2 mM AsA, an increase in the chl a/chl b ratio and in the total chlorophyll-to-carotenoid ratio was observed. Chlorophyll fluorescence parameters were also evaluated, and the results are summarized in Table 3 . Table 3 Chlorophyll fluorescence parameters in maize seedlings cultivated in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are expressed as means of four replicates ± standard deviation. Values within columns followed by the same letter do not differ significantly. Concentration ILs [mg·kg − 1 of soil DW] Concentration AsA [mM] F 0 F m F v F v /F m F v /F 0 0 (control) 0 199 ± 4 e 970 ± 40 bc 771 ± 38 a 0.794 ± 0.008 ab 3.869 ± 0.170 a 0.5 214 ± 26 e 938 ± 37 cd 725 ± 34 a 0.772 ± 0.024 abc 3.431 ± 0.434 ab 1 219 ± 20 de 1025 ± 42 abc 819 ± 82 a 0.797 ± 0.049 a 3.732 ± 0.126 a 2 201 ± 5 e 989 ± 27 abc 788 ± 26 a 0.796 ± 0.007 ab 3.915 ± 0.153 a 1 0 202 ± 4 e 988 ± 39 abc 786 ± 37 a 0.795 ± 0.007 ab 3.892 ± 0.153 a 0.5 204 ± 5 e 994 ± 25 abc 790 ± 22 a 0.795 ± 0.005 ab 3.884 ± 0.101 a 1 207 ± 12 e 982 ± 44 abc 774 ± 33 a 0.788 ± 0.005 ab 3.739 ± 0.116 a 2 200 ± 11 e 965 ± 70 bcd 764 ± 60 a 0.792 ± 0.007 ab 3.813 ± 0.159 a 50 0 200 ± 5 e 980 ± 30 abc 780 ± 25 a 0.796 ± 0.003 ab 3.903 ± 0.068 ga 0.5 198 ± 18 e 969 ± 60 bc 756 ± 95 a 0.778 ± 0.055 abc 3.808 ± 0.215 a 1 202 ± 5 e 969 ± 55 bc 767 ± 53 a 0.791 ± 0.010 ab 3.805 ± 0.223 a 2 213 ± 13 e 982 ± 34 abc 769 ± 36 a 0.783 ± 0.014 abc 3.624 ± 0.298 a 100 0 211 ± 3 e 1014 ± 22 abc 803 ± 21 a 0.791 ± 0.005 ab 3.807 ± 0.103 a 0.5 206 ± 15 e 953 ± 51 abc 747 ± 60 a 0.782 ± 0.025 abc 3.646 ± 0.487 a 1 210 ± 3 e 1004 ± 28 abc 794 ± 28 a 0.790 ± 0.006 ab 3.787 ± 0.136 a 2 209 ± 7 e 995 ± 51 abc 786 ± 45 a 0.789 ± 0.006 ab 3.752 ± 0.130 a 500 0 325 ± 58 c 1095 ± 65 ab 772 ± 79 a 0.705 ± 0.052 abc 2.473 ± 0.633 c 0.5 312 ± 50 cd 1121 ± 71 a 809 ± 42 a 0.722 ± 0.033 abc 2.648 ± 0.492 bc 1 327 ± 64 c 1036 ± 40 abc 709 ± 68 a 0.686 ± 0.082 abc 2.262 ± 0.609 c 2 350 ± 80 c 1056 ± 85 abc 705 ± 58 a 0.673 ± 0.095 c 2.117 ± 0.618 c 1000 0 315 ± 59 cd 1078 ± 78 abc 763 ± 47 a 0.709 ± 0.040 abc 2.490 ± 0.545 c 0.5 328 ± 59 c 1043 ± 44 abc 715 ± 63 a 0.686 ± 0.055 bc 2.249 ± 0.533 c 1 679 ± 61 a 1062 ± 90 abc 383 ± 55 b 0.311 ± 0.087 d 0.503 ± 0.195 d 2 568 ± 43 b 819 ± 92 d 252 ± 66 b 0.304 ± 0.047 d 0.496 ± 0.151 d Table 3 . Chlorophyll fluorescence parameters in maize seedlings cultivated in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are expressed as means of four replicates ± standard deviation. Values within columns followed by the same letter do not differ significantly. The presence of EMIMCl at 500 and 1000 mg·kg⁻¹ of soil DW significantly increased minimal fluorescence (F₀) and maximal fluorescence (Fₘ). Simultaneously, the ratios F v /Fₘ and F v /F₀ were reduced, indicating impairment of photosystem II efficiency. In soils containing 1–500 mg·kg⁻¹ of soil DW EMIMCl, AsA supplementation did not significantly affect fluorescence parameters. Marked disturbances were observed only when AsA was applied to soil containing 1000 mg·kg⁻¹ of soil DW EMIMCl. Under these conditions, 1 and 2 mM AsA nearly doubled F₀ values, caused approximately a twofold decrease in variable fluorescence (F v ) and in the F v /Fₘ ratio, and led to a several-fold reduction in F v /F₀. Moreover, the addition of 2 mM AsA to soil with 1000 mg·kg⁻¹ of soil DW EMIMCl significantly decreased maximal fluorescence (Fₘ). MDA, H₂O₂, and AsA The levels of H₂O₂, MDA, and AsA in maize seedlings are presented in Fig. 3 . Cultivation of maize in soil contaminated with EMIMCl resulted in elevated concentrations of H₂O₂ and MDA, indicating enhanced oxidative stress. In addition, a slight initial decrease in endogenous AsA content relative to the control was observed, followed by an increase after exposure to the tested ionic liquid. Exogenous application of AsA differentially affected the concentrations of H₂O₂, MDA, and AsA in maize seedlings, depending on the antioxidant dose. Only the 1 mM AsA treatment led to a slight reduction in H₂O₂ content. Both 1 and 2 mM AsA caused a modest decrease in MDA levels. Furthermore, supplementation with 1 and 2 mM AsA slightly increased endogenous AsA concentration compared with plants grown in EMIMCl-contaminated soil without antioxidant addition. In contrast, 0.5 mM AsA did not significantly influence any of the analyzed oxidative stress markers. Figure 3 . Concentrations of MDA, H₂O₂, and AsA in maize seedlings cultivated in soil contaminated with various levels of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are expressed as means of four replicates ± standard deviation. Asterisks indicate significant differences compared with the respective control at the same AsA concentration ( p < 0.05). Multivariate analysis of physiological and biochemical responses of maize seedlings to EMIMCl stress To integrate the results related to growth, photosynthetic performance, and oxidative stress in maize seedlings exposed to EMIMCl and exogenous AsA, principal component analysis (PCA) was performed. This approach enabled simultaneous evaluation of relationships among the measured parameters and identification of the main factors differentiating plant responses to chemical stress. The results are presented in Fig. 4 . Figure 4 . Principal component analysis (PCA) of physiological and biochemical responses of maize seedlings exposed to EMIMCl and treated with AsA. (a) Score plot showing sample distribution along PC1 and PC2; different point shapes indicate AsA concentrations, and ellipses represent 95% confidence intervals for EMIMCl treatments. (b) Loading plot illustrating relationships among the measured variables. (c) Heatmap of PCA loadings showing the contribution of individual variables to PC1 and PC2. The score plot (Fig. 4 a) demonstrates a clear separation of samples along the PC1 axis, which corresponded to the gradient of EMIMCl concentration in the soil. Control samples and those treated with low EMIMCl doses were positioned on the negative side of PC1, whereas samples exposed to higher concentrations (500–1000 mg·kg⁻¹ of soil DW) clustered on the positive side of this axis. The loading plot (Fig. 4 b) revealed positive loadings of H₂O₂, MDA, AsA, and dry weight content along PC1, while growth- and photosynthesis-related parameters—including biomass yield, germination rate, and photosynthetic pigment content—were negatively associated with this component. The PCA loading heatmap (Fig. 4 c) confirmed the patterns observed in both the score and loading plots, supporting the consistency of the multivariate analysis. Discussion Effect of EMIMCl on the growth and development of maize seedlings Seedling emergence, shoot and root length, and fresh biomass yield are among the most straightforward indicators of plant growth. A noticeable decline in any of these parameters is widely recognized as clear evidence that plants have been subjected to stress, whether abiotic or biotic (Chen et al. 2018). Seed germination represents a critical stage in the plant life cycle, and ionic liquids (ILs) may influence this process in different ways. Depending on their chemical structure, ILs can reduce or completely inhibit seed germination—particularly at higher concentrations (Chen et al. 2024)—have no measurable effect (Pawłowska et al. 2023), or even stimulate germination when applied at low doses (Chu et al. 2021). During germination, seeds imbibe water along with dissolved substances present in the soil solution. While some of these compounds are essential for plant development, others, such as heavy metals, may enter the seed and interfere with metabolic processes, thereby affecting germination and seedling establishment. Moreover, ILs containing chloride, tetrafluoroborate, or hexafluorophosphate anions may release HCl or HF through hydrolysis, leading to alterations in soil pH. Soil pH values that are unsuitable for a given plant species can negatively influence germination dynamics (Cvjetko Bubalo et al. 2014a,b; Bagheri et al. 2017; Chu et al. 2021). Regardless of the direction of the effect, the magnitude of IL impact on germination and emergence is closely related to the applied concentration. Low doses often exert negligible or even stimulatory effects, whereas higher concentrations frequently suppress germination and reduce seedling emergence (Chu et al. 2021; Chen et al. 2024; Pawłowska et al. 2023). In the present study, more pronounced effects were observed in shoot and root elongation than in seedling emergence. The results demonstrate that EMIMCl negatively affects maize growth in a concentration-dependent manner. Only the lowest tested concentration (1 mg·kg⁻¹ of soil DW) stimulated seedling growth and increased fresh biomass yield. Previous reports (Li et al. 2022; Liu et al. 2015a) suggest that low IL concentrations may promote plant growth, whereas higher levels can damage root cell membranes. Such damage may facilitate the penetration of toxic substances into root tissues and their subsequent translocation to aerial plant parts. Impairment of root development compromises water and nutrient uptake, ultimately affecting overall plant performance (Chapman et al. 2012; Xu et al. 2018). Habibul et al. (2020) reported that imidazolium-based ILs tend to accumulate predominantly in roots, although they may also be transported to stems and leaves. The extent of accumulation depends on both the applied concentration and the alkyl chain length of the ILs. Disturbances in root function and reduced root growth can lead to insufficient water and nutrient absorption. Consequently, decreased cellular turgor may occur, resulting in an increased proportion of dry weight in plant tissues. Elevated dry weight content is often interpreted as an indicator of impaired water balance, a common response to chemical and osmotic stress (Chen et al. 2018; Pawłowska et al. 2019; Biczak et al. 2020). Alterations in photosynthetic pigment content and chlorophyll fluorescence in maize seedlings exposed to EMIMCl Adequate levels of photosynthetic pigments are essential for proper plant functioning, as these compounds are directly involved in photosynthesis, a process fundamental to plant survival. In the present study, a reduction in chlorophyll content was already apparent during visual assessment of the plants, as higher EMIMCl concentrations induced chlorotic symptoms on the leaves. These observations were subsequently confirmed by quantitative measurements of individual photosynthetic pigments. Previous studies have similarly reported adverse effects of ionic liquids on pigment content in higher plants and algae (Li et al. 2018; Liu et al. 2015b; Xia et al. 2018). Imidazolium-based ionic liquids may disrupt the lipid bilayer structure and impair chloroplast membrane integrity. Exposure to ILs can also promote excessive production of reactive oxygen species (ROS), which damage cellular membranes and thylakoid structures within chloroplasts. Chloroplast impairment may lead to leakage of chlorophyll, while the combined action of ILs and ROS may facilitate penetration into internal chloroplast compartments, further disturbing chlorophyll biosynthesis and photosynthetic machinery organization (Chen et al 2018; Deng et al. 2017, Reddy et al. 2017; Li et al. 2018; Liu et al. 2018a; Liu et al. 2018b). In addition to pigment concentration, chlorophyll fluorescence is considered a sensitive indicator of oxidative and photosynthetic stress. The light-dependent reactions of photosynthesis are initiated in photosystem II (PSII). An increase in minimal fluorescence (F₀) may reflect either reversible or irreversible inactivation of PSII or structural damage to thylakoid membranes. A reduction in variable fluorescence (F v ) indicates decreased PSII efficiency. The ratios F v /Fₘ and F v /F₀ provide information about the functional state of the PSII reaction center. Alterations in these parameters may result from EMIMCl-induced disturbances in the PSII electron transport chain or in the primary electron acceptor. A decline in F v /Fₘ is commonly interpreted as evidence of PSII reaction center damage. Such changes suggest the occurrence of photoinhibition under stress conditions, often accompanied by increased energy dissipation as heat and enhanced photodamage to the photosynthetic apparatus (Liu et al. 2015a; Li et al. 2018; Gao et al. 2016; Chen et al. 2019). Effect of EMIMCl on oxidative stress in maize seedlings Hydrogen peroxide (H₂O₂), classified as a reactive oxygen species (ROS), plays a dual role in plants. Under physiological balance, it functions as a signaling molecule involved in stress perception and response. However, a rapid increase in H₂O₂ concentration is a clear indicator of oxidative stress. Such accumulation may result from enhanced superoxide dismutase (SOD) activity and intensified dismutation of superoxide radicals, or it may reflect a situation in which the stress level exceeds the detoxification capacity of the antioxidant defense system (Sánchez-Rodríguez et al. 2010; Kumar et al. 2013; Demidchik et al. 2015; Di Baccio et al. 2017). The elevated H₂O₂ levels observed in maize seedlings cultivated in soil containing EMIMCl demonstrate that this ionic liquid induces oxidative stress in maize. Moreover, the magnitude of this effect increased with rising EMIMCl concentrations. Comparable increases in H₂O₂ content following exposure to ionic liquids have been reported by Zhang et al. (2013) in duckweed treated with C₈MIMBr, by Xu et al. (2018) in wheat exposed to three C₈MIM ionic liquids with different anions, and by Cvjetko Bubalo et al. (2014b) in barley seedlings subjected to four imidazolium-based ionic liquids. Reactive oxygen species, including H₂O₂, are capable of damaging proteins and DNA. They can also attack polyunsaturated fatty acids, initiating lipid peroxidation, with malondialdehyde (MDA) formed as one of its end products. MDA can subsequently interact with functional groups in proteins, lipoproteins, and nucleic acids, leading to cellular injury. Disruption of membrane integrity may have serious physiological consequences for plants (Zhang et al. 2013; Xu et al. 2018). The increased MDA content detected in maize seedlings exposed to EMIMCl further confirms the occurrence of oxidative stress. As with H₂O₂, the intensity of these changes was positively correlated with the applied concentration of the ionic liquid. Similar detrimental effects of ionic liquids on crop species have been documented for barley (Cvjetko Bubalo et al. 2014b), wheat (Liu et al. 2014; Liu et al. 2016), and rice (Liu et al. 2015a). Role of exogenous ascorbic acid in alleviating EMIMCl-induced stress Application of L-ascorbic acid (AsA) at low and moderate concentrations partially mitigated the adverse effects caused by EMIMCl. This protective response was reflected in improved plant growth, higher levels of photosynthetic pigments, and reduced accumulation of H₂O₂ and MDA. These findings indicate that AsA enhanced the antioxidant capacity of maize, thereby limiting oxidative damage. Exogenous supplementation with L-ascorbic acid clearly attenuated the inhibitory effect of the ionic liquid on seedling emergence as well as shoot and root growth. AsA plays a central role in maintaining both extracellular and intracellular redox balance, which directly influences multiple signaling pathways, including those associated with abscisic acid (ABA), auxin, and reactive oxygen species (ROS). Properly regulated redox homeostasis and signaling networks enable plants to respond rapidly and effectively to environmental disturbances, thereby protecting cells against abiotic stress (Xiao et al. 2021). The growth-promoting effect of AsA has also been documented in maize exposed to cadmium (Xiao et al. 2021), wheat subjected to lead stress (Alamri et al. 2018), rapeseed under drought conditions (Shafiq et al. 2014), peach trees experiencing water deficit (Panella et al. 2017), and wheat exposed to salinity (Ishaq et al. 2021). The beneficial impact of AsA on plant development may result from stimulated synthesis of amino acids, proteins, and photosynthetic pigments. Moreover, AsA regulates cell division, differentiation, and senescence, and by strengthening antioxidant defenses, it protects lipids and proteins from oxidative injury (Xiao et al. 2021; Zhang et al. 2019; Chen et al. 2021; Zong et al. 2023; Li et al. 2025; Wang et al. 2024). Ascorbic acid is one of the principal non-enzymatic antioxidants in plant cells. It can directly neutralize hydroxyl radicals and superoxide anions and serves as a cofactor for enzymes involved in ROS detoxification (Liu et al. 2015b; Li et al. 2018). Approximately 90% of cellular AsA is localized in the cytoplasm, and owing to its strong reducing properties, it is regarded as one of the most effective antioxidant molecules. In cooperation with vitamin E, ascorbate participates in the quenching of excited or intermediate reactive oxygen species, either directly or through enzyme-mediated reactions. In addition, together with glutathione, it forms the core of the ascorbate–glutathione cycle, a crucial system responsible for maintaining cellular redox balance and regulating ROS levels, thereby influencing plant growth and development (Noctor and Foyer 1998; Asada 1999; Shao et al. 2008; Akram et al. 2017). External application of AsA—via seed priming, foliar spraying, or soil supplementation—leads to an increase in endogenous ascorbate content, which can positively modulate antioxidant metabolism in plants (Akram et al. 2017). In the present study, maize seedlings grown in EMIMCl-contaminated soil exhibited a marked rise in AsA levels. This increase may represent a stress-induced response, as oxidative stress triggered by the ionic liquid likely stimulated the synthesis and accumulation of low-molecular-weight antioxidants, including ascorbate (Kumari et al. 2020). Under abiotic stress conditions, plants are known to upregulate genes encoding enzymes of the AsA biosynthetic pathway as well as those involved in the ascorbate–glutathione cycle (Xiao et al. 2021). Furthermore, membrane damage—indicated by elevated MDA content—activates defense mechanisms that may also contribute to increased AsA accumulation (Kumari et al. 2020). The effectiveness of exogenously applied AsA in reducing oxidative stress in maize seedlings was concentration-dependent. This observation is consistent with findings by Khazaei et al. (2020), who demonstrated that acetylsalicylic acid applied at optimal concentrations enhanced drought tolerance in pepper ( Capsicum annuum L.), and by Hassan et al. (2021), who reported similar concentration-dependent protective effects of AsA against drought stress in barley ( Hordeum vulgare L.). Biphasic effects of AsA under severe EMIMCl-induced stress One of the key and novel findings of this study is the demonstration that the action of exogenous L-ascorbic acid under EMIMCl-induced stress follows a biphasic pattern and is strongly dependent on both its concentration and the severity of chemical stress. Low and moderate doses of AsA exerted a protective effect, whereas higher concentrations—particularly when combined with elevated levels of EMIMCl in the soil—not only failed to alleviate stress symptoms but markedly intensified them. This detrimental response was evidenced by a further increase in oxidative stress markers (H₂O₂ and MDA), along with deterioration of growth parameters and impairment of photosynthetic performance. The dual nature of AsA activity is supported by numerous reports indicating that, despite its central role as an antioxidant, ascorbate may under certain conditions display pro-oxidative properties. AsA is a pivotal component of the plant redox network and functions within the ascorbate–glutathione cycle, where it modulates ROS levels and maintains cellular redox balance (Foyer and Noctor 2011; Akram et al. 2017). However, under intense stress conditions that overwhelm ROS-detoxifying systems, this balance may become disrupted. At elevated concentrations, AsA can promote ROS generation through redox reactions, particularly in the presence of transition metal ions such as Fe²⁺ and Cu⁺, which catalyze Fenton-type reactions. In such circumstances, ascorbate may shift from acting solely as a ROS scavenger to facilitating the formation of highly reactive hydroxyl radicals, thereby enhancing lipid peroxidation and cellular damage (Smirnoff 2018; Halliwell and Gutteridge 2015). This mechanism is especially relevant in plant cells exposed to xenobiotics that already compromise membrane integrity and organelle function. Imidazolium-based ionic liquids have been shown to interact with biological membranes and proteins containing thiol groups, leading to disturbances in mitochondrial and chloroplast function and consequently to increased ROS production (Pham et al. 2010; Cvjetko Bubalo et al. 2017). Under such conditions, the application of high doses of AsA may aggravate redox imbalance rather than counteract it. This explains the intensified oxidative stress symptoms observed in maize seedlings simultaneously exposed to high concentrations of EMIMCl and elevated levels of exogenous AsA in the present study. Comparable pro-oxidative effects of externally supplied antioxidants have been reported in studies involving heavy metal toxicity, herbicide exposure, and salinity stress, where excessive AsA application resulted in worsened physiological performance instead of improvement (Gill and Tuteja 2010; Anjum et al. 2014). These findings highlight that antioxidant-based mitigation strategies are not universally beneficial and must be carefully tailored to the type and intensity of stress encountered. Multivariate analysis of the physiological and biochemical responses of maize seedlings to EMIMCl stress Principal component analysis (PCA) demonstrated that EMIMCl was the dominant factor shaping the physiological response of maize seedlings. This was reflected in the strong association between oxidative stress indicators and the positive values of PC1. In contrast, growth-related traits and photosynthetic parameters were positioned oppositely along this axis, indicating a close relationship between oxidative stress induction and suppression of photosynthetic efficiency. The distribution of samples along PC2 suggests a modulatory influence of exogenously applied AsA; however, this effect was clearly concentration-dependent. Under conditions of high EMIMCl contamination, elevated AsA levels were positively correlated with increased stress markers, supporting its potential pro-oxidative role under disrupted redox homeostasis, as reported previously (Smirnoff 2018; Chen et al. 2021). These findings clearly indicate that the external application of AsA as a strategy to alleviate stress caused by ionic liquid contamination, such as EMIMCl, should be approached with caution. The biphasic nature of AsA action suggests that uncontrolled antioxidant supplementation may produce effects opposite to those intended, leading to enhanced oxidative damage and physiological dysfunction. Such disturbances may result in inhibited plant growth, yield reduction, and in extreme cases, plant death. The dual response pattern of AsA is of considerable importance both for understanding plant tolerance mechanisms to xenobiotics and for environmental risk assessment and the development of mitigation strategies in agroecosystems exposed to chemical stress. Conclusion In summary, exogenous AsA may attenuate oxidative damage in plants, at least partially restoring antioxidant balance through direct ROS scavenging and stimulation of antioxidant enzymes. However, the effectiveness of AsA in mitigating stress induced by ionic liquids depends on both the applied AsA concentration and the level of IL contamination. When IL exposure severely disrupts cellular function, exogenous AsA may exhibit limited protective capacity, and an improperly selected dose may further intensify oxidative stress rather than alleviate it. Given the scarcity of studies addressing the use of exogenous AsA to counteract IL-induced oxidative stress in plants, further comprehensive research in this area is warranted. Declarations Funding Sources This research was funded by Polish Ministry of Education and Science for The Faculty of Science and Technology of Jan Dlugosz University in Czestochowa (SBR/WNSPT/KBBE/18/2023). Author Contributions B.P. – Conceptualization, methodology, software, formal analysis, validation, investigation, resources, data curation, visualization, supervision, project administration, writing—original draft preparation, A.L: investigation; R.S.: writing—review and editing; R.B.: funding acquisition, validation, data curation, writing—review and editing. All authors have read and agreed to the published version of the manuscript. Data availability Data will be made available on request. Ethics approval This is not applicable. Consent to participate This is not applicable. Consent for publication This is not applicable. Declaration of Competing Interest The authors declare that there are no conflicts of interest in the present experiment. Acknowledgements This is not applicable. References Akram NA, Shafiq F, Ashraf M (2017) Ascorbic acid—A potential oxidant scavenger and its role in plant development and abiotic stress tolerance. Frontiers in Plant Science 8:613. Alamri SA, Siddiqui MH, Al-Khaishany MYY, Khan MN, Ali HM, Alaraidh IA, Alsahli AA, Al-Rabiah H, Mateen M (2018) Ascorbic acid improves the tolerance of wheat plants to lead toxicity. Journal of Plant Interactions 13:409–419. 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Zheng G, Webster TF, Salamova A (2021) Quaternary ammonium compounds: Bioaccumulation potentials in humans and levels in blood before and during the COVID-19 pandemic. Environmental Science & Technology 55:14689–14698. Zong D, Liu H, Gan P, Ma S, Liang H, Yu J, Li P, Jiang T, Sahu SK, Yang Q, Zhang D, Li L, Qiu X, Shao W, Yang J, Li Y, Guang X, He C (2023) Chromosomal-scale genomes of two species provide insights into genome evolution and ascorbate accumulation. The Plant Journal 117:1264–1280. Supplementary Files Supplementarymaterials.pdf Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-8938488","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":600612381,"identity":"f50dbe20-bd08-4789-97d1-20016456a1a3","order_by":0,"name":"Barbara Pawłowska","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYBACAzBZAMQ8DIwPGHiYgawEqAheLQZgLcwGCC0GxGlhk2BgIEKLOQPvwc88BjaJ83sOP6vmkbFm4GfPMWAuwKPFsoEvWZrHIC1xw9k2s9s8POkMkj1vDJhn4HPYAR4D6RyDw7kb+BlAWg4zGNwA2sKDX4vxb5CW+f3s34pBWuyJ0GIGtqXhbI8ZM9gWCQJaLJv50qz/GKTVbzhzplhyDk86j8SZZwWH8fnFnL338M0ZFTbG8j3pGz+87bGW429P3vi4oAK3FgZmHiQOYw8DmHsYjwYGBgZkLQw/oObg1zIKRsEoGAUjDAAAX99GtFOoYxMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5314-920X","institution":"Jan Dlugosz University in Czestochowa Faculty of Science and Technology: Uniwersytet Humanistyczno-Przyrodniczy im Jana Dlugosza w Czestochowie Wydzial Nauk Scislych Przyrodniczych i Technicznych","correspondingAuthor":true,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Pawłowska","suffix":""},{"id":600612382,"identity":"ce636170-cf6a-4006-8be3-e8c5f95a2fd8","order_by":1,"name":"Aleksandra Lechowska","email":"","orcid":"","institution":"Jan Dlugosz University in Czestochowa Faculty of Science and Technology: Uniwersytet Humanistyczno-Przyrodniczy im Jana Dlugosza w Czestochowie Wydzial Nauk Scislych Przyrodniczych i Technicznych","correspondingAuthor":false,"prefix":"","firstName":"Aleksandra","middleName":"","lastName":"Lechowska","suffix":""},{"id":600612383,"identity":"af98750c-7c67-4396-bdeb-7cab673466e7","order_by":2,"name":"Radomír Ščurek","email":"","orcid":"","institution":"VŠB-Technical University of Ostrava Faculty of Safety Engineering: Vysoka Skola Banska-Technicka Univerzita Ostrava Fakulta Bezpecnostniho Inzenyrstvi","correspondingAuthor":false,"prefix":"","firstName":"Radomír","middleName":"","lastName":"Ščurek","suffix":""},{"id":600612384,"identity":"4e72b2b5-2737-4de7-a5fc-2eaed98b86f5","order_by":3,"name":"Robert Biczak","email":"","orcid":"","institution":"Jan Dlugosz University in Czestochowa Faculty of Science and Technology: Uniwersytet Humanistyczno-Przyrodniczy im Jana Dlugosza w Czestochowie Wydzial Nauk Scislych Przyrodniczych i Technicznych","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Biczak","suffix":""}],"badges":[],"createdAt":"2026-02-22 10:27:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8938488/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8938488/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104222407,"identity":"438ba98b-9b01-4dc1-9879-9c867c9c2c47","added_by":"auto","created_at":"2026-03-09 10:34:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":844988,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8938488/v1/97b9a36deb0089495c6af706.png"},{"id":104222405,"identity":"76a0f56d-2ea9-4f64-8b55-f754a6578a73","added_by":"auto","created_at":"2026-03-09 10:34:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":941515,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8938488/v1/fa6771b1631133468256b4e1.png"},{"id":104404656,"identity":"11332b7c-40d9-488c-b08b-3b65ef6ba4ec","added_by":"auto","created_at":"2026-03-11 12:20:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":581471,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8938488/v1/ebd50cc4c19a2bf66ccbd711.png"},{"id":104405270,"identity":"db67d093-9b80-4783-83bc-ed6d5a91faf0","added_by":"auto","created_at":"2026-03-11 12:22:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112384,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8938488/v1/784b07c1847d55a487a3d080.png"},{"id":108804145,"identity":"6c2b8830-03f6-4cd4-b54c-cfe760529a0b","added_by":"auto","created_at":"2026-05-08 15:16:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2793270,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8938488/v1/fc84e83e-a113-4a10-8899-bf8f70bb8d6c.pdf"},{"id":104222409,"identity":"4d13e2ba-64f9-42e8-8f4f-b6a681c0c5e0","added_by":"auto","created_at":"2026-03-09 10:34:08","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":628491,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8938488/v1/97c235523e45736e2910af47.pdf"}],"financialInterests":"","formattedTitle":"The effect of 1-ethyl-3-methylimidazolium chloride on oxidative stress and the functioning of the photosynthetic apparatus in maize seedlings – the modulatory role of exogenous ascorbic acid","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe rapid advancement of chemical technologies over recent decades has resulted in a substantial increase in both the production and application of ionic liquids (ILs). Ionic liquids constitute a vast and structurally diverse class of compounds. Owing to their distinctive physicochemical properties, these substances have been widely implemented across numerous industrial sectors. In particular, imidazolium-based ionic liquids, including 1-ethyl-3-methylimidazolium chloride (EMIMCl), have found applications in chemical synthesis, lignocellulosic biomass processing, and electrochemical technologies (Bae et al. 2024; Maculewicz et al. 2024; Rowan et al. 2025; Egorova and Ananikov 2018).\u003c/p\u003e \u003cp\u003eMarket forecasts indicate that the global ionic liquids market is expected to reach USD 7,266.2\u0026nbsp;million by 2030 (Industry ARC 2025). The continuous expansion of ILs production and their growing range of applications across various industries raise concerns regarding their potential environmental impact. An increasing body of literature suggests that, despite their advantageous technological properties, many ionic liquids exhibit significant biological activity, which may pose risks to living organisms following their release into the environment. Scientific investigations have confirmed that ionic liquids have already entered environmental compartments and have been detected, among others, in river waters. Due to their water solubility and limited susceptibility to biodegradation, imidazolium-based ILs may accumulate in soils and surface waters, thereby increasing the exposure risk for terrestrial organisms, including crop plants. Numerous studies report that ionic liquids can exert toxic effects on bacteria, fungi, algae, crustaceans, fish, and plants (Maculewicz et al. 2022; Cvjetko Bubalo et al. 2014a; Matzke et al. 2007; Cvjetko Bubalo et al. 2014b). Previous research has demonstrated that ILs, including EMIMCl, may inhibit seed germination, impair plant growth and development, and disrupt metabolic processes in various plant species, including agriculturally important crops. Moreover, ionic liquids may display strong binding affinity to human blood proteins, which could contribute to their bioaccumulation in the human body (Maculewicz et al. 2022; Zheng et al. 2021; Hrubec et al. 2021; Neuwald et al. 2021).\u003c/p\u003e \u003cp\u003eDespite numerous studies conducted to date, the precise mechanisms underlying the phytotoxic effects of ionic liquids have not yet been fully elucidated. Growing evidence suggests that disruption of biological membrane integrity and the induction of oxidative stress play central roles in their mode of action. Imidazolium cations are capable of interacting with membrane phospholipids as well as thiol-containing proteins, which may result in destabilization of cellular structures and impaired functioning of mitochondria and chloroplasts. These disturbances can lead to excessive generation of reactive oxygen species (ROS), triggering lipid peroxidation and damage to the photosynthetic apparatus (Pham et al. 2010; Cvjetko Bubalo et al. 2017).\u003c/p\u003e \u003cp\u003ePlants have evolved complex defense strategies to maintain redox homeostasis under environmental stress conditions. A key component of these protective systems is the antioxidant network, which includes both enzymatic antioxidants and low-molecular-weight reducing compounds. Ascorbic acid (AsA) plays a particularly important role within this system by directly scavenging ROS and regenerating other antioxidants through the ascorbate\u0026ndash;glutathione cycle. In addition, AsA functions as a cofactor for certain oxidases and participates in the biosynthesis of several phytohormones, including abscisic acid (ABA) and ethylene. It also contributes significantly to maintaining intracellular and extracellular redox balance in plants. Through these functions, AsA enables plants to respond rapidly and effectively to environmental fluctuations, thereby enhancing their tolerance to various abiotic stresses (Ding et al. 2020; Xiao et al. 2021; Wu et al. 2024; Zhang et al. 2019). Numerous studies have demonstrated that exogenous application of AsA may exert beneficial effects on plants exposed to drought, high salinity, or heavy metal stress (Zhang et al. 2019; Ali et al. 2018; Alves et al. 2022).\u003c/p\u003e \u003cp\u003eIt should be noted, however, that the effects of AsA are not invariably protective. Under conditions of severely disrupted redox balance or when applied at excessive concentrations, ascorbic acid may exhibit pro-oxidant properties. In particular, through participation in redox reactions\u0026mdash;especially in the presence of transition metal ions\u0026mdash;AsA can promote the overproduction of reactive oxygen species (ROS) (Smirnoff 2018). This phenomenon indicates that the effectiveness of exogenous antioxidants is strongly dependent on their concentration and on the intensity of environmental stress.\u003c/p\u003e \u003cp\u003eDespite the increasing number of studies addressing the toxicity of ionic liquids, comprehensive analyses integrating their simultaneous effects on plant growth, photosynthetic performance, and oxidative stress\u0026mdash;along with the potential modulation of these responses by exogenous antioxidants\u0026mdash;remain limited. In particular, there is a scarcity of data concerning the impact of EMIMCl on economically important crop species such as maize and the potential implications for agroecosystems.\u003c/p\u003e \u003cp\u003eMaize is the third most important cereal crop worldwide, following rice and wheat. Compared with these two cereals, however, maize has a broader range of applications. In addition to its role in human nutrition, it serves as a major component of livestock feed and is widely used in industrial and bioenergy production systems (Erenstein et al. 2022; Zhang et al. 2019).\u003c/p\u003e \u003cp\u003eThe objective of the present study was to evaluate the effects of 1-ethyl-3-methylimidazolium chloride (EMIMCl) on early growth and development, photosynthetic efficiency, and oxidative stress parameters in maize seedlings. Furthermore, the potential role of exogenously applied ascorbic acid in modulating plant responses to this chemical stressor was investigated. Particular emphasis was placed on dose\u0026ndash;response relationships and on the possibility of biphasic effects of AsA under progressively increasing EMIMCl-induced stress. To the best of our knowledge, this is the first study to examine the influence of ascorbic acid on the phytotoxicity of ionic liquids.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003e1-Ethyl-3-methylimidazolium chloride (EMIMCl; \u0026ge;95% purity) was obtained from Sigma-Aldrich Chemical Co. L-ascorbic acid (analytical grade) was purchased from Chempur. All other chemicals and reagents used for the respective analyses were of at least analytical reagent grade (\u0026ge;\u0026thinsp;AR grade).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003ePhytotoxicity assays were conducted under controlled greenhouse conditions. The study was performed in accordance with the guidelines outlined in OECD/OCDE (2006). Ten uniform maize (\u003cem\u003eZea mays\u003c/em\u003e L.) seeds of the cultivar \u0026lsquo;Rywal\u0026rsquo; were sown in plastic pots containing 250 g of control soil (without ionic liquids) or soil amended with EMIMCl at concentrations of 1, 50, 100, 500, and 1000 mg\u0026middot;kg⁻\u0026sup1; of soil dry weight (DW). Maize seeds were obtained from the Plant Breeding and Production Station in Nieznanice, belonging to Małopolska Hodowla Roślin \u0026ndash; HBP Sp. z o.o. The soil used in the experiment was classified as sandy loam, containing approximately 11% particles\u0026thinsp;\u0026lt;\u0026thinsp;0.02 mm, 8.5 g\u0026middot;kg⁻\u0026sup1; organic carbon, and a pH (KCl) of 6.0. EMIMCl was applied to the soil in the form of aqueous solutions and thoroughly homogenized prior to sowing. Immediately after seed sowing, the soil was irrigated either with distilled water (control treatment) or with L-ascorbic acid solutions at concentrations of 0.5, 1, and 2 mM. Throughout the experiment, growth conditions were maintained at constant levels: soil moisture was kept at 70% of water-holding capacity, temperature at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and light intensity at 170 \u0026micro;mol\u0026middot;m⁻\u0026sup2;\u0026middot;s⁻\u0026sup1; under a 16 h light/8 h dark photoperiod. All measurements were performed in four independent replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 14 days of growth, chlorophyll fluorescence was measured and plant morphology was visually assessed and documented using digital photography. Subsequently, the following parameters were determined: shoot and root length, fresh biomass yield, dry matter content, photosynthetic pigment concentration, and levels of H₂O₂, malondialdehyde (MDA), and ascorbic acid (AsA).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Experimental scheme for assessing EMIMCl phytotoxicity in maize under controlled conditions\u003c/p\u003e\n\u003ch3\u003eDetermination of basic phytotoxicity parameters\u003c/h3\u003e\n\u003cp\u003e Inhibition of shoot and root growth was evaluated according to the procedure described by Wang et al. (2009). Fresh biomass yield was also determined for each treatment.\u003c/p\u003e \u003cp\u003eDry wieght content was assessed following the method of Kowalska (2004). Approximately 1 g of leaf tissue was dried at 105\u0026deg;C until a constant weight was achieved.\u003c/p\u003e\n\u003ch3\u003eChlorophyll fluorescence\u003c/h3\u003e\n\u003cp\u003eChlorophyll fluorescence parameters were measured using an OS1p chlorophyll fluorometer (Opti-Sciences, Inc., Hudson, USA). The following parameters were recorded: initial (minimal) fluorescence (F₀), variable fluorescence (F\u003csub\u003ev\u003c/sub\u003e), maximal fluorescence (Fₘ), the maximum quantum efficiency of photosystem II (F\u003csub\u003ev\u003c/sub\u003e/Fₘ), and the more sensitive ratio F\u003csub\u003ev\u003c/sub\u003e/F₀.\u003c/p\u003e\n\u003ch3\u003eDetermination of photosynthetic pigments\u003c/h3\u003e\n\u003cp\u003eThe concentrations of photosynthetic pigments, including chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids, were determined according to the method proposed by Oren et al. (1993). Briefly, 200 mg of maize leaf tissue was homogenized in 80% acetone (4\u0026deg;C) and subsequently centrifuged. Pigment concentrations were quantified spectrophotometrically by measuring absorbance at 470, 647, and 664 nm, followed by calculation using the appropriate equations.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and ascorbate (AsA)\u003c/h2\u003e \u003cp\u003eFresh plant material (0.5 g) was homogenized in 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged. The resulting supernatant was used for the determination of MDA and H₂O₂ concentrations.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMDA assay.\u003c/em\u003e The reaction mixture consisted of the supernatant, 0.5% thiobarbituric acid (TBA) prepared in 20% TCA, and phosphate buffer (pH 7.6). Samples were incubated in a water bath at 95\u0026deg;C for 30 minutes and then rapidly cooled in an ice bath. After cooling, absorbance was measured at 532 and 600 nm, following the method described by Hodges et al. (1999).\u003c/p\u003e \u003cp\u003e \u003cem\u003eH₂O₂ assay.\u003c/em\u003e For hydrogen peroxide determination, the supernatant was mixed with 1 M potassium iodide (KI) and phosphate buffer (pH 7.6). The reaction mixture was incubated in the dark for 1 hour, after which absorbance was recorded at 390 nm according to Singh et al. (2007).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAsA assay\u003c/em\u003e. To determine ascorbate content, 0.5 g of fresh maize leaf tissue was homogenized in 10% TCA and centrifuged. An aliquot of 0.2 cm\u0026sup3; of the supernatant was combined with 2% TCA, 1 M H₃PO₄, 0.8% bipyridyl, and 0.15% FeCl₃. The prepared reaction mixture was incubated at 37\u0026deg;C for 60 minutes in darkness. Absorbance was subsequently measured at 525 nm, and AsA concentration was calculated using a standard calibration curve, as described by Law et al. (1983).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed using two-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s post hoc test, employing STATISTICA 13.3 software. Differences were considered statistically significant at \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05. All measurements were performed in at least four independent replicates (n\u0026thinsp;=\u0026thinsp;4). Results are presented as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003cp\u003ePrincipal component analysis (PCA) was performed using standardized data (z-score normalization). The analysis was based on the correlation matrix, and the first two principal components were used for graphical interpretation. PCA calculations and visualizations were carried out in the R statistical environment (R Foundation for Statistical Computing, Vienna, Austria).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhytotoxicity\u003c/h2\u003e \u003cp\u003eThe effect of different concentrations of 1-ethyl-3-methylimidazolium chloride (EMIMCl) and L-ascorbic acid (AsA) on maize emergence is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The presence of EMIMCl in the soil reduced seedling emergence only at the highest tested concentration (1000 mg\u0026middot;kg⁻\u0026sup1; of soil DW). In contrast, soil supplementation with AsA at all applied concentrations mitigated the adverse effect of the ionic liquid on seed germination capacity. Even at the highest EMIMCl level, exogenous AsA application restored emergence to values that did not differ significantly from the control treatment.\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\u003eShoot and root length of maize seedlings and percentage emergence of plants grown in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are presented as means of 10 replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Values within columns followed by the same letter do not differ significantly.\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=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration ILs [mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of soil DW]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration AsA [mM]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoot length [cm]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShoot length [cm]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNumber of plants that germinated\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0 (control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003csup\u003ebcde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003csup\u003ebcdef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003csup\u003ebcde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003csup\u003ebcde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003csup\u003ebcdefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003csup\u003eefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003csup\u003ebcde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003csup\u003ecdefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003csup\u003efgh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003csup\u003edefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003csup\u003ebcde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003csup\u003ebcdefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003eefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003csup\u003ebcde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003csup\u003egh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Shoot and root length of maize seedlings and percentage emergence of plants grown in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are presented as means of 10 replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Values within columns followed by the same letter do not differ significantly.\u003c/p\u003e \u003cp\u003eApplication of EMIMCl resulted in a clear, concentration-dependent inhibition of maize seedling growth, affecting both shoots and roots. The presence of AsA in the soil promoted faster shoot and root development compared with plants grown without AsA supplementation. However, AsA did not fully prevent the negative effects of the ionic liquid. Moreover, the addition of 2 mM AsA to soil containing 1000 mg\u0026middot;kg⁻\u0026sup1; of soil DW EMIMCl slightly intensified growth inhibition of both shoots and roots (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProper germination and seedling establishment strongly influenced subsequent plant morphology and root system development (Suppl. Figures. 1 and 2). The photographic documentation shows that EMIMCl inhibited plant and root growth in a dose-dependent manner. Furthermore, exposure to 500 and 1000 mg\u0026middot;kg⁻\u0026sup1; of soil DW EMIMCl induced chlorotic symptoms in maize leaves. When AsA was applied to soils containing the highest EMIMCl concentrations, chlorosis was more pronounced compared with plants exposed to EMIMCl alone.\u003c/p\u003e \u003cp\u003eThese findings were reflected in fresh biomass production. The presence of EMIMCl at 1\u0026ndash;50 mg\u0026middot;kg⁻\u0026sup1; of soil DW did not significantly affect maize fresh weight. Higher concentrations led to progressively stronger biomass reduction. In control soil, supplementation with 0.5 and 1 mM AsA visibly increased fresh biomass yield. Similarly, in EMIMCl-contaminated soil, AsA application improved fresh weight compared with plants grown in the presence of EMIMCl alone. Importantly, following AsA addition, a significant reduction in fresh biomass was observed only from 500 mg\u0026middot;kg⁻\u0026sup1; of soil DW EMIMCl onwards. The extent of AsA-mediated mitigation of EMIMCl toxicity depended on the applied AsA concentration. Under the highest EMIMCl level (1000 mg\u0026middot;kg⁻\u0026sup1; of soil DW), a beneficial effect on fresh biomass was observed exclusively at 1 mM AsA (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFresh biomass yield and dry weight content of maize seedlings cultivated in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are expressed as means of four replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Values within columns marked with the same letter are not significantly different.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration ILs [mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of soil DW]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration AsA [mM]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYield [g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDry weight\u003c/p\u003e \u003cp\u003e[g\u0026middot;g\u003csup\u003e\u0026minus;1\u003c/sup\u003eFW]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0 (control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.837\u0026thinsp;\u0026plusmn;\u0026thinsp;0.281\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0993\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0028\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.330\u0026thinsp;\u0026plusmn;\u0026thinsp;0.269\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0965\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0031\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.184\u0026thinsp;\u0026plusmn;\u0026thinsp;0.208\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1018\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.862\u0026thinsp;\u0026plusmn;\u0026thinsp;0.322\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1027\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0005\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.111\u0026thinsp;\u0026plusmn;\u0026thinsp;0.133\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0026\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.498\u0026thinsp;\u0026plusmn;\u0026thinsp;0.079\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0990\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0030\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.171\u0026thinsp;\u0026plusmn;\u0026thinsp;0.231\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1008\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0020\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.137\u0026thinsp;\u0026plusmn;\u0026thinsp;0.335\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1022\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0041\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.622\u0026thinsp;\u0026plusmn;\u0026thinsp;0.107\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0986\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0010\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.125\u0026thinsp;\u0026plusmn;\u0026thinsp;0.133\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0988\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0016\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.762\u0026thinsp;\u0026plusmn;\u0026thinsp;0.185\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0980\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0026\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.649\u0026thinsp;\u0026plusmn;\u0026thinsp;0.388\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0987\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0021\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.058\u0026thinsp;\u0026plusmn;\u0026thinsp;0.054\u003csup\u003eefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0979\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0008\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.339\u0026thinsp;\u0026plusmn;\u0026thinsp;0.176\u003csup\u003edef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0999\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0017\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.807\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0995\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0005\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.707\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0983\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0029\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.474\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003csup\u003ehi\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1157\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0050\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.438\u0026thinsp;\u0026plusmn;\u0026thinsp;0.063\u003csup\u003ehi\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1171\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0020\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.997\u0026thinsp;\u0026plusmn;\u0026thinsp;0.045\u003csup\u003efgh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1082\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.592\u0026thinsp;\u0026plusmn;\u0026thinsp;0.147\u003csup\u003eghi\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1134\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0015\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.078\u0026thinsp;\u0026plusmn;\u0026thinsp;0.076\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1254\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0025\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.127\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1214\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0012\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.608\u0026thinsp;\u0026plusmn;\u0026thinsp;0.111\u003csup\u003eghi\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1223\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0026\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.041\u0026thinsp;\u0026plusmn;\u0026thinsp;0.143\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1242\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0040\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Fresh biomass yield and dry weight content of maize seedlings cultivated in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are expressed as means of four replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Values within columns marked with the same letter are not significantly different.\u003c/p\u003e \u003cp\u003eThe presence of the ionic liquid also affected dry weight accumulation in maize seedlings. Exposure to 500 and 1000 mg\u0026middot;kg⁻\u0026sup1; of soil DW EMIMCl significantly increased dry weight content. The addition of AsA did not significantly alter dry weight levels compared with plants exposed to EMIMCl without antioxidant supplementation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhotosynthetic pigment content and chlorophyll fluorescence\u003c/h2\u003e \u003cp\u003eThe concentrations of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids), as well as the ratios of chlorophyll a to chlorophyll b (chl a/chl b) and total chlorophyll to carotenoids ((chl a\u0026thinsp;+\u0026thinsp;chl b)/car), are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Photosynthetic pigment content in maize seedlings grown in soil contaminated with different concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Values represent means of four replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Asterisks indicate significant differences compared with the respective control at the same AsA concentration (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eExposure of maize seedlings to EMIMCl resulted in a significant reduction in all analyzed photosynthetic pigments. The magnitude of this decline increased with rising concentrations of the ionic liquid. In soil not contaminated with EMIMCl, AsA supplementation led to a slight decrease in pigment content. However, when 0.5 mM AsA was applied to soil containing 1\u0026ndash;100 mg\u0026middot;kg⁻\u0026sup1; of soil DW EMIMCl, pigment levels were higher than in plants grown without antioxidant supplementation. At higher EMIMCl concentrations (500 and 1000 mg\u0026middot;kg⁻\u0026sup1; of soil DW), beneficial effects on pigment accumulation were observed following the application of 0.5 and 1 mM AsA. In contrast, 2 mM AsA consistently reduced pigment content relative to plants grown without AsA. When high EMIMCl levels were combined with 2 mM AsA, an increase in the chl a/chl b ratio and in the total chlorophyll-to-carotenoid ratio was observed.\u003c/p\u003e \u003cp\u003eChlorophyll fluorescence parameters were also evaluated, and the results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChlorophyll fluorescence parameters in maize seedlings cultivated in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are expressed as means of four replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Values within columns followed by the same letter do not differ significantly.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration ILs [mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of soil DW]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration AsA [mM]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003csub\u003em\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF\u003csub\u003ev\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF\u003csub\u003ev\u003c/sub\u003e/F\u003csub\u003em\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eF\u003csub\u003ev\u003c/sub\u003e/F\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0 (control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e199\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e970\u0026thinsp;\u0026plusmn;\u0026thinsp;40\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e771\u0026thinsp;\u0026plusmn;\u0026thinsp;38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.794\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.869\u0026thinsp;\u0026plusmn;\u0026thinsp;0.170\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e214\u0026thinsp;\u0026plusmn;\u0026thinsp;26\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e938\u0026thinsp;\u0026plusmn;\u0026thinsp;37\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e725\u0026thinsp;\u0026plusmn;\u0026thinsp;34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.772\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.431\u0026thinsp;\u0026plusmn;\u0026thinsp;0.434\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e219\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1025\u0026thinsp;\u0026plusmn;\u0026thinsp;42\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e819\u0026thinsp;\u0026plusmn;\u0026thinsp;82\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.797\u0026thinsp;\u0026plusmn;\u0026thinsp;0.049\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.732\u0026thinsp;\u0026plusmn;\u0026thinsp;0.126\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e 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align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e327\u0026thinsp;\u0026plusmn;\u0026thinsp;64\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1036\u0026thinsp;\u0026plusmn;\u0026thinsp;40\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e709\u0026thinsp;\u0026plusmn;\u0026thinsp;68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.686\u0026thinsp;\u0026plusmn;\u0026thinsp;0.082\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.262\u0026thinsp;\u0026plusmn;\u0026thinsp;0.609\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e350\u0026thinsp;\u0026plusmn;\u0026thinsp;80\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1056\u0026thinsp;\u0026plusmn;\u0026thinsp;85\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e705\u0026thinsp;\u0026plusmn;\u0026thinsp;58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.673\u0026thinsp;\u0026plusmn;\u0026thinsp;0.095\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.117\u0026thinsp;\u0026plusmn;\u0026thinsp;0.618\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e315\u0026thinsp;\u0026plusmn;\u0026thinsp;59\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1078\u0026thinsp;\u0026plusmn;\u0026thinsp;78\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e763\u0026thinsp;\u0026plusmn;\u0026thinsp;47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.709\u0026thinsp;\u0026plusmn;\u0026thinsp;0.040\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.490\u0026thinsp;\u0026plusmn;\u0026thinsp;0.545\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e328\u0026thinsp;\u0026plusmn;\u0026thinsp;59\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1043\u0026thinsp;\u0026plusmn;\u0026thinsp;44\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e715\u0026thinsp;\u0026plusmn;\u0026thinsp;63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.686\u0026thinsp;\u0026plusmn;\u0026thinsp;0.055\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.249\u0026thinsp;\u0026plusmn;\u0026thinsp;0.533\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e679\u0026thinsp;\u0026plusmn;\u0026thinsp;61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1062\u0026thinsp;\u0026plusmn;\u0026thinsp;90\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e383\u0026thinsp;\u0026plusmn;\u0026thinsp;55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.311\u0026thinsp;\u0026plusmn;\u0026thinsp;0.087\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.503\u0026thinsp;\u0026plusmn;\u0026thinsp;0.195\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e568\u0026thinsp;\u0026plusmn;\u0026thinsp;43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e819\u0026thinsp;\u0026plusmn;\u0026thinsp;92\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e252\u0026thinsp;\u0026plusmn;\u0026thinsp;66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.304\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.496\u0026thinsp;\u0026plusmn;\u0026thinsp;0.151\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Chlorophyll fluorescence parameters in maize seedlings cultivated in soil contaminated with various concentrations of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are expressed as means of four replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Values within columns followed by the same letter do not differ significantly.\u003c/p\u003e \u003cp\u003eThe presence of EMIMCl at 500 and 1000 mg\u0026middot;kg⁻\u0026sup1; of soil DW significantly increased minimal fluorescence (F₀) and maximal fluorescence (Fₘ). Simultaneously, the ratios F\u003csub\u003ev\u003c/sub\u003e/Fₘ and F\u003csub\u003ev\u003c/sub\u003e/F₀ were reduced, indicating impairment of photosystem II efficiency. In soils containing 1\u0026ndash;500 mg\u0026middot;kg⁻\u0026sup1; of soil DW EMIMCl, AsA supplementation did not significantly affect fluorescence parameters. Marked disturbances were observed only when AsA was applied to soil containing 1000 mg\u0026middot;kg⁻\u0026sup1; of soil DW EMIMCl. Under these conditions, 1 and 2 mM AsA nearly doubled F₀ values, caused approximately a twofold decrease in variable fluorescence (F\u003csub\u003ev\u003c/sub\u003e) and in the F\u003csub\u003ev\u003c/sub\u003e/Fₘ ratio, and led to a several-fold reduction in F\u003csub\u003ev\u003c/sub\u003e/F₀. Moreover, the addition of 2 mM AsA to soil with 1000 mg\u0026middot;kg⁻\u0026sup1; of soil DW EMIMCl significantly decreased maximal fluorescence (Fₘ).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMDA, H₂O₂, and AsA\u003c/h2\u003e \u003cp\u003eThe levels of H₂O₂, MDA, and AsA in maize seedlings are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Cultivation of maize in soil contaminated with EMIMCl resulted in elevated concentrations of H₂O₂ and MDA, indicating enhanced oxidative stress. In addition, a slight initial decrease in endogenous AsA content relative to the control was observed, followed by an increase after exposure to the tested ionic liquid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExogenous application of AsA differentially affected the concentrations of H₂O₂, MDA, and AsA in maize seedlings, depending on the antioxidant dose. Only the 1 mM AsA treatment led to a slight reduction in H₂O₂ content. Both 1 and 2 mM AsA caused a modest decrease in MDA levels. Furthermore, supplementation with 1 and 2 mM AsA slightly increased endogenous AsA concentration compared with plants grown in EMIMCl-contaminated soil without antioxidant addition. In contrast, 0.5 mM AsA did not significantly influence any of the analyzed oxidative stress markers.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Concentrations of MDA, H₂O₂, and AsA in maize seedlings cultivated in soil contaminated with various levels of EMIMCl and supplemented with AsA at 0.5, 1, and 2 mM. Data are expressed as means of four replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Asterisks indicate significant differences compared with the respective control at the same AsA concentration (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMultivariate analysis of physiological and biochemical responses of maize seedlings to EMIMCl stress\u003c/h2\u003e \u003cp\u003eTo integrate the results related to growth, photosynthetic performance, and oxidative stress in maize seedlings exposed to EMIMCl and exogenous AsA, principal component analysis (PCA) was performed. This approach enabled simultaneous evaluation of relationships among the measured parameters and identification of the main factors differentiating plant responses to chemical stress. The results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Principal component analysis (PCA) of physiological and biochemical responses of maize seedlings exposed to EMIMCl and treated with AsA. (a) Score plot showing sample distribution along PC1 and PC2; different point shapes indicate AsA concentrations, and ellipses represent 95% confidence intervals for EMIMCl treatments. (b) Loading plot illustrating relationships among the measured variables. (c) Heatmap of PCA loadings showing the contribution of individual variables to PC1 and PC2.\u003c/p\u003e \u003cp\u003eThe score plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) demonstrates a clear separation of samples along the PC1 axis, which corresponded to the gradient of EMIMCl concentration in the soil. Control samples and those treated with low EMIMCl doses were positioned on the negative side of PC1, whereas samples exposed to higher concentrations (500\u0026ndash;1000 mg\u0026middot;kg⁻\u0026sup1; of soil DW) clustered on the positive side of this axis.\u003c/p\u003e \u003cp\u003eThe loading plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) revealed positive loadings of H₂O₂, MDA, AsA, and dry weight content along PC1, while growth- and photosynthesis-related parameters\u0026mdash;including biomass yield, germination rate, and photosynthetic pigment content\u0026mdash;were negatively associated with this component. The PCA loading heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) confirmed the patterns observed in both the score and loading plots, supporting the consistency of the multivariate analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of EMIMCl on the growth and development of maize seedlings\u003c/h2\u003e \u003cp\u003eSeedling emergence, shoot and root length, and fresh biomass yield are among the most straightforward indicators of plant growth. A noticeable decline in any of these parameters is widely recognized as clear evidence that plants have been subjected to stress, whether abiotic or biotic (Chen et al. 2018). Seed germination represents a critical stage in the plant life cycle, and ionic liquids (ILs) may influence this process in different ways. Depending on their chemical structure, ILs can reduce or completely inhibit seed germination\u0026mdash;particularly at higher concentrations (Chen et al. 2024)\u0026mdash;have no measurable effect (Pawłowska et al. 2023), or even stimulate germination when applied at low doses (Chu et al. 2021). During germination, seeds imbibe water along with dissolved substances present in the soil solution. While some of these compounds are essential for plant development, others, such as heavy metals, may enter the seed and interfere with metabolic processes, thereby affecting germination and seedling establishment. Moreover, ILs containing chloride, tetrafluoroborate, or hexafluorophosphate anions may release HCl or HF through hydrolysis, leading to alterations in soil pH. Soil pH values that are unsuitable for a given plant species can negatively influence germination dynamics (Cvjetko Bubalo et al. 2014a,b; Bagheri et al. 2017; Chu et al. 2021). Regardless of the direction of the effect, the magnitude of IL impact on germination and emergence is closely related to the applied concentration. Low doses often exert negligible or even stimulatory effects, whereas higher concentrations frequently suppress germination and reduce seedling emergence (Chu et al. 2021; Chen et al. 2024; Pawłowska et al. 2023).\u003c/p\u003e \u003cp\u003eIn the present study, more pronounced effects were observed in shoot and root elongation than in seedling emergence. The results demonstrate that EMIMCl negatively affects maize growth in a concentration-dependent manner. Only the lowest tested concentration (1 mg\u0026middot;kg⁻\u0026sup1; of soil DW) stimulated seedling growth and increased fresh biomass yield. Previous reports (Li et al. 2022; Liu et al. 2015a) suggest that low IL concentrations may promote plant growth, whereas higher levels can damage root cell membranes. Such damage may facilitate the penetration of toxic substances into root tissues and their subsequent translocation to aerial plant parts. Impairment of root development compromises water and nutrient uptake, ultimately affecting overall plant performance (Chapman et al. 2012; Xu et al. 2018). Habibul et al. (2020) reported that imidazolium-based ILs tend to accumulate predominantly in roots, although they may also be transported to stems and leaves. The extent of accumulation depends on both the applied concentration and the alkyl chain length of the ILs.\u003c/p\u003e \u003cp\u003eDisturbances in root function and reduced root growth can lead to insufficient water and nutrient absorption. Consequently, decreased cellular turgor may occur, resulting in an increased proportion of dry weight in plant tissues. Elevated dry weight content is often interpreted as an indicator of impaired water balance, a common response to chemical and osmotic stress (Chen et al. 2018; Pawłowska et al. 2019; Biczak et al. 2020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAlterations in photosynthetic pigment content and chlorophyll fluorescence in maize seedlings exposed to EMIMCl\u003c/h2\u003e \u003cp\u003eAdequate levels of photosynthetic pigments are essential for proper plant functioning, as these compounds are directly involved in photosynthesis, a process fundamental to plant survival. In the present study, a reduction in chlorophyll content was already apparent during visual assessment of the plants, as higher EMIMCl concentrations induced chlorotic symptoms on the leaves. These observations were subsequently confirmed by quantitative measurements of individual photosynthetic pigments.\u003c/p\u003e \u003cp\u003ePrevious studies have similarly reported adverse effects of ionic liquids on pigment content in higher plants and algae (Li et al. 2018; Liu et al. 2015b; Xia et al. 2018). Imidazolium-based ionic liquids may disrupt the lipid bilayer structure and impair chloroplast membrane integrity. Exposure to ILs can also promote excessive production of reactive oxygen species (ROS), which damage cellular membranes and thylakoid structures within chloroplasts. Chloroplast impairment may lead to leakage of chlorophyll, while the combined action of ILs and ROS may facilitate penetration into internal chloroplast compartments, further disturbing chlorophyll biosynthesis and photosynthetic machinery organization (Chen et al 2018; Deng et al. 2017, Reddy et al. 2017; Li et al. 2018; Liu et al. 2018a; Liu et al. 2018b).\u003c/p\u003e \u003cp\u003eIn addition to pigment concentration, chlorophyll fluorescence is considered a sensitive indicator of oxidative and photosynthetic stress. The light-dependent reactions of photosynthesis are initiated in photosystem II (PSII). An increase in minimal fluorescence (F₀) may reflect either reversible or irreversible inactivation of PSII or structural damage to thylakoid membranes. A reduction in variable fluorescence (F\u003csub\u003ev\u003c/sub\u003e) indicates decreased PSII efficiency. The ratios F\u003csub\u003ev\u003c/sub\u003e/Fₘ and F\u003csub\u003ev\u003c/sub\u003e/F₀ provide information about the functional state of the PSII reaction center. Alterations in these parameters may result from EMIMCl-induced disturbances in the PSII electron transport chain or in the primary electron acceptor. A decline in F\u003csub\u003ev\u003c/sub\u003e/Fₘ is commonly interpreted as evidence of PSII reaction center damage. Such changes suggest the occurrence of photoinhibition under stress conditions, often accompanied by increased energy dissipation as heat and enhanced photodamage to the photosynthetic apparatus (Liu et al. 2015a; Li et al. 2018; Gao et al. 2016; Chen et al. 2019).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffect of EMIMCl on oxidative stress in maize seedlings\u003c/h2\u003e \u003cp\u003eHydrogen peroxide (H₂O₂), classified as a reactive oxygen species (ROS), plays a dual role in plants. Under physiological balance, it functions as a signaling molecule involved in stress perception and response. However, a rapid increase in H₂O₂ concentration is a clear indicator of oxidative stress. Such accumulation may result from enhanced superoxide dismutase (SOD) activity and intensified dismutation of superoxide radicals, or it may reflect a situation in which the stress level exceeds the detoxification capacity of the antioxidant defense system (S\u0026aacute;nchez-Rodr\u0026iacute;guez et al. 2010; Kumar et al. 2013; Demidchik et al. 2015; Di Baccio et al. 2017). The elevated H₂O₂ levels observed in maize seedlings cultivated in soil containing EMIMCl demonstrate that this ionic liquid induces oxidative stress in maize. Moreover, the magnitude of this effect increased with rising EMIMCl concentrations. Comparable increases in H₂O₂ content following exposure to ionic liquids have been reported by Zhang et al. (2013) in duckweed treated with C₈MIMBr, by Xu et al. (2018) in wheat exposed to three C₈MIM ionic liquids with different anions, and by Cvjetko Bubalo et al. (2014b) in barley seedlings subjected to four imidazolium-based ionic liquids.\u003c/p\u003e \u003cp\u003eReactive oxygen species, including H₂O₂, are capable of damaging proteins and DNA. They can also attack polyunsaturated fatty acids, initiating lipid peroxidation, with malondialdehyde (MDA) formed as one of its end products. MDA can subsequently interact with functional groups in proteins, lipoproteins, and nucleic acids, leading to cellular injury. Disruption of membrane integrity may have serious physiological consequences for plants (Zhang et al. 2013; Xu et al. 2018). The increased MDA content detected in maize seedlings exposed to EMIMCl further confirms the occurrence of oxidative stress. As with H₂O₂, the intensity of these changes was positively correlated with the applied concentration of the ionic liquid. Similar detrimental effects of ionic liquids on crop species have been documented for barley (Cvjetko Bubalo et al. 2014b), wheat (Liu et al. 2014; Liu et al. 2016), and rice (Liu et al. 2015a).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRole of exogenous ascorbic acid in alleviating EMIMCl-induced stress\u003c/h2\u003e \u003cp\u003eApplication of L-ascorbic acid (AsA) at low and moderate concentrations partially mitigated the adverse effects caused by EMIMCl. This protective response was reflected in improved plant growth, higher levels of photosynthetic pigments, and reduced accumulation of H₂O₂ and MDA. These findings indicate that AsA enhanced the antioxidant capacity of maize, thereby limiting oxidative damage.\u003c/p\u003e \u003cp\u003eExogenous supplementation with L-ascorbic acid clearly attenuated the inhibitory effect of the ionic liquid on seedling emergence as well as shoot and root growth. AsA plays a central role in maintaining both extracellular and intracellular redox balance, which directly influences multiple signaling pathways, including those associated with abscisic acid (ABA), auxin, and reactive oxygen species (ROS). Properly regulated redox homeostasis and signaling networks enable plants to respond rapidly and effectively to environmental disturbances, thereby protecting cells against abiotic stress (Xiao et al. 2021). The growth-promoting effect of AsA has also been documented in maize exposed to cadmium (Xiao et al. 2021), wheat subjected to lead stress (Alamri et al. 2018), rapeseed under drought conditions (Shafiq et al. 2014), peach trees experiencing water deficit (Panella et al. 2017), and wheat exposed to salinity (Ishaq et al. 2021). The beneficial impact of AsA on plant development may result from stimulated synthesis of amino acids, proteins, and photosynthetic pigments. Moreover, AsA regulates cell division, differentiation, and senescence, and by strengthening antioxidant defenses, it protects lipids and proteins from oxidative injury (Xiao et al. 2021; Zhang et al. 2019; Chen et al. 2021; Zong et al. 2023; Li et al. 2025; Wang et al. 2024).\u003c/p\u003e \u003cp\u003eAscorbic acid is one of the principal non-enzymatic antioxidants in plant cells. It can directly neutralize hydroxyl radicals and superoxide anions and serves as a cofactor for enzymes involved in ROS detoxification (Liu et al. 2015b; Li et al. 2018). Approximately 90% of cellular AsA is localized in the cytoplasm, and owing to its strong reducing properties, it is regarded as one of the most effective antioxidant molecules. In cooperation with vitamin E, ascorbate participates in the quenching of excited or intermediate reactive oxygen species, either directly or through enzyme-mediated reactions. In addition, together with glutathione, it forms the core of the ascorbate\u0026ndash;glutathione cycle, a crucial system responsible for maintaining cellular redox balance and regulating ROS levels, thereby influencing plant growth and development (Noctor and Foyer 1998; Asada 1999; Shao et al. 2008; Akram et al. 2017).\u003c/p\u003e \u003cp\u003eExternal application of AsA\u0026mdash;via seed priming, foliar spraying, or soil supplementation\u0026mdash;leads to an increase in endogenous ascorbate content, which can positively modulate antioxidant metabolism in plants (Akram et al. 2017). In the present study, maize seedlings grown in EMIMCl-contaminated soil exhibited a marked rise in AsA levels. This increase may represent a stress-induced response, as oxidative stress triggered by the ionic liquid likely stimulated the synthesis and accumulation of low-molecular-weight antioxidants, including ascorbate (Kumari et al. 2020). Under abiotic stress conditions, plants are known to upregulate genes encoding enzymes of the AsA biosynthetic pathway as well as those involved in the ascorbate\u0026ndash;glutathione cycle (Xiao et al. 2021). Furthermore, membrane damage\u0026mdash;indicated by elevated MDA content\u0026mdash;activates defense mechanisms that may also contribute to increased AsA accumulation (Kumari et al. 2020). The effectiveness of exogenously applied AsA in reducing oxidative stress in maize seedlings was concentration-dependent. This observation is consistent with findings by Khazaei et al. (2020), who demonstrated that acetylsalicylic acid applied at optimal concentrations enhanced drought tolerance in pepper (\u003cem\u003eCapsicum annuum\u003c/em\u003e L.), and by Hassan et al. (2021), who reported similar concentration-dependent protective effects of AsA against drought stress in barley (\u003cem\u003eHordeum vulgare\u003c/em\u003e L.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBiphasic effects of AsA under severe EMIMCl-induced stress\u003c/h2\u003e \u003cp\u003eOne of the key and novel findings of this study is the demonstration that the action of exogenous L-ascorbic acid under EMIMCl-induced stress follows a biphasic pattern and is strongly dependent on both its concentration and the severity of chemical stress. Low and moderate doses of AsA exerted a protective effect, whereas higher concentrations\u0026mdash;particularly when combined with elevated levels of EMIMCl in the soil\u0026mdash;not only failed to alleviate stress symptoms but markedly intensified them. This detrimental response was evidenced by a further increase in oxidative stress markers (H₂O₂ and MDA), along with deterioration of growth parameters and impairment of photosynthetic performance.\u003c/p\u003e \u003cp\u003eThe dual nature of AsA activity is supported by numerous reports indicating that, despite its central role as an antioxidant, ascorbate may under certain conditions display pro-oxidative properties. AsA is a pivotal component of the plant redox network and functions within the ascorbate\u0026ndash;glutathione cycle, where it modulates ROS levels and maintains cellular redox balance (Foyer and Noctor 2011; Akram et al. 2017). However, under intense stress conditions that overwhelm ROS-detoxifying systems, this balance may become disrupted.\u003c/p\u003e \u003cp\u003eAt elevated concentrations, AsA can promote ROS generation through redox reactions, particularly in the presence of transition metal ions such as Fe\u0026sup2;⁺ and Cu⁺, which catalyze Fenton-type reactions. In such circumstances, ascorbate may shift from acting solely as a ROS scavenger to facilitating the formation of highly reactive hydroxyl radicals, thereby enhancing lipid peroxidation and cellular damage (Smirnoff 2018; Halliwell and Gutteridge 2015). This mechanism is especially relevant in plant cells exposed to xenobiotics that already compromise membrane integrity and organelle function.\u003c/p\u003e \u003cp\u003eImidazolium-based ionic liquids have been shown to interact with biological membranes and proteins containing thiol groups, leading to disturbances in mitochondrial and chloroplast function and consequently to increased ROS production (Pham et al. 2010; Cvjetko Bubalo et al. 2017). Under such conditions, the application of high doses of AsA may aggravate redox imbalance rather than counteract it. This explains the intensified oxidative stress symptoms observed in maize seedlings simultaneously exposed to high concentrations of EMIMCl and elevated levels of exogenous AsA in the present study.\u003c/p\u003e \u003cp\u003eComparable pro-oxidative effects of externally supplied antioxidants have been reported in studies involving heavy metal toxicity, herbicide exposure, and salinity stress, where excessive AsA application resulted in worsened physiological performance instead of improvement (Gill and Tuteja 2010; Anjum et al. 2014). These findings highlight that antioxidant-based mitigation strategies are not universally beneficial and must be carefully tailored to the type and intensity of stress encountered.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMultivariate analysis of the physiological and biochemical responses of maize seedlings to EMIMCl stress\u003c/h2\u003e \u003cp\u003ePrincipal component analysis (PCA) demonstrated that EMIMCl was the dominant factor shaping the physiological response of maize seedlings. This was reflected in the strong association between oxidative stress indicators and the positive values of PC1. In contrast, growth-related traits and photosynthetic parameters were positioned oppositely along this axis, indicating a close relationship between oxidative stress induction and suppression of photosynthetic efficiency.\u003c/p\u003e \u003cp\u003eThe distribution of samples along PC2 suggests a modulatory influence of exogenously applied AsA; however, this effect was clearly concentration-dependent. Under conditions of high EMIMCl contamination, elevated AsA levels were positively correlated with increased stress markers, supporting its potential pro-oxidative role under disrupted redox homeostasis, as reported previously (Smirnoff 2018; Chen et al. 2021).\u003c/p\u003e \u003cp\u003eThese findings clearly indicate that the external application of AsA as a strategy to alleviate stress caused by ionic liquid contamination, such as EMIMCl, should be approached with caution. The biphasic nature of AsA action suggests that uncontrolled antioxidant supplementation may produce effects opposite to those intended, leading to enhanced oxidative damage and physiological dysfunction. Such disturbances may result in inhibited plant growth, yield reduction, and in extreme cases, plant death.\u003c/p\u003e \u003cp\u003eThe dual response pattern of AsA is of considerable importance both for understanding plant tolerance mechanisms to xenobiotics and for environmental risk assessment and the development of mitigation strategies in agroecosystems exposed to chemical stress.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, exogenous AsA may attenuate oxidative damage in plants, at least partially restoring antioxidant balance through direct ROS scavenging and stimulation of antioxidant enzymes. However, the effectiveness of AsA in mitigating stress induced by ionic liquids depends on both the applied AsA concentration and the level of IL contamination. When IL exposure severely disrupts cellular function, exogenous AsA may exhibit limited protective capacity, and an improperly selected dose may further intensify oxidative stress rather than alleviate it. Given the scarcity of studies addressing the use of exogenous AsA to counteract IL-induced oxidative stress in plants, further comprehensive research in this area is warranted.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Polish Ministry of Education and Science for The Faculty of Science and Technology of Jan Dlugosz University in Czestochowa (SBR/WNSPT/KBBE/18/2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB.P. \u0026ndash; Conceptualization, methodology, software, formal analysis, validation, investigation, resources, data curation, visualization, supervision, project administration, writing\u0026mdash;original draft preparation, A.L: investigation; R.S.: writing\u0026mdash;review and editing; R.B.: funding acquisition, validation, data curation, writing\u0026mdash;review and editing. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e This is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e This is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e This is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest in the present experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eThis is not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAkram NA, Shafiq F, Ashraf M (2017) Ascorbic acid\u0026mdash;A potential oxidant scavenger and its role in plant development and abiotic stress tolerance. 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The Plant Journal 117:1264\u0026ndash;1280.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"phytotoxicity, oxidative stress, chlorophyll fluorescence, maize, 1-ethyl-3-methylimidazolium chloride","lastPublishedDoi":"10.21203/rs.3.rs-8938488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8938488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIonic liquids (ILs) are widely used chemical compounds that may pose potential risks to the environment. In the present study, the effects of 1-ethyl-3-methylimidazolium chloride (EMIMCl) on growth, photosynthetic performance, and oxidative stress in maize (\u003cem\u003eZea mays\u003c/em\u003e L.) seedlings were evaluated, and the role of exogenous L-ascorbic acid (AsA) in modulating plant responses to this stress was investigated. Plants were cultivated in soil contaminated with EMIMCl at concentrations ranging from 1 to 1000 mg\u0026middot;kg\u003csup\u003e-1\u003c/sup\u003e of soil dry weight and treated with AsA at concentrations of 0.5\u0026ndash;2 mM. EMIMCl significantly inhibited plant growth, reduced photosynthetic pigment content, and impaired chlorophyll fluorescence parameters, accompanied by increased hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) levels, indicating the induction of oxidative stress. Moderate doses of AsA partially alleviated EMIMCl-induced toxicity, whereas higher AsA concentrations under severe EMIMCl contamination intensified stress symptoms. These findings demonstrate a dose-dependent and biphasic role of AsA in maize responses to EMIMCl-induced stress.\u003c/p\u003e","manuscriptTitle":"The effect of 1-ethyl-3-methylimidazolium chloride on oxidative stress and the functioning of the photosynthetic apparatus in maize seedlings – the modulatory role of exogenous ascorbic acid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 10:34:03","doi":"10.21203/rs.3.rs-8938488/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ee10de4-0985-413b-bb0a-b3848eddca87","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Reject with possible Resubmission","date":"2026-05-04T10:23:37+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T14:25:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 10:34:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8938488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8938488","identity":"rs-8938488","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}