Zeolite application contributes to remediation of lead-polluted soil by spinach

Zeolite application contributes to remediation of lead-polluted soil by spinach | 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 Article Zeolite application contributes to remediation of lead-polluted soil by spinach Tao Liu, Xuemei Zhang, Rui Wu, Zhenqi Shi, Lishan Duan, Changcun Lin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4611742/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 Remediation of lead (Pb)-contaminated soils is increasingly important because production of food crops in these soils is associated with human health risk. The present study investigated use of natural zeolite to remediate Pb-contaminated soil by quantifying changes in soil properties, soil nutrients and distribution of Pb in spinach plant parts. Natural zeolite was added to pots containing four different soil media that were artificially amended with Pb in a greenhouse. Zeolite was mixed with the media at four rates plus a zero control, and for three of the Zeolite rates a second application was made to the soil surface in the pot 30 days later for a total of eight zeolite treatments. The 32 treatments were replicated four times. Pb immobilization by zeolite was dose dependent, with greater immobilization at higher zeolite rates. Heterogeneous soil humus components exerted both mobilizing and stabilizing effects, so the medium nutrition was most effective for phytostabilization. So,split applications of zeolite were more effective than once, because the lag-phase time of split zeolite treatment applications was longer than for one application, to the same total dosage. This method might be an efficient way to remediate the lead-polluted soils with zeolite application twice. Biological sciences/Ecology Biological sciences/Plant sciences Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences Spinach (Spinacia oleracea) Nutrient Zeolite Pb immobilization Phytoremediation Figures Figure 1 Figure 2 Figure 3 Introduction Lead (Pb) is the most important pollutants and widely found in surface soil, emanating from geological processes, mining, burning fossil fuels, and manufacture of fertilizers (Ryan et al. 2004; Cui et al. 2007; Z. Li et al. 2014). Sustainable agricultural ecosystems are at risk from harmful trace elements that arise from mining, landfilling, and overuse of pesticides and fertilizers（Jadwig et al.2024). Because of Pb’s non-degradable nature, Pb-contaminated soils seriously affects quality of vegetables growing in those fields (Huang et al. 2007; Breck 1974; H. Li et al. 2009). Methods for remediation of Pb-contaminated soils have recently become agricultural focus and concern because the production of crops in Pb-enriched soils is serious threat to human health (Kumpiene et al. 2008; Castaldi et al. 2005). Conventional remediation of Pb-contaminated soils is expensive and ineffective, involving excavation and chemical/physical treatment of contaminated soil (Delkash et al. 2015). Phytostabilization is considered a cost-effective and environmentally friendly approach which is a state-of-the art remediation technique used for heavy metal immobilization in polluted environment.The technique uses heavy-metal-resistant plants to stabilize heavy metals in soil or restore heavy metals in plant roots so as to restrict the entry into the human food chain (Salt et al. 1998). In order to enhance the efficiency of phytostabilization, many chemical amendments have been utilized. However, these amendments can dramatically inhibit soil fertility and physicochemical property, which negative impacts on the environmental safety (Lasat 2002). Natural zeolite has been widely utilized for the removal of heavy metals (Chlopecka and Adriano 1996; Querol et al. 2006; Nguyen et al. 2015; Wen et al. 2016) and decrease heavy metal concentration in vegetables because of its special physico-chemical property and low cost (Castaldi et al. 2005). Zeolite is a high-water-retention material that can be used as a soil conditioner to improve soil electrical conductivity, water absorption, and nutrient conservation(Harhash et al.2022). There are various large or small holes and channels in the zeolite's structure, which make the zeolite selectively absorb molecules of appropriate size. In additional, natural zeolite has a negative charge that can be neutralized by exchanging heavy metal for sodium and potassium (Breck 1974; Eroglu et al. 2017). mobility. There are complex interactions between heavy metals and soil organic matter, involving synergistic and antagonistic. Soil organic matter is evaluated to reduce the solubility of trace metals because of the tendency of forming stable complexes between heavy metal with organic ligands (Lin et al. 2009; Madejon et al. 2006). Thus humus is the most abundant fraction of decomposed organic matter, which may decrease the potential availability of metals by redistributing them into oxides and carbonate precipitate forms (Halim et al. 2003; Clemmente and Bernal 2006; Shi et al. 2009), but greater amounts of humus may increase heavymetal accumulation in plant shoots because of greater plant biomass and lower solution pH (Yang et al. 2005). Although many experiments have studied the effectiveness of zeolite and soil organic matter for immobilization of heavy metals (Clemmente and Bernal 2006; Najafi-Ghiri and Rahimi 2016; Jiwan et al. 2013), the application of different rates of zeolite in soils varying in proportion of humus has never been investigated. This study evaluated use of spinach ( Spinacia oleracea L.) plants to remediate Pb-contaminated soils. Therefore, the aims were: (1) to study the effect of greater soil organic matter, that likely will increase spinach biomass accumulation, combined with exogenous zeolite, on plant Pb uptake and (2) to test whether timing of the zeolite application affects plant uptake of Pb. Material and methods Components of soil mixtures and amendments Pure sand and humus were mixed for bulk soil samples. Physico-chemical characteristics of the humus are shown in Table 1 . The sand and humus were air-dried and passed through a 2-mm sieve prior to mixing. Lead nitrate solution, Pb(NO 3 ) 2 , was added to the air-dried mixture of sand and humus at a concentration of 500 mg Pb kg − 1 , based on the Chinese Environmental Quality Standard for Soils (GB 15618 − 1995) (2006). The treated soils were aged for two weeks with deionized water at 60% of the soil water-holding capacity. Soils were then dried at about 25 o C for two weeks. The pot experiment was initiated after the artificially contaminated soil was subjected to three wetting and drying cycles before (Blaylock et al. 1997). Table 1 Physical and chemical characteristics of humus substrate Parameter Concentration pH 6.2 ± 0 Organic matter concentration % 15 ± 1.6 Total N g kg − 1 15.7 ± 1.3 Extr. P P 2 0 5 Olsen (mg kg − 1 ) 6113 ± 104 Cd mg kg − 1 1.30 ± 0.2 Cr mg kg − 1 21.2 ± 0.5 Cu mg kg − 1 38.9 ± 2.0 Ni mg kg − 1 17.5 ± 1.1 Pb mg kg − 1 24.9 ± 1.1 Zn mg kg − 1 38.1 ± 2.0 Plant growth experiment Selection of spinach in this study was based on the results from our previous study (Lin et al. 2010). After seeds were sown in compost next week, seedlings with the first pair of true leaves were replanted in pots filled with the various soil media, at five seedlings per pot. Each pot (25-cm diameter, 18-cm high) contains 18 kg of treated soil. During the experiment, to maintain close to 80% of soil water-holding capacity, plants were irrigated every three days with deionized water based on water loss by weight. Pots remained in controlled greenhouse conditions for two months, with day/night temperatures of 25/20°C and, 16 h of day and 8 h of night. Soil media and Zeolite treatments The humus and sand compositions of different treatments are given as follow: 1:1, 1:2, 1:3 and 1:4 humus:sand by weight. Before the media were put in pots, Zeolite was blended into the treatment soils at concentrations of 0 (control), 5, 10, 20, and 40 g zeolite kg − 1 of media. Three additional treatments included two equal applications of Zeolite; the first was the initial mixing of either 5, 10, or 20 g Zeolite kg − 1 of media, and it was followed by a second application of the same amount of Zeolite to the soil surface 30 days after the initial mixing. The 32 treatments (four media x 8 Zeolite levels) were arranged in a completely randomized design with four replications. Seedlings with the first pair of true leaves were replanted in pot one week after the first zeolite application (five seedlings per pot). After 60 days, all plants were harvested, oven-dried at 60°C and weighed. Chemical analysis After harvest, plants were separated into root, stem and leaf fractions, and oven-dried at 60°C for 72 h. For measurement of Pb in plant tissues, 1 g of dried tissue was digested with 15 mL HCl/HNO 3 /HClO 4 mixture (3:1:2 v/v) at 150°C until the solution became transparent. The resultant solutions were filtered and diluted to 25 mL in volumetric flasks and determined using flame atomic absorption spectrometry (SpectrAA-220FS, USA). Statistical analysis All statistical analysis was performed using SPSS 16. The zeolite treatments, soil medias and their interactions’ effects were analyzed using wo-way ANOVA. The effects of soil media on plant tissue biomass and Pb concentration were analyzed using one-way ANOVA. Differences in plant tissue Pb concentration and pools due to zeolite application were compared using t -tests. Differences among means were compared by Duncan’s multiple range tests( P < 0.05). Results Biomass As can be seen from Fig. 1 , whole plant biomass was affected by soil media ( P < 0.0001). The maximum aboveground and belowground spinach biomass was in the 1:2 humus:sand ratio, which was 10–22% greater than other treatments. Moreover, in the high humus levels (humus:sand ratios of 1:1 and 1:2), aboveground and belowground biomass were significantly more than those for 1:3 and 1:4 treatments. Patterns of biomass accumulation in plant fractions were similar for different soil media. Biomass of leaf and stem tissue were comparable between 1:1 and 1:2 humus:sand treatments; with similar patterns in 1:3 and 1:4 media except that leaf and stem biomass were 20% greater in 1:1 and 1:2 media than 1:3 and 1:4 ( P < 0.0001 and P < 0.001, respectively). Soil-media induced reductions in biomass occurred in all tissues, and there was a significant effect of soil media on the sum of above and belowground biomass. However, the magnitude of the soil media effect was much greater for belowground than aboveground biomass. Pb concentrations in plant tissue There were significant interactions ( P < 0.01) between soil media and zeolite levels in leaves and roots but not stems (Table 2). In the 1:1 humus:sand medium, all zeolite treatments reduced Pb concentration in aboveground tissues compared with the no zeolite control. Although the 5 g zeolite kg − 1 of medium treatment reduced aboveground tissue Pb concentration, greater zeolite rates decreased tissue Pb concentration further (Fig. 2 ). Aboveground tissue Pb concentration was least in the double application of 20 g zeolite kg − 1 , 30–250% lower than the other zeolite treatments except for two application of 10 g zeolite kg − 1 for the 1:1 medium ( P < 0.01). There were similar patterns of response to zeolite in both 1:2 and 1:3 media (Fig. 2 ), and Pb concentration decreased in all tissues with increasing zeolite levels. In the 1:2 medium, all multiple application treatments of zeolite decreased Pb concentration more than the single doses of 10, 20, or 40 g kg − 1 ( P < 0.01). In the 1:3 soil medium treatment, the lowest shoot Pb concentration was 0.33 mg g − 1 for the zeolite treatment of two applications of 20 g kg − 1 . For the 1:4 soil medium, the pattern of aboveground tissue Pb concentration was similar to 1:3, and Pb concentration in belowground tissue was not affected by treatment, in part because the coefficient of variation was large (28%, Fig. 2 -D ) . In comparison to the single application treatment, all the two applications treatments showed little additional effect on Pb concentration of all tissues. With addition of zeolite, Pb concentration in aboveground tissues was lesser in soil medium 1:1 (0.298 ± 0.0299 mg g − 1 ) and 1:2 treatments (0.301 ± 0.0352 mg g − 1 ), than in 1:3 and 1:4 treatments (0.573 ± 0.1515 and 0.499 ± 0.0921 mg g − 1 ). Pb concentration of belowground tissues in soil media 1:1 and 1:2 (0.325 ± 0.0329 and 0.333 ± 0.0375 mg g − 1 , respectively) was less than in 1:3 and 1:4 (0.666 ± 0.0452 and 0.693 ± 0.0391 mg g − 1 , respectively). With decreasing proportions of humus in the soil media, Pb concentration in leaves and roots increased accordingly. Addition of two high doses of zeolite significantly decreased Pb concentration of aboveground tissues in 1:1, 1:2, and 1:3 soil media treatments ( P < 0.05). Similar to the trend for various nutrient levels, there were no detectable differences of Pb concentrations between two low doses of zeolite and a single addition. df Pb concentration in roots Pb concentration in stems Pb concentration in leaves F P F P F P Soil medium Zeolite Zeolite × Med. 3 7 21 115.2 6.24 5.45 0.000 ** 0.000 ** 0.000 ** 21.72 9.18 2.71 0.000 ** 0.000 ** 0.120 NS 21.72 30.19 3.27 0.000 ** 0.000 ** 0.006 ** * P < 0.05; ** P < 0.01; NS : Not significant. Table 2. Two-way ANOVA’s for the Pb concentrations in individual tissue types (roots, stems and leaves) for spinach following addition of different rates of zeolite (Zeolite) and use of different soil humus:sand ratios in the growth media. Pools There were significant interactions ( P < 0.01) between soil media and zeolite in tissues other than stems (Table 3). Aboveground shoot Pb content in all zeolite treatments was greater than in belowground tissues. Compared with the zero zeolite control, addition of zeolite decreased Pb content in aboveground biomass. In soil medium 1:1, all zeolite treatments decreased Pb content in all tissues ( P < 0.01; Fig. 3 − 1:1). Stems had the smallest Pb content among plant parts. The aboveground biomass was greater than belowground mass, such that the proportion of total Pb content in the aboveground fraction ranged from at 56–82%. With increasing zeolite dose from 5–40 g kg − 1 , total Pb content in the whole plant decreased, but total Pb content was lowest with application of the two 20 g kg − 1 doses. In the 1:2 soil medium, the pattern of Pb content was similar to that in 1:1, with the lowest total Pb content when using the two 20 g kg − 1 doses ( P < 0.01) (Fig. 3 − 1:2). In contrast to the 1:1 medium, Pb content of whole plant was less for the 1:2 medium. In 1:3 medium, all zeolite treatments varied little in total Pb content except for the 40 g kg − 1 dose which had lowest Pb content. In the 1:4 soil medium, the 40 g kg − 1 single application and two applications of 10 g kg − 1 and two applications of 20 g kg − 1 decreased total Pb content ( P < 0.01; Fig. 3 − 1:3;Fig. 3 − 1:4). The effect of increasing rate of zeolite on total plant Pb content was more pronounced for single than double applications of zeolite. Pb content of aboveground tissue in 3:1 and 4:1 treatments was greater ( P < 0.05) than in the 1:1 and 1:2 soil media. Similarly to aboveground tissues, total Pb content of belowground tissue in 1:3 (1.00 ± 0.21 mg plant − 1 ) and 1:4 soil media (0.80 ± 0.15 mg plant − 1 ) were greater than in 1:1 and 1:2 media (0.57 ± 0.15 and 0.57 ± 0.14 mg plant − 1 , respectively). Application of two doses of 20 g kg − 1 of zeolite had greater impact on Pb content in 1:1 and 1:2 soil media than application of one dose of 40 g kg − 1 ( P < 0.05). With decreasing humus proportion in soil, the two doses of 20 g kg − 1 had less effect on Pb content than one dose of 40 g kg − 1 . Over all soil media types, addition of two applications of 5 or of 10 g zeolite kg − 1 had less effect on Pb content than one dose of 10 or 20 g kg − 1 . df Pb pool in roots Pb pool in stems Pb pool in leaves F P F P F P Nut. Zeolite Zeolite× Nut. 3 7 21 22.78 5.21 3.91 0.000 ** 0.001 ** 0.000 ** 4.57 7.41 1.01 0.021 * 0.000 ** 0.821 NS 7.87 20.57 4.52 0.000 ** 0.000 ** 0.009 ** * P < 0.05; ** P < 0.01; NS : Not significant. Table 3. Two-way ANOVAs for the Pb pools in individual tissue types (roots, stems and leaves) for spinach with the effects of various concentrations of zeolite and different soil media. Discussion Heavy metal immobilization Pb immobilization by zeolite appeared to be dose dependent, as evidenced by the increasing immobilization observed with the high dose. Additionally, Pb concentration in the shoots was found to be directly correlated with the amount of zeolite or related agents added to particular soils in earlier studies (El-Eswed et al. 2015; H. Li et al. 2009; Shi et al. 2009). Generally speaking, the rise in plant Pb concentration matched the soil's soluble Pb concentration. Furthermore, earlier research has shown a positive correlation between plant and soil Pb concentrations (Schmidt 2003; H. Li et al. 2009; Shi et al. 2009; Shen et al. 2002). Metals from various soil components or their surfaces are extracted or desorbed by cation exchange regulated by particle diffusion with zeolite.Additionally, metal promotes the creation of complexes, oxides, and metal-carbonate precipitates, all of which reduce metal mobility (Chlopecka and Adriano 1996; Hamidpour et al. 2010; H. Wang et al. 2016). This is explained by the poor adsorption of lead (Pb) by soil and the formation of sediment with zeolite, which increases Pb stabilization more readily (Sarkar et al. 2008; Hamidpour et al. 2017). Pb content in 1:3 and 1:4 humus:sand ratio soils in the current study was more than that of 1:1 and 1:2 ratio soils in almost all zeolite treatments (Fig. 2 ), suggesting that soils with different humus:sand ratios had different stabilization patterns. Regarding the function of humus in the remediation of heavy-metal-contaminated soils, opinions are currently divided. Humus is a heterogeneous substance, and small structural and chemical differences can concurrently exert mobilizing and stabilizing effects. due to the improvement of the cation exchange capacity of soils, the adsorption of Pb ions to the humic acid in the complex will be increased, which results in reducing mobility of Pb, thus permitting the re-establishment of vegetation at contaminated sites (Chaturvedi et al. 2007; Narwal and Singh 1998). These opinions are supported by more research, and our results are similar to these reports. However, acid humus reduces soil pH, which could increase the soluble Pb fraction concentration in soil (Schmidt 2003). This is opposite to the experimental results of the present study, so that this explanation should be discarded. At the lowest and highest zeolite doses, respectively, zeolite clearly immobilized more soluble Pb at 1:1 and 1:2 than at 1:3 and 1:4 humus:sand media, ranging from 5–40 g kg-1. These results imply that there were substantial differences in the distribution patterns following zeolite treatment amongst the different humus treatments. It is well known that the complex structure of humus can interact, via complex formation or chelation, with heavy metals. The adsorption in a metal-humus-zeolite system is influenced by the complexing of metal ions with humus and the competitive adsorption of heavy metal ions and humus on zeolite surfaces. The results of this experiment show that there is no difference in Pb immobilization between the 1:1 and 1:2 humus:sand media. This indicates that Pb and humus complexing is considerably weaker and that Pb and humus competitive adsorption is stronger. According to Wang et al. (2008), Pb > humus is the sequence in which the adsorptive affinity of Pb and humus to the zeolite is followed. It follows that Pb immobilization is greatly increased by Pb adsorption on zeolite and Pb complexing with humus in the 1:1 ration soil. Strong Pb adsorption on zeolite prevents Pb from complexing with humus in the presence of low humus, which may be the cause of the greater insoluble Pb in the soil with a 1:4 ratio. On the zeolite, there is a kinetic curve showing dynamic Pb uptake. Pb adsorption on zeolite is progressively enhanced during the adsorption process, reaching equilibrium adsorption after 140 hours (S. B. Wang et al. 2008). At 1:1, 1:2, 1:3 humus:sand media, Pb immobilization increased and plant uptake in aboveground tissues decreased for two applications of 20 g zeolite kg − 1 moreso than one application of 40 g kg − 1 (Fig. 2 ). The mechanism is not known for greater Pb stability in soil associated with repeated application of zeolite. The difference in Pb immobilization between one and two zeolite applications might be due to the effective adsorption capability of zeolite. Effective adsorption capability is defined as the change in heavy metal immobilization by soil amendments. For all treatments, Pb adsorption on the zeolite in binary component systems, which Pb immobilization initially continued to increase with time after zeolite treatment and then tended to be stable. This lag-phase, which was the decrease in cation exchange effect before the second sampling point onwards, must be taken into account. For zeolite this lag-phase is about 3 weeks (H. Wang et al. 2016) or 9 weeks (Shi et al. 2013). With a lag-phase of 9 weeks, the leachate Pb concentration of 20 g·kg − 1 zeolite was reduced to about 25% of highest Pb concentration with on zeolite treatment. There was lesser decrease for 10 g kg − 1 zeolite treatment and rate of decrease was about only 40%. The high persistence of the cation exchange effect at higher dosage may have been present because of more suface area of sorption. The immobilization within this initial period is assumed to be caused by forming insoluble Pb fraction. The water soluble fraction, exchangeable fraction and carbonate-bound fraction of Pb were the main Pb solubility-controlling phases in the treated soil, nevertheless zeolite treatment caused a reduction of the above three fractions in the Pb- contaminated soil (H. Li et al. 2009; Wen et al. 2016). According to previous research (Lin et al. 2010), Pb carbonate, Pb Fe-Mn oxide and Pb organically bound fractions account for as much as 5%, 43%, and 52% of the total Pb, respectively. Although the Pb Fe-Mn oxide and Pb organically bound fraction accounts for the greatest proportion, both are immobilizable- only if they transform into the Pb water soluble fraction, Pb exchangeable fraction, and Pb carbonate fraction (Querol et al. 2006). However, the mechanism that zeolite treatment resulted in a reduction of the carbonate bound Pb fraction is not thoroughly understood (Castaldi et al. 2005; Basaldella et al. 2007; Lei et al. 2008). For an equal dose of zeolite, the lag-phase of two zeolite applications is longer than a single application, which makes Pb immobilization with two applications more effective. From the lag-phase, zeolite is a sufficiently long-lived substance to be appropriate for phytostabilization: its longevity will increase Pb immobility, even long after harvest. Therefore, it is suitable option for phytostabilization that high dose and second application after the initial treatment. Plant uptake Composition of the soil medium significantly affected biomass production, but there was no effect of zeolite (Fig. 1 ). This was attributed to detoxication to plants with zeolite and humus treatment. Pb content in the controls was 3.11 ± 0.16 mg plant − 1 . Pb uptake was significant in all zeolite treatments, 30–70% less than in the untreated controls. Zeolite amendments resulted in significant differences, and the observed differences are considered sufficient for enhanced phytostabilization. Compared to low nutrient levels, significantly less of the Pb pool was allocated to aboveground (leaves and stems) in high nutrient levels. Also, the effect on Pb uptake by zeolite was dose dependent, except for the low nutrient treatment. Because the fraction of Pb absorbed by zeolite varies considerably in high nutrition soil (Sarkar et al. 2008; Schmidt 2003), previous studies have also noted that the concentration of soluble Pb should correlate well with plant Pb concentration (Cui et al. 2007; Hamidpour et al. 2010; Shen et al. 2002). Humus has a high organic matter content, which forms stable complexes with heavy metals, which increase with zeolite application (Shi et al. 2009). Soil with high humus content was significantly affected by zeolite. Besides humus directly affecting Pb solubility in soils, humus can promote plant growth in poor soils, providing a higher nutrient and water supply. The higher Pb accumulations may have been a side effect of higher biomass (Lin et al. 2010). However, the level of uptake in our experiment may be unduly high because of the short time between soil zeolite treatments and transplanting of seedlings (7 d). Wang et al. (2013) found that there were the similar leachate Pb concentration patterns with and without zeolite addition for the first 3 weeks, which increased the damage to seedlingswith heavy metal stress. Longer periods between zeolite addition and transplanting of seedlings may be required to redistribute the amendment throughout the upper soil profile and subsequent heavy metal translocation. Therefore, it is absolutely essential that the interval between zeolite addition and transplanting of seedlings exceeds 3 weeks. Post-harvest effects A majority of metals are immobilized by zeolite and biodegradation of the ligand, and only a limited fraction of mobilized Pb was absorbed and translocated by plants. Post-harvest effects are inescapable because the absence of an actively transpiring plant greatly increases the risk for leaching of the mobilized metals (Lin et al. 2010). Due to lag-phase accumulation, post-harvest effects decreased with two applications of zeolite. There was a trade-off between enhanced Pb phytostabilization with two applications of zeolite and reduced plant biomass because of plowing disturbance with zeolite application. Conclusion This research was carried out to observe spinach growing on Pb-contaminated soil to evaluate humus and zeolite’s possibility for phytoremediation purpose. The results obtained highlighted the notable ability of zeolite to immobilize Pb from different soil media, and Pb immobilization by zeolite appeared to be dose dependent. By cation exchange with zeolite, Pb is adsorbed from soil components or their surfaces, thus preventing the impact of Pb in the food chain. The heterogeneity characteristic of humus can concurrently exert stabilizing and mobilizing effects, thus there was a trade-off (Lin et al. 2010), and the 1:2 soil medium was most effective. The application of twice high dose zeolite instead of single application is recommended to be a better option to enhanced Pb accumulation in plants; inasmuch as lag-phase time of application of two high zeolite applications was longer than in a single application. The premature zeolite application may cause the risk of post-harvest metal mobility and leaching. Thus, it is likely that the risk of Pb leaching must be accurately checked at field scale. The present results need to be confirmed with further studies at field scale, where several agronomic strategies can be adopted to enhance the feasibility of phytostabilization technology. Declarations Statement The spinach seeds used in the article have been saved in the National Crop Germplasm Resource Bank of China and can be purchased in the market as commercial seeds.( https://www.cgris.net/).Vouche r samples have been preserved in the National Germplasm Repository and identified by Lu Xinxiong. Author Contribution Tao Liu wrote the main manuscript text and participated in all experiments .Z and W prepared figures 1-2. S and D prepared figures 3.All authors reviewed the manuscript Data Availability All data generated or analyzed during this study are included in the article. References Basaldella, E. I., Vazquez, P. G., Iucolano, F., & Caputo, D. Chromium removal from water using LTA zeolites: effect of pH. Journal of Colloid and Interface Science, 313 , 574–578(2007). 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Soil amendment application frequency contributes to phytoextraction of lead by sunflower at different nutrient levels. Environmental and Experimental Botany, 65 , 410–416(2009). Lin, C., Liu, L., Wang, D., & Zhu, T. Influences of major nutrient elements on Pb accumulation of two crops from a Pb-contaminated soil. Journal of Hazardous materials, 174 , 202–208(2010). Madejon, E., de Mora, A. P., Felipe, E., Burgos, P., & Cabrera, F. Soil amendments reduce trace element solubility in a contaminated soil and allow regrowth of natural vegetation. Environmental Pollution, 139 (1), 40–52(2006). Najafi-Ghiri, M., & Rahimi, T. Zinc Uptake by Spinach (Spinacia oleracea L.) as Affected by Zn Application Rate, Zeolite, and Vermicompost. Compost Science & Utilization, 24 (3), 203–207(2016). Narwal, R. P., & Singh, B. R. Effect of organic materials on partitioning, extractability and plant uptake ofmetals in an alum shale soil. Water Air Soil Pollut., 103 , 405–421(1998). Nguyen, T. C., Loganathan, P., Nguyen, T. V., Vigneswaran, S., Kandasamy, J., & Naidu, R. Simultaneous adsorption of Cd, Cr, Cu, Pb, and Zn by an iron-coated Australian zeolite in batch and fixed-bed column studies. Chemical Engineering Journal, 270 , 393–404(2015). Querol, X., Alastuey, A., Moreno, N., Alvarez-Ayuso, E., García-Sánchez, A., Cama, J., et al. Immobilization of heavy metals in polluted soils by the addition of zeolite material synthesized from coal fly ash. Chemosphere, 62 (2), 171–180(2006). Ryan, J. A., Scheckel, K. G., Berti, W., Brown, S. L., Casteel, S., Chaney, R. L., et al. Reducing children's risk from lead in soil. Environmental Science and Technology, 38 , 18–24(2004). Salt, D. E., Smith, R. D., & Raskin, I. Phytoremediation. Annual Review of Plant Physiology and Plant Molecular Biology, 49 , 643–668(1998). Sarkar, D., Andra, S. S., Saminathan, S. K. M., & Datta, R. Chelant-aided enhancement of lead mobilization in residential soils. Environmental Pollution, 156 (3), 1139–1148 (2008). Schmidt, U. Enhancing phytoextraction: the effect of chemical soil manipulation on mobility, plant accumulation, and leaching of heavy metals. Journal of Environmental Quality, 32 , 1939–1954(2003). Shen, Z., li, X., Wang, C., Chen, H., & Chua, H. Lead phytoextraction fromcontaminated soil with high-biomass plant species. Journal of Environmental Quality, 31 , 1893–1900(2002). Shi, W. Y., Li, H., Du, S., Wang, K. B., & Shao, H. B. Immobilization of lead by application of zeolite: Leaching column and rhizobox incubation studies. Applied Clay Science, 85 , 103–108(2013). Shi, W. Y., Shao, H. B., Li, H., Shao, M. A., & Dua, S. Co-remediation of the lead-polluted garden soil by exogenous natural zeolite and humic acids. Journal of Hazardous materials, 167 , 136–140(2009). Wang, H., Wang, X., Chen, J., Xia, P., & Zhao, J. Recovery of nutrients from wastewater by a MgCl 2 modified zeolite and their reuse as an amendment for Cu and Pb immobilization in soil. Royal Society of Chemistry Advances, 6 , 55809–55818(2016). Wang, S. B., Terdkiatburana, T., & Tade, M. O. Adsorption of Cu(II), Pb(II) and humic acid on natural zeolite tuff in single and binary systems. Separation and Purification Technology, 62 , 64–70(2008). Wen, J., Yi, Y., & Zeng, G. Effects of modified zeolite on the removal and stabilization of heavy metals in contaminated lake sediment using BCR sequential extraction. Journal of Environmental Management, 178 , 63–69(2016). Yang, B., Lan, C. Y., & Shu, W. S. Growth and heavy metal accumulation of Vetiveria zizanioides grown on lead/zinc mine tailings. Acta Ecol. Sin., 25 , 45–50(2005). Additional Declarations No competing interests reported. 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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-4611742","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":325689566,"identity":"e8a0ca7f-6ae1-42c6-84fa-8e98d2e15752","order_by":0,"name":"Tao Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Liu","suffix":""},{"id":325689567,"identity":"6c68f8a9-fe3d-45ad-b990-3955b46a78fb","order_by":1,"name":"Xuemei Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xuemei","middleName":"","lastName":"Zhang","suffix":""},{"id":325689568,"identity":"f5d1682f-31d1-469a-81b2-7594d75d6d88","order_by":2,"name":"Rui Wu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Wu","suffix":""},{"id":325689569,"identity":"d647690f-b52b-4bb5-80bc-3537599f0b7a","order_by":3,"name":"Zhenqi Shi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhenqi","middleName":"","lastName":"Shi","suffix":""},{"id":325689570,"identity":"604cea12-5263-4663-9c64-e24e882918bd","order_by":4,"name":"Lishan Duan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lishan","middleName":"","lastName":"Duan","suffix":""},{"id":325689571,"identity":"1ee8ec01-570a-4ad9-a721-5517990fc5e2","order_by":5,"name":"Changcun Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYBACPmYGBmYom/ExA48ciGGAVwsbkhZmYwYeYyK0MCC0sEkzMBCjhZ334OeCmjt2/TNyj1UXyBgkNrA3b5NgqLmDx2F8ydIzjj1LnnEjL+32DB6gFp5jZRIMx57h0cJjxszDdjjZQCLH7DYPz5/EBiBDgrHhMAEt/yBainlAtsi/IUILb9thO5AWZrAWCR6CWoylefsOJ0iceWMsDfSLcRtPWrFFwjHcWvj5zxh+5vl22J6/Pcfwc2GPgWw/++GNNz7U4NYCA4kNIJKxBxJTDAkENTAw2EOoH0QoHQWjYBSMghEHAKdFRbjonWb1AAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Changcun","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2024-06-20 12:22:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4611742/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4611742/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60149288,"identity":"75dd9901-60a7-49f7-b7d5-09a45d20bce5","added_by":"auto","created_at":"2024-07-12 10:38:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93997,"visible":true,"origin":"","legend":"\u003cp\u003eBiomass of plants grown in different soil media. Histograms are subdivided into individual tissues (roots, stems and leaves). Errors bars denote standard error for aboveground biomass (stems and leaves) and belowground biomass (root), respectively. Alphabetic characters denote results of LSD post hoc tests for total aboveground biomass. Significance accepted at P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4611742/v1/08edfbfff29aa8403f6fb1c7.jpg"},{"id":60148538,"identity":"a1657511-6810-4bf4-b1df-cf8c13265062","added_by":"auto","created_at":"2024-07-12 10:30:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":394677,"visible":true,"origin":"","legend":"\u003cp\u003ePb concentration in aboveground and belowground of spinach grown with various zeolite treatments in different soil medias. Error bars represent error for aboveground Pb concentration. Alphabetic characters denote results of LSD post hoc tests for aboveground Pb concentration in different zeolite treatments. Significance accepted at P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4611742/v1/c21cfc45846d62d9b6ac6244.jpg"},{"id":60148540,"identity":"505e74d3-e949-4844-a1ec-26241abcdb26","added_by":"auto","created_at":"2024-07-12 10:30:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":335337,"visible":true,"origin":"","legend":"\u003cp\u003eStanding stock of Pb in individual tissues (roots, stems and leaves) of spinach grown with various zeolite treatments in different soil medias. Error bars represent error for aboveground Pb content (stems and leaves) and belowground Pb content (root), respectively. Alphabetic characters denote results of LSD post hoc tests for aboveground Pb content. Significance accepted at P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4611742/v1/3023d28e9b949d2278472bdc.jpg"},{"id":62338711,"identity":"9f13646a-73e9-4bd2-84e5-fb3ea455f396","added_by":"auto","created_at":"2024-08-13 05:57:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1319571,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4611742/v1/f291c437-27b4-4772-9079-3ba2b18f4bfe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zeolite application contributes to remediation of lead-polluted soil by spinach","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLead (Pb) is the most important pollutants and widely found in surface soil, emanating from geological processes, mining, burning fossil fuels, and manufacture of fertilizers (Ryan et al. 2004; Cui et al. 2007; Z. Li et al. 2014). Sustainable agricultural ecosystems are at risk from harmful trace elements that arise from mining, landfilling, and overuse of pesticides and fertilizers（Jadwig et al.2024). Because of Pb\u0026rsquo;s non-degradable nature, Pb-contaminated soils seriously affects quality of vegetables growing in those fields (Huang et al. 2007; Breck 1974; H. Li et al. 2009). Methods for remediation of Pb-contaminated soils have recently become agricultural focus and concern because the production of crops in Pb-enriched soils is serious threat to human health (Kumpiene et al. 2008; Castaldi et al. 2005).\u003c/p\u003e\n\u003cp\u003eConventional remediation of Pb-contaminated soils is expensive and ineffective, involving excavation and chemical/physical treatment of contaminated soil (Delkash et al. 2015). Phytostabilization is considered a cost-effective and environmentally friendly approach which is a state-of-the art remediation technique used for heavy metal immobilization in polluted environment.The technique uses heavy-metal-resistant plants to stabilize heavy metals in soil or restore heavy metals in plant roots so as to restrict the entry into the human food chain (Salt et al. 1998). In order to enhance the efficiency of phytostabilization, many chemical amendments have been utilized. However, these amendments can dramatically inhibit soil fertility and physicochemical property, which negative impacts on the environmental safety (Lasat 2002).\u003c/p\u003e\n\u003cp\u003eNatural zeolite has been widely utilized for the removal of heavy metals (Chlopecka and Adriano 1996; Querol et al. 2006; Nguyen et al. 2015; Wen et al. 2016) and decrease heavy metal concentration in vegetables because of its special physico-chemical property and low cost (Castaldi et al. 2005). Zeolite is a high-water-retention material that can be used as a soil conditioner to improve soil electrical conductivity, water absorption, and nutrient conservation(Harhash et al.2022). There are various large or small holes and channels in the zeolite\u0026apos;s structure, which make the zeolite selectively absorb molecules of appropriate size. In additional, natural zeolite has a negative charge that can be neutralized by exchanging heavy metal for sodium and potassium (Breck 1974; Eroglu et al. 2017). mobility. \u003c/p\u003e\n\u003cp\u003eThere are complex interactions between heavy metals and soil organic matter, involving synergistic and antagonistic. Soil organic matter is evaluated to reduce the solubility of trace metals because of the tendency of forming stable complexes between heavy metal with organic ligands (Lin et al. 2009; Madejon et al. 2006). Thus humus is the most abundant fraction of decomposed organic matter, which may decrease the potential availability of metals by redistributing them into oxides and carbonate precipitate forms (Halim et al. 2003; Clemmente and Bernal 2006; Shi et al. 2009), but greater amounts of humus may increase heavymetal accumulation in plant shoots because of greater plant biomass and lower solution pH (Yang et al. 2005).\u003c/p\u003e\n\u003cp\u003eAlthough many experiments have studied the effectiveness of zeolite and soil organic matter for immobilization of heavy metals (Clemmente and Bernal 2006; Najafi-Ghiri and Rahimi 2016; Jiwan et al. 2013), the application of different rates of zeolite in soils varying in proportion of humus has never been investigated. This study evaluated use of spinach (\u003cem\u003eSpinacia oleracea \u003c/em\u003eL.) plants to remediate Pb-contaminated soils. Therefore, the aims were: (1) to study the effect of greater soil organic matter, that likely will increase spinach biomass accumulation, combined with exogenous zeolite, on plant Pb uptake and (2) to test whether timing of the zeolite application affects plant uptake of Pb. \u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eComponents of soil mixtures and amendments\u003c/h2\u003e \u003cp\u003ePure sand and humus were mixed for bulk soil samples. Physico-chemical characteristics of the humus are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The sand and humus were air-dried and passed through a 2-mm sieve prior to mixing. Lead nitrate solution, Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, was added to the air-dried mixture of sand and humus at a concentration of 500 mg Pb kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, based on the Chinese Environmental Quality Standard for Soils (GB 15618\u0026thinsp;\u0026minus;\u0026thinsp;1995) (2006). The treated soils were aged for two weeks with deionized water at 60% of the soil water-holding capacity. Soils were then dried at about 25\u003csup\u003eo\u003c/sup\u003eC for two weeks. The pot experiment was initiated after the artificially contaminated soil was subjected to three wetting and drying cycles before (Blaylock et al. 1997).\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\u003ePhysical and chemical characteristics of humus substrate\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganic matter concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtr. P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003csub\u003e2\u003c/sub\u003e0\u003csub\u003e5\u003c/sub\u003e Olsen (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6113\u0026thinsp;\u0026plusmn;\u0026thinsp;104\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e38.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e38.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth experiment\u003c/h2\u003e \u003cp\u003eSelection of spinach in this study was based on the results from our previous study (Lin et al. 2010). After seeds were sown in compost next week, seedlings with the first pair of true leaves were replanted in pots filled with the various soil media, at five seedlings per pot. Each pot (25-cm diameter, 18-cm high) contains 18 kg of treated soil. During the experiment, to maintain close to 80% of soil water-holding capacity, plants were irrigated every three days with deionized water based on water loss by weight. Pots remained in controlled greenhouse conditions for two months, with day/night temperatures of 25/20\u0026deg;C and, 16 h of day and 8 h of night.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSoil media and Zeolite treatments\u003c/h3\u003e\n\u003cp\u003eThe humus and sand compositions of different treatments are given as follow: 1:1, 1:2, 1:3 and 1:4 humus:sand by weight. Before the media were put in pots, Zeolite was blended into the treatment soils at concentrations of 0 (control), 5, 10, 20, and 40 g zeolite kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of media. Three additional treatments included two equal applications of Zeolite; the first was the initial mixing of either 5, 10, or 20 g Zeolite kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of media, and it was followed by a second application of the same amount of Zeolite to the soil surface 30 days after the initial mixing. The 32 treatments (four media x 8 Zeolite levels) were arranged in a completely randomized design with four replications. Seedlings with the first pair of true leaves were replanted in pot one week after the first zeolite application (five seedlings per pot). After 60 days, all plants were harvested, oven-dried at 60\u0026deg;C and weighed.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eChemical analysis\u003c/h2\u003e \u003cp\u003eAfter harvest, plants were separated into root, stem and leaf fractions, and oven-dried at 60\u0026deg;C for 72 h. For measurement of Pb in plant tissues, 1 g of dried tissue was digested with 15 mL HCl/HNO\u003csub\u003e3\u003c/sub\u003e/HClO\u003csub\u003e4\u003c/sub\u003e mixture (3:1:2 v/v) at 150\u0026deg;C until the solution became transparent. The resultant solutions were filtered and diluted to 25 mL in volumetric flasks and determined using flame atomic absorption spectrometry (SpectrAA-220FS, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analysis was performed using SPSS 16. The zeolite treatments, soil medias and their interactions\u0026rsquo; effects were analyzed using wo-way ANOVA. The effects of soil media on plant tissue biomass and Pb concentration were analyzed using one-way ANOVA. Differences in plant tissue Pb concentration and pools due to zeolite application were compared using \u003cem\u003et\u003c/em\u003e-tests. Differences among means were compared by Duncan\u0026rsquo;s multiple range tests(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiomass\u003c/h2\u003e \u003cp\u003eAs can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, whole plant biomass was affected by soil media (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The maximum aboveground and belowground spinach biomass was in the 1:2 humus:sand ratio, which was 10\u0026ndash;22% greater than other treatments. Moreover, in the high humus levels (humus:sand ratios of 1:1 and 1:2), aboveground and belowground biomass were significantly more than those for 1:3 and 1:4 treatments.\u003c/p\u003e \u003cp\u003ePatterns of biomass accumulation in plant fractions were similar for different soil media. Biomass of leaf and stem tissue were comparable between 1:1 and 1:2 humus:sand treatments; with similar patterns in 1:3 and 1:4 media except that leaf and stem biomass were 20% greater in 1:1 and 1:2 media than 1:3 and 1:4 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). Soil-media induced reductions in biomass occurred in all tissues, and there was a significant effect of soil media on the sum of above and belowground biomass. However, the magnitude of the soil media effect was much greater for belowground than aboveground biomass.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePb concentrations in plant tissue\u003c/h2\u003e \u003cp\u003eThere were significant interactions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) between soil media and zeolite levels in leaves and roots but not stems (Table\u0026nbsp;2). In the 1:1 humus:sand medium, all zeolite treatments reduced Pb concentration in aboveground tissues compared with the no zeolite control. Although the 5 g zeolite kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of medium treatment reduced aboveground tissue Pb concentration, greater zeolite rates decreased tissue Pb concentration further (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Aboveground tissue Pb concentration was least in the double application of 20 g zeolite kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 30\u0026ndash;250% lower than the other zeolite treatments except for two application of 10 g zeolite kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the 1:1 medium (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eThere were similar patterns of response to zeolite in both 1:2 and 1:3 media (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and Pb concentration decreased in all tissues with increasing zeolite levels. In the 1:2 medium, all multiple application treatments of zeolite decreased Pb concentration more than the single doses of 10, 20, or 40 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the 1:3 soil medium treatment, the lowest shoot Pb concentration was 0.33 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the zeolite treatment of two applications of 20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor the 1:4 soil medium, the pattern of aboveground tissue Pb concentration was similar to 1:3, and Pb concentration in belowground tissue was not affected by treatment, in part because the coefficient of variation was large (28%, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-D\u003cb\u003e)\u003c/b\u003e. In comparison to the single application treatment, all the two applications treatments showed little additional effect on Pb concentration of all tissues.\u003c/p\u003e \u003cp\u003eWith addition of zeolite, Pb concentration in aboveground tissues was lesser in soil medium 1:1 (0.298\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0299 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and 1:2 treatments (0.301\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0352 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), than in 1:3 and 1:4 treatments (0.573\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1515 and 0.499\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0921 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Pb concentration of belowground tissues in soil media 1:1 and 1:2 (0.325\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0329 and 0.333\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0375 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively) was less than in 1:3 and 1:4 (0.666\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0452 and 0.693\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0391 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively). With decreasing proportions of humus in the soil media, Pb concentration in leaves and roots increased accordingly. Addition of two high doses of zeolite significantly decreased Pb concentration of aboveground tissues in 1:1, 1:2, and 1:3 soil media treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similar to the trend for various nutrient levels, there were no detectable differences of Pb concentrations between two low doses of zeolite and a single addition.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"12\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c3\" namest=\"c2\" rowspan=\"2\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003ePb concentration in roots\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003ePb concentration in stems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003ePb concentration in leaves\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil medium\u003c/p\u003e \u003cp\u003eZeolite\u003c/p\u003e \u003cp\u003eZeolite \u0026times; Med.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e7\u003c/p\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e115.2\u003c/p\u003e \u003cp\u003e6.24\u003c/p\u003e \u003cp\u003e5.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.72\u003c/p\u003e \u003cp\u003e9.18\u003c/p\u003e \u003cp\u003e2.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.120\u003csup\u003eNS\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21.72\u003c/p\u003e \u003cp\u003e30.19\u003c/p\u003e \u003cp\u003e3.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.006\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e*\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u003csup\u003eNS\u003c/sup\u003e: Not significant.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;2.\u003c/b\u003e Two-way ANOVA\u0026rsquo;s for the Pb concentrations in individual tissue types (roots, stems and leaves) for spinach following addition of different rates of zeolite (Zeolite) and use of different soil humus:sand ratios in the growth media.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePools\u003c/h2\u003e \u003cp\u003eThere were significant interactions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) between soil media and zeolite in tissues other than stems (Table\u0026nbsp;3). Aboveground shoot Pb content in all zeolite treatments was greater than in belowground tissues. Compared with the zero zeolite control, addition of zeolite decreased Pb content in aboveground biomass. In soil medium 1:1, all zeolite treatments decreased Pb content in all tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026thinsp;\u0026minus;\u0026thinsp;1:1). Stems had the smallest Pb content among plant parts. The aboveground biomass was greater than belowground mass, such that the proportion of total Pb content in the aboveground fraction ranged from at 56\u0026ndash;82%. With increasing zeolite dose from 5\u0026ndash;40 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, total Pb content in the whole plant decreased, but total Pb content was lowest with application of the two 20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e doses. In the 1:2 soil medium, the pattern of Pb content was similar to that in 1:1, with the lowest total Pb content when using the two 20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e doses (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026thinsp;\u0026minus;\u0026thinsp;1:2). In contrast to the 1:1 medium, Pb content of whole plant was less for the 1:2 medium.\u003c/p\u003e \u003cp\u003eIn 1:3 medium, all zeolite treatments varied little in total Pb content except for the 40 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dose which had lowest Pb content. In the 1:4 soil medium, the 40 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e single application and two applications of 10 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and two applications of 20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e decreased total Pb content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026thinsp;\u0026minus;\u0026thinsp;1:3;Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026thinsp;\u0026minus;\u0026thinsp;1:4). The effect of increasing rate of zeolite on total plant Pb content was more pronounced for single than double applications of zeolite.\u003c/p\u003e \u003cp\u003ePb content of aboveground tissue in 3:1 and 4:1 treatments was greater (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than in the 1:1 and 1:2 soil media. Similarly to aboveground tissues, total Pb content of belowground tissue in 1:3 (1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mg plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and 1:4 soil media (0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 mg plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were greater than in 1:1 and 1:2 media (0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 and 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 mg plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively). Application of two doses of 20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of zeolite had greater impact on Pb content in 1:1 and 1:2 soil media than application of one dose of 40 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). With decreasing humus proportion in soil, the two doses of 20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e had less effect on Pb content than one dose of 40 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Over all soil media types, addition of two applications of 5 or of 10 g zeolite kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e had less effect on Pb content than one dose of 10 or 20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"12\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c3\" namest=\"c2\" rowspan=\"2\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003ePb pool in roots\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003ePb pool in stems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003ePb pool in leaves\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNut.\u003c/p\u003e \u003cp\u003eZeolite\u003c/p\u003e \u003cp\u003eZeolite\u0026times; Nut.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e7\u003c/p\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e22.78\u003c/p\u003e \u003cp\u003e5.21\u003c/p\u003e \u003cp\u003e3.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.001\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.57\u003c/p\u003e \u003cp\u003e7.41\u003c/p\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.021\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.821\u003csup\u003eNS\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e7.87\u003c/p\u003e \u003cp\u003e20.57\u003c/p\u003e \u003cp\u003e4.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.009\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e*\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u003csup\u003eNS\u003c/sup\u003e: Not significant.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;3.\u003c/b\u003e Two-way ANOVAs for the Pb pools in individual tissue types (roots, stems and leaves) for spinach with the effects of various concentrations of zeolite and different soil media.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHeavy metal immobilization\u003c/h2\u003e \u003cp\u003ePb immobilization by zeolite appeared to be dose dependent, as evidenced by the increasing immobilization observed with the high dose. Additionally, Pb concentration in the shoots was found to be directly correlated with the amount of zeolite or related agents added to particular soils in earlier studies (El-Eswed et al. 2015; H. Li et al. 2009; Shi et al. 2009). Generally speaking, the rise in plant Pb concentration matched the soil's soluble Pb concentration. Furthermore, earlier research has shown a positive correlation between plant and soil Pb concentrations (Schmidt 2003; H. Li et al. 2009; Shi et al. 2009; Shen et al. 2002). Metals from various soil components or their surfaces are extracted or desorbed by cation exchange regulated by particle diffusion with zeolite.Additionally, metal promotes the creation of complexes, oxides, and metal-carbonate precipitates, all of which reduce metal mobility (Chlopecka and Adriano 1996; Hamidpour et al. 2010; H. Wang et al. 2016). This is explained by the poor adsorption of lead (Pb) by soil and the formation of sediment with zeolite, which increases Pb stabilization more readily (Sarkar et al. 2008; Hamidpour et al. 2017).\u003c/p\u003e \u003cp\u003ePb content in 1:3 and 1:4 humus:sand ratio soils in the current study was more than that of 1:1 and 1:2 ratio soils in almost all zeolite treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting that soils with different humus:sand ratios had different stabilization patterns. Regarding the function of humus in the remediation of heavy-metal-contaminated soils, opinions are currently divided. Humus is a heterogeneous substance, and small structural and chemical differences can concurrently exert mobilizing and stabilizing effects. due to the improvement of the cation exchange capacity of soils, the adsorption of Pb ions to the humic acid in the complex will be increased, which results in reducing mobility of Pb, thus permitting the re-establishment of vegetation at contaminated sites (Chaturvedi et al. 2007; Narwal and Singh 1998). These opinions are supported by more research, and our results are similar to these reports. However, acid humus reduces soil pH, which could increase the soluble Pb fraction concentration in soil (Schmidt 2003). This is opposite to the experimental results of the present study, so that this explanation should be discarded.\u003c/p\u003e \u003cp\u003eAt the lowest and highest zeolite doses, respectively, zeolite clearly immobilized more soluble Pb at 1:1 and 1:2 than at 1:3 and 1:4 humus:sand media, ranging from 5\u0026ndash;40 g kg-1. These results imply that there were substantial differences in the distribution patterns following zeolite treatment amongst the different humus treatments. It is well known that the complex structure of humus can interact, via complex formation or chelation, with heavy metals. The adsorption in a metal-humus-zeolite system is influenced by the complexing of metal ions with humus and the competitive adsorption of heavy metal ions and humus on zeolite surfaces. The results of this experiment show that there is no difference in Pb immobilization between the 1:1 and 1:2 humus:sand media. This indicates that Pb and humus complexing is considerably weaker and that Pb and humus competitive adsorption is stronger. According to Wang et al. (2008), Pb\u0026thinsp;\u0026gt;\u0026thinsp;humus is the sequence in which the adsorptive affinity of Pb and humus to the zeolite is followed. It follows that Pb immobilization is greatly increased by Pb adsorption on zeolite and Pb complexing with humus in the 1:1 ration soil. Strong Pb adsorption on zeolite prevents Pb from complexing with humus in the presence of low humus, which may be the cause of the greater insoluble Pb in the soil with a 1:4 ratio. On the zeolite, there is a kinetic curve showing dynamic Pb uptake. Pb adsorption on zeolite is progressively enhanced during the adsorption process, reaching equilibrium adsorption after 140 hours (S. B. Wang et al. 2008).\u003c/p\u003e \u003cp\u003eAt 1:1, 1:2, 1:3 humus:sand media, Pb immobilization increased and plant uptake in aboveground tissues decreased for two applications of 20 g zeolite kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e moreso than one application of 40 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The mechanism is not known for greater Pb stability in soil associated with repeated application of zeolite. The difference in Pb immobilization between one and two zeolite applications might be due to the effective adsorption capability of zeolite. Effective adsorption capability is defined as the change in heavy metal immobilization by soil amendments. For all treatments, Pb adsorption on the zeolite in binary component systems, which Pb immobilization initially continued to increase with time after zeolite treatment and then tended to be stable. This lag-phase, which was the decrease in cation exchange effect before the second sampling point onwards, must be taken into account. For zeolite this lag-phase is about 3 weeks (H. Wang et al. 2016) or 9 weeks (Shi et al. 2013). With a lag-phase of 9 weeks, the leachate Pb concentration of 20 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e zeolite was reduced to about 25% of highest Pb concentration with on zeolite treatment. There was lesser decrease for 10 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e zeolite treatment and rate of decrease was about only 40%. The high persistence of the cation exchange effect at higher dosage may have been present because of more suface area of sorption.\u003c/p\u003e \u003cp\u003eThe immobilization within this initial period is assumed to be caused by forming insoluble Pb fraction. The water soluble fraction, exchangeable fraction and carbonate-bound fraction of Pb were the main Pb solubility-controlling phases in the treated soil, nevertheless zeolite treatment caused a reduction of the above three fractions in the Pb- contaminated soil (H. Li et al. 2009; Wen et al. 2016). According to previous research (Lin et al. 2010), Pb carbonate, Pb Fe-Mn oxide and Pb organically bound fractions account for as much as 5%, 43%, and 52% of the total Pb, respectively. Although the Pb Fe-Mn oxide and Pb organically bound fraction accounts for the greatest proportion, both are immobilizable- only if they transform into the Pb water soluble fraction, Pb exchangeable fraction, and Pb carbonate fraction (Querol et al. 2006). However, the mechanism that zeolite treatment resulted in a reduction of the carbonate bound Pb fraction is not thoroughly understood (Castaldi et al. 2005; Basaldella et al. 2007; Lei et al. 2008). For an equal dose of zeolite, the lag-phase of two zeolite applications is longer than a single application, which makes Pb immobilization with two applications more effective. From the lag-phase, zeolite is a sufficiently long-lived substance to be appropriate for phytostabilization: its longevity will increase Pb immobility, even long after harvest. Therefore, it is suitable option for phytostabilization that high dose and second application after the initial treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlant uptake\u003c/h2\u003e \u003cp\u003eComposition of the soil medium significantly affected biomass production, but there was no effect of zeolite (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This was attributed to detoxication to plants with zeolite and humus treatment. Pb content in the controls was 3.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 mg plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Pb uptake was significant in all zeolite treatments, 30\u0026ndash;70% less than in the untreated controls.\u003c/p\u003e \u003cp\u003eZeolite amendments resulted in significant differences, and the observed differences are considered sufficient for enhanced phytostabilization. Compared to low nutrient levels, significantly less of the Pb pool was allocated to aboveground (leaves and stems) in high nutrient levels. Also, the effect on Pb uptake by zeolite was dose dependent, except for the low nutrient treatment. Because the fraction of Pb absorbed by zeolite varies considerably in high nutrition soil (Sarkar et al. 2008; Schmidt 2003), previous studies have also noted that the concentration of soluble Pb should correlate well with plant Pb concentration (Cui et al. 2007; Hamidpour et al. 2010; Shen et al. 2002).\u003c/p\u003e \u003cp\u003eHumus has a high organic matter content, which forms stable complexes with heavy metals, which increase with zeolite application (Shi et al. 2009). Soil with high humus content was significantly affected by zeolite. Besides humus directly affecting Pb solubility in soils, humus can promote plant growth in poor soils, providing a higher nutrient and water supply. The higher Pb accumulations may have been a side effect of higher biomass (Lin et al. 2010). However, the level of uptake in our experiment may be unduly high because of the short time between soil zeolite treatments and transplanting of seedlings (7 d). Wang et al. (2013) found that there were the similar leachate Pb concentration patterns with and without zeolite addition for the first 3 weeks, which increased the damage to seedlingswith heavy metal stress. Longer periods between zeolite addition and transplanting of seedlings may be required to redistribute the amendment throughout the upper soil profile and subsequent heavy metal translocation. Therefore, it is absolutely essential that the interval between zeolite addition and transplanting of seedlings exceeds 3 weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePost-harvest effects\u003c/h2\u003e \u003cp\u003eA majority of metals are immobilized by zeolite and biodegradation of the ligand, and only a limited fraction of mobilized Pb was absorbed and translocated by plants. Post-harvest effects are inescapable because the absence of an actively transpiring plant greatly increases the risk for leaching of the mobilized metals (Lin et al. 2010). Due to lag-phase accumulation, post-harvest effects decreased with two applications of zeolite. There was a trade-off between enhanced Pb phytostabilization with two applications of zeolite and reduced plant biomass because of plowing disturbance with zeolite application.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis research was carried out to observe spinach growing on Pb-contaminated soil to evaluate humus and zeolite\u0026rsquo;s possibility for phytoremediation purpose. The results obtained highlighted the notable ability of zeolite to immobilize Pb from different soil media, and Pb immobilization by zeolite appeared to be dose dependent. By cation exchange with zeolite, Pb is adsorbed from soil components or their surfaces, thus preventing the impact of Pb in the food chain. The heterogeneity characteristic of humus can concurrently exert stabilizing and mobilizing effects, thus there was a trade-off (Lin et al. 2010), and the 1:2 soil medium was most effective. The application of twice high dose zeolite instead of single application is recommended to be a better option to enhanced Pb accumulation in plants; inasmuch as lag-phase time of application of two high zeolite applications was longer than in a single application. The premature zeolite application may cause the risk of post-harvest metal mobility and leaching. Thus, it is likely that the risk of Pb leaching must be accurately checked at field scale. The present results need to be confirmed with further studies at field scale, where several agronomic strategies can be adopted to enhance the feasibility of phytostabilization technology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eStatement\u003c/h2\u003e \u003cp\u003eThe spinach seeds used in the article have been saved in the National Crop Germplasm Resource Bank of China and can be purchased in the market as commercial seeds.(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cgris.net/).Vouche\u003c/span\u003e\u003cspan address=\"https://www.cgris.net/).Vouche\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003er samples have been preserved in the National Germplasm Repository and identified by Lu Xinxiong.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eTao Liu wrote the main manuscript text and participated in all experiments .Z and W prepared figures 1-2. S and D prepared figures 3.All authors reviewed the manuscript\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBasaldella, E. I., Vazquez, P. G., Iucolano, F., \u0026amp; Caputo, D. Chromium removal from water using LTA zeolites: effect of pH. Journal of Colloid and Interface Science, \u003cem\u003e313\u003c/em\u003e, 574\u0026ndash;578(2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlaylock, M. J., Salt, D. E., Dushenkov, S., Zakharova, O., Gussman, C., Kapulnik, Y., et al. Enhanced accumulation of Pb in Indian mustard by soil-applied chelating agents. Environmental Science and Technology, \u003cem\u003e31\u003c/em\u003e, 860\u0026ndash;865(1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBreck, D. W. \u003cem\u003eZeolite Molecular Sieves: Structure Chemistry and Use\u003c/em\u003e (J. Hebei Agric. Univ.). New York: John Wiley and Sons Press(1974).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCastaldi, P., Santona, L., \u0026amp; Melis, P. Heavy metal immobilization by chemical amendments in a polluted soil and influence on white lupin growth. Chemosphere, \u003cem\u003e60\u003c/em\u003e(3), 365\u0026ndash;371(2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaturvedi, P. K., Seth, C. S., \u0026amp; Misra, V. Selectivity sequences and sorption capacities of phosphatic clay and humus rich soil towards the heavy metals present in zinc mine tailing. 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The present study investigated use of natural zeolite to remediate Pb-contaminated soil by quantifying changes in soil properties, soil nutrients and distribution of Pb in spinach plant parts. Natural zeolite was added to pots containing four different soil media that were artificially amended with Pb in a greenhouse. Zeolite was mixed with the media at four rates plus a zero control, and for three of the Zeolite rates a second application was made to the soil surface in the pot 30 days later for a total of eight zeolite treatments. The 32 treatments were replicated four times. Pb immobilization by zeolite was dose dependent, with greater immobilization at higher zeolite rates. Heterogeneous soil humus components exerted both mobilizing and stabilizing effects, so the medium nutrition was most effective for phytostabilization. So,split applications of zeolite were more effective than once, because the lag-phase time of split zeolite treatment applications was longer than for one application, to the same total dosage. This method might be an efficient way to remediate the lead-polluted soils with zeolite application twice.","manuscriptTitle":"Zeolite application contributes to remediation of lead-polluted soil by spinach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-12 10:29:59","doi":"10.21203/rs.3.rs-4611742/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":"971d9510-120b-4aa5-a97f-4d2a2f38a6df","owner":[],"postedDate":"July 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34457031,"name":"Biological sciences/Ecology"},{"id":34457032,"name":"Biological sciences/Plant sciences"},{"id":34457033,"name":"Earth and environmental sciences/Ecology"},{"id":34457034,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2024-08-13T05:41:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-12 10:29:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4611742","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4611742","identity":"rs-4611742","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}