Crucial role of iron plaque on thallium uptake by rice plant

Iron plaque is a Fe-containing oxide film produced by the oxidation of Fe(II) in the rice root system under the combined action of oxygen infiltration and other microorganisms. Owing to its special surface structure and physio-chemical properties, the iron plaque has a strong absorption capacity for a variety of heavy metal ions. This study aimed to first investigate the effects of Fe species on the geochemical fractionation of Tl in typical paddy soil systems affected by industrial activities, followed by pot culture experiments to probe the effects of Fe species on the uptake and translocation of Tl in rice plants. The results of field work preliminarily showed that iron at different valences affected the conversion of the Tl geochemical fraction in the soil. Oxidizable Tl exerted significant positive correlation relationships with Fe2+ and negative correlation relationships with Fe3+, while reducible Tl only displayed a positive correlation with Fe3+. Further analysis by pot culture experiments revealed that the contents of Fe were significantly positively correlated with Tl contents in Fe plaque (R2 = 0.529). In contrast, the water-soluble Tl contents in the soil were significantly negatively correlated with the contents of Fe (R2 = – 0. 90, p < 0.05). It suggests that the iron plaque promoted the absorption and fixation of Tl on the root surface of rice plants, causing Tl to accumulate in the iron plaque. Besides, the Tl content in the Fe plaque on the root surface of rice plants was greater than that in the above-ground tissues, which indicates that most Fe plaque exerts a certain degree of inhibition on Tl migration into the above-ground tissues of rice plants. All these findings indicate that Fe film is also an important carrier of Tl transfer in the soil–rice plant system, which provides new scientific support for the remediation of typical Tl-contaminated rice fields.


Introduction
Thallium (Tl) is a highly toxic heavy metal with extremely low lethality [1][2][3][4][5][6]. In recent years, with the development of mining, metal smelting, and chemical industry in China, Tl polluted wastewater discharged from mining activities and chemical industry processes has often been released into the soil as irrigation water [7][8][9]. Compared with other crops, rice is more likely to accumulate Tl from the growing soil. High content of Tl easily accumulates in agricultural products, causing considerable health risks to humans [6,7,10].
As one of the largest grain crops in the world, rice plays a very important role in agricultural production worldwide. In particular, in China, rice production accountes for 26% of global rice production [11]. With the development of mineral exploitation, a large amount of Tl has been released into the environment, which is accumulated in the farmland soils and eventually biomagnified into human bodies via the food chain [11,12]. Specifically, high levels of Tl have been detected in paddy soil systems. Worse still, rice plants have a strong enrichment capacity for Tl that can easily take up Tl from polluted soil, owing to the identical ion radius between Tl(I) and K(I) [13]. It was found that Tl in rice plants in some specific areas even exceeded Xiaoyin Zhang and Wenhuan Yuan have equally contributed to this work.

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the maximum permissible levels [14], exerting great health risks [11]. Thus, it is critical to minimize Tl accumulation in rice to ensure human health.
It is well known that rice growing on flooded soils liberates oxygen into the rhizosphere to maintain normal respiration. At the same time, Fe(II) is oxidized into Fe(III), which forms precipitation as iron plaque wrapping root surfaces and exerts an important role in metal transport from paddy soil to plant roots [15][16][17][18]. Owing to its sorptive functional groups, iron plaque can adsorb or co-precipitate various metal ions, such as Cr, As, Hg, Ni, Cu, and Pb, thereby affecting their uptake by plants [12,[19][20][21][22][23]. However, little is known about the relationship between iron plaque and Tl accumulation in rice.
Thus, the aims of this study are to investigate (1) the migration and transformation characteristics of Tl in typical paddy soil systems and the effects of Fe species on the geochemical fractionation of Tl; (2) and the effects of Fe species on the uptake and translocation of Tl in rice plants using pot culture experiments.

Fieldwork sampling and pretreatment of paddy soil samples
In total, 18 Tl-contaminated soils were collected from paddy farmland near an industrial zone that utilizes Tlbearing pyrite minerals in Guangdong Province, China. Each soil sample was weighed and loaded into 50 mL centrifuge tubes at a soil-liquid (deionized water and 0.01 mol/L CaCl 2 solution) ratio of 1:10. The supernatant obtained by centrifugal shaking at room temperature for 2 h and centrifugation for 10 min was filtered and used to determine the respective content of Tl water-soluble and CaCl 2 -extracted Tl [24,25].
A total of 10.000 g of soil sample was used to obtain the solution for the determination of Fe ions. 1 mL of the solution to be measured was fixed to 50 mL by adding 10% sodium acetate solution, 0.1% o-phenanthroline solution, and distilled water to determine the Fe 2+ concentration by Ultraviolet Spectrophotometry [26]. Ultimately, the Fe 3+ content was equal to the difference between the known total iron and ferrous iron concentrations.
Three geochemical fractions of Tl, namely, weakly acid soluble (F1), reducible (F2), and oxidizable (F3), were determined by a modified IRMM (Institute for Reference Materials and Measurement, Europe) sequential extraction procedure. For details of the extraction steps, refer to Yang et al. [27]. Soil pH was measured in deionized-water extract at a soil:solution ratio of 1:2.5 (w/v).

Hydroponics experiment
As shown in Fig. 1a, 120 high-quality rice seeds of Yexi-angyou688 were cultivated at the site of our field work in Guangdong Province. Inorganic iron ions can be easily oxidized by oxygen in the air during the cultivation process and can be easily affected by environmental pH variations during water and/or soil culture stages. Therefore, in this study, EDTA (eathylene diamine tetraacetic acid)-Fe(II) was used as the iron source to induce the formation of an iron film with a stable effect and prevent Fe 2+ from being oxidized less than expected during the cultivation process. The rice seeds were selected and cultured until rooting and germination to 2-3 cm (12 days) and then transferred to artificial incubators containing four concentration gradients of EDTA-Fe(II) supply solution at 0, 10, 20, 30, and 40 mg/L, respectively [28,29]. By adding EDTA-Fe(II) solutions of different concentrations, different amounts of iron films were induced. When the seed roots appeared distinctly reddish-brown and remained unchanged for several days (approximately 12 days) (Fig. 1b), they were transferred to the specimen soil for further incubation.

Rice cultivation in soil
Five plastic culture containers were set up according to different concentrations of EDTA-Fe(II), and 600 g of soil containing organic nutrient solution was put in, flooded, and equilibrated for 2 weeks. From the rice cultivated with different concentrations of EDTA-Fe(II), 2-4 rice seedlings with good uniform growth were selected separately and transplanted to the pretreated soil samples for 1 month and then harvested (Fig. 1c).

Preparation and analysis of rice plant samples
Whole rice root systems were carefully removed from the potted containers, and each plant sample was washed at least three times with deionized water until the whole plant surface was completely washed. The roots were cut from the base of the stem and leaves, and after the root system was extracted from the root surface iron film, the remaining stem and leaf parts were dried in an oven at 70 °C for 72 h and weighed, crushed, and prepared for use. 0.1 g of each root and stem and leaf were digested with 5-10 mL of concentrated nitric acid. The contents of Fe and Tl in the extracts were determined by Inductively Coupled Plasma Mass Spectrometer (ICP-MS, Elan 6000, PerkinElmer, Waltham, MA, USA).

Preparation and analysis of iron plaque
The DCB (sodium citrate-sodium bicarbonate-sodium dithionite), was used to leach iron plaque on the root surface [30][31][32] and the contents of Fe and Tl in the extracts were determined by ICP-MS.

Geochemical fractionation of Tl and Fe in the soils from paddy fields
In region A, for the geochemical fractionation, Tl was mostly enriched in the weak acid soluble fraction (F1), followed by reducible fraction (F2) and oxidizable fraction (F3) in most of the soils (Fig. 2). It can be preliminarily suggested that Tl is active in these soils, which can be easily used by plants [33]. For the geochemical fractionation, Tl content in the soils from the region B and region C generally decreased in the order of F2, F1, and F3 (Fig. 2). This may be explained by the fact that Tl can be easily adsorbed to the iron-manganese oxide surface [33]. Previous studies have also shown that the F2 fraction is the main host for Tl fixation in soil [34][35][36][37]. In acidic soils, redox reactions can enable the release of Tl. Overall, Tl was highly bioavailable in all the studied soils.

Characteristics of the distribution of Fe 2+ and Fe 3+ content in paddy soils
As shown in Fig. 3, the average Fe content in the soils decreased in the order of region B, region A, and region C. The soils in Region B contained significantly more Fe 3+ (34,600-59,700 mg/ kg) than Fe 2+ (6500-9300 mg/kg) (Fig. 3). The soils from region A were featured by a higher Fe 3+ content (10,000-15,000 mg/ kg) than Fe 2+ (13,200-28,700 mg/kg). However, soils from region C contained lower Fe 3+ (300-14,600 mg/kg) than Fe 2+ (17,000-24,000 mg/kg).

Relationship between geochemical forms of Fe and Tl in soils
To understand the association between the geochemical forms of Tl and Fe, a correlation analysis was performed. As shown in Table 1, Fe 2+ showed a significant positive correlation with oxidizable Tl, with a correlation coefficient of 0.856 (p < 0.01). No significant correlation was observed between Fe 2+ and weak acid soluble and reducible fractionation of Tl. However, Fe 2+ showed a negative correlation (R 2 = − 0.405) with the reducible fractionation of Tl. Fe 3+ displayed a significantly negative correlation with weak acid soluble Tl and oxidizable Tl, but a significantly positive correlation coefficient of 0.569 (p < 0.05) with reducible Tl. When the soil system is under reducing conditions, obvious reduction and dissolution of Fe oxide will occur [35,37]. Meanwhile, Fe 2+ acts as an important reducing agent in the soil, and the redox reaction of iron ions is not only related to the migration and release of metal pollutants adsorbed by iron [38]. This fully shows that the redox reaction of Fe ions is related to the migration and transformation of metals in the soil, and that Fe 2+ is one of the key factors in the formation and quantity of the root surface iron membrane.
To sum up, different valent states of Fe in the soil affect the geochemical fraction of Tl. The correlation is the most significant between the Fe 2+ and Fe 3+ contents and the oxidizable fraction of Tl. When the organic matter in the soil is oxidized and decomposed, metal in the oxidizable fraction is released into the environment. This finding indicates that the conversion of Fe 2+ and Fe 3+ under oxidative conditions will affect the content of Tl in soil and change the bioavailability of Tl. When soil is flooded in the rice cultivation process, the rice rhizosphere can make the environment in the oxidation stage due to its oxidation capacity, thereby facilitating Tl release.

Physical and chemical properties of soil
The soil for the pot culture experiment was acidic with a pH of 4.79 and Tl content of 11.00 μg/g, which were obviously higher than the background values in Guangdong Province (0.52 μg/g) and China (0.58 μg/g). In addition, the Eh value of the soil sample is 128 mV, which may indicate a high oxidation ability.

Morphological characteristics of Tl in soil
In the soil for the pot culture experiment, Tl was mainly in the reducible fraction (F2), followed by the weak acid soluble fraction (F1) and oxidizable fraction (F3) (Fig. 4), with contents of 0.710 μg/g, 0.564 μg/g, and 0.462 μg/g, respectively. Since the reducible fraction contains Fe-Mn oxide-binding state, which can provide abundant Tl-binding reaction sites, Tl in the reducible fraction is preferentially adsorbed onto the vacancies of the Fe-Mn oxide phase.

Relationship between Fe film and Tl absorption in rice root surface
Two-to-three plants that were hydroponically cultured with different Fe 2+ concentrations to the seedling in the early stage were planted in the soil samples. After growing for 1 month (tillering stage), rice plants were further analyzed for Fe and Tl content in DCB extract of Fe plaque from the root surface. As shown in Fig. 5, Fe content and Tl content in the Fe plaque varied greatly in the rice grown at different Fe 2+ concentrations during the hydroponic stage. The application of different concentrations of Fe 2+ in the early stage of hydroponics is related to the Fe content in Fe plaque from the root surface. Fe 2+ shows a positive correlation with Tl in Fe plaque on the root surface. The formation of Fe plaque on the root surface is derived from the continuous oxidation of Fe 2+ in the soil solution in rhizosphere microenvironment. Since the soil samples of cultivated rice were consistent and only one rice variety was selected in this experiment, the differences in the oxidation conditions of rice roots could be excluded from the reasons that led to the change in Fe plaque quantity. Thus, it can be demonstrated that Fe 2+ is one of the key factors for the formation and quantity of iron film on the root surface.
The correlation analysis indicated that there was a significant positive correlation between the content of iron plaque on the root surface and the content of Tl in iron plaque (R 2 = 0.529) (Fig. 6). Comparing the content of Tl in rice root surface iron plaque with that in rice stems and leaves, the content of Tl in iron plaque was greater than that in stems and leaves (Fig. 5). Based on the control of the aboveground growth environment of rice, it can be explained that the formation of iron plaque reduces the accumulation of Tl in above-ground stems and leaves. The iron plaque on the root surface acts as a physical barrier layer on the root surface to prevent the migration of heavy metal ions into the plant and act as a temporary reservoir [38]. This explains why iron plaque on the root surface plays an important role in the migration of heavy metal ions into the plants. The iron plaque absorbs and fixes part of Tl and wraps a large amount of Tl in the root, thus reducing the migration of Tl to the stems and leaves of the above-ground part. This can also explain the increase in Tl in iron plaque on the surface of rice roots.
In addition, the uptake rate of water-soluble Tl in soil by the iron film on the root surface ranged from 42.52% to  . 4 Geochemical fractions of Tl in the soil used for pot experiments 98.68%, and the uptake rate of CaCl 2 -extracted Tl ranged from 45.13% to 98.70%. The soil flooding conditions caused the activation of Fe-Mn oxides in the soil, which led to an increase in the surface adsorption sites and the adsorption capacity produced for soil metals [39]. This is due to the fact that the adsorption of metal ions by iron oxides under flooded conditions is always accompanied by the release of H+ from the adsorbed surface [40]. Thus, the release of H + from the surface of iron oxides increases the negative charge and, consequently, the adsorption of positively charged metal ionophores in solution, leading to a decrease in the solubility of metals in the soil [41]. There was a significant difference in Tl in the soil compared to the original state, and the amount of iron film was significantly negatively correlated with Tl content in the soil, with a higher utilization of the water-soluble state and a correlation coefficient of −0.90 (Fig. 7). With the increase in ferrous concentration, Tl in the water-soluble and CaCl 2 -extracted fractions of the soil showed a general decreasing trend.

Conclusions
The experimental results of field samples in this study showed that Tl in typical Tl-contaminated rice soils existed mainly as a fraction bound to Fe-Mn oxides/hydroxides, with a high risk of release under acidic conditions. The oxidizable Tl exerted significant positive correlation relationships with Fe 2+ and negative correlation relationships with Fe 3+ , while the reducible Tl only displayed a positive correlation with Fe 3+ . All these findings reveal that variations in Fe(II) and Fe(III) concentrations may be one of the key factors in geochemical transformation of Tl in soil, which affects Tl uptake by rice. The hydroponic experiments revealed that Fe 2+ addition contributed to elevated amounts of Fe plaque on the root surface. Meanwhile, iron plaque promotes Tl uptake and fixation at the root surface of rice, thus hindering further migration of Tl to the stem and leaves. This study provides an important scientific and theoretical reference for initiatives related to reducing heavy metal accumulation in rice grown on Tl-contaminated farmland and further safeguarding human dietary safety.