The contribution of atmospheric deposition of cadmium and lead to their accumulation in rice grains

Over-accumulation of cadmium and lead in rice grain is a global concern as it has adverse health impacts. Atmospheric deposition is an important source of heavy metal accumulation in soil, but contribution to crops has not been quantified and the mechanisms of foliar Cd and Pb uptake via the stomata of rice leaves exposed to atmospheric fallout are unclear. To quantify the contribution of atmospheric deposition on Cd and Pb accumulation in rice grains, a rice pot experiment with four exposure treatments (T1, all day exposure without geotextile membranes; T2, all day exposure with geotextile membranes; T3, daytime exposure with geotextile membranes; and T4, night exposure with geotextile membranes) using severely (ZZ) and moderately (XT) polluted soils was conducted. Cd content in shoots and roots was T1 > T2, T3 > T4 in XT soils, and T1 > T2, T4 > T3 in ZZ soils, while Pb content in both soils was T1 > T2, and T4 > T3. Cd and Pb contents in rice grains showed the same trend. Using the isotope ratios tracing method (114/111Cd, 112/111Cd, and 207/206Pb, 208/206Pb), it can be concluded that the contribution of atmospheric deposition to rice grains was quantified as 63.55% and 18.01% for Cd, and 27.69% and 41.13% for Pb in XT and ZZ soils, respectively. Foliar uptake atmospheric deposition had substantial effect on Cd and Pb accumulation in rice grains and the control of heavy metal foliar uptake should be paid more attention to maintain rice safety production.


Introduction
Cadmium and lead are highly toxic trace elements in the environment and can cause obvious damage to human health even at low concentrations (Bohdálková et al. 2020). Studies have proven that crops contaminated with Cd and Pb can be passed on through the food chain and pose a direct threat to 1 3 Vol:. (1234567890) humans due to biomagnification effect (Keller et al. 2014;Baycu et al. 2016). Therefore, food safety issues caused by over-accumulation of Cd and Pb in crops has drawn increasing attention recently. Rice (Oryza sativa L.) is the most important staple food for half of humanity. China (especially southwestern China) is one of the countries with high consumption and production of rice. Liu et al. (2016) collected and analyzed heavy metals in 101 pairs of soil and rice samples from the main rice-producing areas along the Yangtze River in China, and found that the average content of Cd in paddy soils was 0.73 mg kg − 1 with an increasing rate of 0.01 mg/kg/ year. Du et al. (2013) conducted a monitoring test in paddy fields in northern Hunan Province and found that the average content of Cd in soils was 0.64 (range from 0.13 to 6.02) mg kg − 1 , with 0.46 (range from 0.01 to 2.77) mg kg − 1 in rice grains, exceeding the limits specified in China's Food Safety and Quality Standards (GB/T 1354(GB/T -2009. Therefore, it is critical to pay efforts to identify the source of origin of Cd and Pb and quantify their accumulation in rice grains. Where are the Cd and Pb coming from? Metal elements can be assimilated through both the root and leaf (Schreck et al. 2014). Many studies have proved that the accumulated heavy metals in rice grains are taken up by roots from the soil and then translocated to edible parts through xylem and phloem (Li et al. 2017). Moreover, foliar uptake can also be an important source responsible for heavy metal accumulation in grains, particularly when ultra-fine particles directly interact with plant leaves (Feng et al. 2011;Hu et al. 2011;Schreck et al. 2012). Airborne PM 2.5 can be directly absorbed by leaves through the stomata and then translocated to other parts of plant, which is closely related to the characteristics of stomata. (Gao et al. 2021;Melotto et al. 2017). The stomata are sensitive to light changes, salt stress, and drought. (Xiong et al. 2018;Mekonnen et al. 2016). It was found that the opening and closing of stomata under light and dark conditions had a considerable influence on metal accumulation through foliar uptake (Gao et al. 2021;Zhou et al. 2020) conducted a field experiment and found that atmospheric metal deposition can lead to over-accumulation of Cu and Pb even in rice crop planted in clean soil. De Temmerman et al. (2012a, b) found the levels of As and Cd in carrot and celery were significantly correlated with the amount of heavy metals in atmospheric deposition.
In recent decades, enormous anthropogenic emissions have resulted in a high level of heavy metals in the atmosphere. In 2012, the anthropogenic emissions of Cu, Pb, and Cd in China were approximately 9547 t, 14,397 t, and 527 t, respectively (Tian et al. 2015). The large stock of heavy metals in atmosphere has already become one of the main contributors to heavy metal accumulation in agricultural soils through long-term rain and dust fallout (Luo et al. 2009;Bermudez et al. 2012;Hou et al. 2014;Feng et al. 2018;Zhang et al. 2018). Studies have shown that the activity of Cd and Pb in atmospheric particulate matter (PM) was high (Liu et al. 2019a, b). For example, in a coastal city of northern Spain, the water-soluble exchangeable components of Cd and Cu in atmospheric deposition accounted for 52% and 43%, respectively (Fernández-Olmo et al. 2014). In Asia, 30-84% of Cd was in exchangeable form, while the components of Pb were mainly in carbonate and reduced form, indicating that heavy metals have higher potential of bioavailability in PMs than in soil (Lee et al. 2015). Therefore, except for root uptake, foliar uptake of Cd and Pb from atmospheric deposition can also be an important source of Cd and Pb accumulation in rice grain.
To explore the contribution of atmospheric deposition to Cd and Pb accumulated in rice grain, there have been many attempts by many scholars. Feng et al. (2019) found that Cd and Pb accumulation in rice grains planted in more polluted deposition zones were significantly higher than in zones with less polluted deposition, and the contribution of atmospheric deposition accounted for more than 35% for Pb, and 40% for Cd. Liu et al. (2019a, b) conducted pot experiments with the same soils in three sites of graded atmospheric heavy metal deposition and found 52-62% of Pb accumulation in vegetable shoots was directly taken up through leaf stomata. Zhou et al. (2020) also conducted a similar experiment, and concluded that atmospheric deposition contributed 31-63% (in moderate deposition areas), and 66-84% (in high deposition areas) to Pb accumulation in rice grains. Cao et al. (2020) conducted a simulated wet precipitation experiment with hydroponic culture of rice at seedling stage and found that Cd concentration in rice shoots increases significantly when the annual Cd 1 3 Vol.: (0123456789) precipitation flux ≥ 50 g ha − 1 (amount of the annual Cd flux in industrial areas was about 61.0 g ha − 1 ). These monitoring and simulation experiments prove that foliar uptake can increase Cd and Pb accumulation in rice grains, but they fail to precisely quantify the contribution.
Elements have fixed stable isotope ratios of homologous pollutants and can maintain this composition in the process of migration and reaction. Therefore, stable isotope ratios have been widely used as a "fingerprint" in source apportionment analyses in aerosols (Jaeckels et al. 2007;Yin et al. 2010), sediments (Tang et al. 2010), urban soils (Wong and Li 2004), and agricultural soils (Huang et al. 2015), but rarely in crops. Liu et al. (2019a, b) used Pb isotope ratio analysis to quantify the sources of lead in soil and rice grains and showed that the sources of Pb in soil were background sources (20-84%), chemical fertilizers (0-42%), atmospheric deposition (16-42%), and irrigation water (0-28%), while that in rice grains, was atmospheric deposition (76-98%), and the soil (0-26%). Bi et al. (2009) analyzed Pb isotope ratios in soil and corn crops and concluded that the main source of Pb in corn grains and leaves was atmospheric, while Pb in roots and culms mainly came from the soil. However, the analysis of Cd isotope ratios was not determined.
Therefore, a full growth period, exposed pot experiment in rice was conducted along with longterm monitoring of atmospheric PM (PM 10 and TSP). The content of Cd and Pb in shoot, root, grain, soil, and atmospheric depositions, as well as 112/111 Cd, 114/111 Cd,207/206 Pb,and 208/206 Pb were determined to explore the effects of stomata and exposure time on foliar uptake, and to quantify the contribution of atmospheric deposition on Cd and Pb accumulation in rice grains. The current study aimed to (1) explore the deposition fluxes of Cd and Pb in the study area; (2) to investigate the temporal change of Cd and Pb accumulation in rice with foliar uptake in a full growth period; (3) to assess and quantify the influence of atmospheric deposition on Cd and Pb accumulation in rice grains. The results will elucidate the foliar uptake mechanism of Cd and Pb in rice, and Cd and Pb accumulation in rice leaves through stomata, which can provide technical support to the control and management of heavy metal contamination in crops.

Study area and rice cultivation
The study was conducted at the experimental base of Hunan Agricultural University, Changsha City, southern China. This is a suburban area and not surrounded by typical sources of pollution, such as factories and mines. The leading wind direction prevails southeasterly in summer and northeasterly in winter. The annual average temperature range was 16 ℃-18 ℃, with an annual average humidly of 70-80%. The average annual precipitation ranged from 1400 to 1700 mm.

Treatments design
To explore the contribution of atmospheric deposition on Cd and Pb accumulation in rice grain, a full plant growth outdoor exposure (all day exposure/day exposure/night exposure) pot experiments with two types of contaminated soils was set up. Moderately and severely contaminated soils were collected from paddy fields in Xiangtan city (XT) (the content of organic matter: 32.7 g kg − 1 , content of Cd: 0.33 mg kg − 1 , content of Pb: 61.9 mg kg − 1 , pH: 5.42) and Zhuzhou city (ZZ) (the content of organic matter: 30.6 g kg − 1 , content of Cd: 0.86 mg kg − 1 , content of Pb: 65.2 mg kg − 1 , pH: 5.42). To avoid direct input of Cd and Pb from atmospheric deposition to soil and then direct uptake by root, geotextile membrane was used to isolate atmospheric matters. Therefore, there were four treatments in the current study: T1 (all day exposure without geotextile membrane), T2 (all day exposure with geotextile membrane), T3 (daytime exposure with geotextile membrane) and T4 (night exposure with geotextile membrane). Four treatments with 12 biological replicates per treatment, a total of 48 pots. Four biological replicates could be taken in each period, and all samples were taken in exactly three periods.
The tested variety is Fengliangyou 4 of middleseason rice. The germinated seeds were firstly cultivated in uncontaminated soils for one month, then transplanted to the pots in mid-May 2019, and finally harvested in early-November 2019. Plants were exposed for the entire growth period and separately sampled in tillering (16th July 2019), heading (3rd

Particulate matter (PM) samples
The PM samples were collected by an automatic precipitation and dust fall sampler each day from May 2019 to November 2019. The airflow rate was 28 L min − 1 with 50% cutoff sizes of > 10.0 μm (PM 10 ), and total suspended particles (TSP). The PM samples were digested using a HNO 3 -HCl (3:1) mixture at 105 ± 5 ℃ for 2 h, cooled to room temperature (25 ℃-27 ℃), and diluted with deionized water to 30 mL. The aqueous samples were first acidified by sub-boiling, quartz-distilled 6 N HCl, and then digested using 2 mL HNO 3 (1 + 1) and 1 mL HCl (1 + 1) at 85 ℃ until the sample was evaporated to 20 mL. Concentrations of Cd and Pb were determined with an inductively coupled plasma mass spectrometer (ICP-MS, Model NEXION 350 X). Sample replicates and reagent blanks were included in each batch analyzed to ensure the quality of analysis.

Rice and soil samples
A whole plant in one pot was taken in each growing period, and the samples were rinsed thoroughly with deionized water to remove any attached soil/substrate particles before separating into shoots and roots. At the same time, surface soil (0-15 cm) samples in each pot were collected. The soil samples were dried at room temperature and homogenized using a mortar and pestle. One portion of the sample was sent through 2 mm sieves to measure the pH, while the other portion was passed through 0.145 mm sieves. Subsamples of grains, shoots, and roots were firstly dried in the oven at 105 ℃ for 30 min, then kept in the oven at 80 ℃ until they reached a constant weight, and then crushed into powders.
Soil samples were digested with a mixture of HNO 3 -H 2 O 2 -HF (6:3:3), and plant materials were digested with HNO 3 -H 2 O 2 (2:1). The digested solutions were diluted to 25 ml with 1% HNO 3 solution. All samples were stored in dark at 4 ℃ and were determined within one month. The concentrations of Cd and Pb in solutions were determined using an inductively coupled plasma mass spectrometer (ICP-MS, Model NEXION 350 X).
To ensure the reliability and quality of the data, standard reference soil (GBW07387, Cd: 0.34 mg kg − 1 ; Pb: 28 mg kg − 1 ), and rice (GBW(E) 100,360, Cd: 0.22 mg kg − 1 ; Pb: 0.049 mg kg − 1 ) samples from the Center of National Reference Materials of China were measured together with soil and plant samples. The recovery of the spiked standard for each element ranged between 90 and 110%, and the limits of detection were 0.01 µg mL − 1 and 0.01 µg mL − 1 for Cd and Pb, respectively.

Scanning electron microscopy (SEM) analysis
Small strips of rice leaf (0.5 cm 2 ) were trimmed between margin and mid-rib. The small strips were first kept in 2.5% glutaraldehyde solution, then left overnight for fixation (prefixation), and then kept in osmium tetroxide for post fixation for 1 h. After washing twice with buffer solution for 15 min, these samples were passed through a series of acetone solutions (30%, 50%, 70%, 95%, and 100%) for dehydration. Samples were then dried in a critical point drier (CPD) with CO 2 as a carrier gas. SEM was carried out using a 30 keV, JEOL JSM-6490 LV scanning microscope with standard automated features such as auto focus/stigmator, auto gun, and auto contrast with multiple live image displays. The samples were coated with carbon using a high vacuum system for wet specimens, the upper surfaces of the leaf segments were studied, and micrographs were taken at various magnifications.

Foliar photosynthetic parameter analysis
The foliar photosynthetic parameters (transpiration rate [Tr], and stomatal conductance [Gs]) were recorded simultaneously at tillering and heading stage using a portable infrared gas analyzer (LI-COR6400XT). The infrared gas analyzer system (IRGAs) was equipped with a clamp-on leaf chamber of 6 cm 2 . According to Majer and Hideg (2012), IRGAs measure the reduction in transmission of infrared wavebands caused by the presence of CO 2 between the radiation source and a detector. The reduction in transmission is a function of the concentration of CO 2 . Thus, the measurements of gas exchange are based on the differences in CO 2 and H 2 O in an air stream that is flowing into a leaf cuvette (reference cell) compared to the air stream flowing out of it (sample cell). The rate of CO 2 uptake (µmol (m 2 ·sec) −1 ) is used to assess the rate of photosynthetic carbon assimilation, while the rate of water loss (mol H 2 O (m 2 ·sec) −1 ) is used to assess the rate of transpiration and stomatal conductance. Gas exchange measurements were performed under irradiance at 425 µmol (m 2 ·sec) −1 and at a temperature of 23 ± 2 °C. During measurement, humidity was fixed at 65%, and CO 2 concentration was maintained at a constant level of 380 µmol mol − 1 using a LI-6400-01 CO 2 injector with a high-pressure liquid CO 2 cartridge source.

Cd and pb isotope ratio analysis
To identify the contributions of atmospheric deposition and the soil to the Cd and Pb accumulation in the edible of rice, the stable Cd and Pb isotope ratios 112/111 Cd, 114/111 Cd,207/206 Pb,and 208/206 Pb in the rice samples, atmospheric deposition and soil were determined using Inductively coupled plasma mass spectrometer (ICAP-QC) (Thermo Fisher Scientific, Waltham, MA, USA). The operating conditions of the ICAP-QC were: RF power 1550 W, atomizing chamber rate 40 r/min, atomizing chamber temperature 2.5 ℃, atomized gas 1.0749 L/min, integration time 0.02 s. In consideration of elemental abundance and isobaric elements, 112/111 Cd,114/111 Cd,207/206 Pb,and 208/206 Pb were chosen for isotope ratio analysis. T1 isotope standard solution (SRM NIST 997) was used to correct the instrumental mass bias. Standard solutions of Cd (GBW 04622), and Pb (SRM NIST 981) were measured after five unknown samples to verify the results.
In the current study, we hypothesized that Cd and Pb in rice were mainly from atmospheric deposition and soil. On the basis of the measured abundance of 111 Cd, 112 Cd, 113 Cd, 206 Pb, 207 Pb, and 208 Pb in the samples, the Cd and Pb contributions of the atmospheric PM and soil can be identified. Therefore, the contributions of soil and atmospheric deposition could be calculated as: Each element had two isotope systems ( 112/111 Cd, 114/111 Cd, 207/206 Pb, and 208/206 Pb), and two potential sources. Where δ A , δ s are isotopic signatures of the two major sources (atmosphere and soil), and f A , f s are their relative contributions, respectively.

Statistical analysis
Data analysis was performed using SPSS 16.0, R Studio and Origin Pro 8.0. The datasets of photosynthetic parameters were subjected to a factorial analysis of variance (ANOVA) using a least significant difference test at a significance level of p < 0.05 and p < 0.01. Using forest package, the random forest algorithm was used to calculate the interpretation intensity of the various factors with Cd content in rice grains. . TSP-Pb and PM 10 -Pb were also lower than the first-class standard of the Ambient Air Quality Standard (GB3095-2012, 0.5 µg m − 3 ). The concentrations of TSP-Pb and PM 10 -Pb in the atmosphere were higher from July to September, and then lower in October. Moreover, the concentration of Pb in PM 10 was significantly higher than that in TSP, which indicated that Pb was mainly enriched in fine particles (p < 0.05).

Cd content in rice shoots and roots
The content of Cd accumulated in shoots and roots was significantly higher in severely Cd polluted (ZZ: 0.86 mg kg − 1 of Cd in soil) soils than that in moderately Cd polluted soil (XT: 0.33 mg kg − 1 of Cd in soil) (Fig. 2). An increasing trend was observed in shoot and root concentrations during growth. In XT soil, there was no significant difference in shoot Cd content between T1 (0.43 mg kg − 1 , 1.50 mg kg − 1 ) and T2 (0.42 mg kg − 1 , 1.54 mg kg − 1 ) at tillering stage and heading stage, while T3 (1.57 mg kg − 1 ) > T4 (1.13 mg kg − 1 ) at heading stage (p < 0.05). The trend of shoot Cd content in ripening stage was T1 (2.93 mg kg − 1 ) > T2 (2.06 mg kg − 1 ) > T4 (1.78 mg kg − 1 ) ≈ T3 (1.75 mg kg − 1 ), and both T2 and T3 was significantly higher than T4 (p < 0.05). The Cd content in roots of the three stages were T1 > T2 but with no significant difference; T3 (3.95 mg kg − 1 , 5.58 mg kg − 1 ) > T4 (2.90 mg kg − 1 , 3.95 mg kg − 1 ) and there was a significant difference between the heading and ripening stages (p < 0.05). In ZZ soil, a similar trend was observed in the shoots and roots. The shoot Cd content in the three periods were T1 (0.42-7.57 mg kg − 1 ) > T2 (0.24-4.10 mg kg − 1 ), T4 (0.34-2.81 mg kg − 1 ) > T3 (0.19-1.78 mg kg − 1 ) (p < 0.05). At ripening stage, the Cd content in shoots and roots under T1 treatment was the highest. Therefore, the results indicated that exposure to atmospheric deposition was beneficial to the accumulation of Cd in rice. In addition, T3 was larger than T4 in shoots and roots in XT soils, which was different from the result that T4 > T3 in ZZ soils. This indicates that rice is highly affected by transpiration rate in ZZ soils, and roots absorb more Cd in soils with high Cd content.

Accumulation of cadmium and lead in rice
Plant growth stages exerted significant effects on Pb and Cd accumulation in each rice plant, and the accumulation of Cd increased with higher soil Cd concentration (Fig. 5). In general, the maximum Cd and Pb accumulation in rice occurred at ripening stage. In both XT and ZZ soils, the accumulation of Cd of each plant in T1 (257.57 ug plant − 1 and 507.78 ug plant − 1 ) was higher than that in T2 (146.71 ug plant − 1 and 254.43 ug plant − 1 ), T3 (197.76 ). Moreover, the accumulation of Cd in shoots was higher than in roots in both soils, while the opposite was true for Pb. More specifically, the amount of Pb accumulated in root was several times higher than in the shoots. However, the accumulation of Cd in roots at ripening stage remained at a relatively low level, although an increasing trend was observed for shoots at the same time. The phenomenon of Cd accumulation in root decreased at ripening stage, probably due to transportation to shoot, while Pb mainly accumulated in root probably due to poor mobility. The main reason for these differences more likely resulted from atmospheric-soil-root transfer as well as the direct transfer of contaminants from the atmosphere to foliar uptake.

Cadmium and lead isotope ratio analysis
To find out the source of heavy metal accumulated in rice grains, 114/111 Cd ratios were plotted against values of 112/111 Cd ratio, and 208/206 Pb against 208/206 Pb for the possible pollutant sources (Fig. 6). For XT soils, the closer points were mainly atmospheric deposition samples, which resulting that atmospheric particulate matter was the most likely source of grain Cd accumulation. The 112 Cd/ 111 Cd and 114 Cd/ 111 Cd ratios in grains (ZZ) were close to the soil, indicating that the soil was the main source of Cd in the ZZ grains. In the case of Pb, the cluster of soil was close to the grains (XT), indicating that the accumulation of Pb in rice grains was greatly affected by soil content. For ZZ soils, both soil and atmospheric deposition substantially contributed to grain Pb accumulation. The contribution of atmospheric deposition and soil for Cd was 40.32-86.78% (63.55%), 13.22-59.68% (36.45%) in XT soils, and 4.2-33.56% (18.01%), 66.44-95.8% (81.99%) in ZZ soils, respectively. For Pb, atmospheric deposition and soil accounted Cd/ 111 Cd and 207 Pb/ 206 Pb ratio vs. 208 Pb/ 206 Pb for rice grains, roots, shoots, soil and atmospheric deposition (air) at ripening stage in XT (a, c) and ZZ soils (b, d).

Stomatal characteristics of rice leaves
The stomatal aperture causes PM to easily enter the plants when stomata open in the presence of light (Uzu et al. 2010;Shahid et al. 2017). Primarily, stomata size plays a critical role in jamming fine particles (Mansfield and Majernik 1970). The stomata dimensions of rice leaves vary in the tillering, heading, and ripening stages. According to previous work, there is basically no difference in the stomatal distribution of the adaxial and abaxial epidermis of rice leaves; thus, in this study we only observed the front side of rice in each period. The width of stomata in the tillering, heading, and ripening stages were 1.33 μm, 2.16 μm, and 2.84 μm (Fig. 7). The length of stomata and guard cells was greater than 10 μm at tillering and heading stage, and the pore diameter and opening of stomata increased with plant growth, reaching 20 μm at ripening stage, indicating that it is more possible for atmospheric fine particles (PM 10 and PM 2.5 ) to enter rice leaves directly when the stomata open at ripening stage.
Stomatal conductance (gs) is a good indicator of the plant response to environmental changes (Gao et al. 2021). Fig. S1 shows that gs of rice leaves at heading stage is decreased compared with that at tillering stage. At tillering stage, the gs values of the leaves were 0.15-0.21 mmol mm − 2 s − 1 in XT and 0.30-0.35 mmol mm − 2 s − 1 in ZZ; but there was no difference between treatments. At heading stage, it was found that the gs values of T3 (0.12 mmol mm − 2 s − 1 ) were significantly higher than that of T4 (0.04 mmol mm − 2 s − 1 ) in ZZ (p < 0.05).

Characteristics of atmospheric fallout and its effect
Many studies have proved that Pb is a marker of traffic emissions because the process of fuel combustion as we as engines and catalysts loss would emit Pb to the environment (Guan et al. 2018;Li et al. 2013). However, Cd was more related to coal combustion and electronics industries (Guan et al. 2018;Huang et al. 2018). The experimental base was located at the campus of Hunan Agricultural University, which was far away from any typical heavy metal emitters such as factories or mines. Compared with the limits stated in the Environmental Air Quality Standard (GB3095-2012), the average values of Cd and Pb in PMs (PM 10 , TSP) in the study area were both lower than the secondary and primary class standards, indicating light pollution of atmospheric deposition in this area. Overall, the content of Pb in dry depositions was higher than that of Cd. Moreover, the contents of Cd and Pb in PM 10 were higher than those in TSP. Canepari et al. (2008) monitored the distribution of heavy metals in coarse particles (> 2.5 μm) and fine particles (< 2.5 μm) in Rome, Italy, and found that most heavy metals that came from natural sources (such as calcium, barium, iron, magnesium, and manganese) were located in coarse particles, while other elements, like Cd and Pb, which mainly came from  a, b, c), the size of stomata were shown with arrows. Each treatment was repeated three times. The scale unit in each diagram is 5 μm in length anthropogenic emissions (such as vehicle emissions, non-exhaust sources, and railway emissions) were found adsorbed on fine particles. Among them, more than 50% of Cd and Pb were adsorbed on particles with a size < 1 μm. Louie et al. (2005) made the same conclusion from a long time monitoring of PM2.5 and PM 10 in Hong Kong.
A previous study also indicated that heavy metals present in precipitation were more bioavailable (Sharma et al. 2008), which suggests that heavy metal deposition in the present study may cause a significant impact on the surrounding agro-ecological system even though the total amount was not excessive. In this study, a random forest analysis revealed that the duration of exposure of rice leaves to atmospheric deposition was one of the main factors affecting Cd and Pb content in rice grains in both XT and ZZ soils. On the contrary, the Cd and Pb contents in atmospheric deposition was not significant (Fig. 8). Therefore, the content of Cd and Pb in T2 was significantly higher than that in T3 or T4. More precisely, In XT, the duration of foliar exposure to atmospheric deposition was the main factor affecting Cd accumulation in grains. Different from ZZ, Cd content in soil is the key factor. However, for Pb, in both XT and ZZ soils, rice grains were mainly affected by Pb content in shoots and duration of leaves exposure, but not by soil content. Therefore, Pb in grains was mainly from atmospheric deposition absorbed by leaves.
Besides, metals from the atmosphere can settle into the topsoil, changing soil conditions and these metals can also be absorbed by rice roots. Therefore, in the current study, the content of Cd and Pb accumulated in T1 (all day exposure without geotextile membrane) was higher than that in T2 (all day exposure with geotextile membrane). Feng et al. (2019) used a retrospective model to predict the long-term accumulation of heavy metals in paddy soil for over 30 years and showed that long-term atmospheric deposition significantly increased heavy metal accumulation on the surface layer of paddy soils (p < 0.05Liu et al. (2019a, b) conducted ectopic soil experiments in three areas with different atmospheric deposition pollution gradients and reported that the content of Cd in soil increased significantly from 1.56 ± 0.07 mg kg − 1 to 1.83 ± 0.08 mg kg − 1 after exposure to high deposition inputs for over six months, indicating that the accumulation of heavy metals was significantly higher in areas with serious deposition. Moreover, influenced by acid rain and depositions, the pH values in soil would continuously reduce and further activate heavy metal transfer to more active forms (Schreck et al. 2011). Lower soil pH values can directly promote the migration of heavy metals and indirectly promote the uptake of heavy metals by plants (Bermudez et al. 2012). In recent years, several studies have been carried out on the uptake of heavy metals in atmospheric precipitated particles through leaves, and the potential mechanisms have been discussed. Gajbhuye et al. (2016) suggested that heavy metals may enter leaves in two ways: (1) through the stomatal openings in the form of nanoparticles; and (2) through the hydrophilic pathway by diffusing through the aqueous pores of the stomata and cuticle. Therefore, to explore the accumulation mechanism of foliar uptake, healthy leaf samples were collected at tillering stage, heading stage, and ripening stage for SEM analysis to observe the morphological characteristics of the surface of rice leaves. Gao et al. (2021) found that PM 2.5 -Pb can be transferred from atmospheric fallout to leaves via the stomata, and larger stomata apertures were responsible for high Pb accumulation in the leaves. Gajbhuye et al. (2016) found large amounts of PM deposited on the leaf epidermis, covering the stomata and their openings on the front of leaves, and the open stomata and guard cells were completely covered by fine particles (PM < 2 μm). There were pores in stomata and the stratum corneum, through which PM-bound metals can penetrate to leaf tissues (Fernandez and Eichert 2009;Schreck et al. 2012a). Tomasevic and Anicic (2010) analyzed the surface of plant leaves by SEMenergy dispersive X-ray (SEM-EDX) and found that Pb adsorbed by small PM (< 2.5 μm) could be captured on the leaf surface. Stomata size played a critical role in jamming fine particles (Mansfield and Majernik 1970). Sawidis et al. (2012) studied the structure and density of stomata and glandular hairs on leaf surface (front and opposite) of different plants and found that foliar surface characteristics were crucial factors that determined capture efficiency. Therefore, the observation results based on SEM have provided strong evidence that PM particles and absorbed toxic heavy metals (Cd and Pb) can be effectively transferred through the stomata on leaves (Fig. 7).
Stomata is one of the important ways for leaves to absorb heavy metals and the only way to absorb a large amount of low solubility solid PM. Therefore, the openness of stomata greatly affects the efficiency of metal absorption (Kaiser 2015). Moreover, the stomatal width-to-length ratio indicates the degree of stomatal opening (Bu et al. 2014). When the stomata are opened in the presence of light, the stomata opens allow particulate matter to easily enter the plant (Shahid et al. 2017). In addition, temperature has an effect on stomata, increasing the temperature can improve stomata opening, and stomata can reach the maximum opening at 30-50℃ (Lei et al. 2021). In this study, except for heading stage, Cd and Pb content of rice plants exposed during the day (T3) was higher than that exposed at night (T4) in XT, which was consistent with the previous result that the content of Pb in leaves exposed during the day was higher than that at night (Gao et al. 2022). This is due to more light and higher temperatures during the day than at night. Therefore, stomatal variation could be a key factor in regulating foliar Cd and Pb uptake and translocation in rice.
Temperature not only changes stomatal opening, previous studies have proved that increase in temperature effectively accelerates element migration and transport in rice from roots to shoots. In this study, T3 and T4 were exposed during the day and at night, respectively, and placed in a transparent enclosure for the rest of the time. Tested rice was grown in the study area where the average temperature was 34 ℃ and the highest temperature could reach 40 ℃, and the temperature was as high as 50 ℃ inside the device. Fig. S1 shows that stomatal conductance (GS) and transpiration rate (Trmmol) of rice leaves at heading stage is decreased compared with that at tillering stage. It was found that the gs values of T3 were significantly higher than that of T4 at heading stage in ZZ (p < 0.05) and there was no difference in XT. On the contrary, the transpiration rate of T4 was higher than that of T3 (p < 0.05). It indicated that as temperature rises, to prevent water loss, some stomata of rice leaves are closed, and stomatal conductance also decreases. Meanwhile, high temperature in T4 accelerates the transpiration rate and promotes the absorption and transport capacity of the root system through the xylem. Moreover, the uptake of Cd by roots from ZZ soil (0.86 mg kg − 1 ) is more than that in XT soil (0.33 mg kg − 1 ), and the amount of Cd transferred upward to the shoots was more obvious. Therefore, the Cd and Pb content of shoots and roots was T4 > T3, especially in ZZ soil with higher Cd content.

Conclusions
To investigate and quantify the influence of atmospheric deposition on Cd and Pb accumulation in rice grains, a rice pot experiment of four exposure treatments (all day exposure, daytime exposure, and night exposure) in both severely and moderately polluted soils was conducted. Although the total Cd and Pb fluxes in the study area were lower than those of the National Environmental Quality Standard, atmospheric deposition was still an important source of Cd and Pb accumulation in rice. The present experiment proved that long-term exposure to atmospheric deposition could significantly increase the content of Cd and Pb in rice shoots and grains. The results of the isotope ratio analysis proved that the contribution of atmospheric deposition in rice grains for Cd was quantified as 63.55% and 18.01%, and was 27.69% and 41.13% for Pb in XT (moderately) and ZZ soils (severe), respectively. These results indicated that the influence of atmospheric deposition on crop Cd and Pb accumulation should be paid more attention, and that foliar control management can be an effective way to maintain food safety.