Effects of microplastics and biochar on soil cadmium availability and wheat plant performance

Biochar is a promising amendment to promote cadmium (Cd) sorption and fixation in agricultural soil, where microplastics are emerging contaminants in soil. Herein, a greenhouse pot experiment was conducted to elucidate the effects on Cd availability in a soil–plant system by biochar and fresh/aged microplastics application. The fresh microplastics led to an obvious increase in soil Cd availability and Cd uptake by wheat plant, while the aged microplastics increased the available Cd in soil but had no effect on Cd uptake by wheat plant, which was likely attributed to the blocking effect of the aged microplastics on Cd transportation from the soil to the wheat plant. Unexpectedly, biochar had increased Cd availability and Cd uptake. The increased soil soluble Cd was because of both decreased soil pH and elevated dissolved organic matter (DOM) content resulted by biochar addition. Also, the unchanged Cd adsorption of the soil was likely responsible for the increased tested soil Cd availability. In addition, the combined effects of a greater decrease in soil pH, an increase in soil DOM content, and a reduction in Cd adsorption after the addition of microplastics to biochar‐amended soil resulted in a significant increase (ranging from 2.63% to 47.73%) in Cd availability compared to soil treated with biochar alone. Moreover, fresh microplastics inhibited wheat growth, and greater inhibition effect was observed for their aged ones. The biochar elevated the wheat biomass; however, the coexistence of microplastics and biochar decreased the wheat plant biomass compared with biochar alone, due to the negative influence of microplastics in plant growth.


| INTRODUCTION
The effects of biochar on Cd availability in soil have been widely reported (Tan et al., 2017(Tan et al., , 2020. Chen et al. (2019) reported that biochar addition at an 8% (w/w) rate significantly reduced the available Cd content in soil, which was mainly due to the changes in soil pH and total organic carbon. In other studies, biochar from orchard prunes decreased the diethylenetriaminepentaacetic acid (DTPA)-extractable Cd, Pb, and Zn amounts by 90%, 38%, and 24%, respectively (Fellet et al., 2011). However, some studies have shown that biochar may have no effect or even increase the availability of heavy metals including Cu and Cd in soils. For example, Yin et al. (2016) suggested that biochar significantly increased the extractable Cd levels using Synthetic Precipitation Leaching Procedure method, which indicated a higher acid solubility for Cd in biochar-amended soils. In addition, Mackie et al. (2015) reported that biochar addition had no significant effect on the reduction of DTPA-extractable soil Cu and the accumulation of Cu by plants. Therefore, the effect of biochar on soil Cd availability has not been definitively concluded, which needs further research.
Soil is also a reservoir for multiple contaminants. One of the emerging contaminants in soil is microplastics. Microplastics are defined as plastic debris smaller than 5 mm in size, which are recognized as emerging contaminants and have attracted increasing concern across the globe . Many studies have shown that microplastics are widely present in aquatic environments, but in recent years, the occurrences and effects of microplastics in terrestrial ecosystems such as agricultural soils have attracted particular interest (Ren et al., 2019;Rillig et al., 2021). In Europe and North America, an estimated annual accumulation of 63,000-430,000 and 44,000-300,000 tons of microplastics, respectively, occurs in farmlands (Nizzetto et al., 2016). Microplastics have properties that are different from soil particles, and microplastic accumulations in soils may alter the soil physicochemical properties, such as increasing the water holding capacity and decreasing the soil bulk density and water-stable aggregates (de Souza Machado et al., 2018). Microplastics often become embedded in soil microstructures by agglomerating with organic matter and microbial secretions . In addition, microplastics have the potential to affect the dissolved organic matter (DOM) in soil environments. It can alter soil DOM composition by decreasing tryptophan-like substances and increasing humic-like substances (Li, Liu, et al., 2022;Li, Xi, et al., 2022), and stimulate enzymatic activity to activate the organic C, N, P pools and increase the DOM levels . The presence of microplastics in soil inevitably changes the soil microenvironment. Until now, the effects of microplastics on plant growing are still not clear, particularly for terrestrial plants. A few studies have shown that the effects of microplastics on plants are intricate and uncertain, and depend on factors such as plant species and the type of plastic involved (Boots et al., 2019;Qi et al., 2020). For example, microplastics can reduce the total biomass of wheat and decrease plant heights and root lengths (Qi et al., 2020) but increase the root biomass of ryegrass (Boots et al., 2019). As a result, the effects of microplastics on plant growing need further study. Furthermore, microplastics undergo aging processes due to environmental exposure (e.g., weathering, UV exposure, and other abiotic or biotic factors), which may change the surface charge, roughness, porosity, polarity, and hydrophobicity of microplastics, and therefore affects their environmental behaviors and adsorption behaviors toward contaminants (Hu et al., 2020).
The association of Cd with microplastics has been reported in both laboratory studies and field surveys, and microplastics can serve as a vector to carry metals and introduce them into the food chain through biological absorption, which causes a greater risk to organisms . Hence, when biochars are used to treat Cdcontaminated soils that also contain microplastics, it is reasonable to speculate that microplastics may affect the sorption of Cd onto biochar and thus influence the bioavailability and toxicity of Cd. In addition, the expandable, low-density and flexible structure of microplastics (Guo et al., 2012), as well as the abundant functional groups on the biochar and microplastics surfaces, make it easy for the microplastics to interact with biochar (Bandara et al., 2019), which would thus influence the ability of biochar to remove Cd. Even though there are growing concerns regarding microplastic pollution in soil, the existing research on microplastics in the soil-organism system, as well as the effects that microplastics could have on Cd removal by biochar, is still very limited.
In this study, we performed a greenhouse pot experiment using wheat as a model plant and the two most frequently used plastics for agricultural mulch (polyethylene [PE] and polypropylene [PP]) and their aged products as the applied plastic residues to investigate the potential effects on Cd availability in the soil containing microplastics and biochar. To the best of our knowledge, this is the first study to investigate the effects on soil Cd availability by the coexistence of biochar and microplastics. This research will provide scientific data for understanding the availability of soil Cd in the presence of microplastics and better guide the remediation applications of biochar for the agricultural soils polluted by Cd and microplastics.

| Facilities and soil
The experimental soil was obtained from the top 0-20 cm soil layer of an experimental station located in the Haidian district, Beijing. Before use, the collected soil sample was thoroughly mixed, air-dried, and sieved through a 2-mm mesh. The soil consists of 61.02% sand, 28.99% silt, and 9.99% clay, with a pH of 8.24 and organic matter content of 4.2 g kg −1 . The initial Cd content in the test soil was 0.16 mg · kg −1 .

| Production and characterization of biochar
The maize straw was pyrolyzed for 2 h at temperatures of 450 and 600°C using a muffle furnace under a pure N 2 atmosphere to produce biochar. Next, the biochar was sieved and passed through a 250-μm sieve for use in the pot experiment. The two biochars that were derived from maize straw at temperatures of 450 and 600°C were abbreviated as PMS450 and PMS600, respectively.
The pH of PMS450 and PMS600 were estimated in a suspension of 1:10 BC:water ratio (w/v) with a pH meter (PB-10; Sartorius) (Lopez et al., 2020). The C, H, and N element content was determined using an elemental analyzer (Elementary Vario ELIII). Total concentration of Cd was determined by acid digestion and estimated with an inductively coupled plasma optical emission spectrometry (Thermo Fisher Scientific, Inc.). The surface structure was characterized by a S-4800 scanning electron microscopy (SEM; Hitachi). The superficial functional groups of the biochar were determined by a Nexus 670 Fourier transform infrared (FTIR) spectrometer (Nicolet Instrument Corporation) with a wavelength range of 4000-400 cm −1 and resolution of 4 cm −1 .

| Preparation and characterization of microplastics
The low-density PE and PP used in this experiment were purchased from the Youngling-TECH Company. The PE has a density of 0.92 g cm −3 and melting point of 105°C. The PP has a density of 0.91 g cm −3 and melting point of 161°C. The particle size of the microplastics was 100 ± 10 μm according to the manufacturer-provided parameters.
Aging treatments of PE and PP with UV were conducted in a customized instrument. The microplastics (5 g) were placed evenly in quartz glass culture dishes and exposed to ultraviolet lamps (4 × 40 W lamps with a wavelength of 313 nm) for 21 days (Wang, Zhang, Wangjin, et al., 2020). During the aging process, the particles were stirred daily to ensure uniform irradiation.
After aging, the surface structures of the microplastics before and after UV aging (21 days) were characterized by SEM (Hitachi), and the functional groups were observed by FTIR (Nicolet Instrument Corporation).

| Greenhouse experiment
The experiment was performed in a greenhouse at the Beijing Academy of Agriculture and Forestry Sciences. We designed an experiment that included (1) four types of microplastics, namely, PE, PP, aged PE, and aged PP, with two doses, for example, 0% and 1% (w/w); (2) two types of biochar, for example, PMS450 and PMS600, with two addition rates, for example, 0% and 2% (w/w); and (3) two addition concentrations of Cd, for example, 0 and 5 mg Cd kg −1 soil. Each treatment was performed with three replicates. The doses of microplastics were designed based on field surveys and literature reviews (Zhang et al., 2018). The Cd concentrations represent environmentally realistic levels that have been reported for China's farmland soils (Wang et al., 2015).
The soil samples were air-dried and passed through a 2 mm sieve to obtain the homogenously mixed samples. A total of 1500 g of air-dried and sieved soil was placed in each pot (16 cm diameter at the top, 15 cm diameter at the bottom, and 17.5 cm in height). All pots received a basic nutrient dressing that consisted of 0.43 g kg −1 of urea and 0.2 g kg −1 of K 2 HPO 4 . The Cd solution was prepared by dissolving Cd(NO 3 ) 2 · 4H 2 O in distilled water, which was then spiked into the soil to obtain the target concentration. The Cd-contaminated soil samples were equilibrated at room temperature for 1 week. Then, the biochars were applied at rates of 2% and were thoroughly mixed with the soil samples according to the experimental design. After equilibrating for 1 week, the microplastics were thoroughly mixed into the soil to achieve the target doses. After all the pots were filled, they were allowed to settle for 1 week prior to growing seeds with a soil moisture content of 20%-22% (v/v) with distilled water . Before being sowed, the mass of each seed was measured, and only those seeds with masses between 0.04 and 0.05 g were used (Qi et al., 2018). Then, 10 surface-sterilized seeds were sown in each pot. After seedling emergence, five seedlings of uniform size were retained from each plot for the experiment. The temperatures were maintained at 28°C during daytime and approximately 23°C during nighttime. During the entire plant growth period, distilled water was added every day to maintain the soil water content at approximately 20%-22%. The pots were randomly placed in the greenhouse.

| Sample collection
The whole wheat plants were removed from the soil at 1 month after seedling emergence. All plant tissues were rinsed with deionized water and dried, and the heights and fresh weights were measured and recorded. The dry weights were determined after dried at 80°C for 2 days until the weight is stable. The soil samples taken from each pot were mixed thoroughly and sieved sequentially through a 2-mm sieve for analysis.

| Sample analysis
The pH values of the soil suspensions (soil:water ratio, 1:2.5, w/v) were measured with a pH meter (PB-10; Sartorius). DOM extractions were performed according to the methods adopted by Gao et al. (2017). Briefly, 7 g of soil was extracted and mixed with 35 mL of Milli-Q water; the mixture was then shaken for 24 h and centrifuged for 15 min at 5000 rpm, which was followed by filtering through a 0.45-μm filter. The DOC contents in the soil solutions were determined with a total organic carbon analyzer (TOC-L; Shimadzu).
The Cd concentrations in the soil samples were digested using a three-acid digestion system of 10 mL HNO 3 , 10 mL HF, and 5 mL HClO 4 at 200°C for 6 h (Gu et al., 2022), while the dried plant materials were ground and then wetdigested by a two-acid digestion system of 18 mL HNO 3 and 6 mL HClO 4 at 200°C for 6 h. The Cd concentrations in the digested solutions of the soil and wheat plant were estimated with inductively coupled plasma-mass spectrometry (ICP-MS; Thermo Fisher Scientific, Inc.).
The available Cd levels in the soil samples were determined by the diffusive gradients in thin-films (DGT) method using Chelex-DGT binding agents (Gao et al., 2017).
The maximum water holding capacity of soil was determined by the method of Veihmeyer and Hendrickson (1949) with some modifications following Verheijen et al. (2019), and then the soil water contents were adjusted to 60% of the maximum water holding capacity by adding Milli-Q water. After 48 h of equilibrium, the soil water content was increased to 100% of the maximum water holding capacity for 24 h. We placed the DGT devices on the soil pastes with a slight amount of pressure to maintain complete contact between the DGT filter membranes and soil surfaces, and this contact was maintained for 24 h. During the deployment, the temperature was kept at 25°C. Then, the DGT devices were washed with distilled water. The resin gels were removed, washed with distilled water, and eluted with 1.3 mL of 1 M HNO 3 . The Cd concentrations in the eluted binding gels were determined by ICP-MS (Thermo). The DGT-measured concentrations of Cd (C DGT ) were determined using Equation (1). where M is the mass of the trace elements that accumulated in the binding gel, △g is the combined thickness of the DGT filter membrane and binding gel, D is the diffusion coefficient of Cd in the diffusive gel at 25°C, T is the deployment time, and A represents the device's exposure area (e.g., 3.14 cm 2 ).

| Statistical analysis
The results were conducted by multifactor analysis of variance to assess the effects of biochar and microplastics application on soil physicochemical property, plant growth, Cd availability, and Cd uptake by wheat plants. Simple effect test was conducted to determine intragroup significance. Statistically significant differences were considered to exist when the p < 0.05. The bivariate Pearson correlation coefficients were used to analyze the data for pH, DOM concentration, and Cd availability under the different treatments. All statistical analyses were subjected to IBM SPSS 26.0 for Windows (SPSS, Inc.). Origin 2021 software was used for data plotting.

| RESULTS
The pH of PMS450 and PMS600 was 10.33 and 10.79, respectively. Total Cd content of PMS450 and PMS600 was 0.31 and 0.34 mg · kg −1 , respectively. The SEM images confirmed that biochar contained abundant micrometer porous structure. Compared with PMS450, PMS600 had a more stable and dense tube bundle structure ( Figure S1). The results of elemental analysis showed that the ratios of H/C, O/C, and (O + N)/C decreased with the increase in pyrolysis temperature (Table S1), indicating that compared with PMS450, PMS600 exhibited increased aromaticity, decreased oxygen-containing functional groups, and decreased polarity. Functional groups of biochar including C-H (1040/1082), -OH (3389), -C=O (1397), and COOH (1577) on the biochar were observed by FTIR ( Figure S1). With the increase in pyrolysis temperature, the strength of -C=O and COOH decreased.
Scanning electron microscopy images of the fresh and aged microplastics are shown in Figure S2. The surfaces of fresh microplastics exhibited relatively homogeneous smooth features, while the surfaces of the microplastics developed small wrinkles and convex structures during the UV aging process. As shown in Figure S2, the fresh and aged microplastics produced similar FTIR absorption bands. The spectra of the aged microplastics exhibited tensile vibration peaks of carbonyl groups at wavenumbers of 1729 and 1716 cm −1 for the aged PP and PE, respectively, which were not present in the spectra of the fresh samples. The formation of carbonyl was mainly due to the reaction of microplastics with oxygen in the air during UV aging. In addition, a strong absorption band ranging from 2800 to 3000 cm −1 was observed for all of the microplastics, which was characteristic of aliphatic C-H . The intensities of these peaks increased after UV aging. The above results indicate that the aged microplastics contained new oxygen-containing functional groups compared to the fresh microplastics. The microplastics decreased soil pH (Figure 1a). The pH levels of the biochar-amended alkaline soil decreased from 0.39 to 0.48 unit (Table S2). Moreover, with the coexistence of microplastics and biochar, the soil pH in this study generally decreased compared with only biochar treatment. Also, the microplastics and biochar increased soil DOM content especially aged PP (Figure 1b; Table S3).
The microplastics generally increased soil C DGT with percentage increases falling in the range of 9.23%-41.68% when compared with the control (Figure 2; Table S3), and biochar caused a more significant increase in C DGT (Figure 2; Table S3). The coexistence of microplastics and biochar increased the soil C DGT values by 2.63%-47.73% compared to the biochar alone ( Figure 2). Additionally, the wheat plants in Cd-contaminated soils contained Cd (Figure 3), and Cd was not detected in the control ones. After addition of the fresh PE and PP, the Cd accumulations in wheat significantly increased from 6.63 to 8.13 mg · kg −1 and from 6.63 to 8.16 mg · kg −1 , respectively (Figure 3). The fresh microplastics could all inhibit wheat growth significantly (Table S4). Biochar in Cd polluted soil exhibited stimulatory effects on the weight of wheat by 4.0%-7.1% and height of wheat by 6.0%-7.7% (Table S4).
F I G U R E 1 pH value (a) and DOM content (b) of soil under different treatments, linear correlation analysis between soil pH (c), DOM content (d) and C DGT in the soil. Control refers to Cd-contaminated soil without any amendments; PMS450/PMS600 refers to the soil amended with 450/600°C biochar, respectively; PE/PP/aPE/aPP represents the Cd-contaminated soil amended with PE/PP/aged PE/aged PP microplastic, respectively. DOM, dissolved organic matter; PE, polyethylene; PP, polypropylene.

| Effects of microplastics and biochar on soil chemical properties
This study specifically tested the soil pH and DOM levels (Figure 1a,b). For the microplastic treatments, significant decreases in soil pH were observed (Figure 1a; Table S3), with the greatest decrease of 0.33 occurring in soil supplemented with aged PP (Table S2). Similarly, Qi et al. (2020) also found that the soil pH clearly decreased after the addition of microplastics. The decrease in soil pH was potentially attributed to the changed status of cation exchange in the soil and free exchange of protons in the soil water. F I G U R E 2 Cd concentration measured by DGT (C DGT ). Control refers to Cd-contaminated soil without any amendments; PMS450/PMS600 refers to the soil amended with 450/600°C biochar, respectively; PE/PP/aPE/aPP represents the Cd-contaminated soil amended with PE/PP/aged PE/aged PP microplastic, respectively. PE, polyethylene; PP, polypropylene.

F I G U R E 3 Cd concentration in plants under different treatments after 30 days cultivation. Control refers to
Cd-contaminated soil without any amendments; PMS450/PMS600 refers to the soil amended with 450/600°C biochar, respectively; PE/PP/aPE/aPP represents the Cd-contaminated soil amended with PE/PP/aged PE/aged PP microplastic, respectively. PE, polyethylene; PP, polypropylene.
For the biochar treatments, unlike the liming effect of biochar on acidic soils (Park et al., 2011;Yuan et al., 2011), this study suggested that the pH levels of the biocharamended alkaline soil decreased for the 2% PMC450 and PMC600 amendments compared with the control, respectively (Table S2). These results were consistent with those of previous studies, which also reported similar decreases in soil pH after adding biochar amendments to alkaline soils (Zhang, Qu, et al., 2020). The decreased pH values in the biochar-amended soil in this study might be explained as follows: (1) biochar addition could improve the buffering capability of soil pH (Zhang, Qu, et al., 2020), and (2) the production of acidic materials (e.g., phenolic and carboxylic acids) as a result of biochar oxidation can reduce the soil pH in biochar-amended soils (Hu et al., 2021). Moreover, with the coexistence of microplastics and biochar treatments, the soil pH in this study generally decreased compared with only biochar amendments. It is necessary to conduct additional extensive investigations on the effects of both microplastics and biochar on the pH values of alkaline soils because most previous studies have focused on acidic soils.
As biochar and microplastics contained a large amount of carbon, the biochar and microplastic application inevitably increase the content soil organic matter (SOM) and alter the composition of SOM Li, Liu, et al., 2022;Li, Xi, et al., 2022). As DOM is the more sensitive indicator of soil structural changes (Gong et al., 2009) and plays a central role in various physical, chemical, and biological processes in soil (Kalbitz et al., 2000), the changes of DOM content in the presence of biochar and microplastics were further investigated. The results showed that the addition of microplastics and biochar increased soil DOM content especially aged PP (Figure 1b; Table S3), which was most likely attributed to the DOC released by the aged PP itself after entering the environment . In addition, soil microorganisms can convert microplastics and the poorly dissolved SOM into soil soluble carbon and increase the DOM contents in soil . Under the coexistence of both microplastics and biochar, the soil DOM contents were higher in aged PP than in biochar amended alone, which further suggested the increasing effect of aged PP on soil DOM contents.

| Effects of microplastics and biochar on Cd availability in soil without wheat plant
Both the fresh and aged microplastics generally increased soil C DGT regardless of wheat planting. Thus, the following discussion applied only to the soil without wheat. Both the fresh and aged microplastics except for fresh PE generally caused obvious increases in soil C DGT with percentage increases ( Figure 2; Table S3), suggesting the microplastics improved the availability of soil Cd. The remarkable increase in Cd availability should be attributed to the following reasons. First, the addition of microplastics decreased the soil pH (Figure 1a; Table S2), which could inhibit the deprotonation of functional groups of minerals and organic matter from soils, decrease the negative charges, and thereby decrease the Cd adsorption by soils and increase Cd availability (Avelar Ferreira et al., 2022). Moreover, a significantly negative correlation was observed between the soil pH and soil C DGT in this study (p < 0.01, Figure 1c), which confirms that the decreased soil pH after the addition of microplastics partly accounted for its higher availability. Second, the soil DOM contents had a significantly positive correlation with soil C DGT (p < 0.05, Figure 1d). Increased DOM levels can reduce Cd adsorption onto soil surfaces, which improves the extractability of Cd (Chappaz & Curtis, 2013). Consequently, the increased DOM in soil after microplastics addition is also a factor for elevating soil Cd availability. In addition, previous study reported that microplastics addition decreased soil adsorption capacity for Cd, but increased Cd desorption (Zhang et al., 2019), which may serve as a potential valid explanation to the elevated soil Cd availability after microplastic addition in this study.
Compared to microplastics, biochar caused a more significant increase in C DGT (Figure 2; Table S3). It has been mentioned above that the decreased soil pH after the addition of microplastics partly accounted for higher Cd availability. Similarly, the reduction of soil pH after biochar application likely led to the increasing C DGT (Figure 1a). In addition, biochar did not increase the adsorption of Cd in soil after being added to soil as previously reported (Gu et al., 2020). In this study, the biochar had no significant influence on the soil adsorption to Cd (p > 0.05, Table S5). Therefore, Cd availability in soil after addition of biochar was not affected by soil adsorption. It can be deduced that compared with the adsorption of Cd by biochar, soil pH and DOM change resulted by the biochar mainly contributed to the increasing soil Cd availability.
After the addition of microplastics and biochar simultaneously to the soil, the soil C DGT values increased in comparison to the case of biochar alone (Figure 2), which suggested that the coexistence of microplastics (especially PP) and biochar resulted in a significant increase in Cd availability. It has been previously reported that biochar was attached to -CHx in microplastics (Li et al., 2017), which may result in the occupation of adsorption sites on biochar and thus inhibit the sorption of Cd on biochar ( Figure S3). In addition, the pH values of the soil with microplastics and biochar were generally lower than those of the soil with biochar alone, and the DOM contents were higher (Figure 1a). Moreover, the negative correlation between soil pH and soil C DGT (p < 0.01, Figure 1c), as well as the positive correlation with DOM content (p < 0.05, Figure 1d), indicated that the changes in soil properties after application of microplastics and biochar were also an important factor affecting the soil Cd availability. The same phenomenon was also observed for the aged microplastics, especially for the aged PP (Figure 2). The coexistence of aged PP and biochar resulted in more significant increases in soil C DGT , which may be attributed to the modification of the microplastic surfaces. Aging leads to larger specific surface areas and the formation of oxygencontaining functional groups on microplastics surfaces ( Figure S2), which consequently results in more complex interactions between microplastics and biochar, hindering the accessibility of Cd to biochar ( Figure S3) and therefore the aged microplastics causes a greater increase in C DGT than the fresh microplastics.

| Effects of fresh or aged microplastics and biochar on Cd uptakes in wheat
Cd was observed in the wheat plants grown in Cdcontaminated soils (Figure 3), and Cd was not detected in the control wheat plants. The Cd contents in all plant samples exceeded the allowable limit (0.2 mg · kg −1 ) specified by the Chinese food security standards. The presence of fresh microplastics significantly increased the Cd uptakes of wheat (Figure 3, p < 0.05). When the soil was treated with a dose of 1% fresh PE and PP, the Cd accumulations in wheat significantly increased from 6.63 to 8.13 mg · kg −1 and from 6.63 to 8.16 mg · kg −1 , respectively ( Figure 3). These fresh microplastics induced higher available Cd concentrations in soil with wheat ( Figure 2); therefore, the increasing availability of soil Cd resulted in higher uptakes in the wheat plant. To date, different effects of microplastics on the Cd uptake by plants have been observed. For example, although the addition of PE induced higher available soil Cd, no effect on the significant Cd uptakes in plant tissues was observed . However, another study showed that the Cu and Cd accumulations in wheat seedlings were reduced in the presence of polystyrene (Zong et al., 2021). The different effects may be caused by the various properties of microplastics and the Cd accumulation abilities of different plants. Additionally, compared with fresh microplastics, the promoting effects of the aged microplastics on the Cd concentrations in wheat were not observed (Figure 3, p > 0.05), with the Cd uptakes remaining no change. Although aged microplastics, especially aged PP, greatly increased the soil C DGT values, and more Cd should be absorbed by the wheat, the aged microplastics had no obvious influences on the Cd uptake of the tested wheat. Similar result was also reported by the previous study . It was demonstrated that microplastics could attach onto the root surface and prevents Cd from contacting wheat roots . Therefore, it can be reasonably deduced that the aged microplastics hinder the transportation of soil Cd to the wheat and reduce the Cd uptake of wheat.
In terms of the effect of biochar on the Cd uptake by wheat, as expected, biochar resulted in significant increased uptake of Cd in wheat because of the elevating soil Cd C DGT values resulted by biochar (Table S4). When 2% doses of 450 and 600°C BC were added to the soil, the Cd concentrations increased from 7.13 to 11.39 mg · kg −1 and from 7.13 to 11.75 mg · kg −1 in wheat, respectively ( Figure 3). The result was in contrary to the previous results that biochar inhibits Cd uptake by plants (Nie et al., 2018). As aforementioned, the decreasing pH led to the improved Cd C DGT values. Therefore, in this study, the pH should be the major factor increasing soil Cd availability compared to other major factor decreasing soil Cd availability by Cd adsorption to biochar reported previously (Gu et al., 2020). Future research should examine the effects of biochar on the Cd uptake levels by different plants in different soils and reveal the associated mechanism. In addition, even though Cd C DGT values were enhanced in the presence of microplastics and biochar compared to the case of biochar alone, the Cd uptake levels by wheat generally were unchanged (Figure 3, p > 0.05). The result was similar to the effect of aged microplastics alone on the soil Cd uptake by wheat, and the microplastics likely also hinder the transportation of Cd in the biochar soil to the wheat and reduce the Cd uptake of wheat.

| Effects of microplastics and biochar on plant biomass
The effects of both microplastics and biochar on the dry weights and heights of wheat under Cd stress are presented in Figure 4a,b, respectively. The addition of fresh microplastics at a dose of 1% decreased wheat growth. Thus, fresh microplastics could all inhibit wheat growth significantly (Table S4). Our findings were consistent with the results of previous studies (Qi et al., 2020). Moreover, the inhibition effect on wheat growth by aged microplastics was generally higher compared to the fresh microplastics. Both the physical and chemical effects of microplastics could possibly explain the plant biomass changes. The inhibitory effect of microplastics on wheat biomass in this study may be attributed to the leaching of toxic additives in microplastics (Pflugmacher et al., 2021), and the reduction resulted by microplastics on both moisture retention and nutrient availability (e.g., magnesium and potassium) to the plant in soil (Boots et al., 2019;Wan et al., 2019). Therefore, the negative impacts of microplastics (especially aged microplastics) on wheat growth under Cd stress in the field deserve more attention.
Biochar in Cd polluted soil exhibited stimulatory effects on the weight of wheat. Biochar has been reported to increase the water holding capacity and improve soil structure (Atkinson et al., 2010), which can provide material conditions that are conducive to plant success. After the addition of microplastics to soil with biochar, the plant biomass was reduced compared with soil amended with biochar alone (Figure 4, p < 0.05). It has been mentioned above that there was no obvious change in Cd uptake of wheat when microplastics were added to soil amended with biochar. Therefore, the reduction in plant biomass caused by the application of microplastics and biochar was not attributed to Cd uptake, but to the negative influence of microplastics themselves on plant growth in soil.

| CONCLUSIONS
This study investigated the influence of microplastics and biochar on Cd availability and plant performance. First, F I G U R E 4 The dry weight (a) and height (b) of wheat under all treatments at 30-day harvests. Control refers to Cd-contaminated soil without any amendments; PMS450/PMS600 refers to the soil amended with 450/600°C biochar, respectively; PE/PP/aPE/aPP represents the Cd-contaminated soil amended with PE/PP/aged PE/aged PP microplastic, respectively. PE, polyethylene; PP, polypropylene. addition of fresh microplastics to the tested soil generally increased available Cd contents in soil and the Cd uptake in plant. However, the aged microplastics generally had no effects on the Cd uptake by wheat plant even though it improved the available Cd contents in soil, which could be likely attributed to the blocking effect of microplastics on Cd transportation from soil to the wheat. Additionally, not as expected, the addition of biochar enhanced the tested soil Cd availability as well as Cd uptake by wheat plant, which was resulted by the increased soil soluble Cd because of both decreased soil pH and elevated DOM content by biochar. Simultaneously, the unchanged Cd adsorption to the soil also supported the increase in Cd availability in tested soil. Moreover, more profound increase in soil Cd availability was observed for biochar soil with microplastics addition. In other words, the coexistence of microplastics and biochar substantially increased the soil Cd availability compared with biochar alone due to the greater decrease in soil pH value and Cd adsorption but increase in soil DOM content after the addition of microplastics. Finally, fresh microplastics inhibited wheat growth; moreover, the inhibition effect on wheat growth by aged microplastics was generally higher. While the biochar exhibited stimulatory effects on the wheat biomass, the coexistence of microplastics and biochar decreased the wheat plant biomass compared with biochar alone, which was attributed to the negative influence of microplastics itself on plant growth in soil but not to the Cd uptake of wheat. This study demonstrated that microplastics should influence the fate of heavy metal in soils as well as the potential of biochar in soil remediation. However, the current work only investigated Cd uptake by 1-month-old wheat seedlings. Future study should further address the effects of microplastics on plant performance regarding more aspects, such as the grain production, which is more closely related to food safety. As microplastics are ubiquitous and tend to steadily accumulate in agricultural soil, the negative effects of microplastics on different soils with and without biochar should not be neglected. Long-term and large-scale experiments should be conducted in the natural environments to determine the potential impacts of microplastics on soil ecosystems.

AUTHOR CONTRIBUTIONS
Jiarui Miao was involved in methodology, investigation, formal analysis, writing-original draft. Yalan Chen was involved in conceptualization and writing-review and editing. Enyao Zhang was involved in methodology and investigation. Yan Yang was involved in conceptualization and formal analysis. Ke Sun was involved in conceptualization, supervision, funding acquisition, project administration, and writing-review and editing. Bo Gao was involved in writing-review and editing.