Joint toxicity of Cadmium (II) and microplastic leachates on wheat seed germination and seedling growth

Cadmium (Cd) and microplastics are widely distributed in soil and pose potential threats to agricultural production and human health. However, the joint toxicity of Cd and microplastics on crop growth remain largely unknown. In this study, the toxic effects of Cd 2+ solution and two kinds of microplastic leachates, polyvinyl chloride (PVC) and low-density polyethylene (LDPE), on wheat seeds germination and seedlings’ growth were explored under the single and combined conditions. The results showed that Cd 2+ solution and two kinds of microplastic leachates stimulated wheat seeds germination process but inhibited the germination rate by 0–8.6%. The combined treatments promoted wheat seeds germination but inhibited the growth of wheat seedlings on different degree. In especial, the combination of 2.0 mg L -1 Cd 2+ and 1.0 mgC L -1 PVC could promote both wheat seeds germination and seedlings’ growth, but they synergistically increased the antioxidant enzyme activity of seedlings. The physiological and biochemical indicators showed that the possible mechanism through which Cd 2+ and microplastics leachates and their combination affected wheat seedlings involved oxidative stress. The toxicity of the PVC leachate to wheat seedlings was stronger than LDPE leachate. The addition of Cd 2+ could alleviate the toxicity of PVC leachate on wheat seedlings, and reduce the toxicity of LDPE leachate on wheat seedlings under the same concentration class combinations but aggravated stress under different concentration class combinations, consistent with the effect on wheat seedlings’ growth. Overall, our study has important implications for the joint toxicity of Cd 2+ solution and microplastic leachates in agriculture.


Introduction
Plastics are widely used all over the world, and the global annual production now exceeds 367 million tons, of which China accounts for 32.0% (Plastics Europe, 2020).Microplastics are a type of plastic with a particle size of less than 5.0 mm (Thompson et al., 2004), which can change the metabolism of organisms and move to various organs, causing neurotoxicity, cancer and other diseases (Rahman et al., 2021).Plastics have low degradability in the environment (Pegram and Andrady, 1989), and as a result, microplastics persist in the environment.However, some additives (such as plasticizers, ame retardants, stabilizers, antioxidants, etc.), which are used to improve the performance and function of plastic products, are usually not combined with polymers (Hermabessiere et al., 2017;Hahladakis et al., 2018), easily disintegrate in water at concentrations ranging from pg L − 1 to µg L − 1 .Studies have pointed out that strong solar radiation and other natural factors may help to enhance the migration rate of microplastics in saturated loam and the migration ability of pollutants and promote the release of its additives (Liu et al., 2019;Luo et al., 2019).
However, these leachates can produce toxic effects on organisms.Lithner et al. (2011) found that microplastic leachates in ltrated into fresh water and produced acute toxicity to Daphnia.Meanwhile, microplastics have a large speci c surface area and can act as an effective vector for metal pollutants.Cadmium (Cd) is one of the main heavy metal pollutants found in soil (Fei et al., 2022) and is of great public concern for its persistence, bioaccumulation, and environmental toxicity (Huang et al., 2023).Cd can easily interact with coexisting microplastics and has strong mobility and is easily absorbed by plant roots and transported to edible parts (Liu et al., 2021b;Zeng et al., 2019), which affects the growth and development of crops and human health (Liu et al., 2021a;Hodson et al., 2017).
Polyvinyl chloride (PVC) is one of the most widely consumed plastics in the world (Ye et al., 2017).It is a durable and corrosion-resistant material, but it is not easy to recycle (Ragaert et al., 2017).The research shows that the adsorption capacity of PVC for heavy metal Cd is signi cantly higher than that of other microplastics due to its bulge, fold, and void structure, and the maximum adsorption capacity can reach 151.4 mg kg − 1  (Guo et al., 2020).Wang et al. (2021) found that the inhibition rate of Cd alone on Chlorella cell growth was 85.14%, but the inhibition rate of combined treatment with micron-sized PVC decreased to 18.89%.Besides PVC, the low-density polyethylene (LDPE) persists in the environment and also is di cult to biodegrade (Sivan et al., 2006).Zou et al. (2022) found that Cd 2+ or LDPE alone inhibited the growth of above ground parts of black eggplant seedlings, but their combined treatment promoted the growth of leaf area in the later stage of seedlings.However, Jia et al. (2022) showed that the root system of rape was damaged and the content of malondialdehyde (MDA) increased under the combined action of polyethylene and heavy metals, which was caused by oxidative stress (Gupta et al., 2019).
Oxidative stress refers to the toxic effect on plants caused by excessive production of intracellular reactive oxygen species (such as superoxide radical and hydrogen peroxide H 2 O 2 ) or damage at the antioxidant level (Li et al., 2007).
Studies have shown that excessive heavy metals and microplastics can lead to the accumulation of reactive oxygen species in crops (Gupta et al., 1999;Gupta et al., 2019).Oxidative stress and excessive reactive oxygen species are the main mechanisms through which microplastics produce toxic effects on plants (Gong et al., 2021).Antioxidant enzymes, such as peroxidase (POD) and catalase (CAT), are important for plants because they can remove reactive oxygen species, prevent damage to cell structure and function, and maintain the dynamic balance of oxidation and reduction, thereby increasing plant stress resistance (Jia et al., 2022;Silva et al., 2018).Different from the action mechanism of POD, CAT directly catalyzes the decomposition of H 2 O 2 (Sun et al., 2007), while POD catalyzes the reaction of H 2 O 2 with other substrates, thus achieving the goal of retaining H 2 O 2 in organisms (Sharma and Dietz, 2009).In addition, MDA is the product of lipid peroxidation, and its content can re ect the damage degree of cell membrane by oxygen free radicals (Li et al., 2007;Halliwell, 1987;Esterbauer et al., 1982).Therefore, we assume that the single or combined pollution of microplastic leachates and Cd 2+ may lead to different oxidative stress reactions in plants, and comparing the effects of both single and combined pollution can help to understand how different mechanisms of microplastics affect plants.
In recent years, considerable concentrations of microplastics have been found in soil all over the world (Zhou et al., 2020).In China, wheat is one of the main food crops, but few studies have examined the physiological and biochemical conditions under which wheat is polluted by aged microplastic leachates and heavy metal Cd 2+ solution.
To address this research gap, this study explores the stress effects of two kinds of aged microplastic (PVC and LDPE) leachates and Cd 2+ solution on wheat seeds germination and seedling growth under single and combined treatments.The results of this study are of great signi cance for in-depth understanding of the coexistence mechanism of heavy metals and microplastic leachates and their environmental risks.
2 Materials and methods

Main reagents and materials
The plant under this study is wheat (Triticum aestivum L.).PVC and LDPE were purchased from Dongguan Huachuang Plasticizing Co., Ltd., CdCl 2 •2.5H 2 O (analytically pure) was purchased from Tianjin Comio Chemical Reagent Co., Ltd., and H 2 O 2 (analytically pure) was purchased from Chengdu Cologne Chemical Co., Ltd.

Preparation of microplastic leachates, Cd 2+ solution and combined treatments solution
To replicate the conditions of in ltration and aging in the natural environment, a sample of water from a natural water body in Xihe River in Nanchong City was ltered through a 0.45 µm membrane.The TOC concentration of the sample was 37.2 mgC L − 1 as detected by the Elementar vario TOC cube analyzer.Four grams of microplastics with a particle size of 0.1 mm was added to each liter of water.The water was illuminated for 30 days, and the microplastic leachates collected following another round of ltration.The solutions were diluted to 1.0, 5.0 and 10.0 mgC L − 1 to make low, medium and high concentration PVC and LDPE leachates (denoted as PVC1, PVC5, PVC10, LDPE1, LDPE5 and LDPE10, respectively).In addition, low, medium and high concentration Cd 2+ solutions of 2.0, 4.0 and 8.0 mg L − 1 (expressed as Cd2, Cd4, Cd8, respectively) were created.

Seed germination experiment
Wheat seeds that were uniform and whole were selected by soaking them in deionized water for 30.0 minutes, discarding oating seeds, soaking the remaining seeds in 1.0% H 2 O 2 solution for 10.0 minutes, and then repeatedly washing them with deionized water to remove H 2 O 2 residues.The sterilized seeds were then soaked in deionized water for 12.0 hours, and surface water was dried with lter paper.
To show the effects of microplastic leachate and Cd 2+ solution on wheat seed germination, a hydroponic method was used for seed germination.There were 18 treatments, including a control group, 9 single treatments and 8 combined treatments.Four groups of parallel treatments were set for each treatment.
First, a 10.0 cm Petri dish was padded with lter paper, and 15 treated wheat seeds were evenly distributed in the Petri dish.For the CK treatment, 10.0 mL of deionized water was added to the culture dish.For the single and combined treatments, 10.0 mL of low, medium or high concentration Cd 2+ solution, PVC leachate, LDPE leachate or the combined treatment solution was added to the culture dishes.The dishes were placed in a constant temperature incubator (NLGH, HerryTech, Chinar) at 20.0°C in the dark for 7 days.During the culture process, deionized water was added after weighing daily to compensate for evaporated water and keep the concentration of the culture solution stable.

Index measurement
A scanner (Epson Expression 10000XL) combined with a root analyzer (Winrhizo Pro 2016a, Regent Instruments Inc., Canada) were used to measure the root parameters of wheat seedlings, including total root length, root surface area, average root diameter, and root volume.Then, the wheat seedlings were dried in a 65.0°C vacuum oven (DZF-6050, Shanghai Yiheng), and the biomass of the buds and roots was measured by an electronic balance (Sartorius BSA224S, Germany).
The germination of wheat seeds was recorded daily, and the standard for germination was when the length of young buds reaches 1/2 of the seed length.After the harvest was nished on the 7th day, the germination rate (GR), germination energy (GE), germination index (GI), vigor index (VI), and average germination time (AGT) of the seeds were calculated using the following formulas (Dong et al., 2022;Bao et al., 2022): Where Gt is the number of new germinated seeds on day t; Dt is the germination days, d.
In line with previous methods, the wheat seedlings was pretreated (Gong et al., 2021), and then the content of MDA and the activities of POD and CAT in the wheat seedlings were determined according to the method in the kit (Suzhou Grace Biotechnology Co., Ltd., Suzhou, China).

Data analysis
In this study, Origin was used for data processing.Under the condition of satisfying the homogeneity of variance (or after data transformation), one-way ANOVA was used to analyze each index, and Tukey method was used to make multiple comparisons.When the homogeneity of variance was not satis ed, nonparametric tests and multiple comparisons were carried out.The experimental data were expressed as mean standard error (Mean ± SD), and when P < 0.05, it showed signi cant difference.At the same time, the correlation between germination and growth indexes of wheat seeds and seedlings under different treatments through principal component analysis (PCA), Mantel test and Pearson correlation analysis using R.
3 Results and discussion

Effects of Cd 2+ and Cd 2+ -microplastic leachates on wheat seed germination
The germination rate (GR), germination energy (GE), germination index (GI), vigor index (VI) and average germination time (AGT) were calculated to characterize the effects of Cd 2+ solutions, two microplastic leachates and their combination treatments (Cd 2+ -microplastic leachates) on wheat seed germination (Table 1).Compared with the CK, the Cd 2+ solutions and two kinds of microplastic leachates inhibited the germination rate of wheat seeds with an inhibition rate of 0-8.6%.The germination energy and germination index of wheat seeds in the medium concentration Cd 2+ GR = Number of germinated seeds within 7 days / Total EquationNumber of seeds tested × 100\% GE = Number of germinated seeds within 3 days / Total EquationNumber of seeds tested × 100\% solution (4.0 mg L − 1 ) decreased signi cantly, but there were no signi cant effects in low and high concentration solutions (2.0 mg L − 1 and 8.0 mg L − 1 ).The vigor index of wheat seeds increased and the average germination time of wheat seeds was shortened to about two days under the pollution of Cd 2+ solution, as well as the two kinds of microplastic leachates, which may be because Cd 2+ solutions and two kinds of microplastic leachates stimulated the germination process.The effect on the germination of wheat seed under Cd 2+ solution and the two kinds of microplastic leachates was different.This may be because Cd 2+ inhibits seed germination through nutrient loss caused by mineral leakage (Wani et al., 2007), while microplastics accumulate in the seed coat and hinder wheat seeds from absorbing water, thus inhibiting seed germination (Ge et al., 2021).The speci c mechanism through which this occurs needs further study.
Although Cd 2+ solutions can signi cantly inhibit the germination of wheat seeds, the GR, GE, GI, and VI under all the Cd 2+ -PVC combined treatments increased (Table 1) and were higher than those of the CK.Under the Cd 2+ -LDPE combined treatments, the GR, GI and VI of the same concentration class combination (Cd2 + LDPE1, Cd8 + LDPE10) increased compared with the CK, while the GR and GE were signi cantly inhibited under different concentration class combination (Cd8 + LDPE1, Cd2 + LDPE10).Similar to the single treatment, the AGT of wheat seeds under combined treatment was also shortened.The bud biomass and bud length (Fig. 1), root biomass, root length, root surface area, average root diameter and root volume (Fig. 2) of wheat seedlings under different treatments were measured to assess the effects of Cd 2+ solution and microplastic leachates on the growth of wheat seedlings.Compared to the CK, the bud biomass was slightly inhibited by the medium concentration of Cd 2+ solution (4.0 mg L − 1 ).However, both low and high concentrations of the single Cd 2+ solutions (2.0 and 8.0 mg L − 1 ) were associated with increased bud biomass of wheat seedlings, which was similar to that of both low and high concentrations of the LDPE leachates (1.0 and 10.0 mgC L − 1 ) and all concentrations of PVC leachates (1.0, 5.0 and 10.0 mgC L − 1 ).All Cd 2+ solutions promoted the root biomass of wheat seedlings with the promotion rate of 2.8-10.6%(Fig. 2a).The combined treatments of the Cd 2+ solution and microplastic leachates increased the growth of bud and root biomass (Figs.1a and 2a), except for the combined treatment of 8.0 mg L − 1 Cd 2+ solution and 1.0 mgC L − 1 LDPE leachate (Cd8 + LDPE1).
All the Cd 2+ solutions promoted bud length, so as to all the PVC leachates and low and high concentrations (1.0 and 10.0 mgC L − 1 ) of LDPE leachates.The bud and root length increased by 0.6-10.7%and 19.0-43.6%under Cd 2+ -PVC combinations compared with the CK (Figs. 1b and 2b), especially the addition of low-concentration PVC leachate (Cd2 + PVC1) signi cantly increased the promotion effect on bud and root length.Meanwhile, the bud and root length also increased by 6.7-10.5% and 21.5-48.2%under Cd 2+ -LDPE combinations (Figs.1b and 2d), except for the slight inhibition under the combined treatment of high concentration Cd 2+ and low concentration LDPE leachate (Cd8 + LDPE1).
The root length and surface area wheat seedlings were promoted under all the single and combined treatments with the promotion rate of 19.0-48.2% and 3.1-43.9%(Figs.2b and 2c), but the average root diameter and root volume were inhibited (Figs.2d and 2e).Speci cally, low concentration of Cd 2+ solution inhibited the average root diameter, while medium and high concentrations promoted growth.All concentrations of Cd 2+ solutions promoted root volume, but the growth in root volume gradually decreased as Cd 2+ concentration increased.Except the combined treatment of Cd2 + PVC1, other Cd 2+ -PVC treatments and all Cd 2+ -LDPE treatments inhibited the growth of root diameter and root volume, which was similar to the effect of microplastic leachate treatments.Because low concentration of PVC leachate (1.0 mgC L − 1 ) increased the average root diameter and root volume, while medium (5.0 mgC L − 1 ) and high concentration (10.0 mgC L − 1 ) PVC leaching solutions inhibited growth (Figs.2d and 2e).The LDPE leachate also inhibited the average root diameter of wheat seedlings, and its effect on root volume of wheat seedlings was similar to that of PVC leachate, with the low concentration promoting and medium and high concentration inhibiting growth.The varying effects of different concentrations of microplastics on wheat seedlings may be determined by microplastics' own chemical and biological toxicity, physical toxicity, and environmental media (Lian et al., 2020b).P ugmacher et al.
(2020) showed that high-concentration microplastic leachate inhibited the growth of soybean seedlings' roots for microplastics hindering the absorption and transportation of essential nutrients.Huang et al. ( 2023) also found that microplastics promote the uptake of Cd by plants and have a signi cant negative effect on roots.
Especially, the combination with high concentration of Cd 2+ solution signi cantly weakened the promotion effect on the root growth.This may be due to the weaker toxicity to wheat seedlings at low concentrations of Cd 2+ and leachate.
On the one hand, PVC and LDPE leachates can be complexed with Cd 2+ .On the other, the combined treatment under  In general, the effect of Cd 2+ -PVC on the growth of wheat seedlings mainly inhibits the growth of root diameter and root volume but promotes the growth of bud and root biomass, while Cd 2+ -LDPE mainly inhibits the growth of roots and the enrichment of buds and roots biomass.The combination of microplastic leachates and Cd 2+ was shown to signi cantly promote the germination of wheat seeds, but different inhibitory effects on the growth of wheat seedlings were observed.This may be because the speci c surface area of microplastics is increased and its hydrophilicity is Cd 2+ solution, 1.0 mgC L − 1 PVC leachate and the combination treatment of Cd2 + PVC1 promoted the growth of wheat seedlings (Figs. 1 and 2), but only the combined treatment of Cd2 + PVC1 promoted both the germination of wheat seeds and the growth of seedlings.

Stress mechanism of Cd 2+ and Cd 2+ -microplastic leachates on wheat seedlings
The activity of the antioxidant enzymes (POD and CAT) in wheat seedlings increased, which indicated that a regulation reaction occurred to avoid stress and toxicity.When the toxicity exceeded the threshold of the wheat seedling's selfregulation ability, the enzymes were destroyed and their activities decreased (Weckx and Clijsters, 1996).And MDA content was positively correlated with the degree of cell membrane damage of wheat seedlings (Lanza and Reis, 2021).
The activity of POD under low concentration Cd 2+ solution is similar with that of the CK, but decreased by 22.6% under high concentration Cd 2+ solution (Fig. 3a).The CAT activity decreased by 0-6.9% under all concentrations of Cd 2+ solutions (Fig. 3b).These results show that the stress due to the high concentration of Cd 2+ solution leads to signi cant inhibition of antioxidant enzyme activity (Figs. 3 and 4), and the expression of related genes might be hindered (Dong et al., 2022).Wheat seedlings can adapt to the pollution of low and medium concentrations of Cd 2+ solutions by maintaining or improving POD activity and maintaining CAT activity.The MDA content also decreased by 4.8-20.7%under all concentrations of Cd 2+ solutions (Fig. 3c), indicating that wheat seedlings might be able to maintain their growth through self-regulation under exposure to solutions with high concentration of Cd 2+ .This also indicates that the regulation mechanism of high concentration of Cd 2+ on wheat seedlings might be different from that of low and medium concentrations.
The activity of the antioxidant enzymes under low concentration PVC leachate is close to 1.5 times that of the CK.With the increase of PVC leachate concentration, the activity of antioxidant enzymes gradually decreased, and when the concentration reached 10.0 mgC L − 1 , the activity of antioxidant enzymes decreased by 24.4% compared to the CK (Figs. 3a and 3b).MDA content have no signi cant difference compared with the CK in low concentration PVC leachate, substantial increases (27.0%) in medium concentration, and decreases (25.4%) in high concentration (Fig. 3c).These results show that wheat seedlings were able to resist the stress of low concentration PVC leachate by increasing antioxidant enzyme activity and the high concentration PVC leachate enhanced stress on wheat seedlings.The stress on wheat seedlings produced by the low and medium concentrations of PVC leachates included signi cant damage to the cell membrane (Lanza and Reis, 2021), while the stress due to the high concentration PVC leachate leads to signi cant inhibition of antioxidant enzyme activity (Weckx and Clijsters, 1996).When Cd 2+ solutions combined with PVC leachates, the POD and CAT activity and the MDA content increased under the combined treatment of Cd2 + PVC1.Under other combined treatments of Cd 2+ -PVC, the POD activity of wheat seedlings increased, CAT activity is similar with that of the CK, and MDA content decreased.These results indicated a synergistic effect between Cd2 and PVC1 and might damage to the cell membrane (Huang et al., 2023), while Cd 2+ could to some extent alleviate the harmful effect of PVC leachate on wheat seedlings under other combined treatments of Cd 2+ and PVC.
The change of LDPE leachate concentration had little effect on the POD activity and MDA content of wheat seedlings, and the POD activity was always higher under treatment than that of CK.There was no signi cant difference in CAT activity and MDA content compared with the CK, which indicates that wheat seedlings can resist and adapt to the stress of LDPE leachate by increasing POD activity and maintaining CAT activity when exposed to pollutants within the concentration range of this experiment (Fig. 3).However, under one of the combined pollution treatments (Cd2 + LDPE1, Cd8 + LDPE10), CAT activity was stable, and POD activity and MDA content were higher than that of CK, but lower than the CK under different concentration levels (Cd8 + LDPE1, Cd2 + LDPE10), which indicated that Cd 2+ could reduce the toxicity effect of the LDPE leachate on wheat seedlings under the same concentration class combination but aggravate stress under different concentration class combinations, consistent with the previous results on the wheat seedlings' growth.Taken together, these phenomena indicate the stress reaction of the POD and CAT and the MDA content in wheat seedlings varied when different microplastic leachates combined with Cd 2+ solutions.
To systematically analyze the relationships between physiological and biochemical indexes with wheat seed germination and seedling growth under single or combined treatments of Cd 2+ and microplastic leachates, a PCA analysis was conducted (Fig. 4).As shown in Fig. 4a, the samples treated with Cd 2+ -PVC were concentrated in the 1st and 2nd quadrants, while the samples treated with PVC leachate alone or Cd 2+ solution alone were concentrated in the 3rd and 4th quadrants.It indicated that the addition of Cd 2+ changed the original effect of PVC leachate on wheat seed germination and seedling growth.However, the addition of Cd 2+ had no signi cant effect on LDPE leachate compared to PVC leachate.
Based on the results of PCA analysis, this study chose the indicators shown in Fig. 5 for the Mantel test and Pearson correlation analysis to further explore the mechanism of Cd 2+ solution, LDPE leachate, PVC leachate, and their combined treatments solution on wheat seeds germination and seedlings growth.Under single treatment, Cd 2+ concentration was negatively correlated with the MDA content of wheat seedlings but positively correlated with root index (Figs.4a and 5a).This explains why the 8.0 mg L − 1 Cd 2+ solution promoted the growth of wheat seedlings (Figs. 1 and 2).There is a signi cant negative correlation between the PVC leachate concentration with the AGT (Fig. 5a), meaning that with the increase of PVC leachate concentration, germination time decreases and the stimulation effect on seed germination is stronger.The PVC leachate is negatively correlated with root and bud biomass, root length and surface area (Fig. 5a).The PVC leachate concentration was negatively correlated with POD and CAT activities in wheat seedlings and positively correlated with MDA content (Fig. 4a), indicating that high concentration PVC leachate can inhibit the related enzyme activities of wheat seedlings and exert strong stress on them.The LDPE leachate is negatively correlated with root indicators (Fig. 4b), and positively correlated with POD and CAT activities and MDA content (Figs.4b and 5b), especially with MDA content (Fig. 5b).This shows that the possibility of an oxidative stress effect of wheat seedlings under the single treatment of PVC leachate is greater than that of LDPE leachate, indicating that the toxicity of the PVC leachate to wheat seedlings' growth is stronger than LDPE leachate, which is similar with the result found by Pignattelli et al. (2020).
Under the combined treatment, the addition of Cd 2+ weakened the correlation between PVC leachate concentration and wheat seed germination index but strengthened the correlation with the wheat root system compared to the single PVC treatment (Fig. 5a).The combined treatments of Cd 2+ -PVC were positively correlated with POD and CAT activities but signi cant negatively correlated with MDA content (Fig. 4a).This indicates that the addition of Cd 2+ could alleviate the toxicity of PVC leachate to wheat seedlings.Previous studies have also shown that the addition of Cd 2+ can reduce the inhibitory effect of plastics on wheat seedlings (Hodson et al., 2017).Meanwhile, the Cd 2+ solution strengthened the negative correlation between LDPE leachate with average root diameter and POD activity of wheat seedlings, but reduced the negative correlation between LDPE leachate concentration with related indexes of wheat seedlings' buds and roots, indicating that the addition of Cd 2+ would stimulate or strengthen the toxic effect of LDPE leachate on wheat seedlings, especially oxidative stress.
The results show that wheat seedling growth is promoted under the high concentration (8.0 mg L − 1 ) of Cd

Conclusion
The average germination time of wheat seeds was shortened to about 2 days under single and combined treatments.
The germination rate of wheat seeds was inhibited by 0-8.6% under single treatments, while the combination of microplastics leachates and Cd 2+ solutions promoted wheat seed germination.This positive effect was more pronounced with combinations of the same concentration class compared to different concentration classes.The combination of 2.0 mg L − 1 Cd 2+ and 1.0 mgC L − 1 PVC promoted both wheat seed germination and seedling growth but increased antioxidant enzyme activity, while Cd 2+ could to some extent alleviate the harmful effect of PVC leachate on wheat seedlings under other combined treatments of Cd 2+ 0 and 8.0 mg L − 1 ; PVC1, PVC5 and PVC10 respectively represent 1.0, 5.0 and 10.0 mgC L − 1 PVC leachate; LDPE1, LDPE5 and LDPE10 respectively represent 1.0, 5.0 and 10.0 mgC L − 1 LDPE leachate.Cd2 + PVC1 and Cd8 + PVC1 respectively represent the combined treatment of 2.0 mg L − 1 or 8.0 mg L − 1 Cd 2+ in 1.0 mgC L − 1 PVC leachate solution; Cd2 + PVC10 and Cd8 + PVC10 respectively represent the combined treatment of 2.0 mg L − 1 or 8.0 mg L − 1 Cd 2+ in 10.0 mgC L − 1 PVC leachate solution; Cd2 + LDPE1 and Cd8 + LDPE1 respectively represent the combined treatment of 2.0 mg L − 1 or 8.0 mg L − 1 Cd 2+ in 1mgC L − 1 LDPE leachate solution.Cd2 + LDPE10 and Cd8 + LDPE10 respectively represent the combined treatment of 2.0 mg L − 1 or 8.0 mg L − 1 Cd 2+ in 10.0 mgC L − 1 LDPE leachate solution.Different superscript letters indicate signi cant differences, P < 0.05.Unlike the single treatments, the combined treatments promoted the germination of wheat seeds and the promotion effect of adding the same class concentration of Cd 2+ low concentrations may reduce the toxicity of Cd 2+ by promoting the metabolism of wheat carbohydrates and amino acids, thus achieving the effect of promoting the growth of wheat seedlings(Ding et al., 2022;Lian et al., 2020a).However, when the concentration of Cd 2+ solution or microplastic leaching solution is high, a large amount of Cd 2+ carried by the microplastics could be released into the body of the wheat seedlings, especially the roots, resulting the toxic effect of the pollutants exceeds the repair ability of the wheat seedlings (P ugmacher et al., 2020).Meanwhile, the enzyme activities involved in the synthesis, transformation, and metabolism of substances related to growth and development are inhibited, and the seedlings grow slowly(Jia et al., 2022;Sil et al., 2019).

Figure 2
Figure 2 Root biomass (a), root length (b), root surface area (c), average root diameter (d) and root volume (e) of wheat seedlings under different treatments enhanced after illumination(Mao et al., 2020;Sun et al., 2020;Li et al., 2020), thereby increasing the adsorbed or complexed Cd 2+ and weakening the harmful effects of the leachates and Cd 2+ on germination.However, with the continuous growth of roots and buds, the root system may gradually absorb the Cd 2+ or toxic substances in leachate and transmit them to the seedlings(Mao et al., 2020; Sun et al., 2020; Li et al., 2020).Zhao et al. (2023) also found that microplastics could promote the synthesis and secretion of rhizosphere amino acids and metabolites, resulting in promoting Cd 2+ accumulation in roots.Therefore, the enhanced bioavailability of Cd 2+ and toxic substances may inhibit the growth and development of wheat seedlings.Within the concentration range set in this study, 8.0 mg L − 1 2+ solution, low concentration (1.0 mgC L − 1 ) of PVC leachate, and the combined treatment of Cd2 + PVC1.The physiological and biochemical indicators show that the possible mechanism of wheat seedling stress involves oxidative stress, and the action mechanisms of varying concentrations of Cd 2+ solution and microplastic leachates are different.

Table 1
Germination indexes of wheat seeds under single and combined treatments : GR, GE, GI, VI and AGT respectively represent germination rate, germination energy, germination index, vigor index and average germination time.Cd2, Cd4 and Cd8 respectively represent Cd 2+ solutions of 2.0, 4. Note (Ding et al., 2022) concentration Cd 2+ solution and a low concentration PVC leachate.This may be because the microplastic leachate combined with Cd 2+ with the same class concentration may have lower toxicity than that of pollutants with different class concentrations, thus weakening the in uence of the leachate and Cd 2+ solution on germination(Ding et al., 2022).At the same time, the promotion of the Cd 2+ -PVC combination on wheat seed germination is greater than that of the Cd 2+ -LDPE combination, which may be because the adsorption capacity of PVC for Cd 2+ is signi cantly greater than that of LDPE (Mohajerani and Karabatak, 2020; Guo et al., 2020) and weakens the toxicity on germination more strongly.In general, the addition of microplastic leachate lessened the damaging effect of Cd 2+ solution on the germination of wheat seeds (Maia de Oliveira et al., 2015), and even achieve the effect of promoting wheat seed germination.Adding the same class concentrations of microplastic leachate to the Cd 2+ solution has a greater promotion impact than adding different class concentrations of microplastic leachates.
solution to microplastic leachates was greater than that of adding different class concentrations of Cd 2+ solutions.For example, the combined treatment of a low concentration Cd 2+ solution and a low concentration PVC leachate promoted the germination of wheat seeds more effectively than the combined and PVC.Biochemical indicators show that microplastic leachates and Cd 2+ solutions have different action mechanisms, and the stress of PVC leachate on the growth of wheat seedlings is greater than that of LDPE leachate.In short, results suggest that Cd 2+ , PVC and LDPE leachates have toxic effects on wheat growth, whether treated under single or combined treatments.Declarations Authorship contribution Ling Du: Lab experiments, Data curation, Visualization, Writing -original draft.Dongming Wu: Methodology, Supervision, Review & editing.Xi Yang: Formal analysis, Visualization.Li Xu: Visualization, Review & editing.Xu Tian: Data curation, Formal analysis, Visualization.Youping Li: Visualization, Review & editing.Yanmei Liu: Conceptualization, Writing -review & editing, Supervision.Project administration, Funding acquisition.