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. 2026 Jul 3;16(35):35808–35821. doi: 10.1039/d6ra03560d

Understanding mechanisms of combined polystyrene and cadmium toxicity in Leonurus japonicus Houtt. across varying pH levels

Qinghai Liu a,e, Canhui Chen b, Youming Dong b,✉, Jingyi Ren b, Yixin Xu b, Weiwen Qiu c, Cheng Qiu a,d, Minling Gao b,✉, Zhengguo Song b,✉
PMCID: PMC13329907  PMID: 42405109

Abstract

The combined toxicological effects of polystyrene (PS) and other pollutants on plants have been a significant focus of recent research. However, the effects of PS and cadmium (Cd) on Leonurus japonicus Houtt. (Leonurus) under varying pH conditions remain unexplored. This study examined the impacts of PS and Cd on the growth and medicinal components of Leonurus, with a focus on how these impacts vary across different pH levels. We selected Leonurus as the experimental subject, used PS and Cd as the test pollutant and conducted the experiment using hydroponic cultivation. The Cd concentration was the main factor affecting the physiological and biochemical indicators of Leonurus. An increase in pH significantly inhibited the absorption of Cd by the roots and leaves of Leonurus and reduced its toxic effects. Under pH 4.5 and 6.5 conditions, 4 mg L−1 Cd reduced root biomass by 60.85% and 41.52%, compared to CK (without Cd and PS). PS and Cd increased the levels of reactive oxygen species (O2−, H2O2) and malondialdehyde (MDA) content in the roots and leaves, leading to oxidative stress and membrane lipid peroxidation, ultimately resulting in decreased biomass as well as lower pyruvate and glutamic acid levels. Compared with the treatment with Cd alone, the combined toxicity of PS and Cd exhibited an antagonism effect. This study confirmed that increasing pH can reduce the bioavailability of Cd to Leonurus, and highlighted the potential negative effects of PS and Cd on its biomass and medicinal components.

The combined toxicological effects of polystyrene (PS) and other pollutants on plants have been a significant focus of recent research.graphic file with name d6ra03560d-ga.jpg

1. Introduction

As a promising therapeutic approach, the medicinal constituents of Chinese herbal medicine have attracted significant attention because of their mild nature and minimal side effects. Leonurus japonicus Houtt. (Leonurus), an herbaceous angiosperm native to China, South Korea, India, and other part of Asia, has been extensively studied (https://www.theplantlist.org/).1 It is widely used in traditional Chinese herbal medicine, particularly as Leonurus injection, which has demonstrated significant efficacy in treating postpartum hemorrhage.2 In Europe and South Africa, Leonurus is commonly employed for managing neurological and cardiovascular disorders, providing anticonvulsant and anxiolytic effects.3 Current research on Leonurus focuses primarily on its medicinal compounds and therapeutic properties. For instance, Nsuala et al. demonstrated that smoke from combusted Leonurus inhibited epileptiform behavior in zebrafish larvae induced by pentylenetetrazol.4 Obikeze et al. used computational and experimental approaches to show that Leonurus extract exhibits cardiovascular effects suggestive of β1 agonism in vivo.5 Wu et al. discovered that Leonurus has the potential to modulate JAK1 and STAT1 gene expression, thereby reducing neuronal apoptosis following cerebral hemorrhage.6 The medicinal functions of Leonurus have been verified and it has made great contributions to human health. However, its quality is profoundly influenced by the growth environment. The effects of environmental pollutants on the growth of Leonurus remain unclear, and the mechanisms underlying its responses to these pollutants require further exploration.

Microplastics have become a prominent focus in environmental science due to their ubiquitous presence and significant impact on plant growth. Microplastics have been detected in the oceans and lakes, in the northwest Himalayas, microplastics in lake surface sediments and water samples from Manasbal Lake range from 840 to 4020 particles per kilogram and 13 to 89 particles per liter, respectively.7,8 Similarly, microplastic concentrations in facility fields reached a alarming 3738 ± 2097 items per kg, predominantly composed of polypropylene and polyethylene, with 45% of these microplastics measuring less than 0.01 mm in size.9 The toxicity of microplastics to plants encompasses various detrimental effects, including the disruption of enzyme activity, elevated reactive oxygen species, reduced chlorophyll levels, impaired photosynthesis and root development, and, a consequent decline in plant dry matter accumulation.10 Zeb et al. reported that microplastics inhibited the stem elongation, photosynthesis, and chlorophyll content in lettuce, accompanied by a decrease in the relative abundance of amino acids, sugars, and sugar alcohols.11 Our previous studies further revealed that polystyrene microplastics reduced carrot biomass and carotene content by impairing root activity and photosynthesis, while also inducing oxidative damage.12 However, current research on the toxic effects of microplastics primarily focuses on common crops and vegetables, such as rice, wheat, lettuce, and carrots.10 It has been well documented that microplastics induce oxidative explosions in plants (such as wheat, rice, lettuce, etc.), disrupt metabolic functions, and inhibit nutrient absorption, thereby reducing crop yield and quality. However, to date, no studies have investigated the impact of microplastics on the growth and biochemical indicators of Leonurus.

Cd is an extremely toxic heavy metal and its adverse effects on plant growth have been extensively studied.13 The toxicity of cadmium in plants is characterized by stunted growth, chlorosis, necrotic tissue, DNA damage, compromised photosynthetic activity, and disruption in nutrient absorption and metabolic processes.14 Research on cadmium toxicity has primarily focused on mitigating its effects in essential crops such as wheat, rice. For example, Shahzad et al. utilized Bacillus thuringiensis and Salix alba to remediate cadmium-contaminated soil, leading to a substantial increase in wheat dry root weight, proline, and sugars content, while simultaneously alleviating the toxic effects of cadmium.15 Many studies have investigated the toxicity of Cd to plants, primarily focusing on grain and economic crops such as wheat, rice, and vegetables. Given the potential toxic effects of Cd on Leonurus, it is necessary to conduct research on its Cd tolerance and associated physiological responses.

Current research on the toxic effects of heterologous substances on plant growth increasingly focuses on the combined toxicity of multiple pollutants, analyzing their synergistic or antagonistic effects.16 Hong et al. observed that microplastics and enrofloxacin jointly reduced nitrogen and phosphorus absorption and inhibited photosynthesis in Myriophyllum verticillatum.17 Moreover, their combined effects on algae were synergistic, intensifying the negative impacts on growth and metabolism. Similarly, our previous research showed that microplastics inhibited arsenic (As) uptake in rice, reducing its adverse impact on yield.18 In contrast, Gan et al. found that polyethylene and microplastics in the environment significantly increased the bioavailability of chromium (Cr), promoting the absorption and transport of both Cd and Cr in wheat, which ultimately inhibited wheat growth.19 It has been hypothesized that microplastics may adsorb heavy metals such as Cd, thereby reducing their free ion concentration and bioavailability to plants. Despite these insights, the combined toxicological impact of PS and Cd on Leonurus need to be explored. Given the edible, medicinal and economic values of Leonurus, as well as the current environmental contamination by PS and Cd and their toxicological effects on plants, it is necessary to investigate their combined toxicological effects on Leonurus. Such a study would offer valuable insights into the environmental behavior and toxicological risks of PS and heavy metals, while also contributing to a deeper understanding of their broader ecological implications.

pH is an important factor affecting the toxicity of environmental pollutants to plants, Li et al. discovered that within a pH range of 4.5 to 7, a decrease in pH mitigated the toxicity of neodymium to wheat.20 Shen et al. found that the physiological stress induced by ciprofloxacin on duckweed intensified as the pH increased to 8.5.21 However, the impact of pH on the toxicity of PS and Cd to Leonurus has not yet been studied. In this study, Leonurus was chosen as the experimental subject, with PS of varying sizes and Cd serving as the test pollutant. The objectives of this study are as follows: (1) to investigate how PS affects the internalization of Cd in Leonurus; (2) to clarify the combined toxic mechanism of PS and Cd on Leonurus; (3) to examine the influence of pH on the toxicity of PS and Cd in Leonurus. It is the first to explore the combined toxic effects of PS and Cd on Leonurus japonicus, while also exploring the role of pH in modulating these toxic effects from a new perspective. Understanding the toxic impacts of PS and Cd on medicinal plants is critical, as it not only expands research on the combined toxicity of PS and heavy metals but also provides a more comprehensive perspective on the ecological and health risks posed by PS.

2. Materials and methods

2.1. Regents

Cadmium nitrate tetrahydrate, used to prepare Cd solution, was purchased from Xilong Science Co., Ltd. PS microspheres with particle sizes of 100 nm and 300 nm were provided by Wuxi Ruige Biotechnology Co., LTD. Ultrapure water (resistivity = 18 MΩ cm). Produced by Millipore water purification systems, was used for solution preparation. The kit used for determining the biochemical indexes of Leonurus was purchased from Suzhou Keming Biotechnology Co., Ltd. Leonurus seeds were obtained from the seed market in Anguo City, Hebei Province.

2.2. Hydroponics experiment

A 1/4 strength Hoagland nutrient solution was used as the base medium. The stress concentrations of PS and Cd were set according to the following research: Fuller and Gautam discovered that the abundance of microplastics in the surface soil could reach up to 7%.22 Zhou et al. reported that the concentrations of soil microplastics ranged from 0 to >10 mg kg−1.23 Dawood et al. discovered that soil Cd pollution could reach 50 mg kg−1, with the available Cd content being approximately 10%.24 Yu et al. set the Cd concentration at 2.8 mg L−1 when conducting toxicological experiments.25 PS solutions of 100 nm and 300 nm were prepared at concentrations of 5 mg L−1 and 10 mg L−1, respectively, while Cd solutions were prepared at concentrations of 2 mg L−1 and 4 mg L−1. Combined treatments of PS and Cd with varying particle sizes and concentrations were established, and a control (CK) without PS or Cd was included. The pH of the treatment solutions was adjusted to 4.5, 5.5, 6.5, resulting in a total 30 treatment combinations. Each treatment was replicated three times.

The Leonurus seeds were immersed in a 5% sodium hypochlorite solution for 15 minutes for disinfection, then rinsed with distilled water and placed in a seedling tray filled with vermiculite. The Leonurus plants were cultivated in a greenhouse under the following conditions: a 12-hour light/12-hour dark cycle, relative humidity of 60%, and a temperature of 25 ± 1 °C. When the Leonurus seedlings developed two true leaves, seedlings of similar size and shape were selected and transplanted into the 30 treatment solutions described above for 14 days, after which they were collected for subsequent analysis.

2.3. Laser scanning confocal microscopy (LSCM) detection

Fresh and clean Leonurus leaf samples were cut into longitudinal sections approximately 2 cm in length and embedded in 4% low melting point agarose. The samples were then sectioned into 20 µm slices using a cryosectioner (CM1950, Leica, Germany). Phosphate buffer solution (PBS) was applied drop by drop and the coverslips were sealed. Fluorescently labeled PS microspheres (excitation/emission 620/680 nm) were used for LSCM observation (TCS-SP5, Leica, Germany).

2.4. Leonurus biomass and Cd content

The roots and leaves of Leonurus were immersed in a 20 mmol L−1 disodium ethylenediamine tetraacetic acid (EDTA) solution for 10 min to dissolve Cd adsorbed on the surface, and then rinsed with ultra-pure water. The leaf and root samples were first dried at 110 °C for 0.5 h, and then dried at 65 ± 1 °C until a constant weight achieved, representing the dry weight of the samples. After drying, the samples were ground and mixed with a solution of HNO3 and H2O2 in a 7 : 3 ratio. The mixture was dissolved in using an electric digestion device at 115 ± 1 °C, heated until nearly dry, and then washed with 1% nitric acid solution. The resulting solution was transferred to a 25 mL volumetric flask, and the volume was adjusted to 25 mL. The Cd content in the solution was determined using an atomic absorption spectrophotometer (GGX-830, Beijing Haiguang Instrument Co., Ltd).26

2.5. Physiological and biochemical indicators

Extraction of crude enzyme solution: fresh roots or leaves (0.1 g) was placed in a mortar, and extracted with 1 mL of extraction solution. The extraction solution for O2˙−, malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD) consisted of Na2HPO4·12H2O and Na2HPO4, while the extraction solution for hydrogen peroxide (H2O2) was acetone. The mixture was homogenized on ice and transferred to a 2 mL centrifuge tube. The sample was centrifuged at 8000×g at 4 °C for 10 minutes, and the supernatant was collected as the crude enzyme solution for analysis. The contents of O2˙−, H2O2 and MDA, as well as the activity of SOD and CAT in crude enzyme solution, were analyzed according to the manufacturer's instructions.12

2.6. Glutamic acid and pyruvate determination

Leaf samples (0.1 g) were combined with 1 mL of extraction solution (sourced from Suzhou Keming Biotechnology Co., Ltd.), homogenized in an ice bath, and centrifuged at 10 000 rpm for 10 minutes at 4 °C. The supernatant was collected for further analysis of glutamic acid and pyruvate contents. The contents of glutamic acid and pyruvate were determined using UV-vis spectrophotometer based on the standard curve method, with measurements conducted at 340 nm and 520 nm, respectively.

2.7. Gaussian calculation

The Gauss package was used to analyze the interactions between PS and Cd, PS + Cd complexes and glutamic acid and pyruvate. Gaussian view software (V32128445015899W-5295 N) was used to establish molecular models of PS, Cd, glutamic acid and pyruvate. Gaussian view (G32131694014899W-5295 N) software optimized the structure with the b31yp/lanl2mb base set. Multiwfn software was used to analyze the checkpoint files generated by Gaussian software to calculate the molecular surface electrostatic potential. The results were visualized using Visual Molecular Dynamics software.

The effects of PS and DBP on the tertiary structure of enzymes were analyzed by molecular docking. The tertiary structure model of the SOD and CAT enzyme were obtained from the Structural Bioinformatics Research Collaboration (RCSB: https://www.rcsb.org/).27 PDB files and images before and after molecular docking were generated using PyMOL (version 0.99rc6), and molecular models were established using GaussView (V32128445015899W-5295 N). Molecular docking was performed using AutoDock (version 1.5.6 Sep\u17\u14). The Discovery Studio visualization tool (version 4.5.0.15071) was employed to analyze molecular interactions and generate two-dimensional diagrams, allowing evaluation of the effects of PS on the tertiary structure of SOD and CAT enzyme.

2.8. Combined toxicity

To evaluate the toxicity effects of Cd and PS on plants and to identify their interactions, the independent action (IA) model was employed for the calculations.28 The equation for the IA model is as follows:

2.8. 1

where “E(Cmix)” represents the anticipated outcome under combined toxicity action. “n” signifies the existence of n pollutants, each with a distinct mode of action and whose effects are mutually independent. “E(Ci)” is the effect caused by the substance of the ith component at concentration Ci. If the predicted outcome is lower than the experimental result, the combined toxicity is defined as synergistic. While it is classified as antagonistic if it exceeds the experimental result. If the predicted and experimental results are equal, the interaction is considered additive.

2.9. Adsorption experiment

2.9.1. Kinetics

A total of 308.48 mg of Cd(NO3)2·(H2O)4 was dissolved in 1 liter of water to prepare a Cd2+ solution with a concentration of 1 mmol L−1. Subsequently, 0.5 g of PS, either 100 nm or 300 nm in size, was added into Cd solution and stirred at 25 °C and 200 rpm, Samples of 1 mL were extracted at the following time intervals: 5, 10, 30, 60, 120, 240, 360, 480, 720, 960, 1200, 1440 minutes. The extracted samples were filtered with 0.22 µm filter (Millipore) before further analysis.

2.9.2. Isothermal

Cd2+ solutions at 2, 4, 6, 8, 10, and 20 mg L−1 (10 mL each) were placed in brown glass bottles and mixed with 10 mg of 100 nm or 300 nm PS. The mixtures were shaken at 200 rpm and 25 °C for 24 hours. The solutions were determined after filtering with 0.22 µm filter (Millipore).29

2.9.3. Detection and calculation

The Cd content in the solutions was determined by atomic absorption spectrophotometer (GGX-830, Beijing Haiguang Instrument Co., Ltd). The Zeta potential and particle size distribution before and after Cd adsorption by PS were analyzed using the Malvern Zetasizer Nano ZS90 and Malvem Mastersizer 2000 equipment (Malvern Panalytical, England).30

The adsorption capacity of Cd was calculated as follows:

qe = V(C0 − Ce)/m 2

In-particle diffusion model:

qt = kpt1/2 + xi 3

Langmuir isothermal model:

Qm = qebCe/(1 + bCe) 4

Freundlich isothermal model:

qe =kfCen 5

C e (mg L−1): the equilibrium concentration, C0 (mg L−1): the initial concentration, V (L): the volume of the solution, m (g): the mass of the adsorption material, qe (mg g−1): the equilibrium adsorption capacity, qt (mg g−1): the adsorption capacity at time t, and kp (g mg−1 min−1/2): the coefficient of diffusion rate in the particles, Qm (mg g−1): the maximum adsorption capacity of the Langmuir adsorption isotherm, and kf, b, and n are the constants in the corresponding model, respectively.

2.10. Statistical analysis

The “counta” and “stdev.p” functions in Excel were used to calculate the mean and standard deviation. In addition, one-way analysis of variance (ANOVA) and Duncan's test were applied using Statistical Package for the Social Sciences (20.0) for significant difference analysis (p < 0.05). Origin 2016 was used for constructing graphs. The R Programming Language (R version 4.1.2) and RStudio were used to perform multiple regression and redundancy analysis (RDA).

3. Results

3.1. Internalization of PS in Leonurus

The internalization of PS in Leonurus was demonstrated by fluorescence intensity. No red fluorescence was found in CK treatment (no PS and Cd). The red fluorescence intensity of Leonurus root treated with SPS (small polystyrene microspheres) was higher than that in roots treated with LPS (large polystyrene microspheres) (Fig. 1B and C). Cross-sectional fluorescence images showed that the SPS fluorescence was mainly concentrated in the xylem catheter region of the root, while as the LPS fluorescence was mainly located in the phloem and vascular bundle regions (Fig. 1B and C). The fluorescence intensity in the PS + Cd composite treatment was lower than that in PS-only treatment, indicating that Cd inhibited the internalization of PS in Leonurus.

Fig. 1. Laser confocal image of Leonurus roots (A and a) CK, (B and b) 100 nm polystyrene, (C and c) 300 nm polystyrene, (D and d) 100 nm polystyrene and Cd, (E and e) 300 nm polystyrene and Cd. (Uppercase letters denote the longitudinal section and lowercase letters denote the transverse section).

Fig. 1

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3.2. Cd content

Factors affecting Cd absorption of Leonurus were classified as PS size, PS concentration, Cd concentration and pH. Among these, Cd concentration (p < 0.001) and pH (p < 0.05) were the main factors affecting Cd content in roots and leaves. pH was negatively correlated with Cd content in both roots and leaves treated with PS and Cd (Table 1). PS inhibited the uptake of Cd by Leonurus. At pH = 4.5, the addition of 5 mg L−1 SPS (SPS5) to treatments with Cd2 (2 mg L−1 Cd) and Cd4 (4 mg L−1 Cd) resulted in a maximum reduction of 9.2% and 10.3% in root Cd content, compared to Cd2 and Cd4 treatments without SPS. Similarly, the addition of SPS to Cd2 reduced leaf Cd content by a maximum of 5.5% compared to Cd2 alone (Fig. 2). At pH = 5.5, the addition of SPS5 to Cd2 and Cd4 resulted in a maximum reduction in root Cd content by 11.9% and 12.6%, respectively. Corresponding reductions in leaf Cd content were 35.2% and 12.6%, compared with single Cd2 and Cd4 treatment. At pH = 6.5, SPS5 + Cd4 treatment led to the highest reduction in root Cd content, decreasing by 12.4% compared to Cd4 alone. In leaves, SPS5 + Cd treatment reduced Cd content by up to 29.5% and 33.1% compared to Cd2 and Cd4 treatments alone, respectively.

Table 1. Multivariate regression analysis of various indicators of Leonurusa.

Multivariate regression equation R 2
LCd y = −13.19*x1 + 2.34x2 −2.66x3 + 72.54***x4 0.667
RCd y = −56.35*x1 −1.67x2 −15.09x3 + 727.36***x4 0.814
LW y = 0.06*x1 −0.02x2 + 0.01x3 −0.22***x4 0.704
RW y = 0.01x1 + 0.02x2 −0.01x3 −0.03*x4 0.699
LMDA y = −9.25**x1 + 2.30x2 −4.03*x3 + 23.79***x4 0.873
RMDA y = −5.26**x1 + 1.29x2 −1.78x3 + 16.01***x4 0.748
LCAT y = −0.03x1 −0.29x2 + 0.02x3 −5.49***x4 0.668
RCAT y = 0.18x1 + 0.09x2 + 0.22x3 −4.04***x4 0.784
LSOD y = 1.23x1 + 3.71x2 + 0.60x3 −35.57***x4 0.809
RSOD y = 0.90x1 + 3.22x2 + 0.34x3 −33.18***x4 0.695
LO2˙− y = −0.65x1 −1.89x2 + 4.36**x3 + 17.03***x4 0.752
RO2˙− y = −0.69x1 −1.89x2 + 4.30**x3 + 16.98***x4 0.662
LH2O2 y = 0.10x1 + 0.13x2 + 0.43x3 + 1.91***x4 0.718
RH2O2 y = −0.07x1 −0.27x2 + 1.92***x3 + 1.32***x4 0.659
Glutamate y = −0.03x1 + 0.17***x2 + 0.02x3 −0.08***x4 0.773
Pyruvic acid y = −1.15*x1 −0.48x2 −0.61x3 −3.77***x4 0.805
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a

x 1, x2, x3, x4 mean influencing factors of pH, PS concentration, PS size, Cd concentration. Coefficient confidence intervals was 0.95, *, **, and *** respectively indicate p < 0.05, 0.01, and 0.001.

Fig. 2. Cd content in roots (A) and leaves (B) of Leonurus. 2 and 4 after Cd represent concentrations of 2 and 4 mg L−1, SPS and LPS represent polystyrene with particle sizes of 100 nm and 300 nm, and 5 and 10 after SPS and LPS represent polystyrene concentrations of 5 and 10 mg L−1. The significance analysis was the analysis at the same pH and Cd concentration. A single-factor analysis was conducted based on the PS concentration. The comparison was made between the small-particle-sized PS at 5 and 10 mg L−1 and the PS-free treatment, as well as between the large-particle-sized PS at 5 and 10 mg L−1 and the PS-free treatment. *, **, and *** respectively indicate p < 0.05, 0.01, and 0.001.

Fig. 2

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3.3. Effect of PS and Cd on Leonurus biomass

Factors influencing Leonurus biomass included PS size, PS concentration, Cd concentration and pH. pH was positively correlated with leaf biomass (p < 0.05), while Cd concentration was negatively correlated with root biomass (p < 0.05) and leaf biomass (p < 0.001). At pH = 4.5, compared to the control (CK, no PS or Cd), Cd2 and Cd4 reduced roots biomass by 34.9% and 60.8%, respectively, and leaf biomass by 47.4% and 55.6%. PS also decreased both root and leaf biomass. A concentration of 5 mg L−1 SPS and LPS had a more significant impact on root biomass compared to 10 mg L−1 SPS or LPS, whereas 5 mg L−1 SPS and LPS caused a smaller reduction in leaf biomass compared to 10 mg L−1 SPS and LPS. In the case of combined pollution of Cd and PS, the toxicological effect was antagonistic (Table S1 and S2). Specifically, 10 mg L−1 SPS (SPS10) and LPS (LPS10) mitigated the effects of Cd2 and Cd4 on roots biomass. Similarly, SPS10, LPS5 (5 mg L−1 LPS), and LPS 10 reduced the effects of Cd2 on leaf biomass, SPS5 and LPS5 reduced the effects of Cd4 on leaf biomass (Fig. S1).

At pH = 5.5, the effects of individual Cd and PS treatments on Leonurus biomass were to those observed at pH = 4.5, and their combined pollution remained antagonistic. SPS5, SPS10, and LPS5 alleviated the effects of Cd2 on roots and leaf biomass, while LPS10 reduced the effects of Cd2 and Cd4 on roots biomass. At pH = 6.5, SPS5, SPS10, LPS5, and LPS10 mitigated the effects of Cd2 and Cd 4 on roots biomass, and reduced the effects of Cd2 on leaf biomass.

3.4. Effect of PS and Cd on Leonurus pyruvate and glutamic acid content

pH was negatively correlated with pyruvic acid concentration (p < 0.05). PS concentration was negatively correlated with glutamate content (p < 0.001), and Cd concentration was negatively correlated with both pyruvate and glutamate contents (p < 0.001). At pH = 4.5, compared to the control (CK), Cd2 and Cd4 reduced pyruvate content by 3.3% and 7.2%, respectively, but increasing glutamate content by 7.9% and 14.0%. PS did not cause significant changes in pyruvate content (p > 0.05), but significantly increased the glutamate content (p < 0.001). The highest increases in glutamate content caused by SPS and LPS were 54.4% and 51.6% respectively. Under combined SPS10 and Cd4 pollution, pyruvate content was significantly lower than that of Cd4 alone. The glutamate content was higher under SPS and LPS combined with Cd2 than Cd2 alone, and it also exceeded that of Cd4 when combined with LPS and Cd4 (Fig. S2).

At pH = 5.5, except for LPS10, which significantly reduced pyruvate content by 23.6%, the effects of individual Cd or PS treatments on pyruvate and glutamate content were similar to those observed at pH = 4.5. Under the combination of SPS5, LPS5, LPS10 and Cd2, the pyruvate content was lower than in Cd2 alone. SPS10 combined with Cd4 alleviated the inhibitory effect of Cd4 on pyruvate content. The glutamate content under SPS5, LPS5, SPS10 and LPS10 combined with Cd4 was higher than that of Cd4 alone. At pH = 6.5, except for SPS5, LPS5, LPS10, which significantly reduced pyruvate content by 8.4%, 11.9%, 11.8%, respectively, the effects of individual Cd or PS treatments on pyruvate and glutamate content were similar to those at pH = 4.5. The combined effect of PS and Cd did not induce significant changes pyruvate content compared to Cd alone. However, SPS5, LPS5, LPS5, LPS10 combined with Cd increased glutamate content compared to Cd2 or Cd4 alone.

3.5. Effect of PS and Cd on Leonurus reactive oxygen species and antioxidant enzymes

The levels of O2˙−, H2O2 in roots and leaves increased with rising pH, while MDA levels decreased as pH increased. Factors affecting Leonurus reactive oxygen species and antioxidant enzymes in Leonurus included PS size, PS concentration, Cd concentration and pH. Cd concentration was positively correlated with roots and leaves O2˙− (p < 0.001) and H2O2 (p < 0.001). PS size was positively correlated with O2˙− levels in roots and leaves (p < 0.01) and with H2O2 content in roots (p < 0.001).

At pH = 4.5, compared to the CK, Cd2 and Cd4 increased O2˙− levels in roots by 77.6% and 48.0%, respectively, and in leaves by 61.8% and 39.1%. LPS caused a greater increase in O2˙− levels in roots and leaves than SPS. LPS5 and LPS10 increased O2˙− levels in roots by 12.2% and 28.9%, and in leaves by 11.2% and 24.1%, respectively, compared to CK. The effects of both single and combined pollution of PS and Cd on the roots of Leonurus were greater than those on the leaves. Under combined pollution of SPS10, LPS10 with Cd2, the O2˙− content was significantly lower than that of Cd2 alone. However, under combined pollution of SPS5, LPS5, SPS10, LPS10 with Cd4, the O2˙− content was significantly higher than that of Cd4 alone. At pH = 5.5 and pH = 6.5, the effects of single and combined pollution of PS and Cd on O2˙− content in roots and leaves were similar to those observed at pH = 4.5 (Fig. S3).

The effects of PS and Cd on H2O2 levels followed a similar trend as those observed for O2˙−. Cd concentration was positively correlated with MDA content in roots and leaves (p < 0.001), while pH was negatively correlated with MDA content (p < 0.01). At pH = 4.5, Cd2 and Cd4 increased MDA content in roots by 65.3% and 141.2%, respectively, and in leaves by 61.9% and 140.0%, compared to CK. SPS5, SPS10, LPS5, LPS10 increased MDA content in roots by 4.0%, 31.8%, 10.8%, and 27.8%, respectively, and in leaves by 16.5%, 30.6%, 2.5%, and 30.9%, compared to CK. The effect of PS at 10 mg L−1 on MDA content in roots and leaves was greater than that of PS at 5 mg L−1. SPS5, SPS10, LPS5, LPS10 reduced the effects of Cd2 on MDA content in roots and leaves, but did not significantly change the effects of Cd4 on MDA content in roots and leaves.

At pH = 5.5, the effects of individual Cd or PS treatments on MDA content in roots and leaves were similar to those at pH 4.5. Under the combined pollution of PS and Cd, changes in MDA content in roots and leaves were not greatly different from those observed with Cd alone. At pH = 6.5, the effects of both single and combined pollution of PS and Cd on MDA content in roots and leaves remained similar to those at pH = 4.5.

Cd concentration was negatively correlated with SOD and CAT activities in roots and leaves (p < 0.001). At pH = 4.5, compared to the CK, Cd2 and Cd4 decreased SOD activity in roots by 13.1% and 14.0%, respectively, and in leaves by 10.3% and 11.1%. SPS5, SPS10, LPS5, LPS10 increased SOD activity in roots by 17.1%, 10.0%, 13.9%, 9.4%, respectively, and in leaves by 19.8%, 12.8%, 16.6%, 12.2%, compared to CK. The effect of PS on SOD activity in roots was less pronounced than its effects in leaves, and 10 mg L−1 PS had a smaller impact on SOD activity in both roots and leaves compared to 5 mg L−1 PS. Under the combined pollution of PS and Cd2, SOD activities in roots and leaves were lower than that observed with Cd2 alone. Similarly, under the combined pollution of PS and Cd4 at 5 mg L−1, SOD activities in roots and leaves were lower than that of Cd4 alone. At pH = 5.5 and pH = 6.5, the effects of both single and combined pollution of PS and Cd on SOD activities in roots and leaves were consistent with those observed at pH = 4.5. The effects of PS and Cd on CAT activity were similar to those found for SOD activity (Fig. 3).

Fig. 3. Effect of polystyrene (PS) and Cd on Leonurus (A) SOD and (B) CAT. (CK indicates no PS or Cd was added, 2 and 4 after Cd represent concentrations of 2 and 4 mg L−1, PS and LPS represent polystyrene with particle sizes of 100 nm and 300 nm, and 5 and 10 after PS and LPS represent polystyrene concentrations of 5 and 10 mg L−1).

Fig. 3

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3.6. Correlation between physiological and biochemical indicies of Leonurus

The first axis explained 98.9% of the variation and was positively correlated with SOD and CAT activities, indicating these enzymes were the main factors affecting biomass and nutrient composition. The second axis explained 0.94% of the variation. Leaf and root biomass, along with glutamic acid and pyruvate contents were positively correlated with SOD and CAT activities, and were negatively correlated with O2˙−, H2O2, MDA and Cd contents (Fig. 4A). Cd content in roots and leaves was positively correlated with O2˙−, H2O2, MDA content, while being negatively correlated with SOD and CAT activities. This suggested that Cd induced reactive oxygen species (ROS) production and inhibited enzyme activity (Fig. 4B). Furthermore, O2˙−, H2O2, MDA levels were positively correlated with one another, highlighting the role of ROS in triggering membrane lipid peroxidation.

Fig. 4. (A) RDA analysis of dry weight, glutamic acid, pyruvate content and biochemical indexes of Leonurus and (B) correlation analysis of different indexes. (L and R mean leaves and roots, RB means roots biomass, LB means leaves biomass, GLU means glutamic acid, PA means pyruvate acid).

Fig. 4

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Regression analysis of root and leaf biomass with other physiological and biochemical indicators showed that biomass was negatively correlated with the contents of O2˙−, H2O2, and MDA, and positively correlated with SOD and CAT activities. The F test results of the regression equation were all <0.05, indicating that the models were statistically significant. All five variables (O2˙−, H2O2, MDA, SOD and CAT) had significant effects on root and leaf biomass (Table S3). When biomass was used as the independent variable (X) and the other indicators as dependent variables (Y), the regression equations for leaf indicators were as follows: O2−: Y = −64.06X + 135.50, H2O2: Y = −7.35X + 22.76, MDA: Y = −406.02X + 92.81, SOD: Y = 116.25X + 175.66, and CAT: Y = 19.74X + 42.89. The regression equations of root indicators were: O2−: Y = −376.06X + 117.56, H2O2: Y = −29.65X + 19.41, MDA: Y = −332.02X + 66.94, SOD: Y = 587.40X + 95.90, CAT: Y = 81.26X + 33.81.

3.7. Interaction between PS and Cd

The adsorption of Cd by PS occurred three stages, including mass transfer, intra-particle diffusion and equilibrium (Fig. S4A). The slope of the adsorption was related to the adsorption rate, with a higher rate observed during the mass transfer stage compared to the intra-particle diffusion stage (Table S4). The adsorption rate of Cd was greater for 100 nm PS than for 300 nm PS, and the equilibrium was reached within 400 minutes. The isothermal adsorption model for Cd adsorption by PS aligned more closely with Langmuir model. The maximum adsorption capacity of Cd by 100 nm (300 nm) PS at pH = 4.5, 5.5, 6.5 was 0.738 (0.577), 0.741 (0.581), 0.745 (0.590) mg g−1, respectively (Table S5).

At pH = 4.5, the zeta potential of 100 nm PS (SPS) and 300 nm PS (LPS) was −6.56 and −5.95 mV, respectively. Following Cd adsorption, the zeta potential increased to 7.34 for SPS and 7.52 mV for LPS, respectively (Fig. S5). The average sizes of SPS and LPS were 101.20 and 313.82 nm, respectively. After Cd adsorption, these sizes increased to 141.6 nm for SPS and 382.65 nm for LPS (Fig. S6). The results of computational chemistry analysis indicate that after the reaction between PS and Cd, the area with negative charge in the surface electrostatic potential was on the PS side, while the area with positive charge is on the Cd side (Fig. 5).

Fig. 5. Gaussian calculation analysis of interaction of PS, Cd, glutamic acid and pyruvate. (A) PS and Cd molecular cluster models before simulation, (B) PS and Cd reaction intermediate state, (C) PS and Cd molecular cluster models after simulation, (D) surface electrostatic analysis of PS and Cd, (E) PS + Cd and glutamic acid molecular cluster models before simulation, (F) PS + Cd and glutamic acid molecular cluster models after simulation, (G) surface electrostatic analysis of PS + Cd and glutamic acid (H) PS + Cd and pyruvate molecular cluster models before simulation, (I) PS + Cd and pyruvate molecular cluster models after simulation, (J) surface electrostatic analysis of PS + Cd and pyruvate. The red, blue, yellow, gray, and white spheres represent atoms O, N, Cd, C, and H, respectively. Blue area was the negatively charged area and red area was the positively charged area.

Fig. 5

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4. Discussion

4.1. Internalization of PS and Cd in Leonurus

Microplastics can enter the plant vascular column through cracks at the sites of lateral root formation, subsequently entering the xylem ducts in the roots. Finally, under the combined effects of root pressure and transpiration, they are transported through the ducts to the stems and leaves.31 Microplastics with smaller particle sizes are more likely to be absorbed by plants. For instance, Li et al. found that lettuce absorbed smaller-sized PS particles more effectively than large ones, which aligns with our observation that the fluorescence intensity in Leonurus root treated with 100 nm PS was higher than that in roots treated with 300 nm.32 The absorption of microplastics by plants is influenced by the surface charge of microplastics. Plant root exudates, primarily composed of negatively charged organic acids, amino acids and sugars, can promote the agglomeration of positively charged particles in the rhizosphere, reducing their entry into plant roots.33 Sun et al. found that negatively charged microplastics were more likely to be taken up by Arabidopsis than positively charged ones.34 This is because, in response to stress, plant roots secrete large amounts of oxalate, which induces aggregation of positively charged microplastics, increasing their particle size and reducing their uptake. In contrast, negatively charged microplastics are not affected by oxalate and remain more readily absorbable. The zeta potential of PS shifted from negative to positive after Cd adsorption, indicating surface charge neutralization and reversal. This change likely reduces the internalization of PS into Leonurus roots, as negatively charged microplastics are more readily taken up than positively charged ones.

Cd can enter plant root system through two pathways: the apoplast (movement along cell wall spaces from the epidermis and cortex to the endodermis, and then into the xylem and phloem) or the symplast (via root hair cell membranes, through plasmodesmata for cell-to-cell transport, and passing through the cortex, endodermis, and pericycle to reach the root duct cells).35,36 During Cd uptake and transport, the casparian strip in root endodermis functions as a barrier that partially restricts Cd transport, causing lower Cd content in leaves compared to roots.37 In acidic environment with low pH, plant root damage can occur, exacerbating the accumulation of heavy metals.38 For example, Wang et al. found a negative correlation between pH and Cd content in the roots and shoots of Solanum nigrum L., which was consistent with our findings.39 The inhibition of Cd absorption by PS in Leonurus may be attributed to two possible mechanisms: first, PS adsorbs Cd, reducing the bioavailable Cd in the solution; second, PS decreases root activity, thereby limiting the plant's capacity to absorb Cd.40 This study found that the fluorescent intensity of smaller PS particles (100 nm) was higher, while their inhibitory effect on cadmium absorption was lower than that of larger particles. We speculate that larger PS particles, being less readily internalized, accumulate on the root surface and may impair root vigor, thereby more effectively inhibiting Cd uptake. However, direct measurements of root activity are needed to confirm this hypothesis.

4.2. Interaction between PS and Cd

During the synthesis of polystyrene, the styrene oligomer forms a straight chain with vinyl at both ends. When polymerized, these electron-rich vinyl groups are exposed on the surface, imparting a negative charge to the polystyrene. As a result, polystyrene typically has a point of zero charge (pHPZC) below 7 and is negatively charged in water at near neutral pH.41 This negative charge facilitates interaction with Cd2+ ions. In this study, PS attracted Cd2+ through electrostatic interaction and van der Waals force, disrupting the molecular structure of cadmium nitrate, and finally formed a PS + Cd complex. In this complex, the maximum positive charge was contributed by Cd ions (Fig. 6A–D). This interaction also explained the increase in the zeta potential of PS after Cd adsorption. The electrostatic repulsion caused by the negative charge on the PS surface was neutralized by Cd ions, altering the surface charge strength and resulting in a higher zeta potential.42 He and Xie reported an increase in Zeta potential following the adsorption of Pb(ii), Cd(ii) and Ni(ii) by hydrated manganese dioxide.43 Electrostatic interaction and van der Waals forces can also lead to agglomeration of materials, thus an increase in particle size after adsorption.44 This phenomenon is consistent with the findings of Gao et al. who found a significant increase in particle size after PS adsorbed Cd.40

Fig. 6. The influence of PS and Cd complexes on the tertiary structure of (A) SOD and (B) CAT enzymes.

Fig. 6

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The adsorption of Cd by PS can be divided into three stages: (1) mass transfer stage: Cd passed through the liquid–solid interface to reach the surface of PS; (2) in-particle diffusion stage: Cd diffused into the inner layer or pores on the PS surface; (3) equilibrium stage: Adsorption/desorption reached kinetic equilibrium.45 The intersection of the tangent line with the Y-axis during the mass transfer stage was close to the origin, indicating that the mass transfer stage was the rate-limiting step in the overall adsorption process. The adsorption of Cd by PS aligned more closely with the Langmuir isothermal model, suggesting that Cd was uniformly adsorbed onto the PS surface. Electrostatic interaction and van der Waals force are likely the main mechanisms driving Cd adsorption by PS.46 Yu et al. studied the adsorption effect Cd by PS using Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS).47 The morphology of PS and PS–Cd complexes was further analyzed using scanning electron microscopy (SEM). The results showed no significant differences in surface functional groups between PS and PS–Cd complexes, suggesting that the adsorption process was primarily governed by physical interactions.

4.3. Effects of PS and Cd on physiological and biochemical properties of Leonurus

When plants are stressed by exogenous substances, the supply of NADP is insufficient, leading to an overloaded in the electron transport chain. This causes electrons leakage from chloroplasts, mitochondria, and the plasma membrane, greatly increasing the production rate of O2˙−.48 Excess O2˙− can be spontaneously removed by cells through the action of SOD, which catalyzes dismutation reaction to produce H2O2.49 H2O2 is then decomposed into H2O and O2 by CAT, thereby mitigating excess reactive oxygen species (ROS) and protecting plants from oxidative damage.40 However, when ROS are not effectively removed, they cause lipid peroxidation of membrane phospholipids with unsaturated double bonds, leading to the production of MDA.50 In this study, it was found that under the of PS and Cd stress, the activities of SOD and CAT were inhibited, while the levels of O2˙−, H2O2, and MDA were positively correlated. This suggests that the inhibition of enzyme activity by PS and Cd, combined with increased O2˙− production, contributed to oxidative damage in Leonurus. Jin et al. discovered that nano-plastics increased the contents of H2O2 and MDA in rice, causing oxidative damage.51 The PS–Cd complex can interact with amino acid residues on enzyme proteins through non-covalent interactions, thereby affecting enzyme function. The binding energies of the PS–Cd complex with SOD and CAT were −122.91 kcal mol−1 and −109.30 kcal mol−1, respectively. In the PS–Cd–SOD interaction, the PS component primarily interacted with arginine, leucine, proline, and threonine, while the Cd component mainly bound to α-amino acids and asparagine. In the PS–Cd–CAT interaction, the monomers of PS mainly interacted with arginine, aromatic amino acids, and phenylalanine, whereas the Cd component primarily bound to glutamic acid (Fig. 6). An increase of pH was found to mitigate the rise in MDA content, possibly due to reduced bioavailability of Cd at high pH value, thus lessening its toxicity to Leonurus. Malik et al. similarly reported that a decrease in effective Cd content in the growth medium reduced the extent of MDA accumulation in melon plants.52

Oxidative damage caused by exogenous substances ultimately manifests as reduced biomass. ROS promote the degradation of Ribulose-1,5-bisphosphate carboxylase (RuBisCO), thereby imparing dry matter accumulation. For instance, exposure of mung bean germ leaves to UV-B radiation has been shown to increase H2O2 levels, resulting in a decline in RuBisCO activity and biomass.53 Xia et al. found that nano-plastics reduced the root length, stem length and chlorophyll content of rice.54 In this study, the combined exposure to PS and Cd inhibited the biomass of Leonuru, with a significant negative correlation observed between biomass and the levels of ROS and MDA. The production of pyruvate mainly results from the degradation of glucose and the fixation of carbon dioxide by the calvin cycle in photosynthesis.55 Pyruvate subsequently enters the tricarboxylic acid cycle to form α-ketoglutarate, which is then converted to glutamic acid by reductive amination. In this study, pyruvate content was positively correlated with glutamic acid and negatively correlated with ROS levels. This relationship may be attributed to ROS inhibiting dry matter accumulation in the Calvin cycle, hence suppressing pyruvate production. Computational chemical analysis also indicates that the influence of PS + Cd on the molecular structure of pyruvic acid is greater than that on the molecular structure of glutamic acid, which may result in the inhibition of PS + Cd on pyruvic acid being greater than that on glutamic acid. An increase in pH was found to enhance the biomass of Leonurus leaves under PS and Cd stress, suggesting that pH mitigated the toxicity of PS and Cd to Leonurus, likely by affecting the bioavailability of Cd. Khan et al. reported that when pH = 5.5 and 7.5, the toxicity of aluminum to submerged plants was lower compared to pH = 4.5, indicating that higher pH levels can reduce the toxicity of certain metals to plants.56 This finding is consistent with the results of this study. It is essential to avoid microplastic and Cd contamination during Leonurus cultivation. If avoidance is not feasible, increasing the pH of the growing environment may help mitigate the toxic effects of these pollutants.

5. Conclusion

PS and Cd can trigger oxidation bursts, leading to an increase of ROS, which caused membrane lipid peroxidation. Eventually, this manifests as changes in biomass, pyruvate, and glutamate levels. In the absence of Cd, PS alone promoted SOD and CAT activities. However, under combined PS + Cd exposure, PS did not alleviate Cd-induced inhibition of these antioxidant enzymes. PS and Cd suppressed pyruvate content but significantly increased glutamic acid content, likely as a compensatory stress response, which may be related to the generation of ROS and the structural damage caused at the molecular level. Both PS and Cd reduced the biomass of roots and leaves under individual and combined conditions, with their combined toxicological effect being antagonistic. This antagonism was mainly due to the reduced bioavailability of Cd and the decreased internalization of PS attributed to the interaction between PS and Cd. Among the influencing factors (Cd concentration, pH, PS concentration, PS size), Cd concentration has the greatest influence on these indices. pH was significantly negatively correlated with Cd content, MDA content and pyruvate content in roots and leaves, while positively correlated with leaf biomass. At pH = 6.5, the dry weight of CK leaves was 13.6% higher compared to pH = 4.5, and MDA content in CK roots and leaves were 27.0% and 31.0% lower, respectively, than those at pH = 4.5. Under pH = 4.5, 5.5 and 6.5, the maximum reductions of Leonurus root (leaf) biomass were 60.8% (55.6%), 38.3% (52.9%) and 48.6% (56.9%), respectively, compared to CK.

Based on this, in moderately acidic soils (pH 4.5–6.5) contaminated by Cd or combined with Cd and PS microplastics, it is recommended that soil pH management be regarded as one of the core strategies for reducing plant toxicity. Specifically, by applying amendments such as lime, calcium carbonate, or silicate magnesium fertilizers, the soil pH can be adjusted to above 6.5 to effectively inhibit the absorption of Cd by the roots, alleviate oxidative stress and membrane lipid peroxidation damage, thereby increasing plant biomass and medicinal quality.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

RA-016-D6RA03560D-s001

Acknowledgments

This work was financially supported by the Key scientific and technological research topics of Xizang (No. XZ202501ZY0028), Key research and development program of Xizang (XZ202601ZY0114), Earmarked Fund for CARS-05, the Innovation Team Project of Guangdong Provincial Department of Education, China (2023KCXTD012), and supported by Guangdong Natural Science Foundation (No. 2023A1515110486 and No. 2026A1515011256), Agricultural and Animal Husbandry Science and Technology Innovation Project of XZAAAS (XZNKY-2025-CXGC-T16).

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra03560d.

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Associated Data

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Supplementary Materials

RA-016-D6RA03560D-s001
RA-016-D6RA03560D-s001.pdf (647.8KB, pdf)

Data Availability Statement

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra03560d.

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