Combined Exposure to Polyethylene Microplastics and Copper Affects Growth and Antioxidant Responses in Rice Seedlings

Abstract

The co-existence of polyethylene microplastics (PE-MPs) and heavy metals in aquatic ecosystems poses emerging threats to crop systems, yet their combined phytotoxic effects remain insufficiently understood. In this study, hydroponic rice (Oryza sativa) seedlings were exposed to PE-MPs (50 mg/L) and copper (Cu, 20 mg/L) individually and in combination. The results showed that PE-MPs alone had no significant impact on shoot or root elongation, while Cu exposure slightly reduced root length (from 6.2 cm in the control to 5.8 cm) without affecting shoot growth (~37 cm). Combined PE+Cu treatment resulted in intermediate biomass values, suggesting that microplastics partially mitigated but did not eliminate Cu toxicity. Antioxidant responses displayed organ specificity: shoot peroxidase (POD) activity dropped sharply from >10,000 U/g in the control to ~1200 U/g under Cu exposure, while root POD activity decreased from >11,000 U/g in the control to ~1500 U/g under combined exposure. Cu accumulation was markedly elevated under co-exposure, reaching ~450 mg/kg, about 25% higher than Cu alone and more than 12 times greater than control. These findings demonstrate that PE-MPs can enhance Cu bioavailability and uptake, thereby intensifying oxidative stress in roots while altering shoot defense responses. The study highlights the ecological risks of microplastic–metal co-contamination in agricultural systems and underscores the need for further investigation into long-term impacts on crop productivity and food safety.

1. Introduction

With the intensification of global plastic pollution, microplastics (MPs) have emerged as ubiquitous contaminants in the environment. Defined as plastic particles under 5 mm, microplastics arise primarily from the physical weathering of plastic goods, the mechanical fragmentation of plastic litter, and the intentional incorporation of plastics into various industrial applications [1]. Polyethylene (PE), one of the most widely used plastics globally, is extensively applied in packaging, agriculture, construction, and various industrial sectors. As a result, polyethylene microplastics (PE-MPs) have become one of the most prevalent types of MPs in the environment [2,3]. PE-MPs are not only commonly detected in aquatic systems, soils, and the atmosphere, but can also bioaccumulate through the food chain, potentially exerting long-term adverse effects on organisms [4,5].

In natural environments, due to their chemical stability and large specific surface area, PE-MPs can adsorb and transport various toxic pollutants, particularly heavy metals such as copper (Cu) [6,7,8]. Cu, while being a common environmental contaminant originating from agricultural fertilization, industrial discharges, and municipal wastewater, is also an essential micronutrient for plants, where it participates in photosynthesis, respiration, and antioxidant defense. However, when present in excess, Cu can accumulate in crops, inducing oxidative stress and impairing growth [9]. When accumulated in plants, Cu may induce oxidative stress, interfere with nutrient uptake, and ultimately impair plant growth and development [10]. While a growing body of research has focused on the impacts of MPs on aquatic and soil organisms [11,12], evidence suggests that MPs can alter soil physicochemical properties, influence rhizosphere microbial communities, and interact with co-existing contaminants, thereby exerting complex effects on plant growth and antioxidant systems [10,11]. Moreover, prolonged exposure to MPs may enhance metal uptake in plants, exacerbating oxidative stress responses and disrupting physiological functions [12]. However, when present in excess, Cu can accumulate in crops, inducing oxidative stress and impairing growth [13,14]. Although the following studies primarily refer to soil studies, they provide insights into the interactions of Cu with microplastics, which are relevant to our hydroponic study.

However, the combined effects of PE-MPs and heavy metals, particularly Cu, on plants remain insufficiently explored. Recent studies have demonstrated that the combined exposure of microplastics with chemical elements can produce synergistic or antagonistic effects in plants. For instance, Dong et al. [3] reported that microplastic particles increased arsenic toxicity to rice seedlings by enhancing uptake and oxidative stress. Zong et al. [4] found that polystyrene microplastics altered the uptake of copper and cadmium in wheat, aggravating heavy metal accumulation in plant tissues. Similarly, Jin et al. [10] observed that co-contamination with polystyrene microplastics and copper ions slightly inhibited root growth of rice seedlings, although no significant synergistic effects were noted on shoot development. Tang et al. [12] showed that polyethylene microplastics influenced the absorption and accumulation of mineral elements in rice under hydroponic conditions. Collectively, these findings highlight that microplastics can interact with essential or toxic elements, modulating their bioavailability and toxicity in plants [15].

Rice is one of the most important staple crops worldwide, providing food for over half of the global population. Moreover, rice is generally cultivated under flooded or semi-aquatic conditions, making it especially susceptible to contamination from microplastics and potentially toxic elements present in water and sediments. Due to its global significance for food security and its frequent use as a model species in pollutant uptake and plant stress studies, rice offers an ideal system to investigate the combined effects of polyethylene microplastics and copper.

Therefore, this research aims to systematically explore the combined impact of co-exposure to PE-MPs and Cu on the hydroponic growth and antioxidant responses of rice (Oryza sativa) seedlings. By assessing growth parameters, antioxidant enzyme activities, and biochemical responses, we seek to elucidate the specific impacts and potential mechanisms underlying the interactions between PE-MPs and Cu in rice. The results will contribute to a better understanding of the potential ecological risks posed by PE-MPs in agricultural environments, providing important scientific insights into the effects of microplastic and heavy metal pollution on crop development, food security, and ecosystem health.

2. Materials and Methods

2.1. Experimental Design

The plant material for the study was rice seedlings (variety Guangxingyou 1380), sourced from Hainan Shennong Gene Technology Co., Ltd. (Haikou, China). Polyethylene microplastics (PE-MPs), with an average particle size of 15 µm, were supplied by Kexinda Polymer Materials Co., Ltd. (Dongguan, China). CuSO₄·5H₂O of analytical grade was employed as the copper ion source. The experiment was structured into four treatment groups: a control group (CK), a PE-MPs group, a Cu group, and a combined PE-MPs+Cu group. Each group consisted of five biological replicates to ensure statistical robustness.

The rice seedlings were planted hydroponically in 500 mL Erlenmeyer flasks, with three healthy seedlings per flask. Each flask held 250 mL of culture solution with the following treatments: (1) CK group (control): nutrient solution without additives; (2) PE group: nutrient solution containing 50 mg/L PE-MPs; (3) Cu group: nutrient solution supplemented with 20 mg/L Cu²⁺; (4) PE+Cu group: nutrient solution containing a combination of 50 mg/L PE-MPs and 20 mg/L Cu²⁺.

After transplantation, the roots of the rice seedlings were completely submerged in the nutrient solution, and the openings of the Erlenmeyer flasks were sealed with aluminum foil to minimize contamination and evaporation. The flasks were then transferred to an environmental pollution simulation incubator (model BRS-WHM-2000G; Ningbo Pulante Instrument Co., Ltd., Ningbo, China), maintained at a constant temperature of 25 °C, under an 8 h light cycle per day, for one week. In order to maintain consistent solute concentration throughout the entire experiment, a micropipette was used to supplement nutrient solution to 250 mL at noon every day to balance the losses caused by transpiration and evaporation.

On the seventh day, the fresh and dry biomass, copper accumulation of the rice seedling, length and peroxidase (POD) activity of root and shoot were measured.

2.2. Solutions Preparation

The Hoagland nutrient solution and Cu²⁺ stock solution were prepared according to methods described in our previous publications [10]. The components of the nutrient solution are as follows: 0.51 g KNO₃, 1.18 g Ca(NO₃)₂·4H₂O, 0.14 g KH₂PO₄, 0.49 g MgSO₄·7H₂O, 2.86 mg H₃BO₃, 1.81 mg MnCl₂·4H₂O, 0.22 mg ZnSO₄·7H₂O, 0.08 mg CuSO₄·5H₂O, and 0.02 mg H₂FMnO₄·H₂O, pH 5.8–6.0. To formulate the PE-MPs suspension, 0.0500 g of polyethylene microplastic powder was introduced into 1 L of the prepared nutrient solution. The mixture was then subjected to ultrasonic treatment using a 400 W, 20.5 kHz ultrasonic homogenizer for 30 min to achieve uniform dispersion, yielding a final concentration of 50 mg/L.

2.3. Measurement Methods

Morphological and physiological measurements were conducted on the rice seedlings to assess treatment effects. Root and shoot lengths were measured using a standard ruler. For biomass analysis, seedlings were first rinsed with distilled water to remove surface debris, followed by air drying to eliminate residual moisture. Fresh weight was recorded using an analytical balance, and samples were subsequently placed in labeled paper envelopes and dried at 80 °C in an oven until a constant weight was reached to determine dry biomass. POD activity was measured by POD Kit (Solarbio, Beijing, China) in accordance with the manufacturer’s instructions. Detailed procedures are available in our previously published study [10].

To determine copper content in rice seedlings, dried plant tissues were finely ground into powder using a mortar. Precisely 0.5000 g of the homogenized sample was transferred into a polytetrafluoroethylene digestion tube. Subsequently, 10 mL of concentrated HNO₃ was added, and the mixture was gently agitated before being left to stand overnight in a fume hood for pre-digestion. The following day, the tubes were heated at 120 °C for 1 h using a digestion apparatus. After allowing the contents to cool for 10 min, 3.5 mL of HF was introduced, followed by gentle shaking and further digestion at 140 °C for 1 h. Upon a second 10 min cooling phase, 1 mL of HClO₄ was added, and the digestion was continued at 160 °C for 1 h. The final stage involved increasing the temperature to 180 °C and maintaining digestion until the emission of white fumes ceased, indicating completion. After cooling for 30 min, 1 mL of a 1:1 (v/v) HNO₃ solution was added. The digested sample was then transferred to a 50 mL volumetric flask and diluted to volume with deionized water. The solution was filtered into a clean polyethylene flask and analyzed for Cu concentration using inductively coupled plasma mass spectrometry (ICP-MS, X Series 2, Thermo Fisher Scientific, Waltham, MA, USA) [11]. Quality control of ICP-MS was ensured by using certified standards for calibration (R² > 0.999) (National Nonferrous Metals and Electronic Materials Analysis and Testing Center, National Standard (Beijing) Inspection and Certification Co., Ltd., Beijing, China), analyzing blanks and duplicates, and performing spike recovery tests (92–106%). Internal standards and QC samples were also measured regularly to confirm analytical stability.

2.4. Data Analysis

Data organization and visualization were conducted using Microsoft Excel and Origin 2024. Statistical analyses were performed via one-way analysis of variance (ANOVA) using SPSS version 27.0.

3. Results and Discussion

3.1. Effects of PE-MPs and Cu on the Growth of Rice Seedlings

The effects of PE-MPs and Cu on the growth of hydroponic rice seedlings were assessed by measuring shoot and root lengths under single and combined treatments. As shown in Figure 1, the shoot length of rice seedlings remained relatively stable across all treatments, with an average value of approximately 37 cm, and no statistically significant differences were detected (p > 0.05). Similarly, Figure 2 shows that root length exhibited only minor variation among treatments without significant differences. The control group (CK) had the longest roots (6.2 cm), followed by the PE treatment (6.0 cm), whereas Cu exposure reduced root length to 5.8 cm, and the combined PE+Cu treatment further decreased it to 5.6 cm. Although these reductions were not statistically significant, the consistent trend suggests that Cu exposure, either alone or in combination with polyethylene microplastics, may slightly inhibit root elongation under hydroponic conditions.

When PE-MPs and Cu were co-exposed (PE+Cu), the rice seedlings showed no significant changes in shoot length compared to the Cu-only treatment. However, root growth was slightly more inhibited compared to the single Cu exposure, suggesting a potential interaction between PE-MPs and Cu. Despite the absence of significant differences in shoot elongation, the reduction in root length under the combined exposure highlights the complex nature of plant responses to mixed contaminants. The lack of synergistic effects on shoot growth implies that the observed reduction in root length may be due to physical interference by PE-MPs, potentially limiting root exploration of nutrients or exacerbating Cu toxicity at the root surface. These results are consistent with existing related studies. Jin et al. investigated the effects of co-pollution from polystyrene microplastics (PS-MPs) and copper ions (Cu²⁺) on rice seedling growth, finding that the treatment group exposed solely to Cu²⁺ showed slight inhibition in root growth, while the combined exposure to PS-MPs and Cu²⁺ did not exhibit significant synergistic toxic effects [13]. Tang et al. analyzed the rice seedlings exposed to four different concentrations of polyethylene microplastics (PE-MPs). After 21 days of laboratory exposure, no differences in shoot growth were observed under the treatments of various dosages of PE-MPs [12].

Overall, these findings suggest that PE-MPs and Cu, when present individually or together in a hydroponic system, do not severely impact the shoot growth of rice seedlings in the short term. However, the observed root growth inhibition, particularly under combined exposure, warrants further investigation into the long-term effects of such contaminants, especially in terms of nutrient uptake and overall plant health.

3.2. Effects of PE-MPs and Cu on the Biomass of Rice Seedlings

Figure 3 and Figure 4 present the fresh weight and dry weight data of rice seedlings under different treatments, respectively. Both figures of data exhibit a consistent trend, where Cu treatment leads to a significant reduction in biomass accumulation compared to the other treatments.

As shown in Figure 3, the fresh weight of rice seedlings varied across treatments. The PE treatment resulted in the highest fresh weight (1.45 g), which was not significantly greater than the CK group (1.31 g). In contrast, Cu exposure was 1.18 g, which was the lowest among all treatments. The combined PE+Cu treatment showed an intermediate value of 1.24 g, which was not significantly different from either the control or PE treatment. These results suggest that while PE alone may promote fresh weight, Cu exposure inhibits biomass accumulation. The combination of PE and Cu did not exacerbate the negative effects of Cu on fresh weight, indicating that the positive effect of PE was partially offset by the presence of Cu. For dry weight in Figure 4, the data further support the observed trend. The Cu treatment resulted in a significant decrease in dry weight, which was markedly lower than all other treatments (p < 0.05). The dry weight of seedlings in the CK, PE, and PE+Cu groups were similar, and no significant differences were observed between these treatments. These findings reinforce the conclusion that copper exposure inhibits dry weight accumulation in rice seedlings, while PE alone or in combination with Cu does not have a significant negative impact on dry weight.

The results demonstrate that Cu exposure significantly inhibited rice seedling biomass, while PE-MPs alone had no negative impact and even slightly promoted fresh weight. Similar observations have been reported in hydroponic wheat and spinach, where microplastics modified plant nutrient uptake and partially alleviated metal toxicity [4,6]. The reduction in biomass under Cu stress is consistent with established findings that excess copper interferes with photosynthesis and nutrient assimilation, leading to growth inhibition [5]. Interestingly, co-exposure to PE+Cu groups did not exacerbate biomass loss compared with Cu alone, but it did significantly increase Cu accumulation in tissues. This suggests that microplastics may act as carriers, enhancing Cu bioavailability through surface adsorption and retention in the rhizosphere. Similar mechanisms have been proposed in studies with arsenic and cadmium, where microplastics facilitated metal uptake into plants [3,8].

In conclusion, both the fresh and dry weight data highlight the significant inhibitory effect of copper exposure on rice seedling growth, with Cu-treated seedlings exhibiting reduced biomass accumulation compared to all other treatment groups. Notably, PE treatment alone had a positive effect on fresh weight, but did not significantly affect dry weight. The PE+Cu treatment did not exacerbate the negative effects of Cu, indicating that microplastics might not intensify copper toxicity in terms of biomass accumulation.

3.3. Effects of PE-MPs and Cu on Peroxidase Activity in Rice Seedlings

As shown in Figure 5, the POD activity in shoots of rice seedlings differed significantly among treatments. The CK group exhibited the highest POD activity, reaching values above 10,000 U/g. In comparison, the PE treatment significantly reduced shoot POD activity to approximately 6500 U/g, while Cu exposure caused a drastic decline to about 1200 U/g, which was the lowest among all groups. Interestingly, the combined PE+Cu treatment partially alleviated the Cu-induced inhibition, with POD activity increasing to around 3000 U/g, though still markedly lower than the CK and PE groups. These results indicate that Cu strongly suppressed antioxidant enzyme activity in shoots, whereas PE alone reduced activity moderately, and the combined exposure led to an intermediate response, suggesting that PE may influence Cu bioavailability and modulate its oxidative impact.

In Figure 6, root POD activity of rice seedlings exhibited significant differences among treatments. The CK group maintained the highest activity, exceeding 11,000 U/g, followed by the PE treatment at approximately 9000 U/g. Cu exposure alone led to a marked reduction in root POD activity, reaching about 4800 U/g. The combined PE+Cu treatment further suppressed root POD activity to nearly 1500 U/g, which was the lowest among all treatments. These results indicate that Cu exposure strongly inhibited antioxidant enzyme activity in roots, and that the presence of PE microplastics further exacerbated this suppression under co-exposure conditions. This suggests a potential synergistic effect of PE and Cu in impairing the root antioxidant defense system.

The organ-specific antioxidant responses observed here further illustrate the complexity of co-contamination effects. In roots, POD activity increased more than twofold under combined exposure, indicating severe oxidative stress at the primary site of contact. In contrast, shoot POD activity was suppressed, suggesting an impaired antioxidant defense. These contrasting patterns align with previous reports that microplastics can differentially modulate antioxidant enzyme activities depending on tissue type and contaminant interactions [10,12].

Overall, these results suggest that copper exposure significantly reduces POD activity in both shoots and roots, highlighting its role in inducing oxidative stress. The addition of PE-MPs appears to exacerbate the effect of copper in the root tissue, while the shoot tissue shows a relatively milder response to the combined stressors. These findings provide critical insights into the physiological and biochemical interactions between microplastics and heavy metals in plants, emphasizing the potential for synergistic effects in terms of oxidative damage and the impairment of antioxidant defenses.

3.4. Accumulation of Cu in Rice Seedlings

Figure 7 illustrates that copper content in rice seedlings differed markedly among treatments. Both the CK and PE-alone groups exhibited very low Cu accumulation (<50 mg/kg) with no significant difference between them. In contrast, Cu treatment alone led to a sharp increase in Cu concentration, reaching nearly 370 mg/kg. The PE+Cu co-exposure group showed the highest Cu accumulation, approximately 450 mg/kg, which was significantly greater than the Cu-only treatment. These results indicate that while PE alone did not affect Cu accumulation, the presence of PE microplastics substantially enhanced Cu uptake under co-exposure conditions, suggesting that microplastics may act as carriers that increase the bioavailability of copper in plants.

Interestingly, the PE+Cu co-exposure group exhibited a significant increase in Cu content, which was 12 times higher than the control and PE alone groups, and significantly greater than the Cu-alone treatment. This suggests that PE-MPs may enhance the bioavailability of copper, likely through surface adsorption and retention in the rhizosphere, which could facilitate its uptake by the plants. The elevated Cu concentration in the PE+Cu group supports the hypothesis that microplastics may act as vectors for heavy metals, leading to increased bioaccumulation in plants.

In summary, the data demonstrate that copper exposure leads to significant accumulation in rice seedlings, with a notable synergistic effect when microplastics are present. The PE+Cu treatment showed the highest Cu content, highlighting the potential for microplastics to amplify the uptake of heavy metals in aquatic agricultural systems. These findings provide important insights into the interaction between microplastics and heavy metals and their implications for environmental and agricultural safety.

4. Conclusions

This study demonstrates that the co-exposure of rice seedlings to polyethylene microplastics and copper produces distinct physiological and biochemical effects with important implications for crop health. While Cu alone significantly inhibited biomass accumulation, PE-MPs at the tested concentration did not cause overt toxicity. However, when combined, microplastics enhanced Cu uptake by approximately one quarter compared with Cu alone, leading to elevated metal accumulation in plant tissues. This increased bioavailability, together with the observed tissue-specific oxidative responses—root peroxidase activity being stimulated while shoot activity was suppressed—indicates that microplastics not only act as carriers of potentially toxic elements but also alter plant defense mechanisms against stress. Although short-term growth inhibition was not further exacerbated under co-exposure, the intensified copper accumulation raised concerns about long-term risks, including bioaccumulation in the food chain and potential impacts on yield and food safety. Taken together, our findings highlight the need to consider microplastic–metal interactions when assessing agricultural contamination, and point to the importance of future field-based and chronic studies that evaluate consequences for grain development, nutritional quality, and ecosystem health.