Charged polystyrene microplastics inhibit uptake and transformation of ¹⁴C-triclosan in hydroponics-cabbage system

Graphical abstract

Highlights

• •PS-MPs affected uptake and metabolism of triclosan in a hydroponics-cabbage system.
• •Compared to triclosan, PS-MPs combined with triclosan reduced biomass of cabbage.
• •BCFs of triclosan in cabbage decreased in the order control > PS-COO^- > PS > PS-NH₃⁺.
• •PS-MPs suppressed the translocation of triclosan from roots to shoots in cabbage.
• •PS-NH₃⁺ greatly inhibited the sulfonation, nitration, and nitrosation of triclosan.

Abstract

Introduction

Since the outbreak of COVID-19, microplastics (MPs) and triclosan in pharmaceuticals and personal care products (PPCPs) are markedly rising. MPs and triclosan are co-present in the environment, but their interactions and subsequent implications on the fate of triclosan in plants are not well understood.

Objective

This study aimed to investigate effects of charged polystyrene microplastics (PS-MPs) on the fate of triclosan in cabbage plants under a hydroponic system.

Methods

¹⁴C-labeling method and liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (LC-QTOF-MS) analysis were applied to clarify the bioaccumulation, distribution, and metabolism of triclosan in hydroponics-cabbage system. The distribution of differentially charged PS-MPs in cabbage was investigated by confocal laser scanning microscopy and scanning electron microscopy.

Results

The results showed that MPs had a significant impact on bioaccumulation and metabolism of triclosan in hydroponics-cabbage system. PS-COO^-, PS, and PS-NH₃⁺ MPs decreased the bioaccumulation of triclosan in cabbage by 69.1 %, 81.5 %, and 87.7 %, respectively, in comparison with the non-MP treatment (control). PS-MPs also reduced the translocation of triclosan from the roots to the shoots in cabbage, with a reduction rate of 15.6 %, 28.3 %, and 65.8 % for PS-COO^-, PS, and PS-NH₃⁺, respectively. In addition, PS-NH₃⁺ profoundly inhibited the triclosan metabolism pathways such as sulfonation, nitration, and nitrosation in the hydroponics-cabbage system. The above findings might be linked to strong adsorption between PS-NH₃⁺ and triclosan, and PS-NH₃⁺ may also potentially inhibit the growth of cabbage. Specially, the amount of triclosan adsorbed on PS-NH₃⁺ was significantly greater than that on PS and PS-COO^-. The cabbage biomass was reduced by 76.9 % in PS-NH₃⁺ groups, in comparison with the control.

Conclusion

The uptake and transformation of triclosan in hydroponics-cabbage system were significantly inhibited by charged PS-MPs, especially PS-NH₃⁺. This provides new insights into the fate of triclosan and other PPCPs coexisted with microplastics for potential risk assessments.

Introduction

Plastic products have widespread applications in various industries, agriculture, and daily human life. Owing to their excessive use, poor management, and environmental sustainability, an amount of approximately 6.3 billion tons of plastic waste was generated from 1950 to 2015, of which 79 % were discharged into landfills or in the natural environment [1]. Plastic materials can be decomposed into tiny plastic particles such as microplastics (MPs, less than 5 mm) through chemical and biological processes, solar radiation, and other physical processes, while also altering the surface charge of MPs [2]. During the photodegradation and biodegradation of MPs in the environment, different functional groups (e.g., –COOH and –NH₂) can be introduced onto MPs’ surfaces and thus alter their surface charge [3]. For example, propagation reactions occur through hydrogen transfer or following the creation of alkoxy radicals, ultimately leading to the creation of hydroxyl groups [4]. Sequential reactions lead to either chain scission or crosslinking, generating oxygenated functional groups such as aliphatic carboxylic acids, aldehydes, and ketones [5]. Furthermore, Wang et al. [6] found that MPs consistently carry a negative charge when in an alkaline solution. Conversely, in an acidic solution, the polymer surface becomes protonated or positively charged. Polystyrene (PS) is one of the most dominant materials in the plastic market [7], with an industrial production of several billion kilograms per year. It is utilized in the manufacturing of styrofoam, a common material used in food containers and packaging products. Moreover, it ranks as one of the most prevalent microplastics detected in the marine environment [8]. Some studies have used PS as a representative type of MPs to investigate the uptake process and localization of MPs in plants, like Arabidopsis thaliana [9], Triticum aestivum, and Lactuca sativa [10]. In a study conducted by Henseler et al. [11] using a normative simulation model, it was found that the maximum concentration of microplastics in agricultural soil could potentially reach 30–50 mg kg⁻¹ by 2020. However, applications of PS result in tremendous environmental pollution. For instance, MPs serve as a threat to organisms and humans [12], causing damage to metabolism system of animals [13], [14], and reducing the chlorophyll content and photosynthetic activity of plants [15]. In addition, an aquatic environment is a major sink for various contaminants, such as MPs [16] and organic pollutants [17]. The plastic debris accumulated in the aquatic (like marine) environment is considered to be one of the most serious environmental threats facing the world, because water is one of the key vectors for MPs transport [18]. However, the interactive effects of MPs and organic pollutants have not been well investigated.

Pharmaceuticals and personal care products (PPCPs) have been increasingly utilized and are now recognized as emerging environmental pollutants on a global scale in recent years [19]. Triclosan, an effective bactericide, has been widely used in personal care, household, veterinary, and industrial products [20]. An annual global production of 1500 tons and application of triclosan have led to its inevitable release into the environment, and the highest recorded concentrations of triclosan were 86 μg L⁻¹ in wastewater, 40 μg L⁻¹ in surface water, 53 mg kg⁻¹ in sediments, and 133 mg kg⁻¹ in biosolids [21]. It is on the list of ten most common pollutants of terrestrial and aquatic environments [22], [23]. These elevated concentrations have raised concerns regarding ecological toxicity [24]. Due to its high lipophilicity and bioaccumulation, triclosan can permeate aquatic environments via irrigation with wastewater and surface water, potentially entering food chains via plant uptake, thereby affecting human health [20]. Triclosan exhibits toxic effects on the reproductive system, immune system, muscle function, and genetics of aquatic species and vertebrates [25]. Triclosan was detected in human urine and breast milk [26]. Triclosan can adversely affect the human metabolic system. Bioconcentration factors (BCFs) of triclosan are in the range of thousands in common vegetables, such as lettuce, cucumber, and potato [27], [28]. Some studies reported the metabolism of triclosan in carrot and celery, and phase I and II metabolites were detected in the plant tissues [29], [30]. Current use of triclosan has been markedly increasing owing to the COVID-19 pandemic [31]. Thus, it is imperative to study the high release, rapid accumulation, and environmental effects of the model contaminant for PPCPs (such as triclosan), particularly in co-presence with MPs, for the concerns of human health.

MPs have high specific surface areas and abundant surface functional groups. MPs can, therefore, affect the environmental behaviors of pollutants. One key issue is their multiple effects on the bioaccumulation and toxicity of organic pollutants [32], [33]. For instance, MPs were reported to be carriers for transporting organic pollutants to lanternfish in aquatic environments, consequently, affecting their environmental behaviors [32]. Polyvinyl chloride (PVC) MPs increased the accumulation of venlafaxine in duckweed [34], whereas PS-MPs reduced the bioavailability of phenanthrene into soil-grown soybean [33]. Polyethylene (PE) and PVC MPs inhibited the accumulation of phenanthrene in a marine diatom, possibly attributed to the adsorption of phenanthrene on MPs [35]. In addition, differentially charged MPs exhibit effects on the environmental behaviors of organic pollutants. For instance, different gene expression patterns were induced by triphenyl phosphate in the presence of charged MPs (such as PS (neutral), PS-COO^-, and PS-NH₃⁺) in the marine medaka [36]. In addition, due to MP’s polymer properties and irregular surfaces, triclosan can be adsorbed on various microplastics such as polystyrene, polyvinyl chloride, polyethylene, and polyhydroxybutyrate via hydrophobic interactions, π-π interactions, non-covalent interactions, and electrostatic interactions [37], [38], [39]. Previous studies have found that MPs play a role in transporting triclosan and influencing its fate in aquatic organisms like Acartia tonsa and Daphnia magna. This interaction within the aquatic environment leads to an intensification of triclosan’s toxicity [40], [41]. Thus far, however, little is known about effects of MP surface charges on the bioaccumulation, translocation, distribution, and metabolism of triclosan in a hydroponic-vegetable system, as well as the environmental fate of triclosan.

Cabbage is a common leaf vegetable and is a common vegetable model for studying bioaccumulation, translocation, distribution, and metabolism of pollutants. Thus, the overall aim of this study was to investigate the effects of differentially charged PS-MPs (i.e., PS-COO^-, PS, and PS-NH₃⁺) on the fate of triclosan in a hydroponics-cabbage system. The uptake, bioaccumulation, distribution, and metabolism of triclosan in a hydroponics-cabbage system with differentially charged PS-MPs were investigated with ¹⁴C-triclosan and liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (LC-QTOF-MS) analysis. Furthermore, the distribution of differentially charged PS-MPs in cabbage was investigated by confocal laser scanning microscopy and scanning electron microscopy. Our findings highlighted the effects of MPs on the environmental behaviors of triclosan in the hydroponics-vegetable system.

Materials and methods

Chemicals and plants

¹⁴C-triclosan was synthesized in our laboratory with a radiochemical purity of > 98 % and a specific activity of 10 mCi mmol⁻¹ (Fig. S1a). Green fluorescent-labeled PS (200 nm) PS-COO^- (negative charge), PS (neutral), and PS-NH₃⁺ (positive charge) MPs were purchased from Da’e Scientific Co., Ltd. (Tianjin, China) (Fig. S1b). Scintillators of 1,4-Bis[2-(5-phenyloxazolyl)] benzene (POPOP) and 2,5-diphenyloxazole (PPO) were used to measure the radioactivity of samples were purchased from Arcos Organics (TCI, Geel, Belgium). Cabbage (Brassica campastris ssp. chinensis ‘Youqing 49′) seeds were purchased from Puyang Shihe Gardening Company (Suqian, Jiangsu, China).

Fig. 1.

(a) Adsorption dynamics of ¹⁴C-triclosan in PS-COO^-, PS, and PS-NH₃⁺ treatments. (b) Biomass (fresh weight) dynamics of cabbage under hydroponics in the control, PS-COO^-, PS, and PS-NH₃⁺ groups. Images of the cabbage plants exposed to (c) the control, (d) PS-COO^-, (e) PS, and (f) PS-NH₃⁺ groups for 10 days. Radioautograph images of the ¹⁴C distribution of cabbage in the (g) control, (h) PS-COO^-, (i) PS, and (j) PS-NH₃⁺ groups for 10 days.

Plant uptake of ¹⁴C-triclosan

To prepare cabbage plants, the seeds were germinated in distilled water at 25 ℃ for 24 h in the dark. The germinated seeds were cultivated into seedlings in a greenhouse, and the conditions were set as light/dark: 12 h/12 h, 25 ℃/20 ℃, and humidity: 65 %. One seedling with 3–4 leaves was transplanted in a brown glass bottle containing 30 mL Hoagland nutrient solution (pH = 6.5) for cultivation, and the bottles were wrapped with tin foil to avoid the light degradation of triclosan. After one week of pre-culture, the cabbage plants were transferred to the hydroponic medium from the nutrient solution and divided into four groups: (1) ¹⁴C-triclosan (the control group), (2) ¹⁴C-triclosan + PS-COO^-, (3) ¹⁴C-triclosan + PS, (4) ¹⁴C-triclosan + PS-NH₃⁺. PS-MPs and triclosan concentrations were selected to be 50 mg L⁻¹ and 1 mg L⁻¹, respectively, according to the previous studies [9], [30]. The cabbage were sampled at 0.1, 0.5, 1, 2, 5, 10, 20, and 30 days. Five replicates (n = 5) were conducted in each group of the cabbage samples at each time set. Finally, the cabbage tissues were separated and collected as roots, stems, and leaves. Each tissue fraction was washed using distilled water and then freeze-dried for analysis.

Determination of the concentration and bioavailability of ¹⁴C-triclosan

¹⁴C-triclosan was determined by a biological oxidizer (OX-501, RJ Harvey Instrument Co., NJ, USA), and a Liquid Scintillation Counter (LSC, Perkin-Elmer Inc., Downers Grove, IL, USA). First, 0.1 g of each cabbage tissue was subjected to combustion in the biological oxidizer, and the released ¹⁴CO₂ was trapped using scintillation cocktails. Second, 1.0 mL of hydroponic medium (rhizosphere) was added into the scintillation cocktails after filtration using a 0.10-μm filter membrane. Radioactivity of cabbage tissues and the hydroponic medium was measured on LSC. The concentration, bioconcentration factor (BCF), and transportation factor (TF) of ¹⁴C-triclosan were calculated according to the method reported by Nie et al. [30]. To visualize and localize ¹⁴C-triclosan and its metabolites in cabbage, radioautographic images were recorded on a Bioimaging Analyzer System (Typhoon FLA 9500, GE Healthcare, Chicago, IL, USA) according to the method reported by Nie et al. [30].

Analysis of ¹⁴C-triclosan metabolites

To determine the effect of PS-MPs on the metabolism of triclosan in the hydroponic-plant system, a high-performance liquid chromatograph-tandem mass spectrometer (HPLC-MS/MS) was employed for the analysis of metabolites according to the method as previously described by Nie et al. [30]. Briefly, 5.0 g of fresh cabbage samples were weighed and placed into 100-mL centrifuge tubes, followed by successive extraction using 50 mL of acetonitrile, methanol, and acetone. Next, the tubes were shaken at 200 rpm for 1 h using an orbital shaker, followed by ultrasonic extraction for 1 h and then centrifugation at 5000g for 5 min to collect the supernatant. The supernatant was then passed through a carb solid-phase extraction column (Waters, Milford, MA), eluted with 10 mL of methanol. Finally, the purified solution and hydroponic medium were dried with a vacuum rotary evaporator. The residues were redissolved in 1.0 mL of methanol for analysis.

¹⁴C-triclosan metabolites were separated on an Agilent Zorbax SB-C₁₈ column (250 mm × 4.6 mm, 5 μm) coupled with an Agilent 1260 HPLC system at 30 ℃. The mobile phase (A: water; B: acetonitrile) gradient was set as follows: 0–1 min, 5 % B; 1–45 min, 5 to 75 % B; 45–46 min, 100 % B; 46–50 min, 100 % B; 50–51 min, 100 to 5 % B; and 51–55 min, 5 % B. The flow rate was maintained constant at 1.0 mL min⁻¹. The injection volume of the samples was 40 μL. 70 % of the eluates from HPLC analysis were collected into vials at an interval of 1 min. The collected eluates were divided into two parts: the first part of 70 % eluates was used to determine the amounts and retention times (t_R) of metabolites via radioactivity measurement, and the remaining eluates were used for MS analysis. The metabolites were determined with an Agilent 6530 Q-TOF-MS system, equipped with an electrospray ionization source (Agilent, Santa Clara, CA) according to the method previously reported [30].

Adsorption experiment of ¹⁴C-triclosan.

Experiments were conducted to investigate the adsorption of triclosan on PS-MPs in the hydroponic medium. Differentially charged PS-MPs (PS-COO^-, PS, and PS-NH₃⁺) and ¹⁴C-triclosan were placed in a 50-mL glass conical flask containing 20 mL of the hydroponic medium. The triclosan and PS-MP concentrations were adjusted to 1.0 mg L⁻¹ and 50 mg L⁻¹, respectively. The conical flasks were then shaken on an orbital shaker (200 rpm, 25 ℃). The solution was sampled at time sets of 1, 2, 6, 12, and 24 h. After filtration through a 0.10-μm nylon filter membrane, 100 µL of the solution was transferred for radioactivity measurement. The adsorption amount of ¹⁴C-triclosan on PS-MPs was calculated based on different concentrations of triclosan before and after adsorption.

Distribution of microplastics in cabbage

To visualize and localize the PS-MPs in cabbage, a laser confocal microscope was used to obtain fluorescent images according to the method reported by Sun et al. [9]. Fresh cabbage collected at 30-day cultivation was cut into 100-μm pieces using a slicer, and fluorescent images of cabbage tissues were observed under a laser confocal microscope (LSM710nlo, Zeiss, Germany). The emission wavelength and reception wavelength of green fluorescence of PS-MPs were set at 488 nm and 518 nm, respectively. Distribution of PS-MPs in cabbage tissues was observed under a SU-8010 field-emission scanning electron microscope (Hitachi, Japan).

Data analysis

The contents of ¹⁴C-triclosan in plant tissues and hydroponic medium were obtained according to equation (1). The bioaccumulation factor (BCF) represented the efficiency of the cabbage plant to take up ¹⁴C-triclosan from the soil, which was calculated with equation (4). The translocation factor (TF) was used to evaluate the translocation efficiency of triclosan from roots to shoots in cabbage and calculated with equation (3).

$$C_{\mathit{plant}\mathit{or}\mathit{hydroponic}\mathit{medium}}=M\times R/(A\times C\times S)$$ (1)

$$\mathit{BCF}=C_{\mathit{plant}}/C_{\mathit{hydroponic}\mathit{medium}}$$ (2)

$$\mathit{TF}=C_{\mathit{shoot}}/C_{\mathit{root}}$$ (3)

where C_{plant or hydroponic medium} is the concentration of ¹⁴C-triclosan in plants (mg kg⁻¹, dry weight, dw) or hydroponic medium (mg L⁻¹). M (mg mmol⁻¹) is the molar mass of triclosan (289.5 mg mmol⁻¹); R (DPM) is the radioactivity of ¹⁴C-triclosan in plant or hydroponic medium; A is the amount of plant (kg, dw) or hydroponic medium (L). C is a conversion unit 2.22 × 10⁹ DPM mCi⁻¹, and S (mCi mmol⁻¹) is the specific activity of ¹⁴C-triclosan (10.0mCi mmol⁻¹). C_shoot and C_root is the concentration (mg kg⁻¹) of ¹⁴C-triclosan in the shoot and root of cabbage plants.

All data were presented as mean values ± standard deviations. Concentrations of triclosan in cabbage and the hydroponic medium under control and PS treatment groups were compared by one-way analysis of variance (ANOVA) using the SPSS software (Version 19.0, SPSS Inc., Chicago, IL). The level of significance for all comparisons was 95 % (p < 0.05). The mass spectra data of the samples were analyzed using Agilent MassHunter Workstation Software Qualitative Analysis B.06.00 (Agilent, Santa Clara, CA).

Results and discussion

Adsorption of triclosan onto PS-MPs

Fig. 1a shows the adsorption dynamics of triclosan on differentially charged PS-MPs over the adsorption time. The first-order models were selected to fit the adsorption kinetic, and the parameters were listed in Table S1. PS-MPs can adsorb triclosan in an aquatic environment, and the adsorption capacity of PS-MPs for triclosan followed the order of PS-NH₃⁺ > PS > PS-COO^-. The considerable adsorption of triclosan in the water-phase environment on PS-MPs may be attributed to the large surface area, hydrophobicity of MPs, and the π–π conjugation between MPs and triclosan [38]. The results suggest that MP surface charges also affect the adsorption. The final adsorption capacity of triclosan on PS and PS-COO^- did not exhibit a significant difference (p > 0.05), despite the higher adsorption amount of triclosan for the PS group being slightly higher than that for the PS-COO^- group.

This result may be attributed to the hydrophilicity of modified MPs with oxygen-containing groups, which may weaken the hydrophobic interaction between the MPs and triclosan [42], [43]. Furthermore, the amount of triclosan adsorbed on PS-NH₃⁺ was significantly greater than that on PS and PS-COO^- (p < 0.05). Similarly, Zhang et al. [36] reported that the adsorption capacity of naphthalene-NH₃⁺ on PS-COO^- was 1.4 times greater than that of neutral naphthalene, plausibly attributed to the stronger electrostatic interaction between naphthalene-NH₃⁺ and PS-COO^-.

Synergistic effects of PS-MPs on biomass of cabbage in the presence of triclosan

Fig. 1b shows the effect of PS-MPs on the growth of cabbage, and the biomass (fresh weight) dynamics of plants in the control, PS-COO^-, PS, and PS-NH₃⁺ groups. The linear models were selected to fit the growth kinetic, and the parameters were listed in Table S2. In general, the cabbage biomass decreased in the order of control (0.37 g d⁻¹), PS-COO^- (0.40 g d⁻¹) > PS (0.26 g d⁻¹) > PS-NH₃⁺ (0.08 g d⁻¹). The results indicated the growth of cabbage in the PS-NH₃⁺ treatment group was far less than that in the control and the other two PS treatment groups. Fig. 1c–f also shows considerable inhibition of PS and PS-NH₃⁺ to the growth of cabbage in all groups after 10 days. The cabbage biomass was reduced by 38.5 % and 76.9 % in PS and PS-NH₃⁺ groups, in comparison with the control. Similarly, Dong et al. [44] reported the accumulation of neutral PS in the epidermis or phloem of rice roots, which reduced root activity to inhibit nutrient uptake. The binding affinity of neutral and positively charged PS nanoparticles on the cell wall of P. subcapitata was greater than that of negatively charged plastic [45]. Khoshnamvand et al. [15] reported that MPs (200 nm) inhibited biomass content and the amount of photosynthetic pigment (chlorophyll A) of Chlorella viridis, possibly attributed to the adsorption of the PS-NH₃⁺ aggregates on the cell surface, thereby limiting the transfer of nutrients and air as well as exchange of energy between the growth medium and cells. Sun et al. [9] also reported that PS-NH₃⁺ was easier than PS-COO^- to attach on the root of Arabidopsis thaliana due to the electrostatic interactions, damaged root cells, and inhibited nutrient absorption. The presence of PS-NH₃⁺ also reduced the content of chlorophyll in Arabidopsis pods, thereby inhibiting its growth.

Effects of PS-MPs on bioavailability of triclosan in cabbage

Fig. 2a shows the dynamics of ¹⁴C (%) of the applied amount in the hydroponic medium (rhizosphere) treated with differentially charged PS-MPs. The first-order models were selected to fit the decrease kinetic, and the parameters were listed in Table S3. In the whole incubation time, the ¹⁴C amount in the hydroponic medium decreased in the order of PS-NH₃⁺ > PS > PS-COO^- > control. The half-time of ¹⁴C in the control, PS-COO^-, PS, and PS-NH₃⁺ groups were 0.73 d, 1.09 d, 3.52 d, and 4.19 d, respectively. Therefore, PS-MPs inhibited the dissipation of triclosan in hydroponics-cabbage system. Correspondingly, the kinetic of ¹⁴C-triclosan (%) of the applied amount in cabbage among PS-MPs groups fitted the first-order models (Fig. 2b), and the parameters were listed in Table S4 (R² > 0.91). In general, the amount of ¹⁴C in cabbage increased with the extension of incubation time, and it decreased in the order of control > PS-COO^- > PS > PS-NH₃⁺. The results indicated that PS-MPs inhibited the accumulation of triclosan in cabbage. At 30 d, compared to the control, the ¹⁴C contents in treatments of PS-COO^-, PS, and PS-NH₃⁺ were suppressed by 6.7 %, 10.0 %, and 17.0 %, respectively, which was positively related to the adsorption amount of triclosan on PS-MPs in the hydroponic medium, indicating that the accumulation of triclosan in plants is considerably related to the adsorption capacity of MPs. Furthermore, MPs large size particles (≥200 nm) prevent them from entering the plant cell walls [46], thereby inhibiting the bioaccumulation of triclosan adsorbed by PS-MPs in cabbage. Similarly, PS-MPs inhibited the bioaccumulation of phenanthrene in a marine diatom, attributed to the strong adsorption of phenanthrene on PE-MPs; hence, the accumulation of phenanthrene is reduced [35].

Fig. 2.

¹⁴C dynamics of applied radioactivity in (a) hydroponic medium and (b) cabbage hydroponically cultivated at different exposure times in the control, PS-COO^-, PS, and PS-NH₃⁺ groups.

Fig. S2 shows the ¹⁴C concentration (mg kg⁻¹) in cabbage over the cultivation time after treatment with PS-MPs. Generally, the concentration of ¹⁴C in all cabbage increased during the initial period of cultivation (0.1 d to 1 d), and decreased gradually by 7.6 % to 85.4 %. This result was probably attributed to the gradual growth and increase (71.4 %–91.5 %) in the biomass content of cabbage (Fig. 1b). The ¹⁴C concentrations in cabbage decreased in the order of control > PS-COO^- > PS > PS-NH₃⁺ from 0.1 to 5 d, with values of 172.6 ± 20.7 mg kg⁻¹ (control), 107.7 ± 9.9 mg kg⁻¹ (PS-COO^-), 94.4 ± 7.5 mg kg⁻¹ (PS), and 73.5 ± 6.9 mg kg⁻¹ (PS-NH₃⁺) at 5 d. The results revealed that PS-MPs significantly reduce the concentration of ¹⁴C in cabbage (p < 0.05). The concentration of ¹⁴C in cabbage in the PS-NH₃⁺ treatment group was greater than that in the other groups (p < 0.05), but no statistically significant difference in the other groups (p > 0.05) from 20 d to 30 d. This result may be attributed to the limited growth (fresh weight of biomass, 0.08 g d⁻¹) of cabbage in the PS-NH₃⁺ treatment group after 20 days, while the growth of cabbage in the other groups increased at a high rate (fresh weight of biomass in the control: 0.37 g d⁻¹, PS-COO^-: 0.40 g d⁻¹, PS: 0.26 g d⁻¹).

Bioconcentration factors (BCFs) of triclosan in cabbage were calculated to assess the effect of PS-MPs on the bioavailability of triclosan (Table. 1). Generally, the BCFs for the control and PS-COO^- treatment groups increased in the initial stage (1870.6 ± 226.4 L kg⁻¹) and then decreased 3-fold in 2 days (578.2 ± 52.9 L kg⁻¹ at 2 d). However, the BCFs for the PS and PS-NH₃⁺ treatment groups increased gradually with time and reached the maximum values of 447.3 ± 34.8 L kg⁻¹ and 340.3 ± 22.9 L kg⁻¹ at 30 d, respectively. During most of the cultivation period (except 0.1 and 10 d), PS-MPs significantly inhibited the accumulation of triclosan in cabbage, which was especially observed for the PS-NH₃⁺ treatment group. The BCFs of ¹⁴C in cabbage in differentially charged PS-MPs were 799.0 ± 35.0 L kg⁻¹ (control), 518.8 ± 41.3 L kg⁻¹ (PS-COO^-), 393.7 ± 24.0 L kg⁻¹ (PS), and 277.8 ± 24.0 L kg⁻¹ (PS-NH₃⁺) at 20 d, respectively. MP-PS reduced the accumulation of triclosan in cabbage due to its strong adsorption and inhibitory biological activity. Previous studies have also reported that MPs can inhibit the accumulation of organic pollutants in plants. For instance, PS-MPs reduced the concentration of phenanthrene in soybean, possibly attributed to the adsorption of phenanthrene by MPs and inhibition of its bioavailability [33]. In addition, PS and PS-NH₃⁺ inhibited the growth of cabbage, preventing the absorption of substances and energy by plants [15]. However, it's interesting to note that in real greenhouse or open field setups, microplastics (MPs) reside within the soil, where their interactions with plant roots are significantly altered and reduced. Further research is needed on the impact of PS-MPs and other MPs on the bioaccumulation of PPCPs in soil–plant system.

Table 1.

Bioconcentration factors (BCFs) of cabbage cultivated in a hydroponic medium at different exposure times.

Incubation time (d)	Treatments
	Control	PS-COO-	PS	PS-NH3+
0.1	47.24 ± 2.09a	44.58 ± 3.6a	40.68 ± 4.32a	42.05 ± 0.86a
0.2	137.76 ± 7.61a	99.57 ± 5.53b	70.28 ± 4.73c	64.33 ± 4.58c
1	1298.4 ± 93.7a	528.29 ± 32.63b	333.37 ± 10.94bc	209.19 ± 17.37c
2	1870.62 ± 226.37a	578.23 ± 52.92b	346.61 ± 17.9c	230.76 ± 21.38c
5	1272.47 ± 269.5a	394.03 ± 26.99b	301.64 ± 27.05b	178.93 ± 14.74c
10	344.4 ± 7.67a	338.32 ± 19.15a	338.47 ± 27.84a	211.41 ± 12.69b
20	798.97 ± 35.01a	518.79 ± 41.25b	393.72 ± 24.05bc	277.83 ± 24.04c
30	504.41 ± 64.94a	469.45 ± 32.72bc	447.32 ± 34.81bc	340.30 ± 22.92c

Note: Different letters at the same sampling time represent the significant difference (p < 0.05).

Effects of PS-MPs on the distribution and translocation of triclosan in cabbage

Fig. 1g-j shows the radioautographic images indicating the effect of PS-MPs on the distribution of triclosan in cabbage. The concentration of ¹⁴C in the roots was considerably greater than that in the stems and leaves of all cabbage after 10 d of cultivation. In addition, the concentrations of ¹⁴C in the leaves of the PS and PS-NH₃⁺ treatment groups were less than those for the control and PS-COO^- groups.

Fig. 3a-c shows the concentrations of ¹⁴C in cabbage tissues to further quantify the distribution of ¹⁴C-triclosan and its metabolites in cabbage. With the increase in time, the concentration of ¹⁴C in the roots increased first and then decreased (Fig. 3a). After 1-day cultivation, the concentrations of ¹⁴C in the roots already reached the peak values in the control, PS-COO^-, and PS groups, with values of 2059.1 ± 97.0 mg kg⁻¹, 1501.3 ± 146.8 mg kg⁻¹, and 1420.9 ± 151.3 mg kg⁻¹, respectively. However, only after 2 days, the ¹⁴C concentration of the PS-NH₃⁺ group reached the maximum of 771.1 ± 27.1 mg kg⁻¹. In addition, the concentration of ¹⁴C in the roots of the control group was significantly greater than that of the PS and PS-NH₃⁺ groups from 0.5 d to 10 d (p < 0.05). After 10-day cultivation, the ¹⁴C concentration of the cabbage roots in the PS-NH₃⁺ group was significantly greater than that of the other groups (p < 0.05). The ¹⁴C concentrations were 86.7 ± 4.3 mg kg⁻¹ (control), 104.2 ± 12.1 mg kg⁻¹ (PS-COO^-), 107.9 ± 4.9 mg kg⁻¹ (PS), and 322.8 ± 43.6 mg kg⁻¹ (PS-NH₃⁺) at 30 d. At the beginning of the experiment (0.5 d–10 d), PS and PS-NH₃⁺ inhibited the accumulation of ¹⁴C in roots, possibly attributed to the strong adsorption of triclosan on PS and PS-NH₃⁺. The results showed that PS-MPs inhibited the amount of triclosan in roots at the beginning of the culture stage. Similarly, the accumulation of phenanthrene in soybean seedling roots in the control sample was greater than that under the combined pollution of PS-MPs and phenanthrene [33]. Another reason could be that PS-NH₃⁺ damaged the root cells, thereby inhibiting the absorption of nutrients and accumulation of triclosan in cabbage [9]. High ¹⁴C accumulation in the roots was observed for the PS-NH₃⁺ treatment groups after 10-day incubation, possibly attributed to the higher availability of triclosan in the hydroponic medium of the PS-NH₃⁺ group (34.0 %) than that in the other groups (11.0–28.4 %, see Fig. 2a).

Fig. 3.

Concentrations of ¹⁴C (mg kg⁻¹; DW) in the (a) root, (b) stem, (c) leaf, and (d) translocation factor (TF) in cabbage at different exposure times in the control, PS-COO^-, PS, and PS-NH₃⁺ groups. Different letters at the same sampling time represent the significant difference (p < 0.05).

The ¹⁴C concentrations in the stems and leaves of cabbage in different PS-MP treatment groups revealed a dynamic similarity to that in the roots (Fig. 3b and 3c). The ¹⁴C concentrations in the stems and leaves increased first and then decreased during cultivation. In addition, the ¹⁴C concentrations in the stems reached the maximum value at 2 d–5 d, which were 103.6 ± 6.3 mg kg⁻¹ (control), 55.0 ± 2.1 mg kg⁻¹ (PS-COO^-), 42.8 ± 2.7 mg kg⁻¹ (PS), and 40.4 ± 3.6 mg kg⁻¹ (PS-NH₃⁺). Moreover, the maximum concentrations of ¹⁴C in cabbage leaves were 19.9 ± 1.8 mg kg⁻¹ (control), 14.7 ± 0.8 mg kg⁻¹ (PS-COO^-), 10.4 ± 1.0 mg kg⁻¹ (PS), 10.3 ± 1.0 mg kg⁻¹ (PS-NH₃⁺) at 5 d–10 d. The concentrations of ¹⁴C in stems and leaves of the control group were significantly greater than those of the other groups (p < 0.05) at 10 d, which was consistent with the results of radioautographic images (Fig. 1g–j). These results revealed inhibition of PS-MPs on the accumulation of ¹⁴C in stems and leaves, especially upon PS-NH₃⁺ exposure. At 30 d, the ¹⁴C concentration in roots of the PS-NH₃⁺ treatment group was significantly greater than that of the PS-COO^- and control groups (p < 0.05), possibly attributed to the high accumulation in the stems and leaves of the PS-NH₃⁺ treated group at this stage.

To further investigate the effects of PS-MPs on the translocation of ¹⁴C-triclosan in cabbage, translocation factor (TF) was determined (Fig. 3d). With the increasing of incubation time, the TF of ¹⁴C-triclosan in cabbage increased continuously; especially, a considerable increase was observed at 10-day cultivation. At 30 d, the TF of ¹⁴C-triclosan in cabbage of all groups was 0.04–0.11. In all sampling groups, a significant difference was not observed in the TF of triclosan from 0.5 d to 2 d (p > 0.05). After 2-day cultivation, the TF values for all groups decreased in the order of the control > PS-COO^- > PS > PS-NH₃⁺. By the end of cultivation (30 d), TF values of ¹⁴C-triclosan of the control, PS-COO^-, PS, and PS-NH₃⁺ were 0.11 ± 0.01, 0.10 ± 0.01, 0.08 ± 0.01, and 0.04 ± 0.01, respectively.

Compared to the control sample, PS-MPs inhibited the TF of ¹⁴C-triclosan from roots to stems and leaves of cabbage. On one hand, MPs can enter the plants through the root tips, root hairs, or epidermal cracks of lateral roots, but they must overcome a series of chemical and physiological barriers, such as the Kjeldahl zone and plasmodesmata [10]. Thus, it may be difficult to transport triclosan adsorbed on MPs into the aerial parts, especially in the PS-NH₃⁺ group due to the agglomeration effect of PS-NH₃⁺. On the other hand, MPs can cause oxidative stress damage to organisms via the reduction of the chlorophyll content and destruction of the integrity of cell membranes, as reported in maize and herbaceous ornamental plants [47], [48]. Furthermore, MPs reduced plant nutrient absorption and energy transfer [15], which may also explain the inhibited translocation of triclosan from roots to aerial parts in cabbage. In addition, further study should be conducted on the mechanism of PS and other MPs affecting the translocation of triclosan in hydroponics-cabbage system.

Distribution of PS-MPs in cabbage

To analyze the distribution of PS-MPs in cabbage, the fluorescence images were recorded (Fig. 4a). Yellow and green colors indicate the plant tissue and PS-MPs, respectively. The spectral signal of the green area is used to enhance the verification of PS-MPs (Fig. 4b). When the main peak in the spectral signal was at a wavelength of 518 nm, it was considered as PS-MPs [10]. As a result, PS-MP particles were detected in the cabbage roots among all the treatments, and stems for the PS-COO^- group. SEM images of cabbage tissues showed the PS-MP distribution (Fig. S3). The PS-MPs were present in the plant roots, stems, and leaves for the PS-COO^- group, while PS-MPs are only found in the roots and stems for the PS and PS-NH₃⁺ groups. These images suggest that PS-NH₃⁺ can agglomerate easily on the plant root surface, therefore, it might disturb the acculturation of triclosan and nutrients. Furthermore, the translocation of PS and PS-NH₃⁺ from the roots to the aerial parts was more difficult than that of PS-COO^-, probably attributed to the stronger adsorption of PS and PS-NH₃⁺ on the cabbage roots than PS-COO^- [45]. In the apoplast and xylem of A. thaliana, a higher content of negatively charged PS nanoplastics was detected than that of positively charged PS [9].

Fig. 4.

Laser confocal microscopy images (a) and fluorescence signal patterns (b) of the microplastics in cabbage tissues. Fluorescence signal patterns are from regions corresponding to those demarcated by the blue arrowheads. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Effects of PS-MPs on triclosan metabolism in cabbage

Identification of metabolites. The cabbage at the mature stage of 30-day cultivation was selected to investigate the effect of PS-MPs on the metabolism of triclosan. The metabolites of triclosan in the roots, stems, leaves, hydroponic medium, and hydroponic medium without cabbage were separated with HPLC. Fig. 5a–e shows the radioactivity HPLC chromatograms of the eluents. A total of 11 radioactive peaks (M1–M11) were detected by LC-LSC. Compared to the control, PS-MPs did not affect the identities of triclosan metabolites in cabbage but affected the transformation of triclosan in the hydroponic medium. For example, M10 and M11 were not observed in the PS-NH₃⁺ group, indicating that PS-MPs inhibit the transformation of triclosan in the hydroponic medium.

Fig. 5.

Chromatograms of ¹⁴C radioactive eluates from cabbage: (a) roots, (b) stems, (c) leaves, (d) hydroponic medium, and (e) the control group. M0: triclosan; and M1 − M11: metabolites of triclosan. High-collision energy mass spectra (ESI⁻), and proposed fragmentation pathway of metabolite M1 (f) and M9 (g). A red box and a blue box represent the parent ion fragment and daughter ion fragments, respectively. Glu: Glucose; SO₄: Sulfate.

Isotopic patterns of the mass spectra of the metabolites could be divided into two categories of metabolites − containing two and three chlorines (Fig. S4). The metabolites containing three chlorine atoms corresponded to the characteristic peak of chlorine: M: M + 2: M + 4: M + 6 = 27:27:9:1 (M is the molecular ion m/z of the parent compound), including M1 and M3–M11, while M2 containing two chlorine atoms the characteristic peaks of chlorine: M: M + 2: M + 4 = 9:6:1.

Fig. 5f-g shows the structures of the metabolites identified based on MS analysis. The peak at t_R 11.2 min gave a mass [M−H]⁻ of M1 at m/z 713.0434. The metabolite was tentatively identified as a conjugate formed by the hydroxylated triclosan (triclosan-OH) and glucose malonate (Fig. 5f). The molecular formula was C₂₇H₂₉Cl₃O₁₆, which was 0.0014 Da different from the theoretical m/z 713.0448. The mass spectral fragment of m/z 506.9997 (C₂₀H₁₈C_l3O₉) was formed by a cleavage of C₇H₁₀O₇ from the molecular ion. The mass spectral fragment of m/z 464.9950 (C₁₈H₁₆Cl₃O₈) may be formed by a cleavage of groups (C₉H₁₁O₉) from the molecular ion. The fragment m/z 301.9272 (C₁₂H₅Cl₃O₃) was likely formed by a cleavage of a glucose molecule from the fragment C₁₈H₁₆Cl₃O₈, followed by dichlorination.

M9 at t_R 35.5 min was one of the main metabolites in the hydroponic medium. M9 (m/z 366.9016) was tentatively identified as sulfonated triclosan (C₁₂H₇Cl₃O₅S), which was 0.0009 Da different from the theoretical m/z 366.9007 (Fig. 5g). Fragments of m/z 286.9438 and m/z 288.9422 (C₁₂H₆Cl₃O₂) were the ion fragments of triclosan formed by the removal of −HSO₃ from the parent ion (C₁₂H₇Cl₃O₅S). The fragment m/z 96.9592 (−HSO₄) was likely a sulfonic-acid molecular ion, while the fragment ion m/z 34.9699 was the chloride ion. Sulfonated triclosan also was detected in other plants and animals [30], [49].

Based on the same analysis methods, triclosan and its metabolites were tentatively identified by their mass spectra (Fig. S5-14) and categorized into eight groups. Triclosan with glucose, and malonic acid (M1 and M5); 2,4-dichlorophenol with glucose, malonic, and maleic acid (M2); triclosan-OH with glucose, and methyl-malonic acid (M3, M6, and M8); triclosan with glucose, sulfonic and malonic acid (M4); triclosan with glucose and sulfonic acid (M7); sulfonated triclosan (M9); triclosan-OH with nitro and nitroso groups (M10); and nitrated triclosan (M11). Similar structures of triclosan metabolites, such as M4, M5, and M7, were reported in carrot and horseradish [29], [50].

Quantification of metabolites. Fig. 6 shows the percentage (%) of triclosan metabolites in the extractable residues of the roots, stems, and leaves of cabbage, as well as in the hydroponic medium with or without cabbage in the hydroponic medium at day 30. Triclosan exhibited the highest percentage (34.4 %–61.4 %), and the main metabolites were M2 (2.4 %–5.4 %), M5 (6.8 %–10.9 %), M6 (3.8 %–14.8 %), M8 (9.5 %–10.3 %), and M9 (3.5 %–6.6 %) in the roots of cabbage. Triclosan in the stems and leaves of cabbage accounted for 3.7 %–11.9 % of the extractable residues, and the main metabolites were M3 (5.0 %–8.5 %), M5 (4.5 %–6.6 %), M6 (7.7 %–15.7 %), M7 (7.7 %–38.6 %), M8 (21.5 %–42.4 %), and M9 (0.9 %–11.8 %). The results revealed that sulfonation, glycosylation, and conjugation of malonic acid are the main metabolic processes of triclosan in cabbage. Furthermore, PS-MPs inhibited the transformation of triclosan in the cabbage roots. For example, the triclosan content in the control cabbage roots was 34.4 %, which was less than that in the PS-COO^- sample (61.4 %), PS sample (55.1 %), and PS-NH₃⁺ sample (46.9 %). The highest content of triclosan was observed in the hydroponic medium (54.4 %–75.5 %). M9 was the main metabolite accounting for 10.0 %–25.3 % of the total metabolites of triclosan, which suggests that sulfonation is the main metabolic process in the medium. Similar results also were observed in the hydroponic medium without cabbage, in which triclosan accounted for 45.8 %, followed by 28.9 % of sulfonated triclosan, and 17.9 % of nitrates. This result may be attributed to the direct reaction of triclosan with sulfate ions and nitrate ions in the Hoagland culture medium [51]. In addition, the contents of triclosan were 46.2 % (control), 71.9 % (PS-COO^-), 82.2 % (PS), and 92.6 % (PS-NH₃⁺), while sulfation and nitrification metabolites were not detected in the PS-NH₃⁺ group.

Fig. 6.

Percentage of triclosan and its metabolites in extractable residues in cabbage: (a) roots, (b) stems, (c) leaves, (d) hydroponic medium, and (e) hydroponic medium without cabbage in the hydroponics system. MPs inhibited the transformation of triclosan in root and hydroponic medium (rhizosphere). M0: triclosan, and M1–11: the metabolites of triclosan.

These results indicated that PS-MPs considerably inhibit the transformation of triclosan in hydroponics-cabbage system, especially the PS-NH₃⁺ group. Kwon et al. [52] reported that the addition of biosolids in soil increases the adsorption of triclosan on soil particles, thereby inhibiting the transformation of triclosan. In addition, microorganisms were the main factor for the metabolism of triclosan in environmental media [53]. Our study revealed that it may be difficult for microorganisms to use triclosan due to the strong adsorption interaction between triclosan and PS-NH₃⁺. Some studies reported that PS-MPs could inactivate microorganisms via the increase in the active oxygen in cells, or direct destruction of the integrity of cell membranes [54]. Furthermore, the phytoxicity of PS-NH₃⁺ was greater than that of PS-COO^- because of the facile entry of positively charged PS-NH₃⁺ into the negatively charged biofilm of cells via electrostatic interactions [55].

Metabolic pathways of triclosan. Fig. 7 shows the proposed metabolic pathway of triclosan in the hydroponics-cabbage system with MPs. Phase I and phase II metabolism of triclosan were observed in the system. The phase I metabolites in the hydroponic-cabbage system mainly included hydroxylation, nitrosification, and nitrification. Hydroxylation was a typical metabolic process in the biodegradation of triclosan, as reported for diatoms [56], bacteria (Sphingopyxis) [57], and sewage environment [58]. Thus far, the nitrification and nitrosification of triclosan were only reported in a previous study [30].

Fig. 7.

Proposed metabolic pathways of triclosan in the hydroponics-cabbage system in the presence of PS-MPs. PS-NH₃⁺ inhibited the sulfonation, hydroxylation, nitrification, and nitrosification of triclosan in hydroponics-cabbage system (red dashed arrow). Dashed frame: no detectable intermediate metabolites. The several metabolites of triclosan in the yellow box have been largely unreported in previous studies.

The phase II metabolic process of triclosan involved the formation of conjugates between triclosan and sulfonic acid, glucose, malonic acid, and methyl-malonic acid. O-1-sulfation with the phenolic hydroxyl groups of triclosan was mainly observed in animals [59] and humans [60], while only a few studies were reported in plants [29], [50]. In addition, conjugation with glucose was an important route for the detoxification of phenolics in plants [61]. Enzymes play a vital role in the glycosylation of pollutants. Glycosyltransferase can directly participate in the coupling reaction of hydroxylated diclofenac and uridine diphosphoglucose [62]. After conjugation with glucose, triclosan can then conjugate with sulfonic acid and malonate. Due to the high chemical or enzyme stability, the toxicity of the conjugates was less than that of the pollutants [63], thus the conjugation reaction increases the detoxication pathways of triclosan in plants. Interestingly, the conjugates of triclosan-OH with glucose and methyl-malonic acid, namely M1, M2, M3, M6, and M8, were not reported. Further analytical work would be required for their identification.

Overall, the results indicated that the MPs can inhibit the metabolism of triclosan in the aquatic environment and that the highest inhibition effect is observed for the PS-NH₃⁺ group. Similarly, PE-MPs exhibited considerable effects on the degradation and transformation of ciprofloxacin in the soil environment [64]. Enzymes play vital roles in the metabolism of triclosan in plants. For example, Li et al. [65] reported that the highly metabolized triclosan was found in both intact radish and root enzyme extracts. The hydroxylation of triclosan in horseradish could be attributed to the presence of P450 monooxygenase [49]. The expression of Cytochrome P450 was inhibited at a high concentration level of PS [66]. Thus, PS affected the metabolic pathway of ¹⁴C-triclosan in cabbage, which may be due to affecting the enzyme system in plants. Further investigation is needed to understand how PS-MPs affect triclosan metabolisms. The results warrant an interest in the further evaluation of the effect of microplastics on the transformation of organic pollutants in the environment.

Conclusion

PS, PS-COO^-, and PS-NH₃⁺ MPs showed distinct effects on the fate of triclosan. The bioaccumulation and translocation of triclosan in cabbage followed in the order of the control group > PS-COO^- > PS > PS-NH₃⁺. PS-NH₃⁺ significantly inhibited the bioavailability of triclosan in cabbage, which is possibly attributed to the strong adsorption of triclosan on PS-NH₃⁺ and its high phytoxicity. Furthermore, PS-NH₃⁺ considerably inhibited the transformation of triclosan, including sulfonation, hydroxylation, nitrification, and nitrosification process. However, more further studies should be needed to explore how the surface charges of MPs affected the uptake, translocation, and metabolism of other PPCPs in the aquatic environments, as well as in actual greenhouse or open field production systems. The findings help a better understanding of the potential effects of MPs and co-existed pollutants in the environment.

Compliance with ethics requirements

This chapter does not contain any studies with human participants or animals performed by any of the authors.

CRediT authorship contribution statement

Enguang Nie: Investigation, Data curation, Writing – original draft. Yandao Chen: Methodology, Data curation, Formal analysis. Shengwei Xu: Investigation, Data curation, Software. Zhiyang Yu: Supervision, Validation. Qingfu Ye: Validation, Writing – review & editing. Qing X. Li: Validation, Writing – review & editing. Zhen Yang: Conceptualization, Writing – review & editing. Haiyan Wang: Funding acquisition, Conceptualization, Supervision, Project administration, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article: