Uptake, accumulation, and degradation of dibutyl phthalate by three wetland plants

Phthalate esters (PAEs), especially dibutyl phthalate (DBP), are one of the most important additives used in the production of plastics (Becky Miriyam et al. 2022). In recent years, DBP has received increased attention due to its ubiquitous occurrence in the natural environment and adverse effects on animals and humans (Lu et al. 2019; Xu et al. 2019). The extensive use of plastic products in industrial manufacturing, agricultural film covering, and daily necessities has led to the detection of high concentrations of PAEs in aquatic environments, atmosphere, and soil ecosystems. DBP may have adverse effects on the human reproductive and renal systems and also cause obesity problems. DBP has been widely detected in aquatic environments, with concentrations up to 35.65 μg/L in the Yangtze River of China (Wang et al. 2008) and 2,705 μg/L in the Ogun River catchment in Nigeria (Adeniyi et al. 2011). Di (2-ethylhexyl) phthalate (DEHP) and DBP have a detrimental influence on the nitrification of black soils in China, reducing the abundance of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria (NOB) (Tao et al. 2022). As a toxic exogenous substance to plants, DBP can inhibit the growth of some plants at excessive concentrations (Sun et al. 2015; Gao et al. 2016). DBP can also be accumulated by humans through inhalation, skin contact, and ingestion, which can disrupt the cardiovascular system and lipid metabolism and also have adverse effects on human thyroid function due to its endocrine disrupting effects (Hauser & Calafat 2005; Miodovnik et al. 2014).

Natural and constructed wetlands can capture pollutants from stormwater runoff, rivers, floods, sewage, and wastewater treatment plant effluents. Phytoremediation is an ecologically friendly treatment method for contaminated wastewater, and wetland plants are commonly used (Farid et al. 2014). Previous studies have demonstrated that wetland plants have high removal efficiencies for nitrogen, phosphorus, and heavy metals in water (Rai 2008; Sartori et al. 2016). Lythrum salicaria, Thalia dealbata, and Canna indica are widely planted in constructed wetlands in China due to their excellent landscape effect and high pollutant removal efficiency. Brunhoferova et al. (2021) found that Phragmites australis, Iris pseudacorus, and L. salicaria can remove 27 kinds of micropollutants (pharmaceuticals, pesticides, herbicides, fungicides, and others), with L. salicaria exhibiting the highest micropollutant uptake. Li et al. (2014) reported that T. dealbata and C. indica had high tolerance and bioaccumulation capability to triazophos. L. salicaria is an invasive plant in North America and occupies a significant niche in wetlands (Uveges et al. 2002). It can uptake and degrade fluoroquinolones, indicating its potential to be used in phytoremediation (Migliore et al. 2000). C. indica is a tolerant plant that can metabolize chlorophenols and fluorides into less toxic metabolites, making it a low-cost phytoremediator for chlorophenols in water (Enyoh & Isiuku 2021; Khandare et al. 2021). In a floating wetland in Jiaxing, China, L. salicaria, T. dealbata, and C. indica achieved high removal efficiencies for COD, total nitrogen, and total phosphorus in urban runoff stormwater, with T. dealbata showing the best seasonal adaptability (Ge et al. 2016). Liu et al. (2021) found that the removal efficiency of six neonicotinoids by nine wetland plants including T. dealbata and C. indica ranged from 9.5 to 99.9%. The study by Lu et al. (2020) showed that the removal efficiency of levofloxacin by eight wetland plants (including T. dealbata and C. indica) was 87.29–96.69%, with no significant variation across different emergent plants. Due to the widespread use of PAEs, DBP has been frequently detected in water (He et al. 2019). However, there are few studies on the efficiency and mechanism of DBP accumulation and biotransformation by typical wetland plants.

An important sink for DBP in the environment is the wetland ecosystem. Constructed wetlands can efficiently remove six PAEs in wastewater (Tang et al. 2015). The removal rate of DBP in domestic sewage tailwater by vertical flow constructed wetlands can reach more than 90% (Li et al. 2020). This removal is mainly attributed to biodegradation, which comprises bacteria, fungi, and wetland plants. Many bacteria and fungi that can efficiently degrade DBP have been isolated from wetlands. Emergent macrophytes play an important role in wetlands, but the mechanisms of uptake, accumulation, and transformation of DBP in a typical wetland are poorly understood.

Hydroponics is a widely acknowledged technology for plant growth (Zhu et al. 2019), as a substrate or soil has been identified as a limiting factor for plant development. Furthermore, it can avoid the adsorption of pollutants by soil or substrate and reduce the impact of the microbial degradation of pollutants, allowing the effect and mechanism of phytoremediation of pollutants in aqueous solutions to be studied.

In this study, hydroponic experiments were used to investigate the growth of L. salicaria, T. dealbata, and C. indica in 500 mL Erlenmeyer flasks after exposure to DBP. The study also examined the uptake and metabolism of DBP, as well as the enzymatic activities in plant tissues. A DBP dose of 0.5 mg/L was chosen based on the discharge limit for DBP in the Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant (GB 18918-2002, China) and the results of our previous study (Li et al. 2020). The objectives of this study were to (1) elucidate the uptake and accumulation of DBP by the three typical wetland plants using hydroponics and (2) decipher the biotransformation mechanism of DBP through the analysis of enzyme activities and metabolites of DBP in the plants.

DBP (analytical reagent) used in the experiment was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Standards of DBP, mono-n-butyl phthalate (MBP), and phthalic acid (PA) and internal standards of DnBP-d4, MBP-d4, and PA-d4 were provided by Dr Ehrenstorfer GmbH (Germany). The experimental stock solutions of PAEs were prepared in methanol and stored in amber glass vials at −20 °C before use. The organic solvents including n-hexane, methanol, dichloromethane, acetone, and acetonitrile were of HPLC grade and obtained from Fisher Scientific, Fair Lawn, NJ. An Ultra water purification system (Hitech-Sciencetool, China) was used for getting deionized water.

The seeds of L. salicaria and T. dealbata were purchased from Jiangsu Tangfeng Agricultural Technology Co. Ltd. After sterilization in H₂O₂, the seeds were germinated in the culture dish on the filter paper moistened with deionized water. After 2 weeks, the seedlings of L. salicaria and T. dealbata were transferred to a glass crystallization dish (diameter 18 cm, height 8 cm) equipped with sterilized silver sand. For seedling development, 200 mL of sterilized 1/4 Hoagland nutrient solution was added to the crystallizing dish. After 1 month of cultivation, the seedlings were removed from the silver sand and washed carefully with deionized water before being planted in a 100 mL glass jar containing 1/2 Hoagland nutrient solution. Because of the poor germination rate of C. indica seeds, seedlings of C. indica purchased at this stage and equivalent in height to those of L. salicaria and T. dealbata at this stage were used for the experiment.

After 4 weeks, the plants were cultivated in 500 mL Erlenmeyer flasks, and experimental treatments were performed. Briefly, in the DBP exposure group, the plants were suspended in a Hoagland nutrient solution containing 0.5 mg/L DBP (DBP_0.5). To negate the possible cross-contamination, a no-spiked control group with plant hydroponics cultivation was also undertaken (DBP_0). To study the effects of microbial degradation and photodegradation on DBP, a no-plant control group spiked with 0.5 mg/L DBP was conducted simultaneously. Plant cultivation was carried out in an artificial growth chamber with a 14 h light/10 h dark cycle, constant 80% relative air humidity, a temperature of 25 °C, and a photosynthetic photon flux density of 350 mmol/(m²·s⁻¹). Every other day, plants were transferred to Erlenmeyer flasks with fresh nutrient solution containing DBP to restore nutritional levels and lower microbial load.

Plants were harvested after 28 days of growth and divided into three groups for the determination of growth indicators (the dry biomass and height of the three wetland plants), DBP and its metabolites, and enzymatic activities.

After 28 days of exposure, the concentration of DBP and its metabolites in the roots, stems, and leaves of all the three wetland plants were determined. Following the blot with tissue paper, the roots were washed three times with Milli-Q water and then rinsed with methanol to assess DBP adsorbed on the root surface. Subsequently, the plants were separated into roots, stems, and leaves. These samples were freeze-dried and ground homogeneously for analysis. DBP and its metabolites were extracted and purified from plant tissues following the methods of Sun et al. (2015) and (Zhu et al. 2019) with some modifications, and details are presented in Supplementary Material, S1 and S3. In this study, DBP was detected by GC–MS (Agilent 7890A-5975C), while its metabolites (MBP and PA) were determined by the Waters Xevo TQ LC–MS/MS system. The instrument operational parameters for GC–MS and LC–MS/MS are presented in Supplementary Material, S2 and S4.

Carboxylesterase (CXE) activity was determined according to the method of Lin et al. (2017). Briefly, 1.0 g of fresh plant tissue was homogenized in an ice bath with 10 mL of phosphate buffer (0.1 M, pH 7.0) containing 0.1 mM ethylene diamine tetraacetic acid (EDTA) and 1% (w/v) polyvinyl pyrrolidone. The homogenate was then centrifuged at 15,000 g for 10 min at 4 °C, with the supernatant reserved for the enzymatic assay. About 0.2 mL of the enzyme extract was added into 2.5 mL of phosphate buffer (0.1 M, pH 7.0) containing 0.3 mM 1-naphthyl acetate and 1 μM physostigmine as a substrate. After incubation in a water bath at 25 °C for 30 min, 0.5 mL of Fast Blue B salt-sodium and dodecyl sulfate solution was added. After 10 min of chromogenic reaction, the absorbance was measured at 600 nm using an ultraviolet spectrophotometer (UV3600,Thermo Fisher Scientific). One unit of enzyme activity (U) is defined as the amount of enzyme releasing 1 μmol/L 1-naphthol per minute.

Polyphenol oxidase (PPO) activity was determined by the method of Zhu et al. (2019). In brief, 0.2 mL of the enzyme extract was mixed with 2.0 mL of phosphate buffer (0.01 M, pH 6.0) and 1.0 mL of o-dihydroxybenzene (0.1 M). Absorbance values were read continuously at 420 nm for four times at 1-min intervals. An enzyme activity unit (U) is defined as the amount of enzyme that causes an increase in absorbance (optical density) per minute.

The activities of superoxide dismutase (SOD) and peroxidase (POD) were measured using kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

The total protein content of each fresh plant tissue was also assessed using kits procured from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All enzymatic activities are expressed as units of enzyme activity per milligram of protein (U/mgprot).

The mean and standard deviation of three replicates were computed using R-4.1.2 for Windows. All statistical analyses and graphics were created in R-4.1.2 for Windows. A one-way ANOVA with a significance threshold of 0.05 was performed to compare differences in target chemical concentrations between various plants and treatments.

DBP is a toxic exogenous substance to plants that is tolerated variably by different plants, and it can inhibit the growth of some plants at excessive concentrations (Sun et al. 2015; Gao et al. 2016). For L. salicaria and C. indica, ANOVA tests revealed no significant differences (P > 0.05) in plant height and dry biomass between the DBP-exposed group (DBP_0.5) and their corresponding control groups (DBP_0) (Table 1). However, for T. dealbata, although there was no substantial difference in plant height, the dry biomass was significantly lower in DBP_0.5 (P = 0.01). DBP dosing reduced T. dealbata dry biomass by 24.2% as compared to the control. This indicated that L. salicaria and C. indica were more tolerant to DBP as their growth was not significantly affected at a DBP concentration of 0.5 mg/L. In contrast, T. dealbata was susceptible to the significant inhibition of growth by DBP. Hence, T. dealbata was less suitable for treating wastewater containing DBP in constructed wetlands as compared to the other two wetland plants.

The DBP concentration in the roots of different plants also varied significantly (Figure 1). It was 8.47 ± 3.88 mg/kg in the roots of L. salicaria, which was significantly higher than that in the roots of T. dealbata (5.67 ± 1.75 mg/kg) and C. indica (5.45 ± 2.29 mg/kg). However, there was no significant difference between the roots of T. dealbata and C. indica (P > 0.05). The lowest concentration of BDP was accumulated in the stems and leaves of T. dealbata, while no significant variations were found in the stems or leaves between L. salicaria and C. indica (P > 0.05). Additionally, the total amount of DBP accumulation from the entire plant was 35.6 ± 10.4 μg in L. salicaria and 29.7 ± 6.3 μg in C. indica, both of which were remarkably higher than 16.5 ± 4.8 μg in T. dealbata (P < 0.05). BCF and TF values among the three wetland species reflected a pattern similar to that of plant accumulation (Table 2). In general, plants take up pollutants mainly through their root system, and thus, the more developed the root system, the better the absorption of pollutants (Li et al. 2014). The study findings indicated that the root system of L. salicaria was particularly well developed, which contributed to the uptake of DBP. Although T. dealbata had a rich root system, a considerable amount of DBP was adsorbed on the root surface (3.12 ± 0.82 mg/kg) (Table 3), instead of direct absorption. All these findings suggest that the uptake and accumulation capacity of DBP is the highest for L. salicaria, followed by C. indica, and the lowest for T. dealbata.

As shown in Figure 2(a), MBP concentration in all tissues of L. salicaria was significantly higher than that in the other two species (P < 0.05), which was 23.1 ± 1.4, 15.0 ± 2.7, and 13.6 ± 1.8 mg/kg in roots, stems, and leaves, respectively. Except for the stems (the stems of C. indica contained only 0.5 ± 0.1 mg/kg MBP), the MBP concentrations in the tissues of T. dealbata and C. indica were comparable. Additionally, the MBP concentration in L. salicaria was significantly higher than the DBP content in its equivalent tissues, but not in T. dealbata and C. indica. Likewise, for PA (Figure 2(b)), the highest content was also found in the roots of L. salicaria. These results confirmed that among the three wetland plants, L. salicaria was the most efficient in degrading DBP, mostly through its roots.

After DBP exposure, the CXE activity increased significantly in the stems of L. salicaria (P < 0.05) while numerically elevated and decreased in the roots and leaves, respectively (Figure 3(a)). For T. dealbata, compared to DBP_0, CXE activity in the roots of DBP_0.5 was significantly reduced by 31.9% (P < 0.05). However, in the leaves, it was remarkably elevated by 30.7% (P < 0.05), while it was not significantly altered in the stems. A marked activity increase was also observed in both roots and stems of C. indica after DBP exposure (P < 0.05), but no significant change was observed in the leaves. CXE has been proved to hydrolyze PAEs, especially short-side-chain PAEs (e.g. DBP) (Ozaki et al. 2017; Zhu et al. 2019), and has the active site required to participate in DBP catabolism (Mahajan et al. 2019). Compared to rice (Zhu et al. 2019) and pumpkin (Lin et al. 2017), relatively higher CXE activities were detected in the tissues of the three wetland plants, indicating that all three plants are capable of metabolizing DBP.

In addition to C. indica, the PPO activity detected in the roots, stems, and leaves of both L. salicaria and T. dealbata was very low in the DBP_0 group (below 1.2 U/mgprot) but was significantly elevated in the DBP_0.5 group (P < 0.05) (Figure 3(b)). For roots, stems, and leaves, the increase was 155-, 101-, and 18-fold in L. salicaria, and 43-, 841-, and 520-fold in T. dealbata, respectively. No substantial increase was observed in the roots of C. indica (P > 0.05), while the stems and leaves exhibited a significant increase (P < 0.05). PPO is an enzyme that is involved in the catabolic conversion of aromatic organic compounds. Research has shown that PPO can catalyze the oxidation of polycyclic aromatic hydrocarbons to open their rings and convert them into more easily degradable intermediates, such as quinones, thus accelerating their degradation (Gao et al. 2012; Taranto et al. 2017). Therefore, the enhanced PPO activity in the tissues of the three plants after DBP exposure (apart from the roots of C. indica) indicated that all three plants can further open the ring to degrade DBP.

Figure 3(c) and 3(d) depicts the changes in the enzyme activities of SOD and POD in the plant tissues of DBP_0 and DBP_0.5, respectively. Compared to DBP_0, a significant increase in SOD activity was observed in the roots of L. salicaria (P < 0.05), while a significant reduction was observed in the roots of T. dealbata and the leaves of C. indica (P < 0.05) (Figure 3(c)). There were no significant alterations in any of the other tissues of the three plants (P > 0.05). Nevertheless, there was a significant difference in POD activity between DBP_0 and DBP_0.5 only in the roots of L. salicaria (P < 0.05), which decreased from 186.3 ± 58.8 to 76.2 ± 21.7 U/mgprot after DBP exposure (Figure 3(d)). SOD and POD are two important classes of antioxidant enzymes that eliminate reactive oxygen species from cells, thereby maintaining the oxidation–reduction balance in the cells and protecting plants from damage (Mascher et al. 2002; Gao et al. 2017). The SOD and POD activities in the roots of T. dealbata decreased dramatically, suggesting that the roots may have been damaged by DBP, so that their growth was somewhat inhibited (Table 1).

In this study, the enzyme activities in different plant tissues responded differentially to DBP exposure. Since the TF values of all three plants to DBP were less than 1 (Table 2), the transport ability of BDP was poor in all three plants, resulting in that the majority of the absorbed DBP accumulated in the roots (Figure 1). Hence, roots' capacity to metabolize and tolerate DBP is crucial. The roots of L. salicaria had a strong metabolic and tolerant ability to DBP, with the enzymatic activities of CEX, PPO, SOD, and POD increased by 19.4%, 155-fold, 21.7%, and 12.6%, respectively. Consequently, L. salicaria absorbed a large amount of DBP in its roots and hydrolyzed it to produce large amounts of MBP and PA (DBP, MBP, and PA concentrations were highest in the roots of L. salicaria (Figures 1 and 2)). For C. indica, the CXE activity of the roots was significantly higher, but its PPO activity was reduced and its antioxidant capacity against DBP was normal (SOD and POD did not change dramatically). Thus, the amount of DBP absorbed was small and its metabolic capacity was limited. After DBP exposure, although there was a significant increase in PPO activity in T. dealbata (Figure 3(b)), the significant decrease in root SOD and POD activities indicated that the roots might have been oxidatively damaged by DBP. This process resulted in a significant decrease in root CXE activity and a poor capacity for DBP uptake and initial hydrolytic metabolism.

This study investigated the uptake, accumulation, and degradation of DBP by three wetland plants, as well as the impact of DBP on enzyme activities and plant growth. The results showed that:

• (1)

  DBP at 0.5 mg/L exhibited no significant effect on the short-term growth of L. salicaria and C. indica but dramatically inhibited the growth of T. dealbata.

• (2)

  DBP could be absorbed and accumulated by the three wetland plants, with most of the DBP accumulating in roots and low transfer to stems and leaves. Among the three species, L. salicaria had the highest DBP uptake and accumulation capacity, followed by C. indica and the lowest by T. dealbata.

• (3)

  DBP could be degraded to MBP and further metabolized to PA in all three wetland plants. However, L. salicaria had the highest DBP degradation ability due to its well-developed root system and the elevated activity of metabolic and antioxidant enzymes in the roots after DBP exposure.

Summarily, the findings of this study suggested that among the three wetland plants, L. salicaria could be the most suitable for the phytoremediation of DBP-contaminated water in constructed wetlands. Moreover, in the future, further research is needed to investigate the practical effectiveness of L. salicaria in constructed wetlands for treating DBP-contaminated water.

This work was supported by grants from the National Natural Science Foundation of China (No. 51578538).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.