Uptake, phytovolatilization, and interconversion of 2,4-dibromophenol and 2,4-dibromoanisole in rice plants.

The structural analogs, 2,4-dibromophenol (2,4-DBP) and 2,4-dibromoanisole (2,4-DBA), have both natural and artificial sources and are frequently detected in environmental matrices. Their environmental fates, especially volatilization, including both direct volatilization from cultivation solution and phytovolatilization through rice plants were evaluated using hydroponic exposure experiments. Results showed that 2,4-DBA displayed stronger volatilization tendency and more bioaccumulation in aboveground rice tissues. Total volatilized 2,4-DBA accounted for 4.74% of its initial mass and was 3.43 times greater than 2,4-DBP. Phytovolatilization of 2,4-DBA and 2,4-DBP contributed to 6.78% and 41.7% of their total volatilization, enhancing the emission of these two contaminants from hydroponic solution into atmosphere. In this study, the interconversion processes between 2,4-DBP and 2,4-DBA were first characterized in rice plants. The demethylation ratio of 2,4-DBA was 12.0%, 32.0 times higher than methylation of 2,4-DBP. Formation of corresponding metabolites through methylation and demethylation processes also contributed to the volatilization of 2,4-DBP and 2,4-DBA from hydroponic solution into the air phase. Methylation and demethylation processes increased phytovolatilization by 12.1% and 36.9% for 2,4-DBP and 2,4-DBA. Results indicate that phytovolatilization and interconversion processes in rice plants serve as important pathways for the global cycles of bromophenols and bromoanisoles.

Introduction

Bromophenols and bromoanisoles are naturally biosynthesized by marine organisms (i.e., algae, marine sponge, and bacteria) in the oceans (Ballschmiter, 2003). They also have anthropogenic sources from industrial production (i.e., polymer intermediates, flame retardant intermediates, and wood preservatives) (Gribble, 2003; Howe et al., 2005). 2,4-Dibromophenol (2,4-DBP) and 2,4-dibromoanisole (2,4-DBA) always coexist (Ballschmiter, 2003) and are frequently detected in various environment matrices, such as river water, seawater, sediment, and air (Bidleman et al., 2014, 2017; Chung et al., 2003; Fuhrer and Ballschmiter, 1998; Koch and Sures, 2018; Reineke et al., 2006; Schwarzbauer and Ricking, 2010). Concentration of 2,4-DBP in surface water was in the range of 10–4000 ng L−1 (Ren et al., 2013; Vetter et al., 2018). 2,4-DBA was ranged from 8.1 to 192 pg L−1 and 3.7 to 20 pg m−3 in water and air, respectively (Bidleman et al., 2014, 2017). Bromophenols can undergo O-methylation to form brominated anisoles in some marine algaes and mussels (Lofstrand et al., 2010; Vetter et al., 2007), and several branches of bacteria in the natural environment (Allard et al., 1987). Correspondingly, methylated phenol contaminants (i.e., methyl triclosan, methoxylated polychlorinated biphenyls, 6-methoxy-2,2′,4,4′-tetrabromodiphenyl ether (6-MeO-BDE-47), as well as tetrabromobisphenol A mono- and di-methyl ethers) can be biologically demethylated back to their parent compounds in high plants (Fu et al., 2018; Hou et al., 2018; Sun et al., 2016; Xu et al., 2016). For instance, a significant amount (more than 15.0%) of methyl triclosan could be back converted to triclosan in A. thalina cells (Fu et al., 2018). Similar demethylation process may occur for bromoanisole to yield bromophenols in the environment. However, the interconversion processes between bromophenol and bromoanisole in intact plants, in particular, their contributions to volatilization and the environmental cycling of bromophenol and bromoanisole are unknown.

Methylation and demethylation of organic and inorganic compounds are pervasive biotransformation processes in various plants (Fu et al., 2018; Hou et al., 2018; Li et al., 2018; Sun et al., 2016; Xu et al., 2016; Zhang et al., 2019). Plant uptake, accumulation as well as volatilization of the contaminants can be significantly altered when methylation and demethylation processes occurred. Some researchers found that 6-MeO-BDE-47 was readily accumulated in maize shoots compared with 6-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (6-OH-BDE-47) (Xu et al., 2016). Selenite and arsenic were inclined to form more volatile methylation products in wetlands which were liable to diffuse into the air phase (Chen et al., 2014; Jia et al., 2012; Terry et al., 2000). With regard to the two target compounds in this study, 2,4-DBA is more volatile than 2,4-DBP in seawater-air exchange system (Limmer and Burken, 2016). Recent studies showed that over 80.0% of 2,4-DBA volatilize from surface seawater and reach a level of 97 pg L−1 in atmosphere (Bidleman et al., 2016; Koch and Sures, 2018). Although vegetation plays an important role in the exchange of volatile and semi-volatile organic compounds (VOCs and SVOCs) between contaminated soil or groundwater and atmosphere in terrestrial ecosystem (Barber et al., 2004; Desalme et al., 2013; Li and Chen, 2014; Nizzetto et al., 2008), limited information is available to quantitatively evaluate the volatilization of 2,4-DBP and 2,4-DBA mediated by vegetation. Moreover, the roles of interconversion processes also need to be considered in the exchange of bromophenols and bromoanisoles between contaminated sites and atmosphere.

Rice (Oryza sativa Japonia cv. Nipponbare) is widely cultivated in Asia (Kim et al., 2018). A large amount of irrigation water is introduced into paddy fields during rice growth, and thus, many unintended compounds such as 2,4-DBP and 2,4-DBA could be uptaken into rice plants (Lopez et al., 2009). In this context, the plant uptake, bioaccumulation, interconversion, as well as volatilization of 2,4-DBP and 2,4-DBA in rice plants were systematically investigated in hydroponic exposure experiments. Rice plants were significantly facilitated the volatilization of 2,4-DBP and 2,4-DBA from culture solution into air phase. Concurrently, methylation and demethylation processes help to volatilize 2,4-DBP and 2,4-DBA from rice plants. These results show that interconversion and phytovolatilization processes greatly affect the global cycles of 2,4-DBP and 2,4-DBA.

Materials and methods

Chemicals and plant material

2,4-DBP (98.0%) and 2,4-DBA (98.0%) applied for hydroponic exposure were acquired from Tokyo Chemical Industry (Tokyo, Japan) and J&K Scientific (Shanghai, China). The native standards (2,4-DBP (98.3%) and 2,4-DBA (98.0%)) for calibration curves and quantitative analysis, and one of surrogate standards ([13C6]-2,4,6-tribromophenol ([13C6]-2,4,6-TBP, isotope purity, 98.0%)) were purchased from AccuStandard (Connecticut, USA). Another surrogate standard [13C6]-2,4,6-tribromoanisole ([13C6]-2,4,6-TBA, isotope purity, > 98.0%) was obtained from Cambridge Isotope Laboratories, Inc. (Massachusetts, USA). Supelclean™ LC-Florisil (1000 mg, 6 mL) cartridges were purchased from Sigma-Aldrich (Philadelphia, USA). Commercial reagents (chromatographic grade of hexane, acetone, dichloromethane and ethyl acetate) were supplied by J.T. Baker (Phillipsburg, NJ), and ultrapure water was produced by Milli-Q system (Massachusetts, USA).

Rice (Oryza sativa Japonia. cv. Nipponbare) seeds were obtained from Nanjing Agricultural University (Nanjing, China) in 2018. Detailed information about the germination and cultivation (16 h/30 °C day and 8 h/25 °C night cycle; a constant relative air humidity (70%)) was the same as we reported earlier (Zhang et al., 2019). In brief, germination was conducted in deionized (DI) water at 30 °C for 3 days. The obtained rice seedlings were sequentially cultivated in half and full-strength Hoagland nutrient solution for one week and two weeks (renewed every 2 days), respectively. Then, rice seedlings with similar height (~15 cm) were selected for exposure experiments. To avoid an abundance of ions in nutrient solution (i.e., the oxidative transformation of bromophenols to form hydroxylated polybrominated diphenyl ethers (OH-PBDEs) by manganese ion (Lin et al., 2014)) affecting the biotransformation of the two target compounds in rice plants, deionized water was chosen for the hydroponic exposure.

Individual hydroponic exposure of 2,4-DBP and 2,4-DBA to rice plants

To systematically evaluate the plant uptake and bioaccumulation of 2,4-DBP and 2,4-DBA, as well as their interconversion, two target compounds were individually exposed to rice seedlings. In consideration of the transformation rate for phenol compounds, such as the methylation of hydroxyl polychlorinated biphenyls, were generally less than 1.0% in rice plants (Sun et al., 2016), a relatively high initial exposure concentration (4.0 mg/L) was selected in exposure experiments. Five rice plants were planted in each reactor with the exposure solution which was prepared by adding 45 μL working solution of individual parent chemical (4000 mg/L) into deionized water and finally constant to 45 mL for each parent chemical. All exposure reactors were 55 mL brown glass bottles and wrapped with aluminum foil to avoid any photolysis of the target compounds. To minimize cross contaminations between reactors and ambient environment, three parallel reactors (n = 3) were treated in an independent space (around 5.0 L) isolated by a pair of glass beakers which connected mouth to mouth, and the connection was sealed with commercial polytetrafluoroethylene film as a treatment (Fig. 1). Similar sealed exposure systems placed with three pieces of 1.50 g polyether-type polyurethane foam (PUF) inside the headspace were set as PUF treatments (Fig. 1). PUF has been used as the sorbent traps to successfully capture bromophenols and bromoanisoles from atmosphere (Bidleman et al., 2014, 2017). The existence of PUF could affect the equilibriums of target compounds among different compartments in the sealed systems to some extent due to the strong adsorption capacity. Therefore, quantitative determination of PUF samples was introduced to evaluate the total amount of target compounds (i.e., parent compounds and metabolites) volatilized from hydroponic solution (direct volatilization) and from rice plants (phytovolatilization) into the headspace.

Fig. 1.

The scheme of treatments without and with PUF. All the brown glass reactors were wrapped with aluminum foil.

Unplanted treatments with PUF for 2,4-DBP and 2,4-DBA were used to evaluate their direct volatilization from culture solution, which were treated with parent chemicals (2,4-DBP or 2,4-DBA) and incubated with PUF but without rice plants in a sealed independent space. The differences between the volatilization of pollutants in unplanted treatments with PUF (defined as direct volatilization) and the planted treatments with PUF (including direct volatilization and phytovolatilization) were attributed to the phytovolatilization process in these sealed systems. Planted blank controls (five whole rice plants without exposure of compounds, n = 3) were simultaneously conducted with and without PUF in the same sealed mode. Blank controls were used to evaluate any potential background contamination to plant and PUF samples. All the PUFs were prewashed with dichloromethane/hexane (50/50, v/v) in Dionex ASE 350 before use. All treatments were placed in an exposure chamber with the same conditions as the cultivation chamber (16 h/30 °C day and 8 h/25 °C night cycle; a constant relative air humidity (70%)) for 5 days.

Sampling and extraction

Distributions of parent and daughter compounds in the exposed plants were characterized in plant treatments, which are incubated without PUF. Their hydroponic solutions and rice seedlings (rice root, leaf sheath and leaf) were sampled at time intervals of 6 h, 12 h, 24 h, 48 h, 72 h and 120 h. Treatments with PUF was sampled for hydroponic solutions, plant samples and PUF samples at the end of exposure (after 120 h incubation). The PUF samples were used to determine the target compounds and corresponding methylated or demethylated metabolites entering into the headspace.

Root samples were washed with DI water before harvest, and then those rinse water was combined with the exposure solution for further analysis. And all solution samples were directly stored at −20 °C before further pretreatment. Plant samples were vacuum freeze-dried in a lyophilizer at −50 °C for 2 days (Boyikang Instrument Ltd., Beijing, China) and stored at −20 °C. PUF samples were all sampled from planted and unplanted treatments and blank control. To avoid the release of analytes from PUF samples which might occur in storage process, PUF samples were immediately extracted after sampling. The extraction of plant tissues, PUF samples and culture solution, as well as cleanup procedure for rice tissues were described in supporting information (Text S1).

Instrumental analysis

2,4-DBP and 2,4-DBA were served as parent compounds or metabolites in different exposure experiments, respectively. When 2,4-DBP was used as the parent compound, its methylation metabolite was 2,4-DBA. When rice seedlings were exposed to 2,4-DBA, the demethylation metabolite was 2,4-DBP. Thus, those two contaminants were concurrently existed in planted exposure systems. All the target chemicals, including 2,4-DBP, 2,4-DBA and two surrogate standards, were analyzed by gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS) in DFS system (Thermo Fisher Scientific, USA) with an electron impact (EI) ion source. Separation of four compounds was performed on a DB-5 MS column (60 m × 0.25 mm × 0.1 mm, J&W Scientific, Folsom, CA) with 1 μL injection volume and splitless mode. The column temperature was programmed initially at 40 °C for 1 min, increased to 220 °C at 15 °C/min and held for 3 min, then ramped to 250 °C at 15 °C/min and held for 1 min, and finally ramped to 300 °C at 25 °C/min and held for 3 min. The HRMS was performed in VSIR mode at a resolution more than 6000, source temperature was 250 °C, and the electron emission energy was 45 eV. Quantification ions of 2,4-DBP, 2,4-DBA, [13C6]-2,4,6-TBP and [13C6]-2,4,6-TBA were 251.8603, 265.8759, 335.7908 and 349.8065, respectively.

Quality control and quality assurance

Procedural blanks and solvent blanks were analyzed for every batch of samples, while no background interference and carryover were observed. Purities of commercial exposure compounds, as well as their native and surrogate standards using for calibration and quantification, were also tested by GC-HRMS. No other impurities were found to interfere in the detection of target chemicals. The average spiking recoveries of 2,4-DBP, 2,4-DBA, 13C-2,4,6-TBP and 13C-2,4,6-TBA were 81.3–90.7%, 84.5–96.2%, 79.2–89.6% and 86.1–97.3%, respectively. Dry weight basis was used to calculate plant samples’ concentrations unless otherwise specified. All the concentrations were corrected according to the recoveries of corresponding surrogate standards. The method detection limits (MDLs) of 2,4-DBP and 2,4-DBA was 2.21–3.32 ng L−1 and 0.22–0.33 ng L−1 for culture solutions, 4.47–6.13 ng kg−1 and 0.43–0.61 ng kg−1 for rice tissues, and 1210–2655 ng kg−1 and 123–261 ng kg−1 for PUF, respectively.

Data analysis

The percentage of total volatilized parent compounds in PUF treatments were calculated by the amount of parent compound (mp) detected in PUF dividing the initial mass of parent compound (mp0, the exposure amount of target compounds) as Eq. (1):

The phytovolatilized parent compound (Vp) was the difference of total volatilized parent compound detected on PUF between planted treatment (including direct volatilization and phytovolatilization, Vt-planted) and unplanted treatments (just including direct volatilization, Vt-unplanted) as Eq. (2). where mp-planted and mp-unplanted are the amounts of parent compounds detected on PUF samples of planted treatments and unplanted treatments with PUF, respectively. The Vt-unplanted here can indicate the direct volatilization.

Transformation ratio was calculated by the mole mass ratios of the total amount of metabolites at the end of exposure (120 h) to the initial amount of parent compound as in Eq. (3): where Tm is the total amount of metabolite (μg) in each separate reactor, including the amounts of metabolite in culture solution, plant tissues and one piece of PUF sample, Mp and Mm are the molecular weight of parent compounds and metabolites.

In the PUF samples of planted treatments, the mole mass ratios of methylated and demethylated metabolites to their corresponding parent compounds were calculated to evaluate the contributions (%) of transformation processes (methylation and demethylation) to the volatilization (TV) of 2,4-DBP and 2,4-DBA in rice plants (Eq. (4)). Moreover, as shown in Eq. (5), contributions of those transformation processes to the phytovolatilization (TVp) of 2,4-DBP and 2,4-DBA were evaluated by the mole mass ratios of methylated and demethylated metabolites detected in PUF (volatilized metabolites) to the phytovolatilized parent compounds which were the different mole masses of 2,4-DBP and 2,4-DBA between planted and unplanted treatments. where mm is the amount of metabolites (μg) in PUF sample.

Results and discussion

Bioaccumulations of parent 2,4-DBP and 2,4-DBA in rice plants

For the planted blank controls in the absence of PUF, none of 2,4-DBP and 2,4-DBA were detected in any of the samples, suggesting that no background contamination influenced the determination of hydroponic solutions and plant samples. Variations of parent 2,4-DBP and 2,4-DBA in culture solution and plant tissues during exposure period are summarized in Fig. 2. For the 2,4-DBP exposure system in the absence of PUF, a rapid disappearance of parent compound within the first 24 h was followed with a slow decrease observed in the culture solution (Fig. 2A). Only 0.40% of the initial amount of 2,4-DBP was left in the hydroponic solution after exposure for 120 h. The photo-degradation of parent chemicals was avoided by wrapping aluminum foil outside the brown glass reactors, so major losses of parent compounds from solution resulted from plant absorption and adsorption as well as direct volatilization. Concurrently, a significant amount of 2,4-DBP was detected in rice root. The highest root concentration (1239 mg kg−1) was occurred in the first 6 h, and then decreased rapidly to around 304 mg kg−1 after 12 h of exposure (Fig. 2B). 2,4-DBP was also found in rice leaf sheath and leaf. However, the concentrations of 2,4-DBP in leaf sheath and leaf was 6.99–54.4 and 95.9–358 times lower than those in the rice root during the period of exposure. It was reported that plants can take up contaminants through the developing root system (Limmer and Burken, 2016; Zhu et al., 2016). Once uptaken into the root, those pollutants were translocated from the rice root to upper plant parts (i.e., stem and leaf) (Liu et al., 2019). Some volatile pollutants were concurrently phytovolatilized into atmosphere. Detection of parent compound in leaf sheath and leaf confirmed that plant uptake and translocation of 2,4-DBP in rice plants. Levels of 2,4-DBP in the leaf sheath and leaf showed similar trends-initially increasing and then decreasing (Fig. 2C and D). But the maximum concentration of 2,4-DBP (49.4 mg kg−1, 24 h) in leaf sheath was later than in leaf (3.86 mg kg−1, 6 h). As well known, distributions and bioaccumulations of pollutants in different tissues are the results of comprehensive processes within the rice plant. Translocation of 2,4-DBP from rice root into leaf sheath was higher than their loss in leaf sheath (i.e., biotransformation, phytovolatilization from leaf sheath and translocation from leaf sheath to leaf) before 24 h, and then those elimination processes were predominant, making the highest concentration occurred at 24 h. While, the amount of 2,4-DBP translocated from rice root and leaf sheath into leaf was lower than those elimination processes just after 6 h. Therefore, the highest concentrations in different rice tissues were observed in different time. Furthermore, the sum of 2,4-DBP decreased in rice plants over time indicating that the losses of the parent compound probably resulted from plant phytovolatilization and metabolism (Sun et al., 2016, 2018; He et al., 2017; Limmer and Burken, 2016; Liu et al., 2019; Xu et al., 2016).

Fig. 2.

Concentration of 2,4-DBP (A, B, C and D) and 2,4-DBA (E, F, G and H) in culture solution and rice tissues in individual exposure experiment. None of 2,4-DBP and 2,4-DBA were detected in planted blank controls without PUF.

For the exposure group of 2,4-DBA in the absence of PUF, similarly, high concentrations of parent compound were detected in rice roots compared with other plant tissues. This phenomenon was consistent with the bioaccumulation of organophosphate esters in various plants (Liu et al., 2019). Concentrations of 2,4-DBA associated with the root gradually increased during 0–72 h, then slightly decreased at 120 h (Fig. 2F) and showed comparable levels to that of 2,4-DBP during 12–120 h (Fig. 2B and F). Meanwhile, the level of 2,4-DBA in leaf sheath (Fig. 2G) and leaf (Fig. 2H) continuously increased during the whole exposure period (6–120 h), which differed from the trends of 2,4-DBP. The concentrations of 2,4-DBA in rice leaf sheath and leaf increased up to 75.2 mg kg−1 and 9.24 mg kg−1 after 120 h exposure, respectively, which were 3.11 and 5.96 times higher than those of parent 2,4-DBP at 120 h. Concentrations of 2,4-DBA in the culture solution displayed a decreasing trend (Fig. 2E), but the decrease was slower and less than that for 2,4-DBP. Finally, 33.6% of initial amount of 2,4-DBA remained in the culture solution, which was considerably higher than that of 2,4-DBP (p < 0.001). Overall, compared with 2,4-DBP, 2,4-DBA showed less loss in hydroponic solution and more remaining in the rice plants, especially in the aboveground tissues of rice. This might be attributed to their different properties and biotransformation abilities. In comparison, 2,4-DBA was less water-soluble and more volatile, making it was probably less taken up by rice roots and more volatilized from the solution. Those comprehensive processes (volatilization and uptake) finally resulted in less loss of 2,4-DBA in the hydroponic solution. Once 2,4-DBA and 2,4-DBP were uptaken into rice plants, numerous enzymes within the rice plants would make them metabolized. 2,4-DBP contains active phenolic hydroxyl group which was reported being readily catalyzed by glycosyltransferase and other Phase II enzymes to form saccharide and amino acid conjugates (Sun et al., 2018). Thus, it was inferred that less 2,4-DBA was biotransformed within rice plant due to the lack of active hydroxyl group but more 2,4-DBP was metabolized by rice plants in the exposure period. Therefore, more 2,4-DBA was remained in the rice plant tissues (Fig. 2G and H).

Direct volatilization and phytovolatilization of parent 2,4-DBP and 2,4-DBA in PUF treatments

Same as plant samples in the planted blank control without PUF, no 2,4-DBP and 2,4-DBA was detected in plant samples for the planted blank control with PUF. However, concentrations of PUF samples in blank control (with PUF) were 0.01 mg kg−1 for both 2,4-DBP and 2,4-DBA due to the strong adsorption capacity of PUF for bromophenols and bromoanisoles. The amounts of 2,4-DBP and 2,4-DBA in blank control were only accounted for 0.73–1.16% and 0.12–0.13% of PUF samples in unplanted treatments (with PUF) and exposure groups (with PUF). Therefore, the average amount of 2,4-DBP and 2,4-DBA in PUF sample of blank control was considered as the background contamination and deducted from the results of concentrations from the PUF samples in unplanted treatments and exposure groups.

Direct volatilization of parent 2,4-DBP and 2,4-DBA from culture solution to headspace was determined by PUF samples in unplanted treatments with PUF. As shown in Fig. 3, the measured fluxes of 2,4-DBA (Henry’s law constant (25 °C), 4.78 Pa m3 mol−1) (Bidleman et al., 2016) through direct volatilization are 8006 ng, 5.66 times higher than that of 2,4-DBP (1415 ng, Henry’s law constant (25 °C), 0.037 Pa m3 mol−1) (Kuramochi et al., 2004), consistent with the finding that volatile compounds with low octanol-air partitioning coefficients are more likely to be volatilized from water (Limmer and Burken, 2016). The direct volatilization accounted for 0.80% and 4.42% of the initial amounts of 2,4-DBP and 2,4-DBA after 120 h exposure (Table 1), respectively. Those results indicated that the properties of compounds, such as Henry’s law constant and octanol-air partitioning coefficients, related the volatilization of 2,4-DBA and 2,4-DBP in exposure system.

Fig. 3.

Direct volatilization and phytovolatilization of parent 2,4-DBP and 2,4-DBA in planted treatments.

Table 1

Direct volatilization, phytovolatilization as well as total volatilization of 2,4-DBP and 2,4-DBA after 5 days exposure in planted treatments.

Incubation experiments	Direct volatilization (%)	Volatile percentage from rice seedlings (%)	Total volatilization (%)
Plant treatment with 2,4-DBP exposure	0.80 ± 0.09	0.58 ± 0.10	1.38 ± 0.12
Plant treatment with 2,4-DBA exposure	4.42 ± 0.26	0.32 ± 0.10	4.74 ± 0.27

The total volatilization of 2,4-DBA (4.74%) was 3.43 times higher than that of 2,4-DBP (1.38%) by comparing the results of PUF samples from their separate planted exposure groups with PUF. Then, it was calculated that 0.32% and 0.58% of initial amounts of 2,4-DBA and 2,4-DBP (Table 1) entered into the air phase through phytovolatilization though only three reactors with fifteen rice plants were incubated in the sealed exposure systems for just 5 days. 2,4-DBA and 2,4-DBP was phytovolatilized at rates of 7830 and 4422 mg kg−1 fresh biomass (leaf and leaf sheath) day−1 which were higher than that of mercury with estimated values between 14.4 and 85.0 mg Hg kg−1 fresh biomass day−1 from tobacco plants (Kuramochi et al., 2004). Overall, 6.78% and 41.7% of total volatilization of 2,4-DBA and 2,4-DBP was attributed to rice plants, respectively, distinctly larger than the contribution of phytovolatilization to the total volatilization of arsenic (0.4–3.2%) (Jia et al., 2012). Obviously, phytovolatilization facilitated the volatilization process between culture solution and atmosphere within the hydroponic exposure systems. Although the experiments were conducted under hydroponic conditions without rice paddy soil which have the sorption capacities to 2,4-DBP and 2,4-DBA to limit their direct volatilization and bioavailability, it still could be concluded that phytovolatilization was an effective process for the exchange of 2,4-DBP and 2,4-DBA between contaminated sites and the air phase. Moreover, phytovolatilization as well as total volatilization of 2,4-DBP and 2,4-DBA in planted treatments were closely related to the exposure time, biomass and growing status of the rice plants. With regarded to the long term exposure of those two contaminants, the biomass of rice seedlings gradually increased with growth, and the phytovolatilization would be significantly enhanced and become more important in the long distance transportation of 2,4-DBP and 2,4-DBA.

Interconversion of 2,4-DBP and 2,4-DBA in planted treatments

Methylation and demethylation metabolites of 2,4-DBP and 2,4-DBA were detected in their individual exposure systems. Xenobiotic contaminants are generally metabolized under the biocatalysis of plant enzymes after being taken up by plant. O-methyltransferase and demethyltransferase have been extensively observed and mediate the methylation and demethylation of contaminants in high plants. None of daughter 2,4-DBP and 2,4-DBA were detected in both planted blank controls without PUF and the unplanted treatments with PUF, confirming that methylation of 2,4-DBP and demethylation of 2,4-DBA were all mediated by rice plants, not chemical reactions in exposure systems. Similar interconversion was also reported between OH-PCBs and MeO-PCBs within rice plants (Sun et al., 2016).

For the 2,4-DBP exposure system without PUF, the methylation product was rapidly formed in rice roots after 6 h exposure (Table S1), and then constantly decreased. The methylation product could also be detected in both leaf and leaf sheath. The concentration was firstly increased and then decreased in leaf sheath (Table S1) but always increased in rice leaf during the exposure (Table S1). The total amount of methylation metabolite increased during 6–48 h, and then decreased from 155.3 ng (48 h) to 97.78 ng (120 h) (Fig. 4A). Majority of methylation metabolite was distributed in the culture solution, accounting for 68.6–83.8% of the total amount of metabolite. While for exposure of 2,4-DBA, as seen from Fig. 4B, no demethylation metabolite was detected in exposure solution. The demethylation product was extensively detected in various rice tissues. The concentrations of demethylation product in rice leaf, root and leaf sheath and the total concentration in whole rice plant were all constantly increased during the exposure period, and reached to 16.34, 42.66, 61.78 and 39.53 mg kg−1 at the end of exposure (120 h) (Table S2). A majority (50.5–70.8%) of the total demethylation metabolite was accumulated in rice roots before 72 h, while demethylation product became dominant in the leaf sheath and leaf (4846 ng, accounting for 74.3%) at 120 h.

Fig. 4.

Mass of methylation metabolites (ng) for planted exposure system of 2,4-DBP without PUF (A) and demethylation metabolites (ng) for planted exposure system of 2,4-DBA without PUF (B) over time. No metabolites were detected in the solutions of 2,4-DBA planted exposure systems without PUF.

Likewise, the formed metabolites, 2,4-DBP and 2,4-DBA, were phytovolatilized from rice seedlings into the headspace. And the methylation metabolite (2,4-DBA) which observed in hydroponic solution could also be directly volatilized into the headspace. Herein, all of metabolites in exposure solution, plant tissues and those volatilized into the headspace were included to evaluate the transformation ratio (TR) between 2,4-DBP and 2,4-DBA in the sealed system. The total volatilized methylation and demethylation metabolites detected in air phases of exposure systems with PUF were 126.8 ng and 218.5 ng (Fig. 5A). Including these volatilized metabolites, the demethylation ratio of 2,4-DBA was 12.0%, that is 32.0 times higher than the methylation ratio of 2,4-DBP (0.38%) after exposure for 120 h (Fig. 5B). The demethylation process was obviously faster than the methylation process. A similar phenomenon was previously reported on the interconversion of OH– and MeO-PCBs in rice seedlings, in which the demethylation process was 7.70–18.2 times greater than methylation (Sun et al., 2016). Results illustrate that bromoanisoles are readily demethylated within rice plants and subsequently released as bromophenols into environment. Unfortunately, bromophenols are precursors to form more lipophilic and bioactive products (unintended potential pollutants) in rice plants, such as hydroxylated polybrominated diphenyl ethers (OH-PBDEs) and polybrominated dibenzo-p-dioxins/dibenzofurans (PBDD/Fs) (Zhang et al., 2019). Therefore, the occurrence of bromophenols and bromoanisoles needs to be considered as a potential risk factor in consumption of food plants.

Fig. 5.

Amounts of methylation and demethylation metabolites in PUF samples (A). The transformation ratios for the methylation of 2,4-DBP and demethylation of 2,4-DBA (B). The contributions of methylation and demethylation processes to the total volatilizations (C) and phytovolatilizations (D) of corresponding parent chemicals in the planted treatments.

Contribution of methylation and demethylation processes to the volatilization in the exposure systems

The contributions of interconversion processes to the volatilization of parent contaminants were further quantitatively evaluated and summarized in Fig. 5C and D. According to the equimolar reaction between parent and daughter compounds, the volatilized methylation and demethylation metabolites were transformed from 0.48 and 0.87 nmol of parent 2,4-DBP and 2,4-DBA accounting for 0.07% and 0.13% of their initial amounts (180 μg), respectively. These amounts enhanced 4.95% and 2.69% of total volatilization mass (Fig. 5C), and 12.1% and 36.9% of phytovolatilization mass of parent 2,4-DBP and 2,4-DBA, respectively (Fig. 5D). Obviously, methylation and demethylation processes served as important strategies to volatilize their corresponding parent chemicals out of intact plants. Furthermore, the volatilization of metabolites was also an important process to reduce bioaccumulation of parent chemical in plants. These observations indicate that interconversion provides another pathway to volatilize phenolic chemicals and methyl-phenols pollutants from contaminated sites into the air phase.

After calculation the mass balance of 2,4-DBP and 2,4-DBA in the treatments and exposure systems with PUF (Fig. S1), it was found that the total recovered 2,4-DBP and 2,4-DBA in the unplanted treatments with PUF were only 88.5% and 87.0%, respectively. The photo-degradation of target chemicals was avoided in incubation system by wrapping aluminum foil outside the brown glass reactors, and microbial transformation (biotic transformation) was minimized by pre-autoclaving the solutions and glass bottles before exposure experiment. Therefore, we inferred that those unrecovered target compounds were resulted from the incompletely sampling of the volatilized chemicals in air phase. Although PUFs were placed in the systems, the analytes detected on PUFs only represent a fraction of the volatilized amounts. Namely, the total volatilization and the contribution of the interconversion to the volatilization are all underestimated. In planted exposure systems with PUF, the recoveries of 2,4-DBP (27.3%) and 2,4-DBA (62.6%) were significantly lower than those of unplanted treatments with PUF. Though non-sterilized rice plants were used for exposure experiments, our former study has shown that the microbial transformation (biotic transformation) of 2,2′,4,4′-Tetrabromodiphenyl Ether (BDE-47) was far lower than pumpkin plant in the similar cultivation and exposure conditions (Sun et al., 2013). Thus, the significant differences between unplanted and planted treatments were inferred with the occurrence of other metabolic pathways or bound residues in rice plants (Wang et al., 2016; Zhang et al., 2019).

Conclusions

In this paper, we characterize the processes for methylation of 2,4-DBP and demethylation of 2,4-DBA in rice plants for the first time. And the demethylation ratio of 2,4-DBA was 32.0 times higher than methylation of 2,4-DBP in rice plants. The quantitative evaluation showed that phytovolatilization of parent 2,4-DBP and 2,4-DBA were enhanced by the volatilization of corresponding metabolites through methylation and demethylation processes in rice plants. Considering the extremely large cultivation area and long growing period (totally six months per year with two major crop cultivation seasons in March-July and August-December) of rice plants, the phytovolatilization of 2,4-DBP and 2,4-DBA play an important role on the long range transportation of these global pollutants. Though hydroponic exposure with a relatively high exposure concentration was carried out in this study, which was different from the real environment, the results reflect the environmental process in real conditions to some extent. Here, we also generalize that methylation and demethylation processes occurred in plants may provide another pathway for volatilization and cycling of these classes of contaminants in the environment.