Article pubs.acs.org/est
Degradation and Metabolism of Tetrabromobisphenol A (TBBPA) in Submerged Soil and Soil−Plant Systems Feifei Sun,† Boris Alexander Kolvenbach,‡ Peter Nastold,‡ Bingqi Jiang,§ Rong Ji,*,†,∥ and Philippe Francois-Xavier Corvini†,‡ †
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, 163 Xianlin Avenue, 210023 Nanjing, People’s Republic of China ‡ Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, Muttenz CH-4132, Switzerland § Fujian Provincial Academy of Environmental Science, No. 10, Huan Bei San Cun, Fuzhou 350013, People’s Republic of China ∥ Institute for Marine Sciences & Institute for Climate and Global Change Research, Nanjing University, 22 Hankou Road, 210093 Nanjing, People’s Republic of China S Supporting Information *
ABSTRACT: Contamination by tetrabromobisphenol A (TBBPA), the most widely used brominated flame retardant, is a matter of environmental concern. Here, we investigated the fate and metabolites of 14C-TBBPA in a submerged soil with an anoxic− oxic interface and planted or not with rice (Oryza sativa) and reed (Phragmites australis) seedlings. In unplanted soil, TBBPA dissipation (half-life 20.8 days) was accompanied by mineralization (11.5% of initial TBBPA) and the substantial formation (60.8%) of bound residues. Twelve metabolites (10 in unplanted soil and 7 in planted soil) were formed via four interconnected pathways: oxidative skeletal cleavage, O-methylation, type II ipso-substitution, and reductive debromination. The presence of the seedlings strongly reduced 14C-TBBPA mineralization and bound-residue formation and stimulated debromination and O-methylation. Considerable radioactivity accumulated in rice (21.3%) and reed (33.1%) seedlings, mainly on or in the roots. While TBBPA dissipation was hardly affected by the rice seedlings, it was strongly enhanced by the reed seedlings, greatly reducing the half-life (11.4 days) and increasing monomethyl TBBPA formation (11.3%). The impact of the interconnected aerobic and anaerobic transformation of TBBPA and wetland plants on the profile and dynamics of the metabolites should be considered in phytoremediation strategies and environmental risk assessments of TBBPA in submerged soils.
■
amounts of bound residues.11 In oxic clay soils, TBBPA has a T1/2 of 65−93 days.12 Under oxic conditions, it may be bacterially O-methylated to mono- and dimethyl ethers (MeOTBBPA and diMeO-TBBPA, respectively) and then mineralized.13,14 However, the fate and metabolism of TBBPA in soil with an anoxic−oxic interface, such as flooded soils, is unknown. Sequential anoxic−oxic treatment has been proposed for the removal of TBBPA and other halogenated compounds found in contaminated sites, based on the debromination of TBBPA under anoxic conditions and the rapid degradation of BPA under oxic conditions.11,15 However, because of bound-residue formation during its anoxic incubation, TBBPA cannot be
INTRODUCTION Tetrabromobisphenol A (TBBPA) is one of the most widely used flame retardants in the world, with applications in printed circuit boards and the production of several types of polymers.1 TBBPA can be released into the environment during the production, use, and disposal of flame-retardant-containing products.1,2 Importantly, it has been detected in various environmental media, e.g., air, sewage sludge, sediment, and soil, and in organisms such as mussels, birds, and even human adipose tissue, breast milk, and plasma.1,3−9 The undesirable effects of TBBPA include its activity as a potential endocrine disrupter and as a source of oxidative stress in a wide variety of organisms.3,10 Once released into the soil environment, TBBPA tends to accumulate because of its high lipophilicity and poor water solubility. In anoxic soils, TBBPA may be reductively debrominated to bisphenol A (BPA), with a half-life (T1/2) of 36−430 days,11,12 accompanied by the formation of large © XXXX American Chemical Society
Received: July 13, 2014 Revised: November 12, 2014 Accepted: November 17, 2014
A
dx.doi.org/10.1021/es503383h | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Article
soil), and soil without plant growth (unplanted soil). For treatments with plant growth, four rice or reed seedlings of similar sizes were transplanted into 50 g of soil (dw) in 100 mL flasks. For treatment without plant growth, 20 g of soil (dw) was placed into flasks of smaller diameter, thus allowing the formation of an oxic−anoxic interface in the flasks during the incubation. Three replicates were prepared per treatment. Each flask was placed in a 1000 mL beaker connected successively to bottles containing ethylene glycol (200 mL, to trap radioactive volatile organic compounds) and NaOH (2 M, 200 mL, to trap 14 CO2) and to a vacuum pump (Supporting Information, SI, Figure S1). The flow-through incubation systems were set up in a growth chamber with cycles of light and temperature that mimicked field conditions: 16 h of light and an incubation temperature of 34 °C followed by 8 h of darkness and an incubation temperature of 26 °C. A water layer of ∼1 cm was maintained above the soil in all the flasks during the incubation. At incubation days 0, 10, 22, 35, 48, and 66, aliquots of the three soil types were sampled to monitor TBBPA degradation and metabolite formation. On days 10 and 66 (reed seedlings) and 10, 35, and 66 (rice seedlings), the seedlings were sampled to analyze the amount and distribution of the radioactivity in the plants. Soil Fractionation. The soil samples were centrifuged (10 000g, 5 min) to remove the water and then freeze-dried. Radioactivity in the water supernatant was determined by liquid scintillation counting (LSC) (see SI). About 1.5 g of the soil pellet was extracted three times using 15 mL of methanol under continuous shaking (150 rpm, 1 h). This extraction procedure led to a recovery of 96.2 ± 0.4% of the applied TBBPA. After centrifugation (3600g, 10 min) of the suspension, the supernatants were combined and the radioactivity was measured by LSC. The methanol-extracted soil was air-dried at room temperature, and the amount of radioactivity was determined by combusting 150 mg in an oxidizer (see SI). After organic solvent extraction of the soil, the remaining, nonextractable radioactivity was defined as the bound residues.24 These were further fractionated into humic acid (HA)-bound, fulvic acid (FA)-bound, and humin-bound residues by alkaline extraction and acidification, as described previously. 11 The radioactivity in these fractions was determined by LSC (see SI). The concentrations of TBBPA and its metabolites in organic extracts of the soil samples prepared on various days during the experiment were analyzed using high-performance liquid chromatography (HPLC) with an online radio flow detector (see SI). To identify the TBBPA metabolites, the extracts at day 66 were purified by HPLC and analyzed using gas chromatography−mass spectrometry (GC−MS) (see SI). Uptake and Distribution of Radioactivity in Plants. The distribution of radioactivity was analyzed by autoradiography of freeze-dried plant material, carried out by its exposure for 1 week to imaging plates. The plates were then scanned by a Fuji scanner (Fuji FLA-9000, Japan). Total plant radioactivity and the radioactivity in the roots and aboveground were determined by an oxidizer (see SI). Data Analysis. SigmaPlot 12.0 was used to fit the TBBPA degradation data to the first-order kinetics equation C = C0 e−kt, where C0 and Ct are the concentration at time 0 and t, respectively, and k is the degradation rate constant. The half-life (T1/2) was calculated using T1/2 = ln 2/k. Significance was analyzed using an ANOVA or Student’s t-test. Differences were considered significant if P < 0.05.
completely removed from the soil; instead, repeated anoxic− oxic incubations have been suggested.11 Yet, whether the presence of adjacent anoxic and oxic layers in flooded soils accelerates the transformation of TBBPA is unknown. Wetland plants transport O2 to their roots from aboveground, thereby forming oxic zones within anoxic soil compartments.16,17 In the rhizosphere, root exudates provide substrates for microbial growth and cometabolism but also surfactants that increase the bioavailability of pollutants as well as inducers of degrading enzymes.18 Thus, at anoxic−oxic interfaces the transformation of pollutants may be stimulated. Indeed, the common reed (Phragmites australis) and cord-grass (Spartina alternif lora), two plants frequently found in wetlands, were shown to accelerate the debromination of TBBPA in salt marshes,19 whereas the effects of freshwater wetland plants on the fate of TBBPA in soil have yet to be examined. The objectives of the present study were (1) to investigate the degradation, metabolites, and residue distribution of TBBPA in both submerged soil and a soil−plant system and (2) to elucidate the effects of the anoxic−oxic interface and wetland plants on the fate and metabolism of TBBPA in soil. A paddy rice soil and soil−plant system were chosen because paddy fields constitute one of the most important flooded ecosystems in the world, especially in Asia,20 and both TBBPA and BPA have been detected in paddy soils in China.21 In addition to paddy rice (Oryza sativa), P. australis, another wetland plant, was chosen, as it is commonly used for the in situ remediation of saltwater and freshwater sediments contaminated by organic pollutants such as polyaromatic hydrocarbons and phenols.19,22,23
■
MATERIALS AND METHODS Chemicals. Uniformly 14C-ring-labeled TBBPA (14CTBBPA) with a specific activity of 1.48 × 109 Bq/mmol and a radiochemical purity of 99% was synthesized from uniformly 14 C-ring-labeled phenol via BPA.11 Nonlabeled TBBPA and other chemicals were purchased from Sigma-Aldrich (Switzerland). Soil. A silty clay loam soil with a pH 8.3, organic matter content of 6.7%, and cation exchange capacity 46.2 cmol(+)/kg was sampled from a paddy rice field located in Jiangning, Nanjing, China. The soil was air-dried, separated from plants residues and stones, and then sieved through a 2 mm sieve before use. Plants. Seeds of O. sativa and P. australis were obtained from Nanjing Agriculture University, China. The sterilized seeds were germinated at 30 °C in the dark and the seedlings were grown in the soil for 1 week and 3 weeks, respectively. Seedlings with similar biomasses were used in the incubation experiments. Incubation Experiments. Incubation experiments were carried out as follows: 100 mg P (KH2PO4)/kg and 100 mg N (NH4NO3)/kg were added as nutrients to 600 g of soil, which was then flooded with 780 mL of H2O. This water-saturated (submerged) soil was stored in the dark for 1 week to activate resident soil microorganisms. About 20 g of dried soil was spiked with 14C-TBBPA in 0.2 mL of methanol. After removal of the methanol by evaporation in a fume hood for 30 min, the mixture was thoroughly combined with the submerged soil, resulting in a final TBBPA concentration of 5 mg/kg soil (dry weight, dw) and a radioactivity of 3 MBq/kg soil (dw). Three treatments were tested: soil planted with O. sativa growth (rice soil), soil planted with P. australis growth (reed B
dx.doi.org/10.1021/es503383h | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
■
Article
layer of the top soil. In the soils with plant growth, only ∼5% of the 14C-TBBPA was mineralized (Figure 1B,C). This reduced mineralization in planted submerged soils could be attributed to (1) plant-mediated alteration of TBBPA metabolism in the soil and (2) a reduction in the amount of mineralizable 14C-labeled residues (parent and metabolites) because of their substantial uptake by the plants (see below). Because the headspace of the incubation system was replaced continuously by fresh air, 14 CO2 reassimilation was likely to be negligible. In addition, the amount of volatile radioactive organic compounds was not significant in any of the treatments during the incubation, ruling out the phyto-volatilization of TBBPA and its metabolites. Degradation and Metabolites of TBBPA. During the incubation, organic-solvent-extractable radioactivity in the unplanted soil decreased to 20.2 ± 3.3% (SI Figure S2A). The amount was slightly lower in soil planted with reed seedlings (16.2 ± 1.6%), whereas there was no effect on the amount of extractable radioactivity in soil planted with rice seedlings (20.8 ± 0.6%, SI Figure S2A). Radio-HPLC analysis of the organic extracts showed the formation of 14C-labeled TBBPA metabolites (SI Figure S3). At the end of the incubation, >90% the labeled TBBPA was removed from the unplanted soil and the half-life (T1/2) of TBBPA under this condition was 20.8 ± 0.1 days (Figure 1A), which was faster than the reported dissipation of TBBPA under anoxic conditions in a rice paddy soil (T1/2 36 days),11 an estuarine sediment (T1/2 30−40 days),25 salt marshes (T1/2 70 to >130 days)19 and a heavy clay soil (T1/2 430 days).12 Loss of TBBPA in the unplanted soil was also faster than the dissipation of TBBPA under oxic conditions in a freshwater sediment (T1/2 40 days),14 a sandy soil slurry (T1/2 41 days),13 and a heavy clay soil (T1/2 65−93 days)12 but slower than the dissipation in a river sediment under anoxic (T1/2 11 days)26 or oxic (16.8 days)27 conditions. Although the types of soil and sediment can affect the transformation of TBBPA in environmental matrices, our results suggest that the oxic−anoxic interface of submerged soils provides a favorable environmental niche for the rapid transformation of TBBPA. TBBPA dissipation was slightly enhanced in rice soil after 35 days of plant growth but was strongly accelerated in reed soil (T1/2 11.4 ± 0.4 days) throughout the incubation. In fact, under the latter conditions, TBBPA had completely dissipated in the soil by day 48 of the incubation (Figure 1C). The different effects of the two wetland plants on TBBPA transformation in soil can be explained by differences in root exudate composition, root morphology, and biomass, and the developing rhizosphere microbial community, as suggested in a previous study examining the dissipation of TBBPA in salt marshes containing reed and cord-grass.19 In that study, cordgrass significantly stimulated the dissipation of TBBPA, while reed, in contrast to our results, had either no or negative effects. This discrepancy between our results and those reported by Ravit et al.19 suggests that in addition to the particular plant species, rhizosphere effects on TBBPA transformation depend on environmental matrix properties and the initial soil community from which the rhizosphere community is recruited. The metabolites in the soil extracts at the end of the incubation were identified by GC−MS, according to their bromine isotope patterns and the resulting characteristic mass fragments of the metabolites (the mass spectra of TBBPA and its metabolites are shown in SI Figures S4−S15). Twelve
RESULTS AND DISCUSSION Both the rice and the reed seedlings were able to grow in TBBPA-contaminated soil (5 mg TBBPA/kg soil). The total radioactivity recovered from the various soil and plant fractions during the experiments was in the range of 93−103%, 95−97%, and 96−105% for unplanted soil, rice soil, and reed soil, respectively (Figure 1).
Figure 1. Total recovered radioactivity (left y-axis) and the radioactivity recovered from CO2, water-soluble fraction (right yaxis), TBBPA, metabolites, and bound residues (left y-axis) during the incubation of 14C-TBBPA in a submerged soil either not planted (A) or planted with rice (B) or reed (C) seedlings. Data are the means of three individual experiments ± one standard deviation.
Mineralization of TBBPA. In a previous study, the rate of anaerobic cleavage of the benzene ring of TBBPA was very low, such that after 195 days of incubation in anoxic soil only 1.3% of the original amount had become mineralized.11 In another study, only 1.1% of the TBBPA in an oxic sandy soil slurry amended with bacterial growth medium was mineralized after 20 days of incubation.13 By the end of the incubation period of our study (66 days), a significant amount of 14C-TBBPA in the unplanted soil was mineralized (11.5 ± 0.4%) (Figure 1A), likely reflecting its higher rate of aerobic degradation in the oxic C
dx.doi.org/10.1021/es503383h | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Article
Figure 2. Identified metabolites of TBBPA transformation and the proposed pathways leading to their formation in a submerged soil with an oxic− anoxic interface in the absence and presence of plant growth. The compounds in dashed brackets were not detected in the soil extract and represent hypothetical intermediates. The blue and red lines indicate the stimulated transformation pathways and the metabolites in soil planted with rice and reed seedlings, respectively.
metabolites (10, 7, and 5 metabolites from the unplanted soil, rice soil, and reed soil, respectively) were identified. Their structure and the identification of their fragments are summarized in SI Table S1. The structures are also shown in Figure 2. These metabolites included debromination products (compounds 1−4 and 9), O-methylation products of TBBPA and its debromination and cleavage metabolites (compounds 5−8, 10, and 11), and compounds with a single benzene ring (compounds 9−12). Among the O-methylation products, four were methyl ethers of brominated BPAs (bromoBPAs; compounds 5−8) and two were methyl ethers of single-ring metabolites of TBBPA (compounds 10 and 11). Only one ether of bromoBPA (MeO-TBBPA; compound 7) was detected in the reed soil whereas three ethers of bromoBPAs (MeOTBBPA, diMeO-TBBPA, and MeO-triBBPA; compounds 7, 8, and 5, respectively) were identified in the rice soil and four ethers (compounds 5−8) in the unplanted soil. Dynamics of Metabolite Formation. In both the unplanted soil and the rice soil, no metabolites were detected
before 22 days (Figure 1A,B), and all the radioactivity in the organic extracts was from TBBPA rather than from its metabolites (SI Figure S2). After 35 days of incubation in unplanted soil, about 29.5 ± 6.0% of the initial 14C-TBBPA was transformed to extractable metabolites, an amount that essentially remained constant during the following month of incubation (Figure 1A). By contrast, as shown in Figure 1C, in the reed soil, metabolites were already detected on day 10 (10.6 ± 1.2% of the initial 14C), with the maximum amount reached on day 22 (52.3 ± 8.1% of initial 14C) after which there was a steady decrease over the course of the incubation to 16.2 ± 1.6%. While considerable amounts of extractable 14C-TBBPA remained in the unplanted soil and in the rice soil, no significant amount of 14C-TBBPA could be extracted from the reed soil (SI Figure S2). This rapid formation of metabolites in the reed soil was in agreement with the stimulated transformation of TBBPA by the reed seedlings (Figure 1); at the same time, the mineralization of 14C-TBBPA in reed soil was markedly slower than in unplanted soil (Figure 1). D
dx.doi.org/10.1021/es503383h | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Article
(Figure 3D) were considerably higher in the planted soils (especially reed) than in the unplanted soil. The amounts of the unknown polar metabolites and lower brominated BPAs (Figure 3A,C) decreased after their appearance in the reed soil, while the levels of BPA and the single-ring metabolites remained consistently high up to day 48 of the incubation, suggesting the temporal accumulation of both BPA and the single-ring metabolites in the reed soil. At the end of the incubation there were fewer metabolites (compounds 9−12) in either of the planted soils than in the unplanted soil (SI Table S1), indicating a faster transformation of these metabolites in the former incubations. The abundance of polar metabolites was in agreement with the higher amounts of water-soluble radioactivity in the reed soil (Figure 1). In addition, debromination and single-ring metabolites were present in higher amounts than the O-methylation metabolites of bromoBPAs (Figure 3B−D), but at the end of the incubation the latter became dominant, accounting for 7.9 ± 0.8% and 11.3 ± 2.1% of the initial TBBPA in rice soil and reed soil, respectively (Figure 3D). The presence of the reed and rice plants apparently stimulated anaerobic reduction (Figure 3), such that even monoBBPA and BPA were detected in these soils (SI Table S1). In both of the planted soils, and especially in the reed soil, the unidentified polar metabolites (Figure 3A) might have derived from the further aerobic degradation of BPA in the rhizosphere. During the stimulated transformation of TBBPA by reed growth, O-methylated bromoBPAs (10.6 ± 1.2% of the initial TBBPA) occurred already on day 10 of the incubation (Figure 3D), followed by single-ring metabolites and debromination metabolites, both of which likewise occurred earlier in this soil than in the unplanted or rice soil (Figure 3). This time course of metabolite development suggests that in the reed soil aerobic O-methylation occurred more rapidly than anaerobic debromination. The presence of the reed plants strongly stimulated the transformation of TBBPA in the soil. Root exudates have been shown to support the growth of dehalogenating microorganisms19and heterotrophic O-methylating microorganisms in the rhizosphere; however, depending on their origin they may differentially affect the functions of the associated microbial communities.29,30 This would explain the different rates of debromination and O-methylation of bromoBPAs in the rhizosphere of rice vs reed seedlings and therefore the temporal differences in the appearance of the debromination and methylation metabolites in the two planted soils. Further studies on the exudate composition, oxygen content, and microbial community in the rhizosphere may provide detailed insights into the different behaviors of rice and reed seedlings in stimulating TBBPA transformation. Transformation Pathways. On the basis of the detected metabolites and the sequence of their occurrence under the three conditions of the study (unplanted, rice-planted, and reed-planted; Figure 3; SI Table S1) and because the state of the unplanted soil during the incubation changed from oxic to anoxic, we propose the following pathways for TBBPA transformation in submerged soil with and without plant growth (Figure 2). In the unplanted soil, four pathways likely participate in the transformation of TBBPA: (I) oxidative skeletal cleavage, (II) O-methylation, (III) type II ipso-substitution, and (IV) reductive debromination (Figure 2). Pathways I and III produce single-ring metabolites that can be further mineralized in the soil under both oxic and anoxic conditions. Oxidative
The dynamics of the formation of the different metabolites are shown in Figure 3. In the unplanted soil, the lower-
Figure 3. Relative amounts of unidentified polar metabolites (A), BPA and single-ring metabolites (compounds 9−12) (B), lower-brominated BPAs (compound 1−4) (C), and O-methylation metabolites of bromoBPAs (compounds 5−8) (D) in organic extracts of soil incubated with 14C-TBBPA without and with plant growth. The structures of the metabolites are shown here and in Figure 2. For details, see SI Table S1. Values are the means of three individual experiments ± one standard deviation.
brominated BPAs and the O-methylation products of bromoBPAs were detected on day 35 of the incubation (Figure 3C,D), indicating that anaerobic and aerobic transformation of TBBPA took place simultaneously in the soil. These Omethylated metabolites were the dominant metabolites at the end of the incubation, accounting for 8.8 ± 0.3% of the initial TBBPA. After their initial occurrence in the soil their total amount remained nearly stable (Figure 3D), which suggests that, together with debromination, O-methylation of bromoBPAs was an important process in the soil. These findings are also consistent with the recalcitrance of O-methylated bromoBPAs to aerobic degradation processes, unlike lowerbrominated BPAs, the amounts of which decreased after day 35 of the incubation (Figure 3C). Bacteria capable of Omethylating halogenated phenolic compounds are ubiquitous in the environment.28 Indeed, 10% of aerobic heterotrophic microorganisms in a freshwater marsh sediment were shown to O-methylate TBBPA.14 The O-methylation of bromoBPAs determined in the present study underlines the significance of the O-methylation of halogenated phenols in soil environments. Given that we did not detect further debromination products of diBBPA (i.e., monoBBPA and BPA) in the unplanted soil, the decrease in triBBPA and diBBPA (Figure 3C) suggests that debromination was slow. Single-ring compounds were detected in the unplanted soil only at concentrations too low to monitor their formation/dissipation dynamics (Figure 3A,B). Plant-mediated stimulation altered the amounts of the metabolites in the soil as well as the time course of their occurrence during the incubation. Before day 48, the amounts of unknown polar metabolites (Figure 3A), single-ring metabolites, BPA (Figure 3B), and O-methylated bromoBPAs E
dx.doi.org/10.1021/es503383h | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Article
bacteria, respectively, in the rhizosphere of the rice soil, where oxic and anoxic zones coexisted. Uptake of TBBPA and Its Metabolites by Plants. Both rice and reed seedlings showed a high potential to accumulate TBBPA and its metabolites from the soil. Reed seedlings accumulated significantly (P < 0.05) more radioactivity than rice seedlings (33.1 ± 10.5% and 21.3 ± 1.5%, respectively, Figure 4A). This difference was consistent with the formation
skeletal cleavage (pathway I) is similar to the aerobic bacterial degradation of BPA,31 which results in the cleavage of this compound into two single-ring compounds via oxidative skeletal rearrangement (SI Figure S16). TBBPA and its Omethylation metabolite MeO-TBBPA (compound 7) have a BPA skeleton and are transformed to 1,3-dibromo-2-methoxy5-vinylbenzene (compound 10) via sequential skeletal cleavage, O-methylation, reduction, and dehydration (SI Figure S16). Type II ipso-substitution is involved in the degradation of alkylphenols with an α-quaternary carbon on the alkyl chain (e.g., nonylphenol and BPA) by sphingomonads (Sphingomonas xenophaga Bayram, Sphingomonas sp. TTNP3, Sphingobium sp. IT-4, and Sphingobium sp. IT-5) and by a nonsphingomonad strain (Stenotrophomonas sp. IT-1).22,32−34 Analogous to the degradation of BPA by type II ipso-substitution, generating hydroquinone and carbocationic para-isopropylphenol, 34 TBBPA degradation by type II ipso-substitution would yield 2,6-dibromo-hydroquinone (compound 14) and the carbocation 2,6-dibromo-para-isopropylphenol (compound 15) (Figure 2);13 O-methylation of the former resulted in the formation of 2,6-dibromohydroquinone monomethyl ether (compound 11), and the latter was rapidly deprotonated to 2,6-dibromo4(propen-2-yl)-phenol (compound 12), which was debrominated to 2-bromo-4(propen-2-yl)-phenol (compound 9) (Figure 2) under anoxic conditions. The carbocationic intermediates may be protonated during BPA degradation by strain TTNP334 and hydroxylated during TBBPA degradation in soil slurry,13 but in the present study there was no evidence of either reaction in the degradation of TBBPA in the submerged soil. In the aerobic metabolism of TBBPA in the environment, Omethylation was shown to be an important reaction.14 In the unplanted soil, we detected not only mono- and dimethyl ethers of TBBPA (MeO- and diMeO-TBBPA) but also monoand dimethyl ethers of debrominated TBBPA (MeO- and diMeO-triBBPA) (Figure 2; SI Table S1). We attribute the formation of triBBPA ethers to combined O-methylation and debromination at the oxic−anoxic interface within the soil. These compounds would also be detected in environments characterized by temporally alternating oxic and anoxic states, such as soils or sediments with water fluctuation zones and dry−wet cycles. Debromination of TBBPA under anoxic conditions (pathway IV) may generate BPA, via less-brominated BPAs (triBBPA, diBBPA, and monoBBPA).11,15,25 Since in our study debromination in the soil was slower than O-methylation, only two debromination products of TBBPA (triBBPA and diBBPA) were identified in the unplanted soil, while mono- and dimethyl ethers of triBBPA presumably formed via the reduction of the TBBPA ethers (Figure 2). The stimulated transformation pathways of TBBPA in the soil with rice and reed growth are shown with blue and red lines, respectively, in Figure 2. The plants stimulated the anaerobic debromination and the aerobic O-methylation of TBBPA. In both planted soils, TBBPA was completely debrominated to BPA (compound 4) in a stepwise process, via triBBPA, diBBPA, and monoBBPA. While both plants enhanced the formation of MeO-TBBPA (compound 7), rice seedlings stimulated further O-methylation and debromination of MeO-TBBPA to diMeO-TBBPA (compound 8) and MeOtriBBPA (compound 5), respectively (Figure 2), in reactions that may have been carried out by aerobic and anaerobic
Figure 4. Radioactivity in the total plants (A) and in the leaves and roots (B) of rice and reed seedlings grown in soil containing 14CTBBPA, assayed on days 35 and 66 of incubation. Values are the means of three individual experiments ± one standard deviation.
of larger amounts of polar metabolites in the reed rhizosphere (Figure 3A), since plants more readily take up polar than nonpolar compounds.35 A previous study also showed that the accumulation of TBBPA in terrestrial vegetables was plantspecies-specific,36 probably reflecting the microbial community and its activity in the respective rhizosphere. The considerable accumulation of radioactivity detected in the plants was in agreement with the lower amounts of both bound-residue formation (see below) and TBBPA mineralization in the planted than in the unplanted soil (Figure 1). During the incubation, most of the radioactivity that accumulated in the plants was localized to their roots (Figure 4B). 14C radio-imaging of the plants (SI Figure S17) showed the distribution of radioactivity within the roots and in the above-ground parts (stems and leaves) of the plants. At the end of the incubation, 18.4 ± 1.7% and 28.1 ± 5.9% of the initial radioactivity was recovered, respectively, from the roots of the rice and reed seedlings and 1.2 ± 0.2% and 5.0 ± 1.5% from the above-ground parts (Figure 4B). The radioactivity translocation factor (TF), defined as the ratio of radioactivity in the aboveground to that in the below-ground plant tissue, was 0.21 on day 35 in the rice seedlings, but 0.067 and 0.18 on day 66 in the rice and reed seedlings, respectively, indicating: (1) a decreasing TF with increasing plant growth, attributable to the formation of the hydrophobic O-methylated bromoBPAs in the soil during the incubation (Figure 3) and the tendency of F
dx.doi.org/10.1021/es503383h | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Article
an oxic−anoxic interface, such as in wetland soils and paddy rice fields, provides a suitable environment for the rapid removal of TBBPA. The pattern and timing of TBBPA transformation in the submerged soils could be explained by several interconnected pathways. Of these, oxidative skeletal rearrangements and type II ipso-substitution seemed to be responsible for its mineralization. Bacterially mediated O-methylation also played an important role in TBBPA metabolism, resulting in the formation of relatively stable metabolites in the soil. In addition to MeO-TBBPA and diMeO-TBBPA, two metabolites previously reported in freshwater and marine sediments14,39,40 and in soil slurry,13 our study is the first to detect methyl ethers of TBBPA debromination products (MeO-triBBPA and diMeO-triBBPA) in environmental samples. These O-methylated derivatives are more lipophilic and more persistent in the environment than the parent compound.4,14 DiMeO-TBBPA was shown to be toxic to zebra fish embryos,41 suggesting that the formation of O-methylated derivatives poses a risk to the environment and to human health. The accelerated transformation of TBBPA and the reduction in bound-residue formation in planted vs unplanted submerged soil demonstrate the potential of wetland plants, especially reed, in the remediation of TBBPA-contaminated soils. However, before phytoremediation can be effectively implemented, the mechanisms by which plants alter the dynamics of TBBPA, and the amounts of its metabolites in the soil must be better elucidated. Also unclear are the effects of the markedly increased amounts of lipophilic MeO-TBBPA, whose environmental behavior is not clear. In addition, the plants accumulated considerable quantities of uncharacterized residues in their below- and above-ground tissues. The nature of these compounds, including their environmental safety, must be determined in further studies.
these compounds to adsorb onto plant roots, and (2) the greater accumulation of TBBPA and its metabolites in the above-ground parts of reed rather than rice seedlings. In vegetable cabbage, TBBPA accumulation was higher in the roots (2−3 times) than in the shoots, while in radish it was almost the same.36 The chemical nature of the radioactivity that accumulated in the rice and reed seedlings remains to be investigated. Distribution of Bound Residues in Soil. In all treatments, the residual radioactivity bound to the soils increased over time such that by the end of the incubation it accounted for the majority of the radioactivity in the soils (Figure 1). From the initially applied radioactivity in the unplanted soil, 60.8 ± 3.0% was associated with bound (nonextractable) residues (Figure 1A). A previous study showed that the formation of TBBPA-derived bound residues in soil was low (
