In Vivo Metabolism of 2,2′,4,4′-Tetrabromodiphenyl Ether (BDE-47

Article pubs.acs.org/est

In Vivo Metabolism of 2,2′,4,4′-Tetrabromodiphenyl Ether (BDE-47) in Young Whole Pumpkin Plant Jianteng Sun, Jiyan Liu,* Miao Yu, Chang Wang, Yuzhen Sun, Aiqian Zhang, Thanh Wang, Zhen Lei, and Guibin Jiang State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing, 100085, China S Supporting Information *

ABSTRACT: Polybrominated diphenyl ethers (PBDEs) are widely distributed persistent organic pollutants. In vitro and in vivo research using various animal models have shown that PBDEs might be transformed to hydroxylated PBDEs, but there are few studies on in vivo metabolism of PBDEs by intact whole plants. In this research, pumpkin plants (Cucurbita maxima × C. moschata) were hydroponically exposed to 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47). A debromination product (BDE-28) and four hydroxylated metabolites (5-OHBDE-47, 6-OH-BDE-47, 4′-OH-BDE-49, and 4-OH-BDE-42) were detected in different parts of the whole plant. In addition, 4methoxylated-2,2′,3,4′-tetraBDE (4-MeO-BDE-42) was observed as a methoxylation product. Root exudates in solution were found to play an important role in metabolizing BDE-47 to a specific OH-PBDE: 4′-OH-BDE-49. BDE-28 was found to translocate more easily and accumulate in shoots than BDE-47 due to the lower hydrophobicity and molecular weight. The concentration ratio between metabolites and parent compound BDE-47 was lower for OH-PBDEs than that for both BDE-28 and 4-MeO-BDE-42. The metabolism pathway of BDE-47 in young whole plants was proposed in this study. three BDE congeners in maize.27 However, control samples and the purity of stock standards were insufficient. BDE-47 is one of the predominant BDE congeners detected in the environment.28 The corresponding OH-tetraBDEs and MeO-tetraBDEs analogues have also been frequently found in various environmental compartments.29−31 The relationship among the three analogues is still under discussion. Therefore, the metabolism of BDE-47 was studied using young whole pumpkin plants by hydroponic exposure, and a possible metabolism pathway was proposed.

1. INTRODUCTION Polybrominated diphenyl ethers (PBDEs) are manufactured as brominated flame retardants and widely distributed in the environment as persistent, bioaccumulative, and toxic organic pollutants.1−5 It is important to understand the fate of PBDEs in the environment, such as transformation, bioaccumulation, and elimination. Hydroxylated (OH−) and methoxylated (MeO−) PBDEs that have no known anthropogenic source but have been widely found in marine biotic samples (algae, mussel, and fish), abiotic samples (sludge, surface water, snow, and rain), and humans are considered as potential metabolites of PBDEs.6−11 In vivo and in vitro studies focused on the metabolism of PBDEs generally use animal models. Mono-, di-, and even trace tri-OH-BDE metabolites were detected in vivo after rats and mice were exposed to PBDE mixtures or individual PBDE congeners.12−18 In vitro PBDEs exposure studies showed the formation of OH-PBDEs in microsomes of rat and beluga whale (Delphinapterus leucas) and in hepatocytes of humans.19−21 However, there are limited papers on the metabolism of PBDEs in plants. Plants play an important role in the global cycling of various compounds and serve as a food source for higher order organisms. OH- and MeO-PBDEs have been found in plants and soil collected surrounding a seafood processing factory and a seafood market.22 Uptake and translocation of polychlorinated biphenyls (PCBs) in poplar plants have been proven, and hydroxylated metabolites were detected after whole poplar plants exposure to individual CB-3 and CB-77.23−26 Wang et al. investigated the metabolism of © 2013 American Chemical Society

2. EXPERIMENTAL SECTION 2.1. Chemicals and Reagents. Stock standards of PBDEs (50 μg/mL) in isooctane were used, including two mono-BDEs (BDE-1, -3), two di-BDEs (BDE-7, -8), two tri-BDEs (BDE-17, -28), three tetra-BDEs (BDE-42, -47, -49), and two pentaBDEs (BDE-85, -99). Twelve standards of hydroxylated PBDEs (10 μg/mL in acetonitrile for each) were 4′-OH-BDE-17, 2′OH-BDE-28, 3′-OH-BDE-28, 4-OH-BDE-42, 4′-OH-BDE-49, 3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47, 2′-OH-BDE68, 6-OH-BDE-85, 5′-OH-BDE-99, and 6′-OH-BDE-99. Twelve methoxylated PBDEs (10 μg/mL in methanol for each) were 4′-MeO-BDE-17, 2′-MeO-BDE-28, 3′-MeO-BDE28, 4-MeO-BDE-42, 4′-MeO-BDE-49, 3-MeO-BDE-47, 5Received: Revised: Accepted: Published: 3701

January 22, 2013 March 11, 2013 March 19, 2013 March 19, 2013 dx.doi.org/10.1021/es4003263 | Environ. Sci. Technol. 2013, 47, 3701−3707

Environmental Science & Technology

Article

BDE-47 owning to volatilization and possible chemical transformation. The plants were not sterilized to allow investigation on the contribution of microbes to the biotransformation of BDE-47. Thus, corresponding microbe controls are also included. The root exudate controls (three samples) were included mainly to explore the combined roles of root exudates and microbes and were performed as follows: the pumpkin plants were planted in reactors filled with 40 mL of autoclaved water without BDE-47. The whole plants were taken away after 10 days, leaving only solutions that contained roots exudates and microbes. Then, the solutions were spiked with 200 ng of BDE-47 and exposed for another 10 days. Microbe controls (three samples) were set as follows: the unexposed exudates control solutions were used for germiculture. After growth in solid medium for 48 h, the obtained bacterial colonies were transferred to 40 mL of autoclaved water, and 200 ng of BDE-47 was added for 10 days exposure. 2.3. Microbes Isolation, Counting, and Identification in the Exposure System. To evaluate the microbes in the exposure system, the microbes in the exposure system were primarily isolated, counted, and identified. After pumpkins were planted in the reactors for 10 days, a serial 10-fold dilution of 500 μL of hydroponic solution was established in phosphate buffer for bacterial isolation and counting. Aliquots of 50 μL of dilutions were spread uniformly on 100 mm culture dishes containing LB agar medium. After 48 h cultivation in 37 °C, the bacterial population in hydroponic solutions was determined by the dilution method of plate counting. Three types of bacterial colonies with different colors and forms were observed and collected for Gram stain identification following the instruction of the Gram stain kit. No bacterial contamination occurred in the unplanted control group during the experiment. The concentration of microbes was about 1.5 × 106 CFU (colony-forming unit)/mL in the exposure group reactors and comparable to that of untreated pumpkin plant controls. It was therefore shown that the bacteria in the system were mainly from the plants. Three typical colonies of bacteria were identified by the Gram stain method. Type 1 was a Gram-positive corynebacteria, and the colony was white in color. Type 2 was a Gram-positive bacillus with a yellowish colony. Type 3 was a Gram-negative bacillus, and the colony secreted a green secondary metabolite in the agar medium. The microbial colonies were scraped and mixed with the hydroponic solution when setting microbe controls. The concentration of microbes in these controls was determined by spectrophotometry at an absorption wavelength of 600 nm. 2.4. Sampling and Sample Preparation. The root, stem, and shoot of whole plants and the hydroponic solutions of the exposure group and controls (Figure 1) were sampled separately after exposure. Wet plant samples were gently dried on Kimwipes (Kimberly−Clark, Roswell, GA, USA) and weighed at fresh weight, thereafter frozen at −50 °C overnight, and then, freeze-dried for further treatment. The extraction and cleanup procedures for PBDEs, MeOPBDEs, and OH-PBDEs were modified from a previously developed method.33 In brief, all samples were spiked with three surrogate internal standards: 13C-6-OH-BDE-47, 13C-6MeO-BDE-47, and 13C-BDE-99 (10 ng for each). The freezedried solid samples (root, stem, shoot) were homogenized and extracted with 2-propanol (0.2 mL) and hexane/MTBE (1:1, v/v) (1 mL) using Tissuelyser (QIAGEN, Hilden, Germany). The frequency and time were set 30 Hz and 1 min, respectively.

MeO-BDE-47, 6-MeO-BDE-47, 2′-MeO-BDE-68, 6-MeOBDE-85, 5′-MeO-BDE-99, and 6′-MeO-BDE-99. The stock standards were purchased from AccuStandard (New Haven, CT, USA). Surrogate standards (13C-6-OH-BDE47, 13C-6MeO-BDE47, and 13C-BDE99) were purchased from Wellington (Guelph, ON, Canada). Working solutions of BDE-47 exposure were prepared at 10 μg/mL in acetone. The working solutions of other PBDEs (in hexane), MeO-PBDEs (in hexane), and OH-PBDEs (in acetonitrile) were prepared at 1 μg/mL for quantitative analysis. Silica gel (100−200 mesh size) (Merck, Darmstadt, Germany) was activated at 140 °C for 7 h. Acid silica gel was prepared by combining 56 g of activated silica with 44 g of concentrated H 2SO4. Water-impregnated silica gel was prepared by 95 g of activated silica with 5 g of deionized water. Acetonitrile (HPLC grade), methyl tert-butyl ether (MTBE) (HPLC grade), 2-propanol (HPLC grade), acetone (pesticide grade), hexane (pesticide grade), and dichloromethane (DCM) (pesticide grade) were purchased from J. T. Baker (Phillipsburg, NJ, USA). Deionized water (18.2 MΩ) was obtained from an ultrapure water purification system (Barnstead International, Dubuque, IA, USA). The 100 mm culture dish containing Luria−Bertani (LB) agar medium and the Gram stain kit were purchased from Landbridge Technology Co., China. All other chemicals and reagents used were of analytical reagent grade or higher purity. 2.2. Hydroponic Exposure. Pumpkin (Cucurbita maxima × C. moschata) was purchased from Taigu Yinong Seed Co., Ltd., Shanxi, China. Seeds of pumpkin were germinated on a bed of sterilized perlite with deionized water. When the seedlings were 3−4 cm high, they were transferred into an incubator without perlite and used for exposure after growing to 8−10 cm high. The reactors were 60 mL brown screw top glass conical flasks. The seedlings were not sterilized before exposure, but all the reactors and deionized water were autoclaved to prevent any possible introduction of foreign microorganisms. Each exposure group reactor was filled with 40 mL of deionized water and 200 ng of BDE-47 (dissolved in 20 μL of acetone), giving an initial exposure concentration of 5 ng/mL, which is comparable to the levels found in some contaminated areas such as soil from electronic waste disposal sites.32 Each reactor was planted with one pumpkin plant. The whole pumpkin plants were rinsed by autoclaved deionized water before planting into the reactors. The mouth of the reactors were covered with aluminum foil and sealed with parafilm. All conical flasks were wrapped with aluminum foil to keep the roots in darkness and also to prevent photolysis of BDE-47. All of these procedures were performed in a laminar flow hood. Four reactors acted as one exposure sample, and there were five parallel samples in each exposure group. The exposure time was 10 days. The temperature during plant growth and exposure was maintained at 22 ± 2 °C, and the photoperiod was set at 16 h/day under fluorescent lighting with a light intensity of 250 μmol m−2 s−1. Transpiration of each whole plant was determined daily by weighing the reactors. Four control groups were designed and carried out simultaneously. Untreated pumpkin plant controls (two samples) without BDE-47 but with 20 μL of acetone in the hydroponic solutions were used to control possible crosscontamination due to volatilization of PBDEs during the experiment. Unplanted controls (two samples) with the same amount of BDE-47 in solutions were used to control the loss of 3702

dx.doi.org/10.1021/es4003263 | Environ. Sci. Technol. 2013, 47, 3701−3707

Environmental Science & Technology

Article

an electron impact ion source (Agilent Technologies, Palo Alto, CA, USA). Selected ion monitoring mode was used for quantification. Compounds were separated using a DB-5 MS (J & W Scientific, Folsom, CA, USA) fused silica capillary column (30 m, 0.25 mm i.d., 0.1 μm film thickness). Identification and quantification of OH-PBDEs was carried out using an Agilent 1290 Series LC system coupled with an Agilent 6460 triple quadrupole MS/MS system. Reverse-phase separation was achieved using a C18 column (100 mm × 2.1 mm, 2.2 μm particle size, Thermo Fisher Scientific, Waltham, MA, USA). All parameters for GC-MS and LC-MS/MS analyses were the same as those optimized in our former publication.33 Figure 1. Scheme of the hydroponic exposure reactor and sampling.

3. RESULTS AND DISCUSSION 3.1. Uptake, Translocation, and Debromination of BDE-47. The purity of the BDE-47 standard was checked, and no target OH-PBDE, MeO-PBDE, or other PBDEs were detected as impurities. As shown in Table 1, the distribution of BDE-47 in different compartments of plants was investigated to elucidate the uptake and translocation of BDE-47 in pumpkins. Results showed that the largest portions of BDE-47 (over 74% of the initial amount) were accumulated by the roots after 10 days exposure. The root concentration factor (RCF) was 883. The average mass of BDE-47 decreased progressively in the stem, solution, and shoots, with only about 1% of the initial amount of BDE-47 translocating to the shoots. Mass balance was calculated, and the recovered total BDE-47 was 93.4% ± 12.6% of initial BDE-47 for exposure reactors and 95.6% ± 12.2% for unplanted controls. No BDE-47 was observed in untreated pumpkin controls, suggesting that there was no BDE47 cross-contamination between reactors. The possible debromination of PBDEs by whole plants was also investigated. BDE-209 has previously been reported to be transformed to lower brominated PBDEs in a plant−soil system.34 In the present research, a debromination product, 2,4,4′-triBDE (BDE-28), was identified in different tissues of pumpkin but was not found in the hydroponic solutions. The mass distribution of BDE-28 in each part of the whole plant was quite different from BDE-47. The mass of BDE-28 was found to be largest in the shoot (reaching 3060 ± 480 pg), less in the

The organic extract was transferred to another clean bottle after centrifugation. This extraction procedure was repeated three times. The hydroponic solutions (160 mL) were added with 2 mL of 2-propanol and then extracted twice with 50 mL of hexane/MTBE (1:1, v/v) under vigorous shaking for 30 min. All extracts were evaporated to dryness and redissolved in 20 mL of DCM. Acid silica gel (10 g) was added, and the mixture was shaken vigorously until the extracts were colorless. The acid silica gel was removed through an anhydrous Na2SO4 column (8 g) with DCM (40 mL) as eluent. The collected extracts were concentrated to dryness using a rotary evaporator and redissolved with 1 mL of hexane. Then, a column packed with 5 g of silica deactivated with 5% water (w/w) was used for fractionation. After prewashing with 30 mL of hexane, the extract was loaded on to the column. First, 20% DCM in hexane (60 mL) was applied to coelute PBDEs and MeOPBDEs. Second, DCM (70 mL) was used to elute OH-PBDEs. Fractions of PBDEs and MeO-PBDEs were concentrated to 200 μL prior to gas chromatography−mass spectrometry (GCMS) analysis. Fractions of OH-PBDEs were evaporated to dryness and solvent exchanged with 200 μL of acetonitrile for liquid chromatography−mass spectrometry/mass spectrometry (LC-MS/MS) analysis. 2.5. Instrumentation. Analysis of PBDEs and MeOPBDEs was performed on an Agilent 6890N gas chromatograph coupled with a 5975C mass spectrometer detector using

Table 1. Detection of BDE-47 and Metabolites after Whole Pumpkin Plants Being Hydroponically Exposed to BDE-47 for 10 Days reactor

amount of samplea

sample type

BDE-47 (pg/g)

BDE-28 (pg/g)

4-MeO-BDE42 (pg/g)

4-OH-BDE42 (pg/g)

4′-OH-BDE49 (pg/g)

5-OH-BDE47 (pg/g)

6-OH-BDE47 (pg/g)

exposed pumpkin

5

root

327 400 ± 53 900b

1005 ± 24

769 ± 203

6±5

27 ± 7

54 ± 26

19 ± 6

2

stem shoot solution root

69 000 ± 5320 2593 ± 3550 371 ± 48 n.d.

416 ± 127 850 ± 194 n.d. n.d.

328 ± 29 n.d.c n.d. n.d.

4±3 n.d. n.d. n.d.

16 ± 8 n.d. 0.05 ± 0.01 n.d.

22 ± 5 4±2 0.21 ± 0.05 n.d.

17 ± 7 3±3 0.16 ± 0.07 n.d.

2

stem shoot solution solution

n.d. n.d. n.d. 4847d

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

3

solution

4680 ± 290

n.d.

n.d.

n.d.

1 ± 0.14

n.d.

n.d.

3

solution

4914 ± 224

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

untreated pumpkin control

unplanted control root exudate control microbe control a

One sample consisted of four reactors. bMean value ± standard deviation (n ≥ 3). cn.d. = none detectable. dMean value. 3703

dx.doi.org/10.1021/es4003263 | Environ. Sci. Technol. 2013, 47, 3701−3707

Environmental Science & Technology

Article

Figure 2. Representative chromatograms of 6-OH-BDE-47, 5-OH-BDE-47, 4-OH-BDE-42, and 4′-OH-BDE-49 in a root sample, detected by LCMS/MS.

root (1730 ± 378 pg), and least in the stem (740 ± 190 pg), indicating a significant translocation of BDE-28 from roots to shoot. By taking into account the mass of the plant tissue, the concentrations of BDE-28 in tissues were ranked as root (1005 ± 240 pg/g fresh weight (fw)) > shoot (850 ± 194 pg/g fw) > stem (416 ± 127 pg/g fw). The concentration ratio between BDE-28 and the initial exposed BDE-47 was 0.7% for all tissues. No debromination products were observed in controls, confirming that BDE-28 was derived from the debromination of BDE-47 by pumpkin plants rather than any other sources. The ability of BDE-47 and BDE-28 to be translocated through the plant could be related to their hydrophobicity (log Kow) and molecular weight, where more hydrophobic congeners are usually translocated with more difficulties. BDE-28, having lower hydrophobicity and molecular weight than BDE-47, can be easier translocated within the pumpkin plant. The transpiration stream concentration factor (TSCF) was calculated according to the description by Briggs et al.35 The values of the TSCF were 0.0008 and 0.00003 for BDE-28 and BDE-47, respectively. As a result, a larger proportion of BDE-28 accumulated in the shoots of pumpkin. 3.2. Hydroxylated Metabolites of BDE-47. The hydroxylated metabolites of BDE-47 in different plants tissues were analyzed. Results show that four OH-PBDEs, 5-OH-BDE47, 6-OH-BDE-47, 4′-OH-BDE-49, and 4-OH-BDE-42, were identified in exposed whole pumpkin plants (Figure 2). The concentrations of OH-BDEs in different compartments of the exposure reactors and the controls are listed in Table 1. 5-OHBDE-47 was the predominant hydroxylated metabolite of BDE47, presenting the highest concentration of 22 ± 6 pg/g fw in the whole plants, followed by 6-OH-BDE-47 with a concentration of 13 ± 1 pg/g fw. They were detected in all plant tissues with concentrations decreasing from the roots to shoots, while 4′-OH-BDE-49 (10 ± 4 pg/g fw) and 4-OHBDE-42 (3 ± 2 pg/g fw) were only detected in the root and

stem samples at lower concentrations. The results indicated that pumpkin plants can metabolize BDE-47 into hydroxylated products and translocation of OH-PBDEs could occur in the plant. Among the whole plants, the highest concentration of ΣOH-PBDEs was detected in the roots, reaching 106 ± 39 pg/ g fw, which is about 180 times greater than that in the solutions. The concentration ratios between hydroxylated metabolites 5OH-BDE-47, 6-OH-BDE-47, 4′-OH-BDE-49, and 4-OH-BDE42 and initial exposure levels of BDE-47 were approximately 0.023%, 0.013%, 0.022%, and 0.006%, respectively. The ratios were similar to in vivo reports on BDE-47 biotransformation to OH-BDEs in rats and mice.18,36 However, the ratios of OHBDE metabolites were much lower than those of debrominated metabolites of BDE-28 detected in this work. Formation of hydroxylated metabolites was not detected in untreated pumpkin controls and unplanted controls, which indicated that there was no chemical transformation and no OH-PBDEs cross-contamination between the reactors. OHPBDEs were also not detected in microbe controls, which suggests that root-associated microbes have no significant effect on the metabolism of BDE-47 in this study. However, in solutions of root exudate controls, 4′-OH-BDE-49 was identified at high total mass of 87 ± 23 pg, which is comparable to the sum of 4′-OH-BDE-49 detected in the root, stem, shoot, and solution of exposed pumpkins. Photodegradation of BDE47 was minimized by wrapping the exposure reactors with aluminum foil and further verified by unplanted controls. The result showed that root exudates that generally contain amino acids, organic acids, enzymes, and many other chemicals played an important role in metabolizing BDE-47 to 4′-OH-BDE-49. 3.3. Methoxylated Metabolites of BDE-47. To our knowledge, MeO-PBDEs metabolites have not previously been reported during in vivo and in vitro metabolism studies of PBDEs using animals and cell cultures. However, a MeO-BDE congener, 4-methoxylated-2,2′,3,4′-tetraBDE (4-MeO-BDE3704

dx.doi.org/10.1021/es4003263 | Environ. Sci. Technol. 2013, 47, 3701−3707

Environmental Science & Technology

Article

Figure 3. Scheme of observed metabolism pathways of BDE-47 within pumpkin. Dotted arrows with question marks are the hypothetical pathways that are not demonstrated.

among humans, mice, and plants may be the result of species differences in cytochrome P450 enzyme expression. The different selectivity in oxidation of the halogenated phenyl ring of different P450 subfamilies could be a possible reason.40 In order to explore the hydroxylation pathway of BDE-47, two tetra-BDEs, BDE-42 and BDE-49, were also analyzed but were not found in any of the samples. This suggested the hydroxylation from BDE-47 proceeded without a rearrangement of bromine atoms of parent BDE-47. Another potential metabolite, 2′-OH-BDE-68, was also not found, suggesting that this compound could not be formed from BDE-47 in pumpkins. OH-tri-BDEs and OH-penta-BDEs were also not detected. 4′-OH-BDE-49 was present at high relative mass in root exudate controls, suggesting that BDE-47 tends to be transformed to 4′-OH-BDE-49 under the function of root exudates, which is known to contain a numerous variety of chemicals. The extensive presence of MeO-BDEs in wildlife tissues has prompted a hypothesis that PBDEs may undergo methoxylation. Haglund et al. proposed that MeO-BDEs could be formed via methylation of either PBDEs or OH-BDEs by intestinal microflora or microorganisms in sediments.41 However, MeO-BDEs have not been observed to be formed in controlled exposure studies with PBDEs either in vitro or in vivo using animal models.21,42,43 The methoxylated product was clearly observed in this study, although its formation mechanism in plants is not understood. Both direct methoxylation of BDE-47 and a two-step process with PBDEs hydroxylation followed by methylation may result in MeO-PBDEs detection in young whole pumpkin plant. In addition, since the methylation from OH-PBDE to MeO-PBDE and demethylation from MeO-PBDEs to OH-PBDEs have both been observed in in vivo exposure using Japanese medaka (Oryzias latipes),43 the simultaneous observation of 4-OHBDE-42 and 4-MeO-BDE-42 in pumpkin also suggested potential methylation or demethylation between both compounds, although direct experimental evidence is still needed to support this assumption. Interconversion preference of MeO-PBDEs and OH-PBDEs was then evaluated using structure-based theoretical calculation methods (as shown in the Supporting Information). A simplified reference system was adopted, namely, the reaction of OH-PBDEs with iodomethane and its reverse one, since

42), was identified in pumpkin plants after being exposed to BDE-47 in this study. The mean concentration was 769 ± 203 pg/g fw in the roots and 328 ± 29 pg/g fw in the stems. The concentration ratio between 4-MeO-BDE-42 and exposed BDE-47 was 0.24%, which is higher than that of OH-PBDEs but lower than that of BDE-28. 4-MeO-BDE-42 was not found in the shoots and solutions in exposed pumpkin reactors, which may be due to its higher hydrophobicity. The TSCF of 4-MeOBDE-42 was 0.00002 and was lower than those of BDE-47 and BDE-28. There were no MeO-PBDEs detected in any controls, which indicated that pumpkin plants play a key role in the formation of a MeO-PBDE congener from BDE-47. 3.4. Metabolism Pathways. Information on the pathways and mechanisms of in vivo metabolism in plants is still scarce. The proposed metabolism pathways of BDE-47 in pumpkin are shown in Figure 3. The hypothesized debromination pathways in our study would suggest that oxidative debromination occurred via the removal of a bromine atom in the ortho position. The debromination products at para positions (BDE8 and BDE-17) were not detected, since the Br atoms at the ortho positions may depart more easily to form BDE-28, which was consistent with other reports.37 Several lines of evidence suggest that biotransformation of PBDEs to OH-PBDEs is mediated by cytochrome P450 enzymes.16,17 There are two possible mechanisms explaining the formation of OH-PBDEs from BDE-47. One involves the formation of epoxide intermediates under the function of enzymes and then transformation to different OH-PBDEs via a 1,2-shift of bromine.38 As such, 4-OH-BDE-42 and 4′-OHBDE-49 might have been formed through a 1,2 bromine shift from BDE-47. The other pathway is the formation of OHPBDEs (i.e., 5-OH-BDE-47 and 6-OH-BDE-47) from BDE-47 via direct insertion of the hydroxyl group to a biphenyl. Qiu et al.17 studied the exposure of mice to BDE-47, and 4-OH-BDE42 was found to be the most dominant metabolite, followed by 3-OH-BDE-47, 4′-OH-BDE-49, and 6-OH-BDE-47. In another report on OH-PBDEs in human blood samples,39 the metabolite profile is very different. 5-OH-BDE-47 and 6-OHBDE-47 were more abundant, followed by 3-OH-BDE-47, 4′OH-BDE-49, and 4-OH-BDE-42 at much lower concentrations. Surprisingly, the metabolite profile between humans and plants is more similar except for 3-OH-BDE-47, which was not detected in pumpkins. The difference in the metabolic profile 3705

dx.doi.org/10.1021/es4003263 | Environ. Sci. Technol. 2013, 47, 3701−3707

Environmental Science & Technology

Article

Notes

there is no information on methyl donor and enzyme characteristics available in the present study. In accordance with the methylation of phenol to anisole with a Gibb’s free energy (ΔrG) of 8.24 kcal/mol, the OH-PBDEs methylation was relatively sluggish in the reference system, and the calculated ΔrG data for 4′-OH-BDE-49, 5-OH-BDE-47, 6OH-BDE-47, and 4-OH-BDE-42 were 7.91, 7.49, 7.72, and 8.23 kcal/mol, individually. The standard deviation was 0.31 kcal/mol, and the coefficient of variance was 4.0%. The forward reaction was thermodynamically unfavorable at 25 °C and 1 atm, while the positive value of ΔrG lower than 20 kcal/mol indicates that methylation of OH-PBDEs may be feasible with the right catalyst and necessary conditions. There were few differences among methylation ΔrG data for the detected OHPBDEs due to their extremely similar structure. All the detectable OH-PBDEs products have a 2,4-brominated phenyl ring and another phenyl ring with one hydroxyl group and two bromine atoms. Only the hydroxyl group of 4-OH-BDE-42 has consecutive unsubstituted meta and ortho positions. Clearly, the hydroxyl group in 4-OH-BDE-42 has the least steric hindrance compared with those in other products when interacting with certain methyltransferases, leading to only 4-MeO-BDE-42 being observed after 10 day exposure. At the same time, OH-PBDEs may result from the demethylation of MeO-PBDEs if MeO-PBDEs are a direct product of methoxylation of PBDEs. According to the reference system, the reversed reaction, demethylation of MeO-PBDEs, has a negative change in ΔrG at 298K and 1 atm. The calculated ΔrG value of 4-MeO-BDE-42 was −8.23 kcal/mol. The demethylation reactions of MeO-PBDEs with hydrogen iodine were spontaneous, but it may not be easily achieved due to a rather lower |ΔrG| value and potential kinetic factors. The demethylation of anisole also had a negative ΔrG of 8.24 kcal/ mol and could not be observed until the system was treated by microwave heating with neat iodotrimethylsilane. The bond dissociation energy of the O−C bond of the methoxyl group in 4-MeO-BDE-42 is 56.37 kcal/mol, indicating the easy dissociation of the O−C bond even without hydrogen iodine. Thus, conversion of 4-MeO-BDE-42 to 4-OH-BDE-42 was also possible in young pumpkin. In summary, a debromination product (BDE-28), a methoxylation product (4-MeO-BDE-42), and four hydroxylated metabolites (5-OH-BDE-47, 6-OH-BDE-47, 4′-OH-BDE49, and 4-OH-BDE-42) were identified in different parts of young whole pumpkin plants being hydroponically exposed to BDE-47. The concentration ratio between metabolites and the parent compound was highest for BDE-28, lower for MeOBDE, and lowest for OH-BDEs. The metabolism pathway of BDE-47 in pumpkin plants was proposed for the first time in this study.

■

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was jointly supported by the National Basic Research Program of China (no. 2009CB421603), the National Natural Science Foundation of China (nos. 21277153 and 21077126), and Important Directional Item of the Chinese Academy of Sciences (nos. KZCX2-EW-QN409 and KZCX2-YW-BR-25).

■

(1) Hites, R. A. Polybrominated diphenyl ethers in the environment and in people: A meta-analysis of concentrations. Environ. Sci. Technol. 2004, 38 (4), 945−956. (2) Law, R. J.; Allchin, C. R.; de Boer, J.; Covaci, A.; Herzke, D.; Lepom, P.; Morris, S.; Tronczynski, J.; de Wit, C. A. Levels and trends of brominated flame retardants in the European environment. Chemosphere 2006, 64 (2), 187−208. (3) Darnerud, P. O.; Eriksen, G. S.; Johannesson, T.; Larsen, P. B.; Viluksela, M. Polybrominated diphenyl ethers: Occurrence, dietary exposure, and toxicology. Environ. Health Perspect. 2001, 109, 49−68. (4) Law, R. J.; Alaee, M.; Allchin, C. R.; Boon, J. P.; Lebeuf, M.; Lepom, P.; Stern, G. A. Levels and trends of polybrominated diphenylethers and other brominated flame retardants in wildlife. Environ. Int. 2003, 29 (6), 757−770. (5) Ross, P. S.; Couillard, C. M.; Ikonomou, M. G.; Johannessen, S. C.; Lebeuf, M.; Macdonald, R. W.; Tomy, G. T. Large and growing environmental reservoirs of deca-BDE present an emerging health risk for fish and marine mammals. Mar. Pollut. Bull. 2009, 58 (1), 7−10. (6) Marsh, G.; Athanasiadou, M.; Bergman, A.; Asplund, L. Identification of hydroxylated and methoxylated polybrominated diphenyl ethers in Baltic Sea salmon (Salmo salar) blood. Environ. Sci. Technol. 2004, 38 (1), 10−18. (7) Ueno, D.; Darling, C.; Alaee, M.; Pacepavicius, G.; Teixeira, C.; Campbell, L.; Letcher, R. J.; Bergman, A.; Marsh, G.; Muir, D. Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) in the abiotic environment: Surface water and precipitation from Ontario, Canada. Environ. Sci. Technol. 2008, 42 (5), 1657−1664. (8) Hakk, H.; Letcher, R. J. Metabolism in the toxicokinetics and fate of brominated flame retardantsA review. Environ. Int. 2003, 29 (6), 801−828. (9) Malmvarn, A.; Marsh, G.; Kautsky, L.; Athanasiadou, M.; Bergman, A.; Asplund, L. Hydroxylated and methoxylated brominated diphenyl ethers in the red algae Ceramium tenuicorne and blue mussels from the Baltic Sea. Environ. Sci. Technol. 2005, 39 (9), 2990−2997. (10) Lacorte, S.; Ikonomou, M. G. Occurrence and congener specific profiles of polybrominated diphenyl ethers and their hydroxylated and methoxylated derivatives in breast milk from Catalonia. Chemosphere 2009, 74 (3), 412−420. (11) Sun, J. T.; Liu, J. Y.; Liu, Q.; Ruan, T.; Yu, M.; Wang, Y. W.; Wang, T.; Jiang, G. B. Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) in biosolids from municipal wastewater treatment plants in China. Chemosphere 2013, 90, 2388−2395. (12) Chen, L. J.; Lebetkin, E. H.; Sanders, J. M.; Burka, L. T. Metabolism and disposition of 2,2′,4,4′,5-pentabromodiphenyl ether (BDE99) following a single or repeated administration to rats or mice. Xenobiotica 2006, 36 (6), 515−534. (13) Hakk, H.; Larsen, G.; Klasson-Wehler, E. Tissue disposition, excretion and metabolism of 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99) in the male Sprague-Dawley rat. Xenobiotica 2002, 32 (5), 369−382. (14) Hakk, H.; Huwe, J.; Low, M.; Rutherford, D.; Larsen, G. Tissue disposition, excretion and metabolism of 2,2′,4,4′,6-pentabromodiphenyl ether (BDE-100) in male Sprague-Dawley rats. Xenobiotica 2006, 36 (1), 79−94. (15) Staskal, D. F.; Hakk, H.; Bauer, D.; Diliberto, J. J.; Birnbaum, L. S. Toxicokinetics of polybrominated diphenyl ether congeners 47, 99, 100, and 153 in mice. Toxicol. Sci. 2006, 94 (1), 28−37.

ASSOCIATED CONTENT

S Supporting Information *

Details of structure-based theoretical calculation methods. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 10 62849334; fax: +86 10 62849339; e-mail: [email protected]. 3706

dx.doi.org/10.1021/es4003263 | Environ. Sci. Technol. 2013, 47, 3701−3707

Environmental Science & Technology

Article

and sediment from an electronic waste recycling facility. Chemosphere 2005, 60 (6), 810−816. (33) Sun, J. T.; Liu, J. Y.; Liu, Q.; Qu, G. B.; Ruan, T.; Jiang, G. B. Sample preparation method for the speciation of polybrominated diphenyl ethers and their methoxylated and hydroxylated analogues in diverse environmental matrices. Talanta 2012, 88 (0), 669−676. (34) Huang, H. L.; Zhang, S. Z.; Christie, P.; Wang, S.; Xie, M. Behavior of decabromodiphenyl ether (BDE-209) in the soil−plant system: Uptake, translocation, and metabolism in plants and dissipation in soil. Environ. Sci. Technol. 2010, 44 (2), 663−667. (35) Briggs, G. G.; Bromilow, R. H.; Evans, A. A. Relationships between lipophilicity and root uptake and translocation of nonionised chemicals by barley. Pestic. Sci. 1982, 13, 495−504. (36) Hamers, T.; Kamstra, J. H.; Sonneveld, E.; Murk, A. J.; Visser, T. J.; Van Velzen, M. J. M.; Brouwer, A.; Bergman, A. Biotransformation of brominated flame retardants into potentially endocrine-disrupting metabolites, with special attention to 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47). Mol. Nutr. Food Res. 2008, 52 (2), 284−298. (37) Wang, S.; Zhang, S. Z.; Huang, H. L.; Zhao, M. M.; Lv, J. T. Uptake, translocation and metabolism of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in maize (Zea mays L.). Chemosphere 2011, 85 (3), 379−385. (38) Jerina, D. M.; Daly, J. W. Arene oxides: A new aspect of drug metabolism. Science 1974, 185 (4151), 573−582. (39) Qiu, X. H.; Bigsby, R. M.; Hites, R. A. Hydroxylated metabolites of polybrominated diphenyl ethers in human blood samples from the United States. Environ. Health Perspect. 2009, 117 (1), 93−98. (40) Bogaards, J. J. P.; Vanommen, B.; Wolf, C. R.; Vanbladeren, P. J. Human cytochrome-P450 enzyme selectivities in the oxidation of chlorinated benzenes. Toxicol. Appl. Pharmacol. 1995, 132 (1), 44−52. (41) Haglund, P. S.; Zook, D. R.; Buser, H. R.; Hu, J. W. Identification and quantification of polybrominated diphenyl ethers and methoxy-polybrominated diphenyl ethers in Baltic biota. Environ. Sci. Technol. 1997, 31 (11), 3281−3287. (42) Wan, Y.; Wiseman, S.; Chang, H.; Zhang, X. W.; Jones, P. D.; Hecker, M.; Kannan, K.; Tanabe, S.; Hu, J. Y.; Lam, M. H. W.; Giesy, J. P. Origin of hydroxylated brominated diphenyl ethers: Natural compounds or man-made flame retardants. Environ. Sci. Technol. 2009, 43 (19), 7536−7542. (43) Wan, Y.; Liu, F. Y.; Wiseman, S.; Zhang, X. W.; Chang, H.; Hecker, M.; Jones, P. D.; Lam, M. H. W.; Giesy, J. P. Interconversion of hydroxylated and methoxylated polybrominated diphenyl ethers in Japanese medaka. Environ. Sci. Technol. 2010, 44 (22), 8729−8735.

(16) Hakk, H.; Huwe, J. K.; Larsen, G. L. Absorption, distribution, metabolism and excretion (ADME) study with 2,2′,4,4′,5,6′hexabromodiphenyl ether (BDE-154) in male Sprague-Dawley rats. Xenobiotica 2009, 39 (1), 46−56. (17) Qiu, X. H.; Mercado-Feliciano, M.; Bigsby, R. M.; Hites, R. A. Measurement of polybrominated diphenyl ethers and metabolites in mouse plasma after exposure to a commercial pentabromodiphenyl ether mixture. Environ. Health Perspect. 2007, 115 (7), 1052−1058. (18) Malmberg, T.; Athanasiadou, M.; Marsh, G.; Brandt, I.; Bergmant, A. Identification of hydroxylated polybrominated diphenyl ether metabolites in blood plasma from polybrominated diphenyl ether exposed rats. Environ. Sci. Technol. 2005, 39 (14), 5342−5348. (19) Stapleton, H. M.; Kelly, S. M.; Pei, R.; Letcher, R. J.; Gunsch, C. Metabolism of polybrominated diphenyl ethers (PBDEs) by human hepatocytes in vitro. Environ. Health Perspect. 2009, 117 (2), 197−202. (20) Erratico, C. A.; Szeitz, A.; Bandiera, S. M. Validation of a novel in vitro assay using ultra performance liquid chromatography-mass spectrometry (UPLC/MS) to detect and quantify hydroxylated metabolites of BDE-99 in rat liver microsomes. J. Chromatogr., B 2010, 878 (19), 1562−1568. (21) McKinney, M. A.; de Guise, S.; Martineau, D.; Beland, P.; Arukwe, A.; Letcher, R. J. Biotransformation of polybrominated diphenyl ethers and polychlorinated biphenyls in beluga whale (Delphinapterus leucas) and rat mammalian model using an in vitro hepatic microsomal assay. Aquat. Toxicol 2006, 77 (1), 87−97. (22) Sun, J. T.; Liu, J. Y.; Liu, Y. W.; Jiang, G. B. Levels and distribution of methoxylated and hydroxylated polybrominated diphenyl ethers in plant and soil samples surrounding a seafood processing factory and a seafood market. Environ. Pollut. 2013, 176, 100−105. (23) Zhai, G. S.; Lehmler, F. J.; Schnoor, J. L. Identification of hydroxylated metabolites of 3,3′,4,4′-tetrachlorobiphenyl and metabolic pathway in whole poplar plants. Chemosphere 2010, 81 (4), 523− 528. (24) Zhai, G. S.; Lehmler, H. J.; Schnoor, J. L. Hydroxylated metabolites of 4-monochlorobiphenyl and its metabolic pathway in whole poplar plants. Environ. Sci. Technol. 2010, 44 (10), 3901−3907. (25) Liu, J. Y.; Schnoor, J. L. Uptake and translocation of lesserchlorinated polychlorinated biphenyls (PCBs) in whole hybrid poplar plants after hydroponic exposure. Chemosphere 2008, 73 (10), 1608− 1616. (26) Liu, J. Y.; Hu, D. F.; Jiang, G. B.; Schnoor, J. L. In vivo biotransformation of 3,3′,4,4′-tetrachlorobiphenyl by whole plants− poplars and switchgrass. Environ. Sci. Technol. 2009, 43 (19), 7503− 7509. (27) Wang, S.; Zhang, S. Z.; Huang, H. L.; Lu, A. X.; Ping, H. Debrominated, hydroxylated and methoxylated metabolism in maize (Zea mays L.) exposed to lesser polybrominated diphenyl ethers (PBDEs). Chemosphere 2012, 89 (11), 1295−1301. (28) Wang, Y. W.; Jiang, G. B.; Lam, P. K. S.; Li, A. Polybrominated diphenyl ether in the East Asian environment: A critical review. Environ. Int. 2007, 33 (7), 963−973. (29) Routti, H.; Letcher, R. J.; Chu, S. G.; Van Bavel, B.; Gabrielsen, G. W. Polybrominated diphenyl ethers and their hydroxylated analogues in ringed seals (Phoca hispida) from Svalbard and the Baltic Sea. Environ. Sci. Technol. 2009, 43 (10), 3494−3499. (30) Verreault, J.; Gabrielsen, G. V.; Chu, S. G.; Muir, D. C. G.; Andersen, M.; Hamaed, A.; Letcher, R. J. Flame retardants and methoxylated and hydroxylated polybrominated diphenyl ethers in two Norwegian Arctic top predators: Glaucous gulls and polar bears. Environ. Sci. Technol. 2005, 39 (16), 6021−6028. (31) Valters, K.; Li, H. X.; Alaee, M.; D’Sa, I.; Marsh, G.; Bergman, A.; Letcher, R. J. Polybrominated diphenyl ethers and hydroxylated and methoxylated brominated and chlorinated analogues in the plasma of fish from the Detroit River. Environ. Sci. Technol. 2005, 39 (15), 5612−5619. (32) Wang, D. L.; Cai, Z. W.; Jiang, G. B.; Leung, A.; Wong, M. H.; Wong, W. K. Determination of polybrominated diphenyl ethers in soil 3707

dx.doi.org/10.1021/es4003263 | Environ. Sci. Technol. 2013, 47, 3701−3707