Letter pubs.acs.org/journal/estlcu
Reciprocal Transformation between Hydroxylated and Methoxylated Polybrominated Diphenyl Ethers in Young Whole Pumpkin Plants Jianteng Sun,†,‡ Jiyan Liu,*,† Yanwei Liu,† Miao Yu,† 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 ‡ Department of Environmental Science, Zhejiang University, Hangzhou, Zhejiang 310058, China S Supporting Information *
ABSTRACT: Relationships among polybrominated diphenyl ethers (PBDEs), hydroxylated PBDEs (OH-PBDEs), and methoxylated PBDEs (MeO-PBDEs) have attracted considerable scientific interest. However, few studies have focused on the in vivo metabolism of these compounds by intact whole plants. In this work, interconversion between OH-tetraBDEs and MeO-tetraBDEs in young pumpkins was experimentally proven. Conversion ratios were higher for pumpkins exposed to OH-BDEs than for those exposed to MeO-BDEs. Twelve PBDE analogues showed different metabolic potential. The largest biotransformation ratio (12.4%) was observed for the transformation from 4-OH-BDE-42 to 4-MeO-BDE-42. The lowest interconversion ratios were found between 2′-OHBDE-68 and 2′-MeO-BDE-68. Transformation products were mainly found in roots. Fewer metabolites were detected in stems, while no metabolites were detected in shoots. Root exudates were found to make only a small contribution to the conversion between OH- and MeO-PBDEs. This is the first study to demonstrate the reciprocal transformation of OHand MeO-PBDEs in plants.
BDE-68 are produced naturally in the marine environment.10 OH-PBDEs were also reported as metabolites of PBDEs in many in vivo and in vitro studies.11−15 Recently, Wan et al. found the interconversion of 6-OH-BDE-47 and 6-MeO-BDE47 in Japanese medaka.16 However, there are only a few studies that have considered the metabolism and fate of OH-PBDEs and MeO-PBDEs in plants. Plants serve as a food source for higher-order organisms and play an important role in the metabolism of organic contaminants in the environment.17,18 Uptake, translocation, and transformation were observed in pumpkin plants exposed to BDE-47 in our previous report.19 In this study, relationships between OH-PBDEs and MeO-PBDEs were explored in young whole pumpkin plants by hydroponic exposure. Six OH-tetraBDEs and six homologous MeOtetraBDEs that were the most frequently detected with relatively high concentrations in the environment were selected for this study. The uptake and tissue distribution of OH- and MeO-PBDEs in different parts of pumpkin plants were
1. INTRODUCTION Polybrominated diphenyl ethers (PBDEs) are used as flame retardants in various products, including textiles, furniture, and electronics. Over the past few decades, PBDEs have received a considerable amount of attention because of their ubiquitous presence in the environment. As structural analogues of PBDEs, hydroxylated PBDEs (OH-PBDEs) and methoxylated PBDEs (MeO-PBDEs) are frequently detected in diverse matrices in the environment.1−4 The adverse effects of OH-PBDEs on organisms include the disruption of oxidative phosphorylation, neurotoxicity, and thyroid disruptions.5−8 For some endpoints, the toxicity of OH-PBDEs was considered to be higher than that of PBDEs or MeO-PBDEs. MeO-PBDEs were also reported to have a strong effect on the mRNA abundance of steroidogenic enzymes in the H295R cell line.9 There is considerable interest in determining the relationships among PBDEs, OH-PBDEs, and MeO-PBDEs. Neither OH-PBDEs nor MeO-PBDEs are commercially produced or used. Several origins of OH-PBDEs and MeO-PBDEs have been proposed. It has been demonstrated that many orthosubstituted OH-PBDE or MeO-PBDE congeners such as 6OH-BDE-47, 2′-OH-BDE-68, 6-MeO-BDE-47, and 2′-MeO© 2014 American Chemical Society
Received: November 8, 2013 Accepted: March 13, 2014 Published: March 13, 2014 236
dx.doi.org/10.1021/ez500068q | Environ. Sci. Technol. Lett. 2014, 1, 236−241
Environmental Science & Technology Letters
Letter
Figure 1. Experimental design of exposure and control groups. The colored squares represent root exudates. Circles represent microbes.
one pumpkin plant (Cucurbita maxima × Cucurbita moschata). Two duplicate reactors were combined into one sample for analysis, and there were five parallel samples in each exposure group for each OH-BDE or MeO-BDE. The exposure time was 10 days. Four control groups were designed and assessed simultaneously for each exposure chemical. Untreated pumpkin plant controls (two samples) were used to monitor possible cross contamination. To the hydroponic solutions was added 20 μL of acetone without any test chemicals. Unplanted controls (two samples) were added with the same amount of exposure chemicals and used to monitor any possible loss. Root exudate controls (three samples) were also set up, in which whole plants were taken away after being planted in reactors for 10 days without exposure chemicals. Then, the solutions that contained roots exudates were spiked with the exposure chemicals and exposed for an additional 10 days. Microbe controls (two samples) were conducted by adding unexposed exudate control solutions to the germiculture. The obtained bacterial colonies were transferred to 40 mL of autoclaved water. Exposure chemicals were then added, and the exposure lasted for 10 days. Microbe isolation, counting, and identification in the exposure system were conducted as described previously.19 2.3. Sampling and Sample Preparation. Root, stem, shoot, and hydroponic solutions of exposure groups and controls were separated after exposure. Sample preparation procedures were the same as those described previously.19 In brief, all samples were spiked with surrogate standards. Water
assessed. On the basis of the results obtained in this study and previous works, the transformation and fate of OH-PBDEs, MeO-PBDEs, and PBDEs in plants are discussed.
2. EXPERIMENTAL SECTION 2.1. Chemicals and Reagents. Chemical standards of six OH-PBDEs (4-OH-BDE-42, 4′-OH-BDE-49, 3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47, and 2′-OH-BDE-68) and six MeO-PBDEs (4-MeO-BDE-42, 4′-MeO-BDE-49, 3-MeOBDE-47, 5-MeO-BDE-47, 6-MeO-BDE-47, and 2′-MeO-BDE68) were used. These 12 compounds were used as exposure chemicals and were all purchased from AccuStandard (New Haven, CT). PBDE standards (BDE-28, -42, -47, -68, and -99) and surrogate standards ([13C]-6-OH-BDE-47 and BDE-75) were purchased from Wellington (Guelph, ON). Standards of PBDEs, OH-PBDEs, and MeO-PBDEs were all analyzed to determine their purity. Standards at concentrations of 100 ng/ mL were directly injected into the analytical instruments, and the amounts of impurities and their abundances in the standard were thereafter calculated. Details of other experimental materials and reagents are available in our previous paper.19 2.2. Hydroponic Exposure. The experimental design is similar to that described in our previous report and is shown in Figure 1.19 Briefly, pumkin plants were exposed to individual standards of OH-PBDEs and MeO-PBDEs in exposure reactors. Each reactor of each exposure group was filled with 40 mL of deionized water and 200 ng of a single exposure chemical (dissolved in 20 μL of acetone), giving an initial exposure concentration of 5 ng/mL in water, and planted with 237
dx.doi.org/10.1021/ez500068q | Environ. Sci. Technol. Lett. 2014, 1, 236−241
Environmental Science & Technology Letters
Letter
Table 1. Detection of Parent Compounds and Metabolites after Pumpkin Seedlings Had Been Exposed to OH-PBDEs for 10 Days exposed pumpkin (five samplesa) exposure chemical
detected compound
3-OH-BDE-47
3-OH-BDE-47 3-MeO-BDE-47 5-OH-BDE-47 5-MeO-BDE-47 4-OH-BDE-42 4-MeO-BDE-42 5-MeO-BDE-47 4′-OH-BDE-49 4′-MeO-BDE-49 4-MeO-BDE-42 6-OH-BDE-47 6-MeO-BDE-47 2′-OH-BDE-68 2′-MeO-BDE-68 6-OH-BDE-47
5-OH-BDE-47 4-OH-BDE-42
4′-OH-BDE-49
6-OH-BDE-47 2′-OH-BDE-68
a
rootb (pg/g) 194000 5390 157000 560 129000 23300 613 152000 4140 78 168000 1950 159000 126 273
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
stemb (pg/g)
18800 1120 20100 165 20100 5400 287 11500 1330 5 11100 149 9200 46 16
10300 35 10600 227 7360 318 ndc 38200 51 ndc 24200 47 11800 115 4
± ± ± ± ± ±
shootb (pg/g)
2290 8 2260 68 1510 65
417 ndc 360 ndc 304 ndc ndc 678 ndc ndc 889 ndc 433 ndc ndc
± 5620 ± 12 ± ± ± ± ±
4360 14 1300 15 2
± 135 ± 113 ± 105
± 101
± 164 ± 172
solutionb (pg/mL) 648 ± 170 14 ± 5 830 ± 70 3±2 511 ± 106 32 ± 13 ndc 951 ± 271 24 ± 8 ndc 485 ± 164 ndc 407 ± 112 ndc 0.8 ± 0.4
One sample consisted of two reactors. bMean value ± the standard deviation (n ≥ 3). cNondetectable.
Table 2. Detection of Parent Compounds and Metabolites after Pumpkin Seedlings Had Been Exposed to MeO-PBDEs for 10 Days exposed pumpkin (five samplesa) exposure chemical
detected compound
3-MeO-BDE-47
3-MeO-BDE-47 3-OH-BDE-47 5-MeO-BDE-47 5-OH-BDE-47 4-MeO-BDE-42 4-OH-BDE-42 4′-OH-BDE-49 4′-MeO-BDE-49 4′-OH-BDE-49 6-MeO-BDE-47 6-OH-BDE-47 2′-MeO-BDE-68 2′-OH-BDE-68
5-MeO-BDE-47 4-MeO-BDE-42
4′-MeO-BDE-49 6-MeO-BDE-47 2′-MeO-BDE-68 a
rootb (pg/g) 126000 357 174000 135 142000 411 ndc 152000 964 131000 754 134000 54
± ± ± ± ± ±
14300 56 12700 78 17700 52
± ± ± ± ± ±
16200 189 10300 111 7930 9
stemb (pg/g)
shootb (pg/g)
± ± ± ± ± ±
2640 11 4650 5 1210 56
± ± ± ± ± ±
9190 160 1520 7 1140 8
1330 ± 206 ndc 998 ± 225 ndc 425 ± 109 ndc ndc 1800 ± 367 ndc 311 ± 100 ndc 866 ± 140 ndc
13600 25 19800 15 3130 391 ndc 37500 921 10400 23 9090 21
solutionb (pg/mL) 342 ndc 356 ndc 1450 3 0.4 435 2 477 5 549 1
± 143 ± 63 ± ± ± ± ± ± ± ± ±
283 2 0.2 113 1 88 1 151 0.3
One sample consisted of two reactors. bMean value ± the standard deviation (n ≥ 3). cNondetectable.
Recoveries for the surrogate standards, [13C]-6-OH-BDE-47 and BDE-75, were 78.3−94.7 and 76.1−92.6%, respectively. The concentrations were recovery corrected. The method limits of quantification (MLQs) were estimated on the basis of a signal-to-noise ratio of 10. For OH-PBDEs, MLQs were 14− 27 pg/L in water and 3−25 pg/g in plants. For MeO-PBDEs, MLQs were 12−33 pg/L in water and 5−21 pg/g in plants.
samples were liquid−liquid extracted with a hexane/MTBE mixture [1:1 (v/v)]. Solid samples were extracted with a hexane/MTBE mixture [1:1 (v/v)] while being violently shaken using a Tissuelyser (QIAGEN, Hilden, Germany). After purification with acidified silica gel (10 g, 44% H2SO4 acidified), the collected extracts were concentrated to dryness using a rotary evaporator and redissolved in 1 mL of hexane. Then, a silica column [deactivated with 5% water (w/w), 5 g] was used for fractionation. First, 20% DCM in hexane (60 mL) was used to elute PBDEs and MeO-PBDEs. Second, DCM (70 mL) was applied to elute OH-PBDEs. Analysis of PBDEs and MeO-PBDEs was performed on an Agilent 6890N/5975C gas chromatograph and mass spectrometer. Identification and quantification of OH-PBDEs were conducted using an Agilent 1290/6460 liquid chromatograph and tandem mass spectrometer. Detailed information about sample analysis is provided in the Supporting Information and was provided in previous studies.20,21 2.4. Quality Assurance and Quality Control. Laboratory blank samples were analyzed to monitor contamination and interference, showing an absence of background interference.
3. RESULTS AND DISCUSSION 3.1. Purity of Stock Solutions. Impurities of standards were determined to ensure the authenticity of results. The analysis of purity suggested that 4-MeO-BDE-42 was present as an impurity (0.012%) in the 4-OH-BDE-42 stock standard solution. 6-MeO-BDE-47 was present as an impurity (0.025%) in the 6-OH-BDE-47 stock standard solution. In the stock solution of 4′-OH-BDE-49, very low levels of 4′-MeO-BDE-49 (0.058%) and 4-MeO-BDE-42 (0.041%) were detected and were considered negligible. A trace amount of 6-OH-BDE-47 (0.33%) was detected in the stock solution of 2′-OH-BDE-68. However, the presence of diverse impurities in stock solutions 238
dx.doi.org/10.1021/ez500068q | Environ. Sci. Technol. Lett. 2014, 1, 236−241
Environmental Science & Technology Letters
Letter
Figure 2. Conversion ratios between metabolites and their parent compounds (M/P).
did not affect conclusions drawn from this research. PBDEs were not found in any tested standards. 3.2. Uptake, Translocation, and Distribution of Exposure Chemicals. The distribution of the 12 exposure chemicals in different compartments of pumpkin plants showed that a majority of parent chemicals (>64%) were concentrated by the roots after exposure. The root concentration factors (RCFs) for different chemicals were different and ranged from 97 (4-MeO-BDE-42) to 488 (5-MeO-BDE-47). Parent OHPBDEs and MeO-PBDEs were taken up by the roots, and translocation from the roots to the shoots was observed. The concentration of parent chemicals decreased progressively in the stem, solution, and shoots, with
