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Uptake Pathway, Translocation, and Isomerization of Hexabromocyclododecane Diastereoisomers by Wheat in Closed Chambers Hongkai Zhu, Hongwen Sun, Yanwei Zhang, Jiayao Xu, Bing Li, and Qi-Xing Zhou Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05118 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on January 31, 2016

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Environmental Science & Technology

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Uptake Pathway, Translocation, and Isomerization of

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Hexabromocyclododecane Diastereoisomers by Wheat in Closed

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Chambers

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Hongkai Zhu,† Hongwen Sun,*,† Yanwei Zhang,‡ Jiayao Xu,† Bing Li,† and Qixing

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Zhou†

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†

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Environmental Science and Engineering, Nankai University, Tianjin 300071, China

MOE Key Laboratory of Pollution Processes and Environmental Criteria, College of

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‡

11

China

Agro-Environmental Protection Institute, Ministry of Agriculture, Tianjin 300191,

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*

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Nankai University

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#94 Weijin Road

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Tianjin, China 300071

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Phone: 86-22-23509241

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Fax: 86-22-23509241

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E-mail: [email protected]

20 21 22 23 24 25 26 27 28

Corresponding author: Hongwen Sun

Revision submitted to: Environmental Science & Technology

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ACS Paragon Plus Environment


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To study the uptake pathways of 3 main hexabromocyclododecane diastereoisomers

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(α-, β-, and γ-HBCDs) in wheat, four closed chambers were designed to expose wheat

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to HBCDs via air and/or soil for four weeks. The results showed that HBCDs could be

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absorbed by wheat both via root from soil and via leaf from air. The Rt values (ratio of

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HBCDs from root-to-leaf translocation to the total accumulation in leaves) ranging

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from 14.4 to 29.8% suggested that acropetal translocation within wheat was limited. A

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negative linear relationship was found between log Rt and log Kow of the HBCD

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diastereoisomers (p < 0.05). The bioconcentration factors (BCFs, (µg/g wheat

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tissues)/(µg/g soil)) were in the order α- > β- > γ-HBCD in wheat roots and stems,

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being negatively related to their Kow values. No such correlation was found in leaves,

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where the HBCDs came mainly from air distribution. The results of enantiomeric

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fractions indicated that the (-)-enantiomer of α- and γ-HBCDs and the

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(+)-β-enantiomer were selectively accumulated. Furthermore, β- and γ-HBCDs were

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transformed to α-HBCD in the wheat, with 0.309~4.80% and 0.920~8.40%

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bioisomerization efficiencies at the end of the experiment, respectively, being the

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highest in leaves. Additionally, no isomerization product from α-HBCD was found.

ABSTRACT

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2


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Hexabromocyclododecanes (HBCDs) are used primarily in extruded and expanded

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polystyrene for thermal insulation in buildings, and to a minor extent in upholstery

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textiles and electrical household equipment.1 In 2001, 16 700 metric tons of HBCDs

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were consumed worldwide, and its production has increased steadily over the years.2

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Due to their widespread use, HBCDs have been detected ubiquitously in a wide range

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of environmental matrices and biota samples, including air, dust, soil, birds, marine

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mammals and human breast milk.3-7 Recent studies indicated that HBCDs share the

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major

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bioaccumulation, long-range transport, and toxicity.1,

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amendment of Stockholm Convention, which lists HBCDs in Annex A (Elimination)

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with specific exemptions (decision SC-6/13), has entered into force for most parties in

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November 2014.8

INTRODUCTION

characteristics

of

persistent

organic

pollutants 2

(POPs):

persistency,

According to this, an

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Due to the difference in spatial orientation of bromine atoms, there are 16 possible

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stereoisomers of HBCDs. Technical HBCDs mixtures (t-HBCDs) are dominated by

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three diastereoisomers: α-HBCD (10~13%), β-HBCD (1~12%) and γ-HBCD

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(75~89%), and trace amounts of other diastereoisomers (δ- and ε-HBCDs).2 The

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substantial structural dissimilarities of these stereoisomers lead to differences in their

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physicochemical properties, such as water solubility (Sw), octanol-water partition

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coefficient (Kow), and octanol-air partition coefficient (Koa), etc. (Table S1 in the

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Supporting Information, SI); subsequently, these different properties may result in

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their distinctive fate and behavior in the environment. Additionally, each of the 3



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diastereoisomers has a pair of enantiomers, which have similar physicochemical

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properties but different biological behaviors.9

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Plant uptake of semivolatile organic compounds (SOCs) has attracted extensive

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attention in respect of the important role of plants in terrestrial ecosystems and the

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risks to human health through food chain transfers. The uptake of SOCs into plants

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occurs mainly via two pathways: (i) passive or active uptake from the soil into plant

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roots and subsequent transport from the roots to the shoots; and (ii) gaseous or

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particulate deposition uptake from the air, with possible subsequent translocation into

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other plant parts.10 In order to evaluate the pathway-specific uptake of SOCs into

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plants, various researchers have proposed a number of qualitative frameworks based

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on the physicochemical properties of the respective compounds.11-14 Among these

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frameworks, Cousins and Mackay proposed that for chemicals with log Koa > 6 and

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log Kaw > -6 (dimensionless Henry’s law constant), uptake from the atmosphere

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should be the important pathway; for chemicals with log Kow < 2.5 and log Kaw < -1,

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uptake by transpiration should be the important pathway.12 Based on the reviewed

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frameworks and physicochemical parameters of HBCDs, foliar uptake should be an

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important route for the accumulation of HBCDs in aerial plant parts. Despite its

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importance, research on the plant uptake of HBCD diastereoisomers is insufficient,15,

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16

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limited root uptake studies have reported the diastereoisomers-specific accumulation

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of HBCDs in agricultural plants in both pot and hydroponic experiments.15,

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addition, Zhang et al. speculated that foliar uptake may be the major uptake process

with no studies found regarding HBCDs uptake by plant leaves via air. These

4


16

In

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for HBCDs presented in plant leaves in a field study.17 Regardless, there is a urgent

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need to elucidate the mechanism of plant foliar uptake of HBCDs as individual

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diastereoisomers.

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To evaluate the pathway-specific uptake of SOCs in plants in a soil-air-plant system,

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several types of exposure chambers (ECs) have been designed to provide a

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controllable and relatively stable environment.18-21 Although it is sometimes difficult

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to extrapolate the results from chamber studies to field investigations because

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small-scale chamber experiments cannot entirely mimic the same environmental

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conditions as the outside world, chamber studies have contributed to better elucidation

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of the fate of SOCs, such as polycyclic aromatic hydrocarbons (PAHs),

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polychlorinated biphenyls (PCBs), and formaldehyde in plants.20-23

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Therefore, the purposes of this study included: (1) to evaluate the feasibility of the

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designed ECs; (2) to distinguish foliar or root uptake pathways and their subsequent

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translocation in plants under controlled conditions; (3) to investigate diastereoisomer-

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and enantiomer-specific accumulation of HBCD diastereoisomers in plants; and (4) to

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examine the bioisomerization of HBCD diastereoisomers in plant tissues. Wheat was

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chosen as the test plant because it is widely cultivated and recent data have indicated

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the potential for atmospheric deposition of SOCs into wheat grain.18

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MATERIALS AND METHODS

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Chemicals and Materials. The information of chemicals, solvents, and the

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polyurethane foam (PUF) used to collect air from the ECs is provided in the SI. The

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source and properties of soils, and the source and germination method of wheat seeds

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are also described in the SI.

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Exposure Chambers. Four identical ECs were prepared using synthetic glass. One

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EC served as the control group, and three ECs served as the testing groups and

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separately received α-, β-, and γ-HBCDs-contaminated air. Specific details on the ECs’

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design can be found in the SI. In brief, each EC was composed of a gaseous HBCDs

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generation system, an air pump, a plant growth chamber (50 cm high, 50 cm diameter,

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volume 98 L), an air sampling system, and polytetrafluoroethylene (PTFE) pipes

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(Figure 1). Air was pumped through a column (25 cm length, 4 cm diameter)

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containing glass beads (2 mm diameter) that were coated with HBCDs into the EC by

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a continuous pump (ALITA AL-80) at a flow rate of 4 L/min. The EC was filled with

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contaminated air, and two PUF plugs were placed in series at the air outlet of the EC

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to collect air from the EC.

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Feasibility Evaluation of the ECs and Distribution of HBCDs. Pre-experiments

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were conducted to observe, (1) whether the distributions of HBCDs were uniform in

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the ECs; (2) the exact concentrations of HBCDs in the air; and (3) the partitioning of

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HBCDs between air and soil.

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A detailed description of the pre-experiments is provided in the SI. Briefly, three

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types of soils were tested in this experiment, and their selected physicochemical

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characteristics are shown in Table S2. Eight PUF plugs were placed inside the ECs to

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monitor whether the distribution of gaseous HBCDs in the ECs was uniform. Two

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PUF plugs in series were placed in the outlet of each EC and replaced daily. The 6


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exposure lasted for one week and the HBCDs concentrations in the PUF plugs and

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soil were subsequently measured. Theoretical gaseous HBCDs concentrations in ECs

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were calculated by the amount of HBCDs collected by the PUF plugs (placed in the

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outlet of each EC) according to equation (eq) 1 in the SI.

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Exposure Experiment. Exposure experiments were conducted using the same ECs

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as in the pre-experiments, and pots planted with wheat plants were introduced. To

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elucidate which pathway is the predominant route for HBCDs uptake in wheat, we

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divided potted soils into two groups for each testing EC: one included spiked soil, and

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the other included unspiked soil. During four weeks of exposure, two pots of wheat

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plants and the corresponding soil (one spiked and another unspiked) as well as the

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PUF plugs at the outlet (collector for gaseous HBCDs) were sampled weekly to

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monitor the distribution of HBCD diastereoisomers among different phases. The

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difference in HBCDs concentrations in the leaf of wheat plants between spiked soil

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and unspiked soil groups was assumed to be absorbed from soil and subsequently

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translocated within the plant to the leaf.

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For details, the method used to prepare the spiked soil is readily available.16 The

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HBCDs-spiked soils were then packed into pots and equilibrated at room temperature

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for one week at 60% of the water holding capacity, so did the unspiked soils. At the

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start of the experiment, the initial concentrations of HBCDs in the aged spiked soils

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were 98.4 ± 5.9, 90.8 ± 9.0 and 62.8 ± 5.9 ng/g dry weight (DW) for α-, β-, and

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γ-HBCDs, respectively. These values were within the range of the soil HBCDs

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concentrations around point sources (140~1300 ng/g DW) (Table S3). The pot soil 7



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surface was covered by a layer of 2-cm thickness silica sand (150~380 µm) to prevent

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the spiked soil particles from entering the above air phase and to minimize the

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HBCDs exchange between the soil and the air. Eight pots of wheat plants (4 pots for

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each of the two soil treatments: spiked and unspiked treatment) were moved into the

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testing ECs, and 4 pots of wheat plants with unspiked soil were placed in the control

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EC. Two PUF plugs in series were placed in the outlet of the EC and replaced weekly,

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which sampled approximately 40 m3 of air in the EC each time. The other conditions

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remained the same as the pre-experiment. The exposure lasted for four weeks, while

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one pot from the control EC and two pots from each testing EC, including one spiked

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and another unspiked, were removed every week for HBCDs analysis.

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Sampling and Workup. At the end of the exposure experiment, a total of 32 PUF

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plugs were removed from all ECs outlets. After being collected, the PUF plugs were

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wrapped in foil, sealed in plastic, and stored at -20 °C until workup. The plants were

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gently removed from the soil, and any remaining plant materials were collected. The

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plants were subsequently washed three times with distilled water and divided into

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three fractions: leaf, stem, and root. After being washed, the plant leaves were

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subjected to a brief extraction with organic solvent to separate the waxes together

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with the HBCDs associated with this phase, whereby the plant leaves were dipped

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into 100 mL of chloroform for 15 s.24 A preliminary experiment showed that an

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extraction time of 15 s was required to remove 90% of the wax from the wheat leaves.

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The soil of each pot was divided into two parts: non-rhizosphere soil and rhizosphere

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soil. The freeze-dried soil and plant tissues (root, stem and remaining fractions of the 8


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leaves) samples were ground with an agate mortar and pestle and stored at 4 °C until

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extraction. The details of sample extraction and cleanup are given in the SI.

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Instrumental Analysis. Full details in relation to the analytical methodology used

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for determination of HBCDs can be found in the SI. Briefly, the cleaned extracts were

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analyzed using LC (Agilent 1200)-ESI-MS/MS (Agilent 6410, USA). The separation

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of HBCD diastereoisomers and enantiomers was accomplished using a C18 column

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(150 mm × 4.6 mm i.d., 5 µm particle size, CNW, Germany) and a β-PM chiral LC

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column (200 mm × 4 mm i.d., 5 µm particle size) containing permethylated

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β-cyclodextrin on silica (Macherey-Nagel, GmbH & Co, Duren, Germany),

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respectively. Further details on QA/QC measurements are described in the SI. Limits

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of quantitation (LOQ) and method limits of detection (LOD) for HBCDs analyses are

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summarized in Table S4.

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■

RESULTS AND DISCUSSION

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HBCDs Partitioning between Soil and Air in Pre-experiment. HBCDs were not

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detected in soil and air samples in the control EC; thus the control EC was not

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discussed further. The concentrations of α-, β- and γ-HBCDs in the air of the testing

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ECs were 2.74 ± 0.08, 1.83 ± 0.08, and 4.02 ± 0.10 ng/m3, respectively, and remained

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constant during the 1-week pre-experiment (Figure S1). The ambient HBCDs

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concentrations are highly variable in the environment, ranging from 0.16~71.9 pg/m3

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in urban and remote areas to 0.89~1300 pg/m3 in indoor air; and it is 0.28~9.7 µg/m3

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around potential point sources (Table S3). In this study, the gaseous HBCDs

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concentrations spiked in the testing ECs match well with their concentrations around

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potential point sources and meet the requirements of the exposure experiment.

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At the end of the pre-experiment, the average HBCDs concentrations, recovered

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from the PUF plugs that were placed inside the ECs contaminated with α-, β-, and

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γ-HBCDs, were 10.9 ± 0.60, 9.14 ± 0.47, and 14.7 ± 0.88 ng/sampler, respectively

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(Table S5). The coefficient of variation (CV%) values for the measured

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concentrations recovered from the PUF plugs at various sites within the ECs were less

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than 5.97% (Table S5), indicating there existed adequate atmospheric HBCDs mixing

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in the ECs and the reproducibility of the PUF plugs. These CV% values are less than

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those obtained in earlier studies using other types of passive air samplers, e.g.,

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activated carbon.25 In addition, a significant correlation was established between the

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PUF plugs measurements and Cair, acquired from concentrations in the PUF plugs at

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the outlet, which indicated that PUF plugs at the outlet could reliably reflect the

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HBCDs concentrations in the air phase (R2 = 0.916, p = 0.000; Figure S2).

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As shown in Table S6, gaseous HBCDs deposition was mainly recovered in the top

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soil layer (0~0.5 cm, 50 g DW) with concentrations in the range of 0.434~0.892 ng/g

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DW, whereas the bottom soil layer (0.5~2 cm, 150 g DW) exhibited only sporadic

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detections of HBCDs. This observation indicated that HBCDs were rapidly adsorbed

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by soil particles and did not readily diffuse into the depth. The low mobility of

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HBCDs in soil could be ascribed to their high hydrophobicity (log Kow > 5).26

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Furthermore, we also investigated the relationships between the partitioning of

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individual HBCD diastereoisomers and soil organic matter (SOM) content in the three 10


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types of soil samples. As shown in Figure S3, the HBCDs concentrations in the

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topsoils and SOM contents showed a positive correlation (R2 = 0.879~0.992, p =

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0.041~0.159), which indicated that SOM was an important phase in the adsorption of

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gaseous HBCDs. This finding is in agreement with the conclusions of Meng et al..27

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Additionally, the dimensionless partition coefficients between soil organic carbon

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and air (Koca) were calculated based on the measured data, yielding average values of

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8.16, 10.2, and 8.90 for α-, β-, and γ-HBCDs, respectively. There was a positive

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correlation between the average log Koca values of the three HBCD diastereoisomers

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and their log Koa values (R2 = 0.738, p = 0.342; Figure S4). This indicated that the Koa

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could be a predictor for the distribution tendency of the three HBCD diastereoisomers

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from air to soil. To date, variable HBCD diastereoisomers profiles have been reported

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in soil samples,5, 28 but the mechanism responsible for the significant variations in

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HBCD diastereoisomers profiles remains unclear. Whether the variations in isomer

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profiles is due to different partitioning between the air and environmental

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compartments (e.g., surface soil and atmospheric particulate matter) during

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long-range transport or due to the selective degradation of different isomers is

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tentative and it merits further studies.

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HBCDs Distribution in Soil in Exposure Experiment. At the end of the 4-week

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exposure experiment, 60.9% (37.6%), 59.6% (38.3%), and 61.8% (24.2%) of the

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initial test concentrations of α-, β-, and γ-HBCDs were still detectable in the

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non-rhizosphere (rhizosphere) soils, respectively (Table S7). It is worth pointing out

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that losses via adsorption to pot wall and leaching with irrigation water could be 11



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negligible since we had wrapped the whole pot with aluminum foil. Besides plant

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uptake and biodegradation, the reduction in HBCDs concentrations in soil can partly

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be attributed to the reduction in extraction recovery due to HBCDs sequestration in

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the soil. It was found in parallel experiment using unplanted sterilized soil that the

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sorption to soil matrix resulted in at least 34.8% decline in recovery of HBCDs.

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Similar results have been obtained in earlier studies on highly hydrophobic

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brominated retardant.16,

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penta-BDEs concentrations compared to initial levels in the dissipation experiments.26

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Furthermore, the HBCDs concentrations in the rhizosphere soil were significantly less

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than the corresponding values in non-rhizosphere soils (p < 0.05; Table S7), which

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agrees with earlier studies.29-31 Several recent perspectives can explain this

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phenomenon, which is named the “rhizosphere effect”. First, the presence of root

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exudates can enhance the desorption of residual pollutants, thus improving the

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bioavailability and subsequent plant uptake and biodegradation potential.29, 31 Second,

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plant root exudates contain abundant low molecular weight carbohydrates that tend to

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stimulate overall bacterial populations,32, 33 which may play an important role in the

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microbiological transformation of HBCDs.9, 34

26

Litz found an approximately 50% reduction in total

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Uptake Pathways and Tissues Distribution of HBCDs in Wheat. As shown in

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Figure 2, a fast uptake process for the three diastereoisomers was observed in wheat

263

roots and stems in the whole period of tests, and their accumulation did not reach an

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apparent equilibrium within the 4 weeks. The calculated quasi-equilibrium factor (αR)

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values for HBCDs in wheat roots are listed in Table S8 (see the SI for the calculation 12


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method for αR). Obviously, the αR values were clearly below 1 (0.017~0.030),

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suggesting that the passive uptake of these compounds by wheat roots is far from

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equilibrium at the end of the experiment. Additionally, they were generally consistent

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with the overall hydrophilic-to-hydrophobic trend of the three diastereoisomers,

270

which indicated that root uptake rate of HBCDs depended on their chemical

271

properties.

272

Regarding to wheat leaves, the absorption rate curves of HBCDs in the wax phase

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can be divided into 2 stages: an initial rapid uptake stage in the first two weeks

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followed by a slow stage, while, an opposite HBCDs concentrations trend was

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observed in inner leaf phase (Figure 2). The HBCDs concentrations in the wax phase

276

of the leaves were at least one order of magnitude higher than their concentrations in

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the leaf inner tissues (Table S9). These results are consistent with that were acquired

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for other SOCs.35,

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approximately two orders of magnitude higher than those in inner tissues.35 PCBs

280

were reported to have a much higher affinity for waxes than for the other cuticular

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constituents.36 Overall, limited by the mass fraction of the wax, the absolute amount

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of HBCDs in the wax accounted for 3.16~9.24% of the total amount in leaves at the

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end of the experiment (Figure S5). Furthermore, as shown in Figure S5, these ratios

284

increased in the first two weeks and then decreased, suggesting that the HBCDs that

285

had accumulated in the wheat leaf surface wax diffused deeper into the leaf to reach

286

intracellular storage compartments. At this point, we deduced that these patterns were

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caused by the combined results of reversible translocation of HBCDs between plant

36

The PAH concentrations in cuticles were reported to be

13



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wax and air,23 and the rapid uptake kinetics of HBCDs in wax and the relatively slow

289

chemical migration from the wax phase to the inner leaf.37, 38

290

Total HBCDs in wheat leaves in the spiked soil treatment were slightly higher than

291

those in the unspiked soil treatment, with significant difference occurring only for the

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fourth week (p < 0.05; Table S9, S10), which suggested the translocation of HBCDs

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from the roots to the above-ground parts. In order to confirm the magnitude of the

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contribution between the both uptake pathways, we calculated the quantity of

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acropetal translocation of HBCDs within the plants. The ratio of HBCDs translocation

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from root to leaf to the total accumulation in leaves (Rt) was calculated using eq 2 in

297

the SI. Rt values of 14.4~29.8% for three HBCD diastereoisomers are acquired and

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exhibited in Table 1, indicating that the major portion of the HBCDs in the leaves is

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directly absorbed from the air. The higher log Kow values (> 5) of HBCD

300

diastereoisomers indicate that they may strongly partition onto the epidermis of the

301

roots and cannot be drawn into the inner roots and be translocated within the plant,

302

ultimately resulting in the lower Rt values. A similar result was presented by Lin et al.,

303

where approximately 4.98~50.5% of the PAHs in the leaves of the tea plant were

304

translocated from the roots.39 Notably, most contaminants are less available in the

305

natural soils than in the fresh soils used in laboratory experiments due to the

306

adsorption to SOM or clay minerals during the aging process.40, 41 Therefore, the Rt

307

values will be much lower in the real environment, and the foliar uptake pathway will

308

make a greater contribution to the overall uptake.

309

The Rt values of the three diastereoisomers were following a sequence of α- > β- > 14


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γ-HBCD (Table 1). Significant negative linear relationships between their log Rt and

311

log Kow values and positive linear relationships between their log Rt and log Sw values

312

were found, respectively (R2 = 0.657, p = 0.03 and R2 = 0.713, p = 0.001 for log

313

Rt-log Kow and log Rt-log Sw, respectively; Figure S6). To our knowledge, the present

314

study is the first report of the relationships between log Rt and log Kow or log Sw for

315

the three diastereoisomers. To further demonstrate the capacity of the root-to-stem

316

translocation, we calculated the translocation factors (TFs), i.e. the ratios of HBCDs

317

concentrations in the stem to those in the root, all in dry weight. As shown in Table 1,

318

low TF values (0.177~0.280) were obtained for HBCDs, indicating again that the

319

fraction drawn into the transpiration stream and translocated within the plants was

320

very low. Additionally, the observed linear inverse relationship between log TF or log

321

Rt and log Kow for the three diastereoisomers is consistent with the view that

322

translocation of hydrophobic organic contaminants (HOCs) within a plant is a

323

combination of the solution of the chemicals in water and their association with the

324

organic cell membrane (Table S11).

325

Diastereoisomer Selectivity during HBCDs Accumulation in Wheat. We have

326

calculated the bioconcentration factors (BCFs) for roots (RCFs), stems (SCFs), and

327

leaves (LCFs) based on the ratios of the concentrations in roots, stems or leaves to the

328

measured bulk soil concentrations, all in dry weight, respectively. The time-dependent

329

RCF, SCF, and LCF values are listed in Table 1, and in general, the BCF values were

330

greater than 1 for the roots and less than 1 for the stems and leaves. The accumulation

331

was in the order α- > β- > γ-HBCD in roots (RCF) and stems (SCF) and γ- > α- > 15



332

β-HBCD in leaves (LCF). These results indicated that the wheat showed a preferential

333

bioaccumulation of α-HBCD over β- and γ-HBCDs in the roots and stems. Until now,

334

little has been known about the diastereoisomer-specific behavior of HBCDs in plants,

335

and the diastereoisomer-specific accumulation of HBCDs is inconsistent for plants in

336

the literature.15,

337

diastereoisomers in maize was β- > α- > γ-HBCD in roots and β- > γ- > α-HBCD in

338

shoots based on a hydroponic experiment.15 Meanwhile, Li et al. reported that the

339

dominant HBCD diastereoisomer in cabbage and radish tissues was γ-HBCD in the

340

roots and α-HBCD in the shoots.16

16

Wu et al. reported that the accumulation order of HBCD

341

The log RCFs and log SCFs of HBCDs were all inversely proportional to their log

342

Kow values and positively proportional to their log Sw values (Table S11), illustrating

343

again that the root uptake and subsequent translocation within plant of HBCDs were

344

controlled by successive distributions between water solution and organic components

345

in soil and plant tissues. However, no significant correlation was found between log

346

LCFs of HBCD diastereoisomers and their log Kow or log Sw values (Table S11),

347

indicating again that the source for HBCDs in leaves was different from those in roots

348

and stems. Similar results are also reported in the literature for other HOCs,10, 42

349

whereas an opposite trend can be obtained in other studies.43, 44 The positive linear

350

relationship between log RCF/log SCF/log LCF and log Kow was obtained mainly in

351

hydroponic experiments or for HOCs with lower log Kow values (< 4), where organic

352

compounds partitioning occurs only between roots and solutions, and their uptake by

353

roots depends solely on the hydrophobicity of the compound and root lipids. However, 16


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354

the situation becomes more complicated for plants that are grown in contaminated

355

soils. Due to the strong binding, plants have less accessibility to absorb these

356

associated compounds into the roots via the transpiration stream.45

357

The dimensionless Kpa values were calculated as the ratio of the mass

358

concentrations in plant leaves to those in the ambient air. As shown in Figure S7, the

359

log Kpa factors exhibited positive correlations with log Koa of the diastereoisomers,

360

which is in accordance with the well-accepted conclusion in the literature.18, 38 The

361

logarithmic plots of Kpa against Koa showed slopes less than 1 for HBCDs and

362

increased with time-lapse (0.219~0.334; Figure S7). Theoretically, slopes that differ

363

from 1 indicate that octanol is not an ideal surrogate to model the uptake capacity of

364

lipid fraction of the plant.46 However, as mentioned above, the distribution of HBCDs

365

did not achieve equilibrium between the air and wheat leaves over the 4-week

366

monitoring period used in this study. Therefore, longer periods of exposure

367

experiments are encouraged in this field, especially when considering that the

368

exposure duration for wheat in the field is at least three more months from just come

369

forth to harvest.

370

Enantioselectivity during HBCDs Accumulation in Wheat. The enantiomeric

371

fractions (EFs) values of the prepared individual diastereoisomers from an external

372

standard solution were determined to be 0.503 ± 0.004, 0.506 ± 0.003, and 0.497 ±

373

0.004 (n = 6) for α-, β-, and γ-HBCDs enantiomers, respectively. Deviation from the

374

EF values of the external standard solution means a stereospecific-enantiomeric shift

375

due to biologically mediated processes. As shown in Figure 3 and Figure S8, the EFs 17



376

values in soil and air remained consistent with the external standard solution, whereas

377

the EFs values in wheat tissues (root, stem and inner leaf) were in general

378

significantly different from those of the standards (p < 0.05). For α- and γ-HBCDs in

379

the wheat, the results showed an obvious decrease in the EFs values with respect to

380

the standards (p < 0.05; Figure 3). Therefore, we can speculate that the plant tissues

381

(root, stem and inner leaf) showed selectivity for the (-)-enantiomer uniformly.

382

However, for β-HBCD, plant tissues exhibited a weaker preferential enrichment of the

383

(+)-enantiomer (Figure 3). To date, the only available research on the

384

enantiomer-specific uptake of HBCDs in plants was conducted recently by Zhang et

385

al.,17 who discovered that the leaves and stems of radish, wheat, and reed plants

386

showed selectivity for the (+)-enantiomer for α-HBCD, whereas the roots of reed

387

plants preferentially accumulated (-)-α-HBCD. In general, enantioselectivity of

388

HBCD diastereoisomers occurred in plants, and the selectivity varied with species and

389

even the organs in the same species. The results in this study offer useful information

390

for researchers to investigate the enantiomer-specific accumulation of HBCD

391

diastereoisomers within plants.

392

Bioisomerization of HBCD Diastereoisomers in Wheat. To test the possibility of

393

bioisomerization of HBCD diastereoisomers in wheat, we have measured the other

394

two diastereoisomers in each testing ECs that was contaminated with one specific

395

HBCD diastereoisomer. No other diastereoisomers were found in any plant tissue

396

samples in the α-HBCD-spiked EC. However, α-HBCD was found in both the β- and

397

γ-HBCDs-spiked ECs, and the α-HBCD generated in both ECs increased over time 18


Page 18 of 31

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398

(Table S12). This report is the first one focusing on the bioisomerization of HBCD

399

diastereoisomers

400

bioisomerization from β- and γ-HBCDs to α-HBCD,47,

401

mechanism has remained unclear. The concentration ratio of α-HBCD to β- and

402

γ-HBCDs in each part of the wheat was further calculated (Figure 4). At the end of the

403

experiment, approximately 0.309~4.80% and 0.920~8.40% of the parent β- and

404

γ-HBCDs were bioisomerized to α-HBCD, which suggested that the bioisomerization

405

capacity was greater for γ-HBCD compared to β-HBCD. Compared with the

406

bioisomerization capacity in aquatic organisms,48 earthworms,49 and mice,50 the

407

bioisomerization capacity is lower in plants. Additionally, we determined the

408

bioaccumulation and bioisomerization of HBCD diastereoisomers in two microalgae

409

(Spirulina subsalsa and Scenedesmus obliquus) in our previous work and what we

410

have found is that these algae does not possess the ability to bioisomerize HBCDs.51

in

plant

tissues.

Although

many

studies 48

have

reported

the isomerization

411

The α-HBCD concentrations transformed from β- and γ-HBCDs varied in different

412

tissues, in the order of roots and stems < wax < inner leaves for both β- and γ-HBCDs

413

(Figure 4). This suggested that leaves had the greatest ability to isomerize HBCDs.

414

The isomerization rates in leaves cultivated in spiked soil was slightly higher than

415

those in unspiked soil for both β- and γ-HBCDs (p < 0.05), which suggested that the

416

bioisomerized α-HBCD in the wheat leaf should be derived from a combination of the

417

metabolism in the leaf (direct) and the translocation of metabolites inside the plants

418

(indirect). Through the comparison of the difference between the ratios in the two

419

treatments, we are able to roughly estimate the contribution from both sources. At the 19



420

end of the experiment, the direct source/indirect source ratio was approximately 3:1

421

for (β→α) and 4:1 for (γ→α), which indicated that majority of the α-HBCD was

422

bioisomerized in the leaf rather than translocated inside the plants. Certainly, this is

423

only an approximate estimation. Nevertheless, these observations are helpful for

424

improving our knowledge of the bioisomerization of HBCD diastereoisomers within

425

plant tissues. More studies using in vivo and in vitro assays are encouraged to provide

426

a detailed understanding of bioisomerization of HBCD diastereoisomers within plant

427

tissues.

428

■

429

Supporting Information

430

Details of physical description of chemicals and materials, the exposure chambers,

431

sample extraction and clean up, instrumental analysis and QA/QC, and additional

432

figures and tables as noted in the text.

433

■

434

Corresponding Authors

435

*Phone: + 86-22-23509241; Fax: + 86-22-23509241; e-mail: [email protected].

436

Notes

437

The authors declare no competing financial interest.

438

■

439

This work was supported by Ministry of Science and Technology of China (973

440

program, 2014CB441105), Natural Science Foundation of China (41225014), and

441

Ministry of Education of China (Innovative Research Team Project, IRT13024).

442

ASSOCIATED CONTENT

AUTHOR INFORMATION

ACKNOWLEDGEMENTS

20


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443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486

■

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accumulation, depuration, bioisomerization, and metabolism of hexabromocyclododecanes (HBCDs) in two ecologically different species of earthworms. Sci. Total Environ. 2016, 542, 427-434. 50. Szabo, D. T.; Diliberto, J. J.; Hakk, H.; Huwe, J.; Birnbaum, L. S., Toxicokinetics of the flame retardant hexabromocyclododecane gamma: Effect of dose, timing, route, repeated exposure and metabolism. Toxicol. Sci. 2010, 117, 282-293. 51. Zhang, Y. W.; Sun, H. W.; Zhu, H. K.; Ruan, Y. F.; Liu, F.; Liu, X. W., Accumulation of hexabromocyclododecane diastereomers and enantiomers in two microalgae, Spirulina subsalsa and Scenedesmus obliquus. Ecotoxicol. Environ. Saf. 2014, 104, 136-142.

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585 586 587

FIGURE LEGENDS Figure 1. Scheme of the exposure device.

588 589 590 591

Figure 2. Time-dependent accumulation of HBCDs in wheat plant tissues and corresponding soil and air on a dry weight basis in the spiked soil treatment. Error bars represent SD values.

592 593 594 595 596 597 598 599

Figure 3. Enantiomeric fractions (EFs) of the three HBCD diastereoisomers in wheat tissues and corresponding soil and air at week 4 in the spiked soil treatment. The shaded volume represents the EFs of the external standard solution. Asterisks indicate a significant difference in EFs of the three HBCD diastereoisomers between wheat

600 601 602 603

Figure 4. The mean ratios of bioisomerized α-HBCD to β-HBCD (A) and γ-HBCD (B) in various tissues of wheat. “US” in parentheses represents of the unspiked

tissues, corresponding soil and air and external standard solution (Independent samples T test, p < 0.05). Error bars represent SD values.

soil treatment; “S” in parentheses represents of the spiked soil treatment. Error bars represent SD values.

604 605 606 607 608 609 610

25



611

Figure 1

612 613

614

615

616

617

618

619

620 621 26


Page 26 of 31

Page 27 of 31


622

Figure 2

623

624

625 626 27



627

Figure 3

628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 28


Page 28 of 31

Page 29 of 31


652 653

Figure 4

654 655 656 657

29



Page 30 of 31

658 659 660

Table

661 662 663 664

Table 1. Time-dependent bioconcentration factors (BCFs) and translocation factors (TFs) of HBCDs in wheat in the spiked soil treatment and the ratio of HBCDs from root-to-leaf translocation to the HBCDs concentration in leaves (Rt, %) Bioconcentration Factors (BCFs)

1wk

2wk

3wk

4wk 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685

α-HBCD β-HBCD γ-HBCD α-HBCD β-HBCD γ-HBCD α-HBCD β-HBCD γ-HBCD α-HBCD β-HBCD γ-HBCD

RCF 1.28±0.17 1.10±0.13 0.550±0.043 1.63±0.26 1.36±0.07 0.961±0.082 2.13±0.19 2.07±0.32 1.27±0.13 3.21±0.39 3.08±0.22 1.99±0.42

SCF 0.286±0.039 0.231±0.089 0.100±0.028 0.405±0.045 0.315±0.056 0.203±0.018 0.606±0.029 0.514±0.046 0.284±0.038 0.880±0.079 0.842±0.096 0.472±0.037

LCF 0.141±0.023 0.134±0.032 0.157±0.028 0.225±0.037 0.175±0.024 0.259±0.029 0.377±0.058 0.335±0.054 0.473±0.035 0.663±0.083 0.604±0.099 0.755±0.103

Translocation Factors (TFs)

Rt, %

0.218±0.013 0.202±0.009 0.177±0.021 0.244±0.019 0.224±0.035 0.206±0.012 0.280±0.014 0.242±0.074 0.203±0.011 0.269±0.024 0.264±0.032 0.216±0.015

22.9±1.2 11.4±2.4 7.20±0.19 27.3±3.3 14.7±2.8 9.50±2.43 30.6±3.2 11.0±5.7 9.41±0.68 29.8±3.5 22.5±4.0 14.4±1.9

The bioconcentration factors (BCFs) for root (RCF), stem (SCF), and leaf (LCF) were calculated as the ratios of the concentrations in root, stem or leaf to the measured soil concentrations on dry weight basis. The translocation factors (TFs) were calculated as the ratios of HBCDs concentrations in stem to those in root, all in dry weight. The Rt values were calculated as the ratio of HBCDs from root-to-leaf translocation to the total accumulation in leaves. Data presented as the mean ± standard deviation (n=3).

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Uptake Pathway, Translocation, and Isomerization of

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