Heavy metal exposure alters the uptake behaviour of 16 priority

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Ecotoxicology and Human Environmental Health

Heavy metal exposure alters the uptake behaviour of 16 priority polycyclic aromatic hydrocarbons (PAHs) by pakchoi (Brassica chinensis L.) Songqiang Deng, Tan Ke, Yanfang Wu, Chao Zhang, Zhiquan Hu, Hongmei Yin, Limin Guo, Lanzhou Chen, and Dayi Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01405 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 17, 2018

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Heavy metal exposure alters the uptake behaviour of 16 priority

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polycyclic aromatic hydrocarbons (PAHs) by pakchoi (Brassica

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chinensis L.)

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Songqiang Denga, Tan Keb, Yanfang Wuc, Chao Zhangb, Zhiquan Hua, Hongmei Yind,

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Limin Guoa,*, Lanzhou Chenb,*, Dayi Zhange,*

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a. School of Environmental Science and Engineering, Huazhong University of

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Science and Technology, Wuhan 430079, P.R. China.

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b. School of Resource and Environmental Sciences, Hubei Key Laboratory of

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Biomass-Resources Chemistry and Environmental Biotechnology, Wuhan University,

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Wuhan 430079, P.R. China.

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c. Wuhan Wenke Ecological Environment Ltd., Wuhan 430223, P.R. China

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d. Hunan Institute of Microbiology, Changsha 410009, P.R. China

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e. School of Environment, Tsinghua University, Beijing 100084, P.R. China

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*Corresponding authors:

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Prof. Limin Guo

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School of Environmental Science and Engineering, Huazhong University of Science

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and Technology, Wuhan, 430079, P.R. China; Tel. +86(0)2787792101; E-mail,

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[email protected]

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Prof. Lanzhou Chen

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School of Resource and Environmental Sciences, Wuhan University, Wuhan, 430079,

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P.R. China; Tel. +86(0)2787152713; E-mail, [email protected]

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Dr Dayi Zhang

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School of Environment, Tsinghua University, Beijing 100084, P.R. China; Tel. +86

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(0)1062773232; E-mail, [email protected]

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Table of Contents

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For TOC only

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Abstract

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Polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs) are predominant

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pollutants normally co-existing at electronical waste dumping sites or in agricultural

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soils irrigated with wastewater. The accumulation of PAHs and HMs in food crops has

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become a major concern for food security. This study explored the hydroponic uptake

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of 16 priority PAHs and 5 HMs (Cd, Cr, Cu, Pb and Zn) by pakchoi (Brassica

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chinensis L.). PAHs exhibited stronger inhibition on pakchoi growth and

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physiological features than HMs. Five HMs were categorized into high-impact HMs

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(Cr, Cu and Pb) and low-impact HMs (Cd and Zn) with distinct behavior under the

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co-exposure with PAHs, and low-impact HMs showed synergistic toxicity effects with

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PAHs. Co-exposure to PAHs and HMs slightly decreased the uptake and translocation

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of PAHs by pakchoi, possibly attributing to the commutative hindering effects on root

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adsorption or cation-π interactions. The bioconcentration factors in PAHs+HMs

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treatments were independent on the octanol-water partition coefficient (Kow), owing to

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the cation-π interaction associated change of Kow and induced defective root system.

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This study provides new insights into the mechanisms and influential factors of PAHs

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uptake in Brassica chinensis L. and gives clues for reassessing the environmental

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risks of PAHs in food crops.

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Keywords: Polycyclic aromatic hydrocarbons (PAHs); heavy metals; Brassica

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chinensis L.; hydroponic uptake; acropetal translocation

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1. Introduction

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Polycyclic aromatic hydrocarbons (PAHs) are a large class of environmental

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pollutants of great concerns for their varied toxicity, widespread presence and

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persistence in the environment

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processes (forest and brush fires) and incomplete combustion of organic materials

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(e.g., coal, petroleum and wood) 4. Since PAHs can bioaccumulate through the food

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chain and seriously threaten food security and human health, 16 parent PAHs have

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been classified as priority pollutants by the United States Environmental Protection

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Agency (USEPA) 5.

1-3

. They are primarily generated during biological

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The soil-to-plant (root uptake and translocation) pathway is important for the

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entrance of PAHs from soils into the food chain 3, 6. This pathway is correlated to the

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octanol-water partition coefficient (Kow) of organic pollutants

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works suggest that the uptake and translocation of organic compounds by plant

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depends on Kow. Recent studies find that most organic pollutants (log Kow ranging

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from -0.77 to 8.27) are preferentially taken up by plant roots and translocated to

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shoots 8, since chemicals with higher hydrophobicity are more easily bound to the root

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surfaces but harder to be translocated within the plant tissues 9. Accordingly, the root

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uptake of organic pollutants is generally positively correlated with logarithm Kow,

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while the transportation process, evaluated by the transpiration stream concentration

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factor (TSCF) or translocation factor (TF), follows a bell-shaped relationship

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Although PAHs are hydrophobic compounds with log Kow > 3.0 and are reported to be

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poorly uptaken by roots, their behavior and fate in the soil/water-to-plant system has

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attracted many attentions

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detected in food crops (fruits and vegetables) range from 0.01 to 5 µg/kg, depending

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on the exposure level

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also follows the bell-shaped relationship 15. Nevertheless, bare PAHs contamination is

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seldom found and they normally co-exist with heavy metals (HMs) in soils,

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particularly at electronic waste dismantling sites and in agricultural fields irrigated

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with wastewater

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mainly on the occurrence, source, accumulation and transfer of PAHs or HMs only,

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and how their co-occurrence affects the bioaccumulation behavior in the food chain is

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seldom discussed

3, 7

. Many previous

10-12

.

6, 13

. Some reports point out that priority PAHs usually

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, and their uptake and translocation behavior in food crops

6, 16

. In PAHs-HMs co-contaminated soils, previous studies focus

17-19

. Some evidence shows that the presence of HMs can 4

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significantly alter the adsorption behavior of several parent PAHs on cyanobacteria

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and biochar

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on the uptake behavior of PAHs in plants, particularly food crops.

20, 21

. However, little information is available about the impacts of HMs

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Root is the predominant organ for plant to uptake substances, e.g., water, nutrients,

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etc., behaving as the primary entry of contaminants into the food chain. Root

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physiological status is reported to play important roles during the uptake of organic

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pollutants in plant tissues, particularly under the stress of HMs 8. HMs exposure can

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significantly influence the root architecture, such as root diameter, root volume, root

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surface area, root tips and root crossings

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toxicity, possessing damage to plant roots and changing their ability to uptake other

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pollutants

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e.g., metal-chelating compounds, can be indiscriminately taken up and acropetally

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translocated by plants 24. Previous studies have suggested the important roles of root

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physiology, which explain the HMs-associated mechanisms of organic pollutant

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uptake by plants. Wang et al. found that the uptake of poly brominated diphenyl ether

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(PBDE) was enhanced with the increasing root electrolytic leakage, attributed to the

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Cu damage 8. However, only limited organic pollutants have been explored, such as

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PBDE and polychlorinated biphenyls (PCBs) 8, 25. Little information is available about

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the uptake behavior of a broader range of organic pollutants by plants under the stress

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of HMs and whether different HMs have the same mechanism influencing the uptake

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of organic pollutants. As a class of organic chemicals with similar structure but broad

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values of Kow, PAHs are model substrates to study the HMs induced uptake behavior

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and the underlying mechanism.

22

. PAHs and HMs also have synergistic

23

. In a defective root system, substances with a large structural formula,

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In the present study, we hypothesize that PAHs-HMs co-exposure could

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significantly alter the uptake and translocation of PAHs by plants. Pakchoi (Brassica

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chinensis L., local name: Shanghaiqing) was chosen as the target plant, since it is a

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popular vegetable consumed by Chinese people and widely cultivated all over China.

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Sixteen priority PAHs and five HMs [copper (Cu), cadmium (Cd), lead (Pb), zinc (Zn)

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and chromium (Cr)] were selected. We aimed to explore whether the uptake behaviour

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of PAHs within the water-to-plant system is altered in the presence of HMs, evaluate

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the change in bioaccumulation of PAHs with different Kow, and discuss the impacts of

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HM types. Our work unveils the underlying mechanism of PAHs uptake by plants

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when co-existing with HMs, gives new insights into the behavior and influential 5

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factors of PAHs accumulation in the food chain at the sites co-contaminated with

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PAHs-HMs, and helps in reassessing the risks of PAHs in food crops under HMs

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exposure.

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2. Materials and Methods

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2.1 Chemicals

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Standards of PAHs mix containing 16 priority PAHs (naphthalene, acenaphthylene,

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acenaphthene,

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benz[a]anthracene,

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benzo[a]pyrene,

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benzo[ghi]perylene) were purchased from Aladdin Ltd. (China, catalogue No

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P115311). Surrogated standards (naphthalene-D8, fluorene-D10, pyrene-D10 and

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perylene-D12) and internal standards (phenanthrene-D10, acenaphthene-D10,

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anthracene-D10 and chrysene-D12) were obtained from Sigma Aldrich (USA). CuSO4,

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Cd(NO3)2, Pb(NO3)2, ZnSO4 and K2CrO4 were purchased from Sinopharm Chemical

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Reagent Co., Ltd. (China). All the standards and chemicals used in this study are of

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analytical reagent grade.

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2.2 Hydroponic experiment

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Seeds of pakchoi were sterilized with 0.5% NaClO for 10 min, washed thoroughly

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with deionized water and germinated for 24 hr at 25 °C in dark. The germinated seeds

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were evenly sown on quartz sands and cultivated using the half-strength Hoagland

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nutrient solution in a greenhouse under the regime of light [122 µmol/(m2·s)]/dark

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14/10 hr and 25/15 °C. Two weeks later, healthy and identical pakchoi seedlings were

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selected, transplanted into 1.5-L plastic pots, and cultivated using the complete

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Hoagland nutrient solution for another 2 weeks. During the first 4 weeks, the nutrient

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solution was replenished twice per day and renewed every 3 days. Afterwards, the

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nutrient solution was replaced by the complete Hoagland nutrient solution containing

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pollutants as the following twelve treatments in triplicates: control (no HMs or PAHs),

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Cu, Cd, Pb, Zn, Cr, PAHs, PAHs+Cu, PAHs+Cd, PAHs+Pb, PAHs+Zn and PAHs+Cr.

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The set of concentrations for each treatment was: 10.3 µmol/L CuSO4 (0.3 µmol/L

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from Hoagland nutrient solution), 10 µmol/L Cd(NO3)2, 10 µmol/L Pb(NO3)2, 10.8

fluorene,

phenanthrene,

chrysene,

anthracene,

benzo[b]fluorathene,

indeno[1,2,3-cd]pyrene,

fluoranthene,

benzo[k]fluoranthene,

dibenz[a,h]anthracene

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pyrene,

and

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µmol/L ZnSO4 (0.8 µmol/L from Hoagland nutrient solution), 10 µmol/L K2CrO4 and

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3 µg/L of each 16 priority PAHs. As the solubility of 7 high-molecular-weight PAHs is

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< 3 µg/L, the PAHs mix was dissolved in 1 mL of methanol (as cosolvent) and then

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added to 1.25 L of Hoagland nutrient solution. The exposure to PAHs and HMs was

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carried out under the same cultivation condition described above. Throughout the

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exposure experiment, the nutrient solution was replenished twice per day with the

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complete Hoagland nutrient solution (without pollutants). Cultivation solutions and

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plant tissues were collected after 14-day exposure.

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Passive air samplers were used to measure the atmospheric deposition of PAHs and

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estimate the evaporation of PAHs from the solution into the air during the entire

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exposure period following a previous study 25. Two samplers with polyurethane foam

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(PUF) disks (14 cm diameter, 1.35 cm thickness and 0.035 g/cm3) were hung above or

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under the experimental pots, and another two were placed 400 m from the

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greenhouse.

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2.3 Sampling

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The harvested plants were washed with deionized water. Subsequently, 6 individual

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plants from each treatment were randomly selected for root architecture analysis.

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Then, the shoots and roots were separated and measured for biomass (fresh weight).

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Part of the fresh roots were used for the electrolytic leakage analysis and the others

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along with the shoots were freeze-dried and ground into fine powder. The PUFs were

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collected and shredded. All samples were stored at -20°C before instrumental

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analysis.

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2.4 Root physiology analysis

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Root architecture analysis followed a previous study 22. Briefly, the washed roots were

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placed in a transparent tray filled with deionized water. The rootlets were separated

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from each other softly by hand. Then, the roots of each plant were scanned by

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Imagery Scan Screen (EPSON Expression 1680). The root parameters including

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projected area (cm2), surface area (cm2), average diameter (mm), total volume (cm3),

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number of tips, number of forks, number of crossings, root length (cm) and root

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length per volume (cm/cm3) were calculated by the WinRHIZO image analysis

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system (2005b, Regent Instruments Inc., Canada). 7

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Electrolytic leakage analysis was carried out using an electrical conductivity meter

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according to previous studies

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cm away from the root tip were picked out, cut into 1 cm segments and put in beaker

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containing 10 mL deionized water. The electrical conductivity was measured after

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25 °C water bath for 3 hr (recorded as EC1). Subsequently, the temperature was

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increased to 100°C for 15 min and a second reading was made after the solution was

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cooled to 25 °C (recorded as EC2). Electrolytic leakage (EL) was calculated according

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to EL= (EC1/EC2)×100%.

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2.5 HMs analysis

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About 0.1 g of plant tissue powders were digested by aqua regia (HCl to HNO3, 3:1,

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v/v) according to our previous study 26. The contents of HMs (Cu, Cd, Pb, Zn and Cr)

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in plant tissues and cultivation solutions were measured by Inductive Coupled Plasma

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Optical Emission Spectrometer (ICP-OES, Optima 8300+, PerkinElmer). The

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recovery rates of Cu, Cd, Pb, Zn and Cr for all samples were 89.2-99.5%,

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91.3-105.7%, 90.7-108.2%, 94.0-101.2% and 82.2-89.5%, respectively. All the HMs

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concentrations were corrected according to reference recovery.

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2.6 PAHs analysis

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PAHs extraction, clean-up and analysis followed previous studies

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with

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perylene-D12), certain amounts of shoots (0.5 g), roots (0.05 g) or PUF disks (0.5 g)

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were Soxhlet extracted for 24 hr with 200 mL of hexane/acetone mixture (1:1, v/v).

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The cultivation solutions (100 mL) were extracted by equivolume methylene chloride

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for 15 min for three times. All of the extracts were concentrated and cleaned-up using

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silica gel (Silica 60; Merck, Germany) and anhydrous sodium sulfate. Subsequently,

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the purified samples were concentrated to 1 mL by nitrogen gas blow and then the

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internal standards (phenanthrene-D10, acenaphthene-D10, anthracene-D10 and

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chrysene-D12) were added. Gas chromatograph-mass spectrometry (GC-MS,

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Shimadzu MSQP2010 plus) was used for quantifying PAHs with DB-5MS (30 m ×

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0.25 mm × 0.25 µm, Agilent) and helium (high purity, 99.999%) as the carrier gas.

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GC temperature condition was: an initial 60 °C hold for 2 min; raise from 60 °C to

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120 °C by 30 °C/min; then raise from 120 °C to 300 °C by 4 °C/min and hold for 8

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min. The internal standards were used for quantification through a standard curve

surrogated

standards

8, 24

. About 0.1 g of fresh root sections approximately 1

(naphthalene-D8,

fluorene-D10,

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. After spike

pyrene-D10

and

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(r2>0.999). The limit of detection (LOD) of all the 16 priority PAHs was 0.1-0.2 µg/L.

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The surrogate standard recoveries for all samples were 81.9-121.7%. All the PAHs

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concentrations were corrected according to reference recovery.

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2.7 Data analysis

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The shoot-to-root (R/S) biomass ratio was calculated by dividing the root biomass by

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the shoot biomass for each individual plant. The bioconcentration factors of root

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(RCF) and shoot (SCF) and translocation factors (TF) were used to evaluate the

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bioaccumulation and acropetal translocation of PAHs/HMs in plant tissues, calculated

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according to Equation (1) to Equation (3):

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RCF = 

220

SCF =

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TF =



(1)

  

 

(2)

   

 

(3)



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where Croots (µg/g dry weight, DW), Cshoots (µg/g DW) and Csolutions (µg/mL) represent

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the contents of PAHs/HMs in roots, shoots and cultivation solutions, respectively.

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Human health risk assessment of PAHs in pakchoi was performed according to

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the USEPA model 29. Benzo(a)pyrene was used as a marker to evaluate the potential

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risks of the 16 carcinogenic PAHs to people by intaking the pakchoi tissues. Total

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equivalence benzo(a)pyrene concentration (TEC) of PAHs was calculated according

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to Equation (4):

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TEC = ∑   × 

(4)

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where Ci is the content of PAH congener i in the pakchoi tissues (ng/g dw), and

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REFi is the corresponding toxicity equivalence factor of PAH congener i. The

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calculated

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fluoranthene, phenanthrene, fluorene and pyrene; 0.01 for anthracene, chrysene and

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benzo[ghi]perylene;

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benzo[k]fluoranthene and indeno(1,2,3-cd)pyrene; and 1 for benzo(a)pyrene and

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dibenz[a,h]anthracene.

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is

0.001

0.1

for naphthalene,

for

acenaphthylene,

benz(a)anthracene,

acenaphthene,

benzo[b]fluorathene,

The estimated daily intake (EDI) through the intake of pakchoi was calculated in 9

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Equation (5). Here,  is the daily intake of pakchoi by Chinese residents (39.4 g/d

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fresh weight, according to a previous study in China 30);  is the body weight for

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an adult (60 kg);  is the moisture content of pakchoi.

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EDI =

"#×$%×('() *+

(5)

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The incremental lifetime cancer risk (ILCR) caused by PAHs exposure from

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intaking pakchoi was calculated in Equation (6). Here,  is the exposure frequency

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(365 days/year); - is the exposure duration (70 years); . is the oral cancer slope

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factor of benzo(a)pyrene, 7.27 (mg/kg/d)-1 (US EPA 1997);  is the conversion

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factor (10-6 mg/ng); /0 is the average lifespan (70 years × 365 days/year=25,500

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days).

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ILCR =

#1$×#2×#1×32×2 4"

(6)

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To explore the noncovalent molecular interaction (cation-π interaction), MINTEQ

250

program was used to estimate the metal speciation in culture medium according to the

251

pH value of the Hoagland nutrient solution (pH=5.5). All the data are expressed in

252

mean ± standard deviation (SD). Pearson’s correlation coefficients between fresh

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weight of shoots or roots, root physiological status, SCF, RCF, TF and the contents of

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PAHs or HMs in shoots or roots were calculated by SPSS 21.0. The statistical

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significance of differences and variance analysis of each parameter was performed

256

using one-way analysis of variance (ANOVA) and t-test (p