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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Environmental Science & Technology

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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

3

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

14 15

*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]

26 27 1

ACS Paragon Plus Environment


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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

55

persistence in the environment

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

57

(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

60

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

76

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,

82

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

83

seldom discussed

3, 7

. Many previous

10-12

.

6, 13

. Some reports point out that priority PAHs usually

14

, 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

87

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

103

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

106

and the underlying mechanism.

22

. PAHs and HMs also have synergistic

23

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

107

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

108

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

113

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

119

exposure.

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2. Materials and Methods

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2.1 Chemicals

122

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

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acenaphthene,

124

benz[a]anthracene,

125

benzo[a]pyrene,

126

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

127

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

128

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

129

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

130

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

131

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

132

analytical reagent grade.

133

2.2 Hydroponic experiment

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

135

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

136

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

137

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

138

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

140

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

141

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

142

nutrient solution was replaced by the complete Hoagland nutrient solution containing

143

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

144

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

145

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

146

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

6


pyrene,

and

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

148

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

149

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

150

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

156

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

159

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

167

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

168

analysis.

169

2.4 Root physiology analysis

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

171

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

177

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



178

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Electrolytic leakage analysis was carried out using an electrical conductivity meter

179

according to previous studies

180

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

181

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

182

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

183

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

184

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

185

to EL= (EC1/EC2)×100%.

186

2.5 HMs analysis

187

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

188

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

189

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

190

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

191

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

193

concentrations were corrected according to reference recovery.

194

2.6 PAHs analysis

195

PAHs extraction, clean-up and analysis followed previous studies

196

with

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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,

8


6, 27, 28

. After spike

pyrene-D10

and

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210

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

211

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

212

concentrations were corrected according to reference recovery.

213

2.7 Data analysis

214

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

215

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

216

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

217

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

218

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

219

RCF =

220

SCF =

221

TF =

(1)

(2)

(3)

222

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

223

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

224

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

225

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

227

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

228

to Equation (4):

229

TEC = ∑ ×

(4)

230

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

231

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

232

calculated

233

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

234

benzo[ghi]perylene;

235

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

236

dibenz[a,h]anthracene.

237

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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238

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

239

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

240

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

241

EDI =

"#×$%×('() *+

(5)

242

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

243

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

244

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

245

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

246

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

247

days).

248

ILCR =

#1$×#2×#1×32×2 4"

(6)

249

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

253

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

254

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

255

significance of differences and variance analysis of each parameter was performed

256

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

Heavy metal exposure alters the uptake behaviour of 16 priority

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