Uptake, Translocation, and Subcellular Distribution of Azoxystrobin in

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Agricultural and Environmental Chemistry

Uptake, translocation, and subcellular distribution of azoxystrobin in wheat plant (Triticum aestivum L.) Chao Ju, Hongchao Zhang, Shijie Yao, Suxia Dong, Duantao Cao, Feiyan Wang, Hua Fang, and Yunlong Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00361 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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Journal of Agricultural and Food Chemistry

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Uptake, translocation, and subcellular distribution of azoxystrobin in

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wheat plant (Triticum aestivum L.)

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Chao Ju, Hongchao Zhang, Shijie Yao, Suxia Dong, Duantao Cao, Feiyan Wang, Hua

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Fang, Yunlong Yu*

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Institute of Pesticide and Environmental Toxicology, College of Agriculture and

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Biotechnology, Zhejiang University, Hangzhou 310029, China.

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*Correspondence: Yunlong Yu, Tel/Fax: +86-571-88982433, E-mail address:

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[email protected]

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ACS Paragon Plus Environment


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Abstract

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The uptake mechanism, translocation and subcellular distribution of azoxystrobin (5

25

mg kg-1) in wheat plants was investigated under laboratory conditions. The

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wheat-water system reached equilibrium after 96 h. Azoxystrobin concentrations in

27

roots were much higher than in stems and leaves under different exposure times.

28

Azoxystrobin uptake by roots was highly linear at different exposure concentrations,

29

while the bioconcentration factors and translocation factors were independent of the

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exposed concentration at the equilibrium state. Dead roots adsorbed a larger amount

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of azoxystrobin than fresh roots, which was measured at different concentrations.

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Azoxystrobin preferentially accumulated in organelles, and the highest distribution

33

proportion was detected in the soluble cell fractions. This study elucidated that the

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passive transport and apoplastic pathway dominated the uptake of azoxystrobin by

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wheat roots. Azoxystrobin primarily accumulated in roots and could be acropetally

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translocated, but its translocation capacity from roots to stems was limited.

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Additionally, the uptake and distribution of azoxystrobin by wheat plants could be

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predicted well by a partition-limited model.

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Keywords: Azoxystrobin; Wheat; Uptake; Translocation; Subcellular distribution;

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Partition-limited model

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Introduction

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The broad application of pesticides in modern agriculture has caused profound

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concerns about environmental and food safety worldwide.1 Many studies have shown

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that nearly 70-80% of pesticides inevitably move off from target sites after field

50

application and contaminate soils and water through spray drifts, surface runoff and

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leaching processes.2 Residual pesticides can not only re-enter crops to result in

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translocation and accumulation but also lead to biomagnification at the higher trophic

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levels through food web and further threaten food safety.3,4 Except for direct

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applications, root uptake and penetration of the soil interstitial water or irrigation

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sewage is the predominant pathway for crop plants to absorb and accumulate

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pesticides.3,4 Much of the literature has indicated that pesticides with log Kow values

57

in the range from 1-3 are most likely to be taken up by crop roots and move across

58

cell membranes to translocate upwards.5-7 Thus, special attention should be given to

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these compliant pesticides.

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As a highly effective, broad-spectrum and top-selling (global sales in 2016

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amounted to $1.27 billion) systemic strobilurin fungicide, azoxystrobin (log Kow=2.5)

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has been marketed as a seed dressing, soil treatment or foliar spray fungicide in 72

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countries, including China, the United States, Germany and Brazil.8,9 Azoxystrobin is

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mainly used to control plant diseases caused by pathogens of Deuteromycotina,

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Ascomycotina, Basidiomycotina and Oomycota by inhibiting mitochondrial

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respiration in fungi.8 Due to the excellent antifungal activity, the annual production of 3



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azoxystrobin in China has exceeded 3700 tons since 2014, and global annual

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production in 2017 is approximately 13670 tons.10,11 Until now, there are already 502

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kinds of commercially available azoxystrobin products registered in China, most of

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which are applied to wheat and vegetables.12 The sustained and extensive application

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of azoxystrobin has led to its widespread distribution in soils ( 98%.

108

Preparation of wheat seedlings. Seeds of wheat were surface sterilized with a 5%

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sodium hypochlorite solution for 10 min and then rinsed thoroughly with deionized

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water. The seeds were germinated in seedling trays for five days after soaking for 16 h. 5



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Afterwards, the seedlings were transferred to a nutrient solution to continue growing

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for another week in a growth chamber at a light intensity of 250 μmol m-2 s-1 with a

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photoperiod of 16 h each day, at a 25/20°C day/night temperature and relative

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humidity of 70%. The composition of the nutrient solution is listed in Table S4, and it

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had a pH of 6.5. The nutrient solution was renewed every two days. Seedlings of

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similar size (root length 11±1 cm; shoot height 15±1 cm) were selected and

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transplanted into a polyvinylchloride (PVC) box (20 cm×12 cm×12 cm) for the

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uptake experiments. The moisture, lipid and carbohydrate contents of the wheat tissue

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were measured according to the methods proposed by Yang et al. 23

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Uptake experiment at different times. The incubation conditions were the same

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as above. Each box contained 50 seedlings. The wheat roots were completely exposed

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to the nutrient solution (1.5 L) containing azoxystrobin at a concentration of 5 mg L-1,

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which was equivalent to the recommended field dose.14 The wheat roots were

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immersed just below the surface of the solution. The box was wrapped with foil paper,

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and the open areas between the cap and wheat seedlings were sealed with sponge

126

wrapped with foil paper. The wheat seedlings were removed from the solution at time

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intervals of 6, 12, 24, 48, 72, 96, 120, 144 and 168 h and immediately rinsed with

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deionized water, dried with tissue paper and separated into three portions of roots,

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stems and leaves. The final solution was also collected for analysis of the residual

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azoxystrobin content. Similarly, treatments without seedling were conducted as

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unplanted controls to monitor the loss of azoxystrobin in the absence of seedlings, and

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treatments without azoxystrobin were performed as untreated controls to monitor the 6


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possibility of cross-contamination. All treatments were carried out in triplicate.

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Throughout the incubation period, the weight loss of the solution was monitored, and

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the latter was replenished with fresh nutrient solution containing no azoxystrobin. The

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transpiration rate, defined as the decrease in the amount of solution in one day, was

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calibrated by subtracting the evaporation of the unplanted controls and used to reflect

138

the phytotoxicity of azoxystrobin to wheat.24 All wheat samples were stored at -20°C

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until extraction of the azoxystrobin. The uptake, accumulation and translocation

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tendencies of azoxystrobin from the nutrient solution by wheat roots, stems, and

141

leaves were evaluated by the BCFs and TFs as follows:

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Root bioconcentration factor (RCF) =Croot/Cwater

(1)

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Stem bioconcentration factor (SCF) =Cstem/Cwater

(2)

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Leaf bioconcentration factor (LCF) =Cleaf/Cwater

(3)

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TFstem/root= Cstem/Croot

(4)

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TFleaf/stem=Cleaf/Cstem

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where Croot, Cstem, Cleaf and Cwater represent the concentration of azoxystrobin in wheat

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roots, stems, leaves and solution samples, respectively, on a fresh weight basis (mg

149

kg-1).

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Uptake experiment at different concentrations. Wheat seedlings were exposed

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to nutrient solutions with different azoxystrobin concentrations (1, 2, 3, 4 or 5 mg L-1)

152

and sampled at 96 h. The control settings, incubation conditions and sampling

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methods were consistent with the procedures described above.

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Partition-limited model. The uptake of azoxystrobin by wheat roots and

(5)

7



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distribution in plants was described by a partition-limited model.22 The equations are

156

listed in the Supporting Information. αpt, the quasi-equilibrium factor, represented the

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extent of the approach to equilibrium. Theoretically, when the system reached its

158

equilibrium state, αpt was equal to 1. The system had not reached equilibrium, and the

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solute was passively transported into the plant via water in the case of αpt < 1. It was

160

assumed that the αpt value was concentration-independent and metabolism did not

161

affect the partition equilibrium.22,25 Once the αpt value was calculated at a given time,

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Cpt could be estimated on the basis of the azoxystrobin concentration of the nutrient

163

solution.

164

Root sorption at different concentrations. The uptake pathway of azoxystrobin

165

by wheat roots was explored by a root sorption experiment at different concentrations.

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The wheat roots were rinsed thoroughly with deionized water and dried with tissue

167

paper and then cut from the basal node. Fresh roots were chopped (approximately 2

168

mm), weighed (2 g) and placed in 50-ml conical flasks. Afterwards, half of the

169

samples were heated at 105°C for 40 min and then cooled.26 Both fresh and dead

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wheat roots were transferred into 10 mL of a 0.01 M CaCl2 solution containing 100

171

mg L-1 NaN3 and azoxystrobin at initial concentrations of 1, 2, 3, 4 or 5 mg L-1. The

172

conical flask was then sealed and shaken at 150 rpm at 25°C for 24 h with a shaker to

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achieve sorption equilibrium, according to the preliminary kinetic study (Fig S1). The

174

control solution containing azoxystrobin but no wheat roots was simultaneously

175

analyzed to account for the solute loss by shaking. All the treatments were conducted

176

in triplicate. The sorption concentration of azoxystrobin by wheat roots was expressed 8


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by the difference in concentration between the control solution and treatment solution

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and used to construct the concentration-dependent sorption isotherm:

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CΔ=Cc-Cw

(6)

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CΔ=CwKpw

(7)

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where CΔ represents the sorption concentration of azoxystrobin by wheat roots (mg

182

L-1), Cc is the practical measured concentration (mg L-1) in control solution, Cw is the

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practical measured concentration (mg L-1) in treatment solution, and Kpw represents

184

the plant-water partition coefficient, which was acquired from the slope of the

185

corresponding sorption isotherm.

186

Subcellular fractionation. The wheat tissue used for subcellular fractionation was

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obtained from the time-dependent uptake experiment. The subcellular distribution of

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azoxystrobin in wheat tissue was studied following the method of Xin et al. with

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certain modifications.27 The chopped frozen wheat tissue (1-2 mm, 5 g) was mixed

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with a precooled extraction buffer (50 mM tris-(hydroxymethyl)-aminomethane

191

hydrochloride (Tris-HCl), 500 mM sucrose, 1.0 mM dithiothreitol (DTT), 5.0 mM

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ascorbic acid, and 1.0% [w/v] polyvinylpolypyrrolidone (PVPP), with a pH of 7.5),

193

ground with a mortar and passed through an 80-μm sieve (Huahong Top Dyed

194

Melange Yarn CO., LTD. Shaoxing, China.). The residues on the sieve were washed

195

twice with an extraction buffer and designated as the cell wall fraction. The filtrate

196

was subsequently centrifuged at 10,000 g for 30 min. The supernatant solution was

197

treated as the cell soluble fractions (including apoplastic water, vacuolar solution and

198

sol-like cell matrix), and the sediment was regarded as the cell organelles (excluding 9



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the vacuoles). The entire operation process was performed at 4°C. The subcellular

200

fractions were vacuum freeze-dried at -65°C, and the fraction contents were

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determined gravimetrically. The azoxystrobin concentrations in the cell walls,

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organelles, and soluble fractions in different types of wheat tissue were extracted and

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determined. The proportion (P, %) of azoxystrobin in each subcellular fraction was

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calculated as follows:

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P(%)=Msubcellular fraction/Mtotal

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where Msubcellular

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fraction of the roots, stems or leaves, and Mtotal is the total amount of azoxystrobin

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(mg) in the corresponding wheat tissue. The subcellular fraction-concentration factor

209

(SFCF) was used to describe the partition between the subcellular components and

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defined as follows:

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SFCF = Csolid /Csoluble fraction

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where Csolid represents the concentration of azoxystrobin in the cell walls or cell

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organelles (mg kg-1), and Csoluble fraction represents the concentration of azoxystrobin in

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the water-soluble cellular components (mg L-1).

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Sample extraction and analysis. The extraction procedure for azoxystrobin was

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performed according to the method of Qin et al. with minor modifications.28 The

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detailed processing is included in the Supporting Information. All the sample extracts

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were analyzed with a Shimadzu GC/MS-QP2010 PLUS in selected-ion monitoring

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(SIM) mode. Chromatographic separation of azoxystrobin was carried out using an

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HP-5 MS capillary column (30 m × 0.25 mm, 0.25 μm) at a flow rate of 2.0 mL min−1

fraction

(8)

is the amount of azoxystrobin (mg) in a certain subcellular

(9)

10


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with the following temperature program: 180°C for 1 min, 20°C min-1 to 250°C for 1

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min, 15°C min-1 to 300°C, and holding for 5 min. The masses of 344 (m/z) were

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selected as the quantitative ions of azoxystrobin, while 388 and 372 (m/z) were

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regarded as the qualitative ions (Figs S6-S7). The sample concentration was

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quantified by the external standard method with matrix-matched calibration curves.

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Quality control and quality assurance (QC/QA). A recovery experiment was

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performed to verify the effectiveness of the extraction method of azoxystrobin in

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wheat tissues (roots, stems and leaves) and solution. Five replicates of spiked samples

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at 0.1 and 10 mg kg-1 per matrix were investigated. A solvent and matrix blank were

230

analyzed with each set of samples. Satisfactory recoveries (81.5-108.5%) were

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obtained for azoxystrobin in different matrices with relative standard deviations of

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1.9-7.6% (Table S5). Good linearity was observed in the range of 0.01-5 mg L−1 with

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R2 ≥ 0.995 for all matrices (Fig S8). The limit of detection (LOD) and the limit of

234

quantitation (LOQ) of azoxystrobin were 0.004 and 0.01 mg kg-1, respectively.

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Azoxystrobin was not detected in any of the blank samples.

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Data processing and statistical analysis. The dissipation dynamics of

237

azoxystrobin

238

C(t)=A×exp(−k×t), where C(t) is the azoxystrobin concentration at time t, A is a

239

constant, and k is the first-order rate constant. The dissipation half-life of azoxystrobin

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was obtained with equation t1/2=ln2/k. Statistical analysis including one-way analysis

241

of variance (ANOVA) followed by Duncan’s test was performed to compare the

242

difference between the concentrations of azoxystrobin in different types of wheat

in

solution

were

fitted

with

a

11


first-order

kinetic

model:


243

tissue or the difference between the predicted and detected concentrations of

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azoxystrobin using SPSS statistical software package version 17.0 (IBM Corp.,

245

Armonk, NY, USA). Correlation analysis was performed to observe the relationships

246

among the distribution proportions of azoxystrobin in the subcellular components

247

using the SAS statistical software package version 9.2 (SAS Institute Inc., Cary, NC,

248

USA). The level of statistical significance was defined at p < 0.05. All data are

249

presented in terms of the mean ± the standard deviation of the values from three

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independently performed experiments.

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Results and discussion

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Transpiration rate. The results of the transpiration rate of wheat plants exposed to

253

azoxystrobin are shown in Fig S2. There was no significant difference in the

254

transpiration rate between the control and treatment at different sampling times (p =

255

0.465). There was also no significant difference between treatments with different

256

concentrations (p = 0.225). No notable toxicity symptoms were observed in any of the

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treatments. These results indicated that no clear phenotypic phytotoxic effect was

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observed on the growth of wheat as a result of treatment with azoxystrobin at the

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tested levels.

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Dissipation of azoxystrobin in nutrient solution. The dissipation pattern and

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kinetic parameters of azoxystrobin in the nutrient solution are shown in Fig 1 and

262

Table 1. Azoxystrobin dissipation was initially rapid but subsequently slowed in the

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treatment compared with the unplanted control in the time-dependent uptake

264

experiment. The half-lives of azoxystrobin in the unplanted control and treatment 12


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were 231±0 and 115.5±9.5 h, respectively, with a value of R2 ≥0.8117. The difference

266

between the residual azoxystrobin concentration in the nutrient solution of the

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unplanted control and treatment increased rapidly within the first 6 h and remained

268

stable after 96 h.

269

The initial rapid loss of azoxystrobin (6 h, 27.4%) in the treatment was caused by

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the rapid uptake by wheat plants with a rapid increase in azoxystrobin concentration

271

in wheat roots, stems and leaves (Fig 2A). The slow-decline phase in the later stage

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might have been caused by the reduced uptake of wheat plants. The trend in the

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variation of the concentration difference further confirmed that the uptake of

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azoxystrobin by wheat plants decreased but maintained a continuous uptake state after

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6 h.

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Uptake experiment at different times. The variation in the concentration of

277

azoxystrobin in wheat roots, stems, and leaves at different exposure times is shown in

278

Fig 2A. No cross-contamination was detected in the untreated control throughout the

279

experimental period. The data showed that azoxystrobin was taken up rapidly through

280

the roots within the first 6 h with the concentration of 3.98±0.68 mg kg-1, and then the

281

concentration in the roots decreased at 12 h to 2.94±0.04 mg kg-1, accompanied by an

282

initial increase in the concentration of azoxystrobin in the stems (0.57±0.11 mg kg-1)

283

and leaves (0.47±0.03 mg kg-1) (Fig 2A). After 24 h, the azoxystrobin concentration

284

in all parts of the wheat plants remained stable, and the average concentration of

285

azoxystrobin was much greater in roots (2.80±0.15 mg kg-1) than in the stems

286

(0.55±0.03 mg kg-1) and leaves (0.60±0.07 mg kg-1) by almost 5 times. The higher 13



287

concentration in the roots implied that the roots were the major site for the

288

accumulation of azoxystrobin in wheat plants. Only a small amount of azoxystrobin

289

was detected in stems and leaves. Our results are in line with those of Romeh, who

290

observed that azoxystrobin mainly accumulated in the roots of Plantago major L. and

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Helianthus annus L. and could be partially transported upward to the leaves.5 Ge et al.

292

also observed similar results in the uptake of difenoconazole by rice.29 They found

293

difenoconazole mainly accumulated in rice roots and its concentration in leaves was

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much lower than that in roots.29

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The values of BCF, which indicated the degree to which wheat plants assimilated

296

azoxystrobin from the nutrient solution, are shown in Fig 2B. These data indicated

297

that wheat roots absorbed azoxystrobin continuously from the nutrient solution, and

298

the system did not reach equilibrium until 96 h. The RCF gradually increased and

299

reached a stable state (1.47±0.11 L kg-1) after 96 h. A similar equilibrium between the

300

nutrient solution and barley roots was reached after 24 h of cultivation for the

301

dodemorph and tridemorph by Chamberlain et al.30 García-Valcárcel et al. also

302

observed a stable RCF for lettuce roots for clotrimazole, fluconazole and

303

propiconazole after 9 days of cultivation.31 The continuous absorption of azoxystrobin

304

by wheat roots did not lead to a synchronous increase in the concentration of all parts

305

of the wheat plant, which revealed that a fraction of the absorbed azoxystrobin was

306

metabolized in the wheat body. Myung et al. observed that 14C-azoxystrobin could be

307

demethylated in wheat cell culture but the demethylated metabolite was difficult to

308

accumulate because it could be rapidly degraded into water-soluble unknowns.32 The 14


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balance between uptake and metabolism may be the reason for the equilibrium of the

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wheat-water system, as observed for pyrene, lindane and trifluralin.25,33 Thus,

311

maintenance of the concentration of azoxystrobin in wheat plants depended on the

312

sustained uptake of the fungicide from the nutrient solution. Similar results were also

313

observed for SCF (0.29±0.03 L kg-1) and LCF (0.31±0.05 L kg-1) after 96 h.

314

The TFs were also determined to evaluate the capability of wheat plants to

315

transfer azoxystrobin. The TFstem/root and TFleaf/stem increased rapidly up to 6 h and 24 h,

316

respectively, followed by a stable level (0.19±0.02 for TFstem/root and 1.08±0.15 for

317

TFleaf/stem, Fig 2C). TFleaf/stem was notably much higher than TFstem/root (p < 0.0001)

318

throughout the experiments. These data revealed that azoxystrobin could be

319

acropetally translocated, but the translocation capacity of azoxystrobin from the roots

320

to the stems was limited. The translocation of azoxystrobin from stems to leaves

321

occurred more easily than that from roots to stems. The longer transport distance

322

resulted in a longer rise time (24 h) for TFleaf/stem. Similarly, Hinman et al. found that

323

chlordane had limited translocation capacity from the roots of the aquatic plant

324

Hydrilla verticillata royle to shoots.34 The relatively stable TFs of azoxystrobin after

325

24 h also suggested a possible fixed translocation proportion between the different

326

types of wheat tissue.

327

Uptake experiment at different concentrations. To further examine if the

328

uptake and distribution of azoxystrobin are concentration-dependent, the wheat plant

329

was exposed to different concentrations of azoxystrobin for 96 h. Fig 3A shows that

330

the azoxystrobin concentration in wheat roots (0.67±0.08, 0.96±0.01, 1.50±0.08, 15



331

1.92±0.08, 2.44±0.20 mg kg-1) was always much higher than in stems (0.27±0.04,

332

0.31±0.01, 0.36±0.02, 0.45±0.08, 0.55±0.02 mg kg-1) and leaves (0.27±0.05,

333

0.34±0.01, 0.38±0.03, 0.45±0.08, 0.52±0.06 mg kg-1) at different practical solution

334

concentrations (0.58±0.13, 0.96±0.07, 1.24±0.15, 1.58±0.08, 1.78±0.05 mg L-1). The

335

accumulation of azoxystrobin in wheat roots, stems and leaves was linearly related to

336

its concentration in the medium with R2 values of 0.9682, 0.9193 and 0.9778,

337

respectively. Similarly, Su et al. found that rice showed linear uptake of atrazine at

338

different concentrations.35

339

The RCF was always much larger than the SCF and LCF (Fig 3B). No significant

340

variations in the values of the RCF, SCF and LCF were observed, although there were

341

slight shifts in their values depending on the exposed level of azoxystrobin. This

342

phenomenon suggested that the BCFs of azoxystrobin in wheat plants were

343

independent of the exposed concentration at the equilibrium state. Similar results were

344

also obtained by García-Valcárcel et al., who found that RCFs were not influenced by

345

the treatment dose in the case of fluconazole and propiconazole.31

346

Fig 3C shows the change in values of TFleaf/stem and TFstem/root with the

347

azoxystrobin exposure concentration. The TFleaf/stem was always much larger than the

348

TFstem/root, reflecting that the translocation of azoxystrobin from stems to leaves was

349

always easier than from roots to stems. There was no significant difference between

350

the values of TFleaf/stem and TFstem/root regardless of the level of azoxystrobin. The data

351

indicated the presence of a relatively fixed translocation proportion of azoxystrobin

352

from wheat roots to stems and leaves. Similarly, Li et al. showed a relatively fixed 16


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translocation proportion of thiamethoxam and clothianidin between komatsuna

354

(Brassica rapa var. perviridis) roots and shoots at the later stage of exposure.36

355

It has been reported that compounds with poor water solubility and a log Kow >

356

1.8 are not easily translocated upward and accumulate largely in roots.37,38 In addition,

357

compounds with low pKa values are most likely trapped by the weakly alkaline root

358

cell matrix and show decreased translocation to the stem.39 However, once the

359

compound enters the stem, the intensity of transpiration may be the main factor

360

affecting the translocation of the compound from stem to leaf, especially for passively

361

transported compounds.40 The moderate lipophilicity (log Kow = 2.5), low pKa value

362

(< 0) and poor water solubility (6.7 mg L-1) of azoxystrobin (Table S3) indicated that

363

it was readily taken up and accumulated in plant roots, but the translocation of

364

azoxystrobin from roots to stems was limited, consistent with our results. Furthermore,

365

the relatively stable transpiration rate of wheat plants at different times (after 24 h)

366

and concentrations (Fig S2) may be one of the reasons for the relatively stable

367

translocation of azoxystrobin from stems to leaves.

368

Performance of the partition-limited model. The partition-limited model was

369

used to predict the uptake and distribution of azoxystrobin by wheat plants based on

370

the above results. The compositions of the wheat plants are summarized in Table 2.

371

According to these measured wheat compositions, the calculated values of

372

[flipKlip+fchKch+fpw] were 2.776, 3.629, and 5.06 for roots, stems and leaves,

373

respectively. The changes in αpt with time were calculated and are shown in Fig 4.

374

Similar to the variation pattern of BCFs with time, the value of αpt of azoxystrobin for 17



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375

the roots, stems and leaves gradually increased up to 72 h and then demonstrated

376

relatively fixed values of 0.527, 0.08 and 0.06, respectively. These results indicated

377

the lower-than-limit uptake (αpt90%).47

431

Thus, azoxystrobin preferentially accumulated in organelles. However, due to the

432

greatly reduced content of organelles compared with the soluble fractions (Table 2),

433

the distribution of the absolute mass of azoxystrobin in root cells demonstrated the

434

opposite order of soluble fractions > cell walls > cell organelles (Fig 6C). These

435

results suggested that the cell-soluble fractions were important compartments for the

436

storage of azoxystrobin despite their poor enrichment ability. Similar results were

437

acquired for stems and leaves (Figs S4-S5).

438

Interestingly, the highest proportions (59%) of azoxystrobin distributed in cell

439

soluble fractions of wheat roots did not lead to a large translocation of azoxystrobin to

440

the shoots, which could be explained as follows: cell soluble fractions mainly consist 20


Page 20 of 38

Page 21 of 38


441

of apoplastic water, vacuolar solution, and sol-like cell matrix between cells or

442

organelles.47 The azoxystrobin in apoplastic water could be easily transported upward.

443

However, the potential translocation ability of azoxystrobin in the weakly alkaline cell

444

matrix (pH = 7.2) may be limited, and azoxystrobin may accumulate in cell matrix

445

because of its pKa < 0.39,48,49 Conversely, the potential accumulation ability of

446

azoxystrobin in weakly acidic vacuolar solution (pH = 5.5) may be less than cell

447

matrix but its potential translocation ability in vacuole solution may exceed the cell

448

matrix.49 The different translocation abilities of azoxystrobin among these three

449

components mean that not all azoxystrobin in the cell soluble fractions could be

450

translocated to the shoots. The relative translocation contributions of these three

451

components need further study. In fact, the subcellular distribution of organic

452

compounds varies with the Log Kow values, pKa values, relative content of subcellular

453

components and plant species.47,49,50 Zhao et al. found that the subcellular distribution

454

of phthalate ester varies even among different cultivars of the same plant.50

455

In addition, the proportion of azoxystrobin in cellular organelles gradually

456

increased with a decreasing proportion of azoxystrobin in the soluble fractions in

457

roots up to 12 h (Fig 6C). The correlation analysis suggested that the cell organelles

458

gradually absorbed azoxystrobin from the cell soluble fractions when azoxystrobin

459

was initially introduced into the root cells (p = 0.0424, Pearson’s correlation

460

coefficient = -0.9576). This phenomenon was verified by the clear upward trend of

461

the SFCF values of organelles in roots up to 12 h (Fig 6B). Similar results were

462

obtained for leaf but not stem cells. Furthermore, the relatively stable SFCF values 21



463

and distribution proportions of azoxystrobin in the subcellular components of all parts

464

of the wheat plant after 24 h revealed that azoxystrobin also reached a partition

465

equilibrium between the subcellular components. Similarly, Kang et al. observed that

466

the distributions of phenanthrene and pyrene among subcellular components in

467

ryegrass root cells approached a relatively stable state after 96 h of exposure.47

468

According to the above results, the processes of uptake and distribution of

469

azoxystrobin in wheat roots can be described as follows: azoxystrobin reaches the

470

roots by passive transport, passes through root cell walls primarily via apoplastic

471

water, and a portion is transported to stems and leaves; the portions are absorbed in

472

root cell walls, penetrated into remaining cell soluble fractions, and then assimilated

473

by cellular organelles from the remaining soluble fractions; the intracellular

474

distribution then reaches dynamic equilibrium.47 The soluble fractions except

475

apoplastic water can be considered the intracellular buffering distribution phase

476

between the cell walls and organelles.47

477

In summary, azoxystrobin can be taken up and accumulated in wheat roots

478

mainly by passive transport via the apoplastic pathway and can be acropetally

479

translocated, but the translocation capacity of azoxystrobin from roots to stems is

480

limited; azoxystrobin is more prone to accumulation in organelles with a higher lipid

481

content, but soluble fractions of cells are the largest compartments for the storage of

482

azoxystrobin, and the partition-limited model is a good simulation of the uptake of

483

azoxystrobin by wheat roots from water and subsequent distribution of azoxystrobin

484

in wheat plants. This study provides, for the first time, information regarding the 22


Page 22 of 38

Page 23 of 38


485

process by which wheat roots assimilate and accumulate azoxystrobin from water and

486

the azoxystrobin distribution in different types of wheat tissue and subcellular

487

components. In addition, the excellent evaluation ability of the partition-limited model

488

will considerably benefit risk assessments of azoxystrobin in the environment. For

489

food safety, more attention should be paid to the accumulation of azoxystrobin in

490

wheat cereals in the field environment in the future.

491 492

Acknowledgments

493

This work was supported by The National Key Research and Development Program

494

of China (2016YFD0200201) and the National Natural Science Foundation of China

495

(No. 21777141, 21477112, 41271489).

496 497

Statement

498

The authors declare no competing financial interest.

499 500

References

501

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developmental effects and potential mechanisms of azoxystrobin in larval and adult

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Biochem. 2016, 92, 50-57. 23



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Chemosphere. 2015, 119, 1262-1267.

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1493-1500.

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Uptake and translocation of imidacloprid, thiamethoxam and difenoconazole in rice

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plants. Environ Pollut. 2017, 226, 479-485.

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(30) Chamberlain, K.; Patel, S.; Bromilow, R. H., Uptake by roots and translocation to

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shoots of two morpholine fungicides in barley. Pestic Sci, 1998, 54, 1-7.

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(31) García-Valcárcel, A. I.; Loureiro, I.; Escorial, C.; Molero, E.; Tadeo, J. L.,

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Uptake of azoles by lamb's lettuce (Valerianella locusta L.) grown in hydroponic

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conditions. Ecotox Environ Saf. 2015, 124, 138.

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water by ryegrass. Chemosphere. 2002, 48, 335-341.

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pesticides by the rooted aquatic plant Hydrilla verticillata Royle. Environ Sci Techno.

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1992, 26, 3622-3633.

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co-existing organic compounds on uptake of atrazine from nutrient solution by rice

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seedlings (Oryza sativa L.). J Hazard Mater. 2007, 141, 223-229.

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(36) Li, Y.; Long, L.; Yan, H.; Ge, J.; Cheng, J.; Ren, L.; Yu, X., Comparison of

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uptake, translocation and accumulation of several neonicotinoids in komatsuna

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(Brassica rapa var. perviridis) from contaminated soils. Chemosphere. 2018, 200,

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525-541.

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Contaminants by Hybrid Poplar Trees. Environ Sci Techno. 1998, 32(21), 3379-3385.

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(39) Briggs, G. G.; Renê L. O. R.; Bromilow, R, H., Physico-chemical factors

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affecting uptake by roots and translocation to shoots of weak acids in barley. Pestic

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Sci, 1987, 19(2), 101-112.

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(42) Jachetta, J. J.; Appleby, A. P.; Boersma, L., Apoplastic and symplastic pathways

621

of atrazine and glyphosate transport in shoots of seedling sunflower. Plant Physiol.

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1986, 82, 1000-1007.

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(43) Su, Y. H.; Zhu, Y. G., Transport mechanisms for the uptake of organic

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compounds by rice (Oryza sativa) roots. Environ Pollut. 2007, 148, 0-100.

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(44) Sterling, T. M., Mechanisms of herbicide absorption across plant membranes and

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accumulation in plant cells. Weed Sci. 1994, 42(2), 263-276.

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(45) Jablonkai, I.; Dutka, F., Effects of uptake and translocation on herbicide

628

phytotoxicity. J Radioanal Nucl Ch. 1986, 106, 1-7.

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(46) Collins, C.; Fryer, M.; Grosso, A., Plant uptake of non ionic organic chemicals.

630

Environ Sci Techno. 2006, 40, 45-52.

631

(47) Kang, F.; Chen, D.; Gao, Y.; Zhang, Y., Distribution of polycyclic aromatic

632

hydrocarbons in subcellular root tissues of ryegrass (Lolium multiflorum Lam.). BMC

633

Plant Biol. 2010, 10, 210.

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(48) Carpinteiro, I.; Ramil, M.; Rodríguez, I.; Cela, R., Determination of fungicides in

635

wine by mixed-mode solid phase extraction and liquid chromatography coupled to

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tandem mass spectrometry. J Chromatogr A. 2010, 1217, 7484-7492.

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(49) Hofstetter, S.; Beck, A.; Trapp, S.; Buchholz, A., How to design for a tailored

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subcellular distribution of systemic agrochemicals in plant tissues. J. Agric. Food 29



639

Chem. 2018, 66, 8687-8697.

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(50) Zhao, H. M.; Du, H.; Xiang, L., Variations in phthalate ester (PAE) accumulation

641

and their formation mechanism in Chinese flowering cabbage (Brassica

642

parachinensis L.) cultivars grown on PAE-contaminated soils. Environ Pollut. 2015,

643

206, 95-103.

644 645

Figure captions

646

Fig 1. The dynamics of azoxystrobin dissipation in the unplanted control nutrient

647

solution and treatment nutrient solution. D-value: the concentration difference value.

648

Fig 2. (A) Uptake concentrations of azoxystrobin in roots, stems and leaves of wheat

649

as a function of exposure time on a fresh weight basis (mg kg-1). (B) Bioconcentration

650

factors of azoxystrobin in roots (RCF), stems (SCF) and leaves (LCF) of wheat as a

651

function of exposure time. (C) Translocation factors of azoxystrobin in stems/roots

652

and leaves/stems as a function of exposure time.

653

Fig 3. (A) Uptake concentrations of azoxystrobin in roots, stems and leaves of wheat

654

at different practical measured solution concentrations (mg L-1) on a fresh weight

655

basis (mg kg-1). (B) Bioconcentration factors of azoxystrobin in roots (RCF), stems

656

(SCF) and leaves (LCF) of wheat at different practical measured solution

657

concentrations (mg L-1). (C) Translocation factors of azoxystrobin in stems/roots and

658

leaves/stems at different practical measured solution concentrations (mg L-1).

659

Fig 4. The quasi-equilibrium factor (αpt) of azoxystrobin in roots, stems and leaves of

660

wheat as a function of exposure time. 30


Page 30 of 38

Page 31 of 38


661

Fig 5. Sorption isotherms of azoxystrobin for fresh and dead wheat roots at different

662

practical measured solution concentrations (mg L-1).

663

Fig 6. (A) Concentrations of azoxystrobin distributed in three subcellular fractions of

664

wheat roots as a function of exposure time. (B) Subcellular fraction bioconcentration

665

factor (SFCF) of azoxystrobin in cell walls and organelles in wheat roots as a function

666

of exposure time. (C) Proportions of azoxystrobin distributed in three subcellular

667

fractions of wheat roots as a function of exposure time.

668

Fig S1. The sorption dynamics of azoxystrobin in fresh and dead wheat roots.

669

Fig S2. Transpiration rate of wheat plants at different exposure times (A) and different

670

initial concentrations (B).

671

Fig S3. The predicted and measured concentrations of azoxystrobin in wheat roots (A),

672

stems (B) and leaves (C) at different practical measured solution concentrations (mg

673

L-1) on a fresh weight basis (mg kg-1).

674

Fig S4. (A) Concentrations of azoxystrobin distributed in three subcellular fractions of

675

wheat stems as a function of exposure time. (B) Subcellular fraction bioconcentration

676

factor (SFCF) of azoxystrobin in cell walls and organelles in wheat stems as a

677

function of exposure time. (C) Proportions of azoxystrobin distributed in three

678

subcellular fractions of wheat stems as a function of exposure time.

679

Fig S5. (A) Concentrations of azoxystrobin distributed in three subcellular fractions of

680

wheat leaves as a function of exposure time. (B) Subcellular fraction bioconcentration

681

factor (SFCF) of azoxystrobin in cell walls and organelles in wheat leaves as a

682

function of exposure time. (C) Proportions of azoxystrobin distributed in three 31



683

subcellular fractions of wheat leaves as a function of exposure time.

684

Fig S6. GC-MS chromatograms of azoxystrobin in wheat roots at 24 h.

685

Fig S7. Mass spectrum of azoxystrobin.

686

Fig S8. The calibration curves of azoxystrobin (0.01–5.0 mg L−1) in the nutrient

687

solution.

688 689

Tables

690

Table 1. Dissipation kinetic parameters of azoxystrobin in the nutrient solution at

691

initial concentrations.

692

Table 2. Determined weight percentage of plant compositions of wheat.

693

Table 3. The predicted and measured concentrations of azoxystrobin in wheat roots,

694

stems and leaves at different practical measured solution concentrations (mg L-1).

695

Table 4. The sorption isotherms of azoxystrobin in fresh and dead wheat roots.

696

Table S1. The concentration range of azoxystrobin in surface water samples.

697

Table S2. The concentration range of azoxystrobin in soil samples.

698

Table S3. Physicochemical properties of azoxystrobin at 25°C.

699

Table S4. Composition of the nutrient solution.

700

Table S5. Recoveries and RSD values for azoxystrobin in two spiked levels from 4

701

matrices.

702 703 704 32


Page 32 of 38

Page 33 of 38


705

Table 1. Dissipation kinetic parameters of azoxystrobin in the nutrient solution at

706

initial concentrationsa parameters

control

treatment

Ab

5.6012 0.003 231±0.0 0.9235

4.0498 0.006 115.5±9.5 0.8117

kc t1/2(h)d R2 707

aAzoxystrobin

708

C(t) = A×exp(−k×t), where C(t) = azoxystrobin concentration at t time, bA = constant; ck =

709

dissipation kinetic constant, and dt1/2 = half-life or time required for 50% dissipation of the initial

710

azoxystrobin concentration. The data for t1/2 are the mean value ± the standard deviation, n = 3.

dissipation in the nutrient solution was described by the first-order kinetic model:

711 712

713

Table 2. Determined weight percentage of plant compositions of wheata cell organelles (%)

cell soluble components (%)

8.94±0.17

1.67±0.01

89.39±0.17

0.84±0.02

13.22±0.15

3.07±0.07

83.70±0.22

1.29±0.03

11.72±0.09

3.22±0.08

85.06±0.17

plant part

water content (%)

carbohydrates (%)

lipid content(%)

cell (%)

root

92.29±0.02

7.14±0.16

0.56±0.01

stem

90.65±0.67

8.51±0.71

leaf

88.87±0.75

9.84±0.62

aThe

wall

data presented are the mean value ± the standard deviation, n = 3

714

33



Page 34 of 38

715 716

Table 3. The predicted and measured concentrations of azoxystrobin in wheat roots, stems and leavesa at different practical measured solution

717

concentrations (mg L-1) roots Cwb (mg L-1)

Cpc (mg kg-1)

Cdd (mg kg-1)

0.58±0.13

0.82±0.18f

0.67±0.08f

0.96±0.07

1.41±0.10f

0.96±0.01g

1.24±0.15

1.78±0.22f

1.50±0.08f

1.58±0.08

2.31±0.11f

1.92±0.08g

1.78±0.05

2.60±0.07f

2.44±0.20f

stems SEe

leaves

Cp (mg kg-1)

Cd (mg kg-1)

23.5

0.16±0.04g

0.27±0.04f

47.0

0.28±0.02f

0.31±0.01f

19.1

0.36±0.04f

0.36±0.02f

19.8

0.46±0.02f

0.45±0.08f

6.50

0.52±0.01f

0.55±0.02f

(%)

SE

SE

Cp (mg kg-1)

Cd (mg kg-1)

-39.8

0.17±0.04f

0.27±0.05f

-35.7

-10.2

0.30±0.02f

0.34±0.01g

-14.4

1.09

0.38±0.05f

0.38±0.03f

0.810

2.41

0.48±0.02f

0.45±0.08f

8.02

-5.11

0.54±0.02f

0.52±0.06f

5.41

(%)

(%)

718

cThe

719

solution after 96 h; cCp and dCd are the predicted and detected concentrations of azoxystrobin in roots, stems or leaves; eSE is the deviation of predicted and detected

720

concentrations of azoxystrobin, SE = (predicted value – detected value) × 100 / (detected value). Letters are used to compare the differences between the predicted

721

and detected concentrations of azoxystrobin, and different letters indicate significant differences (p < 0.05, Duncan's test).

data presented are the mean value ± the standard deviation, n = 3. bCw denotes the different practical measured concentrations of azoxystrobin in the nutrient

722 723 724

34


Page 35 of 38

725

726


Table 4. The sorption isotherms of azoxystrobin in fresh and dead wheat roots parameters

fresh roots

dead roots

Kpwa R2

0.549 0.9986

2.5496 0.9892

aK

pw

represents the plant-water partition coefficient

727 728 729

Fig 1

730 731 732

Fig 2

733 734 735 736 35



737

Fig 3

738 739 740 741

Fig 4

742 743 744

Fig 5

36


Page 36 of 38

Page 37 of 38


745 746 747

Fig 6

748 749 750 751 752 753 754 755 756 757 37



758

Table of Contents Graphic

759 760 761

38


Page 38 of 38

Uptake, Translocation, and Subcellular Distribution of Azoxystrobin in

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