Uptake, Translocation, Metabolism, and Distribution of Glyphosate in

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The Uptake, Translocation, Metabolism and Distribution of Glyphosate in Non-target Tea Plant (Camellia sinensis L.) Mengmeng Tong, Wanjun Gao, Weiting Jiao, Jie Zhou, Yeyun Li, Lili He, and Ruyan Hou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02474 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 10, 2017

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Journal of Agricultural and Food Chemistry

1

The Uptake, Translocation, Metabolism and Distribution of

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Glyphosate in Non-target Tea Plant (Camellia sinensis L.)

3

Mengmeng Tong‡ a1, Wanjun Gao‡ a, Weiting Jiaoa, Jie Zhoua, Yeyun Lia, Lili Heb, Ruyan Houa*

4

a

5

Laboratory on Tea Chemisty and Health Effects, School of Tea and Food Science &

6

Technology, Anhui Agricultural University, Hefei, 230036, P. R. China

7

b

8

01003, United States

State Key Laboratory of Tea Plant Biology and Utilization; International Joint

Department of Food Science, University of Massachusetts, Amherst, Massachusetts

9 10 11

E-mail: Ruyan Hou, [email protected] , Tel: +86 -551-65786401

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Mengmeng Tong,[email protected] ;

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Wanjun Gao, [email protected] ;

Weiting Jiao, [email protected] ;

14

Jie Zhou, [email protected] ;

Yeyun Li,[email protected] ;

15

Lili He, [email protected];

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‡

Mengmeng Tong and Wanjun Gao contributed equally to this work.

ACS Paragon Plus Environment


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ABSTRACT

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The uptake, translocation, metabolism and distribution behavior of

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glyphosate in non-target tea plant was investigated. The negative effects

20

appeared to grown tea saplings when the nutrient solution contained of

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glyphosate, above 200 mg L−1. Glyphosate was highest in the roots of the

22

tea plant, where it was also metabolized to AMPA. The glyphosate and

23

AMPA in the roots were transported through the xylem or phloem to the

24

stems and leaves. The amount of AMPA in the entire tea plant was less

25

than 6.0% of the amount of glyphosate. The glyphosate level in fresh tea

26

shoots was less than that in mature leaves at each day. These results

27

indicated that free glyphosate in the soil can be continuously absorbed by,

28

metabolized in, and transported from the roots of the tea tree into edible

29

leaves, and therefore free glyphosate residues in the soil should be

30

controlled to produce teas free of glyphosate.

31

Keywords:

32

distribution; Aminomethyl Phosphonic Acid

glyphosate;

absorption;

translocation;


metabolism;

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INTRODUCTION

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Pesticides can be classified as non-systemic or systemic, based on

35

their transportation abilities in plant tissues.1 Systemic pesticides can

36

penetrate plant tissues, move through the xylem and/or phloem, and be

37

metabolized by the plant. 2 Systemic pesticides and their metabolites may

38

have good effects on the targeted biology, such as insects, fungus or

39

weeds. Glyphosate (PMG, N-[phosphonomethyl]-glycine), a globally sold

40

systemic herbicide, is widely used to remove annual or perennial weeds

41

by killing the whole plant.3 In target weeds, glyphosate can be transported

42

from the fresh shoots to the roots and be metabolized to Aminomethyl

43

Phosphonic Acid (AMPA).

44

AMPA is a phycotoxin4 and genotoxic to aquatic organisms.5

45

Research on human cell lines and in mice has suggested that AMPA is

46

genotoxic to mammalian models.6 Although only few scientific reports

47

have stated that glyphosate may be carcinogenic to humans7, a concern

48

that has been received more attention in recent years. To ensure food

49

safety, many countries and organizations have set maximum residue

50

limits (MRLs) for glyphosate in agriculture products. In commercial tea

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(made from leaves of Camellia sinensis L.), the MRLs for glyphosate are

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2.0 mg kg−1 in the European Union and 1.0 mg L−1 in Japan and China.8

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The uptake, transport, and metabolism of glyphosate has been



54

studied in target plants during the thorough investigation of the

55

mechanism by which it inhibits different weeds.9-12 Glyphosate can be a

56

pollutant to non-target plants through direct contact or through release

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from soil, where it can be aborbed by the roots and then transported to

58

other parts of the plant.13 When glyphosate sprayed in soil, 4% of it can

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be extracted.14 Glyphosate persisted in soil for 30 to 60 days after

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applications of 0.5 to 2.0 kg ha-1 in tea crops and it residues in the tea

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leaves were detected up to 15 days at all three treatment doses.15 There

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has been some research about the metabolism of glyphosate into AMPA

63

in herbaceous weed species.9,

64

concerning the uptake and transport of PMG/AMPA in woody plants.

65 66

16

However, there are few reports

14

C-glyphosate was studied in the leed tree, where the metabolites of

AMPA, sarcosine and other unknown components were detected.17

67

As a perennial woody plant, the tea tree is one of the most important

68

economic crops in China. Glyphosate is an effective herbicide used in tea

69

plantations to control weeds around the roots of tea trees. Glyphosate

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residues have been found by UPLC-MS/MS in 18.6% of a sampling of

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tea products18 at 0.105−3.223 mg kg−1. However, there is no published

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report about the capabilities of the tea tree to uptake, transport, or

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metabolize glyphosate or about the distribution of glyphosate or its

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metabolite AMPA within the tea tree. It is relatively difficult to detect

75

glyphosate and AMPA using conventional methods because they are


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highly polar, non-volatile, and lack chromogenic and fluorescent groups.

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Gas chromatography (GC),19-21 gas chromatography/mass spectrometry

78

(GC/MS),22 high-performance liquid chromatography (HPLC)23-25 and

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liquid chromatography/mass spectrometry (LC/MS)26-31 are the common

80

used methods for determining glyphosate and AMPA. The best selectivity

81

and sensitivity are usually achieved using an HPLC–MS/MS method,

82

based

83

chloroformate (FMOC-Cl).32 This combined method has gradually come

84

to play an important role in the analysis of glyphosate and AMPA.

85

LC-MS/MS-based methods using solid phase extraction (SPE) cleanup

86

coupled with derivatization by FMOC-Cl are available for determination

87

of glyphosate and AMPA levels from tea samples.18 While the

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time-consuming and costly SPE clean-up step may improve method

89

sensitivity, it may also increase the signal variability. The use of a

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QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction

91

approach has been developed with a pretreatment method for the analysis

92

of multiple pesticides in food. GCB, an absorbent of pigments and sterols,

93

was commonly used in the QuEChERS method. Recently, an inexpensive

94

and excellent aborbent, polyvinylpolypyrrolidone (PVPP), has been

95

shown to eliminate the abundant and interfering polyphenols from tea

96

matrices.33

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on

pre-column

derivatization

with

9-Fluorenylmethyl

The aim of the present study was to develop a simple, selective, and



98

reliable method based on a QuEChERS dispersive cleanup approach and

99

derivitization coupled with UPLC-MS/MS for the determination of

100

glyphosate and AMPA levels in different parts of the tea plant.

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Furthermore, this developed method was used to study the uptake,

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transport, metabolism, and distribution of glyphosate in tea plants. This

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data reveals the interaction between glyphosate and the tea plant and

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provides some information about both the source and mitigation of

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glyphosate contamination of tea products. Knowing the distribution of

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glyphosate and AMPA in different parts of the tea plant provides

107

information that can aid decision making regarding safety of tea pre- and

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post-harvest.

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MATERIALS AND METHODS

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Chemicals and Reagents.

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Chromatography grade acetonitrile and dichloromethane (CH2Cl2)

112

were obtained from Tedia Company, OH, USA. Water used for

113

LC–MS/MS was produced in the laboratory with a Milli-Q water

114

purification system (Millipore, Bedford, MA). Graphitized Carbon Black

115

(GCB, 120/400 Mesh) and C18 (230–400 Mesh, 60 Å; SiliCycle, Canada)

116

were obtained from ANPEL Scientific Instrument Co., Ltd. (Shanghai,

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China) and Polyvinylpolypyrrolidone (PVPP) was purchased from

118

Solarbio Science and Technology Co., Ltd. (Beijing, China). An Oasis®


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HLB cartridge for SPE (Oasis® HLB, 3 mL/60 mg) were obtained from

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Waters Corporation (Milford, MA). KOH，acetone，sodium tetraborate

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decahydrate and ammonium acetate were purchased from Sinopharm

122

Chemical Reagent Co., Ltd. (Shanghai, China). HCl was purchased from

123

Shanghai SuYi Chemical Reagent Co., Ltd. (Shanghai, China). The

124

FMOC-Cl was purchased from Alfa Aesar (Tianjin, China). Formic acid

125

was obtained from Aladdin Industrial Corporation (Shanghai, China).

126

Glyphosate

127

Aminomethylphosphonic

128

Ehrenstorfer (Augsburg, Germany). Glyphosate isopropylammonium salt

129

(41%) was obtained from Anhui Sanonda Biological Technology Co., Ltd.

130

(Anhui, China). Standard

131

(98.0%),

stock

Glufosinate acid

(99.0%)

solutions

of

ammonium were

(97.5%)

received

Glyphosate

from

(PMG)

and Dr

and

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Aminomethylphosphonic acid (AMPA) were prepared by weighing 10

133

mg of each analyte and dissolving in 10 ml of water. Working standard

134

solutions were prepared by diluting the standard stock solution with water.

135

All solutions were stored at -4 °C.

136

FMOC-Cl was dissolved in acetone at concentrations of 0.5, 1.0, 10,

137

20, and 40 g L−1. Borate buffer consisted of 5 g of Na2B4O7·10 H2O

138

dissolved in 100 mL of water with the pH adjusted to 9 using 5 mol L−1

139

HCl.

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Annual cuttings of Camellia sinensis cultivar Shu Cha Zao



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(Shucheng County, Anhui province, China) were cultured for six months

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in an automated hydroponic system (Anhui Agricultural University, Hefei,

143

China). The nutrient solution contained (in mg L−1) 30 NH4+, 10 NO3-, 3.1

144

PO4-, 40 K+, 20 Ca2+, 25 Mg2+, 0.35 Fe2+, 0.1 B3+, 1.0 Mn2+, 0.1 Zn2+,

145

0.025 Cu2+, 0.05 Mo+ and 10 Al3+. All tea saplings displayed the same

146

growth rate and were 15-20 cm in height. All cultivation experiments

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were done in the greenhouse at Anhui Agricultural University.

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LC-MS/MS Analysis.

149

Tea sapling sample preparation

150

About 5 g leaves, stems and roots from tea plants were picked, cut

151

into pieces and mixed homogeneity, and then a 0.25 g aliquot of the

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samples were put into mortar, added 10 mL water, and then ground. After

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that, the roots or stem samples mixture were sonicated for 10 min, leaves

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sonicated for 30 min respectively. After ultrasonic extraction, the samples

155

were centrifuged at 5000 rpm for 5 min. The aqueous supernatant was

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transferred to a new centrifuge tube, mixed with 2.5 mL of CH2Cl2 by

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vortex for 2 min and centrifuged at 5000 rpm for 5 min. A 2-ml aliquot of

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supernatant was transferred into a 5-mL centrifuge tube to which 5 mg of

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GCB and 50 mg of PVPP had been added. The mixture was shaken by

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vortex for 2 min and then centrifuged at 10000 rpm for 10 min. The

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supernatant (1.0 mL) was transferred into a 5-mL centrifuge tube and

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mixed with 1 mL borate buffer a by vortex for 2 min. FMOC-Cl (1.0 mL


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of 20 g L−1) was added to the mixture and allowed to react overnight at

164

room temperature. The reaction solution was filtered through a 0.22-µm,

165

hydrophilic PTFE needle filter for subsequent UPLC-MS/MS analysis.

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LC-MS/MS Analysis

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The LC–MS/MS system included an Agilent Series 1290

168

ultra-performance liquid chromatography system (UPLC) and an Agilent

169

6460 triple quadrupole mass spectrometer (QQQ; Agilent Technologies,

170

Palo Alto, CA, USA). The UPLC system was equipped with a quaternary

171

pump, a vacuum solvent degasser, a column oven, and an autosampler.

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A Waters HSS T3 column (particle size: 1.8 µm, length: 100 mm and

173

internal diameter: 2.1 mm) was used with a solvent flow rate of 0.3

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ml·min−1. The column compartment temperature was set at 40 °C and the

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injection volume was set at 5 µL. Mobile phase A was 0.1% formic acid

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in water containing 2 mmol·L−1 ammonium acetate and B was 0.1%

177

formic acid in acetonitrile. The solvent gradient was as follows: 0-0.5 min

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5% B, 0.5-6 min 50% B, 6-7 min 95% B, 7-9 min 5% B, and 9-14 min

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5% B, according to the Chinese standard method SN/T 1923-2007.34

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The mass spectra were acquired using electrospray ionization (ESI)

181

in the positive ionization mode. Analysis of glyphosate and AMPA were

182

performed in multiple reaction monitoring (MRM) mode. The settings

183

were: a drying gas flow of 6 L min−1 with a drying gas temperature of

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325 °C, a nebulizer pressure of 45 psi, a sheath gas temp of 350 °C, and a



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sheath gas flow of 11.0 L min−1. The fragmentor voltage for PMG-FMOC

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and AMPA-FMOC was 135 V, cell accelerator voltage was 7 V, and

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collision energies were 11 and 15 eV, respectively. The mass transition

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ion-pair of PMG-FMOC and AMPA-FMOC were m/z 392 → 88 and 334

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→ 179 respectively.

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Validation of the Analytical Procedure

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Evaluation of the method included fitting to linear equations and

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determining the matrix effect, recovery rate, and limit of quantification

193

(LOQ). The standards produced a linear result between 5 to 500 µg L−1.

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The matrix effect (ME), the change of ionization efficiency in the

195

presence of other compounds, was expressed as the responses of FMOC

196

derivatives of PMG and AMPA in matrix compared to the signal in

197

solvent, calculated by the following equation:35

198 199 200 201 202 203

ME = (

Peak area (spiked extract)

−１)×100%

Peak area (solvent standard)

204

An ME value equal to 0% means that no matrix effect was detected,

205

while positive and negative values indicate enhancements and

206

suppressions, respectively, of the analyte signal by matrix compounds.

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Matrix effects were classified into different categories based on the value

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of this percentage. The matrix effect was not obvious when the values of

209

was within ±20%. 36 The matrix effect in this proposed method was


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evaluated in fresh tea leaves spiked with 0.5 mg kg−1 compared with the

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same concentration of standard sample. Leaves were used because they

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are a more complex matrix than roots or stems. The recoveries of

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glyphosate and AMPA in roots, stems, and leaves at spiked levels of 0.5

214

or 2 mg kg−1 using standard calibration, each concentration level repeated

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6 times. The Limit of Quantitation (LOQ) was calculated as a

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signal-to-noise ratio of 10 (S/N = 10), using the lowest responding

217

concentration for each pesticide at the primary ion transition (quantitation

218

ion transition) obtained from the MS/MS mode.

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Uptake, Transport, Metabolism and Distribution of Glyphosate in

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Tea Saplings.

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Phytotoxicity of Glyphosate to Tea Saplings

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Tea saplings were selected from the hydroponic system and moved

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into one of five red plastic buckets. Five tea saplings were cultivated in

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1.2 L nutrient solution containing 0, 5, 50, 200, or 2000 mg L−1

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glyphosate (41% glyphosate isopropylammonium salt) in different

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buckets. After 1, 3, 5, 7, 10 and 14 days, growth, wilting, and

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phytotoxicity of the tea saplings were noted.

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The uptake, Transport, Metabolism and Distribution of Glyphosate

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Tea saplings were transferred from the hydroponic system into blue

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plastic buckets, with thirty tea saplings cultivated in 6 L nutrient solution

231

containing 5 mg L−1 glyphosate. After 0, 1, 3, 5, 7, 10, 14 and 21 days,



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different parts of the saplings or the whole sapling was collected for

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determination of the content of glyphosate and AMPA.

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RESULTS AND DISCUSSION

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Sample preparation

236

Derivatization reaction

237

The derivatization reaction is shown in Fig. 1. Among the tree

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published methods for derivatization of PMG and AMPA, the

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concentration of FMOC-Cl in solvent differed significantly. One study

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used 0.1 mL of 10 g L−1 FMOC-Cl for derivatization18, another used 1.0

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mL 20g L−1 FMOC-Cl27 and the latest research used 0.3 mL of 1.5 g L−1

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FMOC-Cl.37 In this study, different concentrations (1.0 mL) of the

243

derivation regent FMOC-Cl (0.5, 1.0, 10, 20, or 40 g L−1) were mixed

244

with 0.05 mg L−1 of the pesticide standards. The average peak areas of

245

glyphosate were 188.7, 214.7, 186.3, 212, and 205.7 and of AMPA were

246

202, 234.3, 220.7, 231.7, and 239.7 with the different concentrations of

247

FMOC-Cl. Perhaps 1.0 g L−1 is the economical choice, but 20·g L−1

248

slightly increased the response of them in tea samples compared with the

249

standard samples. Thus, 20 g L−1 FMOC-Cl was used in our proposed

250

method.

251

Extraction

252

Glyphosate and AMPA are strongly polar, water-soluble compounds.


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Water has been used as the extraction solvent in most published methods,

254

whereas different extract methods and treatment times were used, such as

255

sonication or grinding. In this study, glyphosate-treated samples (about

256

3~5 g) were cut to small pieces. A 0.25 g aliquot of the cut pieces of

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sample were ground in mortar and then sonicated for 2, 10 or 30 min to

258

compare the extraction amounts for glyphosate and AMPA. The MS/MS

259

total ion chromatograms for the derived compounds in blank matrix and

260

0.5mg kg-1 spiked samples were shown in Fig. 2. The results showed that

261

there is a baseline separation of glyphosate and AMPA and little

262

interfenrence in the trace of AMPA. The extraction amount of different

263

method was shown in Fig. 3. The amounts of glyphosate and AMPA

264

recovered were significantly lower with the cutting method than with the

265

grinding method. A longer sonication time of 10 min increased the

266

extraction of glyphosate in all samples. However, sonication for longer

267

than 10 min caused a slight decrease in the extraction of PMG from roots

268

and stems, although extraction from leaves still increased with increase

269

sonication time. There were no significant differences in extraction of

270

APMA from roots or stems when sonication increased from 10 to 30 min.

271

From these preliminary extraction tests, the optimized extraction used in

272

the study consisted of grinding all samples and extracting root and stem

273

samples with sonication for 10 min and the fresh leaf samples for 30 min.

274

Clean-up Method



275

Strategies used to minimize matrix interference include improvement

276

of chromatographic selectivity, to avoid interference of co-extracted

277

matrix components, and modification of sample preparation. Tea

278

represents a complex matrix, containing high amounts of free amino acid

279

(1%~2%), polyphenols (18%~36%) and alkaloids (2~4%), which can

280

easily be co-extracted with target pesticides and may interfere with the

281

subsequent derivatization reaction, especially the free amino acids.38 A

282

method has used liquid extraction with CH2Cl2 as solvent combined C18

283

column (alkylsilane bonded to silica gel) to minimize matrix disturbance

284

during determination of glyphosate and AMPA in commercial tea.36 The

285

aborbents in HLB cartridges (m-divinylbenzene and N-vinylpyrrolidone

286

copolymer) have similar characteristics to C18. For example, glyphosate

287

and AMPA residues in tea were determined by alkaline solution

288

extraction and HLB column purification18. The difference between these

289

methods was whether the first water extraction was followed by a

290

re-extraction containing or lacking CH2Cl2. Other studies have shown that

291

the purification effect of CH2Cl2 is better for fresh agricultural products

292

extraction such as soybean,26 rice, maize and soybean,30 olive and other

293

plant materials39. Until now, there is no report about the fresh tea plant.

294

Some co-extracted unknown compounds may greatly affect the pesticide

295

derivatization that follows. Our recent research showed that a QuEChERS

296

extraction method using PVPP combined with GCB effectively and


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297

efficiently cleans up tea samples for detection of pesticide residues.40 To

298

find a suitable cleanup method for fresh samples taken from different

299

parts of the tea plant, three preparation methods were compared, as

300

follows: (A) cleanup the aqueous extract with HLB; (B) re-extract the

301

aqueous extract with CH2Cl2 as solvent and then cleanup with HLB

302

cartridge; (C) re-extract the aqueous extract with CH2Cl2 as solvent and

303

then cleanup with PVPP and GCB as sorbents. As the most complex

304

matrix of our three tissues, tea leaf was chosen to verify the effectiveness

305

of these three clean-up methods. The pesticide standard (4 mg kg−1) was

306

added to the fresh leaf extract after aqueous solution extraction and

307

sonication for 30 min. This mixture was cleaned up by three methods,

308

derivatized with FMOC-Cl, and analyzed by UPLC-MS/MS. To quickly

309

compare the recoveries between the clean-up methods, the standard

310

solutions of glyphosate and AMPA were used rather than matrix match

311

calibration (Figure S1). The lowest recovery of both glyphosate and

312

AMPA resulted from using HLB only. This MS signal decreased possibly

313

because the matrix effect was higher than the other two methods. When

314

the leaf extract was re-extracted with CH2Cl2, the recoveries of

315

glyphosate and AMPA increased. The highest recoveries occurred when

316

the re-extracted sample was mixed with PVPP and GCB. This

317

QuEChERS method resulted in 84.2% and 72.3% recovery rates for

318

glyphosate and AMPA, respectively. It was encouraging to see that the



319

best recovery rate was achieved with the quickest and least expensive

320

sample preparation method.

321

Method validation

322

The standards ranged from 5 to 500 µg L−1 as described in Section

323

2.2. The peak areas for each standard concentration were plotted and fit to

324

a linear equation, from which the correlation coefficients of the two

325

compounds were obtained. As shown in Table S1, the linearity of the

326

standard curves of the two compounds were good and the r2 values were

327

higher than 0.999.

328

The recovery rates and relative standard deviation (RSD) of the two

329

compounds from the roots, stems and leaves of tea plant are as shown in

330

the Table 1. The recovery rate of glyphosate ranged from 82.3 to 116.0%

331

and of AMPA from 72.3 to 94.6%. The RSD (n=6) were 4.70-12.99% and

332

6.50-13.43% respectively. The recovery rate and RSD values meet the

333

requirement for pesticide analysis. The LOQ of glyphosate was 0.1 mg

334

kg−1 for both glyphosate and AMPA in leaf samples and 0.05 mg kg−1 for

335

stem and root samples.

336

The phytotoxicity of glyphosate to tea plant

337

To investigate the phytotoxicity of glyphosate to tea plants, tea

338

saplings were cultivated in a nutrient solution containing different

339

concentrations of glyphosate (0, 5, 50, 200 or 2000 mg L−1). The tea

340

leaves were observed at different times (from 0 to 21 days; Fig. 4). When ACS Paragon Plus Environment

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grown in 2000 mg L−1 glyphosate, mature leaves on 7 DAT and young

342

leaves on 8 DAT showed some dark brown spots. This browning

343

gradually spread over the entire leaves, which began to fall off on 14 DAT.

344

For tea plants cultivated in 200 mg L−1 glyphosate, the mature leaves

345

began to develop dark brown spots on 8 DAT, and the young leaves began

346

to appear dark brown on 10 DAT. The browning increased gradually and

347

the leaves fell off eventually. This negative effect can come from

348

inhibited acquisition of micronutrients such as Mn, Zn, Fe and B, which

349

are involved in plant disease resistance mechanisms. 13, 41 However, the

350

concentrations of 5 mg L−1 and 50 mg L−1 glyphosate, the tea leaves did

351

not show any phytotoxicity over 14 days and showed no significant

352

difference to the control sample. This data indicates the application of

353

glyphosate needs to be controlled under 50 mg L-1 in order to avoid

354

toxicity to the tea plants.

355

The uptake, transport, metabolism and distribution of glyphosate in

356

tea plant

357

After the tea saplings were treated with water nutrient solution

358

containing 5 mg L−1 glyphosate for 21 days in blue box. The uptake,

359

transport, metabolism and distribution of glyphosate and AMPA in tea

360

plants were investigated. Results shown in the Fig. 5-A1, the amount of

361

glyphosate in the roots increased gradually with time, from 113.54 mg

362

kg−1 on day 0 (2 hours) to 294.87 mg kg−1 on day 5, which marked the



363

highest accumulation level, and then decreased to 66.94 mg kg-1 on day

364

21. Compared to the concentration of glyphosate (5 mg L−1) in the

365

nutrient solution, the accumulation coefficient of roots was about 3.5 mg

366

at 2 hours. The amount of glyphosate in stems increased from 17.80 mg

367

kg−1 on day 0 to 46.83 mg kg−1 on day 5 and then decreased to 12.78 mg

368

kg−1 on day 21. The amount of glyphosate in mature tea leaves increased

369

from 0.35 mg kg−1 on day 0 to 14.49 mg kg−1 on day 5 and then

370

decreased to 7.07 mg kg−1 on day 21. The amount of glyphosate in

371

young leaves increased from 0 mg kg−1 on day 0 (after 2 h) to 13.84 mg

372

kg−1 on day 5 and then decreased to 2.17 mg kg−1 on day 21. After

373

glyphosate absorption by the roots, the young leaves accumulated 1.65

374

mg kg−1 glyphosate on the first day. This indicated that glyphosate was

375

transferred from roots to leaves through transpiration pull.42 The

376

cumulative amount of glyphosate in each part of the tea sapling

377

decreased gradually from the 5th day because glyphosate was gradually

378

degraded to AMPA and other metabolites. On days 1, 3, 7, 10, 14 and 21,

379

the cumulative amount of glyphosate in the young leaves (leaves 1-3)

380

were 1.65, 2.61, 3.55, 3.57, 3.86, 2.71 mg kg−1, respectively, amounts

381

that were all less than half of the amounts in mature leaves (4.87, 8.09,

382

12.78, 10.63, 17.31 and 7.07 mg kg−1, respectively) (Figure. 5-A3).

383

Interestingly, the glyphosate level in young and mature leaves was

384

almost equal on day 5 (13.84 and 14.49 mg kg−1). From these results, it


Page 18 of 35

Page 19 of 35


385

seems that teas prepared with young leaves would not only be of higher

386

grade but would also have lower glyphosate content, making these

387

products relatively safer than those made from mature leaves.

388

The levels of the glyphosate metabolite AMPA was determined in

389

roots, stems and leaves of tea saplings (Fig. 5-A2). AMPA was not

390

detected in the young or mature leaves from day 0 to 21, but did increase

391

in stems and roots with time (in roots, from 0 to 2.76 mg kg−1 and in

392

stems from 0 to 0.53 mg kg−1). The absorption of glyphosate and the

393

production of AMPA in the whole plant was also quantified (Fig. 5-B1,

394

B2). The cumulative amount of glyphosate in the plant as a whole

395

ranged from 67.70 to 133.99 mg kg−1 from 0 to 3 days, remained

396

relatively constant during days 3 to 10, and then decreased to 25.26 mg

397

kg−1 by day 21. Meanwhile, the AMPA increased gradually from 0 to

398

1.58 mg kg−1 from 0 to 21 days. The metabolic rate of glyphosate

399

transformation into AMPA in whole plant was calculated (Fig. 5-B3).

400

The rate of glyphosate metabolism into AMPA increased from 0.19% on

401

the 3st day to 5.89% on the 21st day. AMPA represented a very small

402

portion of the PMG/AMPA pool, and so has a lesser impact on tea or tea

403

product safety than does glyphosate.

404

This report represents the first investigation into the uptake, transport,

405

metabolism, and distribution of a systemic pesticide in tea plants over

406

time. This study informs growers that by controlling the free glyphosate



407

residues in the soil one can produce tea product free of glyphosate. It also

408

reminds researchers and growers alike that systemic pesticides such as

409

glyphosate can transfer through the root to leaf. This represents another

410

route generating potential exposure to consumers that differs from the

411

direct contact of pesticide residues applied to edible parts.

412

ACKNOWLEDGEMENT

413

This work was supported by the National Key Research &

414

Development Program (2016YFD0200900) of China, National Natural

415

Scientific Foundation of China (No. 31270728), project of “Nutrition

416

and Quality & Safety of Agricultural Products, Universities Leading

417

Talent Team of Anhui Province” and Natural Science Foundation for

418

Distinguished Young Scholars of Anhui Province (1608085J08).

419

Supporting Information Available: The optimization procedure for

420

different clean-up methods and the linear equations, matrix effect (ME),

421

recoveries of our proposal method were showed in Figure S1.and Table

422

S1.

423

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424

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19. Konar, S. K.; Roy, D. N., Method for the determination of residues of the

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and Its Metabolite, and Glyphosate in Crops. Analyticalences 1997, 13, 283-285. 22. Tsunoda, N., Simultaneous determination of the herbicides glyphosate,

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bialaphos

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chromatography—ion-trap mass spectrometry. J. Chromatogr., A 1993, 637,

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167-173.

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23. Qian, K.; He, S.; Tang, T.; Shi, T.; Li, J.; Cao, Y., A rapid liquid

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chromatography method for determination of glufosinate residue in maize after

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derivatisation. Food Chem. 2011, 127, 722-726.

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24. Hogendoorn, E. A.; Ossendrijver, F. M.; Dijkman, E.; Baumann, R. A., Rapid

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determination of glyphosate in cereal samples by means of pre-column

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derivatisation with 9-fluorenylmethyl chloroformate and coupled-column liquid

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chromatography with fluorescence detection. J. Chromatogr., A 1999, 833,

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67-73.

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25. Hidalgo, C.; Rios, C.; Hidalgo, M.; Salvadó, V.; Sancho, J. V.; Hernández, F.,

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Improved coupled-column liquid chromatographic method for the determination

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of glyphosate and aminomethylphosphonic acid residues in environmental

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waters. J. Chromatogr., A 2004, 1035, 153-157.

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26. Martins-Junior, H. A.; Lebre, D. T.; Wang, A. Y.; Pires, M. A.; Bustillos, O. V.,

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An alternative and fast method for determination of glyphosate and

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aminomethylphosphonic acid (AMPA) residues in soybean using liquid

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chromatography coupled with tandem mass spectrometry. Rapid Commun. Mass

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Spectrom. 2009, 23, 1029-1034.


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27. Wu, X.; Chen, X.; Xiao, H.; Liu, B., Simultaneous determination of glyphosate

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and glufosinate-ammonium residues in tea by ultra performance liquid

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chromatography-tandem

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derivatization. Chinese J. Chromatogr. 2015, 33, 1090-1096.

mass

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with

pre-column

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28. Nagatomi, Y.; Yoshioka, T.; Yanagisawa, M.; Uyama, A.; Mochizuki, N.,

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Simultaneous LC-MS/MS Analysis of Glyphosate, Glufosinate, and Their

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Metabolic Products in Beer, Barley Tea, and Their Ingredients. Biosci.

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Biotechnol. Biochem. 2013, 77, 2218-2221.

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29. Bernal, J.; Martin, M. T.; Soto, M. E.; Nozal, M. J.; Marotti, I.; Dinelli, G.;

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Bernal, J. L., Development and application of a liquid chromatography-mass

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spectrometry method to evaluate the glyphosate and aminomethylphosphonic

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acid dissipation in maize plants after foliar treatment. J. Agric. Food Chem.

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2012, 60, 4017-4025.

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30. Botero-Coy, A. M.; Ibáñez, M.; Sancho, J. V.; Hernández, F., Direct liquid

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chromatography-tandem mass spectrometry determination of underivatized

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glyphosate in rice, maize and soybean. J. Chromatogr., A 2013, 1313, 157-165.

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31. Stefan, E.; Reddy, T. M., Analysis of Glyphosate and Aminomethylphosphonic

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Acid

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Nutritional

Ingredients

and

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Fluorenylmethyloxycarbonyl

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Spectrometry. J. Agric. Food Chem. 2015, 63, 10562-10568.

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Milk

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Chromatography-Mass

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32. Arkan, T.; Molnár-Perl, I., The role of derivatization techniques in the analysis

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of glyphosate and aminomethyl-phosphonic acid by chromatography. Microchem.



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of PVPP in a modified QuEChERS extraction method for UPLC-MS/MS

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analysis of neonicotinoid insecticides in tea matrices. Anal. Methods 2015, 7,

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5521-5529.

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34. SN/T 1923-2007 (in Chinese) Chinese recommendation standard for inprots and

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exports commodity, Determination of glyphosate residues in food for import and

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export-HPLC-MS/MS

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35. Stahnke, H.; Kittlaus, S.; Kempe, G.; Alder, L., Reduction of matrix effects in

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liquid chromatography-electrospray ionization-mass spectrometry by dilution of

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1474-1482.

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36. Ferrer, C.; Lozano, A.; Agüera, A.; Girón, A. J.; Fernándezalba, A. R.,

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37. Oulkar, D. P.; Hingmire, S.; Goon, A.; Jadhav, M.; Ugare, B.; Thekkumpurath,

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A. S.; Banerjee, K., Optimization and Validation of a Residue Analysis Method

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for Glyphosate, Glufosinate, and Their Metabolites in Plant Matrixes by Liquid

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Chromatography with Tandem Mass Spectrometry. J. AOAC Int. 2017, 100,

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631-639.

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38. Wan, X., Chemical constituents and properties in tea. Tea Biochemistry, edition


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3; China Agricultural Publishing House: Beijing, China, 2003; 8-39. 39. Hernández, F.; Hidalgo, C.; Sancho, J. V., Determination of glyphosate residues

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chromatography with fluorescence detection. J. AOAC Int. 2000, 83, 728-734.

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combination of dilution and refined QuEChERS to overcome matrix effects of

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Wood, B. W., Glyphosate effects on diseases of plants. Europ. J. Agronomy

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2009, 31, 144-152.

572 573

42. Shaner, D. L., Role of translocation as a mechanism of resistance to glyphosate. Weed Sci. 2009, 57, 118-123.

574



575

FIGURE CAPTIONS

576

Fig. 1. The derivatization of glyphosate (PMG) and its metabolite AMPA with

577

9-Fluorenylmethyl chloroformate (FMOC-Cl).

578

Fig. 2. MS/MS total ion chromatograms of derived compounds in blank matrix (first

579

column) and from leaves (A), stems (B) or roots (C) spiked (0.5 mg·kg-1) with PMG

580

(second column) or AMPA (third column).

581

Fig. 3. The extraction contents of PMG and AMPA of different part of tea plant by

582

two extraction methods of cutting and grinding.(A1 and A2) Roots; (B1 and B2) Stem;

583

(C) Leaves.

584

Fig. 4. Visual phytotoxicity on tea leaves of different ages, young leaves (leaves 1-3)

585

or mature leaves (leaves 4-6) at different Days After Treatment (DAT) with different

586

concentrations of glyphosate (0, 5, 50, 200, or 2000 mg L−1) delivered in the

587

hydroponic nutrient solution.

588

Fig. 5. The concentration of PMG and AMPA in different parts of tea saplings (A1,

589

A2) and the whole plant (B1, B2) with sustained treatment of 5 mg L−1 glyphosate in

590

nutrient solution. Comparison of the content of glyphosate in young leaves (leaves 1-3)

591

and mature leaves (leaves 4-6) of tea saplings (A3) and the metabolic rate of

592

glyphosate metabolism into AMPA (B3).

593


Page 28 of 35

Page 29 of 35


594

TABLE Table 1 Average recovery rates, Relative Standard Deviation (RSD) and Limit of Quantification (LOQ) of PMG and AMPA compounds from leaves, stems, and roots (n=6). Spiked level Matrix

Leaves

Stem

Roots

Compound

0.5 mg/kg

2 mg/kg

LOQ(mg/kg)

recovery(%)

RSD(%,n=6)

recovery(%)

RSD(%,n=6)

PMG

84.2

5.87

82.3

10.82

AMPA

72.3

10.14

94.6

13.06

0.1

PMG

91.7

5.95

84.9

4.70

0.05

AMPA

91.8

6.50

93.2

8.68

0.05

PMG

116.0

12.99

96.7

7.34

0.05

AMPA

74.8

13.43

85.8

8.73

0.05

595


0.1


596

FIGURE GRAPHICS

Fig. 1. The derivatization of glyphosate (PMG) and its metabolite AMPA with 9-Fluorenylmethyl chloroformate (FMOC-Cl).


Page 30 of 35

Page 31 of 35


Fig. 2. MS/MS total ion chromatograms of derived compounds in blank matrix (first column) and from leaves (A), stems (B) or roots (C) spiked (0.5 mg·kg-1) with PMG (second column) or AMPA (third column).



Fig. 3. The extraction contents of PMG and AMPA of different part of tea plant by two extraction methods of cutting and grinding.(A1 and A2) Roots; (B1 and B2) Stem; (C) Leaves.


Page 32 of 35

Page 33 of 35


Fig. 4. Visual phytotoxicity on tea leaves of different ages, young leaves (leaves 1-3) or mature leaves (leaves 4-6) at different Days After Treatment (DAT) with different concentrations of glyphosate (0, 5, 50, 200, or 2000 mg L−1 ) delivered in the hydroponic nutrient solution.



Fig. 5. The concentration of PMG and AMPA in different parts of tea saplings (A1, A2) and the whole plant (B1, B2) with sustained treatment of 5 mg L−1 glyphosate in nutrient solution. Comparison of the content of glyphosate in young leaves (leaves 1-3) and mature leaves (leaves 4-6) of tea saplings (A3) and the metabolic rate of glyphosate metabolism into AMPA (B3).


Page 34 of 35

Page 35 of 35


Highlights: 1 A modified QuEChERS / UPLC-MS method for glyphosate and AMPA determination in tea plant was developed. 2 Higher concentrations, but not lower, of glyphosate are toxic to tea plants. 3. The interaction between glyphosate and tea plant was investigated. 4. Glyphosate can be rapidly absorbed by tea tree roots and metabolized to AMPA. 5. Glyphosate in fresh tea shoots was always less than that in mature leaves at different DAT.

TOC


Uptake, Translocation, Metabolism, and Distribution of Glyphosate in

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