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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Agricultural and Food Chemistry
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The Uptake, Translocation, Metabolism and Distribution of
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Glyphosate in Non-target Tea Plant (Camellia sinensis L.)
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Mengmeng Tong‡ a1, Wanjun Gao‡ a, Weiting Jiaoa, Jie Zhoua, Yeyun Lia, Lili Heb, Ruyan Houa*
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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, hry@ahau.edu.cn , Tel: +86 -551-65786401
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Mengmeng Tong,18255108995@163.com ;
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Wanjun Gao, wanjunGao6574@163.com ;
Weiting Jiao, 657929028@qq.com ;
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Jie Zhou, 18270823219@163.com ;
Yeyun Li,lyy@ahau.edu.cn ;
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Lili He, lilihe@foodsci.umass.edu;
16
‡
Mengmeng Tong and Wanjun Gao contributed equally to this work.
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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
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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
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tea plant, where it was also metabolized to AMPA. The glyphosate and
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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
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shoots was less than that in mature leaves at each day. These results
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indicated that free glyphosate in the soil can be continuously absorbed by,
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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.
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Keywords:
32
distribution; Aminomethyl Phosphonic Acid
glyphosate;
absorption;
translocation;
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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
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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
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weeds. Glyphosate (PMG, N-[phosphonomethyl]-glycine), a globally sold
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systemic herbicide, is widely used to remove annual or perennial weeds
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by killing the whole plant.3 In target weeds, glyphosate can be transported
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from the fresh shoots to the roots and be metabolized to Aminomethyl
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Phosphonic Acid (AMPA).
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AMPA is a phycotoxin4 and genotoxic to aquatic organisms.5
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Research on human cell lines and in mice has suggested that AMPA is
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genotoxic to mammalian models.6 Although only few scientific reports
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have stated that glyphosate may be carcinogenic to humans7, a concern
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that has been received more attention in recent years. To ensure food
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safety, many countries and organizations have set maximum residue
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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
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studied in target plants during the thorough investigation of the
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mechanism by which it inhibits different weeds.9-12 Glyphosate can be a
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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
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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
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in herbaceous weed species.9,
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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
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As a perennial woody plant, the tea tree is one of the most important
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economic crops in China. Glyphosate is an effective herbicide used in tea
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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
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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
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(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
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used methods for determining glyphosate and AMPA. The best selectivity
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and sensitivity are usually achieved using an HPLC–MS/MS method,
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based
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chloroformate (FMOC-Cl).32 This combined method has gradually come
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to play an important role in the analysis of glyphosate and AMPA.
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LC-MS/MS-based methods using solid phase extraction (SPE) cleanup
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coupled with derivatization by FMOC-Cl are available for determination
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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
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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
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approach has been developed with a pretreatment method for the analysis
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of multiple pesticides in food. GCB, an absorbent of pigments and sterols,
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was commonly used in the QuEChERS method. Recently, an inexpensive
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and excellent aborbent, polyvinylpolypyrrolidone (PVPP), has been
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shown to eliminate the abundant and interfering polyphenols from tea
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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
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reliable method based on a QuEChERS dispersive cleanup approach and
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derivitization coupled with UPLC-MS/MS for the determination of
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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
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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)
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were obtained from Tedia Company, OH, USA. Water used for
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LC–MS/MS was produced in the laboratory with a Milli-Q water
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purification system (Millipore, Bedford, MA). Graphitized Carbon Black
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(GCB, 120/400 Mesh) and C18 (230–400 Mesh, 60 Å; SiliCycle, Canada)
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were obtained from ANPEL Scientific Instrument Co., Ltd. (Shanghai,
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China) and Polyvinylpolypyrrolidone (PVPP) was purchased from
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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
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Chemical Reagent Co., Ltd. (Shanghai, China). HCl was purchased from
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Shanghai SuYi Chemical Reagent Co., Ltd. (Shanghai, China). The
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FMOC-Cl was purchased from Alfa Aesar (Tianjin, China). Formic acid
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was obtained from Aladdin Industrial Corporation (Shanghai, China).
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Glyphosate
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Aminomethylphosphonic
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Ehrenstorfer (Augsburg, Germany). Glyphosate isopropylammonium salt
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(41%) was obtained from Anhui Sanonda Biological Technology Co., Ltd.
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(Anhui, China). Standard
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(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
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mg of each analyte and dissolving in 10 ml of water. Working standard
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solutions were prepared by diluting the standard stock solution with water.
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All solutions were stored at -4 °C.
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FMOC-Cl was dissolved in acetone at concentrations of 0.5, 1.0, 10,
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20, and 40 g L−1. Borate buffer consisted of 5 g of Na2B4O7·10 H2O
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dissolved in 100 mL of water with the pH adjusted to 9 using 5 mol L−1
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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,
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China). The nutrient solution contained (in mg L−1) 30 NH4+, 10 NO3-, 3.1
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PO4-, 40 K+, 20 Ca2+, 25 Mg2+, 0.35 Fe2+, 0.1 B3+, 1.0 Mn2+, 0.1 Zn2+,
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0.025 Cu2+, 0.05 Mo+ and 10 Al3+. All tea saplings displayed the same
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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.
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Tea sapling sample preparation
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About 5 g leaves, stems and roots from tea plants were picked, cut
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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
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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
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room temperature. The reaction solution was filtered through a 0.22-µm,
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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
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ultra-performance liquid chromatography system (UPLC) and an Agilent
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6460 triple quadrupole mass spectrometer (QQQ; Agilent Technologies,
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Palo Alto, CA, USA). The UPLC system was equipped with a quaternary
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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
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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%
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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)
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in the positive ionization mode. Analysis of glyphosate and AMPA were
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performed in multiple reaction monitoring (MRM) mode. The settings
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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
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(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
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presence of other compounds, was expressed as the responses of FMOC
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derivatives of PMG and AMPA in matrix compared to the signal in
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solvent, calculated by the following equation:35
198 199 200 201 202 203
ME = (
Peak area (spiked extract)
−1)×100%
Peak area (solvent standard)
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An ME value equal to 0% means that no matrix effect was detected,
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while positive and negative values indicate enhancements and
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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
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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
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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
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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
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Derivatization reaction
237
The derivatization reaction is shown in Fig. 1. Among the tree
238
published methods for derivatization of PMG and AMPA, the
239
concentration of FMOC-Cl in solvent differed significantly. One study
240
used 0.1 mL of 10 g L−1 FMOC-Cl for derivatization18, another used 1.0
241
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
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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
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202, 234.3, 220.7, 231.7, and 239.7 with the different concentrations of
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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.
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Extraction
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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,
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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
257
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
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Strategies used to minimize matrix interference include improvement
276
of chromatographic selectivity, to avoid interference of co-extracted
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matrix components, and modification of sample preparation. Tea
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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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efficiently cleans up tea samples for detection of pesticide residues.40 To
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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
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best recovery rate was achieved with the quickest and least expensive
320
sample preparation method.
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Method validation
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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.
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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
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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
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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
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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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42. Shaner, D. L., Role of translocation as a mechanism of resistance to glyphosate. Weed Sci. 2009, 57, 118-123.
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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
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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
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Journal of Agricultural and Food Chemistry
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FIGURE GRAPHICS
Fig. 1. The derivatization of glyphosate (PMG) and its metabolite AMPA with 9-Fluorenylmethyl chloroformate (FMOC-Cl).
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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).
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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.
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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.
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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).
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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
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