Rinsing tea before brewing decreases pesticide residues in tea

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Food Safety and Toxicology

Rinsing tea before brewing decreases pesticide residues in tea infusion Wanjun Gao, Min Yan, Yu Xiao, Yaning Lv, Chuanyi Peng, Xiaochun Wan, and Ruyan Hou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04908 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 17, 2018

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Journal of Agricultural and Food Chemistry

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Rinsing tea before brewing decreases pesticide residues in tea

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infusion

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Wanjun Gaoa1, Min Yana1, Yu Xiaoab, Yaning Lvab, Chuanyi Penga, Xiaochun Wana*,

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Ruyan Houa*

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a

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Science & Technology, Anhui Agricultural University, Hefei, 230036, China; and Anhui

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Province Key Lab of Analysis and Detection for Food Safety, Hefei, 230022, China;

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b

State Key Laboratory of Tea Plant Biology and Utilization; School of Tea and Food

Hefei Customs Technology Center, Hefei, 230022, China;

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* Corresponding authors: Ruyan Hou, [email protected], Tel: +86 -551-65786765;

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Xiaochun Wan, [email protected];

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Wanjun Gao, [email protected];

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Min Yan, [email protected];

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Yu Xiao, [email protected];

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Yaning Lv, [email protected];

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Chuanyi Peng, [email protected]

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1

Wanjun Gao and Min Yan contributed equally to this work.

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ACS Paragon Plus Environment


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Abstract: Rinsing dried tea leaves before brewing is a traditional way of preparing rolled

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oolong tea in China. This study analyzes how rinsing green, black and oolong teas before

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brewing affects the levels of pesticide residues in the tea infusion. Eight representative

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insecticides of different polarities were tracked, namely three neonicotinoids (acetamiprid,

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imidacloprid, and thiamethoxam), two organophosphates (dimethoate and malathion) and

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three pyrethroids (bifenthrin, beta-cypermethrin, and fenvalerate). The results showed

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that the 8 pesticides transferred into the rinse water at rates between 0.2% and 24% after

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5, 10, 20, or 30 seconds. Rinsing tea before brewing reduced the pesticide risk levels by 5

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to 59% in the tea infusion. Five functional components, such as epigallocatechin gallate

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and caffeine, were reduced by 0 to 11% in the tea infusion. The results can be used to

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develop an effective method of rinsing tea before brewing that reduces pesticide exposure

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risks.

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Keywords: rinsing tea; kungfu tea; pesticide exposure risk; tea rituals; Chinese tea

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ceremony

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Introduction

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Tea is the second most consumed beverage globally, after water.1-3 Tea has been

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consumed for centuries as a brew of unfermented (green), semi-fermented (oolong), and

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fermented (black) tea.4 Tea has biological activities that often confer health benefits to

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consumers, most attributable to the polyphenols and alkaloids it contains. Studies have

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indicated that tea might reduce the risk of cancer, lower cholesterol and help control body

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weight.5-8 Most tea is consumed by brewing the dried tea leaves. Traditionally, tea is

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brewed by steeping 3 g of tea in 150 mL of boiled water for 5 min, and the same leaves

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will be used two or three times. There is a custom of washing or rinsing the tea

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immediately before brewing, especially for oolong tea.8 To rinse the tea, a small amount

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of boiling water is poured onto the tea leaves, the mix is briefly and gently stirred for a

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few seconds, and the water is poured away. This rinse not only enhances the sensory

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quality as one prepares and drinks the tea, but also removes dust or other contaminants,

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such as polycyclic aromatic hydrocarbons (PAHs).8

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Neonicotinoid, organophosphorus, and pyrethroid insecticides are commonly

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applied during tea cultivation to control insect pests.9, 10 Consumers may be exposed to

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residues of these compounds on or in tea leaves through drinking tea. Numerous studies

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have

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organophosphates, and pyrethroids to brewed tea.11-16 The transfer rate of a pesticide is

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influenced by its physicochemical properties, including water solubility, octanol-water

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partition coefficient (log Kow) and vapor pressure.3, 17, 18 A longer brew time and a higher

investigated

the

transfer

rates

of

pesticides

3


such

as

neonicotinoids,


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temperature also contribute to the transfer rates.19-22 It is possible that rinsing tea prior to

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brewing may reduce the amount of residues initially in the dried tea leaves and thus

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reduce the exposure risk for those consuming the tea infusion. This rinse may also reduce

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nutrients, such as polyphenols, in the infusion. At the present, there are no reports about

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these two possibilities.

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Chromatography tandem mass spectrometry analysis can be used to quantify trace

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levels of pesticide residues due to its precision, robust residue analysis, and high

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sensitivity.10, 23-25 This study used LC-MS/MS and GC-MS/MS to evaluate the transfer of

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8 kinds of pesticides, with different polarities, to both the rinse water and the tea infusion

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made using rinsed or not rinsed tea leaves. The levels of tea functional components

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(epigallocatechin gallate, caffeine, etc) were also measured after rinsing and brewing.

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Samples were taken for extraction of pesticide residues before rinsing, from the rinsate,

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and from the infusion after brewing (Fig. 1). This study provides information regarding

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the risk of exposure to pesticide residues from drinking tea and may be used to develop a

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public health campaign promoting a safer way of drinking tea.

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Materials and methods

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Materials. Primary secondary amine (PSA, 40-63 μm) and Graphitized Carbon Black

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(GCB, 38-125 μm) were purchased from ANPEL Scientific Instrument Co., Ltd.

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(Shanghai, China). Polyvinylpolypyrrolidone (PVPP) was obtained from Solarbio

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Science & Technology Co., Ltd. (Beijing, China). Dimethoate (98.5%), malathion

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(99.4%), acetamiprid (98.1%), imidacloprid (98.0%), thiamethoxam (98.5%), bifenthrin

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(99.0%), beta-cypermethrin (99.0%) and fenvalerate (98.3%) were purchased from Dr.

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Ehrenstorfer GmbH (Augsburg, Germany). HPLC-grade acetonitrile, n-hexane, toluene

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and dichloromethane were purchased from Tedia Company (Farfield, OH, USA). Formic

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acid was obtained from Aladdin Industrial Corporation (Los Angeles, USA). NaCl,

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anhydrous MgSO4 and anhydrous Na2SO4 were purchased from Sinopharm Chemical

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Reagent Co., Ltd. Anhydrous MgSO4 and anhydrous Na2SO4 were heated at 550 °C for 5

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h and kept in a desiccator.

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Standard stock solutions of dimethoate, malathion, acetamiprid, imidacloprid,

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thiamethoxam were each prepared in acetonitrile at 1000 μg mL-1 (ppm) and bifenthrin,

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beta-cypermethrin and fenvalerate were each prepared in n-hexane at 1000 ppm. The

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working solutions were prepared by diluting the standard stock solution with acetonitrile

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or n-hexane. All solutions were stored at 4 °C in a refrigerator. Blank tea samples were

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made from the untreated fresh tea leaves in field trail and they were used to investigate

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the recoveries of pesticide residues in dried tea, rinsate and tea infusion. The values were

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calculated using matrix match solution standards.

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Field trials and sampling. The presence status of pesticide residues in tea may affect its

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pesticide rinsing and leaching behavior, therefore the rinsing and leaching effects of

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pesticide residues in commercial tea can not be truly reflected with widely used spiked 5



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method. However, the positive tea samples with multi-residues are obtained difficultly.

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So, the positive tea samples with multi-pesticides was gotten from field trial. The field

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trial was carried out at the Tea Experimental Farm of Anhui Agricultural University at

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Hefei, Anhui province, China (31° N, 117 °E). The grown tea tree of Camellia sinensis L.

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cultivar Nong Kang Zao were cultivated in the field. The trial (October 2017) consisted

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of plot areas of 166.7 m2 for treatment with a combination of the 8 pesticides. The treated

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plot was partitioned and isolated by two untreated guard rows with untreated plot. The

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8-pesticide combination was sprayed with a hand operated backpack sprayer, using a

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recommended formulation volume of 750 L ha−1, at the dosages of 75 mL active

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ingredient (a.i.) ha−1 (fenvalerate), 225 mL a.i. ha−1 (malathion), 45 g a.i. ha−1

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(acetamiprid), 67.5 mL a.i. ha−1 (beta-cypermethrin), 63 g a.i. ha−1 (imidacloprid), 157.5

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g a.i. ha−1 (thiamethoxam), 300 mL a.i. ha−1 (dimethoate) and 30 mL a.i. ha−1 (bifenthrin).

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Three days after pesticide application, about 6 kg of fresh tea shoots (two leaves and a

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bud) were harvested from treated plot and control plot, then brought to the laboratory.

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Processing of tea leaves. Fresh tea shoots from the field trial were processed in the

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Anhui Agricultural University shared laboratory’s mini-manufacturing unit, using a

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conventional green, black or oolong tea manufacturing process. The manufacturing

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processes for green and black tea were according to our previous study.16 The

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manufacturing process for oolong tea consisted of five steps: withering (step I), the

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shoots are spread out in the sunshine at an ambient temperature of about 30 °C for 1–3 h;

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zuo-qing (step II), the withered leaves are shook, rubbed and collided, then allowed to

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stand at a room temperature of 25 °C and 70 % relative humidity, step II was repeated

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many times to damage the leaf margin and redden the tea; chao-qing (step III), the tea

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leaves are put into a continuous rotary fixation machine to inactivate the oxidative

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enzymes at 200–300 °C for 5 min; rolling (step IV), the tissue is twisted and ruptured to

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express the juice for 30 min; and drying (step V), the rolled leaves are dried in a tea dryer

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using hot air at 110 ± 5 °C for about 10 min, then dried at 70 – 75 °C for 20 min to a final

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moisture content of less than 6%.

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Rinsing and brewing tea. The positive tea samples (3.0 g) were rinsed with boiled water

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(50 mL) by swirling rapidly for 5 s then steeping for 5, 10, 20, or 30 s in a 200 mL glass

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cup. Control positive tea samples were not rinsed before brewing. Samples were treated

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this way in duplicate or triplicate. In order to further investigate the transfer of 8

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pesticides from treated tea leaves to three sequential brew infusion, the samples rinsing

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after 30 s were brewed with 150 mL of boiled water for 5 min, with the cup sealed with a

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cap. This was collected as the first infusion. Fresh boiled water (150 mL) was then added

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to the spent tea and collected as the second infusion. This was repeated again for the third

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infusion. The tea rinsate and three tea infusions after brewing were vacuum filtered

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through microporous qualitative filter paper into a filter flask.

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Sample preparation for pesticide residue analysis using a QuEChERS method.

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Made Tea. To prepare the samples for LC-MS, tea samples after Step V were smashed

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(50 mesh) and weighed (1.0 g) into a 50-mL centrifuge tube. Water (2 mL) was added to

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hydrate the tea and let stand for 30 min. Acetonitrile (20 mL) was added, and the solution

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was rocked for 2 min. Anhydrous MgSO4 (2 g) and 2 g NaCl were added, and the tube

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was vortexed immediately for 2 min. After centrifugation (5000 rpm, 5 min), 2 mL of the

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supernatant was transferred into a 5-mL centrifuge tube containing 150 mg PVPP, 50 mg

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PSA, 10 mg GCB and 150 mg anhydrous MgSO4.26 The mixture was shaken for 2 min

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and centrifuged at 10000 rpm for 10 min. The supernatant (1 mL) was reduced to near

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dryness by evaporating with a weak nitrogen stream. Finally, the residue was dissolved in

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1 mL acetonitrile/water (2:8, v/v) and then filtered through a 0.22 μm PTFE filter for

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HPLC-MS/MS analysis.

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To prepare the samples for GC-MS,27 the tea samples after Step V were smashed (50

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mesh) and weighed (1.0 g) into a 50-mL centrifuge tube, gently mixed with 2 mL water,

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and let stand for 30 min. Acetonitrile (10 mL) was added, and the solution was rocked for

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2 min. Anhydrous MgSO4 (1 g) and 1 g NaCl were added into the solution, and the tube

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was vortexed immediately for 2 min and then sonicated for 10 min. After centrifugation

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(8000 rpm, 5 min), all clarified supernatant was transferred into a round bottom flask.

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Repeated extractions with 10 mL acetonitrile were carried out by shaking for 1 min and

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centrifuging (8000 rpm, 5 min). All supernatants were transferred into the same round

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bottom flask and were reduced to about 2 mL by rotary evaporation. This was transferred

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into a TPT-SPE column containing about 2 cm anhydrous sodium sulfate and was

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activated by 4 mL acetonitrile/toluene (3:1, v/v). The round bottom flask was washed

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three times with 2 mL acetonitrile/toluene, with each rinse transferred into the column.

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The loaded TPT column was washed with 25 mL acetonitrile/toluene. All liquid was

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reduced to about 0.5 mL by rotary evaporator, with the outflow collected and then

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reduced to nearly dryness by evaporating with a weak nitrogen stream. The remaining

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residue in the round bottom flask was rinsed with 2 mL acetonitrile/toluene, from which

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1 mL was transferred into a 5-mL centrifuge tube and reduced to near dryness by

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nitrogen steam. The residue was dissolved in 1 mL n-hexane and then filtered through a

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0.22-μm PTFE filter for GC-MS/MS analysis.

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Tea rinsate and infusions. After washing or brewing tea, the rinsate or infusion liquids

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were filtered into a flask, and mixed with 5 g NaCl. The liquid was transferred into a

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separating funnel. Dichloromethane (50 mL) was added into the separating funnel for

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pesticide extraction (shaking 1-2 min) and then was allowed to stand for 5 min. The

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dichloromethane layer was collected into a round bottom flask, while the emulsified layer

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was collected in a 50-ml centrifuge tube and centrifuged at 8000 rpm/min for 5 min so

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that the subnatant could be transferred to the round bottom flask. The extraction solution

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was reduced to nearly dryness by rotary evaporation. The residue was dissolved with 10

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mL acetonitrile. The reconstituted sample (2 mL) was transferred into a 5-mL centrifuge tube

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containing 150 mg PVPP, 50 mg PSA and 10 mg GCB. The mixture was shaken for 2

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min and centrifuged at 10000 rpm for 10 min. The supernatant (1 mL) was transferred

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and reduced to nearly dryness by nitrogen flow, and then the residue was dissolved by 1

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mL acetonitrile/water (2:8, v:v) and filtered through a 0.22-μm PTFE filter for

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HPLC-MS/MS analysis.

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The remaining 8 mL of the acetonitrile mixture was reduced to nearly dryness by

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rotary evaporation. n-Hexane (2 mL) was added to dissolve the residue, which was then

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filtered through a 0.22-μm PTFE filter for GC-MS/MS analysis.

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Analysis of the functional components of tea infusions. The brewed tea infusion (1 mL)

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was filtered through a 0.22-μm PTFE filter for HPLC analysis.

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HPLC-MS/MS analysis. The LC−MS/MS system included an Agilent Series 1200 high

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performance liquid chromatography system (HPLC) and an Agilent 6430 triple

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quadrupole mass spectrometer (QQQ; Agilent Technologies, Palo Alto, CA, USA). The

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HPLC system was equipped with a binary pump, a vacuum solvent degasser, a column

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oven, and an autosampler. An Agilent SB- C18 column (particle size, 1.8 μm; length, 50

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mm; internal diameter, 2.1 mm) was used with a solvent flow rate of 0.2 mL min−1. The

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column compartment temperature was set at 40 °C. The injection volume was 10 μL.

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Mobile phase A was acetonitrile and B was water. The solvent gradient was as follows:

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0−2 min 80% B, 2−6 min 20% B, 6−8 min 20% B, 8−9 min 1.0% B, 9−10 min 1.0% B,

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10−10.01 min 80% B and 10.01−18 min 80% B.

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The mass spectra were acquired using electrospray ionization (ESI) in positive

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ionization mode. Analyses of pesticides were performed in multiple reaction monitoring

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(MRM) mode. The drying gas flow was 9.5 L min−1 with a drying gas temperature of 300

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°C. Nebulizer pressure was 40 psi. Nitrogen was used as the nebulizer and collision gas.

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The monitored fragment ions of the pesticides were selected as quantitation and

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confirmation ion. The parameters of mass spectrometric analysis for the 5 pesticides were

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optimized as shown in Table S1.

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GC-MS/MS Analysis. Volatile and thermally stable pesticides, such as bifenthrin,

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beta-cypermethrin and fenvalerate, were analyzed by GC-MS/MS using a Shimadzu

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GCMS-TQ8040. Separation was performed on a Rtx-5 MS column (30 m × 0.25 mm ×

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0.25 μm). The column temperature was maintained at 60 °C for 1.0 min and then ramped

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at 40 °C min−1 to 240 °C, held for 0 min, increased at 3 °C min−1 to 253 °C (held for 2

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min), and then at 10 °C min−1 to 280 °C (held for 6 min). The injection volume was 1.0

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μL, and splitless mode was adopted. Helium (99.99%) at a flow rate of 1.0 mL min−1 was

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used as the carrier gas and argon (99.99%) as the collision gas. The injection port and ion

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source were 280 and 230 °C, respectively. The triple quadrupole mass spectrometer was

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operated in electron impact ionization mode. Three mass transitions were acquired for

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each compound, with the one showing the highest sensitivity used for quantification. The

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GC-MS/MS parameters for 3 pesticides are shown in Table S1. 11



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HPLC Analysis. The HPLC determination of polyphenol and alkaloid compounds was

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modified from the published method.28 The Series 1260 HPLC system (Agilent

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Technologies, Palo Alto, CA, USA) included a quaternary pump, integrated vacuum

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degasser, autosampler, thermostated column compartment, and diode array detector. An

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Agilent ZORBAX SB-Aq column (4.6 × 250 mm, 5 μm) was used as the analytical

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column. The mobile phase A was 0.2 % (v/v) formic acid – water and B was methanol.

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The detection wavelength was set at 278 nm. The injection volume was 5 μL for each

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sample extract. The temperature of the column was kept constant at 40 °C. The flow rate

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was 1.0 mL/min, and the gradient elution was as follows: 0–5 min, 5–20% B; 5–18 min,

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20–25% B; 18–25 min, 25–42% B; 25–32 min, 42% B; 32–40 min, 42–100% B; 40–42

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min, 100-5% B and 42–55 min, 5% B.

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Calculating the Transfer Rate of Pesticides. The residue concentration and percentages

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of the transfer of each pesticide were calculated. To calculate the residue concentration of

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pesticide, the residue concentration of each pesticide in the rinsate or the first, second and

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third infusion were added together (eq 1):

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RCi = Ci × Vi / M (1)

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where RCi is the residue concentration of pesticide that can be transferred from a certain

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amount of made tea (μg kg-1), Ci is the concentration of pesticide detected in tea rinsate

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or the 3 infusions (ng mL−1), Vi is the volume of tea rinsate or infusion (mL), M is the

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mass of made tea (g).11

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The transfer rate was an indirect calculation of pesticides in the rinsate or brewed tea

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infusion (eq 2)

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TR = RCi / RCm × 100% (2)

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where TR is the transfer rate (%), RCi is the residue concentration of pesticide that can be

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transferred from a certain amount of made tea (μg kg-1), and RCm is the residue

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concentration of pesticide in the made tea (μg kg−1).11

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Calculating the Risk of Exposure to Each Pesticide Residue through Tea

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Consumption. The risk of exposure to unacceptable levels of each of the 8 pesticides in

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tea infusion after rinsing or brewing was evaluated by comparing the estimated daily

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intakes (EDI) and the acceptable daily intake (ADI) for a 60-kg person. The daily average

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intake of tea was obtained from the GEMS/Food Cluster Diets database.29 Tea belongs to

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the tea and mate beverages group, and China belongs to G09, where daily average intake

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is estimated to be 1.123 g of tea day-1. The estimated daily intakes (EDI) are calculated

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from equation 3 (eq 3); while the dietary risk assessment was performed by calculating

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the hazard quotient (HQ) (eq 4).30-33

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EDI = residue concentration × average intake / 60 (3)

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HQ = EDI / ADI × 100% (4) 13



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where EDI is the estimated daily intake (mg kg-1 bw-1), residue concentration is obtained

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from the residue concentration of each pesticide during rinsing or brewing tea, the

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average intake is obtained from GEMS/Food Cluster Diets database, and 60 kg is the

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average weight for Chinese adult. HQ is the hazard quotient, and ADI is the acceptable

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daily intake (mg kg-1 bw-1). The risk is considered unacceptable when HQ is higher than

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100%.33

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Calculation of decreases in the exposure risk to each pesticide residue through

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rinsing tea. The exposure risks were calculated for each pesticide in tea infusions brewed

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with and without rinsing. The hazard quotient (HQ) of the tea rinsate was subtracted from

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the HQ of tea brewed without a pre-rinse:

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HQd = HQc - HQr (5)

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where HQd is the hazard quotient of a tea infusion after rinsing for different lengths of

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time, HQc is the hazard quotient of control tea infusion without rinsing, HQr is the hazard

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quotient of the tea rinsate.

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The exposure risk decrease level (RDL) for each pesticide residue was calculated as:

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RDL = 1 - HQd / HQc (6)

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RESULTS AND DISCUSSION

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Method Validation. The detection method was evaluated for its selectivity, linearity,

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accuracy, precision, and limits of quantification (LOQs).11 A QuEChERS extraction and

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cleanup method was designed based on current lab protocols26 for both the dried tea

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leaves after Step IV and the tea rinsate and infusion liquids (Fig. 1). Pesticide-free (blank)

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tea (3 g) was brewed with 150 mL of boiling water for 5 min before the infusion liquid

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was collected. To generate calibration curves, smashed blank green tea (1 g) or the tea

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infusion was spiked with pesticide standards and then processed according to the

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extraction methods as above. The targets peaks were observed for HPLC-MS/MS and

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GC-MS/MS (Fig. S1). The HPLC-MS/MS linearity, with correlation coefficients (r2)

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>0.995 for each of the 5 pesticides including three neonicotinoids and two

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organophosphates, ranged from 5 to 500 μg L−1. The linearity for GC-MS/MS, with r2

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>0.998 for 3 pesticides including three pyrethroids analyzed, was 10−500 μg L−1. The

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accuracy of the method was verified by measuring the recovery from blank green tea

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leaves and infusion. The recovery rates from made tea ranged from 70 to 109%, with

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relative standard deviation (RSD, n=5) values ranging from 1 to 20% for the 8 pesticides

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analyzed at the spiked levels of 0.01 (0.1) and 0.02 (0.2) mg kg-1 for the dried tea by

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HPLC-MS/MS and GC-MS/MS; The recovery rates of the 5 pesticides in tea infusions

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were ranged from 70 to 109% at the spiked level of 5 (50) μg L−1, with RSDs ranging

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from 2 to 20 % by HPLC-MS/MS; The recovery rates in tea infusions were spiked at 10

289

and 100 μg L−1 for the 3 pesticides and showed recovery rates of 62 – 67% and 74 –

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101%, with RSDs of 9 – 16% and 10 – 18% by GC-MS/MS. The LOQs of the 8

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pesticides were 1−20 μg kg−1 in processed tea leaves and 0.005−0.010 μg mL −1 in tea 15



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infusions.

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Pesticide Transfer after Rinsing and Brewing Tea. Tea is usually brewed twice,

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because the taste and aroma of the first and second infusions are favorable, but decline in

295

subsequent infusions.11 This study investigated the transfer of 8 pesticides from treated

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tea leaves to water after different lengths of rinsing (5, 10, 20, 30 s) and after three

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sequential brews (Table 1). Only the transfer rates (%) are discussed for clarity, but the

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transfer amount values showed the same trends. In green tea, the transfer rates increased

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with the increase in rinsing time (5 to 30 s) for 5 pesticides, namely dimethoate (10.8 to

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16.7%), malathion (8.4 to 12.6%), acetamiprid (7.1 to 13.3%), imidacloprid (7.8 to 10.5%)

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and thiamethoxam (3.8 to 5.6%). In black tea, a similar increase in transfer during longer

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rinsing time was seen for dimethoate, acetamiprid, imidacloprid and thiamethoxam, the

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malathion transfer rate from black tea increased from 8.5% at 5 s to its highest value of

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13.1% at 20 s and then decreased to 12.0% after 30 sec of rinsing. However, there was no

305

clear upward trend for the transfer rates of the same five pesticides with longer rinsing

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time in the oolong tea. In the green, black and oolong tea, longer rinsing time did not

307

clearly affect the transfer of pyrethroids (bifenthrin, beta-cypermethrin, fenvalerate).

308

The results showed that the transfer rates of the 8 pesticides into the water used to

309

rinse the three types of tea leaves were between 0.2% and 24% after 5 to 30 seconds. The

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transfer rates of the 8 tested pesticides were not related to the type of tea, in accordance

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with a previous study by Chen et al.11 The transfer rates of thiamethoxam, bifenthrin,

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beta-cypermethrin and fenvalerate were lower than those of the other 4 pesticides. For 16


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bifenthrin, beta-cypermethrin and fenvalerate, this is possibly due to their lower water

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solubility and higher log Kow (Table 2).11,

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might be because it binds to suspended organic matter in tea infusion (proteins,

316

carbohydrates, pigments etc.),13 which should be confirmed through further study. In this

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study, the highest transfer rate was 24% for dimethoate from black tea during the 30-s

318

rinse, either because dimethoate has the highest water solubility (2.5×104 mg L-1, 21 °C)

319

among the 8 tested pesticides (Table 2), or because dimethoate was released during the

320

rolling of black tea, as the tissue was broken, and attached to the leaf surface.

18

The low transfer rate of thiamethoxam

321

This study further investigated the transfer of 8 pesticides from treated tea leaves to

322

three sequential brew infusion after 30 sec of rinsing (Table 1). In green, black and

323

oolong tea, for 5 pesticides (dimethoate, malathion, acetamiprid, imidacloprid and

324

thiamethoxam), the transfer rates decreased with each brew. The brew number did not

325

clearly affect the transfer of pyrethroids (bifenthrin, beta-cypermethrin, fenvalerate) in

326

three types of tea. The greatest transfers into the first brew (49.6%) were for imidacloprid

327

from the black tea, so it showed the highest exposure in the infusion of three types of tea

328

after rinsing for 30 s.

329

Risk of Exposure to Pesticide Residues in Brewed Tea Infusion. In order to compare

330

the exposure risk of different catalogue of pesticide residues in tea brew infusion, we

331

evaluated the risk of pesticides exposure in total tea infusion (unrinsed, tea mix-infusion

332

for brewing three times) by calculating the hazard quotient (HQc). The results showed

333

that the HQc to all pesticide residues in the total brew infusion of three types of tea were 17



334

from 0.0018% to 1.2308% which were significantly lower than risk level (=100%)

335

(Table 3). The risk of each pesticides in tea infusion is considered acceptable. Among

336

these 8 pesticide residues, the HQc to dimethoate has the highest value in black tea.

337

In order to show if rinsing these three types of tea lowers the risk of exposure to

338

different pesticides, a dietary risk assessment and a risk decrease level were calculated for

339

each pesticide residue. The risk decrease level was defined as the decrease in the level of

340

potential health risk of the brewed tea infusion through rinsing (Table 3). The risk was

341

expressed by the hazard quotient (HQ) (Fig. 2 and Table 3). The exposure risk decrease

342

level (RDL) to imidacloprid, acetamiprid, dimethoate, malathion, thiamethoxam,

343

bifenthrin, beta-cypermethrin, and fenvalerate in the brewed green tea infusion were 17%,

344

18%, 28%, 18%, 25%, 27%, 28%, and 29% after the 30 s rinse; these were the largest

345

decreases seen for each insecticide in rinses of green tea. In the black tea, increasing the

346

rinsing length from 5 to 30 s decreased the risk in the brewed tea infusion by 19%, 35%,

347

19%, and 27%, for acetamiprid, dimethoate, thiamethoxam, and fenvalerate, respectively

348

(Fig. 2B, 2C, 2E, 2H and Table 3). The imidacloprid exposure risk decreased by 14%

349

after rinsing for both 20 s and 30 s, and beta-cypermethrin exposure risk decreased by

350

24% after rinsing for 10 s or 30 s (Fig. 2A, 2G and Table 3). Rinsing black tea decreased

351

the malathion exposure risk in the brewed tea infusion by 13% after 5 s of rinsing, by

352

20% after 10 s or 20 s of rinsing, and by 19% after 30 s of rinsing (Fig. 2D and Table 3).

353

Rinsing black tea reduced the bifenthrin exposure risk in the brewed infusion by 51% to

354

59% (for 5 s or 10 s rinses), or by 31% after rinsing for 30 s (Fig. 2F and Table 3). 18


Page 18 of 38

Page 19 of 38


355

Rinsing oolong tea decreased the exposure risk to imidacloprid, acetamiprid, dimethoate,

356

malathion, thiamethoxam, bifenthrin in the infusion by 9% - 41% (Fig. 2A - F and Table

357

3). The RDLs to beta-cypermethrin and fenvalerate went from 31% and 30% after 5 s of

358

rinsing to 39% and 38% after 20 s of rinsing, then to 35% and 34% after 30 s of rinsing

359

(Fig. 2G - H and Table 3).

360 361

Levels of Functional Components in Rinse Water and Infusion. Rinsing tea before

362

brewing may also remove functional and sensory components, such as polyphenols and

363

alkaloids. The important polyphenols in tea are gallic acid (GA), epigallocatechin gallate

364

(EGCG), and gallocatechin gallate (GCG), while theobromine and caffeine are the major

365

alkaloids. The levels of these tea components in the rinse water and brewed tea infusion

366

were analysed by HPLC. The results showed that rinsing removed 0% to 11% of these 5

367

functional components (Fig. 3 and Table 4), indicating that people can drink brewed tea

368

after rinsing and expect that most nutritional components are retained.

369

In conclusion, rinsing tea leaves in boiled water removed between 0.2% and 24% of

370

the residues of 8 different pesticides from 3 types of tea. Rinsing tea before brewing can

371

reduce the risk of exposure by 5% - 59% in the brewed tea infusion. While a short

372

pre-rinse with a small volume of boiling water can significantly reduce the health risk

373

from most pesticide residues, the loss of nutrients (0 - 11%) was negligible. Therefore,

374

rinsing tea before brewing is strongly recommended for these three types of tea. While

375

the health risk posed by pesticide residues can be reduced, some methods of rinsing are 19



376

better than others, some pesticides are removed at greater rates, and no method can

377

remove all residues. Therefore, it remains that the overall application of pesticides should

378

be reduced, perhaps with a special focus on those residues that are not removed in great

379

quantities by a simple water rinse.

380

Acknowledgements

381

This work was supported by the National Natural Scientific Foundation of China

382

(No. 31772076 and No. 31270728), the National Key Research & Development Program

383

(2016YFD0200900) of China, the Earmarked fund for China Agriculture Research

384

System (CARS-19 ） and the Natural Science Foundation for Distinguished Young

385

Scholars of Anhui Province (1608085J08).

20


Page 20 of 38

Page 21 of 38


387

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the

grades

(in

of

Chinese).

Keemun

GEMS/Food

E.;

Kurdziel,

Chinese

black

Cluster

A.;

Matyaszek,

National

tea

by

Diets

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Podbielska,

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492

26


Page 26 of 38

Page 27 of 38


Table 1. Residue concentration (main number in each column) and transfer ratio (in parentheses) of eight pesticides from green, black and oolong tea into rinse water or tea infusion.

494 495 496 497

Pesticide residue concentration from green tea leaves to infusion (μg kg-1) ± SD pesticides

rinse time (s) 5

dimethoate

malathion

acetamiprid

10

m bifenthrin

ethrin fenvalerate

first brew

second brew

third brew

concentration without

pesticide in green

rinsing in green tea

tea (μg kg-1) ± SD

infusion (μg kg-1) ± SD

178.1±5.5ab

220.8±24.8b

344.7±19.6

160.5±7.3

70.6±3.0

1321.2

796.8

(10.8)

(12.2)

(13.5)

(16.7)

(26.1)

(12.2)

(5.3)

±70.9

±38.6

87.5±3.8a

107.3±8.3b

111.8±6.0b

130.6±6.0c

327.3±28.3

176.2±16.2

100.5±8.9

1036.3

734.6

(8.4)

(10.4)

(10.8)

(12.6)

(31.6)

(17.0)

(9.7)

±227.4

±57.3

632.8±55.5ab

636.7±44.9a

757.6±29.7b

1181.7±47.1c

2617.0±91.3

1765.2±157.1

1099.2±41.6

8888.5

6663.1

±245.1

±164.4

2134.5

1316.2

±195.3

±27.4

5735.7

1270.4

±263.3

±64.7

(7.2)

165.9±4.1

(8.5)

166.3±25.0

a

219.2±10.9 (3.8)

227.6±52.2

273.3±25.4

(4.0)

15.1±3.6

23.1±2.3ab

(0.4)

(0.5)

21.3±1.1a

(0.4)

(8.7)

28.2±3.8

(0.6)

21.9±0.7

(0.6)

29.0±1.4c

(0.5)

90.8±33.6

(7.7)

28.2±1.4c

26.4±1.1bc

(5.5)

80.1±10.6

(7.6)

(0.5)

23.8±1.4ab

(0.4)

79.0±53.9

25.1±1.7bc

315.3±28.2

(7.7) a

(1.3)

(16.1)

439.3±62.6

(5.6) a

(3.0)

18.9±1.0a

319.7±30.9

14.0±2.3

a

343.8±13.4

(26.1) a

(4.8)

31.5±15.9

a

(10.5) a

(19.9)

557.8±41.6

b

(8.6) a

(29.4)

224.9±13.7

a

(7.8) a

(13.3)

183.0±2.6

a

(1.4) beta-cyperm

30

concentration of

161.1±15.4a

(7.8) thiamethoxa

20

pesticide residue

143.3±0.0a

(7.1) imidacloprid

brew number

residue

(0.4)

27.3±2.1

(0.5)

22.7±0.4

(0.5)

(0.4)

(12.4) 189.7±8.4 (8.9) 196.1±5.5 (3.4) 45.5±12.6 (4.4) 21.8±1.9 (0.4) 22.4±1.2 (0.4)

Pesticide residue concentration from black tea leaves to infusion (μg kg ) ± SD -1

pesticides

rinse time (s) 5

10

brew number

20

30

first brew

second brew

third brew

1044.0

295.4

±67.7

±58.2

5119.5

100.1

±389.0

±6.3

5832.1

101.3

±474.2

±4.1

residue

pesticide residue

concentration of


pesticide in black

rinsing in black tea

tea (μg kg ) ± SD


1896.0

1314.9

-1

dimethoate

258.2±22.5a (13.6)

(16.4)

(21.0)

(24.0)

malathion

46.7±5.2a

70.0±8.4b

71.6±7.4b

65.6±6.2b

(12.8)

(13.1)

(12.0)

(8.5) acetamiprid

968.1±64.1a (11.0)

imidacloprid

m

1112.2±122.3ab (12.6)

190.2±29.8a (7.6)

thiamethoxa

311.2±31.5a

365.0±21.8 (5.6)

221.7±42.2ab

411.6±54.1

bifenthrin

95.6±11.8

beta-cyperm

21.2±1.2a

(3.7)

109.5±51.8 (4.2) 29.5±2.1b

313.2±39.0bc

511.3±32.1

108.8±49.9 (4.2) 26.8±0.2ab

(33.5)

(25.4)

1559.7±168.4c

3511.4±186.2 (39.9)

327.6±47.4c

1232.8±163.7 (49.6)

595.4±84.2

c

(9.2) a

635.6±42.0

138.9±31.3

(13.2) bc

(7.9) a

455.0±19.9b

(17.7)

(12.6) ab

(6.4) a

1407.5±127.3b (16.0)

(8.9) a

398.9±47.0b

1411.7±180.2 (21.8)

57.2±28.3

a

(2.2)

41.2±15.2 (1.6)

29.8±3.9b

26.3±3.1

174.4±19.3 (9.2)

49.8±3.4

±116.1

±43.1

88.3±8.5

56.4±5.5

546.7

349.2

(16.2)

(10.3)

±72.1

±41.0

8795.4

8132.4

±371.1

±316.2

2487.7

2319.5

±52.6

±344.3

6468.0

3113.5

±208.1

±318.4

2597.8

186.5

±214.8

±39.2

9669.9

123.8

1975.1±129.1 (22.5) 429.7±240.0 (17.3) 692.8±97.4 (10.7) 53.7±11.4 (2.1) 37.1±4.1

27


(2.6)

1086.3±38.9 (12.4) 329.5±30.3 (13.2) 413.6±49.6 (6.4) 34.3±9.7 (1.3) 30.5±8.9


ethrin fenvalerate

(0.2)

(0.3)

23.1±0.6a

(0.3)

29.2±2.1b

(0.2)

(0.3)

27.4±0.1ab

(0.3)

(0.3)

30.0±3.0b

(0.2)

(0.4)

25.3±2.2

(0.3)

31.2±2.5

(0.2)

(0.3)

Page 28 of 38

(0.3) 26.4±4.7 (0.2)

Pesticide residue concentration from oolong tea leaves to infusion (μg kg ) ± SD -1

pesticides

rinse time (s) 5

dimethoate

10

271.7±48.7a (17.6)

malathion

803.9±109.7a

208.2±33.7 (7.0)

thiamethoxa m bifenthrin

353.7±55.7 (4.7)

ethrin fenvalerate

60.3±44.9

(0.6)

326.0±41.9

53.3±15.1

(0.5)

42.2±5.6ab

38.6±3.6ab (0.6)

181.8±28.4

317.2±30.6

39.9±11.6

(0.7) 43.1±3.0b (0.6)

862.0±44.3a

218.2±18.7

1066.1±108.5 (36.0)

362.6±10.0

a

1002.9±93.6 (13.3)

41.7±12.1

a

(2.6)

33.5±16.5 (2.1)

42.1±0.4ab (0.7)

29.8±4.7 (0.5)

39.2±1.3ab (0.6)

3069.9±180.2 (29.5)

a

(4.8) a

46.4±3.3b

(35.4)

(7.4) a

(2.5)

128.1±14.9

(8.3) a

(4.2) a

(0.7)

34.8±1.5a

666.1±126.4a

430.9±36.9 (28.0)

(13.0)

(6.1) a

(3.3)

36.8±2.3a

498 499 500 501 502 503 504

204.7±76.7

265.7±29.0a

46.9±11.3a

(6.4) a

(4.3) a

(3.7) beta-cyperm

685.4±75.9a

first brew

(17.2)

(15.6)

(6.9) a

219.1±48.5a

56.4±12.9a

(6.6) a

30

(14.2)

(14.7)

(7.7) imidacloprid

206.1±19.3a

53.3±11.1a

(16.9) acetamiprid

20

(13.4)

61.2±11.8a

brew number

28.5±3.4 (0.4)

second brew

133.2±3.9 (8.6) 73.7±13.6 (20.4) 1883.7±49.9 (18.1) 498.0±13.1 (16.8) 530.6±17.1 (7.0) 30.2±7.8 (1.9) 23.8±1.4 (0.4) 23.3±0.5 (0.3)

±875.7

±12.3

11159.6

112.8

±1262.7

±7.4

residue

pesticide residue

concentration of


pesticide in oolong

rinsing in oolong tea

tea (μg kg-1) ± SD


1541.4

874.6

±66.9

±9.6

41.0±7.0

361.9

289.7

(11.3)

±17.8

±17.8

10420.1

6879.7

±368.1

±270.0

2957.5

2056.2

±161.9

±128.2

7560.7

2213.1

±531.1

±85.8

third brew

44.9±4.2 (2.9)

1064.1±104.0 (10.2) 273.9±44.3 (9.3) 317.0±41.4 (4.2) 40.9±14.6 (2.5) 24.0±1.8 (0.4) 23.4±1.3 (0.3)

1618.0

146.4

±123.9

±25.4

6415.2

119.7

±516.4

±3.6

6927.0

114.4

±709.0

±3.8

Brew number: round of brewing tea after rinsing for 30 s; Numbers in parentheses () are the transfer rates, in %, based on the total pesticides in the tea leaves; a, b, c: significant differences between the rinse time, 5 - 30 s, within the same type of tea are indicated with different letters (P 175.1

13

106

acetamiprid

3.429

223.1 > 126.0

21

223.1 > 56.1

13

102

dimethoate

3.008

230.0 > 199.0

5

230.0 > 171.0

10

80

malathion

10.026

331.1 > 127.0

5

331.1 > 99.0

10

80

thiamethoxam

1.734

292.1 > 211.1

6

292.1 > 181.2

22

76

GC-MS/MS Pesticide

Rt (min)

ion transitions (m/z)

CE (eV)


CE (eV)


CE (eV)

bifenthrin

11.145

181.1 > 166.1

12

181.1 > 153.1

8

181.1 > 179.1

12

beta-cypermethrin

15.711

181.1 > 152.1

22

181.1 > 127.1

22

181.1 > 77.0

24

225.1 > 147.1

10

225.1 > 119.1

20

419.1 > 125.1

6

15.784 fenvalerate

17.125 17.575

584

Rt: retention time; CE: collision energy

36


Page 37 of 38


586

587 588 589 590 591 592

Figure S1. Multiple reaction monitoring chromatograms for blank green tea infusion spiked at 0.005 μg mL-1 of 5 pesticides (acetamiprid, imidacloprid, thiamethoxam, dimethoate and malathion) for HPLC-MS/MS analysis (A) and at 0.01 μg mL-1 of 3 pesticides (bifenthrin, beta-cypermethrin and fenvalerate) for GC-MS/MS analysis (B).

593

37



595

Page 38 of 38

TOC

596

Pesticides Tea Processing

Rinse/Brew

Green tea Black tea Oolong tea

Residue in soup 5s 10s 20s 30s 30s+15min

1200 900 600

Nutrient in soup 5s 10s 20s 30s 30s+15min

32000 24000 16000

300

8000

0

green tea black tea oolong tea

597

40000

EGCG (ug/g)

Dimethoate (ug/kg)

1500

0

green tea black tea oolong tea

598 599

38


Rinsing tea before brewing decreases pesticide residues in tea

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