Lonicera japonica

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Degradation dynamics and dietary risk assessments of two neonicotinoid insecticides during Lonicera japonica planting, drying and tea brewing processes Qingkui Fang, Yanhong Shi, Haiqun Cao, Zhou Tong, Jinjing Xiao, Min Liao, Xiangwei Wu, and Rimao Hua J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04658 • Publication Date (Web): 08 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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Journal of Agricultural and Food Chemistry

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Degradation dynamics and dietary risk assessments of two

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neonicotinoid insecticides during Lonicera japonica planting, drying

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and tea brewing processes

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Qingkui Fang1, a, Yanhong Shi1, b, Haiqun Cao*, a, Zhou Tonga, Jinjing Xiaoa, Min Liaoa, Xiangwei

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Wub, Rimao Huab

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a School of Plant Protection, Provincial Key Laboratory for Agri-Food Safety, Anhui Agricultural

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University, Hefei 230036, China.

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b School of Resource & Environment, Provincial Key Laboratory for Agri-Food Safety, Anhui

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Agricultural University, Hefei, 230036, China.

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* Corresponding Author. Email address: [email protected]

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The first two authors contributed equally to this work.

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ACS Paragon Plus Environment


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Abstract

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The degradation dynamics and dietary risk assessments of thiamethoxam and thiacloprid

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during Lonicera japonica planting, drying and tea brewing processes were systematically

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investigated using high-performance liquid chromatography. The half-lives of thiamethoxam and

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thiacloprid were 1.0-4.1 d in the honeysuckle flowers and leaves, degradation rate constants k

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ranged from -0.169 to -0.696. And the safety interval time was 7 d. The sun- and oven-drying

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(70 °C) percent digestions were 59.4-81.0% for the residues, which were higher than the shade-

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and oven-drying percent at lower temperatures (30, 40, 50 and 60 °C, which ranged from 37.7% to

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57.0%). The percent transfer of thiamethoxam and thiacloprid were 0-48.4% and 0-25.2%,

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respectively, for the different tea brewing conditions. Based on the results of this study, abiding by

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the safety interval time is important and using reasonable drying methods and tea brewing

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conditions can reduce the transfer of thiamethoxam and thiacloprid to humans.

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Key words: Lonicera japonica, neonicotinoid insecticide, degradation dynamics, dietary risk

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assessment, drying method, tea brewing condition

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1. Introduction

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Honeysuckle, the flowering buds of Lonicera japonica Thunb. (Caprifoliaceae), is one of the

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most popular traditional Chinese medicinal herbs and contains a variety of biologically active

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ingredients, including polyphenols, saponins, volatile oils, and iridoids.1,2 Pharmacological studies

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have shown that the bioactive ingredients of honeysuckle possess broad-spectrum antibacterial,

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antiviral, anti-inflammatory, hepatoprotective and choleretic properties that enhance the immune

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system.3-7 Honeysuckle is also used in indigenous beverages in Korea and China.1 Because L.

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japonica is prone to attack by several pests and diseases, many pesticides are used to protect crops,

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which can result in pesticide residues in agriproducts.

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Thiamethoxam and thiacloprid are systemic, contact and chloronicotinyl neonicotinoid

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insecticides that act as an agonist on postsynaptic nicotinic acetylcholine receptors and modify

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insect behavior, resulting in death. They are widely used as insecticides for controlling aphids,

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thrips, diamondback moths and white flies.8,9 Residues of these two pesticides, which are

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potentially hazardous to consumers, can be found in L. japonica and environmental samples due to

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the high prevalence of aphids on L. japonica plantings.10-12 Therefore, it is necessary to study the

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degradation dynamics and dietary risk assessments of thiamethoxam and thiacloprid in L. japonica

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to assure human and environmental safety.

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There have been several studies of the residue behavior and dietary risk assessment of

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pesticides in agriproducts and environmental samples. Yu et al.13 reported the residual dynamics of

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thiacloprid in the medical herbs marjoram, thyme, and chamomile. Abd-Alrahman14 investigated

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the residue and dissipation kinetics of thiamethoxam in potato plants and soil in a field ecosystem.

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Some researchers have reported on the effects of handling and processing pesticide residues in raw



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cucumber,15 button crimini,16 olive oil,17 and camellia oil18. There are recent studies of the effect

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of tea brewing conditions on pesticide residues in herbal tea and tea.19-24 These studies found

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several critical controlling factors, such as tea variety, tea/water ratio, tea brewing time, and

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washed or unwashed tea. However, there is an issue with these studies that must be addressed. The

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entire process, from pesticide application to commercial and home processing to final

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consumption, should be studied rather than examining residue behavior and dietary risk

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assessment separately.

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The aim of this research was to develop a systematic study that (i) investigates the

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degradation dynamics of pesticide residues in L. japonica; (ii) studies the effects of honeysuckle

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drying methods on the percent digestion of pesticide residues; and (iii) evaluates the effects of

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honeysuckle brewing conditions on the percent transfer of pesticides. We hope that this systematic

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research will aid in understanding how the proportions of degradation and percent transfer of the

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pesticides in L. japonica planting, drying and tea brewing processes affect dietary risk. We used

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high-performance liquid chromatography (HPLC) to determine thiamethoxam and thiacloprid

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residues and investigate the degradation dynamics and dietary risk assessments of thiamethoxam

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and thiacloprid in L. japonica planting, drying and tea brewing processes. The results of this work

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will not only contribute to establishing adequate monitoring of thiamethoxam and thiacloprid

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residues and their use in pest management strategies in Chinese herbs but also provide significant

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guidance to the systematic study of degradation dynamics and dietary risk assessments in other

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contaminated agriproducts.

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2. Materials and Methods

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2.1 Chemicals and Instruments


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Thiamethoxam and thiacloprid were purchased from Dr. Ehrenstorfer (Augsburg, Germany).

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Acetonitrile (liquid chromatography grade) as well as acetonitrile, petroleum ether, dichlomethane,

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and acetone (analytical grade) were all purchased from the Chinasun Specialty Products Co., Ltd.

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(Jiangsu, China). Anhydrous sodium sulfate and sodium chloride were obtained from the

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Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China). A Florisil solid-phase extraction

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(SPE) column (1000 mg·6 mL-1) was purchased from Agela Technologies (Tianjin, China).

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High-performance liquid chromatography (HPLC) was performed using a Shimadzu

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LC-20AT instrument (Shimadzu, Japan). An HC-C18 column (4.6×250 mm, 5 µm) was used from

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Agilent Technologies (Palo Alto, CA, USA). An EYELA N-1100 rotary evaporator was used from

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the Shanghai Ailang Instruments Co., Ltd. (Shanghai, China). A SC-3610 low-speed centrifuge

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was used from Anhui Zhongke Zhongjia Scientific Instruments Inc. (Anhui, China). A KQ-5200

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ultrasonic cleaner was used from the Kunshan Ultrasonic Instrument Co., Ltd. (Shanghai, China).

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A SQ-2119B multifunctional food processing instrument was used from the Shanghai Shuaijia

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Electronic Technology Co., Ltd. (Shanghai, China). A DHG-9070A electrothermal oven

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thermostat blast was used from the Shanghai Yiheng Science and Technology Co., Ltd. (Shanghai,

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China). An LM-02 Chinese medicine grinder was used from the Zhejiang Dahai medicine factory

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Instrument (Zhejiang, China).

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2.2 Field trials of residue degradation dynamics

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Field trials were carried out at the Sijiyuyang L. japonica planting base in Tongcheng City

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(31°N, 117°E), Anhui Province, China. During the experiment, the temperature was 20-35 °C and

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the rainfall was light. A plot size of 20 m2 was selected for the control and each treatment of the

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pesticide under study, in a treatment plot is about 10 honeysuckle plants, leaving two rows of



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bushes as guard rows between the control plot and the different treatment plots. The two pesticides

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(thiamethoxam and thiacloprid) were sprayed at two times of the recommended dosages [i.e., the

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recommended doses for aphid control were 56.25 g a.i. hm-2 (thiamethoxam) and 102.5 g a.i. hm-2

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(thiacloprid), leading to 112.5 g a.i. hm-2 (thiamethoxam) and 205 g a.i. hm-2 (thiacloprid)] in three

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replicates using a hand-operated knapsack sprayer with a recommended formulation volume of 15

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L hm-2. The use of five-point sampling method, 0.5 kg honeysuckle flower samples and 0.5 kg leaf

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samples were then picked at 2 h and 1, 3, 5, 7, 14 and 21 days.

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Final residue trials

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The two pesticides were sprayed at two dosages (recommended dosage and two times of the

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recommended dosage; 56.25 and 102.5 g a.i. hm-2 (thiamethoxam) and 112.5 and 205 g a.i. hm-2

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(thiacloprid)) for three replicates. The plants were sprayed once every 7 days, with continuous

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spraying three times. And then honeysuckle samples were collected on the 7th and 14th days after

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the last application.

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2.3. Sample extraction, cleanup, and HPLC analysis

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2.3.1 Honeysuckle flower sample treatment

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Honeysuckle flower samples (2.0 g) were extracted with 20 mL of acetonitrile by thoroughly

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shaking for 30 min, centrifuging at 4,000×g for 5 min, and transferring the supernatant to a 150

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mL separatory funnel. An additional 20 mL of acetonitrile was added to the samples and the

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mixture was thoroughly shaken for 30 min and centrifuged at 4,000×g for 5 min. The supernatant

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was transferred to the separatory funnel. Then, 30 mL of petroleum ether was added to the

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separatory funnel, shaken for 2 min, and left standing for 5 min. The lower-layer liquid was

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transferred to a 150 mL flask and concentrated close to dryness at 40 °C.


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A Florisil SPE was activated with 3 mL of a petroleum ether:acetone (v/v=7:3) mixed

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solution. The honeysuckle flower extract was washed with the mixed solution (2 mL×2) and

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transferred to the SPE column, which was rinsed by the mixed solution (2 mL×2) to remove

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interfering substances. The eluent was discarded. The SPE column was then eluted with

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acetonitrile (3 mL×3), and the eluate was concentrated to dryness at 40 °C. The volume was set to

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5 mL and placed in a volumetric flask with acetonitrile to await measurement.

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2.3.2 Leaf sample treatment

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Leaf samples (5.0 g) were extracted with 30 mL of acetonitrile by thoroughly shaking for 30

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min. The samples were then centrifuged at 4,000×g for 5 min, and the supernatant was transferred

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to a 150 mL separatory funnel. An additional 30 mL of acetonitrile was added to the samples, and

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the mixture was thoroughly shaken for 30 min and centrifuged at 4,000×g for 5 min. The

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supernatant was transferred to the separatory funnel and concentrated close to dryness at 40 °C.

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A Florisil SPE was activated with 3 mL of a petroleum ether:acetone (v/v=7:3) mixed

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solution. The leaf extract was washed with the mixed solution (2 mL×2) and transferred to the

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SPE column, which was rinsed by the mixed solution (2 mL×2) to remove pigments and other

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interfering substances. The eluent was discarded. The SPE column was then eluted with

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acetonitrile (3 mL×3), and the eluate was concentrated to dryness at 40 °C. The volume was set to

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5 mL and placed in a volumetric flask with acetonitrile to await measurement.

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2.3.3 Tea infusion sample treatment

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The boiling water was used for extraction, and after the tea infusion was cooled to room

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temperature, 20 mL of the tea infusion was added to a separatory funnel along with 20 mL of a

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NaCl saturated solution and 30 mL of dichloromethane. The mixture was thoroughly shaken for 2



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min and left standing for 5 min; the organic phase was then transferred to a 150 mL flask. Another

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20 mL of NaCl saturated solution and 30 mL of dichloromethane were added to the samples and

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the mixture was thoroughly shaken for 2 min and left standing for 5 min; the organic phase was

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then transferred to a 150 mL flask and concentrated close to dryness at 40 °C. The volume was set

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to 5 mL and placed in a volumetric flask with acetonitrile to await measurement.

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HPLC analyses of the thiamethoxam and thiacloprid were followed measuring absorbance at

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254 nm and 270 nm (UV), respectively. The mobile phase was acetonitrile:water (v/v=25:75) at a

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flow rate of 1 mL·min-1. An HC-C18 column (4.6×250 mm, 5 µm) was used. The temperature of

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the column oven was maintained at 30 °C with an injection volume of 20 µL.

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2.4 Effect of drying method

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To study the degradation of thiamethoxam and thiacloprid during the honeysuckle drying

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process, the two pesticides were sprayed at two dosages [recommended dosage and two times of

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the recommended dosage; 56.25 and 102.5 g active ingredient per square hectometer (a.i. hm-2)

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thiamethoxam and 112.5 and 205 g a.i. hm-2 thiacloprid] using a recommended formulation

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volume of 15 L hm-2. Honeysuckle samples were collected at 2 h post application when the

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spraying mixture had dried.

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The effects of the three drying methods on the degradation rate of the two pesticides were

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investigated, including sun-, shade- and oven-drying. For shade-drying, 0.5 kg samples of low and

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high concentrations of pesticides were spread over 1 m2 of area. Because the honeysuckle could

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not be turned, proper indoor ventilation was ensured to keep the space relatively dry. For

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sun-drying, 0.5 kg samples of low and high concentrations of pesticides were spread over 1 m2 of

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area (the honeysuckle could not be turned). For oven-drying, the samples were placed in an oven


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with the temperature set to 30, 40, 50, 60 and 70 °C (the honeysuckle could not be turned). All

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treatments were performed until the samples were fully dry (i.e., the weight no longer changed).

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2.5 Effects of tea brewing conditions of honeysuckle samples

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Pesticide concentration, tea/water ratio (TWR), number of infusions, tea brewing time, and

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covered or uncovered cups were investigated as the tea brewing conditions. The pesticide-free

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honeysuckle sample was treated with the standard solution of pesticide. The samples were

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separately sprayed with the pesticide solutions and left to stand at room temperature for 1 h. Tea

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brewing refers to preparing a hot water infusion of the honeysuckle. Infusions were prepared with

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tap water, using a stainless steel electric kettle to boil the tap water. Two grams of treated samples

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were immersed in boiling water and allowed to stand at room temperature for 2-60 min. After tea

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brewing, the infusions were filtered through an ordinary stainless steel tea strainer and cooled.

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Both the infusions and the spent leaves after tea brewing were examined separately for residues.

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The procedure was carried out in three replications.

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Effect of Pesticide Concentration. The pesticide-free honeysuckle samples were separately

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sprayed with three different concentrations of pesticide solutions and left to stand at room

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temperature for 1 h. Two grams of three different concentrations of pesticide treated samples were

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immersed in 100 mL of boiling water and then retained at room temperature for 10 min.

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Effect of Number of Infusions. Two grams of samples were immersed in 100 mL of boiling

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water and left at room temperature for 10 min. The liquid portion was decanted and collected in

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another enclosed cup; this was the first infusion. Then, another 100 mL of boiling tap water was

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poured into the original cup to brew the residual tea and to obtain a second infusion in the same

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manner as the first tea brewing. A third infusion was also obtained by the same process.



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Effect of Tea Brewing Time. Two grams of samples were immersed in 100 mL of boiling water

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and left to stand at room temperature for 2, 5, 10, 15, 20, 30 and 60 min.

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Effect of Tea/Water Ratio. Two grams of samples were immersed in 60, 100, 160 and 200 mL of

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boiling water and left to stand at room temperature for 10 min. The ratios were 1/30, 1/50, 1/80

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and 1/100 (w/v tea infusion).

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Effect of Uncovered Cup. Two grams of samples were infused in 100 mL of boiling water,

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enclosed in a 100 mL covered cup and left to stand at room temperature for 10 min. Another 2.0 g

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sample was infused in 100 mL boiling water in a 100 mL uncovered cup and left to stand at room

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temperature for 10 min.

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2.6 Data analysis

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The relationship between the two pesticide residues and time is Ct = C0e-kt, where Ct (mg/kg)

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is the residue after time t, C0 (mg/kg) is the initial residue, and k is the dissipation rate constant

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(d-1). This equation is widely used to assess the level of pesticides in agriproducts.25,26

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The percent transfer of thiamethoxam and thiacloprid from tea infusion was calculated using the following formula:

transfer (%)

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=

Ct × Vt C 0 × W0

× 100%

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where Ct represents the concentration (µg/mL) of thiamethoxam and thiacloprid in the tea infusion,

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Vt is the volume (mL) of the tea infusion, C0 is the content (µg/g) of thiamethoxam and thiacloprid

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in the brewed tea, and W0 is the weight (g) of the tea used in the infusion.22

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3. Results and discussion

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3.1 Recovery study

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The limits of detection (LOD) were determined as the sample concentration of thiamethoxam


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and thiacloprid at a signal-to-noise ratio of 3:1 by HPLC. The LOD of thiamethoxam and

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thiacloprid were both estimated at 0.01 mg kg-1 for the honeysuckle flower and leaf samples, and

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0.01 mg L-1 for the tea infusion samples. The analyte recoveries and precision values obtained

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from the validation study are summarized in Table 1. Thiamethoxam and thiacloprid were added

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to the untreated control samples at 0.05, 0.1 and 1.0 mg kg-1 for the honeysuckle flower and tea

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infusion and 0.1, 0.5 and 1.0 mg kg-1 for the leaves. For method validation, the control and treated

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samples were analyzed under the same conditions. The mean values of five replicates at each

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spiked level were recorded. The recoveries of thiamethoxam and thiacloprid ranged from 77.8% to

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99.2%. The coefficients of variation (CV) were between 1.2% and 10.5% for the three samples.

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Thus, these results demonstrate that the HPLC method can be used for sample determination.

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3.2 Degradation dynamics of the pesticides in honeysuckle flowers and leaves

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The data on degradation dynamics obtained for thiamethoxam and thiacloprid in the

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honeysuckle flowers and leaves are shown in Figure 1. A gradual and continuous deterioration of

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pesticide residues in the two treated samples was observed. The average residues of thiamethoxam

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and thiacloprid in honeysuckle (Figure 1A) were 3.2 and 4.1 mg kg-1, respectively, after 2 h of the

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application of two times of the recommended dosages and the residual amounts of the two

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pesticides dissipated by 95.3% and 98.4%, respectively, after 7 days. The half-life values (t½) for

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the degradation of thiamethoxam and thiacloprid in the honeysuckle were calculated to be 2.2 d

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and 4.1 d (Table 2), respectively, after application of the recommended dosage, and the dynamics

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are described by the equations C = 2.518e-0.3108t and C = 3.452e-0.169t, with R2 = 0.9184 and R2 =

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0.8639, respectively. The average residues of thiamethoxam and thiacloprid in the leaves (Figure

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1B) were 2.3 and 4.4 mg kg-1, respectively, after 2 h of the application of two times of the



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recommended dosages, and the residual amount of the two pesticides dissipated by 96.5% and

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91.0%, respectively, after 5 days. The half-lives of thiamethoxam and thiacloprid in the leaves was

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1.0 d and 1.1 d (Table 2), respectively, and the dynamics are described by the equations C =

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2.438e-0.696t, R2 = 0.9918 and C = 5.466e-0.613t, R2 = 0.8552, respectively. Based on previously

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published studies27,28, sunlight, evaporation and rainfall elution are important factors affecting the

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degradation of pesticides, especially sunlight photodegradation. The two pesticides faster

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dissipated in leaves, because of the different structure of leaves and honeysuckle, when light,

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rainfall and evaporation occurs, the surface area of the leaves was larger than that of honeysuckle.

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When thiamethoxam and thiacloprid were applied at two times of the recommended dosages,

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the initial deposits of thiamethoxam (2 h) were 3.2 mg kg-1 in the honeysuckle flowers and 2.3 mg

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kg-1 in the leaves and the initial deposits of thiacloprid (2 h) were 4.1 mg kg-1 in the honeysuckle

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flowers and 4.4 mg kg-1 in the leaves. The residuals of the two pesticides were evenly distributed

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in the honeysuckle flowers and leaves.

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3.3 Final residue of pesticides in honeysuckle

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The final residues of thiamethoxam and thiacloprid were measured at 7 and 14 days after the

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last application of the recommended and two times of dosages. The results are shown in Table 3.

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Residues of thiamethoxam in the honeysuckle were undetectable (

Lonicera japonica

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